The development of a novel approach to the design of ......Last but not least, I would like to...

The development of a novel approach to the design of microdevices

Submitted in total fulfillment of the requirements for the degree of

Doctor of Philosophy

by

Yulia Alekseeva

Faculty of Life and Social Sciences

Swinburne University of Technology

December 2011

ii

Abstract

The effectiveness of protein-based microdevices depends on the ability of their

surfaces to provide spatial immobilization and maintain protein bioactivities. Although

methodologies for the construction of microdevices for biomedical applications have

been developed, the manufacturing of microdevices remains expensive due to the high

cost of materials and fabrication processes. As the surfaces display structural

uniformities which restrict protein-surface interactions and consequently protein

immobilization, innovative approaches to the design of surfaces are required. The

approaches need to allow for the minimization of fabrication costs via efficient

amplification and spatial immobilization of multiplex proteins so that the bioactivity

of protein-based microdevices (e.g., microarrays) can be retained.

A novel approach to the design of surfaces for microdevices has been

developed and evaluated in this work. This approach is based on micro/nanostructures

fabricated via laser ablation of a thin metal layer deposited on a transparent polymer.

The structures of a 100 nm-range are represented by „combinatorialized‟ micro/nano-

channels that allow amplified protein immobilization in a highly controlled manner.

The relationship between the properties of the micro/nano-channel surface

topography, physico-chemistry, and protein immobilization, for five, molecularly

different proteins, i.e., lysozyme, myoglobin, alpha-chymotrypsin, human serum

albumin, and human immunoglobulin has been investigated. Using quantitative

fluorescence measurements and atomic force microscopy, protein immobilization on

microstructures has been characterized. It has been found that the combinatorial nature

of the micro/nano-channels allowed a 3 to 10-fold amplification of protein adsorption,

as compared to the protein adsorption on flat, chemically homogenous polymeric

surfaces. An improved methodology allowing in vitro assembled micron- and nano-

scale tracks of proteins (i.e., actin) which support unidirectional translocation of beads

functionalized with motor proteins (i.e., myosin) was also developed. The nanotracks

composed of aligned F-actin/gelsolin bundles were formed by electrostatic

condensation of F-actin/gelsolin with Ba2+.

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The prospects for employment of bacterial ATP producers as replacements for

the energy source, and prokaryotic actin homologues as replacements for eukaryotic

actin in microdevices based on molecular motor-based systems, have been explored. A

search for ATP producers among 86 environmental strains of 17 genera, including 4

species of 3 genera described in this thesis has been performed. Bacteria belonging to

the genera Sulfitobacter, Marinobacter and Staleya and/or Planococcus and Kocuria

have been found to be promising producers of extracellular and intracellular ATP,

respectively. Substitution of eukaryotic actin with inherently stable prokaryotic actin-

related proteins, i.e., MreB or FtsA, may point the way to the development of the next

generation of microdevices for biomedical applications.

iv

Acknowledgments

This thesis resulted from research supported by the Australian Research

Council (ARC) and partially supported by the Defense Advanced Research Projects

Agency (DARPA).

I acknowledge the great support of the Research Higher Degrees Committee

(RHDC) of my alma mater.

I enjoyed working on my project. I had the great honor and pleasure of being

supervised by a person with a very strong interdisciplinary vision, Professor Elena

Ivanova. I acknowledge her incredible support and encouragement. I thank Professor

Michael Gilding for his kind words of encouragement and support. I acknowledge the

support provided by Professor Pam Green. I thank Professor Russell Crawford for his

support.

I also wish to thank Professor Dan V. Nicolau for his support; Dr Vlado Buljan

and Dr Murat Kekic (the University of Sydney) for sharing their experience in

molecular motor protein extraction and handling; Dr Igor Sbarski, Dr Gregory M.

Demyashev, Dr Luisa Filipponi, Dr Andrea Viezzoli, Dr Dan V. Nicolau, Jr.,

Dr Jonathan P. Wright, Dr Duy K. Pham, Marjan Ilkov, Dr Hans Brinkies, Anya

Ilkova, Dr Natasa Mitik-Dineva for assisting in laboratory experiments and data

analysis. I thank all LSS and IRIS staff members who supported this study. I thank my

student teammates who have shared their thoughts with me. I enjoyed a friendly

scientific atmosphere of our group meetings.

I acknowledge the support of Professor Tomoo Sawabe and Dr Karin Hayashi

(Hokkaido University), Professor Richard Christen (the University of Nice Sophia

Antipolis), Dr Nataliya I. Kalinovskaya, Dr Natalia V. Zhukova, Dr Galina M.

Frolova, Professor Valery V. Mikhailov, Dr Nataliya M. Gorshkova, Dr Valeriya V.

Kurilenko, Dr Olga I. Nedashkovskaya (Pacific Institute of Bioorganic Chemistry)

v

and Arkady Kurilenko (Pacific Oceanological Institute). I thank Professor Victor P.

Chelomin (Pacific Oceanological Institute) for fruitful scientific discussions.

I would also like to thank Maryna Mews for her editorial assistance.

Last but not least, I would like to express my greatest appreciation to my

darling parents, Lyubov & Vladimir, for participating in my son‟s upbringing and

supporting this study. Special thanks are extended to all those who helped me in

gaining sole custody of my child. And lastly, I would love to thank my son, Maxim,

for being a good guy.

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This thesis is dedicated to my son, Maxim.

vii

DECLARATION

I certify that the work presented in the thesis contains no material which has been

submitted for another degree of any other university. To the best of my knowledge it

does not contain any material previously published or written by another person

except where due reference is made in the text.

Contributions of the respective researchers to this study: Professor Elena Ivanova

supervised the research project; Professor Dan V. Nicolau organized the project;

Dr Vlado Buljan and Dr Murat Kekic (the University of Sydney) assisted in protein

extraction and handling; Dr Igor Sbarski, Dr Gregory M. Demyashev, Dr Luisa

Filipponi, Dr Andrea Viezzoli, Dr Dan V. Nicolau, Jr., Dr Jonathan P. Wright, Dr Duy

K. Pham, Marjan Ilkov, Dr Hans Brinkies, Anya Ilkova, Dr Natasa Mitik-Dineva

assisted in laboratory experiments and data analysis; Professor Tomoo Sawabe and Dr

Karin Hayashi (Hokkaido University), Professor Richard Christen (the University of

Nice Sophia Antipolis), Dr Nataliya I. Kalinovskaya, Dr Natalia V. Zhukova, Dr

Galina M. Frolova, Professor Valery V. Mikhailov, Dr Nataliya M. Gorshkova, Dr

Valeriya V. Kurilenko, Dr Olga I. Nedashkovskaya (Pacific Institute of Bioorganic

Chemistry), Professor Victor P. Chelomin and Arkady Kurilenko (Pacific

Oceanological Institute) assisted in microbiology-related experiments and data

analysis.

I declare that this thesis has been partially professionally copyedited and proofread by

Maryna Mews, however, the editorial assistance did not affect its substantive content.

Yulia Alekseeva

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List of Publications

Book chapter 1. Critical aspects in microfluidic systems design. Alekseeva YV, Crawford RJ,

Ivanova EP. In: Advances in Chemistry Research 15, Nova Publishers (NY), 2012.

Journal articles 2. Protein immobilisation on micro/nanostructures fabricated by laser

microablation. Nicolau DV, Ivanova EP, Fulga F, Filipponi L, Viezzoli A, Dobroiu S, Alekseeva YV, Pham DK. Biosensors and Bioelectronics 26(4):1337-1345, 2010.

3. “Pseudoalteromonas januaria" SUT 11 as the source of rare lipodepsipeptides.

Kalinovskaya NI, Dmitrenok AS, Kuznetsova TA, Frolova GM, Christen R, Laatsch H, Alexeeva YV, Ivanova EP. Current Microbiology 56(3):199-207, 2008.

4. ATP level variations in heterotrophic bacteria during attachment on

hydrophilic and hydrophobic surfaces. Ivanova EP, Alexeeva YV, Pham DK, Wright JP, Nicolau DV. International Microbiology 9(1):37-46, 2006.

5. A comparative study between the adsorption and covalent binding of human

immunoglobulin and lysozyme on surface-modified poly(tert-butyl methacrylate). Ivanova EP, Wright JP, Pham DK, Brack N, Pigram P, Alekseeva YV, Demyashev GM, Nicolau DV. Biomedical Materials 1(1):24-32, 2006.

6. Characterization of unusual alkaliphilic gram-positive bacteria isolated from

degraded brown alga thalluses. Ivanova EP, Wright JP, Lysenko AM, Zhukova NV, Alexeeva YV, Buljan V, Kalinovskaya NI, Nicolau DV, Christen R, Mikhailov VV. Microbiological Journal 68(4):10-20, 2006.

7. Controlling the covalent and noncovalent adsorption of proteins on polymeric

surfaces in aqueous liquids. Ivanova EP, Pham DK, Alekseeva YV, Demyashev GM, Nicolau DV. Chinese Journal of Light Scattering 17(3):234-236, 2005.

8. Presence of ecophysiologically diverse populations within Cobetia marina

strains isolated from marine invertebrate, algae and the environments. Ivanova EP, Christen R, Sawabe T, Alexeeva YV, Lysenko AM, Chelomin VP, Mikhailov VV. Microbes and Environments 20(4):200-207, 2005.

9. Controlled self-assembly of actin filaments for dynamic biodevices.

Alexeeva YV, Ivanova EP, Pham DK, Buljan V, Sbarski I, Ilkov M, Brinkies HG, Nicolau DV. Nanobiotechnology 1(4):379-388, 2005.

10. Bacillus algicola sp. nov., a novel filamentous organism isolated from brown

alga Fucus evanescens. Ivanova EP, Alexeeva YV, Zhukova NV, Gorshkova NM, Buljan V, Nicolau DV, Mikhailov VV, Christen R. Systematic and Applied Microbiology 27(3):301-307, 2004.

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11. Brevibacterium celere sp. nov., isolated from degraded thallus of a brown alga. Ivanova EP, Christen R, Alexeeva YV, Zhukova NV, Gorshkova NM, Lysenko AM, Mikhailov VV, Nicolau DV. International Journal of Systematic and Evolutionary Microbiology 54:2107-2111, 2004.

12. Formosa algae gen. nov., sp. nov., a novel member of the family

Flavobacteriaceae. Ivanova EP, Alexeeva YV, Flavier S, Wright JP, Zhukova NV, Gorshkova NM, Mikhailov VV, Nicolau DV, Christen R. International Journal of Systematic and Evolutionary Microbiology 54(3):705-711, 2004.

13. Low-molecular-weight, biologically active compounds from marine Pseudoalteromonas species. Kalinovskaya NI, Ivanova EP, Alexeeva YV, Gorshkova NM, Kuznetsova TA, Dmitrenok AS, Nicolau DV. Current Microbiology 48(6):441-446, 2004.

14. Sulfitobacter delicatus sp. nov. and Sulfitobacter dubius sp. nov., respectively

from a starfish (Stellaster equestris) and sea grass (Zostera marina). Ivanova EP, Gorshkova NM, Sawabe T, Zhukova NV, Hayashi K, Kurilenko VV, Alexeeva Y, Buljan V, Nicolau DV, Mikhailov VV, Christen R. International Journal of Systematic and Evolutionary Microbiology 54(2):475-480, 2004.

15. Impact of cultivation conditions on haemolytic activity of Pseudoalteromonas

issachenkonii KMM 3549T. Alexeeva YV. Kalinovskaya NI, Kuznetsova TA, Ivanova EP. Letters in Applied Microbiology 38(1):38-42, 2003.

16. Ecophysiological variabilities in ectohydrolytic enzyme activities of some

Pseudoalteromonas species, P. citrea, P. issachenkonii, and P. nigrifaciens. Ivanova EP, Bakunina IY, Nedashkovskaya OI, Gorshkova NM, Alexeeva YV, Zelepuga EA, Zvaygintseva TN, Nicolau DV, Mikhailov VV. Current Microbiology 46(1):6-10, 2003.

17. Marinobacter excellens sp. nov., isolated from sediments of the sea of Japan.

Gorshkova NM, Ivanova EP, Sergeev AF, Zhukova NV, Alexeeva Y, Wright JP, Nicolau DV, Mikhailov VV, Christen R. International Journal of Systematic and Evolutionary Microbiology 53(6):2073–2078, 2003.

18. Optimization of glycosidases production by Pseudoalteromonas issachenkonii. Alexeeva YV, Ivanova EP, Bakunina IY, Zvaygintseva TN, Mikhailov VV. Letters in Applied Microbiology 35(4):343-346, 2002.

19. Pseudoalteromonas issachenkonii sp. nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens. Ivanova EP, Sawabe T, Alexeeva YV, Lysenko AM, Gorshkova NM, Hayashi K, Zukova NV, Christen R, Mikhailov VV. International Journal of Systematic and Evolutionary Microbiology 52(1):229-234, 2002.

20. Two species of culturable bacteria associated with degradation of brown algae Fucus evanescens. Ivanova EP, Bakunina IY, Sawabe T, Hayashi K, Alexeeva YV, Zhukova NV, Nicolau DV, Zvaygintseva TN, Mikhailov VV. Microbial Ecology 43(2):242-249, 2002.

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Referred Conference proceedings 1. Amplification of protein adsorption on micro/nanostructures for microarray

applications. Ivanova EP, Alekseeva YV, Pham DK, Filipponi L, Nicolau DV. NSTI Proceedings 1:95-98, 2004.

2. Microlithographically fabricated bar-coded microarrays. Ivanova EP, Pham

DK, Alekseeva YV, Filipponi L, Nicolau DV. SPIE Proceedings 5328:49-55, 2004.

3. Actin nanotracks for hybrid nanodevices based on linear protein molecular

motors. Watson GS, Cahill C, Blach J, Myhra S, Alexeeva Y, Ivanova EP, Nicolau DV. MRS Proceedings 820:25-35, 2004.

4. Immobilization of multiple proteins in polymer microstructures fabricated

via laser ablation. Ivanova EP, Viezzoli A, Alekseeva YV, Demyashev GM, Nicolau DV Jr, Filipponi L, Pham DK, Nicolau DV. SPIE Proceedings 4966:37-49, 2003.

Poster presentations with published abstracts

1. Protein adsorption on micro/nano-structures fabricated by laser microablation. Nicolau DV, Ivanova EP, Alexeeva YV, Viezzoli A, Pham DK, Dobroiu S et al. 20th Anniversary World Congress on Biosensors, P2.1.127, 2010.

2. Variations in ATP levels in heterotrophic bacteria during biofilm formation. Ivanova EP, Alexeeva YV, Pham DK, Wright JP, Nicolau DV. “ASM 2004 Sydney National Conference”, 2004.

3. Controlled self-assembly of actin filaments for nanobiotechnological devices.

Alexeeva YV, Ivanova EP, Buljan V, Nicolau DV. “ASM 2004 Sydney National Conference”, 2004.

4. The optimization of fermentation processes of Pseudoalteromonas

issachenkonii. Alexeeva YV, Ivanova EP, Bakunina IY, Zvyagenseva TN, Mikhailov VV. Conference “Marine Bio Shizuoka-2001”, p. 98, 2001.

5. Microbial community of Pseudoalteromonas issachenkonii and Halomonas

marina degrade the tallus of brown algae Fucus evanescens. Ivanova EP, Alexeeva YV, Bakunina IY, Zvyagenseva TN, Mikhailov VV. 9th Int Symposium on Microbial Ecology, Amsterdam, p. 314, 2001.

6. Convertible energy of living organisms. Alexeeva YV, Burceva RА. II regional

conference “Problems of marine biology, ecology and biotechnology”, Vladivostok, FESU, p. 6, 1999.

_____________________________________________________________________ *My surname Alekseeva can be spelled either with “x” or with “ks”.

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TABLE OF CONTENTS Abstract ii

Acknowledgments iv

Declaration vii

List of publications viii

List of abbreviations xix

List of tables xxiii

List of figures xxv

List of schemes xxviii

Chapter 1. Introduction 1

1.1. Overview 2

1.2. Aim of the study 5

1.3. Organization of the thesis 5

Chapter 2. Literature review 7

2.1. Overview 8

2.2. Concept and benefits of biosensors 8

2.3. Types of biosensors 11

2.4. Microfluidic devices 13

2.4.1. Overview 13

2.4.2. Advantages of microfluidic devices 14

2.4.3. Critical aspects of microfluidic devices 17

2.4.3.1. Overview 17

2.4.3.2. Surface properties 17

2.4.3.3. Microfluidic device geometry 20

2.4.3.4. Fluid properties 21

2.4.4. Types of microfluidic devices 23


2.4.4.2. Droplet system 23

2.4.4.3. Continuous system 26

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2.4.5. Control of microfluidic devices 28


2.4.5.2. Control of fluidic movement 28

2.4.5.3. Control of fluidic interactions 30

2.4.5.4. Immobilization of proteins 33

2.4.5.4.1. Overview 33

2.4.5.4.2. Physical adsorption 33

2.4.5.4.3. Covalent binding 35

2.4.5.4.4. Self-assembled monolayers (SAMs) 37

2.5. Concept of protein molecular motors 39

2.5.1. Overview 39

2.5.2. Eukaryotic actin 39

2.5.3. Prokaryotic actin related proteins 42


2.5.3.1.1. MreB 43

2.5.3.1.2. FtsA 46

2.5.4. Evolution/Phylogeny of bacterial actin homologues 48


2.5.4.2. Evolutionary/Phylogenetic comparison of MreB with FtsA 48

2.5.4.3. Use of 16S rRNA as a molecular chronometer 50

2.5.5. Classification of protein molecular motors 52


2.5.5.2. Linear molecular motors 53

2.5.5.3. Rotary molecular motors 55

2.5.6. Native functions of molecular motors 57


2.5.6.2. Cytoskeleton 58

2.5.6.3. Cellular metabolism 60

2.5.6.4. Flagella-based motion 62

2.5.6.5. Tactics of enteric pathogens 65

2.5.6.5.1. Overview 65

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2.5.6.5.2. Common tactics of enteric pathogens 65

2.5.6.5.3. Listeria as a regulator of actin assembly 67

2.5.6.6. Bacterial ATP generation 70

2.5.6.7. Use of MreB and FtsA proteins by bacteria 72

Chapter 3. Methodology 76

3.1. Overview 77

3.2. Methods used to study protein-surface interactions 78

3.2.1. Protein preparation for immobilization on polymeric surfaces 78

3.2.2. Polymeric film preparation 78

3.2.3. Preparation of microfabricated structures 79

3.2.4. Protein adsorption on surfaces 80

3.2.4.1. Protein adsorption on flat surfaces 80

3.2.4.2. Protein adsorption on micro/nano-fabricated structures 80

3.2.5. Protein covalent binding onto surfaces 80

3.2.6. Detection and quantification techniques 81

3.2.6.1. Fluorescence spectroscopy of adsorbed proteins 81

3.2.6.2. X-ray photoelectron spectroscopy 81

3.2.6.3. Goniometry 82

3.2.6.4. Ellipsometry 82

3.2.6.5. Atomic force microscopy (AFM) 83

3.2.6.6. Calculation of protein-surface parameters 83

3.3. Methods of actin/myosin preparation 84

3.3.1. Actin and heavy meromyosin (HMM) preparation 84

3.3.2. Preparation of the electrostatically condensed actin bundles 85

3.3.3. Preparation of the polymeric surfaces 85

3.3.4. Protein immobilization on the polymeric surfaces in the flow cell 86

3.3.5. Beads functionalization 86

3.3.6. Fluorescence microscopy 87

3.3.7. Scanning electron microscopy (SEM) 87

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3.3.8. X-ray photoelectron spectroscopy 87

3.3.9. Rheological measurements 88

3.4. Methods of bacterial taxonomy 88

3.4.1. Bacterial isolation 88

3.4.1.1. Isolation of gram-negative bacteria 88

3.4.1.1.1. Isolation of Marinobacter excellens 88

3.4.1.1.2. Isolation of Sulfitobacter delicatus and Sulfitobacter dubius 89

3.4.1.2. Isolation of gram-positive bacteria 90

3.4.1.2.1. Isolation of Planococcus maritimus 90

3.4.2. Bacterial characterization 90

3.4.2.1. Phenotypic analysis 90

3.4.2.1.1. General phenotypic tests 91

3.4.2.1.1.1. Microscopic examination 91

3.4.2.1.1.2. Utilization of organic substrates 92

3.4.2.1.1.3. Degradation of macromolecules 92

3.4.2.1.1.4. Cytotoxic and antibacterial activities 93

3.4.2.1.1.5. Susceptibility to antibiotics 93

3.4.2.1.2. Species-specific phenotypic tests 93

3.4.2.2. Chemotaxonomic methods 94

3.4.2.2.1. Polar lipid (PL) analysis 94

3.4.2.2.2. Fatty acid (FA) analysis 94

3.4.2.3. Genotypic analysis 95

3.4.2.3.1. DNA GC content determination 95

3.4.2.3.2. DNA hybridization 95

3.4.2.4. Phylogenetic analysis 96

3.4.2.4.1. 16S rRNA gene analysis 96

3.5. Methods used to assess ATP production by bacteria 97

3.5.1. Bacterial strains 97

3.5.2. Polymeric surface preparation 100

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3.5.3. Contact angle measurements 101

3.5.4. Bacterial growth and sample preparation 101

3.5.5. Bioluminescence assay for ATP determination 102

3.5.6. Cell-surface characterization by AFM 102

3.6. Methods used to assess MreB and FtsA proteins 103

3.6.1. Analysis of mreB and ftsA genes 103

3.6.2. Computation of MreB and FtsA protein parameters 104

Chapter 4. Immobilization of proteins on flat surfaces 105

4.1. Overview 106

4.2. Results and discussion 106

4.2.1. PtBMA film characterization 106

4.2.2. Adsorption and covalent binding of selected HIgG on PtBMA surface 110

4.2.2.1. X-ray photoelectron spectroscopy analyses 110

4.2.2.2. Ellipsometry analysis 114

4.2.2.3. AFM analysis 116

4.3. Conclusion 119

Chapter 5. Advantage of immobilization of proteins in microchannels 121

5.1. Overview 122


5.2.1. Characterization of poly(methyl methacrylate) polymeric films 123

5.2.2. Fabrication of microstructures in Au-deposited PMMA films 123

5.2.3. Impact of molecular descriptors on protein adsorption on microstructures 130

5.2.4. Characterization of thickness of polymeric films and attached proteins 134

5.2.5. Protein adsorption in PMMA-based channels and

on native PMMA films 137

5.2.6. Characterization of adsorption properties of selected proteins 139

5.3. Conclusion 142

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Chapter 6. Control of self-assembly of actin filaments

for dynamic microdevices 144

6.1. Overview 145


6.2.1. Polymeric surface characterization 146

6.2.2. Effectiveness and stability of G-actin self-assembly 146

6.2.2.1. Adsorption and self-assembly of G-actin

on selected polymeric surfaces 146

6.2.2.2. Evaluation of covalent bonding of G-actin


6.2.2.3. Covalent bonding and self-assembly of G-actin


6.2.3. Alignment of self-assembled actin filaments

along fabricated microstructures 151

6.2.4. Fabrication of electrostatically self-assembled actin filaments bundles 152

6.3. Conclusion 158

Chapter 7. Characterization of potential ATP,

MreB and FtsA producers 160

7.1. Overview 161


7.2.1. Phenotypic and chemotaxonomic classification 162

7.2.1.1. Gram-negative marine bacteria belonging to the genera Sulfitobacter

and Marinobacter 162

7.2.1.1.1. Phenotypic and chemotaxonomic properties of Sulfitobacter delicatus 162

7.2.1.1.2. Phenotypic and chemotaxonomic properties of Sulfitobacter dubius 165

7.2.1.1.3. Phenotypic and chemotaxonomic properties of Marinobacter excellens 166

7.2.1.2. Gram-positive bacteria belonging to the genus Planococcus 168

7.2.1.2.1. Phenotypic and chemotaxonomic properties of Planococcus maritimus 168

xvii

7.2.2. Genotypic and phylogenetic characterization 173

7.2.2.1. Gram-negative marine bacteria belonging to the genera

Sulfitobacter and Marinobacter 173

7.2.2.1.1. Genotypic and phylogenetic characterization of Sulfitobacter delicatus

and Sulfitobacter dubius 173

7.2.2.1.2. Genotypic and phylogenetic characterization of

Marinobacter excellens 175

7.2.2.2. Gram-positive bacteria belonging to the genus Planococcus 177

7.2.2.2.1. Genotypic and phylogenetic characterization of Planococcus maritimus 177

7.3. Conclusion 179

7.3.1. Classification of gram-negative marine isolates 179

7.3.2. Classification of gram-positive marine isolates 180

Chapter 8. Characterization of ATPases activities of marine bacteria 182

8.1. Overview 183


8.2.1. Levels of ATP detected in heterotrophic bacteria of different taxa 184

8.2.2. Pattern of bacterial growth on surfaces 186

8.2.3. Effect of polymeric surfaces on intracellular ATP generation 188

8.2.4. Variation in extracellular ATP generation 189

8.2.5. AFM investigation of bacterial surface ultrastructure 190

8.3. Conclusion 196

Chapter 9. Evaluation of MreB, FtsA proteins and actin 198

9.1. Overview 199


9.2.1. Comparison/Evaluation of predicted physicochemical

properties of MreB proteins of selected bacterial taxa and actin 200

9.2.1.1. Stability of MreB proteins and actin 200

9.2.1.2. Isoelectric point (pI) and grand average of

hydropathicity (GRAVY) of MreBs and actin 203

xviii

9.2.1.3. Phylogenetic relationships of MreB producers 205

9.2.2. Comparison/Evaluation of predicted physicochemical

properties of FtsA proteins of selected bacterial taxa and actin 209

9.2.2.1. Stability of FtsA proteins and actin 209

9.2.2.2. Isoelectric point (pI) and grand average of hydropathicity (GRAVY)

of FtsAs and actin 213

9.2.2.3. Phylogenetic relationships of FtsA producers 215

9.3. Conclusion 218

Chapter 10. Conclusions and further work 219

10.1. Conclusions 220

10.1.1. Overview 220

10.1.2. Protein immobilization in „combinatorialized‟ micro/nano-channels 220

10.1.3. Controlled self-assembly of actin filaments along microchannels in a

continuous-flow system 221

10.1.3.1. Search for bacterial ATP producers to be used as replacements for

the energy source in microdevices 222

10.1.3.2. Evaluation of prokaryotic actin-related proteins, MreB and FtsA, as

possible replacements for eukaryotic actin 223

10.2. Future work 224

10.2.1. Advancements of surface modification 224

10.2.2. Incorporation of ATP-producers into microdevices 224

10.2.3. Study of MreB and FtsA proteins in vitro 224

List of References 225

xix

LIST OF ABBREVIATIONS ABP Actin-binding protein

AChE Acetylcholinesterase

ADP Adenosine diphosphate

AFM Atomic force microscopy

AI Aliphatic index

AIEC Adherent invasive Escherichia coli

Arp 2/3 Actin-related protein 2/3 complex

ATP Adenosine triphosphate

ATPase Adenosine triphosphatase

BIONJ An advanced version of the neighbor joining (NJ) algorithm

BLAST Basic local alignment search tool

BSA Bovine serum albumin

BW Biological warfare

Cc Critical concentration

CFB Cytophaga–Flavobacterium–Bacteroides

CIP Collection of the Pasteur Institute

DEP Dielectrophoresis

DNA Deoxyribonucleic acid

DSM German Collection of Microorganisms

DTT Dithiothreitol

ECM Extracellular matrix

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EHD Electrohydrodynamics

Ena/VASP Enabled/vasodilator-stimulated phosphoprotein

EPEC Enteropathogenic Escherichia coli

ESI-MS Electrospray ionization mass spectrometry

F-actin Filamentous actin

Fn Fibronectin

G-actin Globular (or monomeric) actin

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GLS Gelsolin

GLUT Glucose transporter

GRAVY Grand average of hydropathicity

GTP Guanosine triphosphate

HBP Heparin-binding peptide

HDMS Hexamethyldisilazane

HGF Hepatocyte growth factor

HIgG Human immunoglobulin G

HMM Heavy meromyosin

HSA Human serum albumin

HPAEC Human pulmonary artery endothelial cell

Hsp Heat shock protein

iDEP Insulator-based DEP

IMF Ion motive force

II Instability index

KMM Collection of Marine Microorganisms

LOC Lab-on-a-Chip

LPS Lipopolysaccharide

LYZ Lysozyme

MAP Multiphoton absorption polymerization

MCJ Multicellular junction

MCM Mini-chromosome maintenance

MF Motive force

MHD Magnetohydrodynamics

ML Maximum-likelihood

MP Maximum-parsimony

MTS Membrane targeting sequence

NC Nitrocellulose

NCIMB National Collection of Industrial and Marine Bacteria

NJ Neighbor-joining

OP Organophosphate

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OTS Octadecyltrichlorosilane

PAG Photoacid generator

PARP Prokaryotic actin-related protein

PBS Phosphate buffered saline

PC Polycarbonate

PCR Polymerase chain reaction

PDB Protein data bank

pDEP Positive dielectrophoresis

PDMS Polydimethylsiloxane

PE Polyethylene

PETG Poly(ethylene terephthalate glycol)

PGMEA Propylene glycol methyl ether acetate

PHYLIP Phylogeny Inference Package

pI Isoelectric point

PMF Proton motive force

PMMA Poly(methyl methacrylate)

PS Polystyrene

PSMA Poly(styrene-maleic acid)

PtBMA Poly(tert-butyl methacrylate)

rRNA Ribosomal ribonucleic acid

RSA Random sequential adsorption

RT Room temperature

PVC Poly(vinyl chloride)

SAM Self-assembled monolayer

SCOEW Single-sided continuous optoelectrowetting

SCS Specialty coating systems

SCVs Small-colony variants

SE Secondary electron

SEM Scanning electron microscopy

SERS Surface-enhanced Raman spectroscopy

Si Silicon

xxii

SMA Shape memory alloy

STR Short tandem repeat

Sulfo-NHS N-hydroxysulfosuccinimide

TCA Trichloroacetic acid

TEA Trapezoidal electrode array

Tg Thyroglobulin

THF Tetrahydrofurane

TIGER Triangulation identification for genetic evaluation of risks

TTF Triphenylsulfonium triftalate

UV Ultraviolet

VASP Vasodilator-stimulated phosphoprotein

VR Virtual reality

WD Working distance

XPS X-ray photoelectron spectroscopy

xxiii

LIST OF TABLES Table 1. Strains and environmental (marine) bacterial isolates used in the

study.

99

Table 2. Atomic concentration ratios (determined by XPS) obtained for

adsorbed and covalently immobilized human immunoglobulin

(HIgG) and lysozyme (LYZ) layers on activated P(tBMA)

surfaces.

108

Table 3. Elemental compositions (determined by XPS) obtained for

adsorbed and covalently immobilized human immunoglobulin

(HIgG) and lysozyme (LYZ) layers on activated P(tBMA)

surfaces.

110

Table 4. Ellipsometric measurements obtained for adsorbed and covalently

immobilized human immunoglobulin (HIgG) and lysozyme

(LYZ) layers on activated P(tBuMA) surfaces.

114

Table 5. Ellipsometric measurements of thicknesses of adsorbed proteins

and correspondent PMMA polymeric films.

137

Table 6. Characteristics of selected proteins. 140

Table 7. Characteristics that differentiate Sulfitobacter delicatus KMM

3584T and Sulfitobacter dubius KMM 3554T from

phylogenetically related species.

164

Table 8. Characteristics that differentiate Marinobacter excellens from

phylogenetically related species.

167

Table 9. Differential phenotypic characteristics of Planococcus maritimus

and other species of the genera Planococcus and

Planomicrobium.

170

Table 10. Levels of extracellular adenosine triphosphate (ATP) detected in

heterotrophic bacteria of different taxa.

185

Table 11. Comparison of theoretical stability parameters (AI, II and half-

life) of MreB proteins of γ-Proteobacteria (1), α-Proteobacteria

(2), Firmicutes (3), Thermotogae (4) and rabbit actin (5).

202

xxiv

Table 12. Comparison of theoretical pI and GRAVY of MreB proteins of γ-

Proteobacteria (1), α-Proteobacteria (2), Firmicutes (3),

Thermotogae (4) and rabbit actin (5).

205

Table 13. Comparison of theoretical stability parameters (AI, II and half-

life) of FtsA proteins of γ-Proteobacteria (1), α-Proteobacteria

(2), CFB group (3), Firmicutes (4), Thermotogae (5) and rabbit

actin (6).

211

Table 14. Comparison of theoretical pI and GRAVY of FtsA proteins of γ-

Proteobacteria (1), α-Proteobacteria (2), CFB group (3),

Firmicutes (4), Thermotogae (5) and rabbit actin (6).

214

xxv

LIST OF FIGURES Figure 1. Representative surface topography of fluorescence images of human

immunoglobulin (HIgG) adsorbed (top, left) and covalently

immobilized (top, right) and lysozyme (LYZ) adsorbed (bottom,

left) and covalently immobilized (bottom, right) on UV-irradiated

PtBMA surfaces. Similar images were obtained in different regions

of at least two different samples.

109

Figure 2. XPS spectra of PtBMA+COOH surfaces: (a) typical C1s; (b) high-

resolution N 1s spectra of samples „activated‟ by treatment with

EDC and NHS; and (c) sample following covalent protein

attachment; (d) high-resolution S 2p spectra of samples „activated‟

by treatment with EDC and NHS; and (e) of samples following

covalent protein attachment.

111

Figure 3. Representative surface topography images and their corresponding

line profile analyses of human immunoglobulin (HIgG) adsorbed

(top) and covalently immobilized (bottom) on UV-irradiated

PtBMA surfaces. Similar images were obtained in different regions

of at least two different samples.

116

Figure 4. Representative surface topography images and their corresponding

line profile analyses of lysozyme (LYZ) adsorbed (top) and

covalently immobilized (bottom) on the UV-irradiated PtBMA

surface.

117

Figure 5. Fabrication of micro/nano-structures for protein arrays using

microablation and directed deposition.

124

Figure 6. AFM mapping of the ablated microchannels. 125

Figure 7. AFM topographical (top left) and lateral force (top right) image of a

channel fabricated via the ablation of a 30 nm Au layer on top of

PMMA.

126

Figure 8. Possible pyrolysis pathways of PMMA localized in micro-regions

leading to the observed lateral distribution of hydrophobicities.

128

xxvi

Figure 9. General concept of the probing of molecular surface of proteins. 129

Figure 10. Modulation of the amplification of protein adsorption in

micro/nano-channels vs. the molecular surfaces of the respective

protein.

132

Figure 11. Correlation between nanothickness and refractive index of PMMA

on glass surface treated with HMDS.

134

Figure 12. Correlation between nanothickness and refractive index of HSA in

double nanolayered sandwich of HSA/PMMA on glass-surface

treated with HMDS.

136

Figure 13. Protein adsorption in microstructured PMMA surface. 138

Figure 14. Protein adsorption in the channels of thin gold layer deposited on a

poly(methyl methacrylate) film and on poly(methyl methacrylate)

films.

139

Figure 15. Adsorption and polymerization of F-actin (23 mM) after 1.5 h in the

continuous flow with the flow rate of 0.06 mL min–1 on polymeric

surfaces: (A) NC, (B) PSMA, (C) PMMA (exposed), (D) P(tBuMA)

(exposed).

147

Figure 16. Estimation of the working buffer viscosity with and without BaSO4

(108 mM).

149

Figure 17. Covalent bonding and polymerization of F-actin (23 mM) after 1.5 h

in the continuous flow with the flow rate of 0.06 mL min-1 on

polymeric surfaces: (A) PSMA, (B) PMMA (exposed), (C)

P(tBuMA) (exposed).

150

Figure 18. Binding of self-assembled F-actin (23 nM) on functionalized PSMA

polymeric surfaces.

152

Figure 19. Fluorescence images of (A) 2-μm actin filaments (23 nM) with their

barbed ends blocked by gelsolin; and (B) their bundles condensed

with Ba2+ (108 mM) during 45 min. Fluorescence images of

electrostatically condensed and aligned actin-filament bundles

assembled from 2-μm actin filaments (23 nM) (C) after 1.5 h; (D)

after 3 h; and intact F-actin filaments: after 1.5 h (E) and 3 h (F) in

154

xxvii

the continuous flow system with the flow rate of 0.06 mL min-1.

Figure 20. SEM images of F-actin/gelsolin bundles formed from

electrostatically condensed F-actin filaments.

156

Figure 21. Translocation of the antiHMM–HMM bead along the bundle formed

from 2-μm-actin Alexa 488-phalloidin–labeled filaments (23 nM)

and condensed with Ba2+ (108 mM).

158

Figure 22. High-resolution AFM topographical images of Planococcus

maritimus F 90 cells and a close-up of the area on the cell surface

(non-contact mode, top) revealing dark spots/pores.

172

Figure 23. Phylogenetic position of Sulfitobacter delicatus KMM 3584T and

Sulfitobacter dubius KMM 3554T according to 16S rRNA gene

sequence analysis.

174

Figure 24. Phylogenetic position of Marinobacter excellens according to 16S

rRNA gene sequence analysis.

176

Figure 25. Phylogenetic position of Planococcus maritimus KMM 3738 based

on 16S rRNA gene sequence.

178

Figure 26. Kinetics of adenosine triphosphate (ATP) production by

Sulfitobacter mediterraneus ATCC 700856T during attachment on

poly (tert-butyl methacrylate) (PtBMA) and mica.

186

Figure 27. Kinetics of ATP production by Planococcus maritimus F 90 during

attachment on PtBMA and mica.

187

Figure 28. High-resolution atomic-force microscopy (AFM) topographical

images of Staleya guttiformis DSM 11458T cells and a close-up of

an area on the cell surface revealing dark spots/porous features.

191

Figure 29. AFM of cells of Formosa algae KMM 3553T. 193

Figure 30. High-resolution AFM topographical images of Marinobacter

excellens KMM 3809T cells and a close-up of the area on the cell

surface revealing dark spots/porous features.

194

Figure 31. Protein neighbor-joining phylogenetic tree shown is based on MreB

sequences from heterotrophic bacteria using Thermotoga maritima

as outgroup.

208

xxviii

Figure 32. Protein neighbor-joining phylogenetic tree shown is based on FtsA

sequences from heterotrophic bacteria using Thermotoga maritima

as outgroup.

217

LIST OF SCHEMES Scheme 1. Reaction scheme for the formation of sulfo-N-hydroxysuccinimide

(sulfo-NHS) activated poly(tert-butyl methacrylate) (PtBMA).

107

1

CHAPTER 1

INTRODUCTION

2

1.1. Overview

Protein-based microdevices are the focus of intensive research (Rajamani and

Sayre, 2011, Zavgorodniy et al., 2010, Lelyveld et al., 2010, Sankaran et al., 2011, Li

et al., 2011a, Campbell et al., 2011). In respect of the rapidity and cost of biosensing,

protein-based microdevices offer an attractive alternative to existing methods allowing

rapid (Chou et al., 2010), efficient (Chandra et al., 2011, Doerr, 2010), and

quantitative (Washburn et al., 2010) protein detection. For example, the detection of

molecularly different proteins, e.g., biomarkers of diseases (Natesan and Ulrich, 2010,

Boja et al., 2011) in multiplex protein-based microdevices as well as in the rapid

detection of pathogens (West et al., 2009, Laue and Bannert, 2010) for ensuring food

safety (Mandal et al., 2011) are the areas of potential application of motor protein-

based microdevices.

Although methods for the construction of protein-based microdevices for

proteomic (Treitz et al., 2008, Cosnier et al., 2009, Kotz et al., 2010), and

immunoassay (Bremer et al., 2009, Li et al., 2011, Chiriaco et al., 2011) analyses have

been developed, the manufacturing of new microdevices remains expensive (Shim et

al., 2011) due to the high cost of materials and fabrication processes (Yan et al., 2011,

Lee et al., 2011). Novel approaches to the design of surfaces are required. These need

to allow for the minimization of the fabrication costs via efficient amplification and

spatial immobilization of multiplex proteins so that the bioactivity of such proteins

(e.g., microarrays, protein-based microdevices) can be retained.

The surface design posed considerable technological challenges arising from

(i) the large variety of proteins that needed to be immobilized on the surface; (ii)

increased density of laterally defined areas with precise immobilization for specific

proteins, required by high-throughput analysis; and (iii) the ever present complexities

of protein-surface and protein-protein interactions, reflected in complex fabrication

and function, respectively. Most technologies for fabrication of protein microdevices

have to ensure the confinement of molecularly different proteins in laterally-defined,

either flat 2D; or profiled ―2D+‖ micro-areas. The profiled features have the advantage

of minimization of inter-spot contamination and the drawback of more difficult access

3

of the recognition of biomolecules (e.g., antigens for antibody microarrays) in a micro-

confined area. An optimal shallow profile feature would take advantage of the benefits

of the former and mitigate the latter (Han and Yoon, 2009).

Among the enabling technologies for the above biomolecule micropatterning

methods, laser beams are capable, depending on the exposure energy and sensitivity or

absorbance of the exposed material, of enabling both photolithography (Wang et al.,

2009) and photo-assisted etching (Gudymovich and Vanifat'eva, 2009). Also, focused

laser beams can solve, in principle, a critical fabrication and operating problem of the

protein chips better than most other alternative methods, i.e. the controlled and

confined variation of the surface properties of the micro-areas where different proteins

are deposited (Uemura et al., 2010, Park and Cho, 2011). Proteins present extremely

complex surfaces (e.g., hydrothilic or hydrophobic; acidic or basic; neutral or charged)

that interact with the surface via electrostatic forces, hydrogen-bonding, van der Waals

or hydrophobic interactions (Heo et al., 2010, Pleskova et al., 2011, Gokarn et al.,

2011). This variety of molecular surface – microdevice surface interactions, can lead

to large variations in protein surface concentrations as well as the possibility of

important changes to protein bioactivity and its denaturation (Hnaien et al., 2011,

Alvarez et al., 2011). Building on this, advanced biomolecule immobilization on the

surfaces has to be developed. One logical approach would result in the spatial

immobilization of multiplexed proteins in micro/nano-channels. This approach will

not only require a very accurate control of the surface properties at the micro- and/or

nano-scale level, but also a priori knowledge regarding the nature of the deposited

proteins and their interactions with surfaces.

Employment of molecular motors in microdevice construction received a

significant amount of attention by the following researchers (Agarwal and Hess, 2010,

Fujita et al., 2011, Fiasconaro et al., 2009). The motor proteins possess many of the

characteristics required to power nanomachines, e.g., generation of force (Linari et

al., 2009), and ability to transport specific cargoes over appropriate substrates

(Takatsuki et al., 2010). The rate of motor protein action can be controlled by the

direct application of in vitro motility assays (Valentine et al., 2006). While motility

assays are easily reproducible, to achieve directional motility is not a simple task. This

4

is due to the fact that motor proteins tend to attach onto the target surfaces at random

locations and consequently move in random directions. A significant number of

studies have focused on solving this problem by modification of surface topography

(van der Meer et al., 2010) and/or surface chemistry (Park et al., 2010b) to fabricate

microstructures of certain geometries for directional movement of molecular motors.

While the latter direction has emerged quite recently, the former has been intensively

explored over several years (Takahashi et al., 2011, Chen et al., 2011). A number of

different methodologies have been applied to align the motility of filaments through a

variety of techniques including protein (myosin) guiding (Butt et al., 2009), magnetic

field (Kaur et al., 2010), electric field (Wigge et al., 2010), UV lithography

(Yamamoto et al., 2008). However, these techniques are not suitable for the

fabrication of aligned proteins (actin) tracks, which can support unidirectional bead

translocation in vitro, due to the lack of precise control over them at the level of either

individual or bundled linear assemblies.

In case of microdevices based on protein molecular motors, there are a few other

challenging aspects that have to be resolved. One of these aspects is the protein

lifetime and in particular the lifetime of actin tracks which would sustain robust

microdevice functioning (Phung et al., 2011, Liu et al., 2010, Pagan and Griebenow,

2010, Oguchi et al., 2010). The actin prokaryotic homologue MreB (Bean and Amann,

2008, Ikeuchi et al., 1990) was found to be a more mechanically robust protein (Popp

et al., 2010b, Shaevitz and Gitai, 2010). It is capable of assembling filaments across a

wide range of temperatures (Bean and Amann, 2008), pH values (Cabeen and Jacobs-

Wagner, 2010), and ionic concentrations (Popp et al., 2010b), and hence MreB

proteins may be useful and replace actin. Functional properties of motor proteins are

directly correlated with the energy suppliers, namely, ATP (Oiwa et al., 1990).

Therefore, a reusable source of ATP such as ATP-producing bacteria may be an

attractive alternative.

5

1.2. Aim of the study

The aim of this study was to develop an approach in the design of advanced surfaces

which can be used for construction of protein-based microdevices, the surfaces that

would be suitable for efficient multiplexed spatial immobilization of proteins which

will be able to retain their bioactivity. The evaluation of the suitability of the

employment of bacterial ATP producers and prokaryotic actin-related proteins as

replacements for the energy source and eukaryotic actin, respectively, in construction

of the next generation of microdevices was also anticipated.

1.3. Organization of the thesis

Chapter 2 starts with a general overview of microdevices followed by an

outline of the various types of biosensors. It looks/reveals several critical aspects of

microfluidic systems before discussing how they can be controlled. The chapter also

explores the prospect of applications of both prokaryotic and eukaryotic molecular

motor proteins and includes an evolutionary/phylogenetic comparison of MreB and

FtsA homologues of eukaryotic actin. Since an understanding of the functions of

bacterial molecular motors is essential for incorporation of prokaryotic motor proteins

in biosensors, the chapter ends by describing the most useful functions, from the

microdevice design point of view.

The following chapters are based on results of this study already published in

peer-reviewed journals, with the exception of Chapter 9.

Chapter 3 contains a description of the methods used for: microdevice surface

design and fabrication; the study of protein-surface interactions; protein handling and

immobilization. It continues with a description of the methods used in bacterial

taxonomy, namely, phenotypic, chemotaxonomic, genotypic, and phylogenetic

analyses. It ends with the methods used to assess MreB and FtsA proteins.

While Chapter 4 covers immobilization of proteins on flat surfaces, Chapter 5

presents comparative immobilization of proteins in micro/nano structures. The latter

describes a newly developed approach for surface design applicable to microdevices.

6

The approach is based on spatial multiplex immobilization of proteins in micro/nano-

channels fabricated via laser ablation.

Chapter 6 is focused on an approach to the design of the surfaces of

microdevices based on self-assembled actin bundles as model protein structures that

retain their bioactivity, i.e., they can support unidirectional movement of cargo

particles.

Chapter 7 describes phenotypic, chemotaxonomic, genotypic and phylogenetic

properties of potential ATP, MreB, and FtsA producers.

Chapter 8 presents a characterization of ATP motor activities discovered in

environmental bacteria. Chapter 9 describes a comparative analysis and evaluation of

MreB and FtsA proteins detected in selected bacterial taxa.

The final chapter of the thesis draws conclusions from the results presented in

this thesis and discusses future work.

7

CHAPTER 2

LITERATURE REVIEW

8

2.1. Overview

This chapter consists of five subsections. It starts with an overview of the

concept and benefits of biosensors (subsection 2.2.), and is followed by a description

of different types of biosensors (subsection 2.3.). As the main goal of this thesis is to

develop an approach to the design of surfaces for biosensors based on motor proteins

(see chapter 6), subsection 2.4. is devoted to microfluidic devices. This subsection

covers the general aspects of microfluidic devices highlighting the importance of

surface properties, geometry and fluid properties (see subsections 2.4.3.1.–2.4.3.4. and

chapters 4–6). Subsection 2.4. continues with an overview of the types of microfluidic

systems (see subsection 2.4.4.) and ends with a description of methods of control of

microfluidic devices (see subsection 2.4.5.). Subsection 2.5. describes the concept of

protein molecular motors including the evolutionary/phylogeny of prokaryotic actin-

related proteins, namely, MreB and FtsA (see subsections 2.5.2.–2.5.4. and chapters

6–9). Classification of molecular motors based on the mode of operation is given in

subsection 2.5.5. Also, the chapter describes native functions of molecular motor

proteins, which can be used for the development of surfaces for biosensors based on

motor proteins (see subsection 2.5.6. and chapters 6, 8 and 9).

2.2. Concept and benefits of biosensors

The unification of science and technology as a distinct nanotechnology area

has been acclaimed as one of the most important events in the history of science and

has deeply influenced ideas on the manipulation of molecules at the micro/nano-scale

level (Chen et al., 2011). Nanotechnology can be best defined as the ability to

understand and control matter of a small size (Ramsden, 2009). The growth of

nanotechnology has led to the fabrication of advanced microdevices (Jiang et al.,

2011, Reedy et al., 2011). In general, it is accepted that an analytical microdevice

composed of a biological sensing component (e.g., bacteria, spores, proteins), a

physico-chemical signal transducer (discussed in the next subsection) and a signal

processing unit (Wildeboer et al., 2010, Lee et al., 2010a), can be considered a

9

biosensor. The common view is that the role of a sensing element is to recognize and

react with a target analyte; the transducer serves as a converter, transferring a

countable output signal from analyte concentration. The history of biosensor creation

dates back to the early 1960s when Leland C. Clark described oxygen electrode.

Continued technological development of this type of biosensor led to the manufacture

of new generations of biosensors for different applications. Biosensors have been

developed for a number of applications ranging from biomedical monitoring (Bachand

et al., 2009) to the detection of biological warfare (Gooding, 2006). Society can

benefit in multiple ways from nanotechnologically improved products. For example,

glucose biosensors are being used in glucose monitoring technology (Chu et al., 2009),

in the food industry (Bordonaba and Terry, 2009), and other biotechnological areas

(Gramsbergen et al., 2003). The construction of a glucose sensor was initially based

on an amperometric enzyme electrode (Pandey et al., 1992). However, during the past

decades, much new research on the improvement of biosensor performance has been

done (Ducloux et al., 2010). Li et al. (2007) have reviewed the development of

implantable electrochemical devices for the management of diabetes mellitus. Their

results indicated that sensors suffer from aging issues that need to be overcome.

Lyandres et al. (2008) have demonstrated that there was potential for detection of

glucose using surface-enhanced Raman spectroscopy (SERS). This optical technique

has been successfully employed for real-time analysis of diverse chemical substances

(Rae and Khan, 2010). Moreover, biosensors based on SERS have been incorporated

into a virtual reality (VR) image-guided surgery unit for accurate targeting of surgical

margins of cancer cells (Reisner et al., 2007). However, there are two major

drawbacks to this system with regard to the precise positioning of the Raman probe

and steady-state scanning.

It is believed that the ability to assess the health risk of different chemicals is

an essential ingredient for preventing life-threatening illnesses (Kovacic and

Somanathan, 2009). Much of the commercial success of environmental biosensors is

attributed to a growing human demand for advanced environmental control. These

sensors offer a simple way of detecting many different compounds. In recent years,

significant progress has been made in constructing cholinesterase (AChE) biosensors

10

that selectively react with hazardous toxins (Pohanka et al., 2009), organophosphate

(OP) pesticides (Istamboulie et al., 2010), and other compounds (Woznica et al.,

2010). A bacterium-based NO2 biosensor that has been applied to the analysis of

freshwater, marine and oxic-anoxic wastewater utilizes Stenotrophomonas

nitritireducens coupled to an electrochemical NO2 sensor to detect nitrite (Nielsen et

al., 2004). Although preferred bacterial species that can be used as bioelements were

clearly identified, the biosensor possessed disadvantages such as a narrow operating

temperature and salinity ranges due to the non-psychrophilic physiology of microbial

candidates. Nowadays, progress has been made in building optical biosensors for

detection of enteric pathogens such as Escherichia coli (Day et al., 2010).

The development of biosensors based on linear molecular motors (see

subsection 2.5.5.2.) allows not only detection of analytes but also their transport to

specific compartments of biosensors, for example, detection units. Of two ways of

biosensor miniaturization, such as microfluidic and ―smart dust‖ approaches, the

former was used by Martinez-Neira et al. (2005) for the construction of a biosensor

based on linear motors (actin and myosin). In so doing, actin and myosin were used as

biosensors for the detection of toxic cations. The latter approach was employed by

Fischer (2009) for building hybrid biosensors based on linear (kinesin and tubulin)

molecular motors ; the energy was provided by caged ATP. Despite unification of

transport and energy supply systems in one microdevice, signal accuracy and strength

remained key issues. Since the terrorist attacks on America in 2001, significant public

concern regarding the possibility of a large-scale bioterrorism event has resulted in the

development of technology for rapid detection and identification of biological warfare

(BW). To accomplish this goal, Hofstadler et al. (Hofstadler et al., 2005) developed

TIGER (Triangulation Identification for Genetic Evaluation of Risks); biosensors

based on electrospray mass spectrometric (ESI-MS) detection of nucleic acids.

Although the TIGER biosensor can be used for identification of various groups of

pathogenic microorganisms, it does not allow real-time analysis due to time-

consuming procedures, e.g. incubation of microorganisms, isolation and purification

of genomic DNA and purification of PCR products.

11

2.3. Types of biosensors

Biosensors can be classified into various groups according to the biological

recognition mechanisms. On the basis of the signal transduction, biosensors may be

divided into four groups: electrochemical, thermal, mass-sensitive and optical sensors.

Of these biosensors, the most publicized are electrochemical, which include

amperometric, potentiometric and conductimetric. Electrochemical biosensors can

respond to the concentration of analytes as small as 10-6 M (Turek et al., 2007). This

detection limit is sufficient for measurement of carbohydrates (glucose, galactose and

fructose), polyphenols, amino acids (glutamate), metabolites (urea and lactate),

cholesterol, and drugs. However, recognition of analytes in the 10-9 M concentration

range remains a problem (Jubete et al., 2008). Thus, this type of biosensor is not

sufficiently sensitive and accurate for the detection of hormones and other serum

components. In 2000, Romani developed the first amperometric biosensor with a

measurement range of 20 to 80 µM polyphenols (Romani et al., 2000). This sensor

could be used for purposes such as the screening of plant materials for polyphenols. A

major drawback of the amperometric kind of sensor is its limitation in the analysis of

biological samples in which endogenous components are present.

Chronopotentiometric ion biosensor with a detection range of 0.1–1 µg/mL for avidin

has been developed by Xu and Bakker (2009). However, this type of biosensor is

dependent on the charge density of the analyte. Therefore, the potential problem is the

inability of potentiometric biosensors to analyze complex biological solutions.

Though conductimetric biosensors measure only small changes in electrical

resistance, they may have applications in the qualititative analysis of chemicals.

Thermal biosensors can be applied to measuring changes in heat and can be used for

detection of different analytes. However, sensitivity of the biosensor remains a major

problem and is associated with the dissipation of heat to the surroundings. Mass-

sensitive biosensors provide a way to detect changes in mass. In this case, optical and

electrical methods can be used to measure the deflection of cantilevers. Compared to

optical techniques, piezo-resistive cantilevers are unaffected by the optical artefacts

and can operate in non-transparent samples.

12

Optical biosensors offer considerable promise for obtaining optical

information in a highly selective, fast and reproducible manner. Many recent advances

in optical technology can be traced to the development of different techniques such as

fluorescence, ellipsometry, bioluminescence, chemiluminescence, phosphorescence,

rotation and polarization for the measurement of analytes. The advantage of using the

fluorescence method is that continuous images from fluorescent groups can be

produced. In addition to providing the fluorescence required to directly observe

biomolecules, the fluorescence approach also allows for quantitative analysis of

molecular motion. The main drawback of this approach is photodestruction of the

biomolecule and of other moieties of the biomolecule such as enzyme sites. This leads

to harmful effects on the performance of the biosensor, apart from decreasing the

reproducibility of results. A number of in vitro motility experiments with assays

containing prokaryotic actin- and tubulin-related proteins were reported in the 2000s,

which formed the foundation for device-oriented research in this area.

According to the biological recognition mechanism, biosensors can be

classified into biocatalytic, bioaffinity and whole-cell systems. In 1962, Clark

developed the first biocatalytic biosensor based on a layer of the glucose oxidase

enzyme ―GOD‖ which was deposited close to the surface of the oxygen electrode

(Clark and Lyons, 1962). Glucose sensors may be used for self-monitoring of the

concentration of glucose in drops of fresh blood. A measurement range of 1x10-6M to

1.5x10-3M for glucose was reported (Ding et al., 2010). The use of enzymes in

biosensing opens the door for a large number of environmental applications.

Enzymatic biosensors have been developed for the detection of environmental

pollutants, such as phenols (Li et al., 2010), cyanide (Mak et al., 2005), nitrate (Quan

et al., 2005), and uranyl (Liu et al., 2007). However, this kind of biosensor suffers

from limitations related to the necessity of using artificial mediators (Kosela et al.,

2002), and substrates (Ges and Baudenbacher, 2010). Bioaffinity sensors, such as

nucleic acid and immunosensors, offer rapidity, simplicity, and selectivity. It has been

shown that DNA recognition layers can be fabricated for frequent utilization (Wang,

2000). Owing to its selectivity, a DNA-based biosensor can be used for targeting of

particular bacterial species (Ng et al., 2008). A sensitive enzyme immunosensor for

13

the detection of Vibrio parahaemolyticus, the food-borne pathogen, with a detection

limit of 6.9 x 103 cfu/ml has been recently developed by Zhao et al. (2010). Although

the main disadvantage of immunosensors is their stability, this biosensor exhibited

only slightly decreased firmness after a week of storage.

For whole cell biosensors, use of living prokaryotic and eukaryotic cells can be

beneficial. In the middle of the 1970s, Divies (1975) suggested that a bacterial cell

could be employed as the sensing element in microbial electrodes for the estimation of

alcohol concentration from the target analyte. Recently, Elad et al. (2008) have

developed a bioluminescence-based bacterial sensor for the detection of toxicants.

These authors indicated that bacterial bioreporter cells were capable of recognizing

analytes within a 30-minute detection time. Even though there is a major drawback in

using bacterial bioreporters, such as the dependence of microbial productivity on the

physiological state, some bioreporters have the potential for becoming a part of

biosensors.

2.4. Microfluidic devices

2.4.1. Overview

The use of microfluidic technology is crucial for the utilization of biological

recognition and/or transporting elements, such as molecular motors, in biosensors.

Microfluidic devices can be constructed in three formats: microarray, droplet and

continuous-flow systems. Furthermore, the growing demand for miniaturization of

large-scale devices along with integration of the sample preparation steps with

biosensing procedures has led to the development of ―Lab-on-a-Chip‖ (LOC) systems.

In doing so, the proposed prototype of biodevice has a high potential for becoming an

essential part of a future LOC. Since the function of this biological motor-based cargo

delivery system mainly depends on a microfluidic environment, the overview of

available formats, droplet and continuous-flow, is given below. It is important to note

that the microarray approach has been used in this study only for the specific purpose

14

of identifying microorganisms. Owing to their distinct applications, DNA as well as

Biolog phenotype microarrays are briefly mentioned in chapter 3.

2.4.2. Advantages of microfluidic devices

Microfluidic technology is an essential instrument-granting transformation to

biosensing in the scale range from pico-to micrometer level. Success in this

technology results from the fact that fluid can be precisely controlled at a micro level.

Early on, scientists realized that the commercialization of microdevices would lead to

financial rewards and so took the first steps to the development of applications. The

ubiquitousness of microfluidics has been witnessed in its utilization in different

applications, with specialists in nanotechnology expanding the borders. Nowadays,

with applications including printing, biomedical analysis and defence, the biodevice

has become the centre of profound scientific attention. The story of microfluidics

began nearly half a century ago with Mack J. Fulwyler‘s report of his construction of a

prototype device (Fulwyler, 1965). His prototype device consisted of a volume sensor

and an ink-writing oscillograph (Robinson, 2005). Ink-jet type printing in its mature

form has evolved from microfluidic technology.

Microfluidics has enabled clinical trials to deliver very accurate results. The

microfluidic device has been successfully used for targeting serum thyroglobulin (Tg),

a cancer biomarker, by loosely adsorbed Immunoglobulin G (IgG); the latter was

evicted from the occupied microfluidic territory by the former through competitive

adsorption (Choi and Chae, 2009). Researchers emphasized that the method used

allowed for the elimination of an antibody probe. However, actual clinical samples

may contain a few different important markers which should be analyzed

concurrently. Murphy et al. (2009) have showed that both metabolic and enzymatic

markers can be detected at the same time. The author‘s findings demonstrated the

possibility of a combination of competitive and non-competitive assay formats. In the

case of lightweight substances, i.e., haptens, such as steroids, the way in which

antibodies identify haptens in immunometric format needs to be ameliorated

(Kobayashi et al., 2007). In addition to giving an overview of new approaches for

15

targeting haptens, the researchers presented an immunometric assay which was

capable of detecting attomolar quantities of a model hapten. It has been found that the

creation of interchanged poly(methyl methacrylate) (PMMA) – polycarbonate and

nanocapillary coats in devices can help separate a mixture of amino acids (Kim et al.,

2009a). Although improved separation of complex analytes was achieved, the

proposed device, as noted by the authors, needed to be upgraded with a cooling

element, new channels and/or optimized fluids. The applicability of a polymer

microfluidic chip for biochemical analysis has been demonstrated by Yang et al.

(2010). It was concluded that the unification of two methods, such as affinity

chromatography and electrophoresis, was necessary for accurate measurement of

biomarkers in biochemically heterogeneous samples. Despite progress in

microfluidics, the need for miniaturization of microfluidic devices, e.g., for drug

discovery applications still exists (Upadhyaya and Selvaganapathy, 2010) Recently, it

has been found that the density of spots (cell layers) on the modified nanomembrane

of a microfluidic structure can be enhanced by means of an electrical field (Upadhyaya

and Selvaganapathy, 2010).

The importance of shear stress in microfluidics has recently been studied by

means of computational (Cioffi et al., 2010) and experimental (van der Meer et al.,

2010) methods. For example, shear-stress was used to examine the interplay of either

plain particles or aptamer-decorated particles and human cells in a microfluidic system

by Farokhzad et al. (2005). These researchers have further developed their approach

for cancer treatment (Farokhzad et al., 2006, Dhar et al., 2011). The authors have

described the use of nanoparticle-aptomer bioconjugates for hunting down malignant

prostate cells. As microfluidics has been extending its borders, novel approaches need

to be developed. Fundamental methods for restoring various types of mammalian

tissues have been reviewed by Lalan et al. (2001). A new approach to investigating

implant-associated bacterial diseases has been proposed by Lee et al. (2010b). They

used a microfluidic system to examine how the presence of an implant-associated

diplococcus affects in vitro behavior of osteoblast cells. The researchers concluded

that osteoblasts were strongly influenced by the settlement of small-colony variants

(SCVs) (Singh et al., 2009), which was described as a biofilm producing phenotype of

16

Staphylococcus epidermidis (Al Laham et al., 2007). A method with potential

applications in cardiovascular tissue engineering has been developed by Suzuki et al.

(2010). The method allowed differentiation of multipotent cells into muscle ones by

means of laminin (LM), a basement-membrane protein.

By incorporating different steps of biochemical analysis performed in

individual devices into one procedure carried out in a single laboratory-on-chip (LOC)

system, researchers moved microfluidics forward. Remarkably, the architecture of a

LOC format can be modified to meet the user‘s needs (Shaikh et al., 2005). For

example, the usefulness of the multicomponent construction for the detection of lead

has been demonstrated (Shaikh et al., 2005).

It should be noted that a microfluidic PCR (polymerase chain reaction) can be

used for detection of various clinical and environmental bacterial species. The most

striking thing about the utilization of the microfluidic chip for PCR is the fact that

different procedures, such as amplification and extraction of nucleic acids from

suspicious samples, can be run in one device (Kim et al., 2010). Recently, a

microfluidic device has been developed for forensic DNA analysis (Aboud et al.,

2010). This study has focused on enabling pentameric short tandem repeat (STR) -

based separation in a miniaturized microfluidic channel.

The issue of chemical contamination of food motivated researchers to build

devices for the screening of toxic chemicals in dairy foods. Using a microfluidic

approach along with ultraviolet (UV) radiation detection, Zhai et al. (2010) have

developed a strategy for detecting melamine in milk samples. Nowadays, concerns

regarding the threat of uncontrolled use of biological weapons come from their

increased production (Coleman and Zilinskas, 2010). In this regard, it has been

demonstrated that biomotors can be successfully employed for the detection of one of

the biological weapon agents, namely, Staphylococcal enterotoxin B in a microfluidic

device (Soto et al., 2008). In fact, microfluidic devices based on molecular motor

proteins have different potential applications (Korten et al., 2010), ranging from

molecular assembly to drug delivery and biosensing (Fischer et al., 2009). These

include military use. Although biomolecular motors may offer a new microfluidic

17

paradigm, a major disadvantage of their in vitro employment lies in their insufficient

stability and poor longevity.

2.4.3. Critical aspects of microfluidic devices

2.4.3.1. Overview

Microfluidic devices provide opportunities for their users to manage fluid

behavior at microscale level. Since the beginning of device manufacturing, substantial

progress has been made in the development of high-grade microfluidic systems. In

order to understand fluid behavior in real conditions for devices, one needs to know

the physical aspects of fluid dynamics. As mentioned above, the success of in vitro

biomolecular productivity depends on being able to provide contact for surfaces in an

optimum environment.

2.4.3.2. Surface properties

Surface preparation is known to play a crucial role in the endurance of all

types of microfluidic systems. Various techniques for fabricating surfaces with

specific properties have been worked out. However, the choice of a method relies on

the suitability of the substrate chemistry for a particular physical and/or chemical

treatment and subsequent application. For example, microfluidic devices can be

constructed from glass (Thiele et al., 2010, Pjescic et al., 2010), or silicon (Si)

(Retterer et al., 2010, Bienvenue et al., 2010). Material features that make glass a

valuable candidate substance are its optical and mechanical qualities (Bach and

Neuroth, 1995, Bach and Neuroth, 1998). In fact, owing to its mechanical properties,

glass remains a widely used construction material. Morever, Bach and Neuroth (1998)

noted that these properties are highly dependent on the composition of glass. Like

glass, quartz is considered to have outstanding optical as well as thermostable

properties (Spassov et al., 2008). However, since quartz usually contains various

admixtures that may affect microdevice performance, the purity of this material

18

should be evaluated by means of gamma-ray technique prior to its utilization (Komov

et al., 1994). In addition to its impressive mechanical features, silicon, a quite popular

electronic material, is considered to have valuable electrical features (Fukuda and

Menz, 2001) as well as biocompatible (Martinez et al., 2009, Erogbogbo et al., 2010)

properties. However, this substrate is known to be brittle (Fukuda and Menz, 2001)

and so is not reliable. Moreover, even though non-carbon based substrates have

valuable properties, they expand more than organic ones.

Growth in demand for disposable devices led to the integration of new

techniques for construction of microfluidic systems. Nowadays, scientific efforts

directed at the utilization of organic substrates have focused on testing living tissues

and synthetic polymers. The engineering of three-dimensional (3D) structures such as

tissues is based on building controllable scaffolds which can support their applicable

cells not only physically but also nutritionally (Chao et al., 2010, Sharifpoor et al.,

2010). The success of this research depends on the degree of availability of

degradable scaffolds for cells. Taylor et al. (2007) have indicated that scaffolds should

be in a very close biological relationship with the cells deposited onto them. As for

polymeric substrates, some of them, namely, poly(styrene-maleic acid) (PSMA),

poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), polystyrene

(PS), poly(tert-butyl methacrylate) (PtBMA), polycarbonate (PC) and polyethylene

(PE) are known to offer user-oriented physical and chemical properties that can be

useful for constructing cheaper microfluidic systems. A major advantage of using

polymeric materials is that native attributes of polymers can be effectively modulated

by suitable methods. Broadly, these fabrication techniques may fall into the following

groups: replication methods (Becker and Gartner, 2000) and direct methods (Liu et al.,

2005a). The master templates can be fabricated using a variety of tools, such as

micromachining (Entcheva and Bien, 2005), electroplating (Burek and Greer, 2010),

embossing (Gan et al., 2010), injection moulding (Liu et al., 2009), and casting (Gitlin

et al., 2009). Replication technologies can be used for the repetitive fabrication of

identical devices in huge quantities. Direct technologies have been found to be useful

for the construction of individual devices. One of the most widely used methods for

the treatment of polymeric substrates is laser ablation. It includes the use of radiation

19

to break down polymer bonds and takes away the demolished portion of polymer from

the ablated spot/pattern of the surface. The success of any ablation depends largely on

optimizing its parameters, mainly energy delivery and polymer removal speed. By

adjusting laser pulse (Gonzalez et al., 2007) and fluency (Amer et al., 2005),

researchers have taken the first step toward minimizing the size of the heat-affected

zones (HAZs) surrounding the ablation area. However, the chemical nature of the

polymers has been known to play a key role in the final morphology of the channel.

Pugmire et al. (2002) have studied surface properties of the channels using laser

ablation under different pressures. The results showed that there was no difference

between a PMMA workpiece and its native film. When poly(ethylene terephthalate

glycol) (PETG), poly(vinyl chloride) (PVC), and polycarbonate (PC) ablated under

different atmospheres, they showed remarkable changes in chemical composition.

To make a particular pattern on polymeric surface, one can utilize the power of

lithography. With techniques adopting SU-8 (an epoxy-based negative photoresist)

(Gao et al., 2008) and similar resists, thick-film technology has attracted a worldwide

attention in the field of microtechnology. Thus, a photosensitive epoxy (SU-8) has

been explored in creation of microchannels of different depths (Edwards et al., 2000).

However, SU-8 fabrication methods include time-consuming multiple step procedures

(Yu et al., 2006). The studies of protein-surface interactions have demonstrated that

lithographically decorated polymeric films can be used as substrates for exclusive

immobilization of nonmotor (Nicolau et al., 2010b) as well as motor proteins, such as

microtubules, and proteins (Turner et al., 1995). Also, see chapters 4 and 5. These

describe immobilization of nonmotor proteins. See chapter 6 for immobilization of

motor proteins on polymer surfaces. The successful deposition of molecular motors on

favourable surfaces and control over their movements, for example, by patterning of

channels for microtubule-based movement (Hiratsuka et al., 2001) is considered to be

a crucial part of the microdevice fabrication process.

20

2.4.3.3. Microfluidic device geometry

Adaptation of any type of fluid in a microfluidic channel, a key part of the

device, depends largely on its having an appropriate geometry. The principal role of a

channel is to provide guidance for a micro-scale fluid flow to the detector. Once

constructed, the system may allow accommodation of specific analytes under optimum

biochemical conditions. Since there is a great range of liquids and analytes, cross-

sections of microchannels should be designed to suit particular microfluidic tasks.

Some fundamental aspects of microfluidic dynamics through channels have been

discussed by Morini (2004). The author used the ―Obot-Jones‖ method to estimate the

value of a Reynolds number for a flow-regime transition.

This method was chosen from a range of channel parameters as its shape is the

most crucial. A cross-sectional profile has been identified as a factor that affects the

stability of condensation processes in microchannels (Wu et al., 2010). For example,

application of circular channels is limited due to the lack of sharp edges (Rahmat and

Hubert, 2006). Channels with elliptical profiles, which can be built by means of a

thermal technique, have been found to be useful for nanofluidic flow modeling

(Czaplewski et al., 2003). Also, other researchers have concluded that such a channel

shape provides an advantage in stabilizing the flow (Huang et al., 2006b). Recently,

attention has been drawn to the distinctive features of fluid flow through a channel

with a triangular profile (Park et al., 2010a). It was noted that the local flow rate was a

critical parameter for the control of solute deposition. A microchannel with a

trapezoidal profile has been designed by Jindal et al. (2005). The authors used

selective filling to introduce polymers onto distinctly specified areas in channels. A

rectangular-shaped channel has been successfully used for alignment of bacterial cells

by means of an electric force (Choi et al., 2010). The feasibility of using rectangular

silicon channels for detection of fluorescently labeled material has been demonstrated

(Kutchoukov et al., 2004). Generation of various channel profiles in a single

polydimethylsiloxane microfluidic device became possible with the use of

multiphoton absorption polymerization (MAP) (Kumi et al., 2010). To accomplish it,

the authors used a photoacid generator (PAG) instead of laser beam. Even though this

21

technique has been demonstrated to be appropriate for SU8, it cannot be employed for

all materials because of their different chemistries.

A microchannel system has been designed and then etched by Hirst et al.

(2005) for production of aligned macromolecular assemblies. Improvement of

interaction between aqueous samples and channel walls was achieved by increasing

hydrophilicity of the sidewalls. Although the functionality of self-assemblies has not

been demonstrated, the guided assemblies in the titanium channel have been

presented. It should be noted that flow rate, a crucial parameter of the fluid flux, may

change channel geometry. For example, relatively elastic PDMS channels with low

aspect ratio (AR) geometry (width is much greater than depth) can become deformed

easily (Gervais et al., 2006). Consequently, it may affect spatial fluid velocity

distributions within a channel. Importantly, the authors suggested that deformation

may be reduced by using more rigid material than PDMS (Gervais et al., 2006). In

fact, Attia and Alcock (2010) have preferred using PMMA to employing elastic

materials owing to the low-shrinkage and rigidity of PMMA. As a result, they created

a structure that was capable of handling flow rate without leakage.

2.4.3.4. Fluid properties

A fluid has three important properties: viscosity, compressibility, and density.

Of these, viscosity is considered the most critical because it reflects a force of the fluid

drag. The analyte-induced viscosity of a fluid can be determined by means of

measurement by the microdevice (Kamholz et al., 1999). Different analytes have been

detected in T-sensor by a competitive immunoassay based on the quantitative

evaluation of molecular diffusion (Hatch et al., 2001). Theoretical estimation of fluid

parameters and hence prognostication of its behavior has been accomplished by using

an analytical model (Kamholz et al., 1999). The transverse diffusion of flow in

microfluidic channels can be analyzed by means of T-sensors (Wang et al., 2005). It

was demonstrated that diffusion relied on channel geometry and dimension. Since a

concentration gradient can be used to control biosensors, researchers have developed

approaches such as distinctive inlet channel profiles (Yang et al., 2007), chemical

22

density differences (Kong et al., 2010), diffusive mixing (Englert et al., 2010), etc., to

build up the gradient. The control of the gradient can be tested by means of

computational fluid dynamics (CFD) simulations (Yang et al., 2007). The fluid

interface location in the microchannel can be adjusted by manipulating the volumetric

flow rates of the two fluid streams (Stiles and Fletcher, 2004). Chung et al. (2009)

have recently reported evidence of viscosity ratio maximization for droplets in

viscoelastic fluids. The authors assumed that it was due to stress build-up in the area

between the droplets and the channel wall. D‘Avino et al. (2010) have studied the

distinctive features of a single particle journeying through confined geometries. It was

demonstrated that a sphere was not always cruising within a main stream. There was a

possibility that it could get off the route and hence change its direction. Moreover, in

the case of continuous flow, a sphere moved fast only at the start of its journey due to

the primary accrual stress.

Another parameter characterizing fluid behavior is compressibility. It is

important to note that if the device is started up properly it may help prevent the

compressibility caused by an air bubble in the microfluidic channel (Cabral and

Hudson, 2006). Townsend et al. (2006) have utilized acoustic radiation to stimulate

bacteria to get closer to the sensor element. The radiation needed for this procedure

depended on the values of the compressibility and density of the particles in fluid.

Wang et al. (2007) have presented a PDMS multilayer device for mammalian cell-

based screening. To optimize density, the cells were captured by sieving, cultivated,

and screened. In this process, one device was used to perform high-density screening

of cytotoxic analytes.

In general, the compressibility of biomolecules is affected by density. Kalinin

et al. (2010) have reported the effects of interdevice bacterial cell density on the

chemotactic strategy of Escherichia coli. Amazingly, the activity of the bacterium

depended heavily on the balance between chemotaxis receptors to judge surrounding

conditions; the relationship between receptors depended on cell density. Ghodssi et al.

(2010) have conducted studies to examine microbial reactions to various stimuli. The

device used analyzed biological samples of high density, for example, products of

bacterial metabolites. Owing to the necessity of supplying some biosensors with

23

energy, microbial fuel cell (MFC) research has become more mature. Recently, a very

small MFC device has been constructed for electric current generation (Qian et al.,

2009). The authors presented evidence showing that some electricity-producing

bacteria, such as Shewanella oneidensis, can be employed as bioenergy suppliers. It is

noted that biofilm was suggested as being in charge of biofuel production. As biofilm

was formed only on the gold anode, it seems that enhancement of cell-anode contact

can shorten the device start-up. Choi et al. (2007) have constructed a system ensuring

viability of high-cell density structures. The authors adapted a perfusion system with a

PMMA pipe to provide fluid flow through microchannels located in microneedles.

Gottschamel et al. (2009) have presented microdevices for prolonged analysis of

variation in the population dynamics of fungi. The device was successfully used to

assess utilization of monosaccharides by Candida albicans. Huang et al. (2007) have

developed a three-tier microfluidic device for kinesin-based transport of microtubules.

Decapitated kinesin was used in order to optimize its density in enclosed

microstructures. However, this system lacked sufficient ATP energy.

2.4.4. Types of microfluidic devices

2.4.4.1. Overview

Nowadays, continuous and droplet-based technologies are explored to

construct microfluidic systems. In continuous-flow-based systems, liquids are treated

as steady flows in appropriate channels. This system can be used for certain

applications, such as biomolecular transport. In droplet-based microfluidic devices,

liquids are treated as discrete droplets on microarray surfaces. This system can help to

improve the efficiency of screening for various analytes, such as different proteins.

2.4.4.2. Droplet system

Droplet-based microfluidic devices provide system scalability and dynamic

reconfigurability. They offer a unique opportunity to support reconfiguration for faulty

24

tolerance. For example, biomolecules in devices can be reconfigured to alter their

properties to go around defective cells. A number of different techniques enable

droplet-based devices to scale down to handy dimensions while increasing the

throughput rate and efficiency of analysis. Electrowetting and dielectrophoresis have

been proven to be the most effective droplet techniques for fast control and handling

of fluid dynamics on a nano/micro-meter scale. Zeng et al. (2004) have studied the

operating principles of electrowetting on dielectric and dielectrophoresis. The authors

demonstrated the applicability of two techniques for droplet generation and

manipulation. Paik et al. (2003) have used electrowetting techniques to mix microliters

of fluids. This mixing process can be configured to fit particular system needs,

resulting in improved performance. A one-dimensional oscillator model has been

proposed by Baret and co-workers, in which the appropriate intrinsic and extrinsic

physical characteristics of the fluid were used to describe drop oscillations (Baret et

al., 2007). The authors employed electrowetting techniques to study the aqueous phase

drops in oil phase surroundings. Park et al. (2010d) have proposed a single-sided

continuous optoelectrowetting (SCOEW) that allowed prolonged active control over

the droplet (e.g. splitting, mixing). Moreover, this technique can be used over a

comparatively wide range of microvolumes. Morimoto et al. (2006) have fabricated a

microfluidic system which had a single pH-sensing site and protease sites.

Furthermore, a gold electrode was joined with an electrowetting-based valve. The

authors performed a trypsin assay, using a bovine serum albumin (BSA), and applied

an enzyme-containing solution to the sensing sites in a micro channel. Nashida et al.

(2007) have presented a microdevice composed of glass and a poly(dimethylsiloxane)

(PDMS) substrates. The authors employed direct electrowetting to operate the working

electrodes of a microdevice constructed for immunoassay analysis. Ohgami et al.

(2007) have shown the use of a micro-electrochemical sensor with Y-shaped PDMS

microfluidic channels to examine the activities of two enzymes. The authors indicated

that the sensitivity of a chip to enzyme concentration depended on the dimension of

the channel. Bahadur et al. (2007) have explained how electrowetting could simulate

droplet shape on rough substrates. The major advantage of the proposed method is that

it can predict the contact angle of a small volume of fluid.

25

Nowadays, numerous studies have focused on the re-evaluation of available

methods as well as the development of new ones. The droplet-based approach was

used for protein immobilization as described in chapters 4 and 5. Recently, Park et al.

(2010c) have provided a method for the electrowetting-controlled transport of

droplets. It is important to note that the benefits of utilization of single-plate

configuration were well explained by theory. In some cases, such as degradation and

high-speed situations, and electrowetting-force values can be used instead of contact-

angle values for characterization of the electrowetting process (Crane et al., 2010).

However, to make an accurate calculation of electrowetting, compensation methods

may need to be applied. In fact, although droplet dynamics can be ignored in this case,

evaporation should be taken into consideration. As for parallel plate microchannels,

disregarding the dynamics of electrowetting in them has been proven to affect

assessment of the basic parameters, such as contact angle and droplet velocity

(Keshavarz-Motamed et al., 2010).

An alternative method for droplet transfer control is dielectrophoresis (DEP).

Non-uniform electric force can be used to polarize and localize the micro- (or sub-

micro-) particles, such as proteins and DNA molecules, by DEP. Yantzi et al. (2007)

have presented a multi-electrode setup for controlled bio-particle locating and

clustering. Scientists employed DEP and AC electrokinetic techniques to control the

positions of particles in solution. Wiklund et al. (2006) have organized physical

competition between dielectrophoretic, ultrasonic and viscous drag forces for

translocation of bioparticles. This device was constructed to provide precise handling

of single particles or structural units of particles. The micro-fluidic sequence of liquid

droplet collisions with a substrate cavity has been described by Chau and co-workers

(2004). The effects of fluid properties have been discussed in terms of microfluidic

characteristics for specific tasks in the droplet deposition process. Taff et al. (2005)

have presented a positive dielectrophoretic (p-DEP) array for controlled manipulation

of single beads. The researchers used unique ―ring-dot‖ p-DEP trap geometry to

achieve single cell capture. Choi et al. (2005) have developed microfluidic systems for

separation of dielectric particles. Trapezoidal electrode array (TEA) was used to

provide dielectrophoretic force for running reactions. Recently, Moncada-Hernandez

26

et al. (2010) have demonstrated concentration and separation of a bacteria-yeast blend

by using insulator-based DEP (iDEP). Although negative dielectrophoretic trapping

was found to be suitable for microbial as well as fungal manipulation, yeast gave a

better result. Over the last decade enormous progress has been made in all aspects of

dielectrophoresis (Pethig, 2010). DEP is considered a promising area due to

elimination of needs for biochemical tags and surfaces.

2.4.4.3. Continuous system

This system supports translocation of continuous flow through a channel

network. External components that can be used to gain precise control of devices

include pumps, valves, and mixers. In addition, various electrokinetic techniques can

be utilized to operate devices. Depending on application, any of basic electrokinetic

forces, namely, electrothermal, electrowetting, electroosmotic and/or electrophoretic

can be employed for actuating fluids. Since electrothermal flow induces a temperature

gradient that runs through the body of the device, proteins are put at risk of

conformational change into a ―soup‖ of molecular debris. On the other hand,

electrothermal flow has been employed to mix landing biomolecules in order to

improve their ability to bind to functionalized substrates (Sigurdson et al., 2005). The

electrowetting technique may be used to successfully manipulate tiny quantities of

liquid.

Electroosmosis is based on an electrical field having a guiding effect on the

motion of microfluids along a charged substrate. With technology involving

electroosmotic flow control, information on channel conditions can be gained.

Electrophoretically guided movement of a conductive fluid, or small particles

enclosed in fluid are important for many applications of microfluidics. It has been

demonstrated that continuous flow devices offer enormous possibilities for applying

motor proteins for carrying out technological tasks. Jia et al. (2004) have presented a

transport system that used electric forces to effectively guide microtubules through

kinesin-coated microfabricated channels. The researchers have provided evidence that

kinesin motors can convey nanowire over relatively long distances. However,

27

unidirectional transport of wire has not been demonstrated. Heuvel et al. (2005) have

developed nanofabricated structures for bringing microtubules to a kinesin-covered

wharf. They improved the microtubule transport rate by applying voltage pulses to the

gold surface. The contribution of the second kinesin head to motion has been

evaluated by Berliner et al. (1995). It was demonstrated that decapitated kinesin was

not able to support uninterrupted movement. On the other hand, headless kinesin can

compete with headed kinesin for the spot on the surface of a microchannel controlling

adsorption (Huang et al., 2007). Kim et al. (2007) have demonstrated controlled

alignment of microtubules in channels using an electric field.

The unique neck domain and K-loop of the Neurospara crassa kinesin was

shown to allow single-headed kinesins to walk in procession (Lakamper et al., 2003).

Kim and co-workers (2007) have shown that different kinesins can perform similarly

to each other. What they suggested is that a weak electric field can cause inefficient

alignment. Li et al. (2005) have fabricated an electrophoretic cell for the analysis of

individual fluorescently labeled macromolecules. Their work has shown that an

approach based on measurement of the electrophoretic properties of biomolecules was

appropriate for filamentous actin. Huang et al. (2006a) applied an electric field to

align actin filaments in a certain direction. The researchers used gelsolin to produce

specifically polarized actin filaments. By using a dielectrophoretic procedure, a study

of the alignment behavior of actin filaments has been carried out (Asokan et al., 2003)

Although short filaments failed to align under dielectrophoretic conditions, long ones

responded well. In addition, the effect of DEP on biomolecules has been employed to

pattern filamentous actin (Asokan et al., 2003). Making polarized actin filaments and

then depositing them in microstructures, Lee and co-workers (2009) constructed a

system that allowed for cargo translocation. The authors coated the channel with the

bacterial protein streptavidin to control the directionality of flow.

28

2.4.5. Control of microfluidic devices

2.4.5.1. Overview

Incorporation of fluidic components into continuous devices has been widely

used to deliver a better analysis. The appropriate components are generally used to

guide a sample through a channel. Actions that can be accomplished by means of

different microactuators include opening/closing, positioning, separating, and

controlling. Although microactuators may be driven by different energy sources

(Sankaranarayanan and Bhethanabotla, 2009, MacDonald et al., 2004), they are often

powered by thermal, electromagnetical and/or mechanical means. Utilizing

technological knowledge of microactuators, one can control fluidic interactions at the

microscale level.

2.4.5.2. Control of fluidic movement

Although it has been generally accepted that fluid molecules cruise along paths

in a laminar state that lets fluids pass through the device without mixing, their final

destination depends on their properties. While bigger molecules are likely to go on

traveling until the end, smaller ones can either follow their initial route, or diffuse

away. Understanding the behavior of fluids in microstructures is the primary step

towards constructing proper devices. The behavior patterns of fluids can be influenced

by use of micropumps, valves, and mixers. Micropumps are known to be essential

elements in many devices due to necessity of maintaining controllable flows of fluids.

As their fundamental parameters such as head pressure and flow rate can be

optimized, micropumps may provide solutions to flow control. Based on the presence

or absence of moving parts, micropumps can be subdivided into two groups: non-

mechanical and mechanical. Non-mechanical micropumps that rely on properties of

fluids include: electrokinetic, magnetohydrodynamic (MHD), bubble based, and

ultrasonic pumps. Since the transition of electrical energy into energy by pumping by

electrokinetic pumps can occur in different ways, this kind of actuator can be further

29

subdivided into two categories: electroosmotic or electrohydrodynamic. Xu and co-

workers (2010) have described experiments that indicated that cell perfusion was

regulated by means of a device with a double chamber micropump. It resulted in the

establishment of a steady-state flow. Electrohydrodynamic (EHD) means themselves

have been recommended as being quite manageable for microfabrication. Kazemi and

co-workers (2009) have successfully introduced an asymmetry in the electrode of the

EHD micropump in order to improve its flow rate. Qian and Bau (2009) have

described advantages and disadvantages of non-mechanical micropumps based on

magnetohydrodynamic principles. Pan et al. (2009) have used numerical simulation to

study a micronozzle-diffuser pump based on the principle of thermal bubble

nucleation. Masini and co-workers (2010) have presented a micropump actuated by

the surface-acoustic-wave (SAW) mechanism. An accurate relocation of the fluids as

well as its splitting was accomplished. Even though it seems easy to impose certain

conditions on microfluids, some fluids can be driven only by means of mechanical

pumps.

The workings of different mechanisms that construct micropumps, which

include, but are not limited to, reciprocating and peristaltic movements, result in

different types of fluid flows. The various types of actuation that deliver reciprocating

movement can be classified as follows: thermopneumatic (Pol van de et al., 1990),

electrostatic (Nakano et al., 2005), electromagnetic (Zordan et al., 2010), shape

memory alloy (SMA) (Benard et al., 1997), and piezoelectric (Wang et al., 2006). Tai

et al. (2007) have used a pneumatic micropump to push cells ceaselessly through

microfluidic channels. Jun and co-workers (2007) have presented a thermopneumatic

micropump that used surface tension to take in fluid. Yun et al. (2002) have employed

the continuous electrowetting (CEW) phenomenon to move a mercury drop in an

electrolyte-filled microchannel. Yamahata et al. (2005a) have reported diaphragm

micropumps based on the electromagnetic actuation principle. Guo et al. (2006) have

developed a prototype micropump that used a solenoid actuator to provide a motion.

Rocha et al. (2009) have presented a micropumping system for potential LOC

applications.

30

There are numerous application-related conditions that can affect the working

performance of micropumping systems. While pneumatic actuation needs improved

flow control (Yang and Hsiung, 2008), thermopneumatic actuation requires efficient

heating to induce vibration of the diaphragm (Pol van de et al., 1990). It has been

demonstrated that one of the hallmarks of electrostatic actuation is a fast response

time. Yet, actuation by electrostatic means appears to have a driving force that works

over short distances (Woias, 2005). In contrast, electromagnetic pumps have a

relatively strong driving force; nevertheless they run on high power consumption

(Yamahata et al., 2005b, Shen et al., 2008). Although piezoelectric and SMA-driven

peristaltic micropumps are also power demanding, they can deliver great actuation

forces (Graf and Bowser, 2008). Because the majority of peristaltic pumps have

advanced programmable and flow control features, they can be relied upon (Graf and

Bowser, 2008). These kinds of pumps may be utilized for transporting heterogeneous

fluids (Hsu and Lee, 2009) including bacterial suspensions (Zhu et al., 2010a). The

method does not require incorporation of valves or mixers.

As mentioned above, some fluidic tasks can be accomplished by using either

passive or active micromixers. Since passive ones lack outer fields, they depend on the

geometrical or chemical properties of channels. Active mixers explore various

phenomena, including electrokinetic (Oddy et al., 2001), electromagnetic (Mohebi and

Evans, 2002), magnetic (Wei et al., 2010), electroosmotic (Jain et al., 2009), thermal

(Kim et al., 2009b), and ultrasonic (Monnier et al., 2000). As active mixers can

contain moving components, such as stir bars or diaphragms, they may be used to

either homogenize or dissolve samples.

2.4.5.3. Control of fluidic interactions

The theoretical insights that promote our understanding of fluid flow

phenomena are essential for design and construction of microdevices. Heterogeneous

fluids containing mixtures of either single biomolecules or self-assembled structures

can make fluid-fluid and/or fluid-solid boundary layers. Mechanical and

electromagnetic interactions have been known to control behavior of particles in

31

complex flows. Freer et al. (2004) have demonstrated the use of interfacial shear and

dilatational deformations to investigate the rheology of proteins at fluid/fluid

interfaces. Du and co-workers (2010) have studied the dynamics of tension for

particles in a fluid/fluid system so as to determine the parameters of the adsorption

process. The effect of the salt content of the particle environment and the structure of

the oil phase on the energetic parameters of particles has been demonstrated. Rathman

et al. (2005) have achieved a synthesis of biocompatible protein films at fluid/fluid

interfaces. The researchers proposed a mechanism of biomolecule self-assembly at the

interface. While the properties of fluid/fluid interfaces can be modified by addition of

different electrolytes (Mellema and Isenbart, 2004), their stability, can be modified by

applied potential (Thaokar and Kumaran, 2005). The leaning of some molecules can

be affected by interfacial curvature. In the case of acetonitrile, it may be due to the

necessity of protruding methyl groups of acetonitrile in a vapor phase (Partay et al.,

2009). It should be noted that there is a substantial difference between the dynamic

response of Newtonian and non-Newtonian samples (Torralba et al., 2005). In later

experiments the researchers observed the formation of nonsymmetric molecular

arrangements at very high amplitudes in the complex fluid (Torralba et al., 2007).

Helton et al. (2007) have reported the flow rate related instabilities of the interface

between the viscoelastic fluid and the Newtonian buffer. Molecular dynamic studies of

the fate of the interchannel bubble by Xie and Liu (2009) have indicated that bubbles

existed in the central part of hydrophilic channels. Furthermore, their velocities were

slightly lower than a single bubble of the main flow.

In order to create a suitable fluid-surface interface, one should identify best

choice surfaces relevant to applications. Using computer simulations, Voronov and his

colleagues (2006) have shown that the hydrophobicity of interfaces can be modified

by adjusting energy and size characteristics of fluid/surface couples. They reported

that drag lessening on hydrophobic substrates can be accomplished by selecting

fluid/substrate couples that have both low energetic and a high size parameter values.

The utilization of the surface nanostructures to achieve the friction control of

interchannel flows was presented by Cao et al. (2006). Pal and co-workers (2005a)

have studied the effect of surface topography on interfacial energy. It is interesting

32

that a higher energy was attributed to the structural features of the surface. In another

study these researchers have shown the use of pits for enhancement of substrate

hydrophobicity (Pal et al., 2005b). Later on, Setny et al. (2006) have reported that the

deportment of water molecules towards the surface in the neighbourhood depends on

the crookedness of the surface. Priezjev et al. (2007) have presented evidence that the

size of a fluid slip in a flow basically depends on local density and temperature. Guo et

al. (2005) have demonstrated that the impact of temperature on the velocity slip can be

eliminated. Iliescu et al. (2007a) have explored effects of both mechanical and

dielectrophoretic forces to transport suspension of round particles through a filter

device. Choi and Park (2007) have described a method of creation of pressure fields

for polystyrene particle manipulation in a microfluidic channel. Shevkoplyas and his

colleagues (2007) have studied the dynamic behavior of superparamagnetic particles

in a microchannel under an applied magnetic field. Cheng et al. (2009) have indicated

that conformations of DNA molecules depend on their positions in the curvilinear

fluid flow. Zhu and co-workers (2010b) have highlighted the role of the interchannel

flow parameters in the enhancement of microbial detection. Wu et al. (2009) have

presented a new microfluidic method for purification of human erythrocytes from the

sample contaminated with Escherichia coli. The authors used asymmetrical flow in a

suitable microchannel to fend off bigger specks.

Understanding the procedure for recruiting bacteria for power generation work

under microfluidic conditions is essential for the development of economical devices.

According to a study conducted by Kaehr and Shear (2009), motile bacteria, for

example Escherichia coli, can be in charge of microtransport in customized

microdevices. Over the past decade, many attempts have been made to use aligned

biopolymers in devices. Asokan et al. (2003) have utilized electrical forces to align

actin filaments during motility. The authors reported that the difference in response of

filaments to DEP torque was related to length of filaments. Recently, Kaur and co-

workers (2010) have provided evidence that actin alignment behavior can be

controlled by applying a weak magnetic field. They noted that filaments should be

deposited onto the surface prior to magnetic treatment. Importantly, Meer et al. (2010)

33

have successfully performed and analyzed the alignment of actin filaments in

microchannels controlled simply by shear stress.

2.4.5.4. Immobilization of proteins

2.4.5.4.1. Overview

There are two main types of relationships between proteins and surfaces that

can be defined: specific and non-specific. A target protein may selectively attach to a

surface either through specific or non-specific interactions. Although surface

modification techniques can be used to control protein adsorption behavior,

undesirable deposition may take place, when, for example, the protein undergoes

surface-triggered conformational changes. Non-specific immobilization may occur via

weak forces such as electrostatic, Van der Waals or hydrophobic. A specific kind of

immobilization can be implemented by the formation of covalent bonds between the

molecule and the channel wall (see chapters 4 and 5). It relies on complementarities of

proteins to surface functional groups. The SAM (self-assembled monolayer) method

can be used to deposit proteins in precise location at certain degrees of adsorption.

2.4.5.4.2. Physical adsorption

Physical adsorption of biomolecules on water-insoluble interfaces is a general

method of non-covalent attachment. The amount of adsorbed protein depends on

several factors including the kind of protein used, its concentration and environment,

and the contact performance of a material surface. A single molecule has the unique

ability to form both specific and non-specific bonds with available surface sites. The

diffusion of protein molecules from a liquid phase towards a surface is a set of

reversible and irreversible processes (Vroman and Adams, 1969, Vroman and Adams,

1986). Depending on the size and structure of the molecule, protein can become a

permanent or a temporary resident of polymeric surface. Erban and Chapman (2007)

have utilized the random sequential adsorption (RSA)-driven approach for the

simulation of irreversible adsorption of objects in real physical time. In the RSA

34

model, the arriving molecule tends to occupy the empty spot on the surface. After

touching down at the surface, the molecule blocks the access to its surface area for the

landing adsorbent (Evans, 1993). However, a later arriving molecule can compete for

a piece of surface with smaller and relatively less attractive settlers. Krishnan et al.

(2003) have highlighted the fact that protein competition for space at the liquid-vapor

interface is based on differences in molecular weight. Upon arrival at the surface,

pliable human immunoglobulin G (HIgG) can be met by early-arriving albumin (BSA)

and delivered to the surface via the Vroman‘s phenomenon (Sahin and Burgess, 2003).

Once attached, a protein may retain or change its native conformation and bioactivity.

For example, protein from almonds was capable of retaining its enzymatic activity

after irreversible adsorption at the water/organic medium boundary (Hickel et al.,

2001). Bower et al. (1999) have studied the effect of net electric charge on the

enzymatic activity of lysozyme at an interface. The study showed that electrostatic

force had a crucial effect on the functional activity of protein at a colloidal silica

substrate. Moulton and co-workers (2003) have demonstrated that electrostatic protein

attraction to an electrode surface is highly affected by an electrode charge. The authors

proved that human serum albumin (HSA) had a better blocking effect on the electron

transfer between an electrical conductor and an electroactive group than

immunoglobulin G (IgG). Kim and Yoon (Kim and Yoon, 2002) have indicated that

protein concentration in a solution affects the fractional surface coverage of polymer

particles. Final surface coverage depends on the protein properties and surface

conditions. Brewer et al. (2005) have explored adsorption of BSA on both citrate-

coated and bare gold substrates. Comparison of two surfaces indicated that protein

binding favors the bare gold one. This implies that protein spreads out on gold

surfaces upon unfolding. In the late 1990s, Cho et al. (1997) have shown that the BSA

protein has slow unfolding kinetics at the air-water boundary. The entire unfolding

journey of a BSA molecule can take on average over 20 hours (Cho et al., 1997). Lu et

al. (1999) have examined effects of BSA concentration and pH on the formation of a

protein layer at the air-water interface. It was found that a high protein concentration

at pH5 causes formation of a thicker adsorption layer due to low electrostatic

repulsion. The authors have noticed that BSA molecules tend to lie on the upper water

35

layer rather than stand on it. In addition to pH, salt concentration and surface

hydrophobicity can be used to control adsorption (Chang et al., 2010a). Noinville et al

(2002) have analyzed interfacial behavior of fungal lipase, BSA, lysozyme and α-

chymotrypsin on hydrophobic surface. This study demonstrates that α-chymotrypsin

exhibits greater attraction toward hydrophobic substrate than BSA and lysozyme.

Changes in the conformation of three model proteins upon adsorption were observed.

Lu et al. (1998) have examined morphology of the adsorbed layer of lysozyme at the

hydrophobic surface. It was concluded that the protein underwent irreversible

structural alteration. Su et al. (1998) have indicated that adsorbed lysozyme remains

safe in a low ionic environment on the hydrophilic surface. With increasing ionic

strength (above 0.5M), all protein molecules are forced to shift from the surface.

Special attention needs to be paid to ―working‖ conditions for self-assembled

proteins and their molecular partners. Numerous studies have demonstrated the

influence of magnesium (Gicquaud et al., 2003) and potassium on globular actin

polymerization (Senger and Goldmann, 1995, Goldmann, 2002). Rioux and Gicquaud

(1985) have noticed that negatively charged actin filaments produce self-assembled

structures on positively charged lipid substrates. St-Onge and Gicquaud (1990) have

suggested that the initial electrostatic attraction between actin and lipids alleviates

subsequent hydrophobic interactions. This type of protein adsorption may occur

anywhere on an appropriate substrate. Recently, Albet-Torres and co-workers (2010)

have proposed different mechanisms to account for heavy meromyosin (HMM)

adsorption. In doing so, the authors compared HMM interaction with two kinds of

substrates: SiO2 and positively-charged ones. It should be noted that despite

considerable research efforts, control of protein interfacial behavior remains

incomplete.

2.4.5.4.3. Covalent binding

Covalent immobilisation of biomolecules onto polymeric surfaces allows for

more precise application and operation of the coating layer. Covalently attached

molecules have a number of advantages over adsorbed ones (see chapters 4 and 5).

36

These advantages include stability, accessibility and security. Biomolecules can offer

a range of external surface groups (e.g., sulfhydryls, phosphates, amines, carboxyles,

etc.) for covalent immobilization. Using surface properties, one can guide a target

molecule toward a supportive matrix. Comparison of the various surface chemistries

such as aldehyde, amino-silane and carboxylic acid-modified glass surfaces revealed

high affinity binding of aminated DNA probes to aldehyde groups (Zammatteo et al.,

2000). These interactions were shown to happen without the participation of a

crosslinking agent.

The employment of the crosslinking couple, 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDC) with N-hydroxysulfosuccinimide (sulfo-

NHS) helped antibodies bind securely to carboxyl substrates (Pei et al., 2010).

Assessment of recruitment of a crosslinking team for immobilization of human

immunoglobulin G (HIgG) and lysozyme (LYZ) on surface-modified poly(tert-butyl

methacrylate) PtBMA has been provided by Ivanova (2006c). In most cases the

efficiency of coupling may be facilitated by the binding environment. There is enough

evidence to show the influence of pH (Weber et al., 1996, Cisneros and Dunlap,

1990), and temperature (Cisneros and Dunlap, 1990, Kao et al., 2010) on the binding

ratios of biomolecules. Although most of the biomolecules can be linked via various

groups, the systems produced by such interactions may become retarded or even

become disabled. Oriented towards substrate, some molecules cannot explore their

active sites. Lu et al. (1996) have demonstrated that the key to improving the binding

capacity of antigen is its orientation. Accordingly, immobilized molecules should meet

their binding partners at the right spot. It has been shown that secure positioning of

DNA molecules on the surface is crucial for obtaining a good DNA-DNA

hybridization yield (Zammatteo et al., 1997). Later studies have proven that one DNA

probe should have enough room on the surface to capture target DNA without

colliding with DNA probe neighbours (Zammatteo et al., 2000). Moreover, a coupled

DNA or protein probes should be able to resist exposure to severe conditions (e.g.,

temperature, pH). A comparative study of immobilization techniques has answered the

question of how much temperature change affects both the kinetics and the efficiency

of hybridization (Hakala and Lonnberg, 1997). The authors demonstrated that rising

37

temperature leads to a decrease in the efficiency of oligonucleotide hybridization. To

stay attached securely, the DNA molecule should be supported by a suitable linker.

Azo-linkers have been developed which can assist in the short-time UV-irradiation

treatment of DNA (Wanga et al., 2008). The bifunctional crosslinker EDC has been

employed to demonstrate that oligonucleotides can be successfully covalently

introduced to the polystyrene-co-maleic acid (PSMA) polymeric surface (Ivanova et

al., 2002c). The same crosslinker has been utilized to support stable binding of DNA

molecules to NucleoLink under DNA-DNA hybridization conditions (Christensen et

al., 2000). Our study provides evidence that the EDC linker not only anchors G-actin

on a PSMA surface, but also allows subsequent self-assembly of the actin filament

(Alexeeva et al., 2005).

2.4.5.4.4. Self-assembled monolayers (SAMs)

Self-assembly of supramolecular structures can be used as a means towards the

fabrication of novel nanomaterials. Controlled chemistry of SAMs allows the

optimization of surface properties that are employed in the construction of biosensors

(see chapter 6). A broad range of subunits (e.g., alkanethioles, proteins, peptides,

nucleotides, lipids) can be successfully utilized for SAM creation. Molecular

interactions in SAMs are mediated by the formation of noncovalent bonds between

building components. Although connection via weak forces is sufficient for SAM

growth, it is not secure for its accommodation. Ataka and Heberle (2008) have noted

that some bulky non-polar molecules tend to dislocate easier than small polar ones. By

applying covalent immobilization, researchers could provide a higher level of SAM

stability for eliminating molecular rearrangements (Darain et al., 2009, Min et al.,

2010).

This ―bottom-up‖ approach, in which novel functional structures (actin

bundles, muscle fibres, ATPase, etc.) are produced from suitable building blocks, is

used by all living organisms. In cell dynamics of major self-assembled networks, such

as pro- and eukaryotic cytoskeletons, it is regulated by both extra- and intracellular

(e.g., temperature, pressure, ion concentration) conditions. Understanding that natural

38

processes can be employed for the production of biomimetic materials, researchers

study the ability of biomolecules to perform nanotechnological tasks on various

artificial surfaces; accordingly, they have molecular candidates checked for

―biomolecular – surface‖ biocompatibility. Baujard-Lamotte et al. (2008) have

demonstrated the effect of SAMs properties on the adsorption of fibronectin (fn),

extracellular glycoprotein. A conformational change from normal to β-sheet-modified

state of fn accompanies its adsorption onto the hydrophobic substrate. The researchers

found that protein concentration acts on SAM capacity to accommodate protein,

letting a certain number of native fn molecules contact SAM. Sagnella et al. (2005)

have developed a system with the surface properties of the extracellular matrix. The

authors created a biomimetic substrate of poly(vinylamine) with adhesive components

on the octadecyltrichlorosilane (OTS) SAM and seeded human pulmonary artery

endothelial cells (HPAEC) on the resulting polymer. Although the stimulator of

adhesion, HBP (heparin-binding peptide), was able to interact with endothelial cells to

provide bonding, the lack of sufficient support for HPAEC adhesion caused untimely

cell cytoskeletal damage and cell-surface detachment. Meanwhile, other researchers

have demonstrated that muscle cells had survived on the cysteamine surface without

anchorage by the extracellular matrix (ECM) regulating components (Coletti et al.,

2009). Furthermore, the study of the cultivation of skeletal muscle cells on surfaces

showed that myotube monolayer morphology was more uniform on cysteamine coated

with gold than when myotubes were on bare gold. However, the ability of muscle

cells grown on the cysteamine SAM to display their differentiation potential was only

about 70 %. Studying the physiology of muscle cells on SAM can help researchers

create hybrid devices that contain rotary motors, ATPase-like muscle cells

(Montemagno and Bachand, 1999), but function much longer. In fact, changes in local

environment (e.g., pH, temperature) may influence ATPase performance. To assemble

a more controllable rotary motor, Tao et al. (2009) combined ATPase with self-

assembled structures. Obviously, ATPase is a promising candidate for implementation

of power management in microdevices.

In addition to the ―bottom-up‖ approach, there is another type of approach, the

―top-down‖ that is used to take away some of the material to create a structure. As the

39

size of any kind of destruction goes up, the potential issues of the method, such as

physical barriers and cost may arise. Despite the potential limits of 3-D structure

construction (e.g., channels of different sizes and geometries), the ―top-down‖

approach may be used to complement the ―bottom-up‖ method. As discussed in

previous subsections, an optimized microfluidic system is able to guide self-assembly

of molecular motor proteins.

2.5. Concept of protein molecular motors

2.5.1. Overview

Molecular motors are protein machines driven by energy coupled to nucleotide

(ATP, GTP) hydrolysis (Liu et al., 2005b), ion motive (IMF) (Bai et al., 2009) or

proton motive forces (PMF) (Nakanishi-Matsui et al., 2010). They can produce

mechanical force and torque and transport cargoes over specific substrates, while the

character and rate of their action can be controlled (Wang and Manesh, 2010,

Bustamante et al., 2001). Although the term motor refers primarily to cytoskeletal

motors like myosin, kynesin or dynein, their partners (actin, tubulin) also produce

force (extend/shrink motion) and may be described as distinct types of motors (Kueh

and Mitchison, 2009). Unlike cytoskeletal motors, the transmembrane F0 motor is

unable to shift membrane. It requires either nucleotide hydrolysis or motive force

(MF) to produce rotary torque. In addition to eukaryotic motors, there are bacterial

nucleotidases such as actin homologues (MreB, FtsA, ParM) that have the structural

and dynamic properties of molecular motors.

2.5.2. Eukaryotic actin

Self-assembly is crucial characteristic of monomeric actin (G-actin) that consumes

ATP energy produced by rotary motors to form a double-stranded, polar, helical,

filamentous actin (F-actin). The polymerization goes though three stages of

development: nucleation, elongation and equilibration and needs sufficient

40

concentration of G-actin (Grintsevich et al., 2010, Husson et al., 2010). Actin in its

monomeric form (G-actin) is composed of two similar domains (Kabsch and Holmes,

1995). Each of the domains consists of two subdomains; two upper subdomains, also

known as the ―barbed end‖, have greater affinity for globular actin, and the two

bottom subdomains (the ―pointed end‖) have lower exchange rates for actin

(Southwick, 2000). The phosphate moiety of a nucleotide (ATP or ADP) sits on the

interdomain clift (Kabsch et al., 1990). Polymeric forms of actin, or thin filaments (or

f-actin) are helical polymers which have 13 actin molecules (42 kDa) arranged on six

left-handed turns repeating every 36 nm. The thermodynamic properties of the self-

assembly/disassembly of actin have been described by Oosawa and Asakura (1975).

The authors suggested that the rate-limited step for polymerization altered with the

joining of the third protomer. Further support for this came when Purich and Allison

(1999) compared kinetic properties of microtubules and actin filaments. The authors

concluded that the third G-actin-ATP protomer takes an important role in the

nucleation process that induces a more stable polymerization ―nucleus‖. Once the

three-meric ―nucleus‖ is built, a thermodynamically favoured elongation stage begins.

Woodrum et al. (1975) studied the growth of F-actin and reported that bidirectional

polymerization occurred from both sides of actin filaments. Moreover, authors

highlighted that topologically distinct ends of the nucleus had different growth

potentials. Thus, the ―barbed‖ tip of an actin trimer grew faster than the ―pointed‖ one.

Pollard (1986b) has demonstrated the importance of the critical concentration of ATP-

actin in a local medium for filament assembly. Thus, as long as it remains above

critical value, both sides of the filament elongate. A decrease in ATP-actin

concentration to a critical concentration is associated with entering the equilibration

stage of polymerization. At equilibrium, the addition of actin subunits at the barbed tip

is in balance with the loss of actin subunits at the pointed tip of the filament. As a

result, F-actin ends are in a state of subunit flux (Kirschner, 1980); it maintains

constant average polymer size.

The destiny of actin polymer (detachment, branching, fragmentation,

crystallization, lifetime, etc.) at any stage of assembly is strongly affected by

numerous factors: temperature, pH, protein, salt and ATP concentration, the presence

41

of actin-binding protein (ABP), etc. Thus, polymerization can be accelerated by

increasing temperature up to room temperature (Grazi and Trombetta, 1985),

adjusting pH to 7.0 (Lin et al., 1997, Wang, 1989), adding ATP (Pollard, 1986b,

Fujiwara et al., 2007), including the most effective bivalent ions Ca++, Mg++

(Bergeron et al., 2010, Carlier et al., 1994), and joining actin binding proteins (ABP)

(Pollard, 1986a).

Wachsstock et al. (1993) have realized that the factors that guided actin alignment

were affinity of ABP, F-actin length and protein concentrations. In the author‘s model,

diffusion can mislead actin filaments. Thus, the formation of aligned actin bundles

does occur, but the behavior of actin in the highly viscous fluid state is so

unpredictable that the influence of diffusion is strong enough to reorganize pre-

bundled filaments. For generating electrostatic association between actin filaments,

the polymerizing medium must contain sufficient amount of divalent cations. Angelini

et al. (2003) have used high (Z) counter ions Ba++ for F-actin bundling. Their later

work continued on the issue of the bundling mechanism, particularly its dynamics

(Angelini et al., 2006). The remarkable fact that has been noticed is that Ba++ bound

between filaments caused acoustic–based dispersion. Furthermore, when divalent

metal ions interacted with nearest-neighbour cages, they produced a liquid-like

correlation phase and dynamic system response. However, both ion and coion

behavior need to be evaluated regarding various areas of the heterogeneous actin

surface.

Undoubtedly, a two-way actin assembly may include interactions with various

members of ABP group. The work reported in this thesis is mainly focused on ABP

representatives from three different families, such as actin, gelsolin and PARPs.

Gelsolin (GLS) is an actin-serving protein that affects both assembly (e.g., capping,

nucleation) and disassembly (e.g., fragmentation) of actin. It contains six structurally

similar domains that differ in regard to actin and calcium affinity (Burtnick et al.,

1997). A schematic model was proposed by Way et al. (1989) to explain the

contributions of individual GLS domains to interactions with actin. It demonstrates

that only GLS 1 remains in touch with actin after the calcium shifts. The authors

further concluded that the GLS 1 segment is responsible for a stable capping of actin

42

after calcium withdrawal. As for segments 2 to 6, GLS 2-3 serve and subsequently cap

actin filaments, while GLS 2-6 support actin nucleation. So the GLS 1 segment has a

specific function. Utilizing the properties of the gelsolin domain (GLS 1), the length

of the actin filament can be controlled via stabilization of the conformation of the

actin‘s domains. In doing so, the length parameter of F-actin can be adjusted (Janmey

et al., 1986).

While actin polymerization in the cell (Meindl et al., 1994), in the solution

(Flanagan and Lin, 1979), and at the surface (Gadasi et al., 1974) was intensively

investigated for its role in a variety of important cellular processes (see sections 2.5.2.

and 2.5.6), the paramount importance of actin assembly along a topographically

patterned surface was realized only in 1990s. For example, it was not until 1995 that

the orientation of kidney fibroblasts at the grooved surface was studied (Wojciak-

Stothard et al., 1995). By exploring patterned substrates – such as poly(methyl

methacrylate) (Suzuki et al., 1997), titanium and silicon (Hirst et al., 2005) – the

researchers have become convinced of the need for development of polymeric

surfaces with built-in channels (Nicolau et al., 1999), and achievement of a better

surface molecular motor alignment (discussed in previous subsections). However,

under device-realistic conditions, eukaryotic actin can stay fit enough to perform its

nanotechnological tasks only for a relatively short period of time. Theoretically, the

best alternative possible is one of PARPs, namely, the MreB or FtsA that the majority

of bacterial species use to stay in a particular body shape and other purposes

(discussed in the following chapter).

2.5.3. Prokaryotic actin related proteins

2.5.3.1. Overview

In the last fifteen or so years, the attention of researchers has been drawn to the

importance of non-molecular motor-based motions. Since then, several families of

actin-related proteins have been defined in organisms as different as humans, yeast,

plants and bacteria; with each ARP assigned to a family on the basis of its primary

43

amino acid sequence identity compared to conventional actin. Actin-related proteins

share 11 to 60 % identity (Schafer and Schroer, 1999, Carballido-Lopez, 2006). Many

bacterial actin relatives share significant sequence similarity but have limited

functional homology and ligand binding specificity. Based on homology in amino acid

sequences, several bacterial proteins are believed to belong to the actin family. These

proteins include heat shock proteins, sugar kinases, and the following proteins

synthesised in microorganisms: the bacterial chaperon DnaK [heat shock protein

(Hsp70)] (Wetzstein et al., 1992, Martinez-Alonso et al., 2010, Rhee et al., 2009), the

cell division protein FtsA (Strahl and Hamoen, 2010); the plasmid-encoded ParM

(Popp et al., 2010a) as well as a constituent of bacterial cytoskeleton Mbl, MreBH

(Schirner and Errington, 2009), and MreB (Wang et al., 2010). In term of the

structural and functional similarity, four bacterial nucleotidases, namely, actin (MreB,

FtsA, ParM) and tubulin (FtsZ) homologues own unique properties of eukaryotic

molecular motors.

2.5.3.1.1. MreB

Among all proteins of the Hsp70/actin/sugar kinases superfamily, MreB

(murein cluster e) protein is the most similar to actin. This actin homologue has a

similar 3D structure and the ability to undergo actin-like polymerisation. It has been

reported that MreB and actin monomers showed significant resemblance at their

atomic level (van den Ent et al., 2001). Both of them had two domains with binding

pockets for ATP molecules. Also, each domain consisted of two subdomains that are

structurally more identical with actin than any other actin-related proteins (the

following subsection provides an example of the FtsA protein structure). The MreB

protein assembles into one-dimensional protofilaments with smaller subunit spacing

(51 Ǻ) than actin (55 Ǻ). Futhermore, in vivo MreB monomers can treadmill (Biteen

and Moerner, 2010) in a directional manner and hence generate polarized assembly

(Kim et al., 2006). Although MreB is structured similarly to actin, it is capable of self-

organizing into straight filaments (Vollmer, 2006, Allard and Rutenberg, 2009),

bundles (Srinivasan et al., 2007, Jiang and Sun, 2010), sheets (Popp et al., 2010b) and

44

ring-shaped structures (Esue et al., 2005). Like actin, MreB polymerization can be

affected by such factors as critical concentration (Cc), the availability of a source of

energy (ATP or GTP), the presence of bivalent cations (Mg++) and temperature

(Mayer and Amann, 2009). On the basis of a report by Esue et al. (2005), there has

been ample evidence that under the same experimental conditions the critical

concentration (Cc) for MreB can be as low as 0.003µM in comparison with 0.25µM

for actin. This means that prokaryotic MreB is roughly 83 times more efficient than its

eukaryotic homologue. In discussing their paper, the authors offered the suggestion

that MreB monomers have not only a much greater affinity for each other but also a

remarkably better affinity for the MreB filament than actin. In this work, MreB sets

phosphate free (Pi) at a rate of 0.10 Pi per actin per min. (Esue et al., 2005), which is

very similar to F-actin (approximately 0.16 times slower). It should be noted that, in

contrast with actin preferences, where ATP is a more potent player than GTP, MreB

catalyses ATP and GTP hydrolysis equally well (Esue et al., 2006).

The fact that some bacteria, namely, Bacillus subtilis owns three isoforms of

the MreB protein (Mbl, MreBH and MreB) (Schirner and Errington, 2009), while

other bacteria, for example, Thermotoga maritima (Popp et al., 2010b) and/or

Escherichia coli (Varma and Young, 2009) hold only single MreBs suggests that all

three isoforms could play important roles in growth and morphogenesis of only some

of the bacteria, such as Bacillus subtilis (Kawai et al., 2009a). Additionally, MreB

does not act alone; it collaborates with other actin orthologs, namely, MreC and MreD

(Defeu Soufo and Graumann, 2005). It is important to emphasize that MreB protein

does not have a protein partner, molecular motor (Vats et al., 2009), so the eukaryotic

mechanism of force generation is unlikely to work in bacteria (Erickson, 2001).

However, one protein, RodZ (YfgA), discovered by Bendezu et al. (2009), was found

to be part of the MreB spiral-like structure (van den Ent et al., 2010). The functional

role of the important prokaryotic player MreB in bacteria is discussed in chapter 2.5.6.

The discovery that bacteria contain ―single‖ actin homologues raised new

issues, especially in respect to assembly of MreBs from various bacteria. It has been

demonstrated that MreB works in both gram-positive, namely, Bacillus subtilis

(Mayer and Amann, 2009), Listeria monocytogenes (van der Veen et al., 2007), and

45

gram-negative bacteria, for example, Thermotoga maritima (Esue et al., 2005), Vibrio

parahaemolyticus (Chiu et al., 2008). However, some pathogenic bacteria, such as

Listeria monocytogenes, do not waste their own MreBs on travelling through the host

cells. They would rather explore the host‘s actin than invest in their own material. The

pathogenic strategy of enteric bacteria is discussed in detail in subsection 2.5.6.5.

Studies on the polymerization of Thermotoga maritima MreB revealed that the

temperature and the concentration of divalent cations, namely Ca++ or Mg++ affect

both its assembly and ultrastructure (Esue et al., 2005). The remarkable thing about

MreB is that it can produce straight (Vollmer, 2006), ring-like structures (Vats et al.,

2009), or sheets (Popp et al., 2010b). It is plausible, on the basis of the suggestion of

Esue et al. (2006) in their study of GTPase activity of MreB that the shape of the

assembled MreB depends on the extent of ATP or GTP hydrolysis within the MreB

polymer. An understanding of MreB assembly seems to be not an easy matter. Thus,

Bean et al. (2008) have come across some significant findings when they tested the

biochemical properties of Thermotoga maritima MreB. It turned out that the MreB

protein can assemble across a wide range of temperatures. As for divalent ions, the

authors assumed that Ca++ and Mg++ do not play a vital role at the nucleation stage of

MreB polymerization. Mayer and Amann (2009) have succeeded in examining MreB

from Bacillus subtilis and have understood its biochemical properties. By determining

and evaluating the temperature range of polymerization, nucleotide and ion

preferences, they showed the difference between the MreBs from Thermotoga

maritima and Bacillus subtilis. Unlike these two representatives of quite different

groups of bacteria, such as Thermotogae and Firmicutes, a member of the phylum

Proteobacteria, Caulobacter, can produce long filamentous assemblies (Kim et al.,

2006). However, the mechanism by which MreB structures are built has not been

clarified. To explain uncommon MreB behavior, the authors proposed that bundling of

short MreB filaments in a free global polarity manner causes formation of mysterious

long structures. Even though MreB has received considerable attention with regard to

its assembly (Graumann, 2009), the possibilities and limitations of the use of MreBs

from various bacterial species in vitro have not been explored (discussed in chapter 9).

46

2.5.3.1.2. FtsA

Although there is no biochemical evidence that cytosolic cell division protein

FtsA forms actin-like filaments, there are two highly conserved proteins (FtsA and

tubulin orthologue FtsZ) involved in the cytokinesis of bacteria that are structurally

related to components of the eukaryotic cytoskeleton and they have a central role in

cytokinesis (Beuria et al., 2009). FtsA is recognized as the key cell division

determinant, other than FtsZ, that lacks a clear membrane-spanning sequence (Pla et

al., 1990). The crystal structure of FtsA did confirm that FtsA is a homologue of actin

and the heat shock protein (Hsc70). However, unlike the members of actin family, the

second subdomain of FtsA, namely 1C, is sitting on the opposite site of subdomain 1A

from its location in actin (van den Ent and Lowe, 2000). It has been shown that the 1C

domain of FtsA is engaged in the interplay between its own molecules and the other

proteins that are recruited to the division ring assembly (Rico et al., 2004). The simple

explanation for the unique structural features, such as insertions of entire domains, is

that FtsA carries out specific cellular functions. Thus, the distinction between the

position of the subdomain 2 homologue for FtsA and MreB, can only make MreB the

true orthologue of actin (Egelman, 2003).

Paradis-Bleau et al. (2005) have proved that of the two nucleotides, namely,

ATP and GTP, the former is favored by FtsA. However, interaction between FtsA and

ATP was found to be strongly dependent on the location of FtsA protein (Sanchez et

al., 1994). Thus, only the cytoplasmic phosphorylated form of FtsA has the ability to

bind ATP molecules. Studies on the contribution of FtsA to bacterial cell division

have revealed that the conserved carboxy motif of FtsA operates as a membrane

targeting sequence (MTS) by tying up the Z ring to the cell membrane (Pichoff and

Lutkenhaus, 2005).

The current understanding of the molecular mechanism of actin (described in

subsection 2.5.2.) and/or MreB (described in the previous subsection) is that

polymerization is not applicable to FtsA assembly. The work reviewed above helps us

to understand why it was found that FtsA was unable to produce actin-like filaments

that can be a reliable FtsZ anchor. Despite the lack of data on FtsA assembly

47

properties, some studies have shown that FtsA molecules can interact with each other

(Lara et al., 2005). Strikingly, Lara and co-workers have provided ample evidence that

the FtsA protein of cocci produces very stable corkscrew-shaped spirals. The authors

have suggested that FtsA can form polymers in vivo due to its sufficient concentration,

presence of a large number of ATP molecules and other proteins that can assist it.

Feucht et al. (2001) have reported that both dimeric and multimeric FtsA forms exist

in Bacillus subtilis. In their examination of the cytological and biochemical properties

of FtsA, the authors indicated that FtsA predominantly exists as dimers.

It is also interesting to note that the contributions of FtsA to cell function may

vary depending on cell structure. For example, the number of FtsA molecules in gram-

positive bacteria, namely, Bacillus subtilis, is 5-20 times higher than the number of the

same molecules in gram-negative bacteria, for example, Escherichia coli. Feutch et al.

(2001) have concluded that gram-positive bacteria need more ATP energy for cell

division due to the broad peptidoglycan coat of the cell, greater intracellular osmotic

pressure and/or lack of a ZipA protein. Indeed, Staphylococcus aureus has 40-nm

thick cell wall, which is 7nm thicker than the periplasmic coat of Escherichia coli

(Dubochet et al., 1983). Furthermore, osmotic pressure in gram-positive bacteria, such

as Staphylococcus aureus, was estimated to reach 20-30 atmospheres, which was 6

times as high as in a gram-negative cell of Escherichia coli (Kuczynski-Halmann et

al., 1958). However, it was observed that the ZipA homologues are missing not only

in gram-positive but also in gram-negative bacteria, except for γ-Proteobacteria.

Having established that Escherichia coli can get around the lack of ZipA, Geissler et

al. (2003) proved that gram-negative bacteria can hire slightly modified FtsA for a

ZipA role. In fact, all of these factors may trigger a higher level of expression of FtsA

in gram-positive cells. So, questions related to FtsA assembly properties remain to be

answered.

48

2.5.4. Evolution/Phylogeny of bacterial actin homologues

2.5.4.1. Overview

Over the last decade or so, there has been some interest in the relationship

between actin and PARPs (Egelman, 2010, Lowe et al., 2010). In eukaryotic cells,

actins are major players in the generation of various internal cytoskeletons.

Acquisition of cytoskeletal motility is considered to be one of the major events in

evolutionary history that enabled eukaryotic cells to perform variety of cellular

functions (the contributions of MreB to the cytoskeletal organization is discussed in

detail in chapter 2.5.6.). Except for some cell wall-less bacteria, such as Mycoplasma

insons, which benefits from a harmless commensal interaction with green iguanas

(Relich et al., 2009), all other prokaryotic and eukaryotic organisms have

cytoskeletons. The classical characteristics commonly used to compare cytoskeleton

counterparts, namely PARPs with actins, include atomic structure, biochemical

properties, biophysical and DNA and/or amino acid composition.

2.5.4.2. Evolutionary/Phylogenetic comparison of MreB with FtsA

As has been demonstrated in previous subsections, actin, MreB and FtsA can

use the energy from either ATP or GTP hydrolysis to produce mechanical motion.

However, actin and FtsA prefer the former to the latter. Some subdomains of these

proteins are predicted to adopt tertiary structures identical to ATP-ase subdomains of

hexokinase (Bork et al., 1992). Comparison of MreB with FtsA subdomains

(discussed in the previous subsections) reveals that MreB exhibits more similarity in

atomic composition to actin than FtsA. In terms of polymerization properties, MreB

shows actin-like behavior, except that it displays greater morphological variation and

more efficient polymerization properties, such as a parsimonious critical

concentration, time-saving nucleation (Esue et al., 2005), and flexibility in nucleotide

choice (Esue et al., 2006). In contrast, the assembly behavior of FtsA is completely

49

different from its actin- or MreB-like behavior (discussed above in subsection

2.5.3.1.2.).

A noteworthy feature of actin is that it belongs to a multigene family of

evolutionary conserved proteins. For example, different members of the eukaryotic

actin family, namely, plant and fungi show about 80-85 % identity to mammalian actin

(Doolittle and York, 2002). The number of actin genes, however, can vary among

species. Thus, the unicellular eukaryote Saccharomyces cerevisiae has only one

isoactin (Gallwitz and Seidel, 1980); ascidians, invertebrate chordates, contain four

actin genes encoding one alpha-muscle actin (Beach and Jeffery, 1992). In addition,

the examination of amino-acid composition in vertebrate bovine tissues revealed a set

of six actin isoforms (Vandekerckhove and Weber, 1978a), the set consists of two

cytoplasmic (beta and gamma) (Vandekerckhove and Weber, 1978b) and four muscle

ones, namely skeletal, cardiac, vascular, and nonvascular actins. A comparison of

yeast actin with bovine beta-actin have showed that those proteins shared a high level

(up to 90 %) of sequence similarity (Egelman, 2001). To account for the unique

conservation, various possible explications have been proposed. One explanation for

this phenomenon is that, in actin, a vitally important cellular player, the selection for

subunit-subunit and subunit-filament contact sites, as well as ABP-sites, is sufficiently

strong, so that the tertiary protein structure is maintained (Pollard, 1984). In order to

estimate the extent of DNA conservation, Ponte et al. (1984) have examined human B-

actin cDNA clones. The authors‘ suggestion that the 3‘-UT region of cDNA can drive

the expression of actin genes was made on the basis of high homology between

untranslated regions of cDNA of human and rat β-actins. It was thus concluded that

the considerable high similarity may reflect strong evolutionary pressure on

untranslated portions of cDNA. In fact, if eukaryotic actin was not well conserved, its

structure could be easily affected by any of the ABPs (their influences on actin are

discussed in subsections 2.5.2. and 2.5.6.2.).

In addition, bacterial homologues of actin, MreB and FtsA have neither got

large sets of accessory proteins (e.g., the only one is MreB‘s spiral-like configuration

creator, RodZ (YfgA) so far), nor any introns. Moreover, despite their non ubiquitous

nature, MreB as well as FtsA genes conserved throughout eubacterial lineage. Both

50

proteins display very low levels (e.g., MreB with 15 % similarity) of sequence identity

to actin (Carballido-Lopez, 2006). Interestingly, the ftsA gene is much more

conserved in bacteria than the other cell division genes and is almost always found in

tandem with ftsZ (Dai and Lutkenhaus, 1992). Furthermore, the relationship between

the ftsA and ftsZ gene products reflects the need to maintain a highly accurate

expression ratio of these genes for a normal cell division. An interesting observation

concerning the evolutionary relationships between eukaryotic cytoskeletal components

and their bacterial counterparts has been made by Doolittle and York (2002). The

authors revealed that the evolutionary scale positions of both MreB and actin drift

away from the ubiquitous heat shock protein 70 (Hsp70) and glycolytic enzymes, such

as enolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and triose-

phosphate isomerase (TIM). Based on these results, they suggested that eukaryotes

and prokaryotes shared a common ancestor long ago; the organism owned a protein

with properties common to MreB. Since the majority of actin relatives cannot

assemble filaments, Egelman (2003) has suggested that the ancestor had a monomeric

structure. However, the idea of sharing a common monomeric ancestor by the

members of the actin family ran contrary to Bork et al.‘s (1992) view that the ancestor

is an ATP binding homodimer which carried on via a duplication event preceeded by

structural divergence. Doolittle and York (2002) proposed that the ancestral protein

was capable of self-assembling into plain filaments in an ATP-dependent manner. In

order to understand MreB and FtsA phylogenies, one needs to take a close look at the

phylogenies of bacteria that own them. Furthermore, the correlation between

phylogenetic analyses based on MreB and/or FtsA and 16S rRNA sequences should be

estimated to evaluate the possibility of using MreB and/or FtsA sequences as a

chronometer to help unravel phylogenies of new and/or misplaced bacteria (see

chapter 9).

2.5.4.3. Use of 16S rRNA as a molecular chronometer

With the recognition that ribosomal ribonucleic acid (rRNA) can be used as a

molecular chronometer (Woese, 1987), the method of 16S rRNA sequencing became a

51

standard procedure for identification of any living organisms, including the producers

of MreB and FtsA proteins. By using a molecular phylogenetic analysis method based

on 16S rRNA sequences, Woese and Fox (1977) were able to present evolutionary

relationships among different species. According to their three-domain model of the

universal tree, prokaryotes include two subkingdoms (urkingdoms), namely,

eubacteria and archaebacteria. Distribution of actins, MreB, FtsA, and Hsp70 proteins

among the three domains of life has been investigated by Doolittle and York (2002).

Notably, while actins are present in eukaryotes, they are lacking in prokaryotes. In

contrast, MreBs are unevenly distributed across only two prokaryotic urkingdoms. The

authors concluded that the striking structural similarity in the absence of sufficient

sequence similarity of MreB and actin was due to early divergence. In addition, only

Hsp70 was found in both prokaryotic and eukaryotic urkingdoms of life. As for FtsA,

it was detected in the eubacterial domain of life.

However, some researchers have doubts about the ability of 16S rRNA

analysis to reflect a evolutionary history of life. Thus, Gupta et al. (1997) have

proposed a molecular approach for estimating microbial phylogeny based on the

presence or absence of conserved amino acid segments (called either indels or

signatures). Using this method, the authors developed a model showing the reflection

of hierarchical order of various bacterial groups. Furthermore, Gupta (2000a) has split

bacteria into two major groups: monoderms (with single membranes) and diderms

(with two membranes separated by periplasmic spaces). Four years later, Griffiths and

Gupta (2004) provided evidence for placing Deinococcus into an intermediate group

(its members possess not only two membranes but also a thick peptidoglycan). It

should be emphasized that Gupta (2000b) has recognized the evolutionary value of

members of the proteobacterial phylum. The author was able to demonstrate that not

only mitochondria but also the nuclear cytosolic homologues of some eukaryotic

genes originated from proteobacteria. Examination of protein sequences led to the

finding of striking homology between MreB and the first half of the Hsp70 sequence

(Gupta and Singh, 1992). Interestingly, in contrast to MreB, bovine actin did not

display substantial similarity to Hsp70. This result might indicate that the MreB

derived from a precursor of Hsp70. Although a ―protein signature‖ method can

52

provide valuable evolutionary insights, it cannot be employed for the analysis of

noncoding sequences such as introns. Another limitation is the lack of reliable indels

for all proteins.

Apart from molecular genetic methods, transitional analysis together with the

fossil record has been used for drafting a sequence of life-history events (Cavalier-

Smith, 2006a). Cavalier-Smith (2006b) has offered an interesting explanation for the

molecular events accountable for polarization of transitions. According to his model,

the transformation of the MreB cytoskeleton into an actin one through gene

duplication occurred with the participation of the Arp 2/3 complex (the product of

MreB triplication in the pre-eukaryotic organism). It was noted that such a gene

creation event was crucial for megaevolution (the term refers to quantum evolution).

Though the approach is valuable in regard to gaining a deeper understanding of

evolutionary relationships between phylogenetically-distant species, it is not suitable

for microbial classification due to the lack of experimental evidence.

It goes without saying that tremendous efforts have been devoted to the

development of a precise approach for deducing prokaryotic phylogeny. However,

none of the currently available methods can reflect the knottiness of microbial

connections. Furthermore, the use of one criterion for figuring out the evolutionary

history may lead to misinterpretation. Consequently, the choice of a sufficient number

of reliable molecular chronometers may help us reach a deeper understanding of

phylogeny (see chapter 9).

2.5.5. Classification of protein molecular motors

2.5.5.1. Overview

Motor proteins can be classified into two major groups such as linear and

rotary according to the mode of operation. This subsection starts with structural

comparison of different types of linear molecular motors and their in vitro

performance (see also chapter 6). It continues with a comparison of eukaryotic and

prokaryotic ATP synthases and their in vitro applications (see also chapter 8). While

53

linear molecular motors, namely eukaryotic actin (or its homologues MreB or FtsA

proteins) can be used for building linear nanotracks (see chapters 6 and 9), prokaryotic

producers of ATP may be incorporated into biosensors for supplying molecular

motors with cheap ATP energy (see chapters 6 and 8).

2.5.5.2. Linear molecular motors

Despite principal differences in molecular properties (e.g., weight, dimension),

the major linear motors, namely, myosins, kinesins, and dyneins, share some

similarities. The three motors have two identical heads (or motor domains) that bind to

the polymerized substrates (i.e., actin filaments for myosins, and microtubules for

kinesins/dyneins) and that catalyse ATP hydrolysis in a cytoskeleton-dependent

manner.

It is important to note that these representatives of three superfamilies have one

hallmark, the P-loop, designed by nature for ATP binding (Walker et al., 1982).

Unlike myosin or kinesin, dynein has four consensus P-loops (Ogawa, 1991) and up to

three heads (Gibbons et al., 1991) with numerous ATP binding sites (Gibbons et al.,

1991). Furthermore, due to multisubunit architecture (Bruno et al., 1996), cytoplasmic

dynein relies heavily on the presence of different accessory proteins (Burkhardt et al.,

1997, Lam et al., 2010). In addition, it cannot walk with a uniform step size (Singh et

al., 2005). Despite the possibility of the control of dynein performance at different

levels, it is practical to employ less demanding and simple motors, such as myosin and

kinesin, with their maximum capabilities to transport cargo in microdevices. As for

myosin and kinesin, they are two-headed (except for the single-headed myosin I),

molecular edifices that end with two-arm forks (Rayment, 1996), which are in charge

of pushing. The common feature of kinesin and myosin is the structure of the motor

domain. It has been shown that the kinesin‘s head domain is remarkably similar to the

catalytic core of myosin (Kull et al., 1996).

So, these proteins exhibit some structural similarity even though they share a

low amino acid identity (Kull et al., 1996) and display distinct enzymatic properties

(Johnson and Gilbert, 1995). Half-lying on the microtubule (Gibbons et al., 2001),

54

kinesin walks on two flexible legs with identical small feet (or motor domains) along

the tubulin track (Kull et al., 1996) and takes a break when it meets an obstacle, such

as another kinesin, in its way (Seitz and Surrey, 2006). Contrary to the assumption that

kinesin remains permanently attached to the microtubule during the entire walk, Block

et al. (1990) have demonstrated the inability of a small team of kinesins to continue

their journey up to the final destination on the microtubule track. So the molecular

motor team came off the track because it could not carry cargo over the expected

distance. The authors suggested that kinesin spent a short time away from its substrate

during any force-generating cycle. Further evidence confirmed kinesin‘s detachment.

By using mutant and wild-type kinesins for creating either roadblocks or obstacles,

Telley et al. (2009) have modulated crowded situations on a microtubule.

Furthermore, the frequency and duration of time spent by kinesin in the waiting state

was shown to depend upon the degree of molecular crowding on the microtubule

track. Presumably, kinesin can go around an obstacle by changing its running path.

However, it is not clear which road motor it is going to take when it faces a jammed

section of a track. Consequently, kinesin movement strategies need to be better

understood to permit an accurate qualitative as well as quantitative interpretation of its

molecular behavior.

In contrast to ―sticky‖ kinesin, which produces processive motion along

microtubules (Seitz and Surrey, 2006), skeletal myosin generates ―rowing‖ movement

along actin filaments (Leibler and Huse, 1993). Tawada and Sekimoto (1991) have

presented the model in which muscle myosin works in collaboration with other

myosin motors in two regimes: productive and nonproductive. According to this

model, there are two major events that occur during the productive cycle: ATP

splitting and force production. Subsequently, nonproductive connection/disconnection

between myosin heads and actin results in the generation of friction drag and

dissipation of heat. It is proven that reduction of the molecular friction may be

achieved by minimizing connection/disconnection time (Lecarpentier et al., 2001).

However, the problem of heat production is still not solved. As mentioned previously,

the key part of the myosin molecule is a motor domain. To understand the

contributions of different parts of this molecule, one should look closer at its structure.

55

The myosin is a product-inhibited ATPase consisting of three parts called a head, a

neck and a tail with communicating functional units: the actin-binding site, the

nucleotide-binding site (Korn, 2000), and the neck domain (or the ―lever arm‖), which

magnifies the small transformations at the active site into the large ones needed to

convey actin (Holmes, 1997). This nanomachine is strongly stimulated by binding to

actin, which is a nucleotide exchange factor for myosin. With the hydrolyzed

nucleotide the myosin binds to the actin filament. After recombining with actin, the

cross-bridge goes through a conformational change allowing Pi and then ADP to be

released, which also brings about the ―power stroke‖ (Holmes et al., 2003) producing

movement along the actin filament. In order to utilize effectively the ability of myosin

to interact with actin polymer, one should optimize the molecular environment of

these biomolecules including the contact surfaces and fluid properties (as discussed in

subsections 2.4.3. and 2.4.5.). However, this biomolecular couple cannot perform any

nanotechnological tasks without an ATP energy supply.

2.5.5.3. Rotary molecular motors

Three protein motors have been described as rotary machines: the bacterial

flagellar motor (BFM) and two portions (Fo and F1) of the ATP synthase (FoF1

ATPase). There are some other molecular motors believed to be driven by a rotary

motion, including a dodecameric portal protein (a part of the genome packaging

machine) (Simpson et al., 2000, Lander et al., 2009) and mini-chromosome

maintenance (MCM) a protein complex that acts as the replicative DNA helicase

(Brewster et al., 2010). Furthermore, we restrict our attention exclusively to three

remarkable rotary machines. Of these, the bacterial flagellar motor, along with the Fo

motor, is driven by the flow of ions across the cytoplasmic membrane – either

hydrogen (H+) or sodium (Na+) ions depending on the organism, whereas the F1 motor

is driven by ATP hydrolysis. Such rotary motor complexes play a major role in

oxidative or photosynthetic phosphorylation, coupling the flow of protons down an

electrochemical gradient to the synthesis of ATP (Mitchell, 1979). ATP synthesis

diverges structurally depending on the source; it consists of eight distinctive subunits

56

in nonphotosynthetic eubacteria (Foster et al., 1980) and nine subunits in

photosynthetic bacteria (Walker et al., 1990). BFM is discussed in detail in subsection

2.5.6.4.

Both eukaryotes and prokaryotes have ATP synthases composed of two

discrete sectors (F1 and F0) that are considered to be separate rotary motors working

cooperatively (Muller and Gruber, 2003). The water-insoluble membrane portion (F0)

and the water-soluble peripheral portion (F1) are joined together by a central shaft

composed of γ (Omote et al., 1999) and ε (LaRoe and Vik, 1992) subunits. The

direction of the shaft rotation affects the way the enzyme works (Diez et al., 2004).

Whilst clockwise rotation is accompanied by ATP synthesis, anticlockwise rotation is

energized by ATP hydrolysis. It is important to note that bacterial ATPase has the

same rotation property, 120˚ step, as the eukaryotic one (Adachi et al., 2000). The

spinning of the heterodimeric stalk is driven by the movement of the F0 rotor which is

energized by a transmembrane ion gradient. There seems to be sufficient evidence

from cross-linking studies that the F0 rotor is a ring oligomer of 12 c subunits (Jones

and Fillingame, 1998). However, the ring stoichiometry may vary in the range

between 10 (Jiang et al., 2001) and 15 c subunits (Pogoryelov et al., 2007) among

living organisms. Additionally, there is data that suggests that the a1b2 subcomplex

may fulfill the function of a stator (Fillingame, 1999) by preventing the shifting of the

α3β3 portion of the F1 domain during catalysis. Moreover, the b subunits (Perlin et al.,

1983) of the a1b2 trimer do not interact directly with the α3β3 spherical subcomplex.

The b dimer has been proven to be connected to an α subunit of the α3β3 hexamer via a

bridging subunit delta (Wilkens et al., 1997). The importance of the b2δ structure has

been indicated by Dunn (2000), showing that it is strongly bound to an α subunit. The

authors believe that the b2δ stalk should be considered as a stator instead of an a1b2

subcomplex.

As for the stator part of the F1 motor, it is composed of an α3β3 hexameric

assembly and a single δ subunit. Although all six homologous subunits of the hexamer

are capable of binding nucleotides (Walker et al., 1982), only β subunits have their

own catalytic sites (Boyer, 1993). It has been demonstrated that α and β subunits are

coupled together to form the smallest functional protomers of enzyme (Hayashi et al.,

57

1989). From the analysis of the α subunit isoform association, Blanco et al. (1994)

have concluded that the α subunits can oligomerize into stable structures. Furthermore,

based on interaction specificity of the α subunits, the authors suggested that αβ

protomerization contributes to stability and physiology of the entire enzyme.

However, the exact contribution of the α subunit to enzymatic performance remains to

be determined. The contribution of the rotor part, namely the γ and the ε subunits, of

the F1 motor to the function of ATPase is discussed above. It is important to

emphasize that integration of energy providers, such as rotary motors, into ATPase-

powered devices is important for producing hybrid devices (Bachand and

Montemagno, 2004).

Using F1-ATPase as the rotary motor, Noji et al. (1997) have assembled a

hybrid structure resembling a propeller by connecting a central rotor of the motor to an

actin filament. By incorporating the F1-ATPase motor into NEMS

(nanoelectromechanical systems), Soon et al. (2000) have succeeded in constructing a

rotory motor-supported nanodevice. They successfully replaced the actin filament with

a nickel bar and mounted the motor protein on a nanometer-sized support structure.

However, the F1-F0 ATPase-powered hybrid device operated at only 50 % efficiency.

Omote et al. (1999) have found that bacterial F1-ATPase is capable of rotating actin

filament in the flow cell with approximately 80 % efficiency. Seeing the potential

applications of a natural ATP supplier, Montemagno and Bachand (1999) have tested

the performance of modified bacterial F1-ATPase on different metal substrates to

estimate its in vitro efficiency. The motor enzyme was shown to work at up to 100 %

efficiency. The researchers concluded that their platform can be used for assembling

devices that employ this kind of rotary motor as a main power source.

2.5.6. Native functions of molecular motors

2.5.6.1. Overview

Molecular motors, as discussed in the previous chapter, are produced in living

organisms and serve vital functions for their owners. The contributions of these

58

natural nanomachines to the anatomy and physiology of cells count on their

biochemical and mechanical aptitudes. Molecular motors play different key roles in

such biological processes as cytoskeletal arrangement, metabolic reactions, flagella

dynamics, bacteria pathogenesis and ATP generation. Although some motors appear

to be sensitive to an in vitro environment, motor utilization seems to hold a great

promise for helping to develop efficient and cheap microdevices. Potential utilization

of motor proteins depends upon the ability to employ the proteins in order to benefit

from their native properties.

2.5.6.2. Cytoskeleton

All living cells have an internal framework called a cytoskeleton (the example

of an unusual organism has been given in chapter 2.5.4.). This big structure is

composed of polarized actin filaments and various ABPs. The actin network allows

the cytoskeleton to re-arrange rapidly, providing a supportive matrix that organizes the

cytoplasm and holds the whole cell together, thereby regulating bacterial motility:

(Mauriello et al., 2010), endocytosis (Suetsugu, 2010), cell division (Wong et al.,

1997b), phagocytosis (Campos-Parra et al., 2010), segregation (Vats and Rothfield,

2007), polarity (Fanto and McNeill, 2004), chemotaxis (Swaney et al., 2010), adhesion

(Hegge et al., 2010), cell migration (Gardel et al., 2010), organelle movement

(Suetsugu et al., 2010), molecular and membrane trafficking (Okamoto and Forte,

2001, Molla-Herman et al., 2010). Furthermore, eukaryotic cells differ from

prokaryotic cells by possessing the presence of a complex cytoskeleton consisting of

an abundant array of proteins. The major ones are actin filaments, microtubules (MTs)

and intermediate filaments (IFs). The filaments provide mechanical support to

eukaryotic cells and serve as tracks for motor molecules to move along. These

filament systems share one essential feature: they are composed of proteins that have

the unique property of being able to self-assemble into linear polymers (Carballido-

Lopez and Errington, 2003). Polymerization occurs at critical monomer concentration,

where non-covalent reversible protein interactions mediate the assembly of

cytoskeleton components into dynamic filaments (the actin assembly is discussed in

59

detail in subsection 2.5.2.). It should be noted that in vivo actin polymerization is not a

fully independent intracellular act for building up a higher-order molecular structures.

This process is controlled through interactions of actin with different members of the

ABP family.

Although the living cell was well recognized for its ability to produce actin/or an

actin-like force generating system, the source of inspiration in this process came from

the master of biomolecular mimicry, the model organism Listeria monocytogenes. The

pathogen derives benefits from using the host‘s cytoskeleton. To stimulate actin

assembly on the back surface of its own body for creating an elastic tail (Gerbal et al.,

2000), the pathogen makes contact with the essential eukaryotic factor, Arp2/3

complex, and utilizes its properties (Boujemaa-Paterski et al., 2001). However, the

contributions of this complex to the regulation of actin assembly and the network

remained incompletely understood by scientists for a decade. Even though Welch et

al. (1998) made quite interesting suggestions regarding the mechanism of Arp2/3

activation, they did not provide a solid explanation for Arp2/3-guided actin assembly.

Later, Footer et al. (2008) shed more light on how actin nucleation can be activated by

the ARP2/3 complex. In addition to the Arp2/3 complex, there is another important

regulator, gelsolin (GLS) that is located both on the bacterial surface and in the ―comet

tail‖ (Laine et al., 1998). The authors assume that the actin-uncapping capability of the

pathogen is closely linked to its gelsolin-serving function. Interestingly, they

heightened Listeria motility by elevating the intracellular concentration of GLS in the

host cell. Despite the lack of experimental evidence for this process, the authors

supposed that either an increase in recycling of monomeric actin or a decrease in the

viscosity of the host cytoplasm was associated with accelerated bacterial movement.

(The properties of this protein are discussed in subsection 2.5.2.).

The dynamic structure, later called a cytoskeleton, was first extracted from

erythrocytes by Yu and co-workers (1973). However, because of the lack of a reliable

technique for non-brutal treatment of cells, the three dimensional arrangement of

cytoskeletal counterparts was not clarified. To prepare muscle cytoskeleton for

scanning electron microscopic (SEM) examination, Wallace and Fischman (1979)

have developed the osmium-TCH method of producing evenly coated actin filaments.

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The authors emphasized that cytoskeletal components could be regarded as protein

assemblies as well as distinct organelles. For a long time, it was believed that the

cytoskeleton was one of the key distinctive features of eukaryotes. That point of view

was revolutionized in the 1990s by the analysis of FtsZ‘s contribution to the life of

bacterium Escherichia coli (RayChaudhuri and Park, 1992). Moreover, structural and

functional homologues of all three main eukaryotic cytoskeleton proteins: actin

homologues, such as MreB (Takacs et al., 2010), FtsA (Shiomi and Margolin, 2007),

ParM (Popp et al., 2010a), MamK (Katzmann et al., 2010); tubulin ones, such as FtsZ

and BtubA/B (Sontag et al., 2009); and intermediate filament protein ones, such as

CreS (Gitai et al., 2004), have been proven to make up cytoskeletal structures in

bacterial cells. These prokaryotic homologues behave in many ways like eukaryotic

cytoskeletal components. Thus, they are involved in a variety of essential cellular

processes in bacteria (Michie and Lowe, 2006).

2.5.6.3. Cellular metabolism

A cell acquires and utilizes energy in order to move anything that a cell needs

(Zimmerman and Walter, 1991) to assemble, develop and survive. A cell performs

metabolic reactions, such as anabolic (Kwast and Hand, 1996) and catabolic ones

(Reggiori et al., 2005), resulting in either the synthesis of complex molecules or

decomposition of complex ones, respectively. The metabolism of substrate by a cell

requires the participation of a transport system that allows for the transport of a certain

organic or ionic molecule. There are two basic types of molecular transport, passive

and active transport (Zeuthen, 1995). The principal means of passive transport is the

diffusion that happens in all living cells spontaneously (Soh et al., 2010). It requires

no energy to transfer a particle downwardly with respect to its concentration gradient.

As an example, the passive transport of glucose (Bell et al., 1990) through the

sarcolemma of the striated muscles (skeletal and cardiac) occurs by means of glucose-

transporting proteins (GLUT1; GLUT4) (Santalucia et al., 1992). To study the insulin-

stimulated transport of glucose in peripheral blood lymphocytes, Piatkiewicz et al.

(2010) have applied a flow cytometry analysis. The authors proposed use of white

61

blood cells as a model due to the difficulty of performing the same experiment in

living cells. Although these cells can be used for gaining a deeper understanding of

insulin-related disorders, the huge difference between various types of cells must be

taken into consideration.

Meanwhile, there are substances that are too large to travel through a cell by

means of diffusion. Furthermore, molecular motors need energy to move along

polarized intracellular tracks. As an example, the active transport of membrane-

enclosed organelles – such as Golgi stacks (Boevink et al., 1998), mitochondria (Tavi

et al., 2010), lysosomes (Sontag et al., 1988, Demirel et al., 2010), peroxisomes (Li

and Nebenfuhr, 2007) or secretory vesicles (Trifaro et al., 2008), as well as protein

complexes – elements of the cytoskeleton, virus particles (Vaughan et al., 2009) – to

their proper place in a living cell is mediated by motor proteins such as myosin and/or

kinesins/dyneins. These proteins use the energy derived from repeated cycles of ATP

hydrolysis. With respect to applications of molecular motors, myosin is useful for

cargo transport. As discussed in subsection 2.5.5.2., the actin/myosin couple can be

considered for participation in nanotechnological experiments. In addition, these

proteins display essential molecular skills in vitro only in the presence of the source of

energy, such as ATP. As discussed in subsection 2.5.5.3., ATP molecules in

eukaryotic and prokaryotic cell membranes are synthesised by rotary motors

(ATPases).

Interestingly, some bacteria, which include pathogens (September et al., 2007)

and marine bacteria (Ivanova et al., 2002b), use the same type of carbon metabolism,

namely heterotrophic, as eukaryotic cells. Thus, heterotrophic species can obtain

energy (ATP) through the fermentation or respiration of such organic compounds as

carbohydrates, lipids, proteins and/or from decaying organic substrate. Furthermore,

due to metabolic plasticity, some pathogens are capable of maintaining both parasitic

and saprophytic lifestyles (Freitag et al., 2009) depending on their circumstances. It

has been proven that the environment has an enormous effect on metabolic states of

heterotrophic bacteria (Alexeeva et al., 2004b, Ivanova et al., 2003a). A recent study

has demonstrated that short thermization has an impact on the metabolism of

heterotrophic microorganisms (Samelis et al., 2009). In doing so, the attempt to

62

deactivate pathogenic bacteria in raw milk caused a significant change in its microbial

community structure. Welch et al. (1995) have used arsenate, a toxic metalloid, that

inhibits binding-protein-dependent transport systems such as PMF-dependent

transport (Richarme, 1988) to check the symmetry of the flagellar motor. The authors

applied two methods: a conventional one and an arsenate-incubation one. The

comparison of these methods showed that motility was permanently lost in the former,

while it was temporary affected in the latter. Although the results of these experiments

indicated that the flagellar motor was asymmetrical, the underlying cause of the

unidirectional rotation remained unclarified. As mentioned in subsection 2.5.5.3.,

unlike other motile cells, swimming bacteria power up flagellar rotary motors by

proton motive force (PMF). It is important to note that metabolic processes, which use

chemical energy to pump protons out of the cell allowing them to return, are the basis

for PMF production.

2.5.6.4. Flagella-based motion

Motility is known to be generated by flagellated cells; however, due to the lack

of understanding of the fine points of this mechanism, the full utilization of this

cellular property for nanotechnological applications in microdevices has not been

accomplished yet. It has been proven that both eukaryotic and prokaryotic motile cells

can either swarm over surfaces or swim in a fluid environment. Although eukaryotic

swarming techniques (Bonner, 2010, Gilbert, 1927), as well as bacterial ones

(McCarter, 2010, Jones and Park, 1967), have been investigated for decades, they still

remain to be studied in greater detail. For example, it has been demonstrated that of

four main swarming strategies, namely, reversing, stalling, lateral or forward moving

used by Escherichia coli swarmer cells to travel over an agar surface, the first one is a

very specific one, which is not used by Escherichia coli swimmers (Turner et al.,

2010). Nowadays, many questions have arisen concerning various aspects of bacterial

swarming including its robustness and effectiveness (McCarter, 2010).

Since the employment of microbial swimmers as energy generators (Zhang et

al., 2010b) or use of their biomimetic actuator in a microfluidic environment may

63

become possible, in this subsection more attention is paid to swimmers. Thus,

swimming microorganisms (Zonia and Bray, 2009) and some eukaryotic cells with

flagellar motors (Woolley, 2010) can exhibit a variety of structures and movement

patterns. These bacteria and eukaryotic cells manifest various locomotion styles,

which are carried out by either rapid rotation or the beating motion of flagellar

filaments that jut out from the swimmers. In animals, flagellated sperm cells propel

themselves forward via symmetric sinusoidal-like and different asymmetric wavelike

movements towards oocytes (Dillon et al., 2007, Gadelha et al., 2010). Many single-

celled organisms related to protists (flagellated protozoa, some algae) use flagella

(Lewin, 1953) or cilia (Manton, 1953, Zhang et al., 2010a) to move through their

aquatic environment. It has been proven that ciliated epithelial cells (with short hair-

like structures), which line body cavities like the respiratory tract (e.g., parts of the

nasal cavities, trachea), need to beat in synchrony to push a mucous blanket and clean

cellular debris off the epithelial surface (Sommer et al., 2010). As for bacteria, it has

been demonstrated that the majority of them swim by rotating their flagella

(Srigiriraju and Powers, 2006). In addition, different bacterial species have different

numbers and arrangements of flagella on or around cell surfaces to inhibit not only

swimming, and swarming but also such modes of locomotion as twitching (Hammond

et al., 2010), and propulsion (Lin et al., 2010). For example, monotrichous bacteria

(have one flagellum at a polar location) change the direction of motor rotation from

forward to backward (Taylor and Koshland, 1974). Moreover, a flagellum can go back

along the path it has swum along as well as move in a zigzag fashion (Goto et al.,

2005). Interestingly, the swimming of lophotrichous bacteria (they have a bunch of

polar flagella at the end of the cell) in a single direction is accompanied by sudden

turns (Harwood et al., 1989). According to recent studies, a representative of

amphitrichous bacteria (they have two flagella, one at each end of the cell)

Helicobacter pylori, swims in a circular fashion (Celli et al., 2009). Peritrichous

bacteria (with a high degree of flagellation) aggregate flagella into posterior bundles

to propel themselves forward. (Darnton et al., 2007).

Although eukaryotic and prokaryotic flagella look similar to each other, they

have completely different structures (Engel et al., 2009). The eukaryotic one is a well-

64

preserved organelle (Hodges et al., 2010) in which an assembly of 9 doublet proteins

is precisely arranged around two separate microtubules (Patel-King et al., 2004,

Mitchell, 2007). It contains over 200 proteins that are arranged into sub-assemblies,

such as dynein arms and radial spokes (Inaba, 2003), etc. It is important to note that

dynein proteins can produce force as linear tension, compression (Lindemann, 2003)

and torque on the doublets through ATP (Nevo and Rikmenspoel, 1970) and ADP

hydrolysis (Lesich et al., 2008). Unlike the eukaryotic flagellum, the single bacterial

one is composed of a hook-basal body (HBB) complex (Kubori et al., 1997, Wosten et

al., 2010), and an extracellular flagellar filament (Hosogi et al., 2010, Macnab, 2003).

A bacterial rotary motor has been proven to power up the corkscrew motion of a

flagellar filament (Dreyfus et al., 2005). From the engineering point of view, the

filament is a helical cylinder attached to the cell surface; it is constructed by means of

self-assembly of 20,000 FliC monomers (Reid et al., 1999, Majander et al., 2005). As

mentioned above, a bacterial motor can rotate filament in both clockwise and

counterclockwise directions (Manson, 2010). The core of the bacterial nanomotor is a

group of rings which rotates flagellar filaments (Sowa et al., 2005) and includes some

13 other proteins (Delalez et al., 2010).

Even though significant efforts towards understanding the structure and

functions of rotary motors have been made, the utilization of their force through

recruitment of their owners still remains far from accomplished. In fact, such

important motor characteristics as the conversion of energy (Nakamura et al., 2009)

and its sustainability (Fukuoka et al., 2010) under various biochemical conditions

(Thormann and Paulick, 2010), for example, change in temperature and/or pH, the

effect of environmental homogeneity as well as shear stress, etc., need to be

understood in greater detail. Furthermore, in order to recruit bacteria for

nanotechnological work, one should find a microbial candidate that would be able to

produce a sufficient amount of ATP in microfluidic conditions (see chapter 8).

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2.5.6.5. Tactics of enteric pathogens

2.5.6.5.1. Overview

As discussed in subsections 2.4.5.4.4. and 2.5.2., the control of molecular self-

assembly can be accomplished by means of different methods. A natural way for

managing self-assembly is used by pathogenic bacteria. It is called the ―comet tail‖

technique. It has been proven that members of the Enterobacteriaceae family, such as

pathogenic representatives of the genera Listeria and Shigella (Gouin et al., 1999, Lin

et al., 2010, Adamovich et al., 2009), employ the method to propel themselves

through host cells. To evaluate the benefits of utilization of pathogenic techniques for

control of actin assembly, one should understand how enteric bacteria achieve high

pathogenicity.

2.5.6.5.2. Common tactics of enteric pathogens

Pathogenic bacteria from the Enterobacteriaceae family can successfully

inhabit intestines of humans and cause diseases such as bacillary dysentery (Fletcher,

1917), typhoid (Baker et al., 2010, Verma et al., 2010) and urinary tract infections

(Yusha'u et al., 2010), etc., which result in three million annual deaths (Nel and

Markotter, 2004). The pathogens can travel from the environment into the host cells

through fecal-oral transmission. Accordingly, it predetermines the choice of therapy

for the disease (Mai et al., 2010). It has been proven that enteric bacteria are not only

physiologically but also metabolically adaptable to their surroundings (Freitag et al.,

2009). Thus, they are able to withstand killing by acid or enzymes during the journey

through the stomach (Barmpalia-Davis et al., 2008, Barmpalia-Davis et al., 2009).

Moreover, pathogens can overcome intestinal peristalsis (which clears the gut), adhere

to (Coconnier et al., 1993), and then invade epithelial cells (Sansonetti, 2002), in spite

of the occupancy of competitors such as host specific faecal bacteria (Sekirov et al.,

2010).

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A pathogenic life cycle starts with host entry and colonization and is followed

by the establishment of infection (Vazquez-Boland et al., 2001). After damaging host

cells, enteric pathogens can continue cruising through the target organ or exit it.

Although much research has been devoted to understanding pathogenic tactics, the

mechanism of interactions of pathogenic Listeria with the surface of intestinal cells

remains to be elucidated (Schuppler and Loessner, 2010). In fact, although several

lines of research have indicated that enteropathogenic bacteria use virulence factors,

such as the pore-forming toxin Listeriolysin O (LLO) (Yin et al., 2010, Meyer-Morse

et al., 2010, Sashinami et al., 2010), ActA (Muller et al., 2010), internalins A and B

(Pentecost et al., 2010), to besiege host defence mechanisms, it is not enough to

convince all researchers. Thus, Conter et al. (2010) have shown that strains of Listeria

monocytogenes differ in their ability to attack the HeLa (human epithelial carcinoma

cell line) cells. Therefore, these researchers believe in the existence of other virulence

factors that have not been discovered yet (Conter et al., 2010). Earlier work by

Donnenberg (2000) has established that two major types of macromolecular

structures, namely, an adhesion system and a secretion system, play a crucial role in

enteropathogenic invasion. Pathogenic bacteria use adhesins (Beachey, 1981) to bind

to the receptors of enterocyte surfaces. It has been demonstrated that adhesins can be

located either at the surface of a gram-positive bacterium (Gilot et al., 1999, Reis et

al., 2010) or at the end of its pili (De Greve et al., 2007). Remarkably, while some

biomolecules, e.g., autolytic amidase (Ami) contribute to cell adhesion (Milohanic et

al., 2001), other bacterial products such as internalins (Parida et al., 1998, Pentecost et

al., 2010) and LapB (Reis et al., 2010) are responsible for both adhesion and invasion.

Gram-negative pathogens are known to have about six secretion systems of different

complexity. Moreover, all of them have specific functions, for example, the type I

secretion system is necessary for transportation of biomolecules from the cytoplasm to

the cell surface (Cescau et al., 2007). The type II secretion system is in charge of

transport of biomolecules through the outer membrane (Francetic et al., 2007). This

secretion system has been proven to be used by the human pathogen Vibrio cholera

for the export of cholera toxin (Camberg and Sandkvist, 2005). Many

enteropathogens, for example, Yersinia spp. (Brodsky et al., 2010), Salmonella spp.

67

(Van Engelenburg and Palmer, 2010), Shigella spp. (Newton et al., 2010), and

enteropathogenic Escherichia coli (EPEC) use the type III secretion apparatus

(Martinez et al., 2010) upon contacting target cells. This secretion system not only

delivers biomolecules but also shoots them into the target cell (Kubori et al., 2000,

Tamano et al., 2002, Galan and Wolf-Watz, 2006). Once transported into host cells,

these effector biomolecules induce actin reorganization. Enteropathogenic bacteria

have been proven to facilitate infection by exploiting host-cell actin (Lu and Walker,

2001).

Of two enteropathogenic masters of harnessing actin dynamics for ―comet tail‖

formation, namely, Listeria monocytogenes and Shigella flexneri, the former has been

chosen as a model organism for study of pathogenicity (Joyce and Gahan, 2010,

Guillet et al., 2010).

2.5.6.5.3. Listeria as a regulator of actin assembly

The gram-positive bacillus Listeria monocytogenes is equipped with a set of

surface proteins, such as internalins: A (InlA) (Van Stelten et al., 2010), B (InlB)

(Auriemma et al., 2010) and J (InlJ) (Sabet et al., 2008, Bublitz et al., 2008); and the

actin nucleator protein ActA (Conter et al., 2010). Remarkably, in order to succeed in

intra/intercellular cruising, the facultative pathogen explores not only host cytoskeletal

actin but also its Arp 2/3 complex (Sousa et al., 2007) and nucleation-promoting

factors (NPFs) (Chong et al., 2009). As mentioned above, Listeria monocytogenes

uses covalently linked internalins to invade the cells of intestinal epithelium.

Travelling through the intestine, the pathogen is looking for the nearest multicellular

junction (MCJ) located between partially separated enterocytes on the top of the villi

in order to get access to hidden epithelial cadherin (E-cadherin) receptors. Though the

study of host cell invasion has gained considerable attention during the last decade

(Parida et al., 1998, Gao et al., 2009), it was not until recently that the contributions of

InlB to this process were recognized. Thus, recent research has shown that InlB does

not play a role in adhesion but triggers its cellular ligand c-Met, a receptor tyrosine

kinase (RTK), to speed up endocytosis (Pentecost et al., 2010). Interestingly, although

68

InlB mimics the hepatocyte growth factor (HGF), it does not compete with HGF for a

site on the cMet receptor (Shen et al., 2000). In contrast to InB, InlJ plays an

important role in adhesion (Sabet et al., 2008). It should be emphasized that InlA

participates in both the adhesion and invasion of pathogens (Schubert et al., 2002) in

order that Listeria monocytogenes may breach host barriers (Lecuit, 2005) and

subsequently multiply inside the host cell. In so doing, Listeria monocytogenes

utilizes InlA to reach the actin cytoskeleton via the E-cadherin receptor; Listeria

monocytogenes uses β-catenin as a link to the actin-connected anchor composed of α-

catenin (Seveau et al., 2007). Furthermore, the intracellular protein ARHGAP10 has

been proven to control the anchor (Sousa et al., 2005). It is important to note that there

are two other key players, namely, myosin Vlla and its transmembrane receptor

vezatin; they not only participate in the formation of direct contacts between cells at

junctions but also contribute to InlA-initiated pathogen internalization (Sousa et al.,

2004). Depending on the availability of a target cell, pathogens can invade either

directly through enterocyte activity or indirectly via Peyer‘s patches (Jensen et al.,

1998, Schuppler and Loessner, 2010, Marco et al., 1997). However, some researchers

have doubts regarding the ability of Listeria monocytogenes to sneak indirectly

through the host barrier (Pron et al., 1998).

One of the key NPF, which is essential for invasion (Suarez et al., 2001) and

motility (Chong et al., 2009, Portnoy et al., 2002), is ActA; it is produced by two

pathogenic species of the genus Listeria only, namely, Listeria monocytogenes and

Listeria ivanovii (Gouin et al., 1995). Even though ActA is not capable of making long

polymers, it can homodimerize in vivo (Mourrain et al., 1997). The model has been

proposed by the authors to explain the contributions of the ActA dimer to actin-

mediated motility of Listeria monocytogenes. However, in contrast to dimerization,

the closely spaced distribution of ActA on the bacterial surface has been recently

proposed to explain its enhancing effect on actin polymerization (Footer et al., 2008).

Strikingly, in order to initiate actin assembly at its own bacterial surface, the pathogen

uses ActA for recruitment of essential host cell factors, such as the Arp 2/3 complex

(Cossart, 2000) and representatives of the enabled homologue/vasodilator-stimulated

phosphoprotein (Ena/VASP) family (Castellano et al., 2001, Lambrechts et al., 2008).

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The Arp 2/3, evolutionary conserved multimeric protein complex, has been

studied extensively due to its importance to nucleation of actin filaments and creation

of the Y-shaped cross-linking actin network (May, 2001, Goley et al., 2010), which is

crucial for actin-based motility (Zalevsky et al., 2001). It is important to note that the

Arp 2/3 complex requires ATP energy to nucleate actin polymerization (LeClaire et

al., 2008). Another key player in Listeria monocytogenes-induced ―comet tail‖

formation is VASP. It was not well understood until the beginning of the last decade

(Loisel et al., 1999) how pathogens can benefit from hiring VASP. Experimental

studies have revealed that Listeria monocytogenes uses VASP to employ profilin, a

nucleotide exchange factor, which serves the barbed ends of actin filaments (Pasic et

al., 2008) to cause hastening of actin growth at its own bacterial surface. In

consequence, bacterium acquires an ―actin cloud‖ (Tilney et al., 1990) composed of

young actin filaments (Lambrechts et al., 2008). By reorganizing the surrounding

cloud at a pole enriched with ActA (Kocks et al., 1993) the pathogen creates a ―comet

tail‖, which supports its directional movement through the host cell. It has been

demonstrated that travelling pathogens display different behaviors. Thus, while gram-

positive Listeria monocytogenes spins around, gram-negative Yersinia

pseudotuberculosis and Escherichia coli do not (Robbins and Theriot, 2003). It has

not been clarified whether this activity of Listeria monocytogenes is due to the

different structure of cell envelopes or the distinctive properties of ActA and IcsA

proteins. In addition, it has been noted that because of spinning, Listeria

monocytogenes can produce various curvatures for its intracellular journey (Shenoy et

al., 2007). Although the authors have shed some light on how Listeria monocytogenes

cruises through the cell, a realistic model still remains to be developed. A remarkable

study stating that ActA-covered beads can produce unidirectional motion in vitro has

been reported (Cameron et al., 1999). The authors concluded that actin-based

movement strongly depends on both the size of the bead and the number of ActA

molecules sitting on it. It should be emphasized that some environmental factors, e.g.,

saturation of the cell extract with protein (Cameron et al., 2004) can affect the

curvature of a bead route. It is important to note that key players, for example, actin

and the Arp 2/3 complex require ATP energy for efficient operation.

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2.5.6.6. Bacterial ATP generation

Owing to the importance of adenosine triphosphate (ATP) energy for various

biological processes, including eukaryotic actin and its prokaryotic homologues,

namely, MreB and FtsA, self-assembly; actin-myosin interaction and actin-based

pathogenic motility, a great deal of research should be devoted to the study of ATP

production by rotary molecular motors in bacterial cells and evaluation of employment

of rotary motors as power suppliers in microdevices. Although eukaryotic organisms

are capable of producing ATP, they utilize different metabolic pathways. Thus, the

key organelles that eukaryotic cells use for energy production are either mitochondria

in animal cells (Hogeboom et al., 1947, Wagner et al., 2010) or chloroplasts in plant

cells (Arnon and Whatley, 1949, Krah et al., 2010). It is believed that eukaryotic cells

acquired the ―power stations‖ for ATP production about 1.5 billion years ago when

bacteria settled down as endosymbionts within eukaryotic cells (van der Giezen and

Tovar, 2005). Those bacteria were free-living α-proteobacteria that developed

endosymbiotic relationship with the ancestor of animals (Lang et al., 1999, Gabaldon

and Huynen, 2007, Chang et al., 2010b) and/or fungal (Bullerwell and Lang, 2005)

cells. Moreover, as a result of endosymbiosis between the ancestor of plants and

cyanobacterium, it was a plant cell gained chloroplast that became the main energy-

converting organelle of the cell (Raven and Allen, 2003, Ran et al., 2010). So,

eukaryotic cells produce energy but they can only do it in vivo or under carefully

optimized conditions. This means that for nanotechnological application they do not

fit into microdevices. Nowadays, bacteria are considered to be more robust (Kitano,

2004, Kitano, 2010) for device-realistic conditions than eukaryotic cells. Thus, some

members of marine bacteria have been shown to possess metabolic plasticity (Ivanova

et al., 2000a, Bong et al., 2009), which is one of the features of biological robustness.

Since both the biomolecular architecture and primary function of ATPase have been

discussed in subsection 2.5.5.3., in this subsection (2.5.6.6.) more attention is given to

the effects of various factors on ATP production by bacteria.

It was not until the beginning of the 1960s that researchers shed some light on

the energetic aspects of bacterial growth (Senez, 1962, Fukui and Hirata, 1968).

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Interestingly, even though Senez did not have enough experimental evidence, he

indicated that bacteria spend ATP as an energy source on biomolecular assembly,

active transport and/or arrangement of cellular structures. Since the 1970s, energy-

related studies have shown that apart from consumption of ATP for self-maintenance,

bacteria can just simply spill it (Neijssel and Tempest, 1976, Neijssel et al., 1990,

Russell, 2007). For example, Streptococcus bovis can waste a lot of energy on

ineffectual transmembrane cycling of ions (Russell and Cook, 1995). In addition, it

has been noted by Neijssel & Teixeira de Mattos (1994) that cultivation conditions can

affect growth energetics. In fact, exposure of bacteria to laser radiation appears to

cause a progressive decrease in ATP production (Nandakumar et al., 2003). The

authors assume that irradiation of bacteria triggers destruction of cellular respiration. It

is well known that both bacterial growth and metabolism can be controlled through

temperature adjustment during cultivation. When the environmental temperature goes

below the optimal level – which is related to bacterial physiology – bacterial

membrane-embedded proteins become incapable of supporting essential molecular

transport because of the altered flexibility of the membrane-associated lipids

(Nedwell, 1999). For example, evidence has been presented that, while a psychrophilic

bacterium produces ATP equally well at 30 °C and at 4 °C, a mesophilic bacterium

struggles to do it at the latter (Theron et al., 1987). In addition, quantitative ATP

analysis must consider bacterial lifestyle because it has a specific impact on the

efficiency of ATP production. Thus, Hong and Brown (2009) have shown that

planktonic bacteria produce less ATP than corresponding ones that go through an

adhesion process. The authors have proposed the charge regulation effect to account

for this observation. So, according to their hypothesis, negatively-charged surface

groups have a positive effect on bacterial ATP production and adhesion, while

positively-charged functional groups have an opposite influence on this kind of

microbial behavior. Phenotypic change has been observed to happen when a bacterium

changes its lifestyle and becomes one of the members of a biofilm community (Sauer

and Camper, 2001). Interestingly, the authors suggest that at the early stage of

adhesion (Dunne, 2002), bacteria may partially respond to their lifestyle change

72

through quorum sensing (QS), or communication by means of specific chemical

language (Allison and Gilbert, 1995, Ren et al., 2010)

It should be noted that bacteria can interact with living organisms at different

levels: intraspecies, interspecies, intrakingdom and interkingdom. For example, a

bacterium can contact members of the same species (Dawid et al., 2009), cross-talk to

bacteria from other species (Kimura et al., 2009), communicate with the members of

the same (Kendall et al., 2007, Weber et al., 2007) or different kingdoms (Kimura et

al., 2009, Subramoni and Venturi, 2009). In order to employ a suitable ATP producer,

one should understand that such cell phenotypes as bioluminescence (Miyamoto et al.,

2000, Nelson et al., 2007), which is driven by ATP production, and biofilm creation

(Ahmed et al., 2009), are dependent on QSs. Nowadays, the focus is on the evaluation

of the possibility of the utilization of efficient, reliable and cheap biomaterials. For

example, on the utilization of microbial-based therapy instead of an antibiotic one

(Defoirdt et al., 2010). Since members of α-, β-, and γ-proteobacteria have been

known to produce not only signal molecules but also enzymes, such as lactonases and

acylases, they can participate in the QS-mediated communication as well as the

inactivate QSs of pathogens (Uroz et al., 2009). So, proteobacteria have been proven

to be promising candidates for ATP generation with regard to their application for

construction of parts of microdevices (see chapter 8).

2.5.6.7. Use of MreB and FtsA proteins by bacteria

Actin homologues appear to play essential roles in the lives of different kinds

of bacteria. It is generally accepted that MreB is in charge of maintaining a rod-shaped

form, while FtsA is responsible for enhancing cell division. Although both proteins are

capable of self-assembly, only MreB displays actin-like structure and polymerization

behavior. Moreover, due to its unique ability to produce morphologically different

structures, it can contribute to a broad range of bacterial cell affairs. Thus, MreB has

been shown to be involved in cell growth (Robertson et al., 2007), shape

morphogenesis (Takacs et al., 2010, Divakaruni et al., 2007, Margolin, 2009), viability

maintenance (Burger et al., 2000, Carballido-Lopez, 2006, Kawai et al., 2009b),

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polarization (Shih et al., 2005), protein positioning (Mauriello et al., 2010), organelle

production (Cowles and Gitai, 2010), sporulation (Mazza et al., 2006), DNA

replication (Defeu Soufo and Graumann, 2005, Munoz-Espin et al., 2009),

chromosome segregation (Soufo and Graumann, 2003), etc. It should be emphasized

that bacterial demand for such a multi-skilled MreB varies across species. The

majority of rod-shaped bacteria lengthen their cells by means of peptidoglycan (PG)

inclusion into the lateral wall, which is coordinated by the MreB helix (Kawai et al.,

2009b, Varma and Young, 2009). Over the last decade, there has been increasing

experimental evidence that the loss of MreB leads to the transition of rod-shaped cells

which were rod-shaped from the beginning into rounded ones. Interestingly, a

spherical shape is a typical characteristic of lack of MreB not only in gram-negative

bacteria, such as Escherichia coli (Wachi et al., 1987), but also in gram-positive

Bacillus subtilis (Soufo and Graumann, 2003). Furthermore, in the absence of MreB,

the gram-negative bacterium Caulobacter crescentus has been observed to acquire a

lemon-like look (Figge et al., 2004).

It is also important to note that MreB plays a key role in handling stress and

aging issues by bacteria. Having both MreB and CreS, which is the intermediate

filament-like analogue (Ingerson-Mahar et al., 2010), Vibrio parahaemolyticus has

been proven to use the former for adaptation to food deprivation and senescence (Chiu

et al., 2008, Chen et al., 2009). Wang and co-workers (2010) have demonstrated that

Escherichia coli may count on the MreB-based cytoskeleton to withstand changes in

external or internal pressure. The researchers have proposed that Escherichia coli can

accomplish it either through building up a thicker MreB cable-like biopolymer or by

varying degrees of its cross-linking with the cell wall. In addition, results from a study

using MreB from Thermotoga maritima have shown that MreB has sufficient rigidity

so that it may manage to maintain its shape under high bending or compressive stress

(Esue et al., 2006). Naturally, there are exceptions to the common view that MreB is a

master of shape management. For example, Helicobacter pylori benefits from

partnership between MreB and intracellular molecules in other ways. It has been

recently demonstrated in Helicobacter pylori that its MreB protein participates in

74

chromosome segregation without being involved in any shape-related activity

(Waidner et al., 2009).

The role of MreB in coordinating localization of proteins has been recognized

via testing dynamics of its whereabouts in Caulobacter crescentus (Gitai et al., 2004).

The observation that MreB can reorganize itself has led to the assumption that such a

dynamic behavior is important for cell polarity. The authors discussed the possibility

of transformation of a molecular polarity into a cell one, though they did not reveal its

mechanism. An investigation of the importance of MreB participation in pilus

assembly in Pseudomonas aeruginosa (Robertson et al., 2007, Cowles and Gitai,

2010), and the gliding motility of Myxococcus xanthus (Patryn et al., 2010, Mauriello

et al., 2010), has led to the conclusion that MreB is responsible for polar positioning of

different proteins including virulence factors. As discussed above, MreB is essential

for some pathogens. Apart from helping them get through environmental stress, it

takes part in the development of predatory bacteria. For example, Bdellovibrio

bacteriovorus uses it for transformation from vibrio-shaped to elongated cells (Fenton

et al., 2010).

It is noteworthy that some bacteria, such as Escherichia coli, have been found

to use both MreB (Wachi and Matsuhashi, 1989, Madabhushi and Marians, 2009), and

FtsA (Strahl and Hamoen, 2010) proteins for cell division. Although FtsA has only

one specific aptitude for reproduction by binary fission (Ping, 2010), it can assemble

into homo-, oligo- (Shiomi and Margolin, 2007) and polymeric (Lara et al., 2005)

structures; (biochemical properties of FtsA are discussed in subsection 2.5.3.1.2.). In

fact, FtsA is the first to support the assembly of the Z-ring, or the major cytokinetic

structure (Adams and Errington, 2009). So, it is capable of making contact with FtsZ,

a homologue of eukaryotic tubulin (Lutkenhaus and Addinall, 1997), and anchoring it

to the cell membrane. Like MreB, it plays the role of leader for certain proteins

(Schmidt et al., 2004, Karimova et al., 2009). However, due to a lack of data on the

differences in the biochemical and the biophysical properties of MreB and FtsA

proteins among different species of bacteria, it is hard to estimate fully the

contributions of MreB and FtsA to bacterial lives. Studies have demonstrated that the

inherent properties of FtsA may vary across species. For example, while FtsA is

75

dynamic in Escherichia coli (Karimova et al., 2005), it is stable in Streptococcus

pneumoniae (Lara et al., 2005). In order to find the suitable actin homologue for

application in microdevices, evaluation should be conducted of possible MreB and

FtsA proteins belonging to different species of marine proteobacteria (see chapter 9).

76

CHAPTER 3

METHODOLOGY

77

3.1. Overview

This chapter consists of five subsections. It starts with a description of

methods used to study protein-surface interactions including detection and

quantification techniques (see subsection 3.2.) for the development of a novel

approach to the design of surfaces for microdevices based on spatial immobilization

of nonmotor proteins in micro/nano-channels fabricated via laser ablation (see

chapters 4 and 5). Reproduced from references (Ivanova et al., 2006c) © 2006 With

kind permission from IOP; (Ivanova et al., 2003b) © 2003 With kind permission from

SPIE; and (Ivanova et al., 2004f) © 2004 With kind permission from SPIE. The

following subsection 3.3. covers methods that allow controlled-self-assembly of actin

filaments along microchannels in a continuous-flow system (see chapter 6).

Reproduced from reference (Alexeeva et al., 2005) © 2005 With kind permission

from Springer + Business Media. As environmental bacteria can be used as

replacements for the energy sorce, and bacterial actin homologues as replacements for

eukaryotic actin, the chapter continues with methods of bacterial taxonomy of

valuable ATP, MreB and/or FtsA producers (see subsection 3.4. and chapter 7). This

subsection starts with bacterial isolation (see subsection 3.4.1.), and is followed by

bacterial characterization (see subsection 3.4.2.) including phenotypic (see subsection

3.4.2.1.), chemotaxonomic (see subsection 3.4.2.2.), genotypic (small subsection

3.4.2.3.) and phylogenetic (see subsection 3.4.2.4.) methods. Reproduced from

references (Gorshkova et al., 2003) © 2003 With kind permission from IJSEM;

(Ivanova et al., 2004e) © 2004 With kind permission from IJSEM; and (Ivanova et al.,

2006b) © 2006 With kind permission from Microbiological journal. The chapter

continues with a description of methods used to assess ATP production by bacteria.

Reproduced from reference (Ivanova et al., 2006a) © 2006 With kind permission from

International Microbiology. The chapter ends with subsection 3.5. devoted to methods

used to assess MreB and FtsA proteins.

http://iopscience.iop.org/1748-605X/1/1/004;jsessionid=1F6978DDE4C10472C9F935AE7CDFA9B4.c1

78

3.2. Methods used to study protein-surface interactions

3.2.1. Protein preparation for immobilization on polymeric surfaces

Prior to depositing onto polymeric surfaces, five proteins with different

biochemical properties, namely human immunoglobulin G (HIgG), human serum

albumin (HSA), lysozyme, myoglobin, and α-chymotrypsin (Sigma), were prepared as

stock solutions (2mg ml-1), and at the later stage, after purification by column

chromatography, were diluted with TBS to working solutions (100 μg ml-1).

Alexa Fluor® 546, an orange-fluorescent phalloidin conjugate, was joined to

the selected proteins (2mg ml-1) with the help of the Fluoro Tag Kit (Molecular

Probes). The labelling procedure was performed strictly in accordance with the

instructions provided by its manufacturer. After labelling, proteins were separated

from unbound fluorescent dye by means of a Sephadex G-25 gel filtration. The

concentrations of labelled proteins were determined by ultraviolet-visible (UV-Vis)

absorption spectroscopy (Cary 50, Varian). Based on both protein adsorption value at

280 nm and the Alexa Fluor® 546 excitation maxima measurements, the fluorescent

dye/protein molar ratio of the purified proteins was estimated. The measurements were

taken in phosphate buffered saline (PBS), prepared by mixing 50 mM phosphate with

150 mM NaCl in filtered (0.2 µm) Nanopure water (18.2 MΏ/cm) and adjusting pH to

7.4 at RT (about 23 ºC).

3.2.2. Polymeric film preparation

Glass slides and cover slips (#1, 0.17 mm thick, 24 x 24 mm, Knittel) were

sonicated in Nanopure water for 30 min and washed copiously with filtered (0.2 µm)

Nanopure water (18.2 MΏ/cm), dried under a stream of high purity nitrogen, and then

primed with hexamethyldisilazane (HDMS). A 4 wt% solution of poly(methyl

methacrylate) (PMMA) and poly(tert-butyl methacrilate) P(tBuMA) in propylene

glycol methyl ether acetate (PGMEA) 99 % (Sigma Aldrich Co.), polysterine-co-

maleic acid (PSMA) (MW~225,000) (Aldrich) in tetrahydrofurane (THF) (99.9 %)

79

XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

were spin-coated at 3000 rpm for 40 s onto HMDS-primed #1 cover glass using a

Specialty Coating System spin coater (Model P6708). The coated substrates were soft

baked at 85 ºC for 1h, and stored in a desiccator prior to and after gold deposition. The

cover slips covered with photosensitive polymeric substrates, namely, (P(tBuMA),

PMMA) were subjected to the λ 254 nm ultraviolet (UV) light for 1 h. When exposed

to the direct irradiation, the original polymer grows into complex phases of

amorphous hydrogenated carbon (a-C:H). This allows the formation of uniform films

of approximately 100-200 nm thick depending on the polymer nature as confirmed by

ELM.

3.2.3. Preparation of microfabricated structures

A Specialty Coating Systems (SCS) spin-coater (Model P6708) was used to

spin-cast a 4 wt % solution of PMMA in propylene glycol methyl ether acetate

(PGMEA) 99 % (Sigma Aldrich Co.) at a high speed of 3000 rpm for 40 s. Once soft

baked at 85 °C for 30 min, the substrates were placed in a desiccator and taken out

only for gold deposition. With the SEM sputter coating unit E5100 (Polaron

Equipment Ltd) depositing gold at 25 mA for 90 s at 0.1 Torr, the gold film coat of 50

nm was formed. To incubate with bovine serum albumin (BSA) required the gold-

layered substrata immersion in a 1 % w/v BSA 10mM PBS solution (pH 7.4) at RT

for 1h, followed by a rinse with PBS and subsequently with Nanopure water.

A laser ablation of the gold and protein coats was carried out by means of a

laser (Cell Robotics workstation, 337 nm, 20 pulses/s, 10 ns/pulses). The proteins

were deposited either by sinking of the entire slides with created surfaces in protein

solution or by spatially addressable deposition with a pipette mounted on an xy

motorized table.

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3.2.4. Protein adsorption on surfaces

3.2.4.1. Protein adsorption on flat surfaces

The proteins, either fluorescently labeled (15 μl of 100 μg ml−1) for the

visualization and quantification of the protein attachment, or unlabeled (100 μg ml−1)

for other experiments (e.g., the thickness estimation) were deposited onto polymeric

surfaces. Adsorption of proteins began with their being incubated on the surfaces in

humidity chambers at RT for 30 min. According to adsorption kinetics experiments

reported elsewhere, the adsorption of relevant proteins was qualitatively the same after

incubation periods of 30 min (Vasina and Dejardin, 2003, Tremsina et al., 1998).

Here, 30 min incubation was considered sufficient to achieve formation of a saturated

protein monolayer on the surface. With proteins adsorbed, slides were washed three

times with 10 mM PBS (pH 7.4), and then two times with filtered Nanopure water

(18.2 MΏ/cm) to take away non-adsorbed proteins.

3.2.4.2. Protein adsorption on micro/nano-fabricated structures

The proteins (20 μl of 70-330 μg/ml), either fluorescently labeled for the

visualization and quantification of the protein attachment or unlabeled for the

thickness estimation were deposited onto micropatterned ablated areas and on native

PMMA polymeric surfaces in a ‗blanket‘ mode, flooding the whole surface of the

micro-assay. For the ‗blanket‘ deposition, the slides were treated as described above.

3.2.5. Protein covalent binding onto surfaces

Protein immobilization via covalent linkage was accomplished by using

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide

(sulfo-NHS) crosslinking couple. The sulfo-NHS ester, a relatively more stable

compound, was first produced by introducing 2 ml of a mixed aqueous solution of

75 mM EDC and 15 mM sulfo-NHS to the selected polymeric substrates for 2 h.

81

The surfaces modified by the reaction allowed protein binding. After a 30-minute

reaction with proteins dissolved in 0.01 mM PBS buffer (pH 7.4), polymeric surfaces

were washed three time with 10 mM PBS (pH 7.4), and then two times with filtered

Nanopure water (18.2 MΏ/cm). The prepared samples were kept in an environmental

chamber prior to analysis.

3.2.6. Detection and quantification techniques

3.2.6.1. Fluorescence spectroscopy of adsorbed proteins

The attachment of fluorescently labelled proteins on the ablated area was

visualised and analysed using two different microscopic systems. One was the

NIKON Microphot FX microscope with a UV light source (Nikon Mercury Lamp,

HBO-100 W/2; Nikon C.SHG1 super high pressure mercury lamp power supply) at

100X objective. The images were captured and recorded by a Nikon camera (FX-

35WA). The second system was a Nikon inverted microscope (Nikon Eclipse TE-DH

100W, 12V) with an attached UV light source (Nikon TE-FM Epi-Fluorescence). The

related images were taken using a Nikon Charged Coupling Device (CCD) camera.

The fluorescence intensities were analysed using Gel-Pro Analyser software, version

4.0.

3.2.6.2. X-ray photoelectron spectroscopy

A Kratos Ultra Imaging X-ray Photoelectron Spectrometer (XPS) with

monochromotised Al Kα (photon energy = 1486.6 eV) radiation at a source power of

150 W was used to perform elemental analysis of polymeric surfaces. The dimensions

of areas analysed were nominally ~700x300 m2. The acquisitions of wide scan and

region spectra were done using 160eV and 20eV pass energies, respectively. Electron

binding energies were calibrated against the C1s emission at 285 eV.

82

3.2.6.3. Goniometry

Contact angle values were used to evaluate the hydrophobic properties of the

films. The measurements were performed on sessile drops (2 l) of Nanopure water at

RT in air employing using a contact angle goniometer. The measurement tool set

included XY stage fitted with a (20 l) micro syringe, a 20 x magnification

microscope (ISCO-OPTIC, Germany) and a fibre-optic illuminator. Six independent

readings were taken to calculate an average value.

3.2.6.4. Ellipsometry

An elipsometry technique was used for measuring changes in light polarization

reflected off polymeric films to determine the thickness of them as a function of 632.8

nm red Helium-Neon laser wavelength. A null-seeking type AutoE1-III ellipsometer

(Rudolph Research, USA) was aligned with respect to the 70º angle of incidence, Φ

(PHI). The data were analysed using elipsometric software, Version 3.9. The film

polymeric/protein thickness was calculated according to De Feijter‘s equation (1978)

that allows a film thickness and refractive index values to be transformed into an

amount of adsorbed protein per unit area (Γ). The mathematical relationship between

these parameters is represented by the following formula:

Γ (ng/mm2) = df[(nf-nm)/(dn/dc)],

where df is the thickness of the adsorbed film (nm), nf is the refractive index of the

adsorbed film, nm is the refractivs index of the ambient, and dn/dc is the refractivs

index increment, a linear function of the protein concentration. Based on this formula,

the same parameter for air/solid interface can be calculated:

Γ (ng/mm2) = K·t,

where K is the density of the protein ≈ 1.36 g/cm3 and t is the protein thickness (nm).

Using the build-in software that evaluates film thickness and its refractive index

concurrently, a polymer/protein coating thickness along with the corresponding values

of the refractive indices were determined. The polymer-covered slides (24x24x5 mm)

were incubated with 600 μl of sample containing 0.1 mg/ml protein in 10 mM

83

phosphate-buffered saline (PBS), pH 7.4, at RT for 1 h, followed by washing with

PBS and Nanopure water.

3.2.6.5. Atomic force microscopy (AFM)

Atomic Force Microscopy (AFM) characterization was carried out on a

TopoMetrix Explorer (Model No. 4400-11) in the non-contact mode using 2 m and

100 m scanners. The analyses were carried out under air-ambient conditions

(temperature of 23 ºC and 45 % relative humidity). Silicon tips and cantilevers with a

spring constant of 42 N/m and resonant frequency of 320 KHz were used. Scanning

direction was perpendicular to the axis of the cantilever and the scanning rate was

typically 4 Hz.

3.2.6.6. Calculation of protein-surface parameters

The distribution of surface-related molecular characteristics, e.g. surface

charge, hydrophobicity at the protein surface was computed using the Protein Surface

Properties Calculator program (Connolly, 1993); estimation of molecular properties

was based on Connolly‘s algorithm. The algorithm was used beyond its original

purpose for the calculation of the surface-related molecular properties (i.e. surface

positive and negative charges; and surface hydrophobicity and hydrophilicity using

Kyte-Doolittle scale of hydrophobicity/hydrophilicity) as well as the molecular

surfaces related to these properties. The program calculated the surface properties

using probing balls with different radius. The charges of individual amino acids have

been calculated using a semi-empirical method (PM3 as implemented in HyperChem

from HyperCube Inc.) for the structures relevant to a particular pH; then averaged

according to acid-base equilibrium equations; then implemented in an input table read

by the program. This procedure allowed the calculation of the charges on the protein

surface as function of the pH of the solution, and therefore accounted for the

modulation of the adsorption by the differences between the pH and the isoelectric

point of the protein. The algorithm used by the program has been reported elsewhere

84

(Cao et al., 2002, Nicolau et al., 2003). The properties of the proteins have been

calculated for a radius of the probing sphere between 1.4 Å and 10 Å. The Protein

Data Bank (PDB) was searched to collect various protein structures (Bernstein et al.,

1977).

3.3. Methods of actin/myosin preparation

3.3.1. Actin and heavy meromyosin (HMM) preparation

Rabbit skeletal muscle myosin and heavy meromyosin (HMM) were prepared

as described by Margossian and Lowey (1982). Actin was prepared from acetone

powder by the method of Pardee and Spudich (1982) and labeled with Rhodamine

(Molecular Probes, R415) - or Alexa 488 (Molecular Probes, A12379)-labeled

phalloidin (Faulstich et al., 1988). Gelsolin was purified from brevin (plasma gelsolin)

according to the protocol described by Kurokawa et al. (1990). Concentrations of

myosin, and HMM were determined from absorption at 280 nm (A280nm) using

extinction coefficients of 0.56 mg-1mlcm-1, 0.65 mg-1mlcm-1, respectively, and the

concentration of G-actin was determined from absorption at 290 nm (A290nm) using

extinction coefficient of 0.62 mg-1mlcm-1.

For the preparation of 2 µm-F-actin filaments (shown in Figure 19), the

procedure adopted from Oda et al. (1998) was used. Briefly, G-actin was dissolved in

buffer containing 0.2 mM CaCl2, 0.5 mM ATP, 1 mM DTT, 0.01 % NaN3, 5mM Tris-

HCl (pH 8.0). Gelsolin segment 1 (GLS 1) was added into G-actin solution, at molar

ratios of 1:800 and protein solution was kept for 30 min on ice. After addition of KCl

up to 100 mM, GLS 1 treated G-actin was incubated at RT for 1 h. In order to remove

large aggregates, the protein solution was centrifuged at 10,000 g for 20 min.

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3.3.2. Preparation of the electrostatically condensed actin bundles

Bundles of actin filaments were formed by electrostatic condensation of

labeled actin filaments (2 µm) with ions of Ba (108 µM) followed by method

described by Angelini et al. (2003).

3.3.3. Preparation of the polymeric surfaces

Glass slides or cover slips (0.17 mm thick, 24 x 24 mm, Knittel) were

sonicated in Nanopure water for 30 min and washed copiously with filtered (0.2 m)

Nanopure water (18.2 M /cm), dried under a stream of high purity nitrogen, and then

primed with hexamethyldisilazane (HMDS). The following polymers were used:

poly(methyl methacrylate), 4 wt % solution of PMMA in propylene glycol methyl

ether acetate PGMEA 99 % (purchased from Sigma Aldrich Co.) with an activator,

0.5 % of triphenylsulfonium triftalate (purchased from Sigma Aldrich Co.);

poly(styrene-maleic acid), 2 wt % solution of PSMA in tetrahydrofurane (THF)

99.7 %; poly(tert-buthyl methacrylate), 4 wt % solution of P(tBuMA) in propylene

glycol methyl ether acetate, PGMEA 99 %. The deposition of the polymers was done

at 2000 rpm for 40 s using a Specialty Coating Systems spin coater (Model P6708).

The coated substrates were then soft baked at 85 °C for 1 h. The photosensitive

polymers [P(tBuMA), PMMA] were activated by irradiation with UV light of λ 254

nm for 1 h. The microstructures were initially fabricated by simple mechanical

scratching of the polymeric surfaces using a stainless steel syringe needle (22 gauge,

Sigma) and later on were readily accomplished by using commercially available

microscope adaptations to ensure the reproducibility. The needle was held in a fix

position vertically in relation to a polymeric surface and moved along the long axis of

the glass slide, which was attached to a computer-controlled XYZ stage. The system

(Cell Robotics, Inc.) was based on a Nikon Eclipse TE300 inverted microscope. All

the operation procedures have been performed according to the Manual provided by

the manufacturer.

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3.3.4. Protein immobilization on the polymeric surfaces in the flow cell

The cell was constructed from a coverslip with fabricated microstructures on

the polymeric surfaces. For covalent attachment, first the N-hydroxysulfosuccinimide,

NHSS, (Pierce) ester was formed by mixing of 75 mM

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC, (Sigma) and

15 mM NHSS on selected polymeric surfaces for 1 h. The slides were rinsed 3 times

with Nanopure water (18.2 M /cm), and then used for flow cell preparation. Two

parallel strips of double sticky tape were placed symmetrically about 20 mm apart on

one coverslip, another coverslip was placed on the top and pressed gently. Labeled G-

actin (54 nM) was repeatedly (3 times) infused in the flow cell from one side while

letting the solution freely pass through the cell from the other side. G-actin was either

adsorbed physico-chemically or covalently bound (depending on the polymer) on

polymeric surfaces and left for polymerization in the flow of buffer A containing 10

mM DTT, 10 mM ATP, pH 7.0 at 4 ˚C, during 1.5 h. The buffer flow rate of 0.06 ml

per min was controlled by peristaltic pump (Ismatec RS 232).

Nitrocellulose-coated coverslip/s was/re used as the reference substrate for the

in vitro motility assay and/or actin immobilisation experiments. The nitrocellulose-

coated glass was prepared as described elsewhere (Kron et al., 1991). The assay

buffer solution contained 5 mM MgCl2, 20 mM KCl, 0.1 mM EGTA, 10 mM MOPS,

10 mM DTT, 2nM ATP, 10 mM MOPS (pH 7.2).

3.3.5. Beads functionalization

Monoclonal anti-skeletal myosin (MY32) (mouse IgG isotype, Sigma M4276)

was covalently grafted via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC)

on the beads (1 μm) Dynabeads M450 (Dynal Biotech), according to Manufacturer‘s

protocol. The Anti-HMM-coated Dynabeads were stored at 4 °C in storage buffer

(10 mM phosphate buffer, pH 7.4, 0.1 % BSA, 150 mM NaCl, 20 mM NaN2). Before

the experiments the beads were incubated in solution of HMM 0.1 mg ml-1 for 60 min

at 4 ˚C.

87

3.3.6. Fluorescence microscopy

Self-assembled actin filaments were observed at room temperature (22-24 ˚C)

with an epifluorescence inverted microscope (Olympus IX71/IX51) and/or NIKON

Microphot FX microscope system with a UV light source (Nikon Mercury Lamp,

HBO-100 W/2; Nikon C.SHG1 super high pressure mercury lamp power supply) at

100 x NA 1.4 oil-immersion objective with Rhodamine or Alexa 488 specific filters.

The images were recorded with an image-intensified CCD camera system (Coolview

FDI) at eight frames per second, 1392 (h) x 1040 (v) pixels, 6.45 μm square. The

recorded images were processed using ImagePro Plus (version 5.0 for Windows).The

velocity of the beads was determined using ImagePro Plus software. Velocities are

reported as the mean and standard deviation for at least six beads.

3.3.7. Scanning electron microscopy (SEM)

A conventional scanning electron microscopy (SEM) JEOL JSM840 was used

for F-actin bundles examination. The cover slides (supporting the filaments) were

mounted on pin-type aluminium SEM mounts with double-sided conducting carbon

tape and then coated in a DYNAVAC CS300 coating unit with carbon and gold to

achieve a conductivity of the specimen surface prior to the SEM examination. The

thickness of the coating was not measured but would be in the order of a few

nanometers. The excitation voltage (kV), the magnification, and working distance

(WD) are given on the lower part of Figure 20. The working distance is the distance

between final aperture and the specimen surface. The images are secondary electron

images (SE). The sample was not tilted, j.e. the electron beam ―hits‖ the specimen

surface at 90°.

3.3.8. X-ray photoelectron spectroscopy

Elemental analyses of polymeric surfaces were carried out on a Kratos Ultra

Imaging X-Ray Photoelectron Spectrometer (XPS), using monochromatised Al Kα

88

(photon energy = 1486.6 eV) radiation at a source power of 150 W. The analysis areas

were nominally ~ 700x300 m2. Wide scan and region scan spectra were acquired

using 160eV and 20eV pass energies, respectively. Electron binding energies were

calibrated against the C1s emission at 285 eV.

3.3.9. Rheological measurements

TA Instruments controlled stress rheometer AR 2000 with cone and plate

measurement geometry (40 mm, 2°) was used to test liquid viscosity. Measurements

were carried out at the controlled temperature 30 °C with accuracy 0.1 °C in shear rate

sweep mode. Results of measurements were processed using ―Rheology Advantage‖

software package provided by company manufacturer.

3.4. Methods of bacterial taxonomy

3.4.1. Bacterial isolation

3.4.1.1. Isolation of gram-negative bacteria

3.4.1.1.1. Isolation of Marinobacter excellens

Bacteria of the genus Marinobacter were isolated from sediments collected in

Chazhma Bay, Sea of Japan. This work was part of the taxonomic investigation of

free-living marine bacteria dwelling in the Bay, sediments of which were

contaminated by radionuclides (Ivanova et al., 2002d). Sediment samples were

collected in 2001 from a depth of 0·5 m (salinity, 32 ‰; temperature, 12 °C) at

Chazhma Bay, Sea of Japan. Bacteria were isolated by plating 0·1 ml of a suspension

of 1 g sediment in 10 ml sterilized natural sea water onto marine 2216 agar plates

(Difco) or plates with medium B, containing 0.2 % (w/v) Bacto Peptone (Difco,

USA), 0.2 % (w/v) casein hydrolysate (Merck, USA), 0.2 % (w/v) Bacto Yeast

Extract (Difco, USA), 0.1 % (w/v) glucose, 0.002 % (w/v) KH2PO4, 0.005 % (w/v)

89

MgSO4 ·7H2O and 1.5 % (w/v) Bacto Agar (Difco, USA), 50 % (v/v) of natural

seawater and 50 % (v/v) of distilled water at pH 7.5-7.8, as described elsewhere

(Ivanova et al., 1996). Plates were incubated aerobically at RT for 5, 7 or 10 days.

Strains were stored at -80 °C in marine 2216 broth (Difco) supplemented with 20 %

(v/v) glycerol. In total, 145 viable bacterial strains have been recovered from sea water

and sediment samples. During isolation studies, bacteria of different taxonomic

groups, including Shewanella, Halomonas, Pseudoalteromonas and Kocuria, have

been isolated (Ivanova et al., 2002d). From this collection, several bacterial strains

with Marinobacter-like phenotypes were identified initially and studied further in

detail.

3.4.1.1.2. Isolation of Sulfitobacter delicatus and Sulfitobacter dubius

This study extends our previous investigations into the biodiversity of marine

proteobacteria from the Sea of Japan, the north-west Pacific Ocean and other

geographical locations (Ivanova et al., 1996, Ivanova et al., 1998, Ivanova et al.,

2000b, Sawabe et al., 2000). During isolation studies, bacteria of various taxonomic

groups, including species of Shewanella, Marinobacter, Halomonas and

Pseudoalteromonas, have been isolated (Kurilenko et al., 2001).. However, only two

strains with Sulfitobacter-like phenotypes have been tentatively identified. The strains

examined in this study were isolated from a starfish (Stellaster equestris) and sea grass

(Zostera marina). The starfish was collected in October 1998 at a depth of 100 m

(salinity 30 ‰, temperature 15 °C) in the South China Sea (26° 28·3' N 122° 29·0' E).

The sea grass was collected in July 1998 at a depth of 5–8 m (salinity 33 ‰,

temperature 12 °C) at the Pacific Institute of Bio-organic Chemistry Marine

Experimental Station, Troitza Bay, Gulf of Peter the Great, Sea of Japan. The starfish

and sea grass were pre-rinsed in sterilized sea water and pieces of tissue (about 3 g)

were aseptically removed. Strains were isolated by plating samples of tissue

homogenate (0·1 ml) onto marine agar 2216 (Difco) plates and medium B plates (the

90

composition of the medium is described in subsection 3.4.1.1.1.) and preserved in

marine broth supplemented with 30 % glycerol at –80 °C.

3.4.1.2. Isolation of gram-positive bacteria

3.4.1.2.1. Isolation of Planococcus maritimus

Brown algae Fucus evanescens were collected by scuba divers in mid-summer

(July 1999) at the Kraternaya Bight, Kuril Islands, N.W. Pacific Ocean, during the

23rd scientific expedition of the R/V ―Akademician Oparin‖. The enrichment

experiments and bacterial isolation were peformed as described elsewhere (Ivanova et

al., 2002a, Ivanova et al., 2002b) with the modification of adding a protein inhibitor

for endo-(1->3)-beta-D-glucanases (Yermakova et al., 2002) to the enrichment

culture. Cultures were maintained on Marine agar plates and medium B (the

composition of the medium is described in subsection 3.4.1.1.1.) and in marine broth

supplemented with 30 % of glycerol at –80 °C. All isolates were streaked on agar

plates from broth cultures every six months to ensure purity and viability.

3.4.2. Bacterial characterization

3.4.2.1. Phenotypic analysis

Unless indicated otherwise, the phenotypic characteristics of gram-negative,

namely, Marinobacter and Sulfitobacter species were studied using standard

procedures (Baumann et al., 1972, Smibert and Krieg, 1994) as described previously

(Ivanova et al., 1996, Ivanova et al., 1998). Phenotypic characteristics of gram-

positive species, Planococcus maritimus, were assessed using standard procedures

(Ivanova et al., 1996, Smibert and Krieg, 1994).

91

3.4.2.1.1. General phenotypic tests

The following physiological and biochemical properties were examined:

oxidation/fermentation of glucose (Hugh and Leifson, 1953); Gram staining; nitrate

and nitrite reduction; catalase (with 5 % H2O2) and oxidase (Kovacs, 1956) activities;

gelatine liquefaction production of arginine dihydrolase, lysine decarboxylase,

ornithine decarboxylase, poly-β-hydroxybutyrate, and acetoin (Voges-Proskauer test);

sodium requirement [0,1,3,6,8,10,12,15, 20) (w/v) NaCl]; indole and H2S production;

the ability to hydrolyse starch, Tween 80, DNA, casein, chitin (1 %, w/v), alginate

(0.1 %, w/v) and agar.

The temperature range for bacterial growth was tested on marine agar plates

incubated at 4, 10, 30, 35, 37, 42 and 45 ºC. To assess the effect of pH on bacterial

growth, bacteria were cultivated in a pH range between 4.5 and 12.0; pH of medium

was adjusted using HCl and NaOH. After 24 h-incubation of bacteria in marine broth,

measurements of the optical density of the cultures at 660 nm were carried out.

Cultures were incubated on a rotary shaker at 160 rpm for 24 h at 25 ºC.

Haemolytic activity of the strains was detected on blood agar containing 40 g

trypticase soy agar in 50 ml sheep blood and 950 ml water. Tests for utilization of

various organic substrates as sole carbon sources (as described in subsection

3.4.2.1.1.2.) at a concentration of 0·1 % (w/v) were performed in 10 ml liquid BM

medium (Baumann et al., 1972).

3.4.2.1.1.1. Microscopic examination

Cellular morphology and gram-stain were examined after 24 h incubation on

medium B. The motility was determined by observing 18-h-old cultures under phase-

contrast light microscope. To test for spreading growth and gliding motility, strains

were grown on medium B with a reduced peptone content (0·2 g l–1). Motility was

verified using phase-contrast microscopy (Nikon) of hanging drop preparations.

Electron micrographs of negatively stained cells were prepared using a Zeiss

EM 10 CA electronmicroscope (80 kV). A drop of particle-free (autoclaved and ultra-

92

centrifuged) distilled water was placed on the culture. The sample (30 μl) of resulting

bacterial suspension was applied to carbon-and Formvar-coated 400-mesh copper

grids, a drop of 1·25 % uranyl acetate was added and the bacteria were allowed to

adhere for 1 min at RT. Superfluous liquid was gently removed using a piece of filter

paper.

Atomic force microscopy (AFM) was employed to characterize the

morphology of the cells, by using a TopoMetrix Explorer (model no. 4400-11;

ThermoMicroscopes) in the non-contact mode, with either a 2 µm liquid scanner

(0·8 µm z-range; model no. 5270-00) or a 100 µm liquid scanner (10 µm z-range;

model no. 5180-00). Silicon cantilevers with a spring constant of 42 N m-1 and

resonant frequency of 320 kHz (model no. 1650.00) were used; all imaging was

performed in ethanol. All samples were prepared on freshly cleaved mica.

3.4.2.1.1.2. Utilization of organic substrates

The tests for utilization of mono- and disaccharides, namely, D-arabinose,

cellobiose, D-fructose, D-galactose, D-glucose, glycerol, lactose, maltose, mannitol,

D-mannose, D-rhamnose, D-ribose, tagatose, L-fucose, sucrose, trehalose and D-

xylose at a concentration of 0.1 % (wt/vol) were carried out in 10 ml per tube of liquid

BM medium (Baumann et al., 1972).

The ability to oxidize 95 carbon sources was tested using both test tubes and

Biolog GN microplates (Rüger and Krambeck, 1994) as described elsewhere (Ivanova

et al., 1998). The range of the substrates utilized according to Biolog profile is

provided in the species descriptions.

3.4.2.1.1.3. Degradation of macromolecules

Degradation of macromolecules was tested using medium B. Chitin

(1 %, w/v), elastin (0.1 %, w/v), and alginate (sodium salt; 0.1 %, w/v), hydrolysis

was determined by development of clear zones around colonies. Cellulose hydrolysis

was tested by using both cellulose overlay plates (1 % carbomethylcellulose), and

93

filter paper strips. The later strips were examined in liquid cell culture for dissolution

(Smibert and Krieg, 1994). Starch, casein and gelatin hydrolysis was tested by the

methods of Smibert and Krieg (1994).

3.4.2.1.1.4. Cytotoxic and antibacterial activities

Cytotoxic and antibacterial activities were assessed by the agar-diffusion assay,

based on methods described elsewhere (Barry, 1980, Sasaki et al., 1985). Cultures

(0·1 ml) of indicator test strains were spread on tryptic soy agar plates in which

circular wells (diameter, 10 mm) had been cut. Areas of inhibited bacterial growth

were measured after incubation for 48 h at 28 °C. Zones of inhibited growth of the

indicator strains surrounding the wells were observed; mean diameters were measured

and 10 mm was subtracted to represent the diameter of the well. Antimicrobial

activities were tested against Staphylococcus aureus CIP 103594, Escherichia coli

ATCC 25290, Proteus vulgaris NBRC 3851T, Enterococcus faecium CIP 104105,

Bacillus subtilis ATCC 6051T and yeast Candida albicans KMM 455.

3.4.2.1.1.5. Susceptibility to antibiotics

Susceptibility to antibiotics was tested by the routine disc-diffusion plate

method using medium B agar and disks (Oxoid) impregnated with following

antibiotics: kanamycin (30 µg), ampicillin (10 µg), benzylpenicillin (10 µg),

streptomycin (30 µg), gentamicin (30 µg), lincomycin (30 µg), neomycin (30 µg),

polymyxin B (25 µg), and tetracycline (30 µg). Agar plates were seeded with light

lawn of bacteria and incubated at 28 ºC for 24 h. A distinct inhibition zone indicated

susceptibility to antibiotic.

3.4.2.1.2. Species-specific phenotypic tests

The ability of Sulfitobacter species to oxidize sulfite was tested as described

by Pukall et al. (1999).

94

3.4.2.2. Chemotaxonomic methods

3.4.2.2.1. Polar lipid (PL) analysis

Lipids were extracted according to Bligh & Dyer (1959). Two-dimensional

micro-TLC of polar lipids was carried out using the method of Svetashev &

Vaskovsky (1972), with chloroform/methanol/benzene/ 28 % NH4OH (65:30:10:6, by

vol.) for the first dimension and chlorophorm/methanol/benzene/acetone/acetic

acid/water (70:30:10:5:4:1, by vol.) for the second dimension (Vaskovsky and

Terekhova, 1979). Non-specific detection of lipids on the TLC was performed with a

10 % solution of H2SO4 in methanol at 180 ºC (Kates, 1986). The following specific

reagents were used: for phospholipids, see Vaskovsky et al. (1975); 2 % ninhydrin in

acetone for amino-containing lipids; Dragendorff's reagent for choline lipids; and

anthrone spray (0·5 % anthrone in benzene and 5 % H2SO4 in water) for glycolipids.

Phosphorus analysis was carried out according to Vaskovsky et al. (1975).

3.4.2.2.2. Fatty acid (FA) analysis

Analyses of fatty acid methyl esters were carried out on a Shimadzu GC-14A

GC with an FID using both a non-polar SPB-5 fused-silica column (30 mx0·25 mm

i.d.) at 210 °C and a polar Supelcowax-10 fused-silica column (30 mx0·25 mm i.d.) at

200 °C. The FID was operated at 240 °C. Helium was used as the carrier gas (Carreau

and Dubacq, 1978, Christie, 1988). Catalytic hydrogenation of fatty acid methyl esters

was carried out as described by Appelquist (1972).

95

3.4.2.3. Genotypic analysis

3.4.2.3.1. DNA GC content determination

DNA was isolated from the strains by following the method of Marmur (1961).

The G+C content of the DNA was determined by using the thermal denaturation

method (Marmur and Doty, 1962).

3.4.2.3.2. DNA hybridization

Reference strains were routinely cultured on marine agar 2216 plates (Difco).

DNA–DNA hybridization was performed spectrophotometrically and initial

renaturation rates were recorded as described by De Ley et al. (1970).

The method described by Christensen et al. (2000) was used to hybridize

DNAs from Marinobacter and Sulfitobacter species. DNA–DNA hybridization

experiments were performed by using covalent attachment of the DNA in micro-wells.

Briefly, 300 ng DNA (400–700 bp fragments) diluted in ice-cold 1-methylimidazole

(Sigma), pH 7·0 and 25 µl 40 mM 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide

(EDC; Sigma) dissolved in sterilized Nanopure H2O (18·2 M cm-1) was added to

each well of NucleoLink micro-well strips (Nalge Nunc International) to bind the

DNA covalently to the NucleoLink surface. After incubation at 50 °C for 18 h

(without shaking), unbound DNA was washed continuously. DNA labelling with

photoactivated biotin, hybridization, detection and quantification were performed as

described by Christensen et al. (2000).

Marinobacter species were hybridized with the following type strains:

Marinobacter hydrocarbonoclasticus (ATCC 49840T), Marinobacter aquaeolei

(ATCC 700491T) and Marinobacter litoralis (KCCM 41591T).

Sulfitobacter species were hybridized with Sulfitobacter pontiacus DSM

10014T, Sulfitobacter mediterraneus ATCC 700856T and Sulfitobacter brevis ATCC

BAA-4T, obtained from the German Collection of Microorganisms and the American

96

Type Culture Collection and Staleya guttiformis DSM 11458T generously gifted by

P. Hirsch (Institut für Allgemeine Mikrobiologie, Kiel, Germany).

3.4.2.4. Phylogenetic analysis

3.4.2.4.1. 16S rRNA gene analysis

DNAs from Sulfitobacter species for PCR were prepared using the Promega

Wizard genomic DNA extraction kit according to the instruction manual. DNA

templates (100 ng) were used for PCR amplification of small subunit rRNA genes as

described previously (Sawabe et al., 1998a, Sawabe et al., 1998b). The PCR

conditions were as follows: initial denaturation step at 94 °C for 180 s, annealing step

at 55 °C for 60 s and extension step at 72 °C for 90 s. The thermal profile consisted of

30 cycles. The amplifcation primers used in this study gave a 1.5 kb PCR product.

PCR products were purified using the Promega Wizard PCR preps DNA purification

kit and were sequenced directly by using a Taq FS dye terminator sequencing kit

(ABI) according to the protocol recommended by the manufacturer. DNA sequencing

was performed with an Applied Biosystems model 373S automated sequencer. The

16S rDNA sequences of Sulfitobacter species were aligned automatically and then

manually by reference to a database of previously aligned relevant bacterial 16S

rDNA sequences. Phylogenetic trees were constructed according to three different

methods (BIONJ, maximum-likelihood and maximum-parsimony). For the neighbour-

joining (NJ) analysis, a distance matrix was calculated according to Kimura's two-

parameter correction. Bootstraps were done using 500 replications, BIONJ and

Kimura's two-parameter corrections. BIONJ was performed according to Gascuel

(1997), and the maximum-likelihood (ML) and maximum-parsimony (MP) data were

from PHYLIP (Phylogeny Inference Package, version 3.573c, distributed by J.

Felsenstein, Department of Genetics, University of Washington, Seattle, WA, USA).

Phylogenetic trees were drawn using NJPLOT (Perriere and Gouy, 1996) and

CLARIS DRAW software for Apple Macintosh computers.

97

The 16S rRNA genes of Marinobacter and Planococcus species were

amplified and sequenced by MIDI Laboratories (Newark, DE, USA). Briefly, primers

used for amplification corresponded to Escherichia coli positions 5 and 1540.

Amplification products were purified by using Microcon 100 (Millipore) molecular

mass cut-off membranes and checked for quality and quantity on an agarose gel. Cycle

sequencing of 16S rRNA gene amplification products was carried out by using

AmpliTaq ES DNA polymerase and rhodamine dye terminators (Applied Biosystems).

Samples were electrophoresed on an ABI Prism 377 DNA sequencer. Related

sequences were selected according to previous phylogenetic analyses of a database of

previously aligned bacterial 16S rRNA gene sequences and BLAST searches against

the latest release of the EBI (European Bioinformatic Institute). In a preliminary

analysis, relevant sequences were selected according to the result of a BLAST query.

The construction of initial tree for each microbial species allowed closely related

sequences to be selected from reference strains when available. When several

sequences were available for a type species, the sequence with the fewest ambiguities

was selected. Phylogenetic trees were constructed using three different methods:

BIONJ, ML and MP. For the BIONJ analysis, distance matrices were calculated using

Kimura's two-parameter correction. BIONJ analysis was performed according to

Gascuel (1997). ML and MP were from PHYLIP (Felsenstein, 1985, Felsenstein,

1993). Phylogenetic trees were drawn using NJPLOT (Perriere and Gouy, 1996).

3.5. Methods used to assess ATP production by bacteria

3.5.1. Bacterial strains

The type strains and environmental (marine) bacterial isolates belonging to the

17 genera were used in this study (Table 1). Type strains were obtained from the

American Type Culture Collection (ATCC, Rockville, MD, USA), the Culture

Collection of Pasteur Institute (CIP, Paris, France), the German Collection of

Microorganisms (DSM, Braunschweig, Germany), the Institute of Molecular and

Cellular Biosciences (IAM, Tokyo, Japan), and the National Collection of Industrial

98

and Marine Bacteria (NCIMB, UK). Other strains were from the Collection of Marine

Microorganisms (KMM Vladivostok, Russia), and kindly provided by U. Simidu

(University of Tokyo, Japan), M. Akagawa-Matsushita (University of Occupational

and Environmental Health, Kitakyushu, Japan), P. Hirsch (Institut für Allgemeine

Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germany), J. Guinea, T. Sawabe

(Hokkaido University Hakodate, Japan), A. Sánchez-Amat (University of Murcia,

Spain), and C. Holmstrom (The University of New South Wales, Sydney, Australia).

Strains used in this study were routinely cultured on Marine Agar 2216 (Difco, USA)

and PYGV agar plates (Labrenz et al., 2000) and stored at -80 °C in marine broth 2216

(Difco) supplemented with 20 % (v/v) of glycerol.

99

Table 1. Strains and environmental (marine) bacterial isolates used in the study.

Genera/species Strain/ isolate Genera/species Strain/isolate Planomicrobium

alkanoclasticum

Planococcus antarcticus

Planomicrobium

koreense

Planomicrobium

mcmeekinii

Planomicrobium

okeanokoites

Planomicrobium

psychrophylum

Planococcus citreus

Planococcus kocurii

Planococcus

maritimus

Kocuria palustris

Kocuria polaris

Kocuria rhizophila

Kocuria rosea

Kocuria varians

Bacillus algicola

Brevibacterium celere

Microbacterium sp.

Formosa algae

Cytophaga lytica

Ruegeria algicola

Ruegeria spp.

Erythrobacter vulgaris

NCIMB 13489T

DSM 14505T

YCM 10704T

ATCC 700539T

NCIMB 561T

DSM 14507T

DSM 20549T

DSM 20747T

KCCM 41587T

KMM 3738,

KMM 3636, F 90

CIP 105971T

DSM 14382T

CIP 105972T

CIP 71.15T,

KMM 3812

CIP 8173T

KMM 3737T

KMM 3637T,

F 81, F 59

F 60

KMM 3553T,

F 83

DSM 7489T

CIP 104267T

1-30, R10SW5

022-2-9

Marinobacter

hydrocarbonoclastis

Marinobacter litoralis

Marinobacter spp.

Marinobacterium

georgiensis

Microbulbifer

hydrolyticus

Cobetia marina

Alteromonas macleodii

‗Alteromonas infernus’

Pseudoalteromonas

atlantica

Pseudoalteromonas

carrageenovora

Pseudoalteromonas citrea

Pseudoalteromonas

distincta

Pseudoalteromonas

elyakovii

Pseudoalteromonas

espejiana

Pseudoalteromonas

haloplanktis

Pseudoalteromonas

issachenkonii

Pseudoalteromonas

maricaloris

Pseudoalteromonas

marinaglutinosa

Pseudoalteromonas

nigrifaciens

ATCC 49840T

KCCM 41591T

2-57, R9SW1

ATCC 700074T

ATCC 700072T

F 6, F 15, F 57

ATCC 27126T

GY785

ATCC 19262T

ATCC 43555T

ATCC 29719T

ATCC 700518T

ATCC 700519T

ATCC 29659T

ATCC 14393T

KMM 3549T

KMM 636T

NCIMB 1770T

ATCC 19375T

100

Sulfitobacter brevis

Sulfitobacter delicatus

Sulfitobacter dubius

Sulfitobacter

mediterraneus

Sulfitobacter pontiacus

Sulfitobacter spp.

Staleya guttiformis

Marinobacter aquaeolei

Marinobacter excellens

ATCC BAA-4T

2-77

(=KMM 3584T)

Z-218

(=KMM 3554T)

ATCC 700856T

DSM 10014T

Fg 1, Fg 36,

Fg 116, Fg 117

DSM 11458 T

ATCC 700491T

KMM 3809T, Fg 86

Pseudoalteromonas

ruthenica

Pseudoalteromonas

tetraodonis

Pseudoalteromonas

undina

Pseudoalteromonas spp.

Shewanella affinis

Shewanella colwelliana

Shewanella gelidimarina

Shewanella japonica

Shewanella pacifica

Shewanella pealeana

Shewanella woodyi

KMM 300T

ATCC 51193T

ATCC 29660T

Z 2/2, SUT 3,

SUT 4, SUT 5, SUT 11, SUT 12,

SUT 13

KMM 3821,

KMM 3586,

KMM 3587T

ATCC 35565T

ACAM 456T

LMG 19691T

KMM 3587T,

R10SW14,

R10SW16

ATCC 700345T

ATCC 51908T

3.5.2. Polymeric surface preparation

Poly(tert-butyl methacrylate) (Sigma-Aldrich, St. Louis, MO, USA) and mica

(Ted Pella, Redding, CA, USA) were used as surfaces. The surfaces were prepared as

described elsewhere (Ivanova et al., 2002e). Briefly, PtBMA (47 kDa, molecular

weight/polydispersity, Mw/Mn = 2.33) dissolved in cyclohexanone (99.9 %) (Sigma-

Aldrich) was spin-coated (substrates: #1 glass cover slips, 10-mm diameter), after

previously being primed with hexamethyldisilasane (HMDS, Sigma-Aldrich). The

substrates were sonicated in PriOH for 30 min, washed with copious amounts of

filtered (0.2 mm) Nanopure water, and dried under a stream of high-purity nitrogen.

The polymeric films were spin-coated on primed glass substrates by using

tetrahydrofuran (THF) solution at concentrations of 2-5 mg/ml. The primer was spun

101

at 1000 rpm and polymers at 3000 rpm with a ramp acceleration of 1000 rpm using a

spin coater (Model P6708, Specialty Coating Systems, Indianapolis, IN, USA).

Finally, polymeric slides were baked at 95 ºC for 60 min. Muscovite mica sheets were

freshly cleaved and used as received.

3.5.3. Contact angle measurements

Advancing contact angles were measured on sessile drops (2 ml) of Nanopure

water at RT in air, using a contact-angle meter constructed from an XY stage fitted

with a (20 ml) microsyringe, a 20× magnification microscope (Isco-Optic, Göttingen,

Germany), and a fiber-optic illuminator. The images were captured using a digital

camera (Aiptek, Tokyo, Japan) and analyzed using PaintShop Pro (Jasc Software).

Observed values were averaged over six different readings. We defined the PtBMA

polymeric surface as being hydrophobic with a measured water contact angle of 91º

and mica as being hydrophilic with a much smaller contact angle of 5º.

3.5.4. Bacterial growth and sample preparation

For initial screening, bacterial suspensions of freshly grown cells (1.0-2.0 × 108

cells/ml, optical density, OD660 = 0.13-0.2) were used for inoculation of 0.5 l of

Marine Broth 2216 (Difco). Bacteria were cultured for 18-24 h at RT without any

growth-limiting factors and were harvested at the late exponential phase of growth.

The growth phases were monitored spectrophotometrically. Bacterial strains were

grown on Marine Agar 2216 plates at 28 °C for 48 h. Polymeric slides and freshly

cleaved mica disks were placed in sterilized Nunc multidishes (12 wells). The

polymer-lined wells were inoculated with exponential-phase cultures (3 ml). The cells

were plated in duplicate for each polymeric surface and the experiment was repeated

12 times to monitor cell growth and ATP generation every 4 h over the course of the

experiment. Cell density was adjusted to OD660 = 0.13 ± 0.05 by the addition of

phosphate-buffered saline (PBS) containing 50 mM phosphate and 150 mM NaCl (pH

7.4). A 300-µl cell aliquot was added into 2700 µl of Marine Broth 2216. The same

102

suspension of each strain (3 ml, in triplicate) was added to an empty well and served

as a control. Every 4 h, a correspondent aliquot (10 µl, in triplicate) of bacterial

suspension was removed and the amount of extracellular ATP was measured. The

optical density of bacterial cells in the wells was also monitored. The biofilms formed

on the polymeric surfaces by statically grown bacteria were rinsed three times with

PBS, and the attached cells were carefully scraped off and resuspended in 1 ml of PBS

to determine the level of intracellular ATP.

3.5.5. Bioluminescence assay for ATP determination

Bioluminescence was monitored with a fluorimeter (FluorStar Galaxy,

Offenburg, Germany) in white opaque 96-well microtiter plates (Nunc, Copenhagen,

Denmark). The internal cellular ATP concentration and the external ATP

concentration in the medium were analyzed separately. ATP generation was detected

using the Enliten ATP Detection Kit (Promega). The homogeneous assay procedure

involves adding a single reagent directly to bacterial cells cultured in medium and

measuring ATP as an indicator of metabolically active cells. The procedure was

carried out according to the manufacturer's protocol. Each well contained 10 µl of the

bacterial suspension sample. Bioluminescence was recorded after the automatic

injection of 90 µl rLuciferase/ Luciferin (rL/L) reagent. Light measurements were

made in triplicate for each sample and for the negative control. ATP values are given

as relative units, which define the amount of light emitted per unit of cell density. The

levels of extracellular ATP were measured directly in bacterial suspension, and the

levels of intracellular ATP in samples prepared via extraction of ATP by 1 %

trichloroacetic acid (TCA, Sigma-Aldrich).

3.5.6. Cell-surface characterization by AFM

AFM characterization of the cell surfaces was carried out on a TopoMetrix Explorer

(model no. 4400-11, Sebastopol, CA, USA) in both the non-contact and normal

contact modes using 2-µm and 100-µm scanners. The analyses were done under air-

103

ambient conditions (23 °C, 45 % relative humidity). Pyramidal silicon-nitride tips

attached to cantilevers with a spring constant of 0.032 N/m were used in the contact

mode, whereas silicon tips and cantilevers with a spring constant of 42 N/m and a

resonant frequency of 320 kHz were used in the non-contact mode. The scanning

direction was perpendicular to the axis of the cantilever and the scanning rate was

typically 4 Hz.…………………

3.6. Methods used to assess MreB and FtsA proteins

3.6.1. Analysis of mreB and ftsA genes

DNAs from 24 bacterial species belonging to the class γ-Proteobacteria,

namely, Cobetia marina LMG 2217T, Marinobacter hydrocarbonoclasticus

ATCC 49840T, Marinobacter aquaeolei, Pseudomonas fluorescens DSM 50030T,

Pseudomonas extremorientalis KMM 3447T, Pseudoalteromonas issachenkonii

KMM 3549T, Pseudoalteromonas nigrifaciens ATCC 19375T, Pseudoalteromonas

haloplanktis ATCC 14393T, Pseudoalteromonas atlantica ATCC 19262T,

Alteromonas macleodii ATCC 27126T, Alteromonas addita R10SW13T, Oceanimonas

doudoroffiin ATCC 27123T, Oceanimonas smirnovii 31-1T, Marinomonas communis

ATCC 27118T, Marinomonas vaga ATCC 27119T, Marinomonas pontica 46-16T,

Aliivibrio fischeri DSM 507T, Idiomarina zobelii KMM 231T, Idiomarina loihiensis,

Idiomarina baltica, Shewanella woodyi ATCC 51908T, Shewanella affinis

KMM 3587T, Shewanella waksmanii KMM 3823T, Shewanella japonica

KMM 3299T, 7 species belonging to the class α-Proteobacteria, namely, Sulfitobacter

pontiakus DSM 10014T, Sulfitobacter mediterraneus ATCC 700856T, Sulfitobacter

delicatus KMM 3584T, Sulfitobacter sp. Fg 107, Sulfitobacter sp. RIOSW6,

Loktanella rosea Fg 1, Loktanella vestfoldensis and one Salegentibacter flavus Fg 69T

relating to the CFB group were isolated following the method of Marmur (1961).

MreB and FtsA genes were amplified, cloned and sequenced using specifically

designed primers by AGRF Ltd (University of Queensland, Australia). Aligned and

translated by AGRF sequences were utilized for phylogenetic analysis. Translated

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sequences were compared with available MreB and FtsAs protein sequences of

Escherichia coli K-12 substr. DH10B (γ-Proteobacteria); Bacillus subtilis, Listeria

monocytogenes (Firmicutes); Thermotoga maritima MSB8 (class Thermotogae) and

rabbit (Oryctolagus cuniculus) actin, alpha skeletal muscle. Also, FtsAs were

compared with Streptococcus pneumoniae (Firmicutes). Treecon software (Van de

Peer and De Wachter, 1994) was used to build and draw NJ phylogenetic trees.

3.6.2. Computation of MreB and FtsA protein parameters

To estimate physico-chemical properties of bacterial MreB and FtsA proteins

including their instability, ProtParam software (Gasteiger et al., 2005) was used.

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CHAPTER 4

IMMOBILIZATION OF PROTEINS ON FLAT

SURFACES

106

4.1. Overview

This chapter presents a practical methodological approach to monitor the

passive adsorption and covalent immobilization of two proteins, human

immunoglobulin (HIgG) and lysozyme (LYZ), on modified poly(tert-butyl

methacrylate) (PtBMA) surfaces. Reproduced from reference (Ivanova et al., 2006c)

© 2006 With kind permission from IOP. As chemistry of surface affects the efficiency

of protein immobilization, chapter starts with a characterization of PtBMA film. The

chapter continues with an investigation of protein–surface interactions via

physicochemical adsorption and covalent immobilization to clarify whether the

protein immobilization behavior of these two attachment processes yields similar

packing densities. Here, results of x-ray photoelectron spectroscopy, ellipsometry and

AFM of two morphologically different proteins, i.e., a big β-sheet structured HIgG

and a small α-helix/β-sheet structured LYZ, are presented and discussed. The chapter

ends with a conclusion that covalent linkage of proteins to homologous polymeric

surfaces can secure formation of reproducible protein layers. It was also concluded

that the density of surface functional groups affected protein immobilization. The

binding efficiency between the substrate and surface is crucial not only for the

development of potential components of microfluidic devices described in the

following chapters but also for the construction of microfluidic device presented in

chapter 6.

4.2. Results and discussion

4.2.1. PtBMA film characterization

PtBMA has been frequently employed as a positive photoresist due to its

excellent mechanical and optical properties, e.g. transparency (>90 % transmission),

stiffness, low water absorption and high abrasion resistance. The PtBMA surface is

typically hydrophobic (77◦ ± 3) due to the presence of the methyl groups on the

polymer backbone and the tert-butyl ester. Following UV irradiation, and subsequent



107

heating (90 ◦C to catalyze the chemical amplification reaction), the modified PtBMA

surface is found to be less hydrophobic (65◦ ± 2) due to the formation of surface

carboxylic acid groups, as determined by water contact angle measurements consistent

with previously reported values (Bahulekar et al., 1998). Exposure to UV (254 nm)

radiation causes the PAG to release protons, which hydrolyze the ester linkages to

produce carboxylic acid groups during the chemical amplification (heating) process.

The tertiary carbon of the ester group (i.e., COO–C∗), with binding energy of 286.6

eV, is absent from the spectrum indicating essentially a complete loss of the tert-butyl

protecting group from the surface esters (Scheme 1).

Activated PtBMA

Scheme 1. Reaction scheme for the formation of sulfo-N-hydroxysuccinimide (sulfo-

NHS) activated poly(tert-butyl methacrylate) (PtBMA).

108

The XPS analysis also confirms the presence of carboxylic groups on the

irradiated PtBMA surface (Table 2, Figure 1). The root-mean-square (RMS) surface

roughness values for the spin-coated polymers were determined from AFM

topographical images using the TopoMetrix Explorer image processing software.

Typical RMS roughness values ranged between 1.05 ± 0.5 nm for a field-of-view of

4.5 × 4.5 μm2 (data not shown).

Table 2. Atomic concentration ratios (determined by XPS) obtained for adsorbed and

covalently immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers

on activated P(tBMA) surfaces.

Surface Protein

attachment

Integrated peak area ratios

(×103)

N/C S/C

P(tBuMA) (as received) - - -

+Lysozyme Adsorbed 146.7 -

+Human immunoglobulin Adsorbed 111.1 -

P(tBuMA)(sulfo-NHS activated) - 20.2 5.05

+Lysozyme Covalent 87.3 4.31

+Human immunoglobulin Covalent 87.3 4.30

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Figure 1. Representative surface topography of fluorescence images of

human immunoglobulin (HIgG) adsorbed (top, left) and covalently

immobilized (top, right) and lysozyme (LYZ) adsorbed (bottom, left) and

covalently immobilized (bottom, right) on UV-irradiated PtBMA surfaces.

Similar images were obtained in different regions of at least two different

samples.

Overall, the surface modification of PtBMA provided moderately less

hydrophobic surfaces than the commercially obtained polymers, which are suitable for

both adsorption and covalent immobilization of biomolecules due to the presence of

the surface carboxylic acid groups.

110

4.2.2. Adsorption and covalent binding of selected HIgG on PtBMA surface

4.2.2.1. X-ray photoelectron spectroscopy analyses

XPS analyses were carried out on the modified PtBMA polymer surfaces after

incubation with either HIgG or LYZ. As mentioned earlier, the XPS analysis reveals

conclusive evidence to support the presence of surface carboxylic acid groups on both

modified PSMA and PtBMA substrates. The typical C 1s XPS spectrum of PtBMA +

COOH surfaces is shown in Figure 2. To achieve chemically active surfaces suitable

for protein immobilization, the standard water-soluble coupling reagents 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-

hydroxysulfosuccinimide (sulfo-NHS) were employed, resulting in the formation of an

amide linkage on incubation with the exposed amines on certain proteins (Pan et al.,

2005). XPS analysis revealed that after chemical treatment, as expected, both nitrogen

and sulfur were present on the ‗activated‘ sample surfaces (i.e. EDC + sulfo-NHS

only, see Tables 2 and 3). The chemical state of the surface after each derivatization

reaction was monitored by XPS as shown in Figure 2, Table 2 and by the contact angle

measurements summarized in Table 3.

Table 3. Elemental compositions (determined by XPS) obtained for adsorbed and

covalently immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers

on activated P(tBMA) surfaces.

Surface Contact angle (°)

Protein attachment

Relative elemental contribution (at%) N C O S

P(tBuMA) (as received)

77 ± 3 - 0 19.5 80.5 0

+(UV-irradiation)a 65 ± 2 - 0 19.6 80.3 0

+Lysozyme Adsorbed 10.2 70.0 19.2 0.4 +Human immunoglobulin

Adsorbed 7.6 68.1 21.1 0.5

P(tBuMA)(sulfo-NHS activated)

- 1.2 59.6 31.5 0.3

+Lysozyme Covalent 3.9 73 20.7 0.3 +Human immunoglobulin

Covalent 6.1 69.9 20.9 0.3

a Since the takeoff angle is 30◦, the data appeared similar.

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Figure 2. XPS spectra of PtBMA+COOH surfaces: (a) typical C1s; (b) high-resolution N 1s spectra of samples ‗activated‘ by treatment with EDC and NHS; and (c) sample following covalent protein attachment; (d) high-resolution S 2p spectra of samples ‗activated‘ by treatment with EDC and NHS; and (e) of samples following covalent protein attachment.

112

Activation of the COOH-functional surface sites of the polymers by NHS/EDC

mediation introduced a nitrogen 1s peak from NHS that is not present in the spectrum.

The nitrogen peak persists in XPS spectra after NHS/EDC treatment: however, the N

1s peak position shifts from 402.2 to 401.1 eV. The N 1s signal centered at 402.2 eV is

consistent with the electron-withdrawing nature of nitrogen in NHS, and the lower BE

(401.1 eV) N 1s is also consistent with the nature of the nitrogen present in NHS/EDC.

XPS-based elemental compositions and reaction yields, the latter calculated from N

1s/C 1s ratios, are summarized in Tables 2 and 3. The N 1s/C 1s ratio was used to

estimate the reaction yield instead of the N/O or O/C ratios because (1) N is only

present after the activation and amidation reactions and (2) most surface contaminants

are of high oxygen content and their presence therefore leads to an overestimate of the

oxygen atomic composition.

The nitrogen level is much higher than that of sulfur. Given that the N:S ratio

of sulfo-NHS is 1:1. As shown in Table 2, the attachment of HIgG and LYZ is

evidenced by the increase in concentration of nitrogen compounds on the polymer

surfaces. The level of sulfur on the samples following covalent immobilization of

either protein suggests that most of the sulfo-NHS still remains on the surface.

Moreover, given that EDC is displaced in favor of the more stable sulfo-NHS group,

and that the N 1s contribution from sulfo-NHS would be minor, it is likely that most

of the nitrogen signal derives from the attached protein molecules. It can be seen in

Table 2 that the N/C values of samples following physicochemical adsorption are

somewhat higher compared to those of their covalently-bound counterparts,

suggesting that the physico-chemical adsorption was more effective than covalent

immobilization in the current experimental regime, i.e. at pH 7.4 which is below the

HIgG and LYZ isoelectric points, pI 7.8 and 11.1, respectively. This may occur due to

the presence of deprotonated carboxylic groups on the modified polymer surfaces,

which increases the electrostatic interactions between the oppositely charged polymer

surface groups and the proteins molecules. Similar interactions of recombinant human

growth hormone and lysozyme with different quartz surfaces containing either silyl

groups, such as silanol, methylsilyl, or quaternary aminopropyldimethylsilyl surface

113

groups were observed by Buijs and Hlady (1997), who concluded that protein

adsorption (in particular LYZ) was mostly affected by electrostatic interactions.

High-resolution S 2p spectra, shown in Figures 2(b) and (c), provide further

insights into the nature of the bonding between the proteins and the polymer surfaces.

The S 2p spectra for the ‗activated‘ samples (i.e., EDC + sulfo-NHS treatment only)

exhibit a doublet at 168.0 eV. This doublet is due to oxidized sulfur and consistent

with the presence of sulfo-NHS. Following the attachment of HIgG or LYZ, the S 2p

spectra exhibited an additional peak at 163.8 eV, which is most likely due to sulfide

species. Data obtained from curve fitting operation show that about 40 % of the

surface sulfur is sulfite, the rest are oxidized species. High-resolution N 1s spectra of

the ‗activated‘ samples exhibited two distinct peaks at 402.2 eV and 400.0 eV in a

ratio of about 0.3, as shown in Figures 2(d) and (e). The peak at 400.0 eV is attributed

to C–N or N–C=O species, whereas the peak at 402.2 eV is most likely due to NH 3 . It

is interesting to note that following the covalent immobilization of the proteins, the

NH 3 peak disappears, and almost all the surface nitrogen is covalently bound. It is

noted that the NH 3 peak still remains on those samples with proteins attached

physico-chemically.

In a previous study, we employed EDC/sulfo-NHS coupling chemistry to

activate the carboxylic acid groups on PSMA. Such ‗activated‘ surfaces were shown to

react with the ε-amino groups of poly(L-lysine) to form amide bonds, with the

concomitant release of sulfo-NHS (Ivanova et al., 2004g). Here, however, the XPS

data indicated that much of the sulfo-NHS ester was still present after reaction with

both proteins, as confirmed by the presence of a significant amount of sulfonyl groups

(168.4 eV). Moreover, a high resolution N 1s analysis revealed that almost all of the

detected nitrogen on the surface corresponds to covalently bound proteins, which

suggests that the protein attachment mechanism described here may follow a different

path to that described in the literature (Ivanova et al., 2004g). It is likely that the

electrostatic attraction between the sulfonyl moiety on the sulfo-NHS ester backbone

and protein amino groups is preferred to amide bond formation resulting from the

subsequent displacement of the sulfo-NHS group.

114

4.2.2.2. Ellipsometry analysis

Ellipsometry yielded estimates of the amount of the proteins and the thickness

of protein layers on the polymer surfaces similar to previous reports (Malmsten and

Lassen, 1995). The amount of physically adsorbed HIgG on PtBMA was 23.0 ± 1.6 ng

mm2, with corresponding protein layer thicknesses of 17.0 ± 1.2 nm, while those after

covalent immobilization were 5.6–8 ng mm2 with a corresponding layer thickness of

5.9 ± 0.6 nm on PtBMA (Table 4).

Table 4. Ellipsometric measurements obtained for adsorbed and covalently

immobilized human immunoglobulin (HIgG) and lysozyme (LYZ) layers on activated

P(tBuMA) surfaces.

Surface Protein

attachment

Protein layer

thickness (nm)

Amount

of protein

(ng mm2)

P(tBuMA) (as received) - - -

+Lysozyme Adsorbed 11.0 ± 3.2 15.0 ± 4.4

+Human immunoglobulin Adsorbed 17.0 ± 1.2 23.0 ± 1.6

P(tBuMA)(sulfo-NHS activated) - - -

+Lysozyme Covalent 7.01 ± 0.6 7.8 ± 0.6

+Human immunoglobulin Covalent 5.9 ± 0.6 8.0 ± 0.8

HIgG is generally considered to adopt a typical Y- or T-shaped conformation

having 3D dimensions of 10 × 15 × 13 nm3 and a thickness of 5 ± 0.5 nm (Day, 1990).

According to these data, it can be inferred that the thickness of the adsorbed HIgG

layers formed on modified polymer surfaces is consistent with the length of the HIgG

arm (7 nm) and the base (6.5 nm), and that of covalently immobilized HIgG may

correspond to proteins lying in a ‗side-on‘ configuration. The results obtained are

consistent with that of Baszkin and Lyman (1980), who calculated theoretical values

115

for a monolayer of adsorbed HIgG molecules attached ‗end-on‘ (18.5 ng mm2) and

‗side-on‘ (2.7 ng mm2).

The adsorption reaction between LYZ on PtBMA surfaces was similar to that

observed for HIgG, where the amount of adsorbed protein was in the range 15 ng mm2

(Table 4), whilst the amount of protein after covalent immobilization was in the range

of 8 ng mm2. The thickness of the adsorbed LYZ protein layers ranged at 11.0 ± 3.2

nm on PtBMA, suggesting the bulk of the compactly adsorbed protein occurred on the

polymer surface (see also the AFM analysis section, Figure 4). It was also found that

the thickness of the covalently immobilized LYZ layer of ~ 5–7 nm could be

construed as a protein monolayer, since the LYZ dimensions [5 × 3.5 × 3.5 nm3]

suggest that the maximum possible height of a single layer would be consistent with

the largest dimension (i.e. 5 nm). Notably, in our previous study we observed that α-

chymotrypsin exhibited a different attachment behavior whilst adsorbing to PMMA.

We found that the amount of α-chymotrypsin adsorbed on PtBMA was typically 4 ng

mm2, with a corresponding protein film thickness of ~ 3–6 nm (Ivanova et al., 2003b).

These values agreed well with, for example, the theoretical value of 3 ng mm2

calculated for an α-chymotrypsin monolayer (based on the size of the protein as

determined from its crystal structure (Aune and Tanford, 1969), while covalent

immobilization resulted in similar or slightly greater amounts of immobilized protein

on the polymer surfaces (Ivanova et al., 2003b). The most likely, on PtBMA HIgG and

LYZ yielded greater adsorption values compared to that of α-chymotrypsin due to

electrostatic interactions being positively charged in the buffer used in experiments

(10 mM PBS, pH 7.4), in contrast to α-chymotrypsin, which is negatively charged

under the same buffer conditions (Aune and Tanford, 1969). Our results correlate with

those reported by Buijs and Hlady (Buijs and Hlady, 1997), who provided evidence to

suggest that greater amounts of adsorbed LYZ are formed on polymeric surfaces when

buffers of low ionic strength (10 mM PBS) are employed, pointing to the important

role electrostatic interactions play on both hydrophobic and hydrophilic surfaces. It

was also reported in previous studies that the build-up of the adsorbed proteins layers

vary significantly for each protein, and LYZ in particular forms a more compact layer

than that of other proteins (Malmsten and Lassen, 1995, Deere et al., 2004).

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4.2.2.3. AFM analysis

AFM imaging was employed to analyze the protein layer formation at the

molecular level following adsorption or covalent immobilization of HIgG and LYZ on

PtBMA (Figures 3 and 4). A careful inspection of the AFM topographical images

revealed that adsorbed/covalently-bound HIgG forms dense layers on PtBMA. A

representative topographical image of surface-immobilized HIgG, together with the

corresponding depth analysis, is presented in Figure 3.

Figure 3. Representative surface topography images and their corresponding line

profile analyses of human immunoglobulin (HIgG) adsorbed (top) and covalently

immobilized (bottom) on UV-irradiated PtBMA surfaces. Similar images were

obtained in different regions of at least two different samples.

117

The adsorbed protein produced a dense surface coverage, with a protein layer

thickness of approximately 15 ± 0.8 nm on PtBMA. The surface roughness of the

covalently immobilized HIgG layer ranged from 4 to 6 ± 0.4nm (Figure 3). This

finding is consistent with the ellipsometric measurements, which suggest that HIgG

molecules physically adsorb in an ‗endon‘ configuration, while the covalently

immobilized protein molecules adopt a ‗side-on‘ configuration. The physical

adsorption of LYZ on the surface-modified PtBMA substrate produced a

heterogeneous surface coverage, with a protein layer had a depth of about 20 ± 0.6 nm

(Figure 4).

Figure 4. Representative surface topography images and their corresponding line

profile analyses of lysozyme (LYZ) adsorbed (top) and covalently immobilized

(bottom) on the UV-irradiated PtBMA surface.

118

Covalently immobilized LYZ on the other hand, formed more homogeneous layers on

polymer surfaces, with depths ranging from 4 to 2 ± 0.3 nm (Figure 4). These depth

values are comparable with those for a single layer of LYZ molecules adsorbed on

mica, as previously imaged by Fritz et al. (1995). Interestingly, the same authors also

noted that in some instances, the depth of the protein layer measured using AFM, was

greater than that determined by x-ray diffraction (Fritz et al., 1995).

The results obtained are consistent with each other, indicating that both the

covalent immobilization and physico-chemical attachment used in the present study

give rise to compatible levels of protein density on the modified surface of the

polymers studied. Fluorescence images of labeled proteins confirmed the formation of

continuous protein layers for both adsorbed and covalently immobilized proteins (data

not shown). Overall, the ellipsometry, XPS and AFM analyses obtained for both

adsorbed HIgG and LYZ on PtBMA, are in good agreement, implying that both

proteins form protein layer(s). This observation is possibly a consequence of the non-

specific nature of adhesion and/or the possible surface diffusion of the protein

molecules (Tsukada and Blow, 1985, Tilton et al., 1990, Tarjus et al., 1990, HÖÖK et

al., 1998), allowing the reorganization of the randomly adsorbed proteins into more

tightly packed layers. One other possibility could result from the close co-existence of

an initial, partially denatured layer of protein molecules, due to the mechanical stress

exerted on the biomolecules during contact with the substrate surface, and a second

(intact) protein layer on the surface (Malmsten and Lassen, 1995, Baszkin and Lyman,

1980, Aune and Tanford, 1969, Ivanova et al., 2003b, Deere et al., 2004, Fritz et al.,

1995, Tsukada and Blow, 1985, Tilton et al., 1990, Tarjus et al., 1990, HÖÖK et al.,

1998, Petrash et al., 1997, Norde et al., 1986, Garrison et al., 1992, Lenk et al., 1989,

Castillo et al., 1984). In addition, the degree of conformational change for each

particular protein can also depend on other factors such as pH and ionic stress, as well

as on the hydrophobic effects mentioned earlier.

It is noteworthy that denser, adsorbed and covalently immobilized HIgG and

LYZ films were found to form on the surface-modified PtBMA substrate. At present,


119

there is no clear explanation for this, however in the light of recent findings, where

certain proteins (e.g. HSA) have been found to bind more tenaciously to surfaces

modified by disordered alkyl-terminated self-assembled monolayers (Petrash et al.,

2001), it is likely that a more flexible surface may conform better to the structure of

the protein molecule (Petrash et al., 2001). Other workers have also noted that the

orientation and packing of certain proteins on substrates is critically dependent on the

surface/substratum characteristics and the immobilization strategy chosen

(Vijayendran and Leckband, 2001, Chen et al., 2003, Zhou et al., 2003, Vikholm and

Albers, 1998). For example, Denis et al. observed the formation of a 6 nm thick

homogeneous collagen layer on smooth hydrophilic substrata, yet on a hydrophobic

surface, this same protein formed a 20 nm thick layer exhibiting elongated, aggregated

structures (Denis et al., 2002). Here, the fact that the modified PtBMA surface is

hydrophobic and from XPS is deemed to consist of greater number of disordered

carboxylic acid groups, could lead to a similar effect allowing a greater

accommodation of protein.

4.3. Conclusion

From the protein thickness results, and given the known three-dimensional size

of the proteins studied, in most cases one would be justified in treating the formed

protein layers as monolayers. Notably, the covalent immobilization in the experiments

performed was translated in good reproducibility in achieving protein monolayers on

both PSMA and PtBMA surfaces. It is also worth noting that a protein concentration

of 0.1 mg ml−1 was sufficient for both proteins to reach saturation point, and coat the

surface completely, which is in good agreement with observations reported elsewhere

(Castillo et al., 1984, Garrison et al., 1992).

Here, the combination of analytical techniques, i.e. XPS, ellipsometry and

AFM, was particularly revealing, providing comparable data during the investigation

of HIgG and LYZ interactions with two surface-modified polymers. We have


120

demonstrated that covalent immobilization can secure the formation of reproducible

protein layers on homologous polymer surfaces. Importantly, this approach can be

effectively applied to proteins with different physical characteristics, e.g., isoelectric

points. The density of substrate surface functional groups, i.e. carboxylic acids,

however, will undoubtedly affect the efficiency of protein–surface interactions.

121

CHAPTER 5

ADVANTAGE OF IMMOBILIZATION OF PROTEINS

IN MICROCHANNELS

© 2004 NSTI http://nsti.org. Reprinted and revised, with permission, from ―Amplification of protein adsorption on micro/nanostructures for microarray applications‖, pp. 95-98, 2004, Boston, U.S.A.

http://nsti.org/

122

5.1. Overview

This chapter describes the newly developed approach for surface design

applicable for microdevices. The approach is based on spatial immobilization of

proteins in micro/nano-channels fabricated via laser ablation. Reproduced from

references (Ivanova et al., 2003b) © 2003 With kind permission from SPIE; and

(Ivanova et al., 2004f) © 2004 With kind permission from SPIE. This work follows

the study of protein immobilization on flat surfaces presented in chapter 4. The

chapter begins with a brief characterization of utilized polymer, i.e., poly(methyl

methacrylate), followed by a discussion of fabrication of microstructures in Au-

deposited PMMA films. The chapter continuous with a comparative investigation of

physico-chemical attachment of five morphologically different proteins, i.e., α-

chymotrypsin, human serum albumin, human immunoglobulin, lysozyme and

myoglobin, immobilized in laser ablated poly(methyl methacrylate)-based channels

and on native poly(methyl methacrylate) films. Because the molecular properties of

proteins and their interactions with surfaces affect protein adsorption, the following

subsection 5.2.3. explains the impact of molecular descriptors on protein adsorption in

microchannels. Subsection 5.2.4. discusses utilization of ellipsometry for evaluation

of the thickness of polymeric films and attached proteins. Subsection 5.2.5. is devoted

to the comparison of protein adsorption on flat surfaces with adsorption in

microchannels, which was performed using fluorescent microscopy and quantified

using Fluor reader. The chapter ends with a characterization of adsorption properties

of selected proteins, and is followed by a conclusion that protein adsorption was

greater in microchannels than that on flat surfaces. As protein immobilization was

affected by microchannel, PSMA strips surrounded by channels were designed for

control of self-assembly of actin filaments, as described in the following chapter.

123


5.2.1. Characterization of poly(methyl methacrylate) polymeric films

Poly(methyl methacrylate) is a rather commonly used polymer due to its

characteristics, e.g. transparency (>90 % transmission), stiffness with excellent UV

stability, low water absorption and high abrasion resistance. The hydrophobicity of

PMMA estimated by contact angle measurements was ranged from 68 θ to 72 θ

indicating its moderate hydrophobic nature.

5.2.2. Fabrication of microstructures in Au-deposited PMMA films

The proposed technology produces surfaces that present to the proteins a large

variation of the properties (in particular hydrophobicity and rugosity) of the surface

concentrated in a small, micron-sized region. The fabrication consists in the ablation

of the opaque thin metallic (e.g., Au) layer deposited on a thick transparent polymer

(e.g., PMMA) layer. The ablation of the thin metallic layer (Figure 5) induces the

pyrolysis and partial ―sculpturing‖ of the polymer, with more hydrophilic surfaces

towards the edges of the channel and a hydrophobic hump in the middle.

124

Figure 5. Fabrication of micro/nano-structures for protein arrays using

microablation and directed deposition.

The higher rugosity of the microstructures (Figure 6) translates in a 3 times

more specific surface in than outside the channels.

125

Figure 6. AFM mapping of the ablated microchannels.

A more important feature than the increased rugosity in the microablated areas

is the large variations in the relative hydrophobicity of different regions, as measured

by AFM in lateral force mode. Figure 7 presents both the topography and the relative

hydrophobicity of the surface of microablated lines.

These structures have micron-size dimensions laterally but tens of nanometers

in depth. The latter dimensions make the structures comparable with medium to large

proteins (Figure 7).

126

Figure 7. AFM topographical (top left) and lateral force (top right) image of a channel

fabricated via the ablation of a 30 nm Au layer on top of PMMA. The middle region

(I) is the most hydrophobic, whereas comparative adsorption of the selected proteins

in the channels of thin gold layer deposited on a poly(methyl methacrylate) film was

visualised using fluorescence and atomic force microscopy and further quantified

using a Fluor reader. Poly(methyl methacrylate) is a rather commonly used polymer

due to its characteristics, e.g. transparency (>90 % transmission), stiffness with

excellent UV stability, low water absorption and high abrasion resistance. The

hydrophobicity of PMMA estimated by contact angle measurements was ranged from

68 θ to 72 θ indicating its moderate hydrophobic nature.

127

The AFM analysis of the microfabricated structures (Figure 7) showed the

presence of a lateral variation of hydrophobicity with the edges of the channels being

the most hydrophilic; and the centre being the most hydrophobic. This variation of

surface chemistry can be attributed to the lateral distribution of the ablation energy,

which translates in different energies delivered to the polymer, and subsequently

different surface chemistries. The expected reactions, which translate to three spatial

regions, would be, in order of increasing pyrolysis temperature, i.e. from the edges

towards the centre, (i) the termination of the side ester groups at one of the C-O bonds,

resulting in a more hydrophilic material; (ii) depolymerization of the main chain,

preserving the same hydrophobicity; and if the pyrolysis process is quick enough (iii)

the breaking of the side bonds, resulting in a more hydrophobic material. Although

there are variations of the dimensions and distribution of hydrophobicity vs. laser

power, the general structure of the microablated channels remains the same. Though

the process can use different polymers and metals, we found that PMMA (and gold)

are so far optimum choices. In particular PMMA can offer large possibilities to

‗combinatorialise‘ the chemistry of the surface upon thermolysis. Figure 8 depicts

possible chemical pathways that would explain the AFM-measured variation in

hydrophobicity.

128

Figure 8. Possible pyrolysis pathways of PMMA localized in micro-regions

leading to the observed lateral distribution of hydrophobicities.

The spatial distribution of the surface chemistries/hydrophobicities reflects in

the topography of the micro-fabricated structures, with an elevated (hydrophilic)

region at the edges; a flat (medium hydrophobic) region between the edges and the

centre; and a central region with a bump (hydrophobic). The micro/nano-topography

of the microchannels, as well as the AFM lateral force mapping validated the

mechanism proposed above. Finally, the rugosity of the surface is also distributed

unevenly, with the region outside the channels and the plateaus (region II in Figure 7)

being flatter than region I in Figure 7.

The strategy behind this method was to allow different proteins, or different

parts of the same large protein, to find the most appropriate in terms of adsorption and

129

preservation of bioactivity surface. Because the different micro/nano-surfaces are co-

located in a small area (channel width around 10 μm or less) the florescence signal

from the proteins would be perceived at the mm-range as being located in the same

areas. Also it has been observed that the concentration of the proteins was apparently

higher in the microstructures than on flat areas with similar material due to a higher

specific surface and larger opportunities for attachment.

The combinatorial character of the surface would in principle also allow the

probing of several patches on the molecular surface of the proteins, or modulate their

bioactivity. Figure 9 presents possible arrangements of IgG-like biomolecules on the

combinatorial surfaces.

Figure 9. General concept of the probing of molecular

surface of proteins.

The proposed technology for the fabrication of microarrays has the following

potential advantages: (i) the combinatorial surfaces would improve the uniformity of

biomolecule surface concentration, in particular for protein microarrays; (ii) the

surface concentration of biomolecules (several very different proteins being tested)

increases, and therefore the sensitivity increases accordingly, by 3-12 times,

depending on the molecular properties of the biomolecules; (iii) it is possible -in

principle- to probe different sides of the biomolecular surface and therefore the

130

bioactivity; and (iv) because the proposed method writes protein lines (that can encode

information in a bar code manner) instead of dots, it can be used for the fabrication of

informationally-addressable, as opposed to spatially addressable microarrays.

5.2.3. Impact of molecular descriptors on protein adsorption on microstructures

The molecular properties of proteins and their interactions with surfaces have

an effect on protein adsorption, which is one of the first and most important events

that occurs when a biological fluid contacts a surface. Interactions based on

hydrophobicity (Tilton et al., 1991) and electrostatics (Lubarsky et al., 2005) have

been found to be driving forces for protein adsorption. As the microstructures

fabricated as described above comprise micro/nano-areas with very different

chemistries, it is expected that both hydrophobicity and electrostatics would contribute

to the adsorption on these ‗combinatorialized‘ micro/nano surface. Indeed an AFM

analysis of the topography of the channels after the deposition of proteins showed that

the initial topography of the channels (Figure 7) is partially smoothed, with IgG

having a more pronounced effect.

Essentially the adsorption of proteins is governed by (i) kinetic processes (i.e.

diffusion of molecules to, and sometimes from the surface); and (ii) thermodynamic

processes (i.e. electrostatic and hydrophobic interactions between the protein and the

surface). These two types of processes are of course interconnected. In particular the

electrostatic interactions which are long range interactions will influence in a larger

degree the protein transport to the adsorbing surface. Also the electrostatic and

hydrophobic interactions will be connected at the protein structure level (e.g. a protein

which presents a hydrophobic molecular surface will, statistically speaking, have a

less charged surface). However, when performing a sensitivity analysis of the ‗tug-of-

war‘ between these three adsorption-relevant parameters, one should try to find the

descriptors that are independent of each other. Three molecular descriptors have been

found to both impact on protein adsorption (on flat and microstructured surfaces) and

be largely independent of each other, namely: (i) total molecular surface (which

modulates the transport of the protein on the surface and also has an effect on the

131

packing of the protein layer on the surface); (ii) ratio between the

hydrophilic/hydrophobic specific density (the specific density is the total property –

e.g. hydrophilicity index- per respective area –e.g. hydrophilic area-); and (iii) ration

between positive and negative areas. The last two descriptors will account for the

thermodynamic factors, i.e. hydrophobic and electrostatic interactions, respectively. It

is worth noting that the hydrophobicity-related descriptors are much more statistically

independent of the size of the molecule, i.e. molecular surface (i.e. R2 = 0.022 is a

maximum), whereas the charge-related descriptors are less independent (i.e. R2 =

0.069 is a minimum).

The transport of proteins to the surface is, to a large extent, diffusion-

dependent as the convection will have a limited role at this scale. In the first

approximation, the diffusion coefficient will be dependent -according to Stokes-

Einstein theory- on the radius of a sphere, r, as D ~ r-1, or dependent on the surface,

S (S ~ r2), D ~ S-0.5. Of course, these simple relationships will be altered by the shape

of the object (apparent increase of the power applied to the size of the object and

subsequently the surface) and environment in which the object diffuses (a confined

environment will reflect in a decrease of power applied to the size of the object and

subsequently the surface, down to -3/2 from -1/2, if an analogy with the Knudsen

diffusion is applied).

A sensitivity analysis of the impact of the total molecular surface and ratio of

the hydrophilic/hydrophobic specific density (Figure 10) revealed a few interesting

relationships.

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Figure 10. Modulation of the amplification of protein adsorption in micro/nano-

channels vs. the molecular surfaces of the respective protein.

First, the protein adsorption is amplified on microstructured rather than on flat

surfaces (the larger coefficient in the fitted function, with a separate analysis to

follow). Second, the adsorption on microstructured surfaces depends on the molecular

surface with a power ~0.33, while the adsorption on flat surfaces is governed by an

almost linear relationship (power ~1.2) versus molecular surface. Indeed on flat

surfaces, for geometric reasons, a linearity of the adsorbed mass with the molecular

surface would be expected, while in confined environments smaller molecules could

capitalize better on newly created areas. Third, the specificity of adsorption on

microstructured surfaces versus the hydrophobicity-related descriptor decreases

dramatically (decrease two times of the respective power), possibly due to the

133

‗combinatorialization‘ of the adsorbing surface in the channels (mechanism proposed

in Figure 7). Fourth, as expected, a higher relative hydrophobicity (i.e. smaller ratio

hydrophilic/hydrophobic specific density) induces a higher adsorption on both types of

surfaces. Fifth, the quality of the fit decreases for microstructured surfaces,

presumably because of the same ‗combinatorialization‘ that adds statistical noise to

the data.

A similar sensitivity analysis of the impact of the total molecular surface and

ratio of the positive/negative areas revealed similar relationships. First, it is confirmed

that the adsorption is much higher on microstructured than on flat surfaces. Second, it

is confirmed that the adsorption is modulated by molecular surface with a power ~1

and ~0.33, for flat and microstructures, respectively. Third, the adsorption on

microstructured surfaces depends less on the charges-related descriptor (decrease of

the power from 0.6 to 0.25, for flat and microstructures, respectively), again possibly

due to the ‗combinatorialization‘ of the adsorbing surface in the channels. Forth,

interestingly, a higher positive charge (area) induces a higher adsorption, possibly due

to the presence of carboxylic groups, created either parasitically or thermo-induced on

flat surfaces and at the edges of the microstructures, respectively. Fifth, the same

degradation of the quality of the fit is observed for the adsorption on flat and

microstructured surfaces.

It appeared that the small proteins can use the combinatorial surface better to

amplify their adsorption, whereas larger proteins are less sensitive to the opportunities

offered by various surfaces at least partially because they exhibit ‗combinatorial‘

molecular surfaces too. Finally, and for the concentrations used in this study, the

adsorption on flat surfaces is proportional with d2-2.5 (where d is the average diameter

of the protein) whereas on microstructured surfaces the adsorption is less sensitive to

the diameter of the protein (proportional with d0.66-0.75).

The role of rugosity on flat and micro-structured surfaces on protein adsorption

can also be quantified. The shallow character of the channels (less than 50nm versus

5-10μm) does not suggest that the additional area created via ablation is large enough

to explain alone the amplification of adsorption. These being said, it is clear that

smaller protein can capitalize better on the newly created surfaces in the mid-valleys

134

in the channels (Figure 7) as well as on the rigidity of the surface than the larger

proteins. Although the statistical relevance decreases for the analysis that considers

only the molecular surface, this parameter is important enough to warrant a separate

analysis. The molecular surface has been calculated using a probing sphere with 10Å

radius, which offers the best statistical fit of the data, as well as being the most

appropriate for the analysis of the adsorption on surfaces. The impact of the total

molecular surface on the amplification of adsorption is presented in Figure 10.

Whatever the circumstances, the amplification of adsorption (i.e., the ratio between the

level of adsorption on micro/nano-structures and on flat surfaces) is important varying

between 3- and 10-fold. The efficiency of the amplification of adsorption decreases

with the molecular surface reaching a plateau at around 3-fold.

5.2.4. Characterization of thickness of polymeric films and attached proteins

Dependence of refractive index for PMMA on its thickness within nanoscale

range between 67 and 259 nm displayed in Figure 11.

Figure 11. Correlation between nanothickness and refractive index of PMMA on

glass surface treated with HMDS.

135

Strong correlation between refractive index and nanothickness for PMMA

polymer films is observed. This relationship was approximated with function of

NPMMA=at-b, where N – refractive index of PMMA, t – nanothickness of PMMA

polymer film, a=188.49 and b=0.88 are constant coefficients for given PMMA

polymer films. This correlation makes sense only within the narrow nanoscale range.

Beyond the nanoscale range, when tPMMA → ∞, refractive index (NPMMA) should come

nearer to 1.4846 that is value for PMMA as the bulk polymer (Wunderlich, 2000).

This nanoscale limit for PMMA is reached starting approximately from ~250 nm. For

thicker PMMA films, the refractive index should be constant (1.4846), and beyond the

thickness of ~250 nm, the PMMA-films should be considered as the bulk PMMA-

polymer. Further decreasing of thickness of the PMMA-nanofilms should lead to the

refractive index of PMMA-monolayer. The refractive index of PMMA-films within

nanoscale thicknesses increases in the factor of 3-4 in comparison with the refractive

index for PMMA as the bulk polymer.

Similar relationships for other polymer nanofilms on glass-surfaces treated

with HMDS were obtained, namely for nitrocellulose (a=264.52, b=0.96), PtBMA

(a=241.91, b=0.93), and PSMA (a=206.51, b=0.9). Values of the constants are defined

with type of materials, which are used for nanofilms manufacturing, and can slightly

vary, approaching to the true value, if number of statistical data increase. Such

behaviour of refractive indexes within nanoscale thicknesses of protein films was

observed as well. For example, correlation between refractive index and

nanothicknesses for HSA is shown in Figure 12.

One can see that the refractive index of HSA films within the nanoscale

thicknesses is far from that for HSA as the bulk protein, i.e. Nbulk=1.465 (Benesch,

2001). Approximation of the relationship by function of NHSA=at-b gives the following

constants a=200.1 and b=0.90. Similar relationships were obtained for myoglobin

(b=1.04), α-chymotrypsin (b=0.99), and human IgG (b=1.1).

136

Figure 12. Correlation between nanothickness and refractive index of HSA in double

nanolayered sandwich of HSA/PMMA on glass-surface treated with HMDS.

The thickness of the protein layers on native PMMA polymeric surfaces according to

ellipsometric measurements significantly varied from ~61 nm for lysozyme and

chicken IgG to ~122 nm for human IgG as shown in Table 5. The relationship

between refractive index and nanothickness for both protein-nanolayers and polymer-

naolayers demonstrates a rivers correlation, namely the thiner nanolayer the higher

refractive index.

137

Table 5. Ellipsometric measurements of thicknesses of adsorbed proteins and

correspondent PMMA polymeric films.

Proteins Protein

thickness (nm)

Refractive

index (Nproteins)

PMMA

thickness (nm)

Refractive

index (NPMMA)

Myoglobin 70.6 ± 2.5 4.519 ± 0.167 170 ± 2.1 2.125 ± 0.020

Human IgG 121.6 ± 0.3 4.841 ± 0.012 78.4 ± 9.0 4.857 ± 0.603

Chymotrypsin 98.9 ± 15.6 4.703 ± 0.904 147.6 ± 3.6 2.362 ± 0.049

HSA 83.3 ± 6.9 4.158 ± 0.336 136.8 ± 2.5 2.518 ± 0.039

Lysozyme 61.2 ± 5.9 5.497 ± 0.504 170.6 ± 1.1 2.119 ± 0.011

5.2.5. Protein adsorption in PMMA-based channels and on native PMMA films

Comparative adsorption of the selected proteins in the channels of thin gold

layer deposited on a poly(methyl methacrylate) film was visualised using fluorescence

microscopy and further quantified using a Fluor reader. Fluorescent images of α-

chymotrypsin, human serum albumin, human and chicken IgG, lysozyme, and

myoglobin adsorbed in microchannels that were fabricated in a bar-code format are

presented in Figure 13.

In addition, in order to estimate the effectiveness of protein attachment in

microfabricated structures we also studied the protein adsorption on native

poly(methyl methacrylate) films with comparative quantification of fluorescently

labelled proteins. The results obtained (Table 5, Figures 13-14) using three different

methodologies are in a good correlation and indicated that in general the protein

adsorption was dependent on protein and on initial concentration of protein solution.

The protein adsorption in microfabricated channels was more effective comparing to

that on native PMMA polymeric surface. Specifically, the adsorption of HSA, HIgG

and myoglobin, in the microfabricated channels was as much as 2.5 times more than

that on native PMMA surface (Figure 14) and the adsorption of lysozyme was

comparable in both cases.

138

Figure 13. Protein adsorption in microstructured PMMA surface. The first row

presents bright field images. The fluorescent images relate to different protein

concentration in solution as follows: 0.014 mg/ml (second from the top); 0.07 mg/ml

(third from the top); 0.14 mg/ml (bottom).

139

Figure 14. Protein adsorption in the channels of thin gold layer deposited on a

poly(methyl methacrylate) film and on poly(methyl methacrylate) films.

5.2.6. Characterization of adsorption properties of selected proteins

Five proteins from the most common three classes, namely, myoglobin (the

class of only-α-helices), IgGs, HSA, α-chymotrypsin (the class of almost exclusively

β-sheets) and lysozyme (the class of α-helix and β-sheet tend to be segregated along

the chain) were selected to compare their attachment behavior. Taking into account the

isoelectric point of myoglobin (7.8) we assume that in our experiments it was mostly

neutral. Our results also indicated that the adsorption yields of myoglobin on native

PMMA polymeric surfaces were rather poor. However, its adsorption in

microfabricated channels was greater. In an attempt to explain this phenomenon we

compared its surface characteristics with those of other proteins studied (Table 6).

The data in Table 6 shows that the solvent-accessible surface area of

myoglobin is very small (7832.6 Å2) relative to its molecular weight.

140

Table 6. Characteristics of selected proteins.

Human IgG α-Chymo

trypsin)

Human

serum

albumin

Myoglobin Lyso

zyme

Mol. weight, D 146000 24000 66500 66000 14000

Size (XxYxZ), Å 74x115x101 60x48x72 129x108x128 47x40x42 42x36x47

Isoelec. point, pI 7.36 4.6 4.7 7.8 10.7

Connolly surface

area, Å2

33677.9 23186 55270 7832.6 5793.5

Area with

positive charge,

Å2

7278.3 4016.9 12474.9 1659.9 1655.5

Total positive

charge

40.6 24.3 57.3 16.9 8.6

Area with

negative charge,

Å2

26398.8 19169.2 42792.8 6172.8 4137.9

Total negative

charge

-255.4 -182.7 -389.2 -34.8 -25.2

Average surface

charge

-6.4 -6.8 -6.0 -2.3 -2.9

Hydrophilic area,

Å2

19670.5 12114.9 30699.6 4662.7 3848.0

Hydrophilicity

index

4,2 4.3 7.3 6.8 5.1

Hydrophobic

area, Å2

14004.6 11070.8 24568 3169.9 1945.4

Hydrophobicity

index

-4.7 -4.7 -4.5 -4.3 -5.1

Total hydrophobicity

-66 -52.4 -111.3 -13.7 -9.9

Total hydrophilicity

82.3 52.1 225.3 31.8 19.6

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Human serum albumin, for example, has molecular weight about 3.5 times greater

than myoglobin but has a solvent-accessible area of 55270 Å2, or around 7 times more.

A comparison of the dimensions of both molecules shows that myoglobin is far more

compact than HSA, with a very smooth molecular surface, free of any atomic-size

clefts. α-Chymotrypsin has a molecular weight of only 24000 D, but a surface area

about 3 times larger than the area of myoglobin.

The proteins from the second structural class were represented by three

proteins, IgGs, HSA, α-chymotrypsin. The IgGs structure is highly asymmetric despite

having two identical heavy and two identical light chains and can be considered a

―snapshot‖ of the broad range of conformations available in solution. The overall

shape is between a Y and a T, with a 143° angle between the major axes of the two

Fabs. The IgG spans 171 Å from the apex of one antigen-binding site to the other.

According to our calculations presented in Table 6, the size of the protein was

estimated as 74 x 115 x 101 Å. It should be noted that IgGs had the high adsorption

yield on both native PMMA polymeric surfaces and in microfabricated channels.

Interestingly that IgGs as well as myoglobin presumably were neutral, yet the

attachment behavior was completely different. This fact might be one more evidence

that proteins adsorption is controlled by a combination of factors. Another protein

from the second structural class was human serum albumin, the most abundant protein

in the blood. Because HSA acts as a fatty acid transporter, it has six binding sites for

fatty acids. Petrash et al. (1997) have shown that specific binding of HSA is one of the

main factors in binding tenacity. α-Chymotripsin contains intricate folding of the

tertiary structure. This folding results in a hydrophobic core and an outer hydrophilic

surface, thus allowing the protein to interact with other proteins in the cytoplasm.

Chymotrypsin consists of three chains. Since the beta barrel is antiparallel, the interior

is expected to be hydrophobic and exterior hydrophilic which favourably interact with

water molecules (Tsukada and Blow, 1985). The adsorption capacity of the latter two

proteins was quite similar (Figures 13-14) on plain surfaces although with greater

yields in microfabricated channels especially for HSA. The isoeletric point of

chymotrypsin and HSA is about 4.7, therefore we assumed that in our experiments

these proteins were negatively charged.

142

Lysozyme belongs to the third structural class of the proteins. It has an

alpha+beta fold, consisting of five to seven alpha helices and a three-stranded

antiparallel beta sheet. The enzyme is approximately ellipsoidal in shape, with a large

cleft in one side forming the active site (Tsukada and Blow, 1985). Since isoelectric

point of lysozyme is 10.7, in our experiments the molecules were slightly positively

charged. However, this protein was poorly attached to plain hydrophobic PMMA

surfaces and in the channels in comparison to other proteins studied. In summary,

there seems to be no single factor but the combination of a few/several that might

control the adsorption of proteins on PMMA polymeric surfaces.

5.3. Conclusion

We propose a method for the fabrication of random ‗combinatorial‘

micro/nano-sized surfaces of 100 nm-range structures that allows a higher adsorption

of proteins and possibly the immobilization of biomolecules on different sides of the

molecular structure. The method, which is based on laser microablation of thin

metal/blocking protein layers deposited on a polymer substrate, has proven to amplify

the protein adsorption between 3 to 10 times depending on the molecular surface of

the protein. It appears that smaller proteins can capitalize better on the newly created

micro-level structure and nano-level rugosity. The fabrication of the microstructures,

achieved by ablating a thin metallic layer deposited on a non-ablatable polymeric

layer, induces the creation of ‗combinatorial‘ surfaces, with different surface

chemistries. This surface ‗combinatorialization‘ makes the adsorption of proteins less

dependent on the local molecular descriptors, i.e. hydrophobicity and charges.

Consequently, molecularly different proteins will adsorb at increased levels with better

chances for the preservation of bioactivity. The amplified and ‗combinatorialized‘

adsorption on micro/nano-structures has the potential of improving the detection of

biomolecular recognition if used for muliplex analysis.

Protein-binding assay with fluorescent detection and quantification enabled

rapidly analyse the adsorption properties of different proteins belonged to three major

structural classes. The physico-chemical adsorption of human serum albumin, human

143

immunoglobulin, α-chymotrypsin, lysozyme, and myoglobin in the microchannels

fabricated via a localized laser ablation of a protein-blocked thin gold layer (50 nm)

deposited on a poly(methyl methacrylate) films and native polymeric surfaces was

2.5-5 times greater than that on the plain PMMA polymeric surfaces. A surface mass

density of adsorbed protein molecules on the latter defined with a protein-film

thickness and a refractive index for the protein layer correlated with data obtained for

fluorescently labeled proteins. So microchannel has an advantage over the flat surface.

144

CHAPTER 6

CONTROL OF SELF-ASSEMBLY OF ACTIN

FILAMENTS FOR DYNAMIC MICRODEVICES

145

6.1. Overview

This chapter presents a simple technique for actin-filament-bundle fabrication

providing a convenient experimental system that is applicable for the development of

a new device technology based on biomolecules. Reproduced from reference

(Alexeeva et al., 2005) © 2005 With kind permission from Springer + Business

Media. The chapter starts with a characterization of PtBuMA, PMMA and PSMA

polymeric surfaces. In addition to adsorption and covalent binding of proteins on

polymeric surfaces, as described in chapters 4 and 5, protein assembly was studied.

Subsection 6.2.2. describes adsorption and self-assembly of G-actin on selected

polymeric surfaces, and is followed by evaluation of covalent bonding of G-actin on

the surfaces. Although the use of covalent binding resulted in more stable bonding and

greater density of rhodamine phalloidin–labeled F-actin on all surfaces,

polymerization under flow field did not cause actin alignment (see subsection 6.2.2.3.

for details). As microchannel can affect protein-surface interactions (see chapter 5 for

details), strips surrounded by microchannels for actin immobilization and alignment

on the most suitable surface, namely PSMA (see subsection 6.2.3. for details), were

constructed. The chapter continues with a description of the fabrication of

electrostatically self-assembled actin filament bundles for the formation of tracks

capable of supporting continuous bead movement, as described in subsection 6.2.4.

The chapter ends with a conclusion that PSMA surface provided sufficient amount of

binding sites for the covalent immobilization of actin. In addition, electrostatically

condensed actin filament bundles can be assembled and aligned in a continuous-flow

system. To provide molecular motor proteins with ATP energy, a search for bacterial

ATP producers among 86 environmental (marine) bacteria belonging to 17 genera was

performed, as described in chapter 8; some of the potential ATP producers are

described in chapter 7.

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6.2.1. Polymeric surface characterization

PSMA is transparent in the visible region and non-fluorescent with appropriate

thermomechanical characteristics, mildly hydrophilic with contact angles of 50°.

There is a possibility that some of the carboxylic groups on the polymeric surface

might have undergone reorientation toward the bulk of the polymer; however, this did

not decrease polymer surface functionality (Ivanova et al., 2002c).

The hydrophobic nature of PMMA is conferred by the methyl groups and the

bonding arrangement around the oxygen (see subsection 5.2.1. for more details). The

irradiation induces the evolution of the original polymer to complex phases of

amorphous hydrogenated carbon (a-C:H). Following UV irradiation, the PMMA

surface became less hydrophobic with a contact angle of 62°.

The surface characteristics of P(tBuMA) are rather similar to those of PMMA,

as these polymers only differ by the number of methyl groups. The non-irradiated

surface was hydrophobic due to tert-butyl-termination (see chapter 4 for more details).

Following UV irradiation, the polymer became hydrophilic due to COOH-termination.

In the presence of H2O P(tBuMA) will release (CH3)3COH. Loss of chemical species,

along with possible bulk densification and surface reconstruction, could presumably

account for the shrinkage of the polymer surface (Watson et al., 2002)

6.2.2. Effectiveness and stability of G-actin self-assembly

6.2.2.1. Adsorption and self-assembly of G-actin on selected polymeric surfaces

In the context of the surface characteristics (discussed above) we selected three

polymeric surfaces PMMA, P(tBuMA), and PSMA, as appropriate substrates to

investigate both adsorption and covalent bonding of G-actin followed by self-

assembly of actin filaments. The results revealed that physicochemical adsorption of

actin filaments in the continuous flow of the buffer for 0.5 h with a flow rate of 0.06

mL min–1 was most effective on PSMA (as monitored visually). The adsorption on

147

PSMA was comparable to that on nitrocellulose, which was used as the positive

control. Actin filament attachment on PMMA and P(tBuMA) was less effective

(Figure 15A–D). UV-irradiated surfaces appeared to have strong inherent fluorescence

as observed in the microscope as bright regions as seen in Figure 15 (C–D), which

probably was a result of an interaction of the buffer with irradiated surfaces. Dried

irradiated surfaces had no or low fluorescence background. In addition, actin filaments

might have been partly disintegrated and therefore washed in greater degree due to

either (CH3)3COH release in water, oxygen release under exposure to light, or both

from the surfaces of PMMA and P(tBuMA). The similar negative effect of PMMA on

mictotubules was reported recently (Brunner et al., 2004).

Figure 15. Adsorption and polymerization of F-actin (23 mM) after 1.5 h in the

continuous flow with the flow rate of 0.06 mL min–1 on polymeric surfaces: (A) NC,

(B) PSMA, (C) PMMA (exposed), (D) P(tBuMA) (exposed). Scale bar, 10 μm.

148

Our efforts to mechanically induce the alignment of the adsorbed F-actin filaments

using the flow field were not satisfactory. When 23 nM of F-actin was introduced into

the flow cell, the initial alignment of actin filaments (up to 50 %) could be observed

(data not shown), but over 1.5 h under the buffer flow, the alignment was not

improved and most of the filaments were washed. At increased concentration, e.g.,

23 mM of F-actin, a bulk of adsorbed filaments was observed. The adsorbing

filaments were gradually and unevenly (depending on the polymeric surface) washing

away over 1.5 h under the buffer flow. The desorption and washing of actin filaments

occurred on P(tBuMA) and PMMA (shown in Figure 15) to a greater extent. Overall,

23 ± 3 %, 19 ± 5 %, 18 ± 3 % and 21 ± 3 % (n = 24) of actin filaments appeared

aligned on nitrocellulose, PSMA, P(tBuMA), and PMMA, respectively. To determine

the influence of fluid stress on the actin filaments in the flow cell, the shear stress at

the wall in a flat-walled (actin-free) chamber (cell), υ, was found as a product of

measured liquid viscosity (Figure 16), ω and shear rate, θ:

υ = ω · θ

Shear rate at the wall of the rectangular chamber is a function of the chamber

width w = 22 mm and height h = 0.1 mm and applied flow rate Q = 0.06 mL min–1

(Han, 1998, Decave et al., 2003).

θ = 6Q/wh2 = 0.6 Pa

The attempts to force the flow-induced orientation of actin filaments by

increasing the hydrodynamic forces in the flow cell have demonstrated that a shear

rate greater than 0.6 Pa would lead to the breakage of filament, fast detachment, and

washing from the surface. Notably, under similar experimental conditions, Fritzsche et

al. (1998), Stracke et al. (2000), and Böhm et al. (2001) were able to align

microtubules using mechanically induced flow fields. Indeed, microtubules can be

more robust in such experiments due to their inherent rigidity and are able to stand

rather strong hydrodynamic force. In contrast to actin, microtubules were able to

149

maintain its motility under 0.5–5 μm s–1 flow velocities applied in the cell (Stracke et

al., 2000).

Figure 16. Estimation of the working buffer viscosity with and without BaSO4 (108

mM).

6.2.2.2. Evaluation of covalent bonding of G-actin on selected polymeric surfaces

To avoid the limitations of unstable physicochemical adsorption, we employed

the cross-linking of G- and/or F-actin filaments with a water-soluble carbodiimide,

EDC, by increasing the strength of the attachment. EDC is commonly used for the

covalent attachment of proteins on surfaces as it catalyzes the formation of amide

bonds between carboxylic groups of the polymeric surface and amine groups of

proteins. The cross-linking reaction is favored by the presence of intermediate, N-

150

hydroxysulfo-succinimide (Grabarek and Gergely, 1990). In contrast to conventional

agents, EDC does not remain as a part of that linkage but simply changes to water-

soluble urea derivatives that have very low cytotoxicity (Tomihata and Ikada, 1997,

Taguchi et al., 2002). The cross-linking reaction was confirmed by XPS analysis.

Initially, XPS analysis of the surface of PSMA and P(tBuMA) + COOH showed peaks

in the C 1s region at 285.0, 286.8 and 288.8 eV, which can be assigned to C–C/C–H,

C–O and O–C = O species, respectively (Beamson and Briggs, 1992, Chastain, 1992).

6.2.2.3. Covalent bonding and self-assembly of G-actin on selected polymeric

surfaces

The actin filament arrays were produced by polymerizing actin filaments

covalently bound to the surface G-actin seeds under the constant flow. The use of

covalent binding resulted in more stable bonding and greater density of rhodamine-

phalloidin-labeled F-actin on all surfaces (Figure 17), which was further polymerized

and aligned under the flow field (Figure 17).

Figure 17. Covalent bonding and polymerization of F-actin (23 mM) after 1.5 h in the

continuous flow with the flow rate of 0.06 mL min-1 on polymeric surfaces: (A)

PSMA, (B) PMMA (exposed), (C) P(tBuMA) (exposed). Scale bar, 10 μm.

151

It is noted that under the same experimental conditions over 1.5 h the amount

of G-actin filaments was roughly three to four times greater on all polymeric surfaces

tested; however, the degree of aligned F-actin filaments remained similar to that of

adsorbed filaments, ranging from 21 ± 3 % to 17 ± 4 %. The degraded F-actin

filaments were particularly noticeable on P(tBuMA), indicating again the poor

biocompartibility of this type of polymeric surface. This observation is in agreement

with the Bernheim-Groswasser et al. (2002) data, which showed that microtubules

degraded rapidly in the presence of poly(dimethylsiloxane) (PDMS) and PMMA. We

therefore excluded P(tBuMA) and PMMA from further experiments. The flow rate of

0.06 mL min–1 appeared to be optimal because the G-actin remained intact on the

surface once it was covalently bound and was available for further polymerization.

6.2.3. Alignment of self-assembled actin filaments along fabricated

microstructures

To concentrate F-actin on specific areas, we fabricated strips 10 μm wide and

about 0.6 μm deep, where actin filaments were attached on the top of such strips (i.e.,

on the PSMA polymeric surface) rather than on the bottom (i.e., glass surface). The

alignment of actin filaments was improved up to 80–90 % of the filaments. However,

we could not achieve absolute selectivity of attachment as some filaments were stuck

on the glass. According to our results (data not shown), the smaller than 5–10-μm-

wide stripes were less effective, perhaps because when microstructures are smaller,

filaments attach across the channels, break under the flow, and wash away. Dark field

image of fabricated microstructures and fluorescence microscope images of

rhodamine-phalloidin–labeled actin filaments along PSMA stipes and HMM-beads

bound on actin filaments are shown in Figure 18.

152

Figure 18. Binding of self-assembled F-actin (23 nM) on functionalized PSMA

polymeric surfaces. Dark field observation shows channels, 10 μm, fabricated on

PSMA polymeric surfaces (A). Fluorescent observation shows self-assembled

rhodamine-phalloidin-labeled F-actin filaments on the same field: (B) F-actin

filaments aligned on the PSMA polymeric surfaces after 1.5 h in the continuous flow

with the flow rate of 0.06 mL min-1; (C) the same as on (B) with antiHMM–HMM

beads binding on F-actin. Scale bar, 10 μm.

6.2.4. Fabrication of electrostatically self-assembled actin filaments bundles

Even though we have demonstrated the feasibility of actin filaments self-

assembly and alignment in the continous-flow system, the density of the filaments was

not sufficient to form filament tracks capable of supporting continous bead movement.

Besides, the directionality of motility is difficult to control as the motility remains

random in such a system. In this regard we have designed the next experiment to

achieve the fabrication of the tracks of actin bundles assembled from either F-actin or

2-μm-actin filaments with their barbed ends blocked by gelosin (Figures 19 A–F).

We used gelsolin as an actin-modulating protein that binds to the plus (or

barbed) end of actin monomers or filaments, preventing monomer exchange (end-

blocking or capping). These F-actin fragments blocked by gelsolin (shown on

Figure 19A) were used as ―seeds‖ for the self-assembly of actin filament bundles.

153

Gelsolin significantly affects the structure of actin polymerization and the rigidity of

the filaments (Prochniewicz et al., 1996). The structural effect of gelsolin could be due

to growth of the filaments on structurally altered nuclei: two monomers directly bound

to gelsolin have been shown to be oriented in a different way than the monomers in

spontaneously nucleated F-actin (Doi, 1992, Hesterkamp et al., 1993). A

homogeneous increase of density in the bridge between two strands of the actin–

gelsolin helix has also been shown (Prochniewicz et al., 1996).

154

Figure 19. Fluorescence images of (A) 2-μm actin filaments (23 nM) with their

barbed ends blocked by gelsolin; and (B) their bundles condensed with Ba2+ (108 mM)

during 45 min. Fluorescence images of electrostatically condensed and aligned actin-

filament bundles assembled from 2-μm actin filaments (23 nM) (C) after 1.5 h; (D)

after 3 h; and intact F-actin filaments: after 1.5 h (E) and 3 h (F) in the continuous

flow system with the flow rate of 0.06 mL min-1. Scale bar, 10μm.

155

In order to assemble actin filament bundles, we adopted the methodology of

like-charge attraction between polyelectrolytes induced by counterion charge density

waves (Angelini et al., 2003, Tang et al., 1996, Butler et al., 2003). Angelini et al.

(Angelini et al., 2003) have demonstrated that at high Ba2+ concentrations, F-actin

condenses into closely packed bundles consisting of parallel arrays of three actin-

filament units. This molecular mechanism is analogous to the formation of polarons in

ionic solids (Wong et al., 1997a). In the case of F-actin, fluctuating counterions drag

along soft helical distortions of the polyelectrolyte, and consequently freeze into static

correlation, providing a transition between the extreme viewpoints of dynamic and

static counterion correlations (Shklovskii, 1999). Electron microscopic images of

electrostatically condensed F-actin/gelsolin bundles show the tightly packed parallel

organization of F-actin filaments in the bundle (Figure 20). However, using SEM we

were unable to visualise a helical structure and/or axial alignment of individual F-actin

filaments (Tang et al., 1996).

156

Figure 20. SEM images of F-actin/gelsolin bundles formed from

electrostatically condensed F-actin filaments. Accelerations voltage:

15 kV; magnification: 10,000x (top) and 20,000x (bottom).

157

In this study we went one step further using F-actin/gelsolin filaments to show

that it can not only be successfully electrostatically condensed, but also form actin-

filament bundles that were covalently bound on the surface. We were able to

progressively form larger bundles aligned in the flow field that could be used in

motility assays to support bead translocation. Intact F-actin filaments (Figure 19F)

could be assembled into larger bundles compared to that assembled from 2-μm

actin/gelsolin bundles (Figure 19D) over 3 h of condensation with Ba2+. We have also

observed that over time, bundles of intact F-actin filaments become less regularly

organized and form ―tree-like‖ tracks (Figure 19F), which, in fact, resulted in

irregular-bead movement. The 2-μm actin/gelsolin fragment condensation after 45 min

of incubation with 108 mM Ba2+ resulted in assembly of 15–20-μm-long bundles,

which grew and aligned under the flow field over 3 h to produce elongated tracks.

Morphologically, F-actin/gelsolin filaments after 3 h of electrostatic condensation

appeared in more compact and organized bundles in contrast to F-actin filament

bundles. This fact correlates with the earlier findings that binding of gelsolin to actin

induces structural changes in the directly bound protomers, and these changes are

subsequently propagated along the whole actin filament during its polymerization

(Doi, 1992). Langford et al. (1994) reported that actin filaments assembled on the

barbed end of acrosomal process of squid Limulus polyphmus maintained

directionality toward the tip of actin filaments. Therefore, it is implied that the

actin/gelsolin filaments in bundles also maintain a uniform polarity and therefore

preserve more organized structure while condensing and aligning under the constant

flow. However, the polar arrangement has not been discerned and unsubstantiated.

Condensed into bundles, actin filaments preserved their functionality to support

HMM-bead translocation. When the bead approached the bundle, it tended to

accelerate along the bundle. The experiments with bead movement (illustrated in

Figure 21) showed that the beads moved unidirectionally along the actin-filament

bundles. The estimated average velocity of a 1 μm bead was 13.8 ± 5.1 μm s–1, which

is faster than the average speed of rabbit actomyosin, 3–4.5 μm s–1 (Suzuki et al.,

1997, Bunk et al., 2005, Sakamaki et al., 2003), and comparable with the sliding

velocity of myosin-coated beads moving on actin filaments and/or bundles of different

158

origin (assembled on acrosomal process, on actin para-crystals, or algal actin bundles):

1.1–60 μm s–1.

Figure 21. Translocation of the antiHMM–HMM bead along the bundle formed from

2-μm-actin Alexa 488-phalloidin–labeled filaments (23 nM) and condensed with Ba2+

(108 mM). Scale bar, 10 μm.

These significant variations depended on samples and experimental setups (Chaen et

al., 1995, Langford et al., 1994, Oiwa et al., 1990, Yamasaki and Nakayama, 1996,

Suda and Ishikawa, 1997, Bernheim-Groswasser et al., 2002). The presence of Ba2+

ions did not affect the viscosity of the buffer (as shown in Figure 16) and hence could

not slow down the bead velocity.

6.3. Conclusion

In summary, we have shown that among poly(styrene-maleic acid),

poly(methyl methacrylate), and poly(t-butyl methacrylate) polymeric surfaces the

former appeared to be more suitable for experiments as it lacked inherent

fluorescence, had appropriate biocompatibility with actin–myosin in supramolecular

manipulations, and provided sufficient amount of binding sites for the covalent

immobilization of actin.

The progressive formation of F-actin/gelsolin bundles by electrostatic

condensation with Ba2+ and the alignment of such bundles can be easily performed

159

and controlled in the flow cell. Long-range cooperative transitions in actin induced by

gelsolin represent a structural perturbation of the barbed end and presumably result in

regularly organized bundles that supported directional bead movement. Our study also

demonstrated that this simple technique for actin-filament-bundle fabrication provides

a convenient experimental system that is applicable for the development of new device

technology based on biomolecules.

160

CHAPTER 7

CHARACTERIZATION OF POTENTIAL

ATP, MreB AND FtsA PRODUCERS

161

7.1. Overview

In order to evaluate possibilities of both substituting eukaryotic linear

molecular motor proteins with more robust bacterial homologues (see chapter 2.5 for

more details) and providing molecular motors in microdevices with ATP energy (see

chapter 6), a search for potential ATP and/or MreB/FtsA producers dwelling in marine

environment was performed. Potential producers were isolated from both associated

and free-living microbial communities. The former included bacteria, namely,

Pseudoalteromonas issachenkonii, a producer of haemolysins, ectohydrolytic

enzymes, biologically active compounds and unusual lipooligosaccharide (Ivanova et

al., 2002b, Ivanova et al., 2002a, Alexeeva et al., 2002, Ivanova et al., 2003a,

Kalinovskaya et al., 2004, Silipo et al., 2004, Alexeeva et al., 2004b), together with

Formosa algae (Ivanova et al., 2004b), Brevibacterium celere (Ivanova et al., 2004c),

Bacillus algicola (Ivanova et al., 2004a) and Planococcus maritimus isolated from the

brown algae Fucus evanescens, Sulfitobacter delicatus from the starfish Stellaster

equestris and Sulfitobacter dubius from the sea grass Zostera marina, while the latter

included Marinobacter excellens isolated from radionuclide-contaminated sediments

of Chazhma Bay, Sea of Japan.

The chapter starts with a characterization of phenotypic and chemotaxonomic

properties of potential marine ATP and/or MreB/FtsA producers, namely,

Sulfitobacter (Reproduced from reference (Ivanova et al., 2004e) © 2004 With kind

permission from IJSEM) and Marinobacter (Reproduced from reference (Gorshkova

et al., 2003)) © 2003 With kind permission from IJSEM) belonging to the gram-

negative group (see Table 10), and is followed by a characterization of the same

properties of a member of the gram-positive group (see Table 10), namely,

Planococcus (Reproduced from reference (Ivanova et al., 2006b) © 2006 With kind

permission from Microbiological journal). The chapter continues with a description of

genotypic and phylogenetic properties of these two groups.

The chapter ends with a conclusion that all members of the gram-negative

group were distinguished from described species by a number of phenotypic,

chemotaxonomic, genotypic and phylogenetic traits. The following names and

162

numbers were assigned: Sulfitobacter delicatus KMM 3584T (=LMG 20554T = ATCC

BAA-321T), Sulfitobacter dubius is KMM 3554T (=LMG 20555T = ATCC BAA-320T)

and Marinobacter excellens KMM 3809T (=CIP 107686T). Remarkably, one member

of the gram-negative group, namely, Marinobacter excellens KMM 3809T was found

to have ―porous‖ features (dark spots in Figure 30) that may contain ATPases

(discussed in subsection 2.5.5.3. and the following chapter). One member of the gram-

positive group, namely, strain KMM 3738 was found to belong to Planococcus

maritimus; it had unusual irregular coccoid shape of cells possessing a single

flagellum. In contrast to Marinobacter excellens (Figures 30), the surface of

Planococcus spp. was found to be smooth with only 3 nm of cell-surface roughness

(Figure 22).

All these members of different microbial communities were screened for ATP

and/or MreB/FtsA production (as described in the following chapters) to find the

potential ATP producer to supply linear molecular motors in microdevices with ATP

energy (see chapters 6 and 8) and/or substitute eukaryotic actin with more robust

MreB or FtsA proteins (see chapters 2.5 and 9 for details).


7.2.1. Phenotypic and chemotaxonomic classification

7.2.1.1. Gram-negative marine bacteria belonging to the genera Sulfitobacter and

Marinobacter

7.2.1.1.1. Phenotypic and chemotaxonomic properties of Sulfitobacter delicatus

Sulfitobacter delicatus (de.li.ca'tus. L. masc. adj. delicatus beautiful).

Rod-shaped cells, single, about 0·7–0·9 μm in diameter. Gram-negative. Non-

motile. Chemo-organotroph with respiratory metabolism. Colonies are uniformly

round, 1–3 μm in diameter, regular, convex, smooth and slightly yellowish after

163

incubation for 48–74 h on marine agar. No diffusible pigment is produced in the

medium. Does not form endospores. Accumulates poly-β-hydroxybutyrate as an

intracellular reserve product. Oxidase- and catalase-positive. Required Na+ or sea

water for growth (Table 7). Growth occurs in media containing 1–8 % NaCl.

Mesophilic. Grows at 12–37 °C and pH 6·0–10·0; optimum growth is observed at

25 °C and pH 7·5–8·0. No growth is detected at 40 °C. Decomposes gelatin and

alginate. Agar, starch, casein, laminarin, Tween 80 and DNA are not hydrolysed.

From the 95 carbon sources tested, according to Biolog, α-cyclodextrin, glycogen,

i-erythritol, psicose, D-raffinose, L-rhamnose, acetic acid, D-galactonic acid lactone,

D-galacturonic acid, D-glucuronic acid, α-ketovaleric acid, glucuronamide, L-leucine,

L-ornithine, D-serine, DL-carnitine, urocanic acid, thymidine, phenylethylamine,

putrescine, 2-aminoethanol, 2,3-butandiol, DL-α-glycerolphosphate and glucose

1-phosphate are not utilized and L-fucose, D-galactose, gentiobiose, α-lactose, D-

mannitol and hydroxyproline are only weakly utilized. Susceptible to ampicillin,

benzylpenicillin, gentamicin, kanamycin, carbenicillin, neomycin, oleandomycin and

streptomycin; not susceptible to polymyxin or tetracycline.

164

Table 7. Characteristics that differentiate Sulfitobacter delicatus KMM 3584T and

Sulfitobacter dubius KMM 3554T from phylogenetically related species. Strains: 1,

Sulfitobacter delicatus KMM 3584T; 2, Sulfitobacter dubius KMM 3554T;

3, Sulfitobacter pontiacus DSM 10014T; 4, Sulfitobacter mediterraneus

ATCC 700856T; 5, Sulfitobacter brevis ATCC BAA-4T; 6, Staleya guttiformis

DSM 11458T. None of the strains tested produced laminarinase or chitinase. +,

Positive; -, negative; W, weak reaction. Data from this study and from Sorokin (1995),

Pukall et al. (1999) and Labrenz et al. (2000).

Phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine are

the major phospholipids. The main cellular fatty acid is cis-vaccenic acid

(approx. 80 %).

165

7.2.1.1.2. Phenotypic and chemotaxonomic properties of Sulfitobacter dubius

Sulfitobacter dubius (du'bi.us. L. masc. adj. dubius doubtful).

Rod-shaped cells, single, about 0·6–0·8 μm in diameter and 1·2–1·5 μm long

with a single subpolar flagellum. Gram-negative. Chemo-organotroph with respiratory

metabolism. Colonies are uniformly round, 1–3 μm in diameter, regular, convex,

smooth, slightly yellowish after incubation for 48 h on marine agar. No diffusible

pigment is produced in the medium. Does not form endospores. Accumulates

poly-β-hydroxybutyrate as an intracellular reserve product. Oxidase- and catalase-

positive. Requires Na+ or sea water for growth. Growth occurs in media containing 1–

12 % NaCl (Table 7). Grows at 10–30 °C and pH 6·0–11·0; optimum growth is

observed at 25 °C and pH 7·5–8·0. No growth is detected at 35 °C. Decomposes

gelatin. Agar, starch, casein, laminarin, alginate, Tween 80 and DNA are not

hydrolysed. From the 95 carbon sources tested, according to Biolog, i-erythritol, D-

raffinose, thymidine, phenylethylamine, putrescine and 2-aminoethanol are not

utilized and α-cyclodextrin, glycogen, L-fucose, D-galactose, L-rhamnose, D-sorbitol,

D-galactonic acid lactone, D-galacturonic acid, glucuronamide, L-phenylalanine,

L-pyroglutamic acid, D-serine and glucose 1-phosphate are only weakly utilized.

Susceptible to ampicillin, benzylpenicillin, gentamicin, kanamycin, carbenicillin,

neomycin, oleandomycin and streptomycin; not susceptible to polymyxin, tetracycline

or lincomycin.

Phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine are

the major phospholipids. The main cellular fatty acid is cis-vaccenic acid

(approx. 80 %).

166

7.2.1.1.3. Phenotypic and chemotaxonomic properties of Marinobacter excellens

Marinobacter excellens (ex'cell.ens. L. masc. adj. excellens remarkable, exceptional).

The majority of cells are rod-shaped, with lengths and widths that vary from 1

to 8 µm and from 0·6 to 1·4 µm, respectively. They are motile, polarly flagellated.

Gram-negative strains that are strictly aerobic heterotrophs. Anaerobic growth occurs

by fermentation of D-glucose by anaerobic respiration of nitrate. No endospores are

formed. Colonies on marine 2216 agar are circular, smooth, and convex with an entire

edge, transparent and 1–3 mm in diameter after 2 days of incubation at RT. Organic

growth factors are not required. Growth occurs at 1–15 % NaCl. No growth at

20 % NaCl. Growth temperature ranges from 10 to 41 °C, with optimum growth at

20–25 °C. No growth is detected at 45 °C (Table 8). pH range for growth is 6·0–10·0,

with optimum growth at pH 7·5. Oxidase-positive and weakly positive for catalase.

Amylase and lipase are hydrolysed, whereas gelatin, casein, chitin, agar, alginate and

laminaran are not. Bacteria are non-haemolytic on mouse blood agar, non-cytotoxic,

do not exhibit antimicrobial activity, are susceptible to polymyxin and resistant to

ampicillin, benzylpenicillin, gentamicin, kanamycin, carbenicillin, neomycin,

tetracycline, lyncomycin, oleandomycin and streptomycin. Positive for lipase and

amylase, but negative for agarase, chitinase, caseinase and gelatinase; able to utilize a

limited range of carbohydrates. Of the 95 carbon sources in the Biolog system, strain

KMM 3809T utilized Tween-85, N-acetyl-D-glucosamine, D-fructose, maltose,

D-mannitol, L-rhamnose, D-sorbitol, methyl pyruvate, monomethyl succinate,

cis-aconitic acid, D-galactonic acid lactone, α-hydroxybutyric acid, γ-hydroxybutyric

acid, succinic acid, L-histidine, L-leucine, L-phenylalanine, L-proline, L-pyroglutamic

acid, D-serine, DL-carnitine, urocanic acid and 2-aminoethanol.

Major respiratory lipoquinone is Q9; PE, PG and DPG are major

phospholipids.

167

Table 8. Characteristics that differentiate Marinobacter excellens

from phylogenetically related species. Taxa: 1, Marinobacter

excellens KMM 3809T; 2, Marinobacter hydrocarbonoclasticus;

3, Marinobacter aquaeolei. All strains are straight rod-shaped

organisms, are oxidase-positive, exhibit lipase, grow in

15 % NaCl, do not hydrolyse gelatin, casein or chitin, are negative

for haemolysis and are susceptible to polymyxin. W, Weakly

positive. Data are from this study, Gauthier et al. (1992) and

Nguyen et al. (1999).

*No. strains tested that are positive.

168

Three components, C16 : 0, C16 : 1ω9c and C18 : 1 ω 9c, accounted for >70 % of

total fatty acids. Minor fatty acids included C12 : 0, C14 : 0, C15 : 0, C17 : 0, C18 : 0 and C17 : 1

ω 8c. In their main features, the fatty acid profiles were similar to those reported for

Marinobacter species (Nguyen et al., 1999, Yoon et al., 2003a). These authors found a

relatively high proportion of hydroxy fatty acids (up to 10 %), whilst in our

experiments, C12 : 0 3-OH was detected in lower amounts (up to 0·7 %). Such variation

in the proportion of fatty acids was observed previously for other Proteobacteria

(Huys et al., 1994, Ivanova et al., 2000c) and can be explained by differing

experimental conditions employed in different laboratories. In addition, a greater

proportion of C16 : 1ω9c in the fatty acid profiles of the new isolates compared to those

of other type strains and a number of differences in distribution of fatty acids present

in minor amounts, i.e. accounting for <5–7 %, namely C15 : 0, C17 : 0 and C17 : 1ω8c,

were also found. Notably, all bacteria of the genus Marinobacter exhibited an

abundance of ω9c isomers of the fatty acids C16 : 1 and C18 : 1, which is in agreement

with results reported previously for type strains grown under different cultivation

conditions (Nguyen et al., 1999, Yoon et al., 2003a). We suggest that ω9c isomers of

fatty acids C16 : 1 and C18 : 1 might be characteristic chemotaxonomic markers of the

genus Marinobacter.

7.2.1.2. Gram-positive marine bacteria belonging to the genus Planococcus

7.2.1.2.1. Phenotypic and chemotaxonomic properties of Planococcus maritimus

Two bacteria were found to be oxidase- and catalase-positive, tolerant to

15 % NaCl levels, but not requiring Na+ ions for growth (Table 9). The effect of

temperature on cell growth was monitored between 5-42 °C, with optimum growth

found to occur at 25 °C, and weak growth around 45 °C. The pH range for growth was

observed between 6.0-11.0, with optimum growth occurring at pH 8.5-9.0. Gelatin,

casein, Tween 80, and alginate were all found to be hydrolyzed, while urea, starch,

and agar were not. Both strains exhibited hemolytic and cytotoxic activities, but only

169

negative reactions towards Voges-Proskauer (acetoin production), indol, arginine

dihydrolase, lysine decarboxylase, and ornithine decarboxylase tests. According to

Biolog results, the following substrates were utilized: Tween 40, Tween 80,

D-mannitol, methylpyruvate, D, L-Lactic acid, L-asparagine, D-serine, glycerol. In

addition, strain KMM 3636 utilized glucose phosphate.

The surface of Planococcus maritimus was found to be smooth with only 3 nm

of cell-surface roughness (Figure 22).

170

Table 9. Differential phenotypic characteristics of Planococcus maritimus and other species of the genera Planococcus and Planomicrobium.

Characteristic 1 2 3 4 5 6 7 8 9 10

Colonies Orange

Orange

Orange

Orange

Yellow

Orange

Orange

Orange

Yellow

/ O

range

Orange

Cell morphology

Irregular cocci

Cocci

Rods

Cocci

Cocci

Cocci

Rods

Cocci/

short rods

Rods

Rods/cocci

Oxidase - - - - - - + - W - Growth at temperature (°C)

5-42 4-41 15-41

0-30 4-37 4-37 0-30 4-38 20-37 0-37

NaCl requirement

no no yes no no no no no yes no

NaCl tolerance, %

15 17 12 12 10 3.3 12 6 15 7

Hydrolysis of: Starch - - + - - - - - - - Casein + + ND ND ND ND ND + + + Gelatin + + + + + + + + + +

Tween 80 + - - +/- - - + - - Nitrate reduction

v - - - - - - - - +

Utilization of: D-Glucose - + - + d v - w - d D-Xylose - - - + - - + - + - Lactose - - - - - - - + - - D-Cellobiose - - - - - - - + - - Melibiose - - - - - - - + - - Glycerole + - - + - + + - - - G+C content (mol%)

48 49 45 41.5 48-51

40-43

44.5 47 46 35

Taxa are identified as: 1 - Planococcus maritimus KMM 3738, KMM 3636; 2 - Planococcus maritimus KCCM 41587T; 3 – Planomicrobium alkanoclasticum NCIMB 13489T; 4 - Planococcus antarcticus DSM 14505T; 5 - Planococcus citreus DSM 20549T; Planococcus kocurii DSM 20747T; 7 – Planomicrobium psychrophilum DSM 14507T; 8 – Planomicrobium koreense JCM 10704T; 9 – Planomicrobium okeanokoites ATCC 700539T; 10 – Planococcus mcmeekinii NCIMB 561T; ―+‖ – positive; ―-‖ – negative, ―v(+)‖ – variable with most positive; d – different data in published articles; w – weak reaction; ND – no data available. Data from this study, Engelhardt et al. (2001), Yoon, et al. (2001, 2003b); Reddy et al. (2002).

171

The most relevant cellular fatty acids were branched chain saturated iso-methyl

and anteiso branched acids, namely 14 : 0i, 15 : 0i, 15 : 0аi, and 16 : 0-i fatty acids the

proportion of those has reached up to 60 %. More precisely, the amount of major fatty

acids ranged as follows: 14 : 0-i - 15.1-16.2 %; 15 : 0-i – 12.9-13.2 %; 15 : 0-аi – 26.0-

25.6 %; 15 : 0 - 4.0-3.0 %; 16 : 0-i - 9.9-12.6 %; 17 : 0-аi – 3.0 - 3.4; 17 : 1ω8 – 3.7 –

2.6.

172

Figure 22. High-resolution A

FM topographical im

ages of Planococcus maritim

us F 90 cells and a close-up of the area on the cell surface (non-contact m

ode, top) revealing dark spots/pores. Correspondent cross-section and line profiles

analysis (bottom) show

s the roughness of the cell surface. Reproduced from

reference (Ivanova et al., 2006a) © 2006 W

ith kind perm

ission from International M

icrobiology.

173

7.2.2. Genotypic and phylogenetic characterization

7.2.2.1. Gram-negative marine bacteria belonging to the genera Sulfitobacter and

Marinobacter

7.2.2.1.1. Genotypic and phylogenetic characterization of Sulfitobacter delicatus

and Sulfitobacter dubius

The type strain of Sulfitobacter delicatus is KMM 3584T (= LMG 20554T =

ATCC BAA-321T). The G + C content of the DNA of the type strain was 60·3 mol%.

Isolated from a starfish (Stellaster equestris) collected from the South China Sea.

The type strain of Sulfitobacter dubius is KMM 3554T (= LMG 20555T =

ATCC BAA-320T). The G + C content of DNA of the type strain was 63·7 mol%

(Table 7). Isolated from sea grass (Zostera marina) collected from the Sea of Japan.

The level of DNA–DNA relatedness between the two strains studied was 33 %

and they were therefore genotypically assigned to separate species. The genetic

similarity of KMM 3584T to type strains of the genus Sulfitobacter, namely,

Sulfitobacter pontiacus, Sulfitobacter brevis, Sulfitobacter mediterraneus and Staleya

guttiformis was rather low (5–24 %); for KMM 3554T, the similarity was 10–41 %.

Based on the generally accepted criterion of the definition of genomic species (Wayne

et al., 1987), strains KMM 3584T and KMM 3554T are representatives of novel

species.

The most similar sequence to those of the novel isolates was that of

‗Oceanibulbus indoliflex‘ (99·5 %), followed by sequences from Sulfitobacter brevis,

Sulfitobacter pontiacus, Sulfitobacter mediterraneus and Staleya guttiformis

(≤97·8 %). An initial first analysis included the 79 most similar sequences as retrieved

by BLAST on EMBL and EMBL new. Removal of sequences pertaining to species

that were uncultured and not validly named led to a dataset of 14 sequences that were

visually aligned and analysed by all three methods (Figure 23). The two KMM

sequences grouped robustly with ‗Oceanibulbus indoliflex‘, a species that is yet to be

described, but not with any species with validly published names, suggesting that each

174

of these sequences represents a novel bacterial species, as confirmed by DNA–DNA

hybridization experiments. These two species clustered with the type species of the

genus Sulfitobacter (Sulfitobacter pontiacus) according to NJ and ML, but not MP,

and the degree of bootstrap replication was low (39 %), suggesting that the genus

Sulfitobacter might be subject to revision in the future, probably to include only the

type species of the genus. Such a revision may require phylogenetic analyses of more

housekeeping genes; it is therefore suggested that the two novel species be assigned to

the genus Sulfitobacter for the time being.

Figure 23. Phylogenetic position of Sulfitobacter delicatus KMM 3584T and

Sulfitobacter dubius KMM 3554T according to 16S rRNA gene sequence

analysis. The unrooted tree shown is the result of a neighbour-joining bootstrap

analysis (1000 replications). Values shown are bootstrap percentages. Branches

that were also retrieved by parsimony (three most parsimonious trees) and

maximum-likelihood (ln=-3506) are respectively indicated by * and X

(P<0·01).

http://ijs.sgmjournals.org/content/vol54/issue2/images/large/IJE36924-1.jpeg


175

7.2.2.1.2. Genotypic and phylogenetic characterization of Marinobacter excellens

The type strain is KMM 3809T(= CIP 107686T). Isolated from sediments

collected in Chazhma Bay, Sea of Japan. DNA G + C content was 55·0–56·0 mol%.

Domains used to construct the final phylogenetic trees (positions 88–1469 of

KMM 3809T) were regions of the small-subunit rRNA gene sequences that were

available for all sequences and excluded positions that were likely to show homoplasy

or notoriously difficult to sequence, i.e. the 5' end of the sequences.

16S rRNA gene sequence analyses revealed that strain KMM 3809T is a

member of the -Proteobacteria and, more precisely, that it is included in the clade

formed by the genus Marinobacter (Figure 24). The topology of the phylogenetic tree

shown in Figure 24 is that of the bootstrap analysis, as it has been demonstrated that

this topology is often better than that of a simple NJ analysis (Gascuel, 1997). As a

result, there is no distance bar in this tree; note also that the distance bar should be

considered with caution in a tree, as it represents distances calculated after corrections

(transversions being accounted for more than transitions) and branch-lengths do not

represent the real number of differences between the sequences themselves. Bootstrap

numbers are indicated only for branches that were also retrieved in the ML and MP

trees (consensus tree). 16S rRNA gene sequence similarities with other available

sequences were calculated by parsing the result of a BLAST analysis of KMM 3809T

on the ‗Bacteria‘ division of GenBank (at 25 November 2002), with the options ‗no

filter‘ and W = 7. The sequence of strain KMM 3809T had 97·3 % or less similarity to

its nearest phylogenetic relatives, i.e. Marinobacter hydrocarbonoclasticus,

Marinobacter aquaeolei and Marinobacter litoralis.

176

Figure 24. Phylogenetic position of Marinobacter excellens according

to 16S rRNA gene sequence analysis. The topology shown was

obtained by using the BIONJ algorithm and 1000 bootstrap

replications with the Kimura two-parameter distance correction.

Bootstrap values are indicated only for branches that were also

retrieved by MP and ML (P<0·01); these branches should be

considered as the only robust clusters identified by this analysis.

DNA–DNA hybridization data revealed a high level of DNA relatedness

among KMM 3809T, KMM 3814, KMM 3817 and KMM 3818, ranging from 93 to

96 º%, which indicated that the strains belonged to the same species (Wayne et al.,

1987). As the phenotypic and chemotaxonomic characteristics of KMM 3815 were

identical to those of KMM 3814, the former was excluded from DNA–DNA

hybridization experiments. Genetic similarity of KMM 3809T with type strains of the

genus Marinobacter was 45–63 %. Based on the generally accepted criterion of the

definition of genomic species (Wayne et al., 1987), strains KMM 3809T, KMM 3814,

KMM 3817 and KMM 3818 are assigned to the novel species.



177

7.2.2.2. Gram-positive marine bacteria belonging to the genus Planococcus

7.2.2.2.1. Genotypic and phylogenetic characterization of Planococcus maritimus

The G + C content of the DNA ranged 48-49 mol%. The level of DNA

hybridization between two novel strains isolated from algae was 98 %, suggesting

these bacteria belong to a single genotypic species. The genetic similarity between the

DNA of both KMM 3738 and KMM 3636 strains compared with that of the type

strains of the genera Planomicrobium and Planococcus ranged between 12-15 % and

16-36 %, respectively, and with DNA from Planococcus maritimus 87 %. According

to generally accepted criteria of the definition of the genomic species (Wayne et al.,

1987), the strains isolated from brown algae Fucus evanescence can be assigned to

Planococcus maritimus (Yoon et al., 2003b).

All phylogenetic analysis revealed that strain KMM 3738 was included in the

clade formed the genera Planococcus and Planomicrobium (Figure 25).

178

Figure 25. Phylogenetic position of Planococcus maritimus KMM 3738

based on 16S rRNA gene sequence.

179

7.3. Conclusion

7.3.1. Classification of gram-negative marine isolates

On the basis of generally accepted criteria for the definition of genomic species

(Wayne et al., 1987), gram-negative bacteria were assigned genotypically to different

species. The following names and numbers were assigned: Sulfitobacter delicatus

KMM 3584T (= LMG 20554T =ATCC BAA-321T), Sulfitobacter dubius KMM 3554T

(= LMG 20555T = ATCC BAA-320T) and Marinobacter excellens KMM 3809T (= CIP

107686T).

The two novel species can be distinguished from other Sulfitobacter species

and Staleya guttiformis by phenotypic features (Table 7). For example, strain KMM

3584T isolated from the starfish Stellaster equestris is able to hydrolyse gelatin and

alginate and does not utilize melibiose, whereas strain KMM 3554T isolated from the

sea grass Zostera marina is more halophilic, hydrolyses only gelatin and utilizes

citrate and melibiose. Both novel species are unable to produce DNase or lipase. The

most similar sequence to those of the novel isolates was that of ‗Oceanibulbus

indoliflex‘ (99·5 %). The two KMM sequences grouped robustly with ‗Oceanibulbus

indoliflex’, a species that was yet to be described, but not with any species with validly

published names (Figure 23), suggesting that each of these sequences represented a

novel bacterial species, as confirmed by DNA–DNA hybridization experiments. The

genetic similarity of KMM 3584T to type strains of the genus Sulfitobacter was rather

low (5–24 %); for KMM 3554T, the similarity was 10–41 %. Based on the generally

accepted criterion of the definition of genomic species (Wayne et al., 1987), strains

KMM 3584T and KMM 3554T are representatives of novel species. The names

Sulfitobacter delicatus and Sulfitobacter dubius are proposed for KMM 3584T and

KMM 3554T, respectively.

Five strains of free-living marine bacteria (KMM 3809T, KMM 3814,

KMM 3815, KMM 3817 and KMM 3818) isolated from radionuclide-contaminated

sediments of Chazhma Bay, Sea of Japan, differed from other Marinobacter species

by a number of phenotypic features (Table 8), for example, susceptibility to only one

180

antibiotic polymyxin. Remarkably, type strain KMM 3809T had ―porous‖ features

(dark spots in Figure 30) that may contain ATPases (discussed in subsection 2.5.5.3.

and the following chapter). Phylogenetic evidence, along with phenotypic and

genotypic characteristics, showed that the bacteria constituted a novel species of the

genus Marinobacter. Thus, phylogenetic 16S rRNA gene sequence-based analysis

placed these bacteria in a clade within the genus Marinobacter in the γ-

Proteobacteria. KMM 3809T showed highest 16S rRNA gene sequence similarity of

97·3 % to Marinobacter litoralis and 96·9 % to Marinobacter hydrocarbonoclasticus

and Marinobacter aquaeolei (Figure 24). DNA–DNA hybridization between the five

isolates was at the conspecific level (94–96 %) and that among the closest

phylogenetic neighbours ranged from 45·0 to 62·5 %. The name Marinobacter

excellens sp. nov. is proposed for this species, with the type strain KMM 3809T (= CIP

107686T).

7.3.2. Classification of gram-positive marine isolates

On the basis of generally accepted criteria for the definition of genomic species

(Wayne et al., 1987), gram-positive bacteria were assigned genotypically to a single

species. The following name and number were assigned: Planococcus

maritimus KMM 3738.

Two orange-pigmented bacteria (KMM 3738 and KMM 3636) isolated from

enrichment culture during degradation of the thallus of the brown alga Fucus

evanescens produced carotenoid pigments, were chemoorganotrophic, alkaliphilic and

halo-tolerant growing well on nutrient media containing up to 15 % NaCl. Growth

temperature ranged from 5 to 45 °C. The DNA base compositions were 48 mol% G +

C and the level of DNA similarity of two strains was conspecific (98 %). A

comparative phylogenetic analysis of 16S rRNA gene sequences (Figure 25) revealed

that the strain KMM 3738 tightly clustered with Planococcus maritimus (99.9 % 16S

rRNA gene sequence similarity). DNA-DNA hybridization experiments revealed that

DNA from the KMM 3738 showed 12-15 % and 16-35 % of genetic relatedness with


181

the DNA of type strains of the genera Planomicrobium and Planococcus, respectively,

and 87 % with DNA from Planococcus maritimus, indicating that new isolates belong

to the later species. Interestingly, bacterium had unusual irregular coccoid shape of

cells possessing a single flagellum. In contrast to Marinobacter excellens, the surface

of Planococcus spp. was found to be smooth with only 3 nm of cell-surface roughness

(Figure 22).

182

CHAPTER 8

CHARACTERIZATION OF ATPASES ACTIVITIES OF

MARINE BACTERIA

183

8.1. Overview

This chapter presents results of screening of several phylotypes of the domain

Bacteria, comprising 86 environmental (marine and freshwater) bacteria of 17 genera.

Reproduced from reference (Ivanova et al., 2006a) © 2006 With kind permission from

International Microbiology. This work follows the study of self-assembly of actin

filaments for microdevices presented in chapter 6. In order to evaluate the possibility

of providing molecular motor proteins in microdevices with a cheap ATP energy, a

search for bacterial ATP producers among strains belonging to different taxa was

performed. The chapter starts with an estimate of the levels of extracellular ATP

generated by bacteria, and is followed by a description of growth patterns of two

selected ATP producers, gram-negative Sulfitobacter mediterraneus and gram-

positive Planococcus maritimus (the latter is described in subsections 7.2.1.2. and

7.2.2.2.), on surfaces of different hydrophobicities: hydrophobic poly(tert-butyl

methacrylate) (PtBMA) and hydrophilic (mica) surfaces (see subsection 8.2.2). The

chapter continues with a discussion of effects of the polymeric surfaces on

intracellular and extracellular ATP productions by two selected strains (see

subsections 8.2.3. and 8.2.4.).

As gram-negative bacteria belonging to the genera Sulfitobacter, Marinobacter

and Staleya generated high amounts of extracellular ATP, while gram-positive strain

belonging to the species Planococcus maritimus produced high amounts of

intracellular ATP, their cell surfaces were examined. AFM (as described in subsection

3.5.6.) was used to reveal distinct features of ATP producers. The chapter ends with a

description of particular features of potential ATP supplies. It was concluded that

gram-negative extracellular ATP producers had porous cell surfaces (Figures 28, 30),

while gram-positive intracellular ATP producer had a rough one (Figure 22). The

former was assumed to have an effective membrane ultrastructure that facilitated the

secretion of ATP.

184


8.2.1. Levels of ATP detected in heterotrophic bacteria of different taxa

An estimate of the levels of ATP generated by 86 microorganisms of the 17

genera analyzed (Table 1) revealed that most of the frequently detected bacteria that

secreted elevated amounts of ATP were members of the α-proteobacteria (e.g.,

Sulfitobacter spp. and related bacteria) and some γ-proteobacteria (in particular

Marinobacter spp.), whereas members of certain gram-positive taxa, e.g., Kocuria

spp. and Planococcus spp., secreted lesser amounts of ATP.

In general, there were significant variations in the levels of secreted ATP,

ranging from 190 μM ATP or 0.1 pM ATP per colony forming unit (cfu), as detected

in Pseudoalteromonas spp., to 1.2–1.9 mM ATP or 6.0–9.8 pM ATP/cfu, as detected

in Sulfitobacter spp., Staleya guttiformis, and Marinobacter spp. (Table 10). From the

screening, two distantly related strains, gram-negative Sulfitobacter mediterraneus and

gram-positive Planococcus maritimus, were selected in order to investigate the

impact—if any—of surface hydrophobicity (in hydrophobic PtBMA and hydrophilic

mica) on ATP production and secretion. The rationale of this selection was based on

the notion that cellular membranes of gram-negative and gram-positive

microorganisms differ significantly, and therefore it is of interest to understand

whether the response of the attached cells reflects this difference. Both of the selected

strains secreted the largest amounts of extracellular ATP in the culture fluid, in

contrast to their counterparts of related and non-related phylotypes (Table 10).

185

Table 10. Levels of extracellular adenosine triphosphate (ATP) detected in

heterotrophic bacteria of different taxa.

Taxon No. strains ATP; μM/ml ATP; pM/cfu*

*cfu: colony forming unit

A genus specific metabolic pattern could be also observed, even though there

were some intra-species and intra-strain variations. Sulfitobacter spp., Staleya

guttiformis, and Marinobacter spp. generated notable amounts of ATP (see Table 10).

To our knowledge, this is the first report in which the ATP levels among diverse taxa

have been estimated; therefore, no data are available that can be comprehensively

compared with our experimental results. Nevertheless, our results are in agreement

with previously reported data on the levels of ATP in microbial cells (Biteau et al.,

2003, Di Tomaso et al., 2001, Fletcher, 1996) and comparable to the amounts detected

in mammalian cells (Tornquist, 1991). For example, Di Tomaso et al. (2001) reported

that recombinant cells of phototrophic Rhodobacter capsulatus (OD660 = 0.5; 3 × 108

cells/ml) contained 1.35–2.64 mM ATP (0.6 pM ATP/cfu), and Biteau et al. (2003)

found that Saccharomyces cerevisiae contained 1.78 mM ATP.

186

8.2.2. Pattern of bacterial growth on surfaces

The relative number of attached cells of Sulfitobacter mediterraneus increased

slowly, and stabilized at 6 × 108 cfu/ml after about 32 h on the hydrophobic PtBMA

surface. The number of attached cells on the hydrophilic mica remained low over the

period studied (Figure 26).

Figure 26. Kinetics of adenosine triphosphate (ATP) production by Sulfitobacter

mediterraneus ATCC 700856T during attachment on poly(tert-butyl methacrylate)

(PtBMA) (top) and mica (bottom). ● number of cells in the culture medium, number

of cells on the surface, production of • extra- and intracellular ATP.

187

In contrast, the number of attached cells of Planococcus maritimus increased

rapidly within the first 20 h, up to 1 × 108 cfu/mlon PtBMA, and continued to increase

over a period of 48 h. On the mica surface, the number of attached cells of this species

reached 2 × 108 cfu/ml after 24 h and it stabilized at this level for the following 24 h

(Figure 27). Notably, the growth pattern of planktonic cells of each strain in the

correspondent wells with different surfaces was identical, although strain-specific

features were retained all the time. Overall, it appeared that both strains showed a

better propensity of attachment to hydrophobic surfaces than to hydrophilic ones.

Figure 27. Kinetics of ATP production by Planococcus maritimus F 90 during

attachment on PtBMA (top) and mica (bottom). ● number of cells in the culture

medium, number of cells on the surface, production of • extra- and intracellular

ATP.

188

Our study of the growth patterns of two bacterial strains on surfaces of

different hydrophobicities and bacterial generation of intracellular and extracellular

ATP revealed a few particular characteristics. Importantly, the generation of

intracellular and extracellular ATP was followed by bacterial growth during

attachment, which, in turn, was controlled by the type of surface. Both strains showed

greater attachment to the hydrophobic PtBMA surface (other characteristics are

discussed in subsections 8.2.3., 8.2.4. and 8.2.5.). This observation is in agreement

with the well-known notion that most bacteria are more prone to attachment to

hydrophobic than to hydrophilic surfaces. Yet, the driving mechanisms of this

phenomenon remain unclear (Davey and O'Toole, 2000, Fletcher, 1996, Maechler et

al., 1998, Pasmore and Costerton, 2003). While the physical environment provided by

the PtBMA and mica surfaces no doubt exerts an effect on Planococcus maritimus and

Sulfitobacter mediterraneus cells, remarkably, these bacteria responded differently, by

producing increasing levels of either intracellular (Planococcus maritimus) or

extracellular (Sulfitobacter mediterraneus) ATP.

8.2.3. Effect of polymeric surfaces on intracellular ATP generation

The levels of intracellular ATP in both species of bacteria were higher than

those of extracellular ATP (Figures 26, 27). In addition, the levels of intracellular ATP

varied during bacterial-cell attachment and biofilm formation over the 48-h

experiments. For example, the level of ATP in Sulfitobacter mediterraneus increased

significantly after 16 h of attachment on PtBMA and after 28 h on mica. Similar

kinetics for intracellular ATP were observed in Planococcus maritimus albeit over a

different time frame. A sharp increase of intracellular ATP production was detected in

the early exponential phase of growth, after 8 h of attachment on PtBMA, and after

28 h on mica after a prolonged exponential phase of growth (Figures 26, 27).

In general, the level of intracellular ATP correlated with the bacterial growth

pattern, i.e., intracellular ATP levels reached maximum values when cells were at the

exponential phase of growth, and they decreased when cells exited this phase and

189

entered the stationary growth phase. The average reduction in the amount of

intracellular ATP produced by the two strains after 44 h was 70–90 %.

It is not surprising that in both strains the levels of intracellular ATP were

higher than those of extracellular ATP. It is logical that the generation of sufficient

amounts of ATP by the cells, in particular during exponential growth, is essential for

metabolic intracellular processes. During the attachment of either strain on hydrophilic

mica, the highest increases in ATP production occurred after a prolonged lag period,

while the same increase in ATP levels on hydrophobic PtBMA occurred earlier. Such

changes in intracellular ATP may indicate that the chemical and/or physical properties

of the surfaces affect cellular metabolism. The increase in the ATP level might be a

reflection of the activities of several intensive metabolic processes as cells adapt, and

then attach to the surface. Recently, it was shown that more than 200 bacterial genes

are involved in the change from planktonic to biofilm life-style (Bassler, 2002, Kotra

et al., 1999, Yan et al., 2003). Notably, the levels of intracellular ATP in the studied

strains differed, in that the successful colonizer, Planococcus maritimus, contained up

to five-fold more intracellular ATP than Sulfitobacter mediterraneus strains.

8.2.4. Variation in extracellular ATP generation

The levels of intracellular ATP of attached cells were in concert with both the

extracellular ATP levels and the planktonic cell density in the same wells above the

surfaces. The increase in the extracellular level of ATP of Sulfitobacter mediterraneus

followed immediately the increase in its intracellular level of ATP. By contrast, some

4 h after intracellular ATP levels increased in Planococcus maritimus, its extracellular

ATP levels increased. Similar patterns of intracellular and extracellular ATP

production were observed on both surfaces.

The levels and proportions of intracellular versus extracellular ATP

significantly differed in the two strains (Figures 26 and 27). For example, the level of

intracellular ATP in Sulfitobacter mediterraneus was 50-55 pM ATP/cfu on both

polymeric surfaces, while Planococcus maritimus produced more intracellular ATP. In

fact, Planococcus maritimus intracellular production was about 2.5–5 times higher and

190

ranged from 120 to 250 pM ATP/cfu depending on the surfaces, e.g., about two-fold

more on mica. The amount of extracellular ATP generated by Planococcus maritimus

planktonic cells was 6 pM ATP/cfu, and about the same for both PtBMA and mica,

while the amount of extracellular ATP generated by Sulfitobacter mediterraneus

ranged from 20 to 50 pM ATP/cfu, and was more than two-fold higher in the wells

with mica (Figures 26 and 27).

Interestingly, higher amounts of extracellular ATP were secreted by

Sulfitobacter mediterraneus on mica, which appeared to be ‗difficult‘ for bacterial

colonization, than on PtBMA. The cell response to this ‗unfriendly‘ physical

environment might therefore have been an increase in the release of extracellular ATP.

In contrast, during attachment on PtBMA, no dramatic changes in extracellular ATP

levels were observed in either strain (Sulfitobacter mediterraneus secreted twice the

amount found in our initial results). This observation can be partially explained by the

fact that the bacterial densities of biofilms formed by both cultures on PtBMA

polymeric surfaces did not reach the saturation level of 1012 cfu/cm3 (Bassler, 2002,

Kuchma and O'Toole, 2000), so that the cell-density-dependent signaling system to

control the production of cellular metabolites might not have been activated yet

(Pasmore and Costerton, 2003). A biofilm-specific signaling system can induce

planktonically grown cells to behave as if they were in a biofilm by regulating the

expression of cellular metabolites (Yan et al., 2003), so that an increase in ATP

production would be also expected.

8.2.5. AFM investigation of bacterial surface ultrastructure

High-resolution AFM images of the cell surfaces at 0.5 μm (lateral dimension)

of two representative strains that secreted high amounts of extracellular ATP, i.e.,

Staleya guttiformis and Marinobacter excellens, are shown in Figures 28 and 30. A

few individual cells were selected and typical cell surfaces were imaged at closer

range.

191

Figure 28. High-resolution atomic-force microscopy (AFM) topographical images of

Staleya guttiformis DSM 11458T cells and a close-up of an area on the cell surface

(non-contact mode, top) revealing dark spots/porous features. Correspondent cross-

section and line profiles analysis (bottom) shows the tentative depth of the pores on

the cell surface.

The surfaces of Staleya guttiformis and Marinobacter spp. cells appeared to be

―porous‖, with a surface roughness of about 11 nm. Although it was rather difficult to

accurately estimate the depth of these surface features because of limitations of the

AFM tip (Binnig et al., 1986, Dufrene, 2001, Dufrene, 2002, Dufrene, 2003,

Grafstrom et al., 1993 ), there is no doubt about the presence of porous features on the

surfaces of these bacteria. High-resolution cell surface images of bacteria that did not

secrete pronounced amounts of extracellular ATP (see Table 10), e.g., Planococcus

192

maritimus and Formosa algae cells, were also obtained. In contrast to bacteria from the

first group, the surface of Planococcus spp. was found to be smooth with only 3 nm of

cell-surface roughness (Figure 22).

Formosa algae produces extracellular polymeric material (most probably

polysaccharides), as revealed by previous AFM analysis (Figure 29). As its surface

appeared to be of amorphous ―gel-like‖ texture, it was not possible to obtain high-

resolution images of those cells.

193

Figure 29. A

FM of cells of Form

osa algae KM

M 3553

T. (a) Deflection im

age of cells deposited on mica. (b)

Deflection im

age and line profile of a cell freshly deposited on mica (30 m

in); the two curves correspond to the

longitudinal and transverse profiles of the cell. Note the occurrence of noise along the edges of the bacterium

. Closer

investigation revealed a fine layer of conditioning film (extracellular polym

eric substances), secreted by the bacterium

to promote gliding and/or attachm

ent. (c) Deflection im

age and line profile of a cell deposited on mica and visualized

after 2 days. (d) Deflection im

age and line profile of a deflated cell deposited on mica and visualized after 2 days.

Reproduced from

reference (Ivanova et al., 2004b) © 2004 W

ith kind permission from

IJSEM.

194

So, the finding that Sulfitobacter spp., Staleya guttiformis, and Marinobacter spp.

generated high amounts of ATP prompted further investigation into whether ATP

generation and secretion might be reflected in distinct features of the cell surface.

AFM imaging of the bacterial cell surface at high resolution revealed topographic

peculiarities of those bacteria that secreted high amounts of extracellular ATP. These

images showed ―porous‖ features on the surface of the studied strains (dark spots on

Figures. 28, 30).

Figure 30. High-resolution AFM topographical images of Marinobacter excellens

KMM 3809T cells and a close-up of the area on the cell surface (non-contact mode,

top) revealing dark spots/porous features. Correspondent cross-section and line

profiles analysis (bottom) shows the tentative depth of the pores on the cell surface.

195

There is no direct evidence yet that the ―porous‖ features found on the cell

surface of Staleya guttiformis and Marinobacter excellens include ATP synthases that

might facilitate ATP secretion. However, recently published research suggests that

ATP synthesis is driven by a trans-membrane electrochemical gradient generated by

light or oxidative reactions via the F0 part of ATP synthases incorporated into the

cellular membrane (Fronzes et al., 2003, Hong and Pedersen, 2003, Müller et al.,

2003). High-resolution AFM and transmission cryoelectron microscopy images of the

ATPase from Ilyobacter tartaricus embedded into a lipid membrane (Lundin and

Thore, 1975, Stahlberg et al., 2001) revealed the native structure and sizing of a single

ATP synthase molecule. An average outer diameter of 5.4 ± 0.3 nm and a vertical

roughness of about 3 nm were reported and are consistent with the sizes of the holes

visualized on the surfaces of Staleya guttiformis and Marinobacter excellens. The

dimensions of the protrusions (―bumps‖) on the cell surface of these strains were about

20–35 nm, with a vertical roughness of 4–11 nm. These measurements correlate well

with the sizes of lipopolysaccharide (LPS) bundles reported recently (Kotra et al.,

1999). While investigating the dynamics of LPS assembly on the surface of

Escherichia coli, Kotra et al. (1999) obtained high-resolution images of the bacterial

surface similar to those obtained in this study. The authors suggested that the spaces

among these LPS bundles might be surface water-filled protein channels (Kotra et al.,

1999). Within the outer membrane of gram-negative bacteria, particular proteins

(antiporters, ABC transporters, symporters, porins, and other energy-transducing

proteins) are incorporated in gated channels that facilitate entry of certain molecules

into the cell (Beveridge, 1999, Biteau et al., 2003, Ferguson and Deisenhofer, 2004).

This assumption does not exclude the possibility of the incorporation of ATPase into

similar channels, which we observed on Staleya guttiformis and Marinobacter

excellens cell surfaces. These bacteria may have an effective membrane ultrastructure

that facilitates the secretion of ATP. Both α- and γ-proteobacteria represent abundant

groups of marine prokaryotes (Buchan et al., 2000, Wagner-Dobler et al., 2003) that

carry out several crucial ecological functions, including the reduction or oxidation of

sulfur compounds (Pukall et al., 1999, Sorokin, 1995), the biodegradation of

hydrocarbons and other compounds (Buchan et al., 2000, Doronina et al., 2000,

196

Gonzalez et al., 1997, Gonzalez et al., 2003), and the development of oxidant-

dependent signal transduction systems (Allgaier et al., 2003, Shiba, 1991). These are

thermodynamically unfavorable processes that are coupled to both an electrochemical

proton gradient and the hydrolysis of ATP. In our experiments, the attachment of the

bacteria onto hydrophilic mica might have imitated somewhat similar

thermodynamically unfavorable/stressful processes, with a subsequent increase in the

generation of ATP.

Conclusion

A survey of the extracellular ATP levels of 86 heterotrophic bacteria showed

that gram-negative bacteria of the genera Sulfitobacter, Staleya, and Marinobacter

secreted elevated amounts of extracellular ATP, ranging from 6.0 to 9.8 pM

ATP/colony forming unit (cfu), and that gram-positive bacteria of the genera Kocuria

and Planococcus secreted up to 4.1 pM ATP/cfu.

The monitoring of variations in the levels of extra- and intracellular ATP-

dependent luminescence in living cells of Sulfitobacter mediterraneus ATCC 700856T

and Planococcus maritimus F 90 during 48 h of attachment on hydrophobic (PtBMA)

and hydrophilic (mica) surfaces demonstrated that bacteria responded to different

polymeric surfaces by producing either extra- or intracellular ATP.

The level of intracellular ATP in Sulfitobacter mediterraneus ATCC 700856T

attached to either surface was as high as 50–55 pM ATP/cfu, while in Planococcus

maritimus F 90 it was 120 and 250 pM ATP/cfu on PtBMA and mica, respectively.

Sulfitobacter mediterraneus ATCC 700856T generated about 20 and 50 pM of

extracellular ATP/cfu on PtBMA and mica, respectively, while the amount generated

by Planococcus maritimus F 90 was about the same for both surfaces, 6 pM ATP/cfu.

The levels of extracellular ATP generated by Sulfitobacter mediterraneus

during attachment on PtBMA and mica were two to five times higher than those

detected during the initial screening. High-resolution atomic force microscopy

imaging revealed a potentially interesting correlation between the porous cell-surface

of certain α- and γ-proteobacteria and their ability to secrete high amounts of ATP.

197

Thus, our results have yielded useful insights in understanding the impact of

hydrophilic and hydrophobic surfaces on bacterial attachment and ATP generation and

further modeling of bacterial metabolism. So, gram-negative extracellular ATP

producers belonging to the genera Sulfitobacter, Marinobacter and Staleya and/or

gram-positive intracellular ATP producers belonging to the genera Planococcus and

Kocuria can be considered as valuable candidates for nanotechnological application in

microdevices.

198

CHAPTER 9

EVALUATION OF MreB AND FtsA PROTEINS

199

9.1. Overview

This chapter presents results of the screening of several phylotypes of the

domain Bacteria, comprising 32 environmental (marine and freshwater) bacteria of 13

genera; 5 reference bacteria of 5 genera including 4 pathogenic and 1 thermophilic

bacteria; and 1 eukaryotic actin for production of thermo- and inherently stable linear

molecular motors with the longest half-lives. This work follows the study of self-

assembly of actin filaments for dynamic microdevices presented in chapter 6 and

study of ATP production by heterotrophic bacteria reported in chapter 8. In order to

evaluate the possibility of substituting eukaryotic molecular motor proteins in

microdevices with cheap prokaryotic homologues, a search for MreB and/or FtsA

suppliers among strains belonging to different taxa was performed.

The chapter starts with an estimate and comparison of the predicted stabilities

of MreB proteins of selected bacterial taxa and actin, and is followed by evaluation of

MreB parameters, namely, isoelectric point (pI) and overall hydrophobicity, that are

important for immobilization of proteins in microdevices (see chapters 4, 5, 6 and

subsection 9.2.1.2. for more details). Furthermore, determination of phylogenetic

relationships based on MreB sequences was performed to evaluate the possibility of

using PARP sequences as chronometers for rod-shaped bacteria (see subsections

2.5.4. and 9.2.1.3. for more details). The chapter continues with an estimate and a

comparison of physicochemical properties of FtsA proteins and actin, and is followed

by a discussion of phylogenetic positions of FtsA producers. The chapter ends with a

conclusion that Pseudoalteromonas atlantica, Loktanella vestfoldensis and

Thermotoga maritima can be used as MreB sources, whereas Marinobacter

hydrocarbonoclasticus, Oceanimonas doudoroffii and Salegentibacter flavus can be

used as FtsA sources for substituting actin in microdevices, however, after preliminary

evaluation of their biochemical properties in vitro. Moreover, it is concluded that FtsA

gene may be used as a chronometer to help unravel phylogenies of new and/or

misplaced bacteria.

200


9.2.1. Comparison/Evaluation of predicted physicochemical properties of MreB

proteins of selected bacterial taxa and actin

9.2.1.1. Stability of MreB proteins and actin

An estimate of stability of MreB proteins of 21 heterotrophic rod-shaped

bacteria belonging to 12 genera and eukaryotic actin of rabbit skeletal muscle was

based on the comparison of the following physicochemical properties: aliphatic index

(AI), instability index (II) and estimated half-life.

Calculation of AIs (Ikai, 1980), or measures of occupancy volumes of aliphatic

side chains in proteins, revealed that all MreBs, including 3 reference MreBs of

pathogens, namely, adherent invasive Escherichia coli (AIEC), Bacillus subtilis,

Listeria monocytogenes and one reference MreB of thermophilic bacterium

Thermotoga maritima, had higher AI values than eukaryotic actin (see Table 11 for

details). The aliphatic indices of all MreBs ranged from 95.96 of Aliivibrio fischeri to

115.42 of Thermotoga maritima. Notably, five members of the γ-proteobacterial

group, namely, Pseudomonas fluorescens, Pseudomonas extremorientalis,

Pseudoalteromonas atlantica, Idiomarina loihiensis, Shewanella waksmanii, and two

members of the α-proteobacterial group, namely, Sulfitobacter mediterraneus and

Loktanella vestfoldensis appeared to have the highest aliphatic indices ranged between

100.89 and 103.58. Evaluation of thermostability of proteins by using AIs confirmed

the dependence of thermostability of some MreBs and actin on thermostability of their

origins. Thus, MreB protein with the highest AI was produced by the thermophilic

bacterium Thermotoga maritima (Table 11). In fact, Thermotoga maritima owns the

most thermostable MreB (see subsection 2.5.3.1.1. for details). Interestingly, all

reference pathogens produced MreBs with similar thermostability ranged between

98.33 and 99.85. However, environmental bacteria produced very thermostable MreBs

regardless of their own thermostabilities. For example, moderately thermophilic

environmental bacteria, namely, Pseudomonas fluorescens, Pseudomonas

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extremorientalis and Idiomarina loihiensis were able to produce MreBs of the similar

thermostability as mesophilic ones, namely, Shewanella waksmanii, Loktanella

vestfoldensis, Pseudoalteromonas atlantica and Sulfitobacter mediterraneus. These

observations are in agreement with results of Ikai (1980).

Assessment of MreB inherent stability based on dipeptide composition

(Guruprasad et al., 1990) categorized 14 proteins including 7 MreBs of γ-

proteobacteria belonging to Pseudoalteromonas atlantica, Alteromonas addita,

Idiomarina zobellii, Idiomarina loihiensis, Pseudomonas fluorescens, Shewanella

waksmanii, Pseudoalteromonas haloplanktis, 2 MreBs of α-proteobacteria belonging

to Loktanella vestfoldensis and Loktanella rosea, 3 MreBs of pathogenic organisms,

MreB of Thermotoga maritima and eukaryotic actin as stable proteins (see Table 11

for details). Once again, the most inherently stable MreB was produced by

Thermotoga maritima. Interestingly, of 7 very thermostable MreBs belonging to

environmental bacteria 5 were inherently stable (IIs < 40), 1 MreB of the α-

proteobacterium Sulfitobacter mediterraneus was slightly unstable (II = 40.97) and 1

MreB of Pseudomonas extremorientalis was unstable (II = 47.49). As candidate

protein for actin (AI = 81.78; II = 36.14) substitution in microdevices must be both

very thermostable (AI > 100) and inherently stable (II < 36.14), only two out of 7

environmental bacteria, namely, Pseudoalteromonas atlantica and Loktanella

vestfoldensis can be considered as the most valuable MreB producers.

The results of evaluation of half-lives of proteins based on the N-end rule

(Varshavsky, 1997) revealed only 3 very stable (up to 100 h in mammalian

reticulocytes, in vitro) proteins: 1 MreB of the γ-proteobacterium Shewanella woodyi

and 2 MreBs of the α-proteobacteria, namely, Sulfitobacter sp. RIOSW6 and

Sulfitobacter sp. Fg 107 (see Table 11 for details). Interestingly, all three stable in

model systems MreBs, but on the other hand, inherently unstable belonged to bacteria

isolated from ecologically unusual environments. Thus, Shewanella woodyi was

isolated from squid ink and intermediate seawater (Makemson et al., 1997);

Sulfitobacter sp. RIOSW6 and Sulfitobacter sp. Fg 107 were isolated from sea water

and sediments, respectively, of radionuclide-polluted area in Chazhma bay (Sea of

Japan, Pacific Ocean). Besides, out of 7 very thermostable proteins, only MreBs of 3

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γ-proteobacteria, namely, Pseudomonas fluorescens, Pseudoalteromonas atlantica,

Idiomarina loihiensis, one α-proteobacterium Loktanella vestfoldensis and

Thermotoga maritima were theoretically capable of living in mammalian reticulocytes

30 hours.

Table 11. Comparison of theoretical stability parameters (AI, II and half-life) of

MreB proteins of γ-Proteobacteria (1), α-Proteobacteria (2), Firmicutes (3),


Bacte rial group

Strain Aliphatic index (AI)

Instabi lity

index, (II)

Estimated half-life, h

Mamma lian

reticulo cytes,

in vitro

Yeast, in vivo

Escheri chia coli,

in vivo

1 Pseudomonas fluorescens DSM 50030T (JF810206)

102.03 36.75 30 h > 20 h > 10 h

Pseudomonas extremorientalis KMM 3447T (JF815021)

100.90 47.49 1.1 h 3 min 10 h

Pseudoalteromonas nigrifaciens ATCC 19375T (JF815022)

99.19 54.98 1.1 h 3 min > 10 h

Pseudoalteromonas haloplanktis ATCC 14393T (JF815023)

97.56 37.50 1.1 h 3 min > 10 h

Pseudoalteromonas atlantica ATCC 19262T (JF815020)

100.89 33.70 30 h > 20 h > 10 h

Alteromonas addita R10SW13T (JF815024)

97.98 37.21 > 20 h > 20 h ND

Aliivibrio fischeri DSM 507T (JF815027)

95.96 40.22 > 20 h > 20 h ND

Idiomarina zobellii KMM 231T (JF815025)

99.04 36.68 5.5 h 3 min 2 min

Idiomarina loihiensis (YP_154773.1)

102.31 39.55 30 h > 20 h > 10 h

Shewanella woodyi ATCC 51908T

97.50 45.79 100 h > 20 h > 10 h

203

Shewanella waksmanii KMM 3823T (JF815026)

103.58 39.54 1.4 h 3 min > 10 h

Escherichia coli UM146 (gb ADN69460.1)

98.33 38.65 30 h > 20 h > 10 h

2

Sulfitobacter mediterraneus ATCC 700856T (JF825543)

102.94 40.97 1 h 30 min

> 10 h

Sulfitobacter delicatus KMM 3584T (JF825544)

96.06 47.73 1.1 h 3 min > 10 h

Sulfitobacter sp. Fg 107 (JF825545)

97.50 48.49 100 h > 20 h > 10 h

Sulfitobacter sp. RIOSW6 (JF825546)

97.50 45.79 100 h > 20 h > 10 h

Loktanella rosea Fg 1 (JF825547)

97.05 37.79 1.1 h 3 min > 10 h

Loktanella vestfoldensis (ZP_01001712.1)

102.35 30.70 30 h > 20 h > 10 h

3 Bacillus subtilis (gb AAA22397.1)

99.85 28.58 30 h > 20 h > 10 h

Listeria monocytogenes (gb CAC99626.1)

98.99 33.52 30 h > 20 h > 10 h

4 Thermotoga maritima MSB8 (gb AAD35673.1)

115.42 27.75 30 h > 20 h > 10 h

5 Oryctolagus cuniculus, rabbit actin (P68135)

81.78 36.14 30 h > 20 h > 10 h

9.2.1.2. Isoelectric point (pI) and grand average of hydropathicity (GRAVY) of

MreBs and actin

Since immobilization of proteins depends on both hydrophobicity of their

amino acid residues and their molecular net charges (Ivanova et al., 2006c, Kyprianou

et al., 2009, Muck et al., 2006), pI and grand average of hydropathicity (GRAVY) of

MreBs and actin were calculated (see Table 12 for details).

According to pI results, the majority of γ-proteobacterial MreBs were acidic,

with the exception of MreBs of Pseudoalteromonas haloplanktis, Idiomarina zobellii

and Shewanella waksmanii. Members of the α-proteobacterial group included 3

producers of acidic MreBs, namely, Sulfitobacter sp. Fg 107, Sulfitobacter sp.

RIOSW6 and Loktanella vestfoldensis; and 3 producers of basic MreBs, namely,

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Sulfitobacter mediterraneus, Sulfitobacter delicatus and Loktanella rosea. Notably, pI

properties of MreBs turned out to be group (α/γ division) and genus independent.

Moreover, while environmental bacteria produced MreBs with pIs ranged from 4.88

of Pseudoalteromonas nigrifaciens to 8.91 of Shewanella waksmanii, all pathogens,

eubacterium and rabbit produced only acidic proteins, MreBs and actin, respectively.

The GRAVY indices provided an evaluation of the overall hydrophobicities of

MreBs and actin (see Table 12). All MreBs, with exception of actin

(GRAVY = -0.232); two α-proteobacteria, Loktanella rosea (GRAVY = -0.049) and

Sulfitobacter delicatus (GRAVY = -0.093); one γ-proteobacterium

Pseudoalteromonas haloplanktis (GRAVY = -0.038) had hydrophobic features. Actin

and Loktanella rosea had hydrophilic characteristics similar to those of two

membrane-spanning proteins: Torpedo californica acetylcholine receptor

(GRAVY = -0.22) and rabbit Ca2+-ATPase (GRAVY = -0.05) reported by Kyte

(1982). As for hydrophobic proteins, all pathogens produced only slightly

hydrophobic MreBs in the range between 0.04 - 0.061, while environmental bacteria

produced MreBs in the range between 0.028 - 0.309. Notably, inherently unstable

MreBs of Pseudomonas extremorientalis and Pseudoalteromonas nigrifaciens

appeared to be the most hydrophobic ones. Besides, Thermotoga maritima produced

moderately hydrophobic MreB (GRAVY = 0.193). To our knowledge, this is the first

study on physicochemical properties of PARP reporting theoretical pIs and GRAVY

indices; therefore, no data are available that can be comprehensively compared with

these results. Nevertheless, our calculations correlate well with previously reported

data on the average hydropathy of membrane-spanning proteins (Kyte and Doolittle,

1982). For example, membrane-spanning proteins such as human anion carrier, bovine

rodopsin and human glucose carrier had 0.04, 0.28 and 0.37 GRAVY scores,

respectively (Kyte and Doolittle, 1982). Kyte (1982) reported that GRAVY values for

membrane-spanning proteins were higher than those for soluble proteins (-0.4). It is

clear that GRAVY scores for membrane-spanning and/or membrane-associated

proteins such as MreB (Defeu Soufo and Graumann, 2005) are higher than for soluble

ones.

205

Table 12. Comparison of theoretical pI and GRAVY of MreB proteins of γ-

Proteobacteria (1), α-Proteobacteria (2), Firmicutes (3), Thermotogae (4) and rabbit

actin (5).

Bacte rial group

Strain Theore tical pI

Grand average of hydropa thicity

(GRAVY)

1 Pseudomonas fluorescens DSM 50030T (JF810206) 5.32 0.028 Pseudomonas extremorientalis KMM 3447T (JF815021) 5.61 0.309 Pseudoalteromonas nigrifaciens ATCC 19375T (JF815022)

4.88 0.300

Pseudoalteromonas haloplanktis ATCC 14393T (JF815023)

8.66 -0.038

Pseudoalteromonas atlantica ATCC 19262T (JF815020) 5.14 0.049 Alteromonas addita R10SW13T (JF815024) 8.28 0.033 Aliivibrio fischeri DSM 507T (DSM 507T) 6.38 0.035 Idiomarina zobellii KMM 231T (JF815025) 8.48 0.132 Idiomarina loihiensis (YP_154773.1) 5.04 0.106 Shewanella woodyi ATCC 51908T 5.27 0.085 Shewanella waksmanii KMM 3823T(JF815026) 8.91 0.198 Escherichia coli UM146 (gb ADN69460.1) 5.19 0.057

2 Sulfitobacter mediterraneus ATCC 700856T (JF825543) 8.00 0.171 Sulfitobacter delicatus KMM 3584T (JF825544) 8.68 -0.093 Sulfitobacter sp. Fg 107 (JF825545) 5.28 0.085 Sulfitobacter sp. RIOSW6 (JF825546) 5.27 0.085 Loktanella rosea Fg 1 (JF825547) 8.66 -0.049 Loktanella vestfoldensis (ZP_01001712.1) 6.03 0.050

3 Bacillus subtilis (gb AAA22397.1) 5.09 0.040 Listeria monocytogenes (gb CAC99626.1) 5.16 0.061

4 Thermotoga maritima MSB8 (gb AAD35673.1) 5.34 0.193 5 Oryctolagus cuniculus, actin, alpha skeletal muscle

(P68135) 5.23 -0.232

9.2.1.3. Phylogenetic relationships of MreB producers

Phylogenetic analysis of MreB sequences of 17 environmental bacteria, along

with published MreB sequences from three pathogenic bacteria and one eubacterium

Thermotoga maritima downloaded from GenBank, was carried out to examine the

relationships among proteins (i.e., stable and unstable MreBs and/or actin). In general,

inherently stable MreB proteins appeared to belong to evolutionary distant bacteria

206

such as the thermophilic eubacterium Thermotoga maritima; γ-proteobacteria

including seven environmental bacteria, namely, Pseudoalteromonas atlantica,

Alteromonas addita, Idiomarina zobellii, Idiomarina loihiensis, Pseudomonas

fluorescens, Shewanella waksmanii, Pseudoalteromonas haloplanktis and the

pathogenic adherent invasive Escherichia coli (AIEC); the α-proteobacteria

Loktanella vestfoldensis and Loktanella rosea; and pathogenic firmicutes. Thus, the

neighbour-joining tree obtained in analysis of the MreB sequences and actin clearly

demonstrated the presence of clades formed by distantly related bacteria such as

Pseudoalteromonas haloplanktis (Ivanova et al., 2001) and Loktanella rosea (Ivanova

et al., 2005). As discussed above, only MreBs of these two bacteria had negative

GRAVY scores like actin. Since actin is distantly related to MreBs, its phylogenetic

position was at the base of two GRAVY-negative sisters.

Although some bacteria produced very thermo- and inherently stable MreBs,

none of those MreBs was capable of living up to 100 hours in mammalian

reticulocytes, with exception of MreBs belonging to three close phylogenetic

relatives: one γ-proteobacterium Shewanella woodyi and two α-proteobacteria, namely

Sulfitobacter sp. RIOSW6 and Sulfitobacter sp. Fg 107. It should be noted that there

was a very strong support for the node uniting those three taxonomically distant

bacteria as bacteria theoretically producing MreBs with the longest half-lives (up to

100 h in mammalian reticulocytes, in vitro).

Furthemore, four very stable environmental bacteria were found to produce

MreBs with 30-hour half-lives. Notably, one out of the four producers was the α-

proteobacterium Loktanella vestfoldensis. This moderately thermophilic (grew at

45 ºC) resident of microbial mats in lake (Van Trappen et al., 2004) owned the most

stable MreB with stability and half-life similar to Thermotoga maritima (see Table 11

for more details). This result correlates with the observation reported by Ikai (1980).

The other very thermostable, but less inherently stable than Loktanella vestfoldensis’s

MreBs, belonged to three γ-proteobacteria, namely, the freshwater inhabitant

Pseudomonas fluorescens (does not grow at 42 ºC) (Lopez-Caballero et al., 2002), the

seaweed-associated marine bacterium Pseudoalteromonas atlantica (does not grow at

40 ºC) (Akagawa-Matsushita et al., 1992) and only one moderately thermophilic

207

hydrothermal vent bacterium Idiomarina loihiensis (grows at 46 ºC) (Donachie et al.,

2003). Importantly, each of these three γ-proteobacteria formed clade with one of

seven environmental γ-proteobacterial producers of thermo- and inherently stable

MreBs.

Interestingly, Loktanella vestfoldensis, a remarkable producer of MreB, had a

phylogenetic brother, Sulfitobacter delicatus, and two distant relatives, namely,

Pseudoalteromonas haloplanktis and Loktanella rosea. Although phylogenetic brother

did not produce stable MreBs, two other members of the common clade of

phylogenetic tree produced MreBs with actin-like overall hydrophobicity and inherent

stability.

It is important to note that two firmicutes, namely Bacillus subtilis and Listeria

monocytogenes, produced MreBs with similar physicochemical properties.

Phylogenetic positions of pathogens dictated by MreBs properties (see Figure 31)

support the conclusion, reported in subsection 2.5.3.1.1., that MreBs of firmicutes

differed from MreB of Thermotoga maritima. Moreover, MreB of the pathogenic

γ-proteobacterium Escherichia coli appeared to have more similarity with MreB of the

γ-proteobacterium Aliivibrio fischeri than with MreBs of pathogenic firmicutes.

208

Figure 31. Protein neighbor-joining phylogenetic tree shown is based on MreB sequences from heterotrophic bacteria using Thermotoga maritima as outgroup. Rabbit actin from Oryctolagus cuniculus is in red. Producers of in vivo stable proteins (instability indices < 40) are indicated by bold font; producers of in vitro stable proteins (half-lives of proteins in mammalian reticulocytes are 100 h) are indicated by underlined letters. Bootstrap values are shown as percentages of 1000 replicates. Bar, 0.05 amino acid substitutions per site.

209

9.2.2. Comparison/Evaluation of predicted physicochemical properties of FtsA

proteins of selected bacterial taxa and actin

9.2.2.1. Stability of FtsA proteins and actin

The same stability parameters as for MreBs (see subsection 9.2.1.1 for details)

were estimated for FtsA proteins of 30 heterotrophic bacteria belonging to 17 genera

and eukaryotic actin of rabbit skeletal muscle.

Calculation of AIs revealed that all FtsAs, including those of the thermophilic

bacterium Thermotoga maritima and 3 reference FtsAs of rod-shaped pathogens and

one FtsA of the pathogenic coccus Streptococcus pneumoniae, had higher AI values

than eukaryotic actin (see Table 13 for details). The aliphatic indices of all FtsAs

ranged from 88.40 of the radionuclide-sediment inhabitant Loktanella rosea Fg 1

(Ivanova et al., 2005) to 112.65 of the hydrocarbon-sediment inhabitant Marinobacter

hydrocarbonoclasticus (Gauthier et al., 1992). Strikingly, nine members of the γ-

proteobacterial group, namely, Cobetia marina, Marinobacter aquaeolei,

Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii, Marinomonas

communis, Marinomonas vaga, Marinomonas pontica, Idiomarina loihiensis and the

pathogenic adherent invasive Escherichia coli (AIEC); one member of the CFB group,

namely Salegentibacter flavus; one firmicute Streptococcus pneumoniae and the

thermophilic bacterium Thermotoga maritima turned out to have the highest aliphatic

indices climbing above 100. Again, similar to results reported in subsection 9.2.1.1,

estimate of thermostability of proteins by means of AIs confirmed the dependence of

thermostability of some FtsAs and actin on thermostability of their owners.

Nevertheless, many questions concerning influence of protein origin on its stability

remain unanswered. For example, not only FtsAs of moderately thermophilic

Marinobacter aquaeolei, Idiomarina loihiensis and Marinobacter

hydrocarbonoclasticus but also FtsAs of mesophilic Marinomonas pontica,

Salegentibacter flavus, Marinomonas vaga and Oceanimonas doudoroffii appeared to

be very thermostable. Moreover, FtsA with the highest AI was produced by a

moderately thermophilic (grows at 45 ºC) Marinobacter hydrocarbonoclasticus

210

(Gauthier et al., 1992) even though Marinobacter hydrocarbonoclasticus is not as

thermophilic as Thermotoga maritima. It is essential to note that MreB of the

thermophilic eubacterium was more thermostable than FtsA (see subsection 9.2.1.1.).

This evidence suggests that this bacterium can produce proteins of the same

superfamily, for example actin superfamily, with different thermostabilities. In

contrast to Thermotoga maritima, the γ-proteobacterium Escherichia coli had more

thermostable FtsA than MreB. In case of protein production by firmicutes, there was

no difference between thermostabilities of two PARPs of Bacillus subtilis and Listeria

monocytogenes. From these results comes the conclusion that not only pathogenic

lifestyle but also cell structure may contribute to thermostability of cell proteins.

Assessment of FtsA inherent stability based on dipeptide composition

(Guruprasad et al., 1990) categorized 13 proteins including 8 FtsAs of γ-

proteobacteria belonging to Marinobacter hydrocarbonoclasticus, Oceanimonas

doudoroffii, Marinomonas communis, Marinomonas vaga, Aliivibrio fischeri,

Shewanella woodyi, Shewanella japonica and a reference pathogen Escherichia coli;

one representative of the Cytophaga–Flavobacterium–Bacteroides (CFB) group

Salegentibacter flavus; two FtsAs of pathogenic firmicutes Listeria monocytogenes

and Streptococcus pneumoniae; one FtsA of Thermotoga maritima and eukaryotic

actin as stable proteins (see Table 13 for details). Interestingly, none of α-

proteobacteria appeared to produce inherently stable and/or very thermostable FtsAs.

Strikingly, 2 out of 13 very thermostable FtsAs belonged to a model pathogen Listeria

monocytogenes (see subsections 2.5.3.1.2. and 2.5.6.5.3. for details) and the

thermophilic eubacterium Thermotoga maritima. Although 6 out of 13 inherently

stable FtsAs (IIs < 40) were very thermostable, candidate protein for actin

(AI = 81.78; II = 36.14) substitution in microdevices must be both very thermostable

(AI > 100) and inherently stable (II < 36.14). So, only three out of 6 environmental

bacteria, namely, Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii and

Salegentibacter flavus can be considered the most valuable FtsA producers.

An estimate of half-lives of proteins based on the N-end rule (Varshavsky,

1997) revealed 10 theoretically very stable (half-lives in mammalian reticulocytes up

to 100 h) proteins: 8 FtsAs of the γ-proteobacteria, namely, Cobetia marina,

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Marinobacter hydrocarbonoclasticus, Alteromonas macleodii, Oceanimonas

doudoroffii, Oceanimonas smirnovii, Marinomonas vaga, Marinomonas pontica,

Aliivibrio fischeri; one FtsA of the α-proteobacterium Loktanella rosea Fg 1 and one

FtsA of the CFB bacterium Salegentibacter flavus (see Table 13 for details). Like

MreBs of the eubacterium Thermotoga maritima and pathogens, FtsAs of the former

and the latter had moderate half-lives (30 h in mammalian reticulocytes, in vitro).

Remarkably, three FtsAs belonging to Marinobacter hydrocarbonoclasticus,

Oceanimonas doudoroffii and Salegentibacter flavus were not only very thermo- and

inherently stable but also the longest living.

Table 13. Comparison of theoretical stability parameters (AI, II and half-life) of FtsA

proteins of γ-Proteobacteria (1), α-Proteobacteria (2), CFB group (3), Firmicutes (4),


Bacte rial group

Strain Alipha tic

index (AI)

Insta bility index

(II)

Estimated half-life, h

Mamma lian

reticulo cytes,

in vitro

Yeast, in vivo

Escheri chia coli,

in vivo

1

Cobetia marina LMG 2217T (JF893438)

102.52 45.71 100 h > 20 h > 10 h

Marinobacter aquaeolei (ABM19523.1)

105.55 43.35 30 h > 20 h > 10 h

Marinobacter hydrocarbonoclasticus ATCC 49840T (JF893439)

112.65 32.02 100 h > 20 h > 10 h

Pseudoalteromonas issachenkonii KMM 3549T (JF893436)

97.45 44.14 30 h > 20 h > 10 h

Pseudoalteromonas nigrifaciens ATCC 19375T (JF893437)

99.51 44.86 30 h > 20 h > 10 h

Pseudoalteromonas atlantica (ABG42017.1)

95.55 41.66 30 h > 20 h > 10 h

Alteromonas macleodii ATCC 27126T

96.09 45.26 100 h > 20 h > 10 h

Oceanimonas doudoroffii ATCC 27123T (JF893440)

110.13 32.02 100 h > 20 h > 10 h

212

1

Oceanimonas smirnovii 31-1T (JF893441)

98.65 44.76 100 h > 20 h > 10 h

Marinomonas communis ATCC 27118T (JF893442)

110.5 38.17 1.1 h 3 min > 10 h

Marinomonas vaga ATCC 27119T (JF893443)

109.35 39.20 100 h > 20 h > 10 h

Marinomonas pontica 46-16T (JF893444)

110.0 47.03 100 h > 20 h > 10 h

Aliivibrio fischeri DSM 507T (JF893445)

98.65 39.32 100 h > 20 h > 10 h

Idiomarina baltica (ZP_01043577.1)

98.15 47.20 30 h > 20 h > 10 h

Idiomarina loihiensis (AAV81283.1)

101.04 47.55 30 h > 20 h > 10 h

Shewanella woodyi ATCC 51908T

95.14 36.94 1.9 h > 20 h > 10 h

Shewanella affinis KMM 3587T (JF893433)

95.71 40.95 1.9 h > 20 h > 10 h

Shewanella waksmanii KMM 3823T (JF893434)

94.60 42.91 > 20 h > 20 h ND

Shewanella japonica KMM 3299T (JF893435)

92.47 36.88 1.9 h > 20 h > 10 h

Escherichia coli UM146 (gb ADN73972.1)

102.29 38.66 30 h > 20 h > 10 h

2 Sulfitobacter pontiakus DSM 10014T (JF893447)

91.51 47.73 0.8 h 10 min 10 h

Sulfitobacter delicatus KMM 3584T (JF893448)

89.78 48.17 1 hour 2 min 2 min

Sulfitobacter sp. Fg 107 (JF893449)

88.45 48.43 1 hour 2 min 2 min

Sulfitobacter sp. RIOSW6 (JF893450)

90.82 49.67 7.2 h > 20 h > 10 h

Loktanella rosea Fg 1 (JF893451)

88.40 47.53 100 h > 20 h > 10 h

3 Salegentibacter flavus Fg 69T (JF893446)

110.13 32.02 100 h > 20 h > 10 h

4 Bacillus subtilis (gb AAA22456.1)

96.09 47.73 30 h > 20 h > 10 h

Listeria monocytogenes (gb CAD00111.1)

98.59 29.19 30 h > 20 h > 10 h

Streptococcus pneumoniae DD39 (YP_816936.1)

100.44 31.50 30 h > 20 h > 10 h

5 Thermotoga maritima MSB8 (NP_229082.1)

106.10 29.21 30 h > 20 h > 10 h

6 Oryctolagus cuniculus, actin, alpha skeletal muscle (P68135)

81.78 36.14 30 h > 20 h > 10 h

213

9.2.2.2. Isoelectric point (pI) and grand average of hydropathicity (GRAVY) of

FtsAs and actin

An estimate of pI and grand average of hydropathicity (GRAVY) of FtsAs of

30 bacteria, including 19 environmental γ-proteobacteria and 1 pathogenic

γ-proteobacterium Escherichia coli (AIEC), 5 environmental α-proteobacteria, 1 CFB

group member, 3 pathogenic firmicutes, 1 thermophilic eubacterium, and eukaryotic

actin of rabbit skeletal muscle revealed that all proteins were acidic (see Table 14 for

details).

The GRAVY indices provided an evaluation of the overall hydrophobicities of

FtsAs and actin (see Table 14). Majority of FtsAs were slightly hydrophilic, with

exception of Marinobacter aquaeolei (GRAVY = 0.125), Marinobacter

hydrocarbonoclasticus (GRAVY = 0.152), Pseudoalteromonas nigrifaciens

(GRAVY = 0.006), Oceanimonas doudoroffii (GRAVY = 0.145), Marinomonas

communis (GRAVY = 0.125), Marinomonas vaga (GRAVY = 0.154), Marinomonas

pontica (GRAVY = 0.096), Aliivibrio fischeri (GRAVY = 0.026), Idiomarina

loihiensis (GRAVY = 0.036), one α-proteobacterium Loktanella rosea

(GRAVY = 0.012) and a member of the CFB group, namely Salegentibacter flavus,

(GRAVY = 0.135). So, the three thermo- and inherently stable FtsAs (see subsection

9.2.2.1. for details) were the most hydrophobic ones (see Table 14). To our

knowledge, no data are available that can be comprehensively compared with these

results except for data on the average hydropathy for membrane-spanning proteins

(Kyte and Doolittle, 1982) discussed in subsection 9.2.1.2., which correlates well with

calculations presented in this subsection.

214

Table 14. Comparison of theoretical pI and GRAVY of FtsA proteins of γ-Proteobacteria (1), α-Proteobacteria (2), CFB group (3), Firmicutes (4), Thermotogae (5) and rabbit actin (6).

Bacte rial group

Strain Theore tical pI

Grand average of hydropathicity (GRAVY)

1

Cobetia marina LMG 2217T (JF893438) 4.7 -0.001 Marinobacter aquaeolei (ABM19523.1) 5.15 0.125 Marinobacter hydrocarbonoclasticus ATCC 49840T (JF893439)

4.82 0.152

Pseudoalteromonas issachenkonii KMM 3549T (JF893436) 4.61 -0.030 Pseudoalteromonas nigrifaciens ATCC 19375T (JF893437) 4.8 0.006 Pseudoalteromonas atlantica (ABG42017.1) 5.0 -0.017 Alteromonas macleodii ATCC 27126T 4.63 -0.096 Oceanimonas doudoroffii ATCC 27123T (JF893440) 4.82 0.145 Oceanimonas smirnovii 31-1T (JF893441) 4.79 -0.099 Marinomonas communis ATCC 27118T (JF893442) 4.4 0.125 Marinomonas vaga ATCC 27119T (JF893443) 4.73 0.154 Marinomonas pontica 46-16T (JF893444) 4.76 0.096 Aliivibrio fischeri DSM 507T (JF893445) 4.83 0.026 Idiomarina baltica (ZP_01043577.1) 4.93 -0.030 Idiomarina loihiensis (AAV81283.1) 4.88 0.036 Shewanella woodyi ATCC 51908T 4.73 -0.139 Shewanella affinis KMM 3587T (JF893433) 4.63 -0.139 Shewanella waksmanii KMM 3823T (JF893434) 4.63 -0.163 Shewanella japonica KMM 3299T (JF893435) 4.62 -0.178 Escherichia coli UM146 (gb ADN73972.1) 5.94 -0.040

2 Sulfitobacter pontiakus DSM 10014T (JF893447) 4.92 -0.024 Sulfitobacter delicatus KMM 3584T (JF893448) 5.71 -0.037 Sulfitobacter sp. Fg 107 (JF893449) 5.62 -0.100 Sulfitobacter sp. RIOSW6 (JF893450) 4.88 -0.018 Loktanella rosea Fg 1 (JF893451) 5.64 0.012

3 Salegentibacter flavus Fg 69T (JF893446) 4.82 0.135 4 4

Bacillus subtilis (gb AAA22456.1) 5.27 -0.249 Listeria monocytogenes (gb CAD00111.1) 4.63 -0.062 Streptococcus pneumoniae DD39 (YP_816936.1) 5.12 -0.063

5 Thermotoga maritima MSB8 (NP_229082.1) 5.21 -0.084 6 Oryctolagus cuniculus, actin, alpha skeletal muscle

(P68135) 5.23 -0.232

215

9.2.2.3. Phylogenetic relationships of FtsA producers

Phylogenetic analysis of FtsA sequences of 25 environmental bacteria,

together with published FtsA sequences of 4 pathogenic bacteria and 1 eubacterium

Thermotoga maritima downloaded from GenBank, was performed to examine the

relationships among proteins (i.e., stable and unstable FtsAs and/or actin). In general,

inherently stable FtsAs appeared to belong to evolutionary distant bacteria such as 8 γ-

proteobacteria, 1 CFB group member, 2 firmicutes and 1 eubacterium.

Thus, the neighbour-joining tree obtained in analysis of the FtsA sequences

and actin clearly demonstrated that producers of inherently stable proteins branched

with their closest phylogenetic relatives. For example, 4 members of the genus

Shewanella, namely, 2 bacteria with inherently stable and 2 bacteria with slightly

inherently unstable FtsAs (see Table 13) constituted one clade (see Figure 32).

Furthermore, actin branched with α-proteobacteria with strong support by bootstrap

analysis. Since the contribution of α-proteobacteria to cell evolution has been under

discussion during the last decade (Vellai et al., 1998, Vellai and Vida, 1999, Vesteg

and Krajcovic, 2008), this phylogenetic relationship may be taken into consideration;

it can give us additional clue to unravel the mystery of cell origins. Moreover,

phylogenetic positions of Aliivibrio fischeri, Escherichia coli and Oceanimonas

doudoroffii shed some evolutionary light on their relationships by supporting the

conclusion that they are phylogenetic neighbors (Ivanova et al., 2004h, Dorsch et al.,

1992).

Furthemore, three environmental bacteria, namely two γ-proteobacteria,

Marinobacter hydrocarbonoclasticus and Oceanimonas doudoroffii, and the CFB

bacterium Salegentibacter flavus, were found to produce FtsAs with 100-hour half-

lives. Despite belonging to different groups, these three bacteria appeared to be three

brothers producing FtsAs with remarkable physicochemical properties.

Phylogenetic positions of pathogens dictated by FtsAs properties (see


216

Figure 32) support the conclusion that FtsAs of all firmicutes and Escherichia coli

differed from FtsA of Thermotoga maritima. Moreover, MreB as well as FtsA protein

of Escherichia coli appeared to have more similarity with MreB and FtsA of the

γ-proteobacterium Aliivibrio fischeri than with those of pathogenic firmicutes.

217

Figure 32. Protein neighbor-joining phylogenetic tree shown is based on FtsA sequences from heterotrophic bacteria using Thermotoga maritima as outgroup. Rabbit actin from Oryctolagus cuniculus is in red. Producers of in vivo stable proteins (instability indices < 40) are indicated by bold font; producers of in vitro stable proteins (half-lives of proteins in mammalian reticulocytes are 100 h) are indicated by underlined letters. Bootstrap values are shown as percentages of 1000 replicates. Bar, 0.05 amino acid substitutions per site.

218

9.3. Conclusion

The information gained from physicochemical and phylogenetic analyses of 21

MreB and 30 FtsA sequences of environmental, pathogenic, thermophilic bacteria and

sequence of eukaryotic skeletal actin presented here provided some valuable insight

into understanding the difference between eukaryotic actin and its prokaryotic

homologues. Thus, some PARPs produced by environmental bacteria had the longest

half-lives and/or were more thermo- and inherently stable than actin.

The study of MreB sequences demonstrated that very thermo- and inherently

stable MreB proteins with moderate half-lives (30 h in mammalian reticulocytes, in

vitro) were produced by three phylogenetically distant bacteria, namely,

Pseudoalteromonas atlantica, Loktanella vestfoldensis and Thermotoga maritima;

these three MreBs can be used instead of actin for building linear molecular

assemblies in microdevices, however, after preliminary in vitro evaluation of their

biochemical properties. Although our results called attention to the finding that three

members of the same phylogenetic clade, namely, Shewanella woodyi, Sulfitobacter

sp. RIOSW6 and Sulfitobacter sp. Fg 107 (Figure 31), produced MreBs with 100-h

half-lives (mammalian reticulocytes, in vitro), the MreB producers were not considered

good candidates for nanothechnological application in microdevices due to moderate

thermostabilities and inherent instabilities of their MreBs.

Remarkably, 3 FtsAs belonging to Marinobacter hydrocarbonoclasticus,

Oceanimonas doudoroffii and Salegentibacter flavus were not only very thermo- and

inherently stable but also the longest living (100-h half-lives of proteins in mammalian

reticulocytes, in vitro), therefore, if they can produce actin-like linear assemblies, they

can be used as replacements for eukaryotic actin in microdevices. Moreover, data

obtained pointed to a correlation between phylogenetic analyses based on FtsA and

16S rRNA sequences, therefore, FtsA gene may be used as a chronometer to help

unravel phylogenies of new and/or misplaced bacteria.

219

CHAPTER 10

CONCLUSIONS AND FURTHER WORK

220

10.1. Conclusions

10.1.1. Overview

A novel approach for designing the surfaces of microdevices has been

proposed. Our approach is based on ‗combinatorialized‘ micro/nano-channels that

allow amplified protein immobilization in a highly controlled manner (Ivanova et al.,

2004d, Ivanova et al., 2003b, Nicolau et al., 2010a, Ivanova et al., 2004f, Nicolau et

al., 2010b). An innovative methodology allowing in vitro assembly of micron- and

nano-scale tracks of protein (i.e., actin) which support unidirectional translocation of

beads functionalized with motor proteins (i.e., myosin) was also developed. It was

further suggested that in order to advance the stability and efficiency of microdevices

based on molecular motor systems: i) substitution of commercial ATP with ATP

produced by bacteria and b) substitution of eukaryotic actin with prokaryotic actin-

related proteins, e.g., MreB or FtsA, may be considered. Employment of bacterial

ATP, rather than a commercial ATP product, and real-time production of ATP are

required to sustain the self-assembly of actin or its homologues and to make the

molecular motor system as a whole more stable and long lasting. This can be achieved

by incorporation of efficient bacterial ATP producers into the next generation of

microdevices. The suitability of the employment of bacterial ATP producers and

prokaryotic actin-related proteins such as MreB or FtsA protein as replacements for

the energy source and eukaryotic actin (see subsection 2.5. and chapter 6 for more

details), respectively, in the construction of the next generation of microdevices was

also evaluated. From all bacteria isolated, bacteria of the genera Sulfitobacter,

Marinobacter and Staleya and/or Planococcus and Kocuria were found to be the most

promising producers of extracellular ATP.

10.1.2. Protein immobilization in ‘combinatorialized’ micro/nano-channels

A comparative study investigating the immobilization pattern of five proteins

belonging to three major structural classes was carried out. The proteins were

221

immobilized along microchannels, fabricated by laser microablation of thin

metal/blocking protein layers deposited on a polymeric substrate. It has been shown

that the protein adsorption was amplified between 3 to 10 times depending on the

molecular surface of the protein (see chapter 5). The results obtained demonstrated

that physicochemical adsorption of HSA, HIgG, α-chymotrypsin, lysozyme, and

myoglobin in the microchannels was at least 2.5 to 5 times greater than that on the

plain PMMA polymeric surfaces (Ivanova et al., 2004d, Ivanova et al., 2003b, Nicolau

et al., 2010a, Ivanova et al., 2004f, Nicolau et al., 2010b). A surface mass density of

adsorbed protein molecules on the latter, defined by a protein-film thickness and a

refractive index for the protein layer, correlated with the data obtained for

fluorescently labeled proteins. Thus, different types of proteins were found to be

immobilized at increased levels retaining their bioactivities. It was concluded that the

amplified and ‗combinatorialized‘ adsorption on micro/nano-structures has the

potential of improving detection of multiplex analytes if used for microdevices.

10.1.3. Controlled self-assembly of actin filaments along microchannels in a

continuous-flow system

Although different methodologies have been applied to align actin and actin-

based motility through a variety of techniques, e.g., myosin guiding (Butt et al., 2009),

magnetic field (Kaur et al., 2010), electric field (Wigge et al., 2010), UV lithography

(Yamamoto et al., 2008), they are not suitable for the fabrication of aligned actin

tracks, which can support unidirectional bead translocation in vitro, due to the lack of

precise control over them at the level of either individual or bundled linear assemblies.

To solve this problem, we developed a methodology allowing assembly of F-

actin filament tracks that can support the movement of cargo particles (Alexeeva et al.,

2004a, Watson et al., 2004, Alexeeva et al., 2005). In this work PSMA polymeric

surfaces were used for the immobilization of self-assembly of the actin filaments in

vitro in a continuous-flow system. Gelsolin was used to induce cooperative transition

in actin via a structural perturbation of the barbed end of monomeric actin resulting in

formation of regularly organized actin/gelsolin bundles that supported directional bead

222

movement. The progressive formation of F-actin/gelsolin filaments by electrostatic

condensation with Ba2+ and alignment of actin/gelsolin bundles was also

demonstrated. This study established that the developed simple technique for actin-

filament-bundle fabrication provides a convenient experimental system that may be

applicable for the next generation of microdevices.

10.1.3.1. Search for bacterial ATP producers to be used as replacements for the

energy source in microdevices

In order to evaluate the possibility of the employment of bacterial producers of

ATP as replacements for the energy source in the construction of the next generation

of microdevices, a search for ATP producers among 86 environmental strains

belonging to several phylotypes of the domain Bacteria has been performed. A

collection of environmental (marine and freshwater) bacteria comprising 17 genera is

maintained at Swinburne University of Technology, Faculty of Life and Social

Sciences.

It was demonstrated that gram-negative bacteria of the genera Sulfitobacter,

Staleya, and Marinobacter secreted elevated amounts of extracellular ATP while

gram-positive bacteria of the genera Kocuria and Planococcus secreted high amounts

of intracellular ATP. Variations in the levels of extracellular and intracellular ATP-

dependent luminescence monitored in living cells of Sulfitobacter mediterraneus

ATCC 700856T and Planococcus maritimus F 90 (the latter is described in subsections

7.2.1.2.1 and 7.2.2.2.1) during 48 h of attachment on different surfaces demonstrated

that bacteria were capable of producing either extra- or intracellular ATP, depending

on the experimental conditions (as described in subsections 8.2.2 – 8.2.4). It was

found that the levels of extracellular ATP generated by Sulfitobacter mediterraneus

during attachment on PtBMA and mica were two to five times higher than those

detected during the initial screening. High-resolution AFM imaging revealed a

potentially interesting correlation between the porous cell-surface of certain α- and γ-

proteobacteria and their ability to secrete high amounts of ATP.

223

Thus, our results have provided important insights into understanding the

impact of surface hydrophobicity on bacterial attachment, ATP generation, and further

modeling of bacterial metabolism. It was concluded that gram-negative extracellular

ATP producers belonging to the genera Sulfitobacter, Marinobacter and Staleya

and/or gram-positive intracellular ATP producers belonging to the genera Planococcus

and Kocuria can be considered as valuable candidates for the replacement of the

energy source in the next generation of microdevices.

10.1.3.2. Evaluation of prokaryotic actin-related proteins, MreB and FtsA, as

possible replacements for eukaryotic actin

Since the lifetime of microdevices depends primarily on the stability of their

biological components, the possibility of replacement of eukaryotic actin with

comparatively more stable prokaryotic homologue/s was evaluated. Several bacterial

taxa were selected and tested as prospective candidates for MreB and/or FtsA

production. The information gained from physicochemical and phylogenetic analyses

of 21 MreB and 30 FtsA sequences of environmental, pathogenic, and thermophilic

bacteria presented here provided some valuable insights into understanding the

differences between eukaryotic actin and its prokaryotic homologues. After

preliminary evaluation of their biochemical properties in vitro, analysis of available

literature indicated that three phylogenetically distant bacteria, namely,

Pseudoalteromonas atlantica, Loktanella vestfoldensis and Thermotoga maritima may

be used as the sources of very thermo- and inherently-stable MreB proteins

(Guruprasad et al., 1990, Ikai, 1980) with moderate half-lives (in mammalian

reticulocytes in vitro up to 30 h) (Bachmair et al., 1986) to replace actin in

microdevices. Similar analysis of FtsA sequences available in public databases

indicated that Marinobacter hydrocarbonoclasticus, Oceanimonas doudoroffii and

Salegentibacter flavus may be used as the sources of thermo- and inherently-stable

FtsA proteins with the longest half-lives (in mammalian reticulocytes in vitro up to

100 h).

224

10.2. Future work

10.2.1. Advancements of surface modification

It is anticipated that the newly developed approach for the fabrication of 100

nm-range microstructures with combinatorial surfaces based on laser microablation of

thin metal/blocking protein layers deposited on a PMMA substrate can be used for

production of surfaces suitable for other types of biomolecule immobilization. The

direction of this research remains to be evaluated. Furthermore, to gain a better

understanding of the degree of independence of molecular behavior on their

descriptors on combinatorial surfaces, the distribution profile of structurally different

molecules within channels should be estimated. Substitution of PMMA with an

optically suitable substratum, e.g., PtBMA may be a useful alternative and should be

tested to evaluate contributions of functional group interactions with the deposited

biomolecules. Accordingly, the laser parameters (e.g., energy, intensity and fluency)

should be adjusted for optimum ablation of other alternative fabrication materials.

10.2.2. Incorporation of ATP-producers into microdevices

The incorporation of bacterial producers in the next generation microdevices is

another possible direction for future work. Bacterial producers can be immobilized on

the suitable surfaces of separate microstructures which are connected directly to the

channels with motor proteins. This is a challenging task as the establishment of a

reliable network between ATP producing bacteria and motor proteins will be required.

10.2.3. Study of MreB and FtsA proteins in vitro

Optimization of prokaryotic actin-related protein extraction and comparative

physicochemical analysis of eukaryotic actin and its prokaryotic homologues (MreB

and FtsA) will be essential for realization of the employment of these proteins for

design of new microdevices.

225

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