Molecular cloning and characterization of

the allophycocyanin gene from Spirulina platensis Cl

MissDauenpen-NIeesapyodsuk B.Sc. (Nursing and Midwifery)

A Thesis Submitted in Partial Fulfillment of the Requirements

for the Degree of Master of Science

Biotechnology Program

School of Bioresources and Technology

King Mongkut’s Institute of Technology Thonburi

1996

Thesis Committee

-5 &L?i,diL LUL 77iLz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chairman

(Asst. Prof. Dr. Supapon Cheevadhanarak)

(&t&J+ c/lzh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Co-Chairman

(Asst. Prof. Suchada Chaisawadi)

h&J f&h ,,a, oy &/-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Member

(Assoc. Prof. Dr. Morakot Tanticharoen)

. . . . . . . . . . . . . . . . . . . p &t?n, h++L..:................................................................ Member

(Dr. Patcharaporn Deshnium)

ISBN 9741621-904-g

Copyright reserved

ii

Thesis Title Molecular cloning and characterization of the

allophycocyanin gene from Spirulina platensis Cl

Thesis Credits 12

Candidate .Mi-ssDauenpen Meesapyodsuk

Supervisors Asst. Prof. Dr. Supapon Cheevadhanarak

Asst. Prof. Suchada Chaisawadi

Degree of Study Master of Science

Department Biotechnology

Academic Year 1996

Abstract

The apcABC genes encoding a, p subunits of allophycocyanin

and partial part of core linker protein were isolated from S. platensis Cl.

Based on sequence comparisons of apt genes from Synechococcus

PCC7002, Synechococcus PCC6301 and Cyanophora paradoxa, two

highly conserved regions were selected for the synthesis of 2

oligonucleotides, primers for amplifying apt gene probe from genomic

DNA of S. platensis Ci by polymerase chain reaction technique. The

amplified probe was used for screening S. platensis Ci genomic DNA

library. By homologous hybridization the upstream sequence of apcA

and partial sequence of the coding region of apcA gene was obtained. In

order to clone the entire apt genes, primer down in the apcC was

designed from the conserved sequence of the corresponding genes from

five cyanobacteria (Synechococcus PCC6301, Synechocystis PCC6803,

Synechococcus elongatus, FremyeZZa diplosiphon and Synechococcus’v

PCC7002) and primer up from a part of the upstream sequence of apcA

. . .1 1 1

isolated from genomic DNA library. By using these two primers, an

amplified DNA fragment of 1.8 kb was obtained, and its nucleotide

sequence was determined and analysed. Two complete open reading

frames of-qcA and-+zB (16 1 amino acids-for -each gene), and a partial

one of apcC (36 amino acids) were found. The identity of the deduced

amino acid sequence of the products of apcA and apcB to those of

Synechococcus elongatus, Cyanophora paradoxa and Synechocystis

PCC6714 were 85,82 and 80% for apcA, and 93, 84 and 88% for apcB,

respectively. For apcC, the identity of amino acid sequences between S.

platensis Ci and Synechococcus elongatus, Synechocystis PCC67 14 and

Fremyella diplosiphon were 92, 92 and 92%, respectively. Expected

chromophore attachment sites were found at position 81 in both amino

acid sequences of apcA and apcB. The sequence analysis also

demonstrated that the obtained genes form an operon, apcABC, with a

single transcription start site and one possible termination site

downstream of apcB. In addition, by sequence comparison of the

upstream region of apcA of S. platensis Ci with those from other

cyanobacteria, it was found that the apcABC promoters lied in the

conserved sequences centered at -50 and -10 with respect to the start of

transcription.

These results suggest that the obtained genes are putative

apcABC operon of S. platensis Cl.

Keywords: Spirulina platensis Ci/ allophycocyanin gene/ apcABC

operon

iV

V

illfil~ElJ (keywords): ~?‘l@El~$7~ULLflU!,~~al Spiruha platensis/ $pd allophycocyanin/

apcABC operon

Vi

Acknowledgements

The au thor i s g ra te fu l to Ass t . Prof . Dr . Supapon

Cheevadhanarak, S.choul~ of Bioresources and Technology, King

Mongkut’s Institute of Technology Thonburi (KMITT), for advice on

all aspects of the work, reading and kindly suggesting improvements of

the manuscript and Asst. Prof. Suchada Chaisawadi, Pilot Plant

Development and Training Institute (PDTI), for her excellent technical

assistance, reading and discussion the manuscript.

I also wish to thank Assoc. Prof. Dr. Morakot Tanticharoen and

Dr. Patcharaporn Deshnium, National Center for Genetic Engineering

and Biotechnology (BIOTEC), for guidance, discussion and reading the

manuscript.

I would like to thank Dr. Christophe Anjard for his valuable

discussion.

Contents

Page

English abstract - - ~._ - ~-

Thai abstract

Acknowledgements

Contents

List of Tables

List of Figures

Abbreviations

Chapter 1 Introduction

1.1 Background

1.2 Objectives

1.3 Scopes

Chapter 2 Literature Review

2.1 Cyanobacteria

2.2 Spirulina

2.3 Phycobilisome and Phycobiliprotein Structure

2.3.1 Phycobilisome Components

2.3.2 Phycobiliproteins

2.3.2.1 PBPs constituing the PBsome core

2.3.2.2 PBPs constituing the rod

elements of PBsome

2.3.3 Linker polypeptides

2.3.4 Pigments

2.3.5 Organization of the genes encoding

the PBsome

ii _

iv

vi

vii

X

xi

xiv

1

1

2

3

4

4

7

1 5

1 5

21

2 6

3 1

38

41

44:r,

. . .Vlll

2.3.6 Energy Transfer in PBsomes

2.4 The molecular biology of cyanobacteria

2.4.1 Genome size of cyanobacteria

-_ - 2.42hlalecularcloning ofphycobilisome

in Cyanobacteria

Chapter 3 Materials and Methods

3.1 Organisms and plasmids

3.1.1 Cyanobacterium strain

3.1.2 Bacterial strains

3.1.3 Plasmids

3.2 Chemicals

3.3 Enzymes

3.4 Media and culture conditions

3.4.1 Spirulina platensis

3.4.2 Bacteria

3.5 Buffers and Solutions

3 .6 Primers/Oligonucleotide synthesis

3.6.1 Primers for PCR

3.6.2 Primers for sequencing

3.7 Molecular biology techniques

3.7.1 Plasmid preparation

3.7.2 Bacterial transformation

3.7.3 Subcloning of DNA fragments

3.7.4 Plating bacteriophages

3.7.5 Preparation of DNA from bacteriophages

3.7.6 DNA sequencing

3.7.7 Southern blotting

4 7

4 8

4 8

48 -

54

54

54

54

55

55

55

55

56

58

59

6 0

6 0

6 1

6 1

6 1

62

63

6 4

65

67k

68

ix

3.7.8 DNA labelling with 32P-dCTP

3.7.9 Dot blotting

3.8 Cloning of allophycocyanin gene from

-_ S. p&ends Cl strain - ~-

3.8.1 Isolation of genomic DNA

3.8.2 Amplification of partial apcAB from genomic

DNA of S. platensis

3.8.3 Screening of genomic library of S. platensis

3.8.4 Southern blot analysis

3.8.5 Characterization of positive clones

3.8.6 Amplification of complete apcAB and partial

apcC from genomic DNA of 5’. platensis

Chapter 4 Results and Discussions

4.1 Construction of apcAB probe from 5’. platensis

4.2 Isolation of allophycocyanin gene from genomic

library of S. platensis

4.3 Cloning of apcAB and partial apcC of 5’. platensis

4.4 Nucleotide sequence analysis

Chapter 5 Conclusion and Suggestion

5.1 Conclusion

5.2 Suggestion

References

Appendix

6 9

6 9

7 0

_

7 0

7 1

73

73

7 4

7 4

7 7

7 7

7 8

86

87

100

100

101

102

116

X

List of Tables

Table

2.1

2.2

2.3

2 .4

2.5

Comparison of-protein content in Spirdina _

and other foods (%)

Chemical analysis of Spirulina from spray dried

Phycobilisome polypeptides and their genes

Abbreviations for the biliprotein subunits and for linker

polypeptides

Genes of 5’. platensis that have been cloned and characterized

as documented in Genbank

Page

10 _

11

2 2

25

5 1

xi

List of Figures

Figure Page

2.1 Schematic diagram&a thin-section ofa cyanobacterial cell 5 _

2 .2 Artist’s representation of the overall three dimensional 6

architecture of Agmenellum quaduplicatum

2.3 Morphology of Spirulinaplatensis 8

2.4 Different morphological types in Spirulina 9

2.5 Life cycle of Spirulina 14

2 .6 Schematic structure of phycobilisomes structure attaching 16

to the photosynthetic membranes or thylakoid membranes

2 .7 Phycobilisomes with two respective three core 1 8

cylinders in cross-section

2 .8 Schematic representation of the hemidiscoidal 1 9

phycobilisomes structure

2 .9 Stereoscopic plot of (@)-monomer

2.10 Schematic side view of a phycocyanin hexamer

2.11 General features of allophycocyanin subunits

2.12 General structure, common features and chromophore

variability of phycocyanins

2.13 General structure, common features and chromophore

variability of phycoerythrins

2.14 A model for rod biogenesis based on

in vitro rod assembly experiments

2.15 Structures of various types of phycobilins

found in cyanobacteria and red algae, linked

by thioether-linkages to PBPs

23

23

2 7

33

37

4 0

43

xii

2.16 Comparison of the organization and transcription of genes

encoding phycobilisome components for the

cyanobacteria Synechococcus sp. PCC7002,

Synechococc~ KC6301 and Anahaena sp. PCC7120

2.17 Energy flow in PBsome of cyanobacteria and red algae

3.1

3.2

3.3

4.1

4 .2

4.3

4 .4

4.5

4 .6

4 .7

Schematic setup of downward capillary transfer of DNA

Comparison of the AP a and p subunits amino acid sequences

of Synechococcus PCC7002, Synechococcus PCC630 1 and

Cyanophora paradoxa

Comparison of the amino acid sequences of

apcC gene products

The amplified PCR products from

genomic DNA of S. platensis

The nucleotide and deduced amino acid sequences

of partial upcAB gene from S. platensis

The deduced amino acid sequence of the amplified

DNA product from S. platensis compared with that of

Synechococcus elongatus, Cyanophora paradoxa and

Synechocystis 67 14

Southern blot analysis of two recombinants hDNAs

isolated from two positive clones, VA1 and VA2 respectively

Southern blot analysis of one positive clone (VAl)

Restriction map 1.6 kb EcoRI-EcoRV fragment of VA1

Lambda phage

The amplified PCR products from

genomic DNA of S. platensis

4 6

4 7

6 8

72

7 6

79

80

8 1

83

84

85

88

. . .x111

4 .8 The nucleotide and deduced amino acid sequences of

the complete apcAB and partial apcC gene of S. platensis Cl

4 .9 The alignment of the amino acid sequence

from-VcAB g - p r o d u c t ~- - . ~

4.10 The alignment of the partial amino acid sequence

from apcC gene product

9 0

9 1

s

92

4.11 Hybridization of the 1.1 kb EC&I-EcoRV fragment

carrying part of the apcAB gene to total genomic

S. platensis DNA

95

4.12 Hybridization of the 1.8 kb EcoRZ fragment of

PCR product carrying apcABC gene to total

genomic S. platensis DNA

96

4.13 Restriction map of a 2 1 kbp EcoRI fragment

of S. platensis composed of the apt gene region

4.14 Alignment of the putative of transcription

start sites and promoters

97

98

4.15 The putative hairpin loop and stem part at the

3’ end of the P-subunit of APC of S. platensis Cl

using GENETYX-MAC program

99

xiv

Abbreviations

DNA

w

*g

k b

O.D.

PCC

PCR

RNase

DNase

rP*

w

=

=

Deoxyribonucleic acid

nanogram ~- ._ - ~-

milligram

kilobase (1 kb = 6.7x lo5 daltons = 1000 base pairs)

optical density

Pasteur Culture Collection

Polymerase Chain Reaction

Ribonuclease

Deoxyribonuclease

revolution per minute

microgram

ul = microlitre

PBsome = phycobilisome

PBPs = phycobiliproteins

APC = allophycocyanin

P C = phycocyanin

P E = phycoerythrin

PEC = phycoerythrocyanin

Chapter 1

Introduction

-

1.1 Background

Spirulina p l a t e n s i s , a phototrophic filamentous

cyanobacterium, has long been used as a staple food in human diet [I].

It is of commercial importance due to its perception as health food

containing high protein content (60% to 70% of dry weight), low fat,

minerals, high vitamin content (particularly Bi2) and essential fatty

acids like gamma-linolenic acid (GLA) Cl 8:3 [2]. Moreover, there are

various kinds of high value substances in Spirulina, e.g. phycocyanin,

allophycocyanin, p-carotene and chlorophylla [3, 41. These substances

including allophycocyanin, a bluish green pigment, are of high value in

their purified forms [5,6].

Allophycocyanin (APC), an accessory pigment in

cyanobacteria, harvests and transfers the solar energy (which has been

transferred from phycocyanin (PC)) to the reaction center of

photosynthesis [7]. In addition, the apcE encoding the core-membrane

linker polypeptide, serves as the terminal transmitter of light energy in

the phycobilisomes core to the reaction center in the thylakoid

membranes [8]. APC is a minor component of the water soluble proteins

constituted macromolecule structures, so called phycobilisomes [9].

Apart from a light-harvesting pigment, allophycocyanin is well suited as;

fluorescent reagent for flow cytometric analysis, since it has a broad

2

excitation spectrum and fluoresces with a high quantum yield.

Moreover, APC has been conjugated to monoclonal and polyclonal

antibodies for use in multicolor FACS (fluorescence-activated cell

sorter) analysis. The-activity of this conjugated antibody remains stable

for at least two years [lo].

Spirulina is the only species of cyanobacteria cultivated

outdoor in large industrial scale as human food or animal feed. Its

growth is affected by outdoor environment extensively. Hence, light

intensity plays a major role in the productivity of cell mass, as well as in

the quantity and quality of the pigments in the cells [ 111. The

knowledge of Spirulina C1 mass cultivation has been developed and

documented widely. However, there is little information concerning the

genetics and mechanisms of gene recombination in this cyanobacterium.

The study of gene(s) involved in light-harvesting protein

pigment(s) of Spirulina may lead to a better understanding in the role of

these pigments in photosynthesis, cell growth and cultivation. As such,

it is the aim of this study to clone and characterize allophycocyanin

operon of S. platensis C1, as the first step to employ molecular biology

as a tool to study allophycocyanin pigment at a molecular level.

1.2 Objectives

1. To clone the allophycocyanin operon from S. platensis Cl

strain.

2. To characterize the obtained operon.

3

1 . 3 Scopes

1. Amplification of allophycocyanin homologous probe by

PCR from -5’. pZateu.sis CL using- the conserved sequence of apt gene

from other organisms for primer designing.

2. Screening of genomic library of 5’. platensis Cr using the

probe from (1).

Cl.

3. Cloning of allophycocyanin gene operon from 5’. platensis

4. Characterization of the obtained gene by restriction

mapping, DNA sequencing and comparison of the gene products with

those of other organisms, as well as analysing the sequence using DNA

analysis program.

Chapter 2

Literature Review

-

2.1 Cyanobacteria

Cyanobacteria or blue-green algae are photoautotrophic

microorganisms [ 121. They are capable of performing oxygenic

photosynthesis similar to that found in eukaryotic algae and higher

plants [ 131. Cyanobacteria have the simplest structural organization

since they are unicellular rods or cocci existing in single or in

aggregated form. Some cyanobacteria exist in a filament of cells or

trichome. The trichomes of cyanobacteria may consist entirely of

vegetative cells, or also contain structurally and functionally

differentiated cells so called heterocyst and akinetes (spores) [ 141. In

cyanobacteria, the outer membrane, plasma membrane and thylakoid

membrane, shown in Fig.2.1, represent three structurally and

functionally distinct membranes [ 151.

The internal organization of the cyanobacterial cell is

prokaryotic, yet it is considerably more complicated structurally than

most bacteria. The structural features of the cell with the light

microscope include a central region (centroplasm or nucleoplasm) rich

in nucleic acid, a peripheral region (chromoplasm) containing the

photosynthetic thylakoid membranes and various inclusions, and several

enveloping layers consisting of plasmalemma, a pellicular wall, and/,

often, a layer of mucilage (Fig.2.1, Fig.2.2) [ 16, 171.

Figure 2.1

G G/

5

Schematic diagram of a thin section of a cyanobacterial cell.

CM, Cell membrane; TH, thylakoid; PBl and PB2, face and

side views of phycobilisomes attached to adjacent

thylakoids; GG, glycogen granules; CY, cyanophycin

granule; P, polyphosphate granule; C, carboxysome,

surrounded by nucleoplasm; R, ribosomes; G, gas vesicles.

(Insert A) Enlarged view of the cell envelope showing the

outer membrane and peptidoglycan wall layers, and the

cytoplasmic membrane. (Insert B) Enlarged view of part of

a thylakoid showing the paired unit membrane with attached;’

phycobilisomes in side view [ 181.

.

Figure 2.2 Artist’s %presentation of the overall three dimensional

architecture of Agmenellum quadruplicatum. 0

carboxysome, (L) lipid body, (P) polyphosphate body, (M)

photosynthetic thylakoid membrane system, and (‘IT)

contacts between thylakoids and cytoplasm membrane.

Thylakoids-cytoplasmic membranes and contact points are

theoretical, as this was not determined precisely (illustration

does not include cell wall, ribosomes, and nuclear material).

(From Nierzwicki-Baur, et al., 1983) [ 171

7

2.2 Spirulina

Spirulina is a multicellular-filamentous cyanobacterium. It

belongs to -phylumLyanophyta, -family Oscillatoriaceae [ 191. Spirulina

can be found in widely differing environments such as soils, marshes,

fresh water, brackish water and sea water. [20]

Under the microscope, Spirulina appears as a blue-green

filament composed of cylindrical cells arranged in unbranched,

helicoidal trichomes (Fig. 2.3) [21]. The filaments are motile, gliding

along their axis, without heterocysts. The helical shape of the trichome

is characteristic of the genus, but the helical parameters (i.e., pith, length

and helix dimensions) vary with the species and even within the same

species (Fig. 2.4). Cell diameter ranges from 1 to 3 pm in the smaller

species and from 3 to 12 urn in the larger ones such as S. platensis and

S. maxima. [20]

Spirulina contains protein as high as 60% of its dry weight and

much higher than that of other agriculture products such as rice, corn,

wheat and soybeans (as shown in Table 2.1) [22]. Besides, Spirulina

contains a rich source of vitamins, especially vitamin B12 and pro-

vitamin A (p-carotene), minerals (especially iron), and pigments (e.g.

phycocyanin and allophycocyanin). Table 2.2 shows the amount of

major compounds found in Spirulina [3].

8

Figure 2.3 - Morphology of Spirulina platensis. (A) Optical microscopy

(x400) of axenic S. platensis. (B) Scanning electroti\’

micrograph of a trichome of axenic S’. platensis [20].

t-,_ \’ ,_

Figure 2.4 Different morphological types in Spirulina (from theFigure 2.4 Different morphological types in Spirulina (from the

collection of the laboratory of Micro-algal Biotechnology atcollection of the laboratory of Micro-algal Biotechnology at

the Jacob Blaustein Institute for Desert Reserch at Sede-the Jacob Blaustein Institute for Desert Reserch at Sede-

Boker, Israel) (a) isolated from the local oxidation pond; (b)Boker, Israel) (a) isolated from the local oxidation pond; (b)

morphological similar trichome as in a developing frommorphological similar trichome as in a developing from

Spirulina platensis typical trichome; (c) Spiruiina pkutensis,Spirulina platensis typical trichome; (c) Spiruiina pkutensis,

nonvacuolated from Lake Chad; (d) straight nonvacuolatednonvacuolated from Lake Chad; (d) straight nonvacuolated

trichomes, isolated from pure culture c, from which theytrichomes, isolated from pure culture c, from which they

have been apparently transformed; (e) S’irulina platensis,have been apparently transformed; (e) S’irulina platensis,

vacuolated; (f) straight vacuolated trichomes isolated fromvacuolated; (f) straight vacuolated trichomes isolated from

pure culture b, (g) Spirulina sp. apparently platensis,pure culture b, (g) Spirulina sp. apparently platensis,

isolated from Lake Bogoria in Kenya, (h) Spirulinaisolated from Lake Bogoria in Kenya, (h) Spirulina

(unidentified), gas vacuolated, appearing during the winter;’(unidentified), gas vacuolated, appearing during the winter;’

in a Spirulina platensis [I2 11.in a Spirulina platensis [I2 11.

10

Table 2.1 Comparison of protein content in Spirulina and other foods

W) PA

-. sours ~.~ - Protein (“A dry-weight)

Beef 1 S-20

Egg 2 8

Fish 16-18

Soybean 41

Wheat 6-10

Rice 7

Chlorella 40-65

Spirulina 60-80

11

Table 2.2 Chemical analysis of Spirulina from spray dried (%dry

weight) [ 31.

Raw protein - - =- .-

Essential amino acids

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Threonine

Tryptophan

Valine

Non essential amino acids

Alanine

Arginine

Aspartic Acid

Cystine

Glutamic Acid

Histidine

Proline

Serine

Available lysine

Lipids

Fatty acids

Laurie

Myristic

- 70%

4.13%

5.80%

4.00%

2.17%

3.95%

4.17%

1.13%

6.00%

5.82%

5.98%

6.43%

0.67%

8.94%

1.08%

2.97%

3.18%

8 5 %

7.00%

5.70%

229 mgkg-’

644 mg.kg-’

:i’

1 2

Palmitic

Palmitoleic

Heptadecanoic

Stearic .- z. .~

Oleic

Linoleic

y-linolenic

Others

Ash

Calcium

Phosphorus

Iron

Sodium

Chloride

Magnesium

Manganese

Zinc

Potassium

Others

Carotenoids

p-carotene

Xanthophylls

Carbohydrates

Ramnose

Glucane

Cyclitols

Glucosamine and muramic acid

21,141 mg.kg-’

2,035 mg.kg-’

142 mg.kg-’

353 mg.kg-’

3,009 mg.kg-’

13,784 mg.kg-1

11,970 mg.kg-’

699 mg.kg-’

9.00%

1,3 15 mg.kg-’

8,942 mg.kg-’

580 mg.kg-’

412 mg.kg-’

4,400 mg.kg-’

1,9 15 mg.kg-’

25 mg.kg-’

39 mg.kg-’

15,400 mg.kg-’

57,000 mg.kg-’

4,000 mg.kg“

1,700 mg.kg-’

1,600 mg.kg-’

16.50 mg.kg-’

9.00 mg.kg-’

1.50 mg.kg-’

2.5 mg.kg-’:I ’

2.00%

1 3

Glycogen 0.50%

Sialic acid and others 0.50%

Nucleic acids 4.50%

Ribonucltiid _ ~. - 3 . 5 0 %

Deoxyribonucleic acid

Vitamins

Biotin

Cyanocobalamin

D-Ca-pantothenate

Folic acid

Inositol

Nicotinic acid

Pyridoxine

Riboflavin

Thiamine

Tocopherol

1 .OO%

0.40 mg.kg-’

2.00 mg.kg-’

11 .OO mg.kg-’

0.50 mg.kg-’

350.00 mg.kg-’

118.00 mg.kg-’

3 .OO mg.kg-’

40.00 mg.kg-’

55.00 mg.kg-’

190.00 mg.kg-’

Spirulina has been produced for commercial purpose in many

countries, according to its special characteristics mentioned above and

its safety in not possessing toxicity. The main production sites are in

USA, Japan, Thailand, Taiwan and Israel [ 1, 221. The pigments

(phycocyanin, p-carotene) of Spirulina also make this algae useful in

aquaculture, particularly as feed for tropical fish. [ 19,20,24].

1 4

The life cycle of Spirulina in laboratory culture is rather simple

[20] (Fig. 2.5). A mature trichome is broken in several pieces through

the formation of specialized cells, necridia, that undergo lysis, giving

rise to biconcave sqxtration disks. The fragmentation of the trichome at

the necridia produces gliding, short chain (two to four cells), the

hormogonia, which give rise to a new trichome. The cells in a

hormogonium lost the attached portions of the necridial cells, becoming

rounded at the distal ends with little or no thickening of the walls.

During this process, the cytoplasm appears less granulated and the cells

assume a pale blue-green color. The number of cells in hormogonia

increase by cell fission while the cytoplasm become granulated and the

cells assume a brilliant blue-green color. By this process trichomes

increase in length and assume the typical helicoidal shape. Random but

spontaneous breakage of trichomes together with the formulation of

necridia assure growth and dispersal of the organism.

Figure 2.5 Life cycle of Spirulina [20].

1 5

2.3 Phycobilisome and Phycobiliprotein Structures

2.3.1 Phycobilisome Components

-. The-accessory ~- light-harvesting apparatus of the

cyanobacteria and red algae is a water soluble multiprotein complex

known as the phycobilisome (PBsome). The PBsome is composed of

both phycobiliproteins (85% of the PBsome by mass) and non-

pigmented linker polypeptides (15% of the PBsome by mass) [25]. The

PBsome is attached to the exterior of the thylakoid membranes or

photosynthetic membranes, as shown in Fig.2.6 [26]. Light energy

harvested by the PBsome is transferred to the reaction center of

photosynthesis [27]. The basic building block of any antenna system is a

complex protein involving pigment molecules, chromophore covalently

or non-covalently associated with a protein component. Each type of

pigment-protein complex exhibits a specific spectral characteristic

depending upon the type of pigment molecule and its interaction with

protein component of the complex. Complexes with varying spectral

quality interact with each other to form the complete antenna. In

general, complexes within an antenna distal to the reaction centre

absorb at shorter wavelengths than those complexes proximal to the

reaction centre. The PBsome is assembled from two types of

polypeptide: (I) phycobiliproteins (PBPs) binding pigment molecules,

the chromophores, those serve to harvest light energy, (II) linkers, those

are involved in arranging the PBPs into a functional PBsome and may

also bind chromophores.

16

17

The pigment-proteins of the PBsome are arranged into

two structures, a core attached to the thylakoid membrane and several

rod fanning out from the core (see Fig.2.7). Glazer (1984) [29] has

described-that the corernay have-two or three cylinders depending upon

the species, and the number of rods attached to the core varies between

four and eight. In the case of bicylindrical cores, the two cylinders lie

side by side, and in the tricylindrical cores the third cylinder lies on top

of the two basal cylinders (Fig.2.8). The core structure of Synechocystis

sp. strain PCC 6701 (Fig.2.7) is one of the studied that the core was

composed of three cylinders lying parallel to the membrane consisting

primarily of allophycocyanin (APC). Glazer et al., (1983) [30] has

reported that some Synechococcus species (strain PCC6301 and

PCC7942) had a core made up of only two cylinders. Most of the

cyanobacterial PBsome have a three-cylinder core but in a few strains

the core may contain only two cylinders [31] (Fig.2.8). The major light-

harvesting polypeptide in the core is APC and in the rods is

phycocyanin (PC), phycoerythrins (PE) and phycoerythrocyanin (PEC).

The rods were radiated from the core in a fan-like array as shown in

Fig.2.8 [32].

The composition of PBsome in cyanobacteria and red algae

change in response to environmental changes such as light intensity,

light quality (only cyanobacteria) and nutrient availability. Grossman et

al., (1993) [5] has reported that cyanobacteria generally increase their

cellular contents of antenna proteins and pigments in response to low

light intensity. One of the most spectacular properties of cyanobacteria

is their ability to modulate the pigment composition of the PBsome ini

1 9

1 : PC1 andLRc

2,3 : PC2 or PE and LR

4 : AP, c?~,-~~*.~,

Lc and LcM

5:APandLc

LCM : 94 kDa

A : Calothrix PCC 7601

1 : PC and LRc

2,3:PCandLR

4 : AP, aAPB, /318.3,

Lc and LcM

LCM : 72 kDa

B : Synechococcus PCC 6301 and PCC 7942

Figure2.8 Schematic representa t ion of the hemidiscoidal

phycobilisomes structure [Tandeau de Marsac, N., 1994,

unpublished] A: Calothrix PCC 7601 B: Synechococcus

PCC 6301 and PCC 7942 For abbreviations, see Table 2.4 i’

20

response to light quality in order to maximise the absorption of

available light; a process described as “chromatic adaptation” [33].

Conley et al., (1988) [32] has found that there are at least two sets of

phycocyanin genes-involved in this process, one transcribes two light-

induced transcrips and the other encodes a single transcript present in

both red and green light in the chromatically adapting species Frenzyella

diplosiphon. Tandeau de Marsac (1977) [34] has demonstrated that

Synechocystis PCC670 1, PE-containing cyanobacterium, grown under

green light results in a stimulation of green light-absorbing PE and the

synthesis of the PE associated rod-linkers.

PBsome size and shape vary with species, and are often

dependent on the light conditions in which cells are grown [ 151. It is

known that in photosynthetic organisms, antenna size is inversely

proportional to the light intensity received by the cultures during

growth. Similarly, cyanobacteria respond to light intensity by increasing

(under low light intensity) or decreasing (under high light intensity)

[W-The polypeptide composition of PBsome varies widely

among strains of cyanobacteria. Adaptation mechanisms occurring

within the peripheral rods of PBsome are presently better understood

than those that occur in the core. [36].

Different morphological types of PBsome, they can be

divided into four classes: (1) hemidiscoidal (2) hemiellipsoidal (3)

bundle-shaped and (4) block-shaped [36]. Hemidiscoidal PBsome are

the most common and best described PBsome structures from various

cyanobacteria [5] (as shown in Fig.2.8). It can be described ad,

organelles, about 70 nm along the base, 30-50 nm in height and 14- 17

21

nm in width, attached to the stromal side of the thylakoid membrane

[37]. These PBsome have a mass of 4.5 to 1.5~10~ Da and contain 300-

800 covalently bound phycobilin chromophore [38].

-_ EssenGalJyall genes encoding structural components of

the PBsome have been isolated and characterized from many diffferent

organisms [S]. Table 2.3 [39] enumerates the different polypeptide

constituents of the PBsome, provides their gene designations and offers

a brief description of the function of each.

2.3.2 Phycobiliproteins

The phycobiliproteins (PBPs) are a family of brilliantly

pigmented water-soluble proteins that may constitute 50% of the soluble

proteins of the cyanobacterial cell [39]. The pigment-protein complexes

consist of linear tetrapyrole chromophores covalently attached to the

cysteine residues of the polypeptide via thioether linkage [27]. PBPs

from cyanobacteria and red algae are hetero-monomers consisting of

two nonidentical subunits, denoted a and p, which are present in

equimolar stoichiometry in the (c@) monomer [26] (as shown in

Fig.2.9). Each subunit differs in molecular mass, amino acid sequence

and chromophore content [36]. The fundamental assembly unit for all

PBsome is a stable phycobiliprotein trimer (c@)j. The PBPs (ap)

monomer of C-phycocyanin looks like a boomerang (see Fig. 2.9) [36].

Three monomers are arranged around a 3-fold symmetry axis in the

trimer (ap)3, while two trimers are assembled face-to-face into the

hexamer (@)6 to form a structure like a daugnut [9]. (see Fig.2.10) :i’

2 2

Table 2.3 Phycobilisome polypeptides and their genes [39]

Protein designation Gene designation Position and function

Rods cl” cpcA-_ .- ~--

PP C cpcB

a P E cpeA

PP E cpeB

LK C cpcG

LR (for PC hexamers) cpcCD

cpcHI

LK (for PE hexamers) cpeCDE

Phycobiliproteins that form the hexameric

buildin; blocks of the - peripheral rod

substructure*

Linker polypeptide that serves as an interface

between the peripheral rods a n d the

phycobilisome core

Different linker polypeptides associated with

PC hexamers of the peripheral rods

Linker polypeptides associated with PE

hexamers in the peripheral rods

Core RAP

PA P

0. A P B

P18 5

apcA

apcB

apcD

apcF

Lc apcC

LCM apcE

Phycobiliproteins that form the building blocks

of the phycobilisome core

Terminal energy acceptor in the core

Associate with LCM in the core as part of the

terminal energy acceptor

Small linker polypeptide of the core

Terminal energy acceptor in the core. May

help stabilize the core/or nucleate its

formation. May also establish a physical

association between the PBS and thylakoid

membranes.

* Not all organisms have phycobilisomes containing PE or PE linker polypeptides.

2 3

Figure 2.9 Stereoscopic plot of @$)-monomer [36]

‘?

. .

x

-1-.

Figure 2.10 Schematic side viewof a phycocyanin hexamer.

The1

molecules are labelled 1 to 6 with respect to the symmetrYi’

operation [13 61.

24

There are four major categories of PBPs, based upon

their spectral characteristecs. These are : (1) phycoerythrocyanin (PEC,

hA.max ~575 nm),(2) phycoerythrins (PE, hA,,,=565-575 nm), (3)

phycocyanins (PCj-3tA;,%=61 5-640 nm) arid(4) allophycocyanin (APC,

hA,,,,=650-655 nm). Glazer (1985) [40] has proposed the following

abbreviations for the biliprotein subunits and linker polypeptides of the

PBsomes (as shown in Table 2.4).

PEC and PE are found at the core-distal ends of the

peripheral rods. PC constitutes the portion of the peripheral rods

adjacent to the core and APC forms the major component of the

PBsome core substructure [36]. The intensely colored PBPs (AP, PC

and PE) have two dissimilar polypeptide chain that each subunit have

molecular weights in the range of 14,000 to 23,000. [ 121

Gantt et al., (1976) [41] proposed the first model of a

PBsome of Porphyridium cruentum, the core was composed of APC

surrounded by rods of PC and PE. However, it has been found in the

cyanophyte Mastigocladus laminosus, that the core of APC was similar

to the previous models, but the rods were composed of PEC as the

terminal hexamers in place of the PE [42]. PBsome from a

nonchromatic adapting cyanobacterium, eg. Spirulina platensis, is

composed of a central core containing allophycocyanin and rods with

phycocyanin and linker polypeptides in a regular array [43].

25

Table 2.4 Abbreviations for the biliprotein subunits and for linker

polypeptides [40].

- -Type oC+mlypeptide~~ A b b r e v i a t i o n

Phycoerythrin subunits aPE, pPE,yPE

Phycoerythrocyanin subunits aPEC, PPEC

R-Phycocyanin subunits a R P C, PWC

C-Phycocyanin subunits cxpc, ppc

Allophycocyanin subunits a? P”

Allophycocyanin B a subunit a A P B

P-type core biliprotein subunit PM W

Rod linker polypeptides LRM W

Linker attaching rod elements to core LRCM W

Core linker polypeptides LcM W

Linker attaching core to membrane LCMM W

Abbreviations: PE, phycoerythrin; PEC, phycoerythrocyanin; RPC, R-

phycocyanin; PC, C-phycocyanin; AP, allophycocyanin; APB,

allophycocyanin-B; L, linker polypeptide; MW, apparent molecular

weight; R, rod; C, core; M, thylakoid membrane.

2 6

Comparisons of partial or complete amino acid

sequences of phycobiliproteins have shown that each subunit is highly

conserved among different cyanobacterial species (about 80% identity)

and that-each phycobiliprotein subunit shares homology with all the

others, especially in the region of the conserved chromophore

attachment site [35]. Houmard et al., (1986) [44] reported that in

Synechococcus PCC6301, the sequences around the cystenyl residue

(Cys81) involved in the linkage of the chromophore (region 70-120)

show 100% identity. Glazer (1984) [45] has described that this portion

of the sequence is the most conserved among all the phycobiliprotein

sequences.

2.3.2.1 PBPs constituing the PBsome core

APC is a light-harvesting pigment-protein complex

found in the phytosynthetic apparatus of red algae and cyanobacteria. It

assembles the PBsome core with the assistance of three types of linker

polypeptides (L CM, LRc, and Lc). APC consists of a and p subunit

polypeptides with covalently attached bile pigment chromophores. Each

subunit contains one phycocyanobilin chromophore (PCB) (as shown in

Fig.2.11). From amino acid sequences, the PCBs are shown to be singly

bound to a- and p-Cys 82 [36]. These subunits are the longest

wavelength absorbing and fluorescing molecule in the PBsome. APC

occurs mainly in the trimeric form (oMcpApc)3. The genes for the a and

p subunits of APC are named apcA and apcB [46].

27

Allophycocyanin

common features

a-APCPCB 82

____________________ I____________________ 160 amino acid residues

P-APCPCB 82

____________________ I_________________--- 158-167 amino acid residues

Figure 2.11 General features of allophycocyanin subunits (a and p

subunits). The broken lines represent the linear polypeptide

chain of allophycocyanin and the PCB-binding sites are

indicated by bars. (one per subunit at homologous positions)

28

The apcA and apcB genes were isolated from an

Anabaena variabilis ATCC 29413 by Johnson et al., (1988) [ 121 by

using the allophycocyanin (apt) genes of Cyanophora paradoxa as

heterologous probe. They-found--that the genes appear to be present in~-

only one copy per genome and the apcA is in the upstream of the apcB.

Northern blot analysis showed that the apt genes gave rise to two

transcripts, a 1.4-kb predominant RNA and a minor 1.75-kb form, one

coding for both the alpha and beta subunits as a dicistronic messenger

and the other coding for the both phycobiliproteins (alpha and beta

subunits) and assosiated linker polypeptides. In addition, DiMagno and

Haselkorn (1993) [7] h ave isolated and characterized genes encoding

the phycobilisome core subunits, allophycocyanin a and p, and two

linker proteins from Synechocystis sp. strain PCC6714. They found that

the apcA, apcB, and gene for a small core linker protein (up&,) form an

operon, apcABC, with a single transcription start site and two possible

termination sites, one following apcB and the other following apcC. The

genes are in the order apcA, apcB and apcC. Northern hybridization

experiments identified two mRNAs of 1.5 kb and 1.8 kb. However, it

has found that there is only one transcription start site. The 1.5 kb

transcript is more abundant than the 1.8 kb transcript. This arrangement

is reasonable because the core requires many more copies of the APC

subunits than it does of the Lc linker protein. For the apcE gene,

encoding the protein that links the phycobilisome core to the thylakoid

membrane (LCM) in this organism, is not linked with apcABC operon

which is similar to that of Synechococcus PCC7002 [47]. However, in

C a l o t h r i x PCC7601 [48], C . paradoxa [47] a n d Synechococcus’~~

PCC6301 [49] the apcE gene is found upstream and close to the apcA

29

gene. Nucleotide sequences of Calothrix PCC7601 were determined by

Houmard et al., (1988), [3 l] the five apt genes, namely apcA1 (ahpl),

apcA2 (aAp2), apcB1 (PAP’), apcC (Lc7.*), and apcE (LcMg2) was

identified.- Four oftkese genes -are adjacent on the chromosome and

form the apcElAlBlC gene cluster and no genes have been found close

to the apcA2 gene. Only a single set of genes encoding the aAPC and

P AP’ subunits was found in Mastigocladus Zaminosus. [38] In addition, a

second gene (apcA2), encoding an aAPC subunit (denoted oApc2) with

59% sequence identity to aAPC1 was found in Calothrix PCC7601 [S],

but the function and location of this subunit is not known.

The core-membrane linker phycobiliprotein, also called

the anchor protein, may play a role in the association of PBsome with

thylakoid membranes. It is important in transferring energy from the

PBsome to the photosynthetic reaction centres [5]. It is the largest

chromoprotein in the PBsome with a molecular mass that varies from

70-128 kDa, depending on the organism. It is present in two copies per

PBsome. Lundell et al., (198 1) [8] suggested the LcM to be a new type

of biliprotein and to be one of the terminal energy emitters of the

PBsome. Bryant (199 1) [26] described that the apcE gene of

Synechococcus PCC7002 has been insertionally inactivated or

completely deletion, and the phenotype of the resulting mutants has

been determined. The mutant grows much slower than the wild type.

Immunological studies, as well as amino-terminal amino acid sequence

analyses, had shown that the L CM linker is highly conserved among both

cyanobacteria and red algae. The LcM proteins were shown to be :i’

30

multifunctional, hybrid polypeptides, that can be divided into 3 to 5

domains, each consisting of approximately 220 amino acids. The

amino-terminal portions of these proteins contain phycobiliprotein

domains- with a cyst&e binding site for a single PCB chromophore

WI.Offner and Troxler (1983) [50] had found that the a

subunit and p subunit of allophycocyanin from the unicellular

rhodophyte, Cyanidium caldarium, contain 160 amino acids and 16 1

amino acids, respectively. The a subunit contains one phycocyanobilin

chromophore attached at residue 80 by a cysteinyl-thioether linkage,

and the molecular weight calculated from the sequence is 18,160. The /3

subunit contains one phycocyanobilin chromophore attached at residue

81 by a cysteinyl-thioether linkage, and the molecular weight calculated

from the sequence is 18,125.

The PBsome core receives the excitation energy from the

peripheral rods and transfers it to the reaction centre in the thylakoid

membrane. A portion of the APC-containing complexes is composed of

different APC subunits, either the aAPB subunit, the p16.5 subunit (in

Mastigocladus Zaminosus) or the phycobiliprotein-domain of the LCM

pro te in . The aAPB subuni t i s encoded by the apcD gene.

Allophycocyanin-B was f i rs t purif ied from the unicel lular

cyanobacterium Synechococcus PCC6301 as a trimeric complex with

the composition (aMBpMC) [51]. AP-B was originally proposed to

function as a terminal energy emitter from PBsome and to play a role in

energy transfer from the PBsome to the chlorophyll-proteins of the,

thylakoids [5 13. Bryant (1991) [26] found that the apcD gene in

3 1

Synechococcus PCC7002 contains a polypeptide of 16 1 amino acids,

which shares considerable sequence homology (45% identity, 64%

similarity) with the aAPC subunit.

-. PMyks-tdmit f 11 -ho a op ycocyanin is encoded by the apcF

gene. The apcF gene encoding the AP-P-like polypeptide denoted p” of

Synechococcus PCC7002 reveals a polypeptide of 160 amino acids,

which is similar in amino acid sequence to the pmc subunit (48%

identity, 69% similarity) [26]. The complete amino acid sequence of

p’6.5 subunit of Mastigocladus Zaminosus was found to have 169 amino

acid residues [36].

Both of these genes (apcD and up@ encode minor

components of the PBsome core which have been believed to play

important roles in energy transfer and in the structural asymmetry which

is required to assemble the core on the thylakoid surface. The

conclusion from the analyses of these two mutants is that the apcD and

apcF gene products are not obligately required for PBsome assembly

and function [26].

2.3.2.2 PBPs constituting the rod elements of PBsome

The peripheral rods of PBsome contain phycocyanin

(PC), phycoerythrin (PE) or phycoerythrocyanin (PEC). The

chromophoric proteins in the rod have their light absorption maxima at

a shorter wavelength than the chromophoric proteins in the core thereby

focusing the excitation energy efficiently down to the chlorophyll

molecules in the reaction centre [52]. :i’

32

The Phycocyanins (PC)

The phycocyanin is a major component in the six rods

attached to the core. They are proximal to the core [53]. The PC

monomer- consists-&two dissimilar subunits, a and p, presented in-

equimolar amounts and assembled into trimers (a& or hexamers (@&

(as shown in Fig.2.10). Both subunits have linear tetrapyrrole

chromophores, designated as phycocyanobilin (PCB), covalently

attached to specific cysteine residues in the protein via thioether linkage

[54]. PCB is the chromophore typically found associated with the

subunits of PC. The blue-colored, deeply red-fluorescent C-phycocyanin

(C-PC) is the predominant PC form and contains three PCB

chromophores per (ap) monomer. All of them are located at Cys a-84,

p-84 and p-155 (see Fig.2.12) [36]. William et al., (1978) [55] found

that the a subunit of PC from Synechococcus PCC6301 has a single

PCB chromophore attached to cysteine residue 84, the p subunit of PC

has two chromophores attached to cysteine residues 84 and 155,

respectively. One, or in some cases two, of the peripheral, sensitizing

chromophores (a-84 PCB and/or p-155 PCB) have been replaced by

PXB (phycobi l iv io l in) , PEB (phycoerythrobi l in) or PUB

(phycourobilin) chromophores in each of the variants in order to adapt

to conditions more enriched on blue and green wavelengths of light.

However, the fluorescing p-84 PCB was conserved in all variants.

Glazer and Hixson (1975) [56] isolated R-Phycocyanin (R-PC or R-PC-

I) (R-, Rhodophytan) from the red alga Porphyridium cruentum as an

(a/3) trimer. The molecular mass of the complex was 103 kDa. The a:i’

subunit contains a single PCB while one of the p subunit chromophores,

33

a-PC

WC

- Phycocyania

common features

1 (84)---------_-_----- I-___________________---- 162 amino acid residues

PCB 84 2 (155)----------------- I----------------_ 1----------172 amino acid residues

Figure 2.12 General structure, common features and chromophore

variability of phycocyanins. The broken lines represent the

linear polypeptide chain and the 3 bilin-binding sites are

indicated by bars. All PCs contain a PCB at position p-84

:1,2=PCB;l=PXB,2=PCB;l=PCB,2=PEB;1,2=

PEB; 1 = PUB, 2 = PCB found in C-PC, PEC, R-PC, P-PC11

and WH8501 Phycocyanin (R-PC-III), respectively. (PCB =

Phycocyanobi l in , PXB = Phycobi l iviol in , PEB =

Phycoerythrobilin, PUB = Phycourobilin) [36].

3 4

that at position Cys 155 normally occupied by a PCB in C-PC, is

replaced by a PEB [56]. Ong and Glazer (1987) [57] isolated R-

Phycocyanin-II (R-PC-II) from the marine Synechococcus sp. strains

WH 8 103, WH 802Qand JVII 7803, as thefirst PEB-containing PC of-

cyanobacterial origin. Ong et al., (1984) [58] studied that the position of

the single PCB was assigned to Cys p-84 position and the two PEBs to

Cys a-84 and Cys p-155 by analysis chromopeptides. The PC isolated

from marine Synechococcus sp. strain WI 8501 was the first PUB-

containing PC. A PUB occurs on the a subunit at position Cys 84 and

the /3 subunit of this protein carries two PCB chromophores at position

Cys 84 and Cys 155 [59].

The presence of PC at the rod-core linkage position is

apparently essential for excitation energy transfer from the rods to the

core. At the rod-core linkage positions in the PBsome, PC and special

rod-core linker polypeptides (LRc) form stable rod-core complexes

together with APC complexes: (aPCpPC)6 . LRc .(aAPCpMC)3 .Lc. Conley

et al., (1986) [60] found that Calothrix PCC7601 has three copies of the

genes encoding the a and p subunits of PC.

In both prokaryotes and eukaryotes the p subunit gene

for PC is followed by the a subunit gene [61]. Certain cyanobacteria are

able to modulate the PC and PE composition of the PBsome under

different light qualities, maximizing absorption of prevalent

wavelengths of light. This phenomenon is called complementary

chromatic adaptation. In organisms that exhibit complementary

chromatic adaptation, there are at least two different sets of PC subunits;,

one set accumulates during growth of cyanobacteria in both red and

green light, while the other accumulates to high levels only in PBsome

from cells grown in red light [32].

Phycocyanins involved in rod-core subcomplexes (C-PC,

R-PC-I, R-PC-II orALPGIII) are constitutive’ PCs and are expressed

under all growth conditions under which PBsome are formed [36].

The Phycoervthrocyanin (PEC)

Phycoerythrocyanin is the shortest wavelength absorbing

rod element of those cyanobacteria which neither contain PE nor

perform complementary chromatic adaptation. The function of PEC is

the extension of the light-harvesting capacity of PBsome into the green

portion of the spectrum under medium or low-light conditions. The PEC

content of PBsome is subjected to regulation by light intensity: under

low light PEC-expression is strongly induced [36]. Bryant et al., (1976)

[62] found that the aPEC subunit contains a purple chromophore

(phycobiliviolin, PXB) whereas the two PCB chromophores of the p-

subunit are identical to those found in PC (see Fig.2.12).

The Phvcoervthrins (PE)

The phycobiliproteins of the red-colored phycoerythrin

(PE) family exhibit a great diversity in spectral properties as well as in

their chromophore and subunit composition. PEs carry only one or two

chromophore types, PEB and/or PUB, instead of the four chromophore

types found among members of the PC family (PCB, PXB, PEB, and

PUB) (as shown in Fig.2.13). Bryant (1982) [63] founded that

cyanobacteria contain either PEC or PE in the PBsome but never both, ”

and many cyanobacteria such as in Synechococcus PCC 7002 posses no

3 6

red-colored phycobiliproteins (i.e., neither PE nor PEC) but only the

blue-colored APC and C-PC. PEC does not occur in red algae. PE is the

only phycobiliprotein present in cyanobacteria, red algae as well as in

cryptomonads, and-is thus most suitablefor comparative studies to-

reveal structural, functional and phylogenetic relationships among these

organisms. In the peripheral rods of the PBsome, PE is located at the

core-distal periphery of the structure.

PE chromophore contents differ among cyanobacteria

living in freshwater and soil, and/or marine environments. The y

subunits found in PE of red algae and in PEII of cyanobacteria are the

second type of bihmctional polypeptide combined phycobiliprotein and

linker polypeptide in addition to the LcM [36].

3 7

Phycoerythrin common features

l(84) 2 (143a)a-PE ________-______ I_---------------------- I---------

3 (50/61) PEB 84 PEB (155)-. p-pE--- =-.-z. ------- ) :I 1 1------------- --z----;--; -_----- _______ _--

-30LR -_--_----_---______-____________________---------------------

l(94) 2 (133) 3 (209) 4 (297)y-PE (red -------------- I---------- I------------- I-----------__- I____algae)PE-II

3 (75) PEB (84) 2 (143a)a-PE _-_____ I_______ I_-___------------------ I---------

3 (50/61) PEB 84 PEB (155)P-PE ---------- l-l------------- I------------------- I-----------

PUB (94)y-PE -___-_-------- I--------------------------------------------

Chroomonas PC-645 (crvptomonad)(19) mesohiliverdin

al-PC-645 ------- ----------_-____-____________

a2-PC-645 ------- I-----------------------------

3 (50/61) PEB 8415,16-dihydrobiliverdin 2PEB (155)

p-PC-645 ---------- l-l------------- I------------------- I----------_

Figure 2.13 General structure, common features and chromophore

variability of phycoerythrins. The broken lines represent the

linear polypeptide chain and the bilin binding sites are

indicated by bars. All PEs contain a PEB at position p-84

and p-1 55. The other three bilin binding sites a-75, a-84, a-

143a and p-50-61 may be occupied by a PUB. PE-I

complexes from soil and marine cyanobacteria contain

linker polypeptides without bound bilins (Lam3’). PE-II from

marine cyanobacteria have one PUB and PE-I from red

algae four PUB or PEB bound to the linker polypeptide

(ysubunits). Cryptomonad PEs are modified with PCB, PEB;1

PUB and other novel chromophores [36].J’

38

2.3.3 Linker polypeptides, the skeleton of the PBsome

The non-chromophoric polypeptides (-15%), termed

linker proteins also make up a group of related proteins [5]. They induce

a face-to-fxe aggrzgationof PE,- PEC and PC trimers, and additionally-

cause the tail-to-tail joining of hexameric assemblies to form larger

aggregates such as peripheral rods and core-cylinders. Lundell and

coworkers (1981) [64] have shown that the ratio of PC to linker is

important in determining rod-length. Based upon experimental data

derived from rod assembly in vitro, they have proposed a model for rod

biogenesis, suggesting that PC interacts with another PC trimer in

preference to a PC hexamer (Fig.2.14). Finally, the hexamers assembly

into rods. These proteins also serve to connect the rods to the core, and

last but not least direct the assembly of the PBsome core and its

attachment to the thylakoid surface [36].

Linker polypeptides are the colorless polypeptides. They

are believed to be located mainly in the central cavity of the torus-

shaped phycobiliprotein hexamers or trimers. They can be divided into

four groups according to their function in the PBsome.

1. Lc, the small core linker polypeptides (a@Y)

In Calothrix PCC7601, the apcC gene (204 bp) located

240 nucleotides downstream from apcB1 encoding the small linker

polypeptide Lc7.* associated with AP in the core of the PBsome [35].

Molecular masses of 8-l 3 kDa have been indicated for other organisms

[36]. The core components from Synechococcus PCC7002, the Lc linker

polypeptide is suggested to be associated with trimeric APC at the

peripheries of the core cylinders. This polypeptide seems not to be”’

absolutely required for PBsome assembly, but its presence considerably

3 9

improves both the stability and energy transfer properties of PBsome

P61.2. LcM, the core-membrane linker polypeptide

-. Redlinger and Gantt (1982) [55] described that LCM-

(apcE gene product) is a high molecular weight. chromophoric

polypeptide, anchor protein attaches to the photosynthetic membranes

and may function in transfer of energy from the PBsome to

photosynthetic reaction centres.

3. LRMW, the rod linker polypeptide

They are involved in the assembly of the peripheral rod

substructure. They can be divided into two groups differing in their

molecular masses:

group I: the small rod linker polypeptide consists of an 8

to 10 kDa polypeptide

group II: the rod linker polypeptide consists of

polypeptides with masses of about 30 kDa.

4. LRC, rod-core linker polypeptides

A key position in the PBsome structure and energy

transfer pathway is the rod-core junction. A special class of linker

polypeptide with a molecular mass of about 30 kDa, the C-PC-

associated rod-core linker polypeptide (LRc), attaches the peripheral

rods to the PBsome core and form different types of C-PC-to-APC

interactions. The rod-core linker polypeptides are found to play two

main roles. Firstly, they associate the peripheral rods with the PBsome

core. Secondly, they impart a strong red-shift in the wavelength of

maximum absorbance and fluorescence emission of the central /3-84”’

40

phycocyanobilin chromophores, to enable an optimal rod-to-core energy

transfer [36].

41

2.3.4 Pigments

PBPs are all relatively small with protomers consisting

of an alpha and a beta subunits that each subunit contains one or more

covalently -bound -chromophore.~- [ 121 The brilliant colors of the PBPs

originate from chromophores, linear tetrapyrrole prosthetic groups,

known as phycobilins [36].

Four main types of phycobilins are present in

cyanobacteria and red algae: the blue-colored phycocyanobilin (PCB);

the red-colored phycoerythrobilin (PEB); the yellow-colored

phycourobolin (PUB); and the purple-colored phycobiliviolin (PXB;

also named cryptoviolin) (as shown in Fig.2.9) [36].

Phycobilin chromophores are generally bound to the

polypeptide chain at conserved positions either by one cysteinyl

thioether linkage on the pyrrole ringA of the tetrapyrrole or occasionally

by two cysteinyl thioether linkages on the A and the D pyrrole rings

S&ram et al., (1971) [ 131 has demonstrated that

phycocyanobilin is the prosthetic group of C-phycocyanin (C-

cyanobacterial) and allophycocyanin. Both of PBPs (C-PC and APC)

belong to the important class of photosynthetically active proteins of

blue-green algae. The structure of phycocyanobilin has been

investigated by means of mass spectral. The result of the mass spectrum

of the acid form of phycocyanobilin is clearly that it is a compound of

molecular weight 588 which contains 40 hydrogen atoms.

In a study of R-phycocyanin-I (R-Rhodophytan) a and p

subunits from the red alga Porphyridium cruentum, Ducret et al., (1994):

[66] showed that the a chain carries a phycocyanobilin pigment”

4 2

covalently linked at Cys84 and the p chain carries a phycocyanobilin

pigment covalently linked at Cys82 and a phycoerythrobilin pigment at

cys153.

4 3

Peptide-linked PHYCOCYANOBILIN Peptide-linked PHYCOBILIVIOLIN

C D

Peptide-6inked PHYCOERYTHROBILINS

E F

&w-HV-C)%-c3-- - -l-IN-Crr-CO- M ~-nN-c;I-C~-rP.-’

” /‘. -H n H H

Peptide-linked PHYCOUROBILINS-~

Figure 2.15 Structures of various types of phycobilins found in cyano-

bacteria and red algae, linked by thioether-linkages to PBPs

(A) phycocyanobilin; (B) phycobiliviolin; (C) and (D)

phycoerythrobilin singly or doubly linked, respectively; (E)

and (F) phycourobilin singly or doubly linked to the poly-‘,,

peptide backbone, respectively [36].

4 4

2.3.5 Organization and transcription of genes encoding

PBsome components

The organization patterns for genes encoding PBsome

components of s-al cyanobacteria were summarized by Bryant

(1991) [26]. In Synechococcus PCC7002, it was found that there were

two transcriptional types of genes encoding PBsome components, one

was polycistronic mRNA and the other was monocistronic mRNA. The

first transcriptional unit in the peripheral rods of the PBsome in this

organism is the cpcBACDEF operon, encodes all components of

peripheral rods except for the 29-kDa rod-core linker polypeptide

(LRc29). The second transcriptional unit is the PC-associated, rod-core

linker polypeptide of apparent mass 29 kDa, the product of the cpcG

gene. Pilot and Fox (1984) [67] found that the cpcB gene, which

encodes the ppc subunit of phycocyanin, lies 105 bp 5’ from cpcA

encoding the cc” subunit. These characterictic was found in

phycocyanin of other cyanobacteria (eg. Synechococcus PCC630 1,

Anabaena PCC7120) (see Fig.2.16) and in cyanelle, Cyanophora

paradoxa. The cpcC and cpcD genes, encoding a PC-associated linker

polypeptide were next identified from the region 3’ to the cpcA gene.

The cpcE and cpcF genes lie 5’ from cpcD, do not encode structural

components of the PBsome but are required for attachment of

phycocyanobilin to the cc” subunit.

The six genes (see Fig.2.16) encoding components of the

PBsome cores of Synechococcus sp. PCC7002 are also arranged in two

transcriptional types. The first of these transcriptional types, those{i’

encoding apcA, apcB, and apcC appear to produce polycistronic

4 5

mRNAs. The apcABC operon encodes the a and p subunits of AP and

the AP-associated core linker polypeptide. It is interesting to note that

the order of genes, with the apcA gene occuring 5’ to the apcB gene , is

opposite-that observed-for alll cpcBA operons and cpeBA operons

(operons of phycoerythrin) [68,69]. The second type appears to produce

monocistronic mRNAs, encoding apcD, apcE, and apcF. The apcD,

apcE, and apcF genes were encoded the oAPSB, the p” subunit, and the

LcM linker, respectively. In Calothrix sp. PCC7601 was found that the

organization of the genes encoding core component similars to that of

Synechococcus 7002

46

Synechococcus PCC7002

__ --

.a

(..........--..... -b

Synechococcus PCC6301

atp~ 1 Igene H 0rfW*...-.....-....

apcE

cpcB2

b b

Anabaem PCC7120-- -

cpcE cpcF cpcG1 cpcG2 cpcG3 cpcG4 .~

bb t-.-.

b b*

Figure 2.16 Comparison of the organization of genes encoding phycobilisome

components for the cyanobacteria Synechococcus PCC7002,

Synechococcus PCC6301, and Anabaena PCC7120. The width of the

arrows is roughly proportional to the steady-state abundance of the

transcripts, and dotted arrows indicate possible transcripts that have

not been detected experimentally. The hatched portion of

Synechococcus PCC7002 orfW indicates a region of this putative gene

that has not yet been sequenced. The box indicated “X” for the cpc

operon of Synechococcus PCC7002 indicates the position of a 38j.

codon open reading frame whose function is not known, [26]

4 7

2.3.6 Energy Transfer in PBsomes

PBsomes transfer absorbed light energy primarily to

reaction centre. The efficiency of energy transfer from the PBsome to

photosynthetic reaction centres approaches 100% [ 121. This implies that

the energy transfer mechanism must proceed rapidly in order to avoid

energy losses by competing radiative or non-radiative decay processes

[36]. Spectroscopic studies on PBsome indicate that the following

scheme of energy transfer is unidirectional from PE/PEC discs at the

periphery of the rods through PC discs to APC and the terminal emitters

in the core (LcM or/and aAPB ), terminating at the photosynthetic reaction

centres (as shown diagramatically in Fig.2.17) [SO].

H20'Phy

\erythrin

:(670 nm) ( (670 ILL) PSI1

chlae-

Figure 2.17 Energy flow in PBsome of Cyanobacteria and red algae.

Radiationless excitation energy transfer from short

wavelength (phycoerythrins) to long wavelength absorbing,

pigment-protein complexes (allophycocyanins). Energy is

finally transferred to and distributed between PSI1 and PSIi’

4 8

2.4 The molecular biology of cyanobacteria

2.4.1 Genome size of cyanobacteria

-. The-genome sizes of 12% strains of cyanobacteria,-

representative of all major taxonomic groups, lie in the range of 1.6~10’

to 8.6x 10’ daltons. They were described by Herdman et al., (1979) [70]

which were measured from the kinetics of renaturation of DNA. The

majority of unicellular cyanobacteria, which reproduce either by binary

fission or by budding, contain genomes of 1.6x 10’ to 2.7x 10’

daltons,comparable in size to those of other bacteria. Most

pleurocapsalean and filamentous strains possess larger genomes than

unicellular cyanobacteria. The genome sizes are discontinuously

distributed into four distinct groups which have means of 2.2x log, 3.6

x log, 5.0x 10’ and 7.4x 10’ daltons. The single strain of the genus

Spirulina (filamentous cyanobacteria that produce motile, helical

trichomes) has a small genome size of 2.53x 10gdaltons. The data

suggested that genome evolution in cyanobacteria occurred by a series

of duplications of a small ancestral genome, and that the complex

morphological organization characteristic of many cyanobacteria may

have arisen as a result of this process.

2.4.2 Molecular cloning of phycobilisome in Cyanobacteria

There were different methods employed to clone various

kinds of genes involving in phycobiliproteins and linker polypeptides

synthesis of cyanobacteria and eukaryotic algae. These cloning:i’

49

strategies can be grouped into three categories: cloning by sequence

similarity, polymerase chain reaction and gene expression.

Cloning by sequence similarity

-. - This--procedure for cloning-specific genes of interest is-

by using sequence similarity of previously cloned genes from other

organisms. This approach simply involves obtaining cloned DNA

fragments of published sequences and using them as hybridization

probes, usually at low stringency. Numerous cyanobacterial genes have

been cloned by this approach and due to its rapidity and simplicity, it is

one that should be explored before undertaking more complicated

cloning procedures. For example, Johnson, et al., (1988) [ 121 isolated

the genes encoding phycocyanin and allophycocyanin of Anabaena

variabilis ATCC294 13 using the phycocyanin (cpc) genes of

Agmenellum quadruplicatum and the allophycocyanin (apt) genes of

Cyanophora paradoxa as heterologous probes.

Cloning by polymerase chain reaction (PCR)

With the powerful method of polymerase chain reaction

technique in molecular biology, various kinds of genes have been

cloned by PCR. This cloning strategy employed oligonucleotide derived

from the conserved sequences of known genes as primers. A fragment

of the gene of interest can be amplified from genomic DNA using

primers mentioned above and used as probe for screening library. For

example, DiMagno and Haselkorn (1993) [7] isolated the genes

encoding allophycocyanin subunits and one linker protein from

Synechocystis 6714 using conserved regions at the N-terminal end of

apcA and C-terminal end of apcB for PCR reactions. The PCR produ&

was used as a probe to screen the genomic library.

50

Cloning by gene expression

Cloning by gene expression is rather complicated than

the other two strategies. A cDNA gene library is constructed in an

expression -vector @Z-&i plasmid or phage) and screened for protein-

production by using specific antibodies. Lemaux and Grossman (1984)

[71] identified the gene encoding the p subunit of phycocyanin of C.

paradoxa. Antibodies to mixtures of phycobiliproteins were used to

screen E. coli colonies containing cDNA of plastid DNA.

In Spirulina, various kinds of genes have been cloned

and their sequences are documented in Genbank (see Table 2.5).

Mostly, they are genes involving in photosynthesis and protein

synthesis. Two of them are genes responsible for fatty acid desaturation.

However, until now, there was no genes or sequences of light harvesting

complex of Spirulina published yet. Up to date, there are 26 genes from

S. platensis isolated and characterized which can be retrieved from

Genbank via electronic mail (retrieve@ncbi.nlm.nih.gov) as shown in

Table 2.5.

51

Table 2.5 Genes of S. platensis that have been cloned and

characterized as documented in Genbank (retrieve

@ncbi.nlm.nih.gov)

Gene name -ReferenceGene product

ribosomal protein S 12

ribosomal protein S7

Translation elongation

factor EF-G

Translation elongation

factor EF-Tu

organism

72rpsL S. platensis

S. platensis

S. platensis

72rpsG

fUS 72

72S. platensis

7316S rRNA 16s ribosomal RNA Spirulina

PCC63 13

Spirulina

PCC63 13

Spirulina

PCC63 13

S. platensis C 1

S. platensis C 1

73tRNA-IIe transfer RNA-IIe

23s rRNA 23s ribosomal RNA 73

serine esterase 74

75

an esterase

ATPase gamma ATPase gamma

subunit gene subunit

rpsI0 ribosomal protein S 10 S. platensis

ribosomal protein S2 S. platens is

76

77

77

rpsB

w- EF-Ts elongation

factor

S. platensis

:i

5 2

Table 2.5 Genes of S. platensis that have been cloned and

characterized. (continued)

Gene name

recA

ilvX

ilvY

fabZ

desA

1euB

desD

glnA

rbcL

rbcS

---Gene product _ organism Reference

recombination protein S. platensis *

strain IAM-

Ml35

Acetohydroxy acid S. platensis C1 7 8

synthase

Acetohydroxy acid S. platensis CI 7 8

synthase

(3R)-hydroxy- S. platensis **

myristoyl acyl carrier strain Italian

protein dehydrase

delta1 2 desaturase S. platensis ***

P-isopropylmalate S. platensis CI 7 9

dehydrogenase

delta6 desaturase S. platensis ****

Glutamine synthetase S. platensis 80

Ribulose-bisphos- S. platensis CI 1 8

phate carboxylase

large subunit

Ribulose-bisphos- S. platensis Cl 1 8

phate carboxylase

small subunit

53

* recA gene (accession U33924)was found by Vachhani, AK.

and Vonshak, A., 1995, unpublished data.

** fab2 gene (accession U4182 1) was found by Los, D.A.and

Murata, N+ 1995, unpublished data. _ _

*** desA gene (accession X86736) was found by Murata, N.,

Deshnium, P. and Tasaka, Y., 1995, unpublished data.

** ** desD gene (accession X87094) was found by Tasaka, Y,

1995, unpublished data.

Chapter 3

Materials and Methods

-. - - :.- -

3.1 Organisms and plasmids

3.1.1 Cyanobacterial strain

S. platensis C1 strain was kindly provided by Prof. Dr. A.

Vonshak, Algal Biotechnology, Ben-Gurian University of the Negev,

Israel. This strain was used as a source of DNA in cloning apt gene by

polymerase chain reaction technique.

3.1.2 Bacterial strains/S. platensis Cl library

The bacterial strain used as recipients for propagation

and subcloning of the different plasmids during this project was

Escherichia coli DH5a (supE4, hsdR17, recA1, endAl, gyrA96, thi-1,

reZA 1).

The host bacterial strain used for transfection and

propagation of recombinant lambda bacteriophages was E. coli LE392

(supE44, supF58, hsdR5 14, galK2, gall22, metB1, trpR55,lacYl).

The library of S. platensis Cl was constructed in

hDASHI1 by Deshnium, 1992 [Sl] and used for screening for the

allophycocyanin gene.

55

3.1.3 Plasmids

pRL498 [82], conferring kanamycin resistance, was

kindly provided by Prof. C. P. Wolk, Michigan State University, USA

and used- for subclonmgthe PCR-fragment-

pGEM4 [83], conferring ampicillin resistance, was used

for cloning and sequencing of apcABC gene.

3.2 Chemicals

All chemicals were of reagent grade and molecular biology

grade.

3.3 Enzymes

Restriction enzymes used in cloning were purchased from

either Boehringer or BRL company (USA). The lysozyme, RNaseA and

DNaseI were obtained from Sigma Co.

3.4 Media and culture conditions

All the media were sterilized for 15 min at 15 PSI, 12 1’C. For

the solid medium, 0.8% (top agar medium) or 1.5% of bacto-agar (agar

medium) was added before sterilization.

56

3.4.1 Spirulina platensis

3.4.1.1 Culture medium, Zarrouk’s medium [84]

-. -- z.- -

Component Concentration (g/l)

NaCl 1 .ooo

MgS04.7H20 0.200

CaClz 0.040

FeS04.7HzO 0.010

EDTA 0.080

K2m04 0.500

NaN03 2.500

K2S04 1 .ooo

NaHC03 16.800

A-5 solution 1 ml

A-6 solution 1 ml

57

A-5 Solution

Component Concentration (g/l)

-. H3B&---- ~- - -~ 2.860-

MnC12.4H20 1.810

ZnS0+7H20 0.222

CuS04.5H20 0.074

Moo3 0.015

B-6 Solution

Component Concentration (g/l)

NH4N03 229.6x 1O-4

K&r2(S0&.24H20 960.0x 1O-4

NiS04.7HzO 478.5x10-’

Na2S04.2Hz0 179.4x1o-4

Ti(SO& 400.0x 1 o-4

CO(NO~)~.~H~O 439.8x 1O-4

Zarrouk’s medium was sterilized for 15 min at 15 PSI. then

cooled slowly in the autoclave to room temperature to prevent

precipitation.

3.4.1.2 Stock and culture conditions

S. platensis C 1 was cultured at 3 5OC in Zarrouk’ si’

medium under illumination with white light of fluorescent lamp (Osram

58

40 watt) at 80 uE.mm2.sec-‘, agitated on a rotary shaker at 150 rpm. Cells

were transferred every two weeks for stock culture. This culture stock

(10 ml) was used to start culture in 250 ml flask containing 100 ml of

Zarrouk’s- medium&Us-in the- mid-log phasem(OD560 =- 0.45 or 4-5

days) were used for isolation of chromosomal DNA.

3.4.2 Bacteria

3.4.2.1 Culture medium, LB-medium (Luria-Bertani

Medium) [ 831

Component Concentration (g/l)

Tryptone 1 0

Yeast extract 5

NaCl 5

adjusted pH to 7.0

3.4.2.2 Culture conditions and storage

Bacteria were stored by adding 15% of steriled glycerol

to overnight culture (grown in liquid medium containing the appropriate

antibiotic) in eppendorf tube. After mixing, the stock culture was kept at

-7O’C until use. This culture stock (20 ~1) was used to start overnight

culture in bottles containing 5 ml of LB (supplemented with antibiotic

when necessary) at 37OC on a rotary shaker, 150 r-pm.

5 9

3.5 Buffers and Solutions (Sambrook, et. al., 1989) [83]

3.5.1 Lysozyme solution (Sigma#L-6876)

-. Lysozyme was dissolved in 10 n-&I Tris-HCl (pH 8.0) at

a concentration of 10 mg/ml.

3.5.2 Ribonuclease A (RNase A) (Sigma#R-5000)

RNase A was dissolved in 10 mM Tris-HCl (pH 7.5), 15

mM NaCl at a concentration of 10 mg/ml. The solution was heated at

1 OO’C for 15 minutes then cooled slowly to room temperature, aliquoted

and kept at -2O’C.

3.5.3 DNase I (Sigma #D-4263)

DNase I was dissolved in TM buffer at a concentration

of 1 mg/ml and kept it at -2O’C.

3.5.4 TM buffer

50 mM Tris-HCl pH7.4,10 mM MgS04

Sterile by autoclave

3.5.5 STET

0.1 M NaCl, 10 mM Tris-HCl (pH S.O), 1 mM EDTA

(pH S.O), 5% Triton X- 100

3.5.6

3.5.7

8 .0

Sterile by autoclave

TE buffer pH8.0

10 n&I Tris-HCl (pH S.O), 1 mM EDTA (pH 8.0)

Sterile by autoclave

TBE buffer

0.089 M Tris base, 0.089 M Boric acid, 1mM EDTA pH:i’

6 0

3.5.8 Denaturing solution

1.5 M NaCl, 0.5 M NaOH

3.5.9 Neutralizing solution

-. 1.5.J&F&Cl, 0.5 M Tris-HCLpH72, 0.001 M EDTA

3.5.102OxSSC

3 M NaCl, 0.3 M Trisodium citrate

3.5.1120xSSPE

pH7.7

3.6 M NaCl, 0.2 M Sodium phosphate, 0.02 M EDTA

3.6 Primers/Oligonucleotide synthesis

Primers for PCR and for sequencing were synthesized by Bio

Service Unit, National Center for Genetic Engineering and

Biotechnology, Bangkok, Thailand.

3.6.1 Primers for PCR

Sequence number : 786: 5’-(GA)TT(GA)TA(ACTG)GT

(TC)TC(TC)TT(ACTG)A(GA)(ACTG)CC(GA)TT-3’

Sequence number : 787: 5’-AA(CT)GC(ACTG)GA(CT)

GC(ACTG)GA(AG)GC(ACTG)CG(ACTG)TA-3’

Sequence number : 1843: 5’-GCCGAATTCAAGTTTTT

CCCCATGAAATG-3’

Sequence number : 1845: 5’-GCCGAATTCC(TG)(TC)

TG(TC)TG(TC)TC(ACTG)C(TG)(AG)AACCA-3’ :i’

6 1

G-3’

GG-3’

3.6.2 Primer for sequencing

Sequence number : 1690: 5’-CTGGCGATGTTACCC-3’

Sequence number : 169 1: 5’-ATCAGGACGTTTTTG

-. -__ I _

Sequence number : 1692: 5’-TGCATCCGTGACCTG-3’

Sequence number : 1693: 5’-AGACTTAGCAACTGC-3’

Sequence number : 1799: 5’-TCATTTTCTACCACAG

Sequence number : 1844: 5’-GCCGAATTCGATTACG

GAAGTGATTGCG-3’

Sequence number : 197 1: 5’-GCTACTGGTGAACTG-3’

3.7 Molecular biology techniques

Most of molecular biology techniques used in this work are as

described by Sambrook et al., (1989) [83] and Davis et al., (1986) [85],

unless otherwise stated.

3.7.1 Plasmid preparation

3.7.1.1 Small-scale preparation

Minipreparation of plasmid DNA was done by the

boiling method (Holmes and Quigley, 1981).

A 5 ml of overnight culture of recombinant bacteria was

centrifuged for 10 min at 6000 rpm. After removing the medium, the

bacterial pellet was resuspended in 450 yl of STET (0.1 M NaCl, 10:i’

mM Tris.Cl pH 8.0, 1 mM EDTA pH 8.0, 5% Triton X- 100). Then

62

suspension was added with 50 ~1 of a freshly prepared lysozyme

solution. The mixture was transferred to an appendorf tube, vortexed for

3 seconds then boiled for 40 seconds. The plasmid solution was clarified

by centrifugation for&l-On&~ at room temperature, The pellet of bacterial

debris was removed by using a sterile toothpick. After adding 50 ~1 of

2.5 M sodium acetate (pH 5.2) and 500 ~1 of isopropanol to the

supernatant, the mixture was incubated for 10 min at room temperature.

The pellet of nucleic acids were recovered by centrifugation for 10 min

at 4’C, 12,000 r-pm. After the pellet of DNA was dried, it was

resuspended in 50 u.1 of TDW.

The DNA solution (0.5 ug/yl) was directly used for

restriction analysis (3 ul per reaction).

3.7.1.2 Large scale preparation

An overnight culture of the recombinant bacteria (0.5

ml) or culture from glycerol stock was inoculated in 100 ml of LB +

ampicillin (100yglml) in 250 ml flask, agitated on a rotary shaker at

37”C, 200 r-pm for 12 to 16 hrs.

After harvesting of cells, the high yield plasmid was

obtained using QIAGEN KIT (Germany), following the method

recommended by the company.

3.7.2 Bacterial transformation

The calcium chloride procedure was used for

transformation of E. coli [ 831. :i’

63

The 1 ml of overnight culture was inoculated to 100 ml

of LB in 250 ml flask, agitated on a rotary shaker at 37’C, 200 rpm.

After 2.5-3 h of incubation (0.D at 560=0.3-0.4), the cells were

incubated- in ice for30 min. The- cells were harvested by centrifugation

at 3,000 rpm for 10 min at 4’C. The cell pellet was resuspended with 40

ml of ice-cold sterile 100 r&l MgC12 and centrifuged again as

previously described. After the pellet was dissolved with 15 ml of ice-

cold sterile 100 mM CaC12 and centrifuged, the pellet was resuspended

in 3 ml of ice-cold 100 mM CaC12. The competent cells were

maintained at 4’C for at least 2 h before use.

The plasmid DNA (10 ~1 containing about 50 ng of

DNA) was added to 200 ~1 of competent cells in a 1.5 ml eppendorf

tube. After 30 min on ice the cells were heat-shocked for 2 min at 42OC.

One millilitre of LB broth was then added and cells were incubated for

45 min at 37OC. The transformed bacteria (200 ~1) were spreaded on LB

agar plates containing the appropriate antibiotic (50 ug ml’ of

kanamycin and 100 ug ml-’ of ampicillin for all plasmids used in this

work). After 12-16 h the transformed bacteria could be picked up and

grown overnight in liquid medium for further screening.

3.7.3 Subcloning of DNA fragments

Plasmids, either pRL498 or pGEM4, were used for

subcloning the fragments of DNA. The fragments of DNA were

separated on a 0.7% agarose gel in 0.5xTBE buffer. The piece of gel

containing the fragment of interest was cut using a scalpel blade, andi,

the fragment was eluted by QIAEX KIT (Germany), following the

64

method recommended by the company. The DNA fragment (0.2 ug)

was then ligated to 0.1 ug of vector DNA using 1 unit of T4 DNA ligase

(BRL company) in a total volume of 10 ~1. All of the ligation mixture

was used to transm -?FcoZi DHSa. Therecombinant colonies were

characterized by small-scale plasmid isolation (section 3.7.1.1) and

restriction analysis.

3.7.4 Plating bacteriophages

3.7.4.1 Preparation of plating bacteria

The plating bacteria were prepared as recommended by

Davis et al., 1986 [85] and Sambrook et al., 1989 [83].

E.coZi LE392 was grown overnight in LB broth

containing 0.2% maltose; the sugar induces the maltose operon, which

contains the gene (ZamB) coded for the bacteriophage h receptor. One

millilitre of the overnight culture was used to inoculate 50 ml of LB

broth containing 0.2% maltose. The culture was grown overnight at

37OC with moderate agitation (250 rpm) until O.D. at 600 nm was

approximately 1.0. The cells were collected by centrimgation at 2,500

xg for 10 min at room temperature. The cell pellet was resuspended in

12.5 ml of sterile 10 mM MgSO4. This yields approximately 1-2x lo9

cells per milliliter. The cell suspension was transferred to a 250 ml

sterile flask and incubate for 1 hour at 37’C, 200 r-pm. The cells could be

stored at 4’C for up to 2 weeks. However, the highest plating

efficiencies were obtained when freshly prepared cells was used.

f

65

3.7.4.2 Plating lambda bacteriophage

The diluted phage suspension was added to lOOu1 of

plating cells. The mixture was incubated at 37’C for 20 min to allow the

bacteriophage particles to-adsorb to E. cati. Then 4 ml molten (45’C)

soft LB agar containing 10 .mM MgS04 was added to the mixture, and

overlaid onto prepoured plates (90 mm) containing 20 ml of LB agar.

The plates were closed and let stand for 5 min at room temperature to

allow the top agar to harden. Plaques appeared after the plates were

inverted at 37’C after about 6 hours of incubation. Plates were stored at

4’C for further screening.

3.7.5 Preparation of DNA from bacteriophage

The method used followed Davis et al., (1986) both for

the growth and preparation of bacteriophage [84].

3.7.5.1 Preparation of clear lysate

With a sterile Pasteur pipette, an agar plaque containing

a single plaque was transferred (one plaque contains approximately lo7

phages) to 50 ml of LB medium supplemented with MgS04 (10 mM

final concentration) and 200 ~1 of plating cells in 250 ml flask. The

culture was shaken vigorously with good aeration (approximately 250

rpm) at 37’C for lo- 12 hrs. The culture then became cloudy and

subsequently clear with lysis. After lysis 500 1-11 of chloroform was

added to the flask, and the flask was shaken further for 5 min at 37’C.

Bacterial debris was removed by centrifugation at 3,000xg for 10 min at

room temperature. The supernatant was transferred to a new tube and\\,

6 6

100 ~1 of 1 M MgS04 was added. The lysate could be stored at 4’C for

several months.

3.7.5.2 DNA preparation from bacteriophage

-. - Ten-mi-lli=kitre of TM buffer@0 -n-&I Tris ~87.4, 10 mM

MgS04) containing 320 ~1 of fresh DNase I solution (80 Kunitz

units/ml in TM) were added to 10 ml lysate. After incubation at room

temperature for 15 min, 2 ml of 5 M NaCl and 2.2 g of solid PEG-6000

were added. The PEG was left to completely dissolve in the lysate

before incubating on ice for 15 min. The phages were centrifuged at

12,000xg for 10 min, 4’C. The pellet was resuspended in 300 ~1 of TM

buffer and transferred into an eppendorf tube. The suspension was

added with 300 ~1 of chloroform, mixed well and centrifuged for 5 min.

The aqueous phase was transferred to a new tube and extracted once

with chloroform. After the addition of 15 ~1 of 0.5M EDTA and 30 ~1 of

5M NaCl, the aqueous phase was extracted with 350 ~1 of phenol,

followed by two more chloroform extractions. The DNA was

precipitated by adding 875 ~1 of ethanol and incubating on ice for 10

min. After centrifugation at 12,000 r-pm for 10 min at 4OC, the pellet was

rinsed with 70% ethanol 2 times. The pellet was dried and resuspended

in 50 ~1 of TDW (triple distilled water).

The DNA obtained still contained a large amount of

bacterial RNA. It was treated with RNaseA (50 ug/ml of final

concentration) for 30 min at 37’C followed by phenol extraction and

ethanol precipitation.

6 7

3.7.6 DNA sequencing

The sequencing of DNA was performed by dideoxy

chain termination method of Sanger et al., [86] using the Sequenase

Version -2;O Kit --(United States Biochemical Corporation, USA).

Double-stranded DNA was used for sequencing, following the protocols

essentially from Sequenase Version 2.0.

The DNA (3-5 pg) (preferably from plasmid miniprep)

was first denatured in an alkaline solution (0.2 M NaOH, 2 mM EDTA)

at 37OC for 30 min. The mixture was then neutralized by 0.1 volume of

3M sodium acetate (pH 4.5-5.5), precipitated with 2-4 volumes of

ethanol at -70°C for 15 min. The DNA pellet was redissolved in 7 pl of

TDW, followed by the addition of 2 pl of sequenase reaction buffer and

1 pl of primer (1 pmollyl). The annealing step was carried out by

warming the mixture to 37’C for 30 min. The primer was extended by

sequenase T7 DNA polymerase with the deoxynucleotide triphosphates

(dGTP, dCTP, dTTP 3.0 pM each of labeling mix). Then the labeling

mixture was added to each termination tube, containing the

deoxynucleotide triphosphate and a dideoxynucleotide triphosphate

(ddGTP, ddATP, ddTTP and ddCTP). The reactions were terminated by

the addition of stop solution (EDTA and formamide). They were

denatured by heating at SO’C for 2 min before loading on sequencing

gels.

6 8

3.7.7 Southern blotting

In Southern blotting, enzyme digested DNA was

transferred onto nylon membrane (HybondTM-N; Amersham, UK) by

downward capilluethod [87]. If the DNA to be transferred

contained fragments larger than 4 kb, the agarose gel was first soaked in

depurination buffer (0.25 M HCl) to just cover the surface of the gel.

After 30 min, the depurination buffer was replaced with denaturation

buffer for 40 min and then by neutralizing buffer for 30 min. The

transfer was set up in a pyramid form as shown in figure 3.1 and carried

out the transfer with 1OxSSC buffer. The membrane transfer was

completed after 1.5 hrs. The buffer was then removed from the

apparatus. Before lifting the gel, the wells of the gel were marked on the

membrane using pencil. Following the transfer, the membrane was

rinsed with 2xSSC and vacuum dried at SO’C for 2 hrs. The DNA was

then ready for hybridization with an appropriate probe.

/-Blotting Membrane

7 p~la&c~;ndxl;

Blotting paper

Paper towels

Figure 3.1 Schematic setup of downward capillary transfer of DNA

6 9

3.7.8 DNA labelling with 32P-dCTP

The labelling of DNA was performed by using Prime-a-

Gene Labelling System (Promega), a random hexanucleotide primers

synthesis, according-to themanufacturer’s instructions.

The DNA template (25 ng/30 ~1) was first denatured by

heating to 95OC for 2 minutes then chilled on ice. After the addition of

10 ~1 of Sxlabelling buffer, 2yl of unlabelled dNTPs, 2 ~1 of BSA and 5

~1 of a-32PdCTP, the mixture was polymerized by using 1 pl of Klenow

enzyme (5 units/pi)/. The reaction tube was mixed gently and incubated

at room temperature for 60 minutes. After the reaction was terminated

by heating at 95-100°C for 2 minutes and subsequently chilling in an ice

bath, the 5 ~1 of 0.2 M EDTA was added into the mixture. Prior to use

the probe was denatured by heating to 95’C for 2 minutes and placed on

ice.

3.7.9 Dot blotting

In dot blotting, DNA was heated to 95’C then chilled on

ice. After adding 1 volume of 2OxSSC, the DNA samples were spotted

onto the nylon membrane which was prewetted with 1OxSSC. Each spot

contained approximately 2 pl (about 0.5 l.tg) of DNA solution. After

allowing of each spot to dry, the membrane was wetted in denaturing

solution for 5 minutes and then in neutralizing buffer for 1 minute.

Following drying with filter paper, the membrane was vacuum dried at

80°C for 2 hrs. The DNA was then ready for hybridization with an

appropriate probe. :i’

7 0

3.8 Cloning of allophycocyanin gene from 5’. platensis Clstrain- -

3.8.1 Isolation of genomic DNA

-. - Gennmic- DNA -was extracted from the cells of S.-

platensis C1, grown to mid-exponential phase, according to the method

of Glatron & Rapoport (1972) [88] with slightly modification. The

harvested cells from 100 ml of the culture of S. platensis Cl (ODsbO =

0.45 or 4-5 days) were resuspended in 650 u.1 of 10 mM sodium acetate

(pH 4.5), 200 n&l Sucrose and 55 mM EDTA. Cell aliquots were

tranferred on to ice cold 15 ml corex centrifuge tube containing : 70 ul

of 20% SDS , 1.5 ml of cold phenol and 1 g of sterile glass beads . Cells

were broken by vortexing 4 to 8 pulses of 15 set at high speed, with an

interval of 30 to 60 set on ice between each pulse to prevent heating.

Then the suspension was centrifuged at 7,500 rpm for 15 min, 4’C. The

aqueous phase was collected and treated with the same volume of

phenol/chloroform/isoamyl alcohol (25:24: 1, by vol.) in order to

remove protein. After centrifugation at 12,000 rpm, 10 min, 4’C,

aqueous phase was extracted with chloroform/isoamyl alcohol (24: 1,

v/v) to remove the remaining phenol. Sodium acetate (pH 5.2) was

added to a final concentration of 0.3 M and DNA was precipitated by

adding 2 volumes of ice-cold ethanol and exposed to -2O’C for at least 2

hrs. DNA was recovered by centrifugation at 12,000 r-pm for 10 min at

4°C. The pellet was washed with ice-cold 70% (v/v) ethanol,

recentrifuged briefly and then dried at room temperature. The dried

DNA was dissolved in 50~1 of TE buffer (pH 8.0) or distilled water and!

kept at 4’C. The obtained DNA concentration was about 0.3 ug ~1~‘.

71

3.8.2 Amplification of partial apt AB from genomic DNA of

5’. platensis Cr by polymerase chain reaction

By comparing the apt AB gene sequences of

Synechowccus PUXOU [26],-- Synechomcus PCC6301 [44] a n d

Cyanophora paradoxa [87], conserved regions at the N-terminal end of

apt A and C - terminal end of apt B could be chosen for designing

oligonucleotide primers for PCR reactions. Two conserved regions at

positions lo- 16 and 110-l 17 (as shown in Fig.3.2), counted from the

amino terminus of the apt A and of the apt B of Synechococcus

PCC7002, respectively were selected. A DNA fragment of 0.9 kb was

amplified by a DNA thermal cycler apparatus (Perkin-Elmer cetus) The

sequences of the designed degenerated primers are as follows :

SAA(CT)GC(ACTG)GA(CT)GC(ACTG)GA(AG)GC(ACTG)CG

(ACTG)TA 3’ contained 2,048 types of primer with 23 bases, and

5’(GA)TT(GA)TA(ACTG)GT(TC)TC(TC)TT(ACTG)A(GA)(ACTG)

CC(GA)TT 3’ contained 4,096 types of primer with 24 bases.

The conditions used for the PCR reactions were: 29

cycles of 1.5 min at 92OC for denaturation, 1 min at 45’C for annealing,

and 1 min at 72’C for polymerization; 1 cycle of 1.5 min at 92’C for

denaturation, 1 min at 45’C for annealing, and 10 min at 72’C for

polymerization. The genomic DNA of 5’. platensis Cl was used as

template. The PCR mixture was then treated with Klenow fragment

enzyme 5 units and incubated at 37’C about 30 minutes for blunt ending

the PCR product . The amplified products were subcloned into pRL 498

and their nucleotide sequences were determined. :i’

72

AP a subunitsSyn 7002 MSIVTKSIW ADAEARYLSP GELDRIKAFV TSGESRLRIA ENLTGSRERI IKSAGDALFQ 6 0Syn 6301 S E T V VG DR QTIAES VKQ NQCyan0 T D S A AS ER QILTDN VRE Q Q

Syn 7002 KRPDWSPGG NAYGEEMTAT CLRDMDYYLR LITYGWAGD VTPIEEIGLV GVREMYKSLG 120Syn 6301 _- V D L V s I I R K-I_ I_Cyan0 I E L V An- L K - N

Syn 7002 TPVDAVAQAV REMKAVATGM MSGDDAAEAG AYFDYVIGAM ESyn 6301 IE EG EL SA AL LT E D A G L SCyan0 VA EG SA SV GL LS D A S ALQ

AP p subunitsSyn 7002 MQDAITSVIN SADVQGKYLD GSAMDKLKAY FTTGALRVRA ASTISANAAA IVKEAVAKSLSyn 6301 A A AS SSALDR S Qs E A S SAL Vcyan0 P A AA TASVEK S QT E A A SSA I

6 0

Syn 7002 LYSDVTRPGG NMYTTRRYAA CIRDLDYYLR YATYAMLAGD PSILDERVLN GLKETYNSLG-~ 120Syn 6301 I E L TCyan0 I D V T

Syn 7002 VPVGSTVQAI QAMKEVTAGL VGADAGREMG VYFDYICSGL SSyn 6301 I A V I S P VL s SCyan0 VA1 A G P IY s G

Figure 3.2 Comparison of the AP a and p subunits amino acid

sequences of Synechococcus PCC7002 [26], Synechococcus

PCC6301 [44] and Cyanophora paradoxa [87]. Selected

amino acid sequences are highly homologous and they are

indicated by underlines and bold. Blank positions indicate

identity with the Synechococcus PCC 7002 sequences at

that position.

73

3.8.3 Screening of genomic library of 5’. platensis Cl

The screening of a genomic DNA library of 5’. platensis

C1 was performed according to the standard method for plaque

hybridization [82],About 1,000 plaques oftherecombinant clones (200

plaques/selection plate) were tranferred to 5 nylon membranes

(HybondTM-N; Amersham UK). The membrane containing hDNAs were

denatured in denaturing solution for 7 minutes, and soaked 2 times in

neutralizing solution for 3 minutes. Then the membranes were washed

once with 2xSSC. The membrane were transferred to dry filter paper

and air dry, plaque side up. The membranes were baked for 2 hours at

8O’C between sheets of filter paper. The baked membranes were pre-

hybridized with SxSSPE, 5xDenhard’s solution, 0.5 %(w/v) SDS and

50% formamide at 42’C for 1 hour. The membranes were hybridized for

at least 12 hours at 42’C with a 32P- labelled probe (amplified partial apt

AB of S. platensis) prepared by using random primer labelling kit

(Promega) as described in section 3.7.8. After hybridization, the

membranes were washed with 2xSSPE, 0.1% SDS at room temperature

for 10 minutes, 2 times and 1 xSSPE, 0.1% SDS at 65’C for 10 minutes

and exposed to X-ray film (Kodak), at -7O’C.

3.8.4 Southern blot analysis

For Southern blot analysis, l-5 pg of each DNA (h or

genomic DNA) was digested with appropriated restriction enzymes. The

digested DNA fragments were fractionated on an 0.7% agarose gel and

transferred to a nylon membrane by downward method and theI ’

membrane was baked for 2 hours at SO’C. The pre-hybridization,

74

hybridization and washing of the membrane were performed as

described in section 3.8.3 and the membrane was exposed to X-ray film

at -70°C.

3.8.5 Characterization of positive clones

The DNA fragment which hybridized with the

appropriate probe was isolated from the positive clones using QIAEX

KIT (Germany), following the method recommended by the company

and subcloning into pGEM 4. The nucleotide sequence of subcloned

DNA fragment was determined by the dideoxy chain termination

method [90] using a DNA sequencing kit as described in section 3.7.6

on single-strand DNA templates according to the protocol of

manufacturer.

3.8.6 Amplification of complete apt AB and partial apt C from

genomic DNA of 5’. platensis Cl by PCR

Two degenerated oligonucleotide primers were designed

from upstream sequences of apt A of 5’. platensis and conserved region

of apcC. The conserved region of apcC was obtained by comparison

and alignment of apcC with sequence of Synechococcus PCC7002 [26],

Synechococcus PCC6301 [44], Synechococcus elongatus [Soga, M.,

1994, unpublished], Calothrix sp. Strain PCC 7601 [3 11, Fischerella sp.

Strain PCC 7603 [89]. The conserved region of apt C at the position

from 37 to 42 (as shown in Fig.3.3), counted from the amino terminus

of apcC of Synechococcus PCC7002. The two oligonucleotides

corresponding to these regions including EcoRI restriction site at 5’ end”

75

(indicated by underlines) were synthesized. The sequences of the

designed primers are as follows :

5’ GCCGAATTCAAGTTTTTCCCCATGAAATG 3’

contained- f type of+imerwith 2-9 bases, and - -

5’ GCCGAATTCC(TG)(TC)TG(TC)TG(TC)TC(ATCG)C(TG)(AG)AA

CCA 3’ contained 256 types of primers with 29 bases.

The oligonucleotides were used as primers (-250 ng) for

amplifying apt AB containing partial apt C by PCR using a DNA

thermal cycler apparatus (Perkin-Elmer cetus).The conditions used for

the PCR reactions were: 29 cycles of 1.5 min at 92’C for denaturation, 1

min at 5O’C for annealing, and 2 min at 72’C for polymerization; 1 cycle

of 1.5 min at 92’C for denaturation, 1 min at 5O’C for annealing, and 10

min at 72’C for polymerization. The genomic DNA of S. platensis Cl (at

various concentration) was used as a template. The amplified products

were subcloned into pGEM4. The nucleotide sequences of the cloned

PCR fragment were determined by using a DNA sequencing kit as

described in section 3.7.6.

76

LC7.8

Syn 7002 MRMEKITACV PSQSRIRTQR ELQNTYFTKL VPYDNWFREO aIMKMGGK1 VKVQLATGKP 60syn 6301 M M R I L PSK F YDA QL I E A R Psyn elan M M K I V QTR Y YEN QM V EF KPCal 7601 --ALKV +--QTS- - ~- Y FEN-m . - M M V EA KQFis 7603 G L K I V QTR Y YDA QM V - E A K Q

Syn 7002 GTNTGLT>

Syn 6301 N T T L>

Syn elan G V T >

Cal 7601 G T T >Fis 7603 GI T A>

Figure 3.3 The comparison of the amino acid sequences of apcC gene

products of Synechococcus PCC 7002[26], Synechococcus

PCC 6301 [44], Synechococcus elongatus [Soga, M. un-

published 19941, Calothrix sp.Strain PCC760 1[3 l] and

Fischerella sp.Strain PCC7603 [89]. Selected amino acid

sequences are highly homologous and they are indicated by

underlines and bold letters. Blank positions indicate identity

with the Synechococcus PCC7002 sequences at that

position.

Chapter 4

Results and Discussions

4.1 Construction of apcAB probe from S. platensis C1 by Polymerase

Chain Reaction

For construction of apcAB probe from S. platensis Cl by PCR,

two amino acid conserved sequences of apcAB gene found in

Synechococcus PCC7002 [26], Synechococcus PCC6301 [44] and

C y a n o p h o r a paradoxa [88] w e r e u s e d f o r d e s i g n i n g t w o

oligonucleotide primers. The sequence NADAEAR (residues lo- 16 in

a-subunits) was chosen for synthesizing the 5’ primer, while

NGLKETYN (residues 110-l 17 in P-subunits) was chosen for the 3’

primer. A 900 bp fragment (as shown in Fig.4.1) was specifically

amplified from S. platensis Cl genomic DNA as template by PCR using

the following primers 5’-AA(CT)GC(ACTG)GA(CT)GC(ACTG)GA

(AG)GC(ACTG)CG(ACTG)TA-3’ (primer up) and 5’-(GA)TT(GA)TA

(ACTG)GT(TC)TC(TC)TT(ACTG)A(GA)(ACTG)CC(GA)TT-3’

(primer down). PCR amplification was performed as described in

materials and methods (chapter 3, section 3.8.2). The amplified DNA

fragment was subcloned into pRL498, (resulting in pMG003), and its

nucleotide sequence was determined by dideoxy nucleotide termination

method as described in materials and methods. The deduced amino acid

sequences were analyzed with the DNA analysis software DNAsis (as’\,

shown in Fig.4.2). A homology search was performed using the BLAST

78

program (National Center for Biotechnology Information (NCBI),

National Library of Medicine, NIH, Bethesda, MD, USA). The analysis

result demonstrated that the amplified fragment contained genes

encoding -parts of thPcxand /3 subunits ofthe allophycocyanin genes.

The deduced amino acid sequence of the a subunit shows 91% (98%),

85% (95%) and 84% (100%) identity (similarity) and the p subunit

shows 88% (97%), 76% (94%) and 82% (92%) identity (similarity)

when aligned with those from Synechococcus elongatus, Cyanophora

paradoxa, Synechocystis PCC6714, respectively (as shown in Fig.4.3).

This result suggests that pMG003 contains a part of apcAB gene of

S. platensis C 1.

4 .2 Isolation of the allophycocyanin gene from a genomic DNA library

of S. platensis C1

The genomic DNA library of S’. platensis Ci constructed in

hDASHI1 by Deshnium, 1992 [8 I] was screened by plaque

hybridization using a 32P-labelled apcAB probe. Two hybridizable phage

clones, namely VAl, VA2 were obtained from 1,000 recombinant

plaques. hDNAs of VA1 and VA2 were isolated and subjected to

Southern blot analysis. The results showed that the two clones were

identical in the restriction and hybridization pattern, and their nucleotide

sequences revealed the sequence of allophycocyanin gene (as shown in

Fig.4.4). As such, VA1 clone was selected for further characterization in

details by Southern blot analysis and nucleotide sequencing. From:i’

7 9

-900 bps

Figure 4.1 The amplified PCR products from genomic DNA of

S. platensis Ci (180 ng) electrophoresed on 1% agarose gel.

A : h-Hind11 (molecular size marker), B : Negative control

(no template), C, D, E, F and G : amplified products from

primer concentrations 300, 250, 200, 100 and 50 ng,

respectively (MgC12 concentration used was 1mM)

7 9

A B C D E F G

-. -

9416 bps-6557 bps-4361 bps-

2322,2027 bps=

-901 D bps

Figure 4.1 The amplified PCR products from genomic DNA of

5’. platensis Ci (180 ng) electrophoresed on 1% agarose gel.

A : h-HindIII (molecular size marker), B : Negative control

(no template), C, D, E, F and G : amplified products from

primer concentrations 300, 250, 200, 100 and 50 ng,

respectively (MgCl, concentration used was 1 mM)

80

a subunit

ATCATCAAGGAAGCAGGJUiACCAACTTTTCC!AAAA?i CGTCCTGATGTAGTCTCTCCCI I K E A G N Q L F Q K R P D V V S P

GGTGGAAATGCCTACGGTGAG-~~TGA~TGCCACCTGCCTGCGGGATCTAGACTACGGNAYGEEMTAT~CL-RD L - D Y

TACCTGCGTCTGATCACCTACGGAATTGTTGTTGCTGGCGATGTTACCCCCATTG~G~Y L R L I T Y G I V A G D V T P I E E

ATCGGGGTTGTAGGTGTTCGCGAAATGTACAAATCTCTCTTGGTI G V V G V R E M Y K S L G

f3 subunit

TCCGTAATCAACTCCTCTGACGTTCAAGGTAAATACCTGGS V I N S S D V Q G K Y L D R S A I Q

AllACTGAAAGCCTATTTCGCTACTGGTGAACTGCGCGTTCGTGCAGCAACCACCATCK L K A Y F A T G E L R V R A A T T I

AGCGCTAATGCAGCTAACATCGTTAAGGAAGCAGTTGCTAGTCTCTGCTGTACTCCS A N A A N I V K E A V A K S L L Y S

GATATCACCCGTCCCGGTGGTAATATGTATGTACACCACTD I T R P G G N M Y T T

Figure 4.2 The nucleotide and deduced amino acid sequences of partial

apcAB gene from S. platensis Cl.

8 1

a subunits

MSVVTKSI YLSPGELDRIKNFVSTGERRLRIAQTLTENRERIVKQAGDQLFQMSIVTKSI YLSPGELDRIKSFAASGERRLRIAQILTDNRERIVREAGQQLFQMSIVTKSI YLSPGELDRIKAFVTGGAARLRIAETLTGSRETIVKQAGDRLFQ

* ** ***IIKEAGNQLFQ

._ - - =--KRPDWSPGGNAYGEEMTATCLRDLDWLRLVTYGIVAGDVTSLGKRPDIVSPGGNAYGEEMTATCLRDLDWLRLVTYGWAGDATSLGKRPDIVSPGGNAYGEEMTATCLRDMDWLRLVTYGWSGDVTRSLG**** ******************* ****** *** * ** l ****** *** *** ***

KRPDWSPGGNAYGEEMTATCLRDLDWLRLITYGIVAGDVTPIEEIGWGVREMYKSLG

Syn elonCyan06714

Sp.Cl

Syn elonCyan06714

Sp.Cl

Syn elon TPIPAVAEGIRAMKNVACSLLSAEDAAEAGSYFDFVIGAMQCyan0 TPVAAVAEGVRSAKSVATGLLSGDDAAEAGSYFDYVIAALQ6714 TPIEAVAQSVREMKEVASGLMSSDDAAEASAYFDFVIGAMSSp.Cl

p subunits

Syn elon MQDAITAVINASDVQGKYLDTAAMEKLKAYFATGELRVRASVISANAANIVKEAVAKSLCyan0 M Q D P I T A V I N A A D V Q G K Y L D T A S V E K L K S Y F Q T G E L R V R A S L6714 MQDAITAVINSADVQGKYLDGAAMDKLKNYFASGELRVRASL

*** ******** *** ** ******** l ** * ********

Sp.Cl SVINSSDVQGKYLDRSAIQKLKAYFATTISASL

Syn elon LYSDITRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDPSILDERV S L GCyan0 LYSDITRPGGNMYTTRRYAACIRDLDYYVRYATYAMLAGDTSILDERV S L G6714 LYSDVTRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDASILDERV S L G

**** *****s-e***

Sp.Cl LYSDITRPGGNMYTT

Syn elon VPIAATVQAIQAMKEVTASLVGADAGKEMGIYFDYICSGLSCyan0 VPVGATIQAIQAAKEVTAGLVGPDAGREMGIYYDYISSGLG6714 VPISSTVQAIQAIKEVTASLVGADAGKEMGWLDYICSGLSSp.Cl

Figure 4.3 The deduced amino acid sequence of the amplified DNA

product from S. platensis Cl (Sp. Cl) compared with

allophycocynin sequences of Synechococcus elongatus (Syn

elon) [Shimazu, T., Soga, M., Hirano, M. and Katoh, S.

Unpublished, 19941, Cyanophora paradoxa (Cyano) [87]

and Synechocystis 6714 (6714) [31]. The identical amino

acids are indicated by asterisks using blast program

(blast@ncbi.nlm.nih.gov). Two conserved amino acid

residues are indicated by boxes which were chosen fori’

designing 5’ and 3’ primers in PCR.

82

Southern blot analysis, a restriction map of VA1 clone could be

determined as shown in Fig.4.5 and Fig.4.6. It was found that the apt

gene of 5’. platensis Cl resided on the 1.6 kb EC&I-EcoRV fragment.

However,- the resultfromnucleotide sequencing and DNA analysis (data

not shown) indicated that only the upstream region along with the upper

part of apcA (252 bp) were found on the 1.6 kb EcoRI-EcoRV fragment.

The physical map of the 1.6 kb fragment is shown in Fig.4.6. By

comparing the protein sequence of the Synechococcus PCC7002 [26],

Synechococcus PCC6301 [44] and Cyanophora paradoxa [88] it was

apparent that the 3’ end of the complete apcAB genes were not

contained on the 1.6 EcoRI-EcoRV fragment. Reprobing the same

library with the either apcA probe (1.6 kb EcoRI-EcoRV fragment) or

apcB probe (EcoRV-BamHI fragment of pMG003), it was found that

there was neither plaques hybridizable with apcB probe nor with both

apcA and apcB probe. Only 2 plaques hybridized with apcA probe was

found. As such, it can be concluded that there was no complete apt

genes in this library, resulting in employing cloning of apt gene from S.

platensis C r by using conserved sequences of apt gene in other

organisms as primers by polymerase chain reaction technique.

83

VA1

12 3

0-- - -

0

0

M V-w)- 21226

- 3530

- 1375

VA2

M @P)

-21226

-

-3530

- 1375

Figure 4.4 Southern blot analysis of two recombinants hDNAs isolated

from two positive clones, VA1 and VA2 respectively.

hDNA from each clone digested with restriction enzymes,

EC&I, lane 1; EcoRV, lane 2; EcoRI and EcoRV, lane 3,

were electrophoresed on 0.7% agarose gel and transferred to

a nylon membrane. The blots of DNA were hybridized with

a 32P-labelled apcAB probe (amplified partial apcAB of

5’. platensis). Molecular marker (M) is hDNA digested

with Hi&III and EcoRI, shown on the right of each picture.

-21226

cl48,4973 .--4268

84

1 2 3 4 5 M @P)

-2027-1904

-1584-1375

Figure 4.5 Southern blot analysis of one positive clone (VAl) which

was digested with EcoRI, lane l;EcoRV, lane 2;EcoRI and

EcoRV, lane 3;BgZII, lane 4;EcoRI and BgZII, lane 5. The

blot of DNA was hybridized with a 32P-labelled apcAB

probe (amplified partial apcAB of S. platensis). Molecular

marker (M) is hDNA digested with Hi&III and EcoFCI,

shown on the right site.

85

ECORI EcoRV

I--9kb-1

Rightarmlambda

Figure 4.6 Restriction map of 1.6 kb EC&I-EcoRV fragment of VA1

lambda phage containing upstream region of apcA region

along with the upper part of apcA of 5’. platensis Cl. The

arrow indicates the direction of the apcA gene. The broken

lines indicate left and right arm of h DASHII.

86

4.3 Cloning of apcAB and par t ia l apcC of 5’. pZatensis Cl by

Polymerase Chain Reaction

After geticDNA library of S”p&tensis Ci was screened

three times by using apcB probe, there was no positive clone. The

genomic DNA library was probably obtained from several

amplification, so the wanted gene may be eliminated. In this strategy,

cloning of allophycocyanin gene operon from S. platensis Cl by PCR

was chosen as an alternative, based on the knowledge of the

organization of the allophycocyanin gene.

Two oligonucleotides were designed for amplification of the

complete apcAB gene by PCR. The upstrand primer sequence was

obtained from upstream region apcA of S. platensis C1 (see Fig.4.6) and

the downstrand primer was designed from conserved regions of the

apcC genes of Synechococcus PCC7002, Synechococcus PCC6301,

Synechococcus elongatus, Calothrix PCC760 1 and Fischerella

PCC7603. The sequence FREQQR (residues 37-42 in Lc, see Fig.3.3)

was chosen for the 3’ primer, resulting in the degenerated primer as, 5’-

C(TG)(TC)TG(TC)TG(TC)TC(ATCG)C(TG)(AG)AACCA-3’. The

sequence of 5’ primer was 5’-AAGTTTTTCCCCATGAAATG-3’. The

facilitate subcloning of the amplified product into the plasmid, and

EcoRI restriction site proceeded by GCC (GCCGAATTC was added to

both primers). The optimal conditions used for the PCR reactions were

perfomed as described in materials and methods (chapter 3, section

3.8.6). After amplification, DNA products were checked on 1% agarose

gel (as shown in Fig.4.7), the band showed the equivalent size (about:’

87

1.8 kb) that corresponded to the expected size of apcABC gene of

Synechococcus PCC7002. These amplified DNA fragments were

subcloned into pGEM4, and sequenced by the dideoxy nucleotide

termination method-theobtained fragment-yas named pMG004.

4.4 Nucleotide sequence analysis of the apcABC operon

The nucleotide sequences of 1.8 kb fragment of the amplified

product (present in Fig.4.8) were determined. The 1.8 kb fragment

contains putative apcA, apcB and partial apcC of S. platensis Cl. The

nucleotide sequences of these genes can be seen in Genbank with an

accession number X95898. The deduced amino acid sequences derived

by translation of the apcA, apcB and apcC genes with the DNA analysis

software DNAsis. They are composed with those of the corresponding

phycobilisome component already known using the BLAST program.

The organization of the genes is in the order apcA , apcB and apcC with

ORFs (open reading frames) of 483 bps, 483 bps and 108 bps,

respectively. Bryant (1988) [47] h as described that the apcA gene

encoding the APCa is found 5’ to the apcB gene that encodes APCp in

Synechococcus PCC7002. Generally, the order of the apt genes in all

cyanobacteria, with the apcA gene occurring 5’ to the apcB gene, is

opposite to that observed for all cpcBA operons and cpeBA operons

[26]. In addition, the apcC gene, encoding the small linker polypeptide

Lc, lies downstream from the apcB gene [36]. However, apcC gene has

not been found in Cyanophora paradoxa [36]. Both apcA and apcB

consist of 162 codons. The apcB and apcC genes locate 84 bp&

I

88

A B C D E

-.1800 bp

21226 bp5 148,4268,35302027, 19041584, 1375

947,83 1

564

Figure 4.7 The amplified PCR products from genomic DNA of

S. platensis C, were electrophoresed on 1% agarose gel. A,

B and C : amplified products from the genomic DNA

template at the concentration of 28,00, 280 and 28 ng

respectively, (1 mM MgC12 and 250 ng primer), D :

Negative control (no template), E : h marker (Hi&III and

EcoRI)

89

downstream from apcA and 251 bps downstream from apcB,

respectively (Fig.4.8). The deduced amino acid sequence of the a

subunit shows 85%, 82% and 80% identity, and the p subunit shows

93%, 84’Sand 88%--&&y when they were-aligned with those from

Synechococcus elongatus, Cyanophora paradoxa and S’ynechocystis

PCC67 14, respectively (Fig.4.9). In addition, Lc polypeptide shows

92% (100%) ident i ty (s imilar i ty) with three organisms of

Synechococcus elongatus, Synechocystis PCC67 14 and Frenzyella

diplosiphon (as shown in Fig.4.10). Closer examination of these

sequences showed that stretches of 100% identity exist in around the

cysteinyl residue (Cys 81) involved in the linkage of the chromophore

(region 70- 120) of a and p subunits. This portion of the sequence is the

most conserved among all the phycobiliprotein sequences.

The position of the chromophore of the a and p subunit of

allophycocyanin in S. platensis Ct were determined by comparison of

the amino acid sequence of the chromopeptides with the putative apcA

and apcB. This located the chromophore at residue 81 of both th a and

p subunits. Offner and Troxler (1983) has determined the complete

amino acid sequence of the a and p subunits of allophycocyanin from

the unicellular rhodophyte, Cyanidium caldarium by automated Edman

degradation of the proteins and peptides derived from them by chemical

and enzymatic cleavages. They found that the a subunit contains 160

amino acids, one phycocyanobilin chromophore attached at residue 80

and j3 subunit contains 161 amino acids, one phycocyanobilin

chromophore attached at residue 81 [50]. :i,

90

-60 -401 CCTCGTCCCTAATTATTAAGTTTTTCCCCATGAAATGTTA CTATTACAAATATACTAATATGTGA

-20 +111 ACATAATGCCTCAAPLATACATTTCGAGGTAGTCATGTCATGAGGTTTCATTTGGGGGACC~TAGGGAC

141 ACCCGAAACTCGTGGCGGCGTATAATCAAATACGCCCGCCCGATCGCGATCGAT~TGACTCGGC~TCTTGG211 TAATAGCCAAAAGTTGCCTGCTCAGGAGAAGTTGCCTGCCTGCT~CCGCCACCTGTGGCAGGTT~TGGTAC280 TTCCCAAAGCTGAGGAGCCACGACACCGGGCTGACCGAAACACATTAGCT351 AAACCCTGTGGTAGAAAATGAGTATCGTTACCAAATCCATGCGCGTTATCT

MetSerIleValThrLysSerIleValAsnAlaAspAlaAspAlaGl~l~rgTyrLem ;-421 GAGC~~TGGTG~TTAGATCGGATCAAATCCTTTGTTACCTCTGGC~~~-GC~GGGTTCGGAT~CTG~

uSerProGlyGluLeuAspArgIleLysSerPheValThrSerGlyGl~rgArgValArgIleAlaGlu

491 ACCATGACAGGTGCTCGTGAGCGCATCATCAAGGAAGCAGCTTTTCC UCGTCCTGATGThrMetThrGlyAlaArgGluArgIleIleLysGluAlaGlyAsnGlnLeuPheGlnLysArgProAspV

561 TAGTCTCTCCCGGTGGAAATGCCTACGGTGAGGAAATGACTCTAGACTACTAalValSerProGlyGlyAs~laTyrGlyGluGl~etThrAlaThrCysLe~rgAspLe~spTyrTy

631 CCTGCGTCTGATCACCTACGGAATTGTTGTTGTTCGGGGTTGTAGGTrLeuArgLeuIleThrTyrGlyIleValAlaGlyAspValThrProIleGluGluIleGlyValValGly

701 GTTCGCGAAATGTACAAATCTCTTGGTACTCCCATCGAAGValArgGluMetTyrLysSerLeuGlyThrProIleGl~laValAlaGluGlyValArgAlaMetLysS

771 GTGTAGCCACTTCCCTGCTGTCTGGAGAAGACGCAGCCGACGCAGCCG~GCAGGTGCTTACTTCGACTACCT~TTGGerValAlaThrSerLeuLeuSerGlyGluAspAlaAlaGl~laGlyAlaTyrPheAspTyrLeuIleGl

841 TGCAATGTCATAAGCACTGGCGATTATCTCTTATTAATCGACCAAGATTTCCTAGATCyAlaMetSer***

911 AAGCGACCATTAGCAAACGAAACCATCATCATGCAACGTTCMetGlnAspAlaIleThrSerValIleAsnSerSerAspValG

981 AAGGTAAATACCTGGATCGTAGCGCTATCCAAAAA CTGAAAGCCTATTTCGCTACTGGTGAACTGCGCGTlnGlyLysTyrLeuAspArgSerAlaIleGlnLysLeuLysAlaTyrPheAlaThrGlyGluLeuArgVa

1051 TCGTGCAGCAACCACCATCAGCGCTAATGCAGCTAACATCGTCTCTGCTGlArgAlaAlaThrThrIleSerAlaAsnAlaAlaAsnIleValLysGluAlaValAlaLysSerLeuLeu

1121 TACTCCGATATCACCCGTCCCGGTGGTAATATGTATGTACACCACTCGTCGCTATGCTGCTTGCATCCGTGACCTyrSerAspIleThrArgProGlyGlyAsnMetTyrThrThrThrArgArgTyrAlaAlaCysIleArgAspL

1191 TGGACTACTACCTCCGCTATGCTACCTATGCTATGCTGGCTGGCGATCCTTCCATCCTGGATGAGCGTGTeuAspTyrTyrLeuArgTyrA1aThrTyrAlaMetLeuAlaGlyAspProSerIleLeuAspGluArgVa

1261 ACTCAATGGCCTGAAAGAAACTTATAACTCTCTTTGGGTGTACCCATTGGCGCTACCGTTC~GCTATCC~lLeuAsnGlyLeuLysGluThrTyrAsnSerLeuGlyValProIleGlyAlaThrValGl~laIleGln

1331 GCTATGAAAGAAGTTACTGCTGGCTTAGTTGGTGCTGATGCTGGT~GG~TGGGCATTTACTTTGATTAlaMetLysGluValThrAlaGlyLeuValGlyAlaAspAetGlyIleTyrPheAspT

1401 ACATCTGCTCTGGCTTGAGCTAAGACTGCTCACTGCTCACAGAGG~GCTAG~TGTAGTCATCCCCTTTGG~~yrIleCysSerGlyLeuSer***

1471 CCTACAGTCTTGGTTCTTCATTCCTATAAACTTAGGGCCGTGAGTGCTAGACCGC1541 CAAAGGCTTGTCTGTATCATTGATAAGTTTTTAGCGAGCTAGTATTGGCTTATGACTCCCGGCCTTTAGTC1611 ATTTGATAAATATTACTGTCAAATACTGTCAAAATTGCTGTC~TTGCTGACTT~CTCAGGAGC~GAT~TCATGAGA

MetArg1681 GTTTTCAAAGTAACAGCTTGCGTTCCCAGCCAAACACGGATACCT

ValPheLysValThrAlaCysValProSerGlnThrArgIleArgThrGl~rgGluLeuGl~snThrT1751 ATTTCACTAAGCTGGTTCCCTATGACAACTGGTTCAGAGACAGCGG

yrPheThrLysLeuValProTyrAspAsnTrpPheArgGluGlnGl~rg

7 0

140210280350420

490 t

560

630

700

770

840

910

980

1050

1120

1190

1260

1330

1400

1470

154016101680

1750

Figure 4.8 The nucleotide and deduced amino acid, sequences of the

complete apcAB and partial apcC gene of 5’. platensis Cl.

The underlining sequences indicate the putative palindromic

motif of terminator. This sequence data has been submitted

to the EMBL/GenBank nucleotide sequence database, and

the accession number is X95898. (+l indicates the putative

of transcription start site);I’

9 1

AP a subunits

Syn elon MSWTKSIVNADAEARYLSPGELDRIKNFVSTGERRLRIAQTLTENRERIVKQAGDQLFQ 60Cyan0 MSIVTKSIVNADAEARYLSPGELDRIKSFAASGERRLRIAEAGQQLFQ6714 _~~IVTKSI~~AEARYLSPGELD~I~F~GG~RLRIAETLTGSRETI~QAGDRLFQ.- = ~sp Cl MSIVTKSIVNADAEARYLSPGELDRIKSFVTSGERRVRIATGARERIIKEAGNQLFQ

** ************************ * * * *** * ** * ** ***

Syn elon KRPDWSPGGNAYGEEMTATCLRDLDrYLRLVTYGIVAGDVPIEEIGLVGVREMYNSLG 120Cyan0 KRPDIVSPGGNAYGEEMTATCLRDLDrYLRLVTYGWAGDASLG6714 KRPDIVSPGGNAYGEEMTATCLRDMDYYLRLVTYGWSGDVTPIEEIGLVGVREMYRSLGsp Cl KRPDWSPGGNAYGEEMTATCLRDLDYYLRLITYGIVAGDVKSLG

**** ****************If** ****** *** * ** ******* *** *** ***

Syn elon TPIPAVAEGIRAMKNVACSLLSAEDAAEAGSYFDFVIGAMQ 161Cyan0 TPVAAVAEGVRSAKSVATGLLSGDDAAEAGSYFDWIAALQ6714 TPIEAVAQSVREMKEVASGLMSSDDAAEASAYFDFVIGAMSsp Cl TPIEAVAEGVRAMKSVATSLLSGEDAAEAGAYFDYLIGAMS

** *** * * ** * * ***** *** * *

AP f3 subunits

Syn elonCyan06714sp Cl

Syn elonCyan06714sp Cl

Syn elonCyan06714sp Cl

MQDAITAVINASDVQGKYLDTAAMEKLKAYFATGELRVRASVISANAANIVKEAVAKSLM Q D P I T A V I N A A D V Q G K Y L D T A S V E K L K S Y F Q T G E L R V R A S LMQDAITAVINSADVQGKYLDGAAMDKLKNYFASGELRVRAASVISANAATIVKEAVAKSLMQDAITSVINSSDVQGKYLDRSAIQKLKAYFATGELRVRASL*** ** *** ******** *** ** ******** * ** * ********

LYSDITRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDPSILDERVLNGLKETYNSLGLYSDITRPGGNMYTTRRYAACIRDLDrrVRYATYAMLAGDTSILDERVLNGLKETYNSLGLYSDVTRPGGNMYTTRRYAACIRDLDWLRYATYAMLAGDASILDERVLNGLKETYNSLGLYSDITRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDPSILDERVLNGLKETYNSLG**** *********************** *********** *******************

VPIAATVQAIQAMKEVTASLVGADAGKEMGIYFDYICSGLS 161VPVGATIQAIQAAKEVTAGLVGPDAGREMGIYYDYISSGLGVPISSTVQAIQAIKEVTASLVGADAGKEMGVYLDYICSGLSVPIGATVQAIQAMKEVTAGLVGADAGKEMGIYFDYICSGLS** * ***** ***** *** *** *** * *** ***

60

120

Figure 4.9 The alignment of the amino acid sequence from apcAB gene

product of 5’. platensis Cl (sp. Cl) and apcAB gene products

of Synechococcus elongatus (Syn elon), Cyanophora

paradoxa (Cyano) and Synechocystis PCC6714 (6714). The

identical amino acids are indicated by asterisks. :1

92

Lc subunits

Syn elon MRMFKITACVPSQTRIRTQRELQNTYFTKLVPYENWFREQQ 41

6714 MRMFRITACVPSQTRIRTQRELQNTYFTKLVPYDNWFREQQ-- - -- : ~

Fre dip RLFKVTACVPSQTRfRTQRELQNTYpTItLVPFENWFREQQ

sp Cl MRVFKVTACVPSQTRIRTQRELQNTYFTKLVPYDNWFREQQ

* * ************************** *******

Figure 4.10 The alignment of the partial amino acid sequence from

apcC gene product of S. platensis Cl (sp. Cl) and apcC

gene products of Synechococcus elongatus (Syn elon),

Synechocystis PCC6714 and Fremyella diplosiphon (Fre

dip) The identical amino acids are indicated by asterisks.

93

The AP a coding sequence extends from nucleotide 368 to

nucleotide 853 while the AP /3 coding sequence extends from nucleotide

937 to nucleotide 1,423. The predicted masses using the program

GENETY-X-MAC -XI+ the APC-m subunit apoprotein are -17,39 1, and

17,414, daltons for APCa and APCP, respectively.The AP a- and AP p-

subunit genes are in the same orientation with APCa located upstream

from the APCp gene. This organization is opposite to that found for the

phycocyanin (PC) genes of Synechococcus PCC7002, in which PCP is

upstream from PCs [32].

Data from genomic Southern hybridization analysis with 1 .l

kbp EcoRI-EcoRV homologous probe of S. platensis Cr (Fig.4.11) and

1.8 kbp EcoRI fragment homologous probe (Fig.4.12), and mapping

studies (Fig.4.13) support the idea that the apcAB genes of S. platensis

C1 are present only a single copy in the genome. Single copy of

phycobiliprotein genes has been found in most in cyanobacteria and

eukaryotic algae. A notable exception is F. diplosiphon of which these

are three sets of cpc genes [32]. Synechococcus sp. strain PCC6301 also

has two sets of cpc genes [61].

A comparison of the promoter sequences found upstream of the

transcription start sites of apcABC from Synechocystis PCC6714,

Calothrix PCC760 1, Synechococcus PCC630 1, Synechococcus

PCC7002 and a putative promoter of S. platensis Cl is shown in

Fig.4.14. The result showed that the untranslated region of S. platensis

Cl apcABC operon extends 271 bp upstream of the apcA start codon.

This region is considerably larger than those found in four organisms

mentioned above and does not appear to be any similarity to the E.coZi’

94

consensus sequences. In the -10 region (with respect to transcription

start site (+l), see Fig.4.14) of the cyanobacteria there are two types of

conserved sequences. The first sequence is C(T/G)GAGAAT(A/G)

found in-Synechom =PCC6714 and Caldrix PCC7601, while the

second one is C(T/G)CAAAAT(G/A) found in Synechococcus

PCC6301, Synechococcus PCC7002 and S. platensis Cr. There are no

sequences conserved in the -35 region. In addition, the consensus

sequence TTA(A/C)AAA(C/A)T(G/A)TTA (in the -50 region) is

conserved among all these five organisms. Although there are no Ecoli

consensus, it does appear that the apcABC genes may be under the same

transcriptional control in all five organisms.

Hypothetical palindromic sequences, possibly the terminator

sequence, could be saught within the DNA sequences of apcAB

presented in Fig.4.8, using the program GENETYX-MAC. It was

apparent that the stem and loop structure could be drawn from

nucleotide 1,446 to 1,478 (AATGTAGTCATCCCCTTTGGAAATACC

TACAGT) with the percentage of matching was 72.72% (as shown in

Fig.4.15). This structure is found within the intergenic region between

apcB and apcC and is not followed by a stretch of T’s characteristic of a

rho-independent terminator. This finding is in accordance to that found

in Synechocystis PCC6714 [7]. DiMagno and Haselkorn (1993) [7] have

reported that the obtained operon apcABC of Synechocystis PCC67 14

has two possible termination sites, one following apcB and the other

following apcC. As such, it seems likely that there is another possible

terminator 5’ to the apcC of S. platensis Cl.

95

A B C D E F G H

-_ -

21226 bp

5 148,4973

4268

3530

2027

Figure 4.11 Hybridization of the 1.1 kb EcoRI-EcoRV fragment of pMG

004 carrying part of the apcAB gene to total genomic S.

platensis Ci DNA. Restriction endonuclease digests of S.

platensis Ci total DNA were electrophoresed, transferred to

nitrocellulose membrane. Lanes: A, h marker digested with

HindIII-EcoRI; B, HincII; C, EcoRI; D, EcoRV; E, EcoRI-

EcoRV; F, EcoRI-HincII; G, EcoRV-HincII; H, uncut DNA.

I

96

A B C

21226

- - -

5 148,4973

4268

3530

1904,2027

1584, 1375

947,83 1

bp

Figure 4.12 Hybridization of the 1.8 kb EcoRI fragment of pMG 004

carrying apcABC gene to total genomic 5’. platensis Cl

DNA. Restriction endonuclease digests of 5’. platensis C,

total D N A w e r e electrophoresed, transferred to

nitrocellulose membrane. Lanes: A, h marker digested with

Hi&III-EcoRI; B, C, total DNA of 5’. platensis Cl digested

with EcoRV, EcoRV-HincII, respectively.

97

Figure 4.13 Restriction map of a 2 1 kbp EcoRI fragment of S. platensis

Ci composed of the apt gene region. Complete open

reading frames of apcA, apcB are shown as solid boxes,

whereas the partial open reading frame of apcC is shown as

an open box. Abbreviation: R, EcoRI; Hc, Hⅈ Rv,

EcoRV; C, CZaI. (- single lines indicates the size of

flanking area of apt genes with known distance, ---- broken

lines indicate the ones with unknown distance). The size of

the genes are shown by the bar denoted 200 bp.

98

SP.CI

Syn 6114

Cal 7601

Syn 6301

Syn 7002

SP.CI

Syn 6714

Cal 7601

Syn 6301

Syn 7002

-60 -40 -20

+1

CGA--GGTAGTCATG

-GGCAGGTTGCT---

CGGAAGGTAACT-GA

CGAAAGGTAATTCG-

CGGATGGCTGTAGGA** **

-2

CATTT

GTTTG

TTTTG

-ATTC

-TTTG** 3

Figure 4.14 Alignment of the putative of transcription start sites (+l)

and promoters (-10, -50 regions in boxes) of apcABC gene

of 5’. platensis Cl to those from Synechocystis PCC6714

(Syn 6714), Calothrix PCC7601 (Cal 7601), Synechococcus

PCC6301 (Syn 6301) and Synechococcus PCC7002 (Syn

7002) [7] Identical nucleotides are indicated by asterisks.

99

[GENE&X-MAC: Hairpin Loop and Stem Parts]1996.05.02Filename : UntitledSequence Size : 1799Sequence Position: 1 - 1799Start Position : 1446StemPafts Size 7 K-

- ~. .-Loop Parts Size : 11Matting Percent : 72.727 %

T T T

C G

C G

C A

C A- A

AT-c :

L :A --TT- A

CT-T - :

GGAAGCTAGk- FCTTGGTTCTTCAATTCCTA

1446 1478

Figure 4.15 The putative hairpin loop and stem part at the 3’end of the p

subunit of apt of 5’. platensis Cl using GENETYX-MAC

program.

Chapter 5

Conclusion and Suggestion

5.1 Conclusion

The complete apcAB and partial apcC genes from 5’. platensis

Cl were cloned by PCR and sequenced. The deduced amino acid

sequences of apcA, apcB and apcC genes of S. platensis Cl were

compared with those of the corresponding known phycobilisome

components. Sequence identities within each class of allophycocyanin

(AP) subunits were very high (80-85% for the a subunits, 84-93% for p

subunits and 92% for Lc polypeptide).

The nucleotide sequences of a 1.8 kb PCR fragment revealed

the coding sequences of genes for the a-, p- subunits and partial small

core linker protein of S. platensis Cr. Two complete open reading

frames of apcA and apcB (16 1 amino acids for each gene), and a partial

one of apcC (36 amino acids) were found. The organization of genes is

in an order as followed: apcA, apcB and apcC. The predicted masses of

the apt subunit apoprotein are 17,391 and 17,414 daltons for apt a and

p, respectively. These genes form an operon, apcABC, with a single

putative transcription start site and one putative termination site,

downstream of apcAB. The apt genes appear to be present in only one

copy per genome. The promoter region, like those of the apcABC

operons of other cyanobacteria, does not resemble the consensus;

promoter sequences of E. coli.

101

5.2 Suggestion

1. To confirm whether the obtained gene is the apt gene of S.

platensis Cl, an--immunological method, -utilizing antibodies to-

allophycocyanin, can be used to screen E.coZi colonies containing

fragment of apt gene, as in the case of phycocyanin. In addition, it can

be simply done by the transformation of the gene into transformable

cyanobacterium, Synechococcus sp., which it has not expression of

allophycocyanin. The occurrence of the allophycocyanin will be

indicator to prove that the obtained genes is the allophycocyanin gene of

S. platensis Cl.

2. Sl nuclease mapping and northern analysis should be

performed to analyse the apcABC operon whether it is composed of one

promoter and two terminators, of which one following apcB another

following apcC.

3. From the knowledge of gene organization of the known

allophycocyanin gene clusters, ie. apcEABC, it is possible to clone apcE

from 5’. platensis Ci by subcloning a DNA fragment upstream of apcA

in the 9 kb EcoRI fragment obtained from the h genomic library.

Moreover, the complete apcC can also be obtained by screening or

chromosome walking the genomic library using the partial apcC as a

probe.

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Appendix

Restriction enzyme of allophycocyanin gene in S. platensis Cl

1 0 2 0 3 0 4 0 5 0 6 05' CC'k!dTCCCTti~T-ficTTTTT?CC-CATGAAA%TT AAAAACTATTACAAATATACT

h

Fin17 0 8 0 9 0 100 110 120

AATAATGTGAACATAATGCCTCAAAATACATTTCGAGGTACATTTCGAGGTAGTCATGTCATGAGGTTTCAT

130 140 150 160 170 180TTGGGGGACCAAATAGGGACACCCGAAACTCGTGGCGTGGCGGCGTAT~TC~TACGCCCGAT

h h h

Fin1 Fin1AflIAsuIAvaIICfrl31Eco471NspIVSau961Sin1190 200 210 220 230 240

CGCGATCGATAAATGACTCGGC~TCTTGGT~TAGCC~GTTGCCTGCTCAGGAG~AA h A

XorII XorIIPVUI PVUI

NruIClaIBan111

PleI250 260 270 280 290 300

GTTGCCTGCTAACCGCCACCTGTGGCAGGTTAAATGGTACGCCA,.. h

BspMI EcoNI310 320 330 340 350 360

CGACACCGGGCTGACCGAAAGTCGTAGGCTTCATCATGAACCCTGTGA

TaqII370 380 390 400 410 420

GTAGAAAATGAGTATCGTTACC~TCCATCGTCAATGGCGCGTTATCTh

AlwNI430 440 450 460 470 480

GAGCCCTGGTGAATTAGATCGGATCAAATCCTTTGTTACCTTTGTTACCTCTGGCG~CGCCGGGTTCGA A

AocIIBan11Bsp12861EcoT381NspIISduI

SecI

117

490 500 510 520 530 540GATTGCTGAAACCATGACAGGTGCTCGTGAGCGCATCATC~GG~GCAGG~CC~CT

HgiEII AocII SfaNIBsp12861HgiAINspII

-. - - _~_ SduI - .~

550 560 570 580 590 600TTTCCAAAAACGTCCTGATGTAGTCTCTCCCGGTGGAAATTGAC

Ah A

SstIII BsmAIMae11

610 620 630 640 650 660TGCCACCTGCCTGCGGGATCTAGACTACTACCTGCGTCTGATCACCTACGG~TTGTTGC

AA A A h A

BspMIXbaIHgaI HphI BspMIBstYI BclIMflIXhoII

670 680 690 700 710 720TGGCGATGTTACCCCCATTGAAGAAATCGGGGGGTTGTAGGTGTTCGCG~TGTAC~TC

A

NruI730 740 750 760 770 780

TCTTGGTACTCCCATCGAAGCAGTAGCTGAAGGTGTACGGTGTACGGGCTATG~GAGTGTAGCCACA A A

AlwNI Ksp6321Ear1

790 800 810 820 830 840TTCCCTGCTGTCTGGAGAAGACGCAGCCGAAGCAGGTGCAGGTGCTTACTTCGACTACCT~TTGG

A A A

GsuI BbvIIBspMI

850 860 870 880 890 900TGCAATGTCATAAGCACTGGCGATTATCTCTTATTAATCG

A

VspIAseI

910 920 930 940 950 960TTCCTAGATCAAGCGACCATTAGCAAACGAAACCATCATGTCACTTCCGT

970 980 990 1000 1010 1020AATCAACTCCTCTGACGTTCAAGGTAAATACCTGGATCGTAGCGCTATCCACTGAA

A h A

SstIII Eco47111Mae11

1030 1040 1050 1060 1070 1080AGCCTATTTCGCTACTGGTGAACTGCGCGTTCGTGCGCATGC

h

Eco47111 :

118

1090 1100 1110 1120 1130 1140AGCTAACATCGTTAAGGAAGCAGTTGCTAAGTCTCTCTGCTGTACTCCGATATCACCCGTCC

h A AA A

BsmAI HphI ECORV Fin1HgiEII

1150 1160 1170 1180 1190 1200CGGTGGTAATATGTACACCACTCGTCGCTATGCTGCTTGCATCCGTGACCTGGACTACTA

-_ - - --A A.~ --~ +. A

BbvI TthlllII SfaNITsp451

1210 1220 1230 1240 1250 1260CCTCCGCTATGCTACCTATGCTATGCTGGCTGGCGATCCTTCCATCCTGGATGAGCGTGT

A

AlwIBin1

1270 1280 1290 1300 1310 1320ACTCAATGGCCTGAAAGAAACTTATAACTCTCTTTGGGTGTACCCATTGGCGCTACCGTTCA

h ,.. A

FokI HaeI TaqII1330 1340 1350 1360 1370 1380

AGCTATCCAAGCTATGAAAGAAGTTACTGCTGGCTTACTGCTGGCTTAGTTGGTGCTGATGCTGGT~GGAA

SfaNI1390 1400 1410 1420 1430 1440

AATGGGCATTTACTTTGATTACATCTGCTCTGGCTTGAGCT~GACTGCTCACAGAGG~

1450 1460 1470 1480 1490 1500GCTAGAATGTAGTCATCCCCTTTGG~TACCTACAGTCTTGGTTCTTC~TTCCTAT~

A

Mb0111510 1520 1530 1540 1550 1560

ACTTAGGGCCAGGGAAGGTCTGAAGTGAGTGAGTGCTAGACCGCC~GGCTTGTCTGTATCATA *

AsuICfrl31NspIVSau961

SecI1570 1580 1590 1600 1610 1620

TGATAAGTTTTAGCGAGCTAGTATTGGCTTATGACTCCCGGCCTTTAGTCATTTGAT~A

PleI1630 1640 1650 1660 1670 1680

TATTACTGTCAAATACTGTCAAZlATTGCTGACTTAACTCTCATGAGAh

SspI1690 1700 1710 1720 1730 1740

GTTTTCAAAGTILACAGCTTGCGTTCCCAGCCAAACACGGGTTAA

TthlllII1750 1760 1770 1780 1790 1800

CAAAATACCTATTTCACTAAGCTGGTTCCCTATGACAACTCAGCGGh

NspBII