Spore Forming Bacteria in Milk Powder

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Accepted Manuscript Title: Challenges and advances in systems biology analysis of Bacillus spore physiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis food isolate and a laboratory strain Authors: Stanley Brul, Johan van Beilen, Martien Caspers, Andrea O'Brien, Chris de Koster, Suus Oomes, Jan Smelt, Remco Kort, Alex Ter Beek PII: S0740-0020(10)00171-1 DOI: 10.1016/j.fm.2010.06.011 Reference: YFMIC 1457 To appear in: Food Microbiology Received Date: 24 November 2009 Revised Date: 2 June 2010 Accepted Date: 24 June 2010 Please cite this article as: Brul, S., van Beilen, J., Caspers, M., O'Brien, A., de Koster, C., Oomes, S., Smelt, J., Kort, R., Beek, A.T. Challenges and advances in systems biology analysis of Bacillus spore physiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis food isolate and a laboratory strain, Food Microbiology (2010), doi: 10.1016/j.fm.2010.06.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Transcript of Spore Forming Bacteria in Milk Powder

Page 1: Spore Forming Bacteria in Milk Powder

Accepted Manuscript

Title: Challenges and advances in systems biology analysis of Bacillus sporephysiology; molecular differences between an extreme heat resistant spore formingBacillus subtilis food isolate and a laboratory strain

Authors: Stanley Brul, Johan van Beilen, Martien Caspers, Andrea O'Brien, Chris deKoster, Suus Oomes, Jan Smelt, Remco Kort, Alex Ter Beek

PII: S0740-0020(10)00171-1

DOI: 10.1016/j.fm.2010.06.011

Reference: YFMIC 1457

To appear in: Food Microbiology

Received Date: 24 November 2009

Revised Date: 2 June 2010

Accepted Date: 24 June 2010

Please cite this article as: Brul, S., van Beilen, J., Caspers, M., O'Brien, A., de Koster, C., Oomes, S.,Smelt, J., Kort, R., Beek, A.T. Challenges and advances in systems biology analysis of Bacillus sporephysiology; molecular differences between an extreme heat resistant spore forming Bacillus subtilis foodisolate and a laboratory strain, Food Microbiology (2010), doi: 10.1016/j.fm.2010.06.011

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.

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Challenges and advances in systems biology analysis of Bacillus spore physiology; 2

molecular differences between an extreme heat resistant spore forming Bacillus subtilis food 3

isolate and a laboratory strain. 4

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Stanley Brul1#, Johan van Beilen1, Martien Caspers2, Andrea O’Brien1*, Chris de Koster3, Suus 6

Oomes4, Jan Smelt1, Remco Kort2 and Alex Ter Beek1 7

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Netherlands Institute for Systems Biology (NISB), 1Department of Molecular Biology and Microbial 9

Food Safety (MBMFS); 2Microbial Genomics Group, TNO Quality of Life, Zeist; 3Department of Mass 10

Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences, University of 11

Amsterdam, Amsterdam; 4 Biosciences, Unilever Research and Development, Vlaardingen; The 12

Netherlands. 13

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*Current address: FEMS Central Office, Keverling Buismanweg 4, 2628 CL Delft, The Netherlands; 22

Tel: +31-15-2693931; Fax: +31-15-2693921; E-mail: [email protected] 23

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#Corresponding author. Molecular Biology and Microbial Food Safety, Nieuwe Achtergracht 166, 1018 26

WV Amsterdam, The Netherlands; Tel: 00-31-20-5257079; Fax: 00-31-20-5256971; E-mail: 27

[email protected] 28

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Abstract 1

Bacterial spore formers are prime organisms of concern in the food industry. Spores from the genus 2

Bacillus are extremely stress resistant, most notably exemplified by high thermotolerance. This 3

sometimes allows surviving spores to germinate and grow out to vegetative cells causing food 4

spoilage and possible intoxication. Similar issues though more pending toward spore toxigenicity are 5

observed for the anaerobic Clostridia. 6

The paper indicates the nature of stress resistance and highlights contemporary molecular approaches 7

to analyze the mechanistic basis of it in Bacilli. A molecular comparison between a laboratory strain 8

and a food borne isolate, very similar at the genomic level to the laboratory strain but generating 9

extremely heat resistant spores, is discussed. The approaches cover genome-wide genotyping, 10

proteomics and genome-wide expression analyses studies. The analyses aim at gathering sufficient 11

molecular information to be able to put together an initial framework for dynamic modelling of spore 12

germination and outgrowth behaviour. Such emerging models should be developed both at the 13

population and at the single spore level. Tools and challenges in achieving the latter are succinctly 14

discussed. 15

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Bacterial spores in food: Introduction 1

Bacterial spores cause major problems in the food industry due to their ubiquitous occurrence and 2

their intrinsic high stress resistance characteristics. In addition some spore-formers are highly 3

toxigenic such as Clostridium botulinum and, though to a lesser extent, Bacillus cereus (Esty and 4

Meyer, 1922; see also e.g. Stringer et al., 2005 and the review by Peck, 2006). Bacterial anaerobic 5

spore formers can be found amongst the Clostridia. Food poisoning due to Clostridium perfringens is a 6

well known disease (see e.g. Rahmati and Labbe, 2008). While Clostridia are a problem the spoilage 7

Bacilli pose an even greater challenge to microbial food stability due to their often extreme levels of 8

thermal resistance. Wild-type isolates of Bacillus subtilis as well as various strains from Bacillus 9

sporothermodurans are causative agents of many cases of food spoilage in which products containing 10

herbs, spices, milk-powder and other dry ingredients of manufactured food are involved (Scheldeman 11

et al., 2006; Oomes et al., 2007). The general observation is that the heat resistance of spores isolated 12

from spoilage isolates is higher than that observed for laboratory strains. Furthermore, it is well known 13

that sporulation conditions of Bacilli may even further enhance the thermal resistance of their spores. 14

Most recently Oomes et al. (2009) reported that enhanced expression of spore coat polysaccharide 15

biosynthesis (sps) genes possibly plays a role in enhanced thermal resistance upon sporulating Bacilli 16

in the presence of high calcium concentrations. The ‘proof of the pudding’ needs to be found in the 17

heat resistance analysis of spores from strains that lack these genes. This work is still to be done. 18

Finally, Bacillus spores display significant heterogeneity with respect to germination and outgrowth 19

behaviour which may be the consequence of heterogeneous sporulation conditions activating 20

sequentially in subpopulations of cells various stress response mechanisms (Hornstra et al., 2009). 21

Most heterogeneity seems to be caused by the initial germination step (Stringer et al., 2005). 22

In order to fulfil the need of providing fresh-like foods that are both nutritious and microbiologically 23

stable, a thorough understanding of not only the generally observed high thermal stress resistance but 24

also this significant heterogeneity in behaviour of spores with respect to the survival efficiency of 25

(thermal) preservation stress is paramount. Sporulation conditions on surfaces of food ingredients are 26

likely to be heterogeneous and play an important role in the generation of heterogeneity in spore 27

physiology such as variable thermal stress resistance and germination behaviour (Veening et al., 28

2006; Rose et al. 2007). 29

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In summary, the current needs are for (1) detection tools that allow for the identification of 1

heterogeneity at the level of species composition in a given ingredient of food manufacturing, (2) of 2

visualisation tools that allow for the study of spatial and temporal heterogeneity in sporulation 3

conditions and (3) of analysis tools that facilitate the study of outgrowth heterogeneity. The latter 4

provides data on the variation in the timing of (out)growth and thus chances of spoilage of food 5

products (Hornstra et al., 2009). The integration of knowledge at all three levels is needed to provide 6

input for Quantitative Microbial Risk Analysis. The efforts in these fields can include both enhanced 7

food safety and minimization of food spoilage as a deliverable since the approaches are analogous. 8

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Detection tools for spore-formers 10

For the detection of microorganisms the use of DNA probes remains one of the most straight forward 11

state of the art choices. In this context the current information on the 16S rRNA genes as well as 12

forthcoming genomotyping data of food spoilage spore formers is relevant (see e.g. Oomes et al., 13

2007; Caspers et al., submitted to this issue of Food Microbiology). Comparative genome sequencing 14

is currently well within reach. Thus, in the case of relevant, closely related bacterial isolates it is 15

increasingly easy to identify unique sequences (see e.g. the discussions in Earl et al., 2008 and Medini 16

et al., 2008). Many of these may then be used to derive sequences amenable to use in DNA chip and / 17

or PCR based detection platforms to the benefit of the safety assessment of food processing. 18

The tools have also been translated to probes that can detect single nucleotide polymorphisms using 19

the oligonucleotide ligation amplification technology (see Wattiau et al., 2008). That platform is in 20

principle suited to discriminate between a large set of different spore formers i.e. for screening and 21

classification. In case a specific organism is sought at low detection levels quantitative PCR (Q-PCR) 22

technology is the better technique of choice. Q-PCR is available for laboratory scale experiments. 23

Although these methods are fast, highly specific and sensitive, the application of molecular-based 24

techniques in the control of food safety is still relatively limited as they suffer from some serious 25

drawbacks. The development of methods that can be uniformly applied is particularly hampered by the 26

fact that all different produce and food products contain their own interfering components. The 27

development of such methods becomes even more difficult due to a constant introduction of new food 28

matrices. While molecular methods are very sensitive, low DNA copy numbers are difficult to detect 29

when the sample size is very small. Introduction of an enrichment step preceding DNA-detection is a 30

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solution, but this makes results qualitative, rather than quantitative. Sample preparation needs close 1

attention. Preferably, such sampling needs to be rapid and as homogeneous as possible. Innovative 2

strategies focus on the use of magnetic beads coated with cell-recognizing molecules, on physical 3

methods such as floatation, and on lysis of whole food matrices (Wagner and Dahl, 2008). The latter 4

was described originally by Hein and co-workers who obtained in a few hours from 5-10 gram of 5

complex structured food matrices such as hard cheese, in a one-step approach, sufficient bacteria for 6

DNA extraction and further study (Rossmanith et al., 2007). In artificially contaminated milk samples 7

the lowest inoculum was 102 Listeria monocytogenes colony forming units per ml. These inoculated 8

samples were found positive in the DNA test. Recovery of bacterial DNA assessed with quantitative 9

PCR as compared to the amount of colony forming units ranged from 30-85%. Similar procedures will 10

have to be assessed for their suitability in the isolation and subsequent analysis of bacterial spores. 11

Finally, a limitation of many contemporary molecular techniques is that they fail to discriminate 12

between viable and inactivated organisms. Thus systems like the recently developed and marketed 13

Salmonella sero-var typing (Wattiau et al., 2008) by Check-points will have to be adapted to the 14

analysis of RNA molecules. Alternatively, propidium monoazide could be included in PCR-based 15

detection procedures as it allows for a selective PCR amplification from cells with intact membranes 16

versus cells with compromised membranes which are mostly no longer viable (Nocker et al., 2007). 17

For now a prototype laboratory system that can assign species based on the DNA hybridization 18

pattern of various Bacillus isolates has been developed (Figure 1). A strain specific system was 19

recently described (Van Zuijlen et al., 2009). Future work should focus on the identified needs to allow 20

for its commercial exploitation. 21

22

Genomics and proteomics of spore formation, structure and origin 23

Spore formation conditions contribute to stress resistance heterogeneity within a spore crop of a given 24

strain. The data from the Setlow group (Rose et al., 2007) show clearly that formation of spores on 25

solid surfaces leads to spores that have a much higher thermal stress resistance than that of spores 26

formed in liquid media. More sophisticated analysis by Veening et al. (2006) had already shown that 27

normal complex colony growth leads to the formation of highly thermal resistant spores. In such more 28

structured colonies spatial heterogeneity in sporulation progression is common (e.g. Veening et al., 29

2008). This was shown with the use of fluorescent reporter proteins specific for various stages of the 30

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sporulation cascade. Veening et al. (2006) showed that spore formation initiates on bundles of cells 1

that are formed on the surface of many wild-type Bacillus colonies. Later data showed that in a micro-2

colony the formation of spores may at any time initiate in a random position of the colony (Veening et 3

al., 2008). The study of the background of these events is currently ongoing. Data from Hornstra et al. 4

(2009) illustrate by using reporter proteins specific for spore formation and the general stress response 5

(regulated by σB) that the initiation of either response occurs heterogeneously in a population of cells. 6

The impact of such heterogeneous responses in groups of cells on the final resulting spore physiology 7

is a current hot topic of research. Not only survival of thermal stress and subsequent observed 8

heterogeneity in germination, but also the heterogeneity in outgrowth may be inferred. One level at 9

which such heterogeneity may be regulated is at the level of germination receptor expression and 10

more in general, the protein composition of spores (Hornstra et al., 2006; 2009). In order to analyze 11

the minimal composition of the spore proteomics techniques are paramount. Such experiments are 12

currently in progress (see further on). 13

It is known that B. subtilis strain A163 generates high thermal resistant spores and the strain has an 14

overall 85% identity to the laboratory strain B. subtilis 168 as evaluated using DNA-DNA hybridization 15

(Kort et al., 2005). In line with this observation, we found that genome-genome hybridization based 16

genomotyping shows that strain 168 and A163 share ~78% core genome markers and contain ~18% 17

accessory genome markers (Figure 2A, defines core and accessory marker). These data were 18

obtained by genome hybridizations of strains 168 and A163 on an array containing a random set of 19

2304 B. subtilis genomic DNA markers, originating from a mix of 7 different B. subtilis isolates 20

including strain 168 and A163 (Caspers et al., 2010). Interestingly, we were able to identify a subset of 21

markers hybridizing significantly stronger with strain A163 than with strain 168 (Figure 2B). 22

This indicates that the corresponding DNA-stretches in the two strains significantly differ in 23

composition or copy number, or that some of those genes even may be absent in strain 168. We 24

highlighted three of these sequenced markers as they encode homologous proteins that can be 25

implicated in spore formation, germination and spore thermal resistance (Figure 2C). The N-26

acetylmuramoyl-L-alanine amidase (xlyB) encodes a member of the autolysins, a group of enzymes 27

involved in the degradation of peptidoglycan occurring in bacterial cell walls and spore matrix. 28

However, autolysins of the Xly-group have not (yet) been reported in connection with sporulation or 29

germination, but seem to be involved in cell lysis following prophage induction (Smith et al, 2000). 30

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Gene wapA is not (yet) reported as sporulation specific, but its product, the wall associated protein A, 1

plays a major role in the cell wall composition, together with other main cell surface proteins as WrpA, 2

LytB and LytC (Antelmann et al, 2002; Serizawa et al, 2005). Notably, preliminary spore coat 3

differential proteomics data show that WapA is probably more abundant in spores of strain A163 then 4

those of strain 168 (our unpublished observations). The final highlighted gene, which shows 5

significantly higher hybridization intensity signals, is spsA encoding a spore coat dTDP-6

glycosyltransferase belonging to inverting GT2-family glycosyltransferases implicated in spore coat 7

formation. Although mutations in the sps locus do not interfere with sporulation or germination, but 8

lead to production of spores with altered surface properties (Ünligil and Rini, 2000; Tarbouriech et al, 9

2001; Stragier and Losick, 1996), Oomes et al. (2009) reported enhanced expression of spore coat 10

polysaccharide biosynthesis (sps) genes in relation to enhanced thermal resistance upon sporulating 11

Bacilli in the presence of high calcium concentrations. Summarizing, we have identified some exciting 12

new candidate genes at least one of which may be directly related with the development of extreme 13

thermal resistance in spores from B.subtilis strain A163. 14

Upon applying a systems approach the gene-composition is the first level in a bottom-up analysis as it 15

provides what might be called ‘the index of cellular capacity’. It is also well established though that 16

analysis of regulation of cellular function at other vertical levels such as protein synthesis and 17

metabolism is paramount to come to a real understanding of biological behaviour. Thus we evaluated 18

various proteomics techniques to analyze bacterial spore protein composition. Here we briefly indicate 19

our attempts to analyse spore proteins using two dimensional gel-electrophoresis coupled to mass 20

spectrometry for protein spot identification. For quantification we used fluorescent staining protocols 21

that allowed for differential in gel (DIGE) analysis to eliminate variability due to the use of different gels 22

and loading conditions. In total the 2D protein profiles from 6 independently generated B. subtilis spore 23

samples were analysed. Each sample was run in duplicate (one labelled with Cy5 and one with Cy3), 24

giving a total of 12 gels for analysis. An immediate observation was that many protein spots could be 25

visualised both from the B. subtilis 168 spore extracts as well as from extracts of B. subtilis A163 26

(~680 in extracts from either sample). Based on DeCyder analysis we observed that over 85% of the 27

spots was similar as was to be expected based on the earlier DNA-DNA hybridisation data. Of the 28

proteins that could be identified from the B. subtilis 168 gels two thirds was directly linked to the 29

sporulation process or cortex synthesis. Interestingly, three proteins YhcQ, YtfJ, YhjR have an 30

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unknown function. YhcQ has homology to spore coat protein CotF and is expressed downstream of 1

yhcN, the gene product of which is in the inner spore membrane. YtfJ expression has been shown to 2

be σF regulated (Kuwana et al. 2002) and to date has been proposed as a candidate spore protein. 3

This study corroborates the notion that the YtfJ protein is indeed most likely a true spore protein. 4

From the labelled gels, DeCyder also enabled the identification of differentially expressed proteins 5

between strain 168 and A163. To that end protein containing gel pieces were excised from the 2D 6

gels, washed with ammonium carbonate, shrunk by adding acetonitrile and dried under vacuum. The 7

proteins were next digested with trypsin and identified using MALDI-TOF and the B. subtilis protein 8

database. It is apparent that many of the proteins in the spores from B. subtilis A163 are post 9

translationally modified. This may be seen in the circled area of the 2D proteomics gel and in more 10

detail in the lower rows of figure 3. Clearly multiple protein spots with different PI values were seen. All 11

of these have been identified as the same (CotA) protein. The modification of CotA can include 12

variation in attached polysaccharide levels in strain A163. While coat protein modifications involving 13

sugars have not been biochemically described, modifications involving amino acid cross-links have 14

been described (Zilhão et al., 2005; Monroe and Setlow, 2006). Finally, the analysis of differential 15

protein presence in highly thermal resistant spores of food spoilage isolate A163 versus laboratory 16

strain derived spores also identified stress responsive proteins such as Clp proteins and proteins 17

belonging to the σB regulon. This suggests that at least some vegetative cells can sporulate after 18

expression of such more typical vegetative stress responses has been activated or visa versa. These 19

events may contribute to the heterogeneous spore stress resistance physiology that is often observed 20

in populations. 21

22

Germination and outgrowth molecular physiology 23

In order to observe events in germination and outgrowth single spore analysis using flow cytometry is 24

state of the art (Smelt et al., 2008). In fact, spores might be capable of growing out months after the 25

original heat treatment had been applied. Processes occurring in these spores are largely unknown, 26

but the heat treatment may have damaged a number of essential proteins or enzymes (Coleman et al., 27

2007). A normal distribution of such damage may already lead to large variation in outgrowth efficiency 28

as discussed in Hornstra et al. (2009). Additionally, possible repair processes may contribute to 29

(heterogeneity in) spore recovery. Once all hurdles have been overcome, the germinating spore will 30

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proceed quickly to normal cell metabolism and division. The overall functional modules have been 1

identified in spore germination using both genome-wide transcript analysis and functional studies with 2

knock-out mutants (Keijser et al., 2007; Ter Beek et al., 2008; Ter Beek, 2009). Remarkable is the 3

observation that while general housekeeping genes are actively transcribed throughout the 4

germination and outgrowth period some of the more stress response specific genes such as yhcA and 5

ymfM are transiently expressed. The latter is involved in shaping a bacterial cell (reviewed in Margolin, 6

2009). Importantly, both genes are known to be operative in weak-organic acid stress response of 7

vegetative cells likely as pump encoding genes or as genes coding for proteins involved in membrane 8

modification (discussed in Ter Beek and Brul, 2010). Also genes operative in the general stress 9

response and (DNA) damage repair are transiently expressed in the absence of the cognate stress 10

(Keijser et al., 2007). The regulation of the transient expression is not understood and it is not clear 11

either whether all outgrowing spores express the same genes. In fact, the heterogeneity in expression 12

of genes at the level of single cells may be significant, particularly when it concerns (thermally) 13

damaged spores or spores that grow out in a sub-optimal environment. The data of Smelt et al. (2008), 14

Cronin and Wilkinson (2008) and earlier Stringer et al. in Clostridia (2005) are exemplary for this. 15

Using specific fluorescent reporter constructs for proteins whose genes are under control of specific 16

sigma factors (i.e. the vegetative general stress response specific σB, the spore specific sporulation 17

sigma factors σF and σG, or the mother cell sporulation sigma factors σE and σK) it is possible to 18

visualize specific molecular events leading to putative heterogeneity at the single cell level (see e.g. 19

Hornstra et al., 2009). A crucial link in matching molecular data to single spore behaviour is the 20

possibility to visually follow and analyze bacterial spore germination and outgrowth. Figure 4 gives an 21

example of how this may be achieved. Single bacterial spores were followed throughout germination 22

from the phase bright stage through to outgrowing rods. In this set up ~30% of the spores did not 23

commence germination within the 11 hours time frame and full outgrowth to cell division was not seen 24

likely caused by limited oxygen availability. Currently, a new set-up is evaluated in which the 25

morphology of the agarose strip is such that aeration is optimized. Additionally, various heat activation 26

regimes and germination triggers are evaluated. Finally, specific fluorescence of reporter proteins will 27

be assessed per spore to visualize relevant physiological parameters such as the intracellular pH or 28

the activation state of specific developmental pathways (Ter Beek, 2009; Hornstra et al., 2009). 29

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General conclusions and emerging questions 1

From the areas discussed a number of key conclusions and derived research questions emerge. A 2

clear conclusion is that the genomotyping approach yields valuable information on strain diversity. For 3

a functional interpretation genomotyping needs to be complemented by other techniques. Proteomics 4

strategies such as the ones indicated here are important further developments. Currently we are 5

setting up a direct proteomic analysis of covalently bound spore coat proteins using liquid 6

chromatography coupled to mass spectrometry that is suitable for metabolic N15 N14 labelling for highly 7

sensitive protein quantification (our unpublished observations). Finally it is crucial to follow spore 8

germination and outgrowth physiology at the single spore level as heterogeneity in spore composition 9

and derived outgrowth characteristics is highly relevant to microbial food stability. 10

The topics of further research can be grouped under three main questions. (a) What is the molecular 11

basis for the difference in spore thermal resistance observed between wild-type Bacilli and the 12

laboratory strain? Is there a role for CotA modification in enhanced thermal stress resistance? What if 13

any is the role of the higher level of the specific spore coat polysaccharide biosynthesis gene spsA in 14

A163? (b) What is the molecular basis of the variation in heat resistance that exists between strains 15

sporulated under different conditions? Which signals govern quantitatively the emergent property ‘heat 16

resistance’? (c) What is the basis of the heterogeneity in spore germination of either non-thermally 17

treated or thermally treated spores? 18

In many cases the spore-forming condition will also heavily impinge on individual spore characteristics. 19

Recently Oomes et al. (2009) showed that culturing spore-forming cells in the presence of high 20

calcium concentrations induces genes that are responsible for the formation of the outer 21

polysaccharide layer of the spore coat. Such enhanced ‘sugar’ synthesis may well play a role in the 22

protection of spore germination enzymes such as CwlJ which are located in outer layers of the spore 23

(Bagyan and Setlow, 2002) Furthermore, it is clear that stress conditions that lead to spore formation 24

often also lead to initial activation of vegetative stress response systems. Again, how homo- or 25

heterogeneous this process takes place will be important ‘guides’ for future research on fundamentals 26

and applications in the field of bacterial spore physiology and its role in food preservation. The 27

generation of a molecular framework for modelling this process quantitatively at the population and 28

single cell level is the most paramount question to tackle. 29

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1

Acknowledgements 2

This research is supported by the Dutch Foundation for Applied Sciences (STW), the EU ERASMUS 3

MUNDUS program and was partially made possible by EET grants from the program Economy 4

Ecology and Technology of the Senter-Novem agency of the Dutch ministry of economic affairs. 5

6

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Rappuoli, R. 2008. Microbiology in the post-genomic era. Nature Rev. Microbiol. 6, 419-430. 15

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Monroe, A. and Setlow, P. 2006. Localisation of the transglutaminase cross-linking sites in 17

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Legends to the figures: 1

2

Figure 1. 3

Image of a typical read out of a prototype of a Check-Points technology based system used for the 4

identification of various spore formers. The DNA chip is in reality less than one centimetre in diameter 5

and can contain species and strain specific oligonucleotide sequences. Here, in short, DNA isolated 6

from spores obtained from milk-powder was incubated with specific probes after which via 7

oligonucleotide ligation dependent amplification reactions sufficient labelled material was generated to 8

obtain a fluorescent signal upon hybridisation to the DNA chip. Technologically the procedure used 9

here was similar to the one described in Nagaoka et al. (2005). Below the image the application 10

scheme of the probes is given as a 7x7 matrix. The chip contains 4 copies of that matrix on the top left 11

and right hand as well as the bottom left and right hand side. An ‘r’ in front of a probe name indicates 12

a signal specific for a 16S-ribosomal RNA gene based probe. The relevant 16S-ribosomal RNA gene 13

sequences have been described before (Oomes et al., 2007). The other probes refer to strain specific 14

genomic sequences for B. subtilis (B.sub), B. coagulans (B.coa), B. licheniformis (B.lich), B. pumilus 15

(B.pum), B. cereus (B.ce) and Geobacillus (Geo). Sequence information for these probes is owned by 16

the partners of a Dutch ministry of Economic Affairs sponsored project and can be requested from the 17

authors. ‘Hyb’ refers to a, positive, hybridisation control and ‘No Amp’ refers to a, negative, ‘No 18

Amplification’ control. The identification of the strains present in the milk-powder is indicated for the top 19

right hand matrix on the chip. Both B. subtilis rRNA gene probes and three of the four Geobacillus 20

rRNA gene probes gave a positive hybridization signal. In this experiment none of the strain specific 21

non 16S-ribosomal RNA gene genomic markers reacted positive. This was at least in part presumably 22

due to their relative low copy number compared to the ribosomal genes. The detection limit was in the 23

experiment shown ~200 spores / gram milk powder. For commercial application in the food industry 24

the system was next adapted for colorimetric detection according to Wattiau et al. (2008) and 25

subjected to commercial evaluation (discussed in Van Zuijlen et al. 2009). See 26

http://www.check-points.com/ for a description of the commercially available Salmonella system. 27

28

29

30

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Figure 2. 1

Hierarchical clustering of genomic DNA hybridization data of B. subtilis laboratory strain 168 and food 2

spoilage isolate strain A163 (strain A163 is described in Kort et al., 2005; see columns in panel A and 3

B) on a mixed genome microarray designed for detection of various Bacilli (Caspers et al. 2010). 4

Strong/weak hybridization is shown as black/white. For each strain 2 independently obtained aliquots 5

of labelled genomic DNA were used. 6

Panel A: Hybridization data of the 4 fluorescently labelled genomic DNA’s (4 columns) on 2304 7

random genomic B. subtilis array markers (2304 rows) originating from random gDNA fragments of a 8

mix of 7 different B. subtilis isolates including strains 168 and A163. Top bar indicates hybridization 9

strength (2log(signal/background), 0 = white, 6 = black). The indicated cluster of “core” genome 10

markers (~78% of entire genome) hybridizes with both strains 168 and A163. The core genome 11

markers are defined as those markers giving hybridization signals for all 4 hybridizations of >50% of 12

the maximum signal intensity of those markers. The cluster of “accessory” genome markers (~18%) 13

was identified as those markers showing differential hybridization between the two strains. For these 14

markers the signals of the duplicate hybridizations have to be for one strain <50% AND the other strain 15

>50% of the maximum signal per marker. Hierarchical clustering on the 4 hybridization data sets (4 16

columns) shows that, despite experimental variations that evidently introduced differences between 17

the two hybridizations of the same strain, these two separate hybridizations cluster per strain together. 18

Panel B: scale-up of data subset from panel A containing differential hybridization markers with 19

significant bias for strain A163. Multiple technical replicates gave identical results with biological 20

duplicates of one strain leading to >50% AND of the other strain <50% of the maximal signal per 21

marker. Markers that did not obey this rule were not evaluated. 22

Panel C: In this panel a table is given in which 3 in strain A163 highly enriched genomic DNA 23

fragments (the fragments labelled with an asteriks in panel B) and their predicted protein functions 24

potentially related with spore formation and thermo resistance are listed. 25

26

Figure 3. 27

A typical set of 2-Dimensional Differential In-Gel Electrophoresis (DIGE) gels comparing total spore 28

extracts of a laboratory wild-type 168 strain and extracts from high heat resistant spores forming food 29

spoilage isolate strain A163. The two strains display an 85% identity based on DNA-DNA hybridization 30

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results. Spores were harvested after 4 days of incubation at 37°C. In brief, the spore pellet was re-1

suspended in 2 ml of a 2% solution of Uragrafin-76 (Bayer-Schering, Belgium) which was then layered 2

above 15 ml of a 50% solution of Uragrafin and centrifuged at 12,000xg for 30 min. The resulting pellet 3

was re-suspended in 1 ml of cold distilled water. After six washes in cold distilled water, the purified 4

spores were stored at 4°C (for up to 4 days) at an A580nm of approximately 20. Microscopic 5

examination confirmed the purity of spore samples and the number of spores in the cell-free spore 6

suspensions was determined with a haemocytometer (Burker-Turk, Marienfeld, Germany). Purified 7

spores were disintegrated with 0.10-mm glass beads in the presence of a bacterial protease inhibitor 8

cocktail in a BioSavant FastPrep 120 machine (Qbiogene). For 2D-DIGE a pooled standard was 9

prepared by pooling 50 µg of protein from each of the samples (168, A163). The protein samples 10

(including the pooled standard samples) were labelled with one of three cyanide dyes (Cy3 or Cy5 for 11

samples, Cy2 for pooled standards) in a ratio of 400 pmol dye: 50 µg protein according to the 12

manufacturer’s instructions. Next the samples (100µl) were added to 100µl of SDS loading dye (4% 13

v/v SDS, 10% v/v β-mercaptoethanol, 1mM dithiothreitol, 0.125 M Tris-HCl (pH 6.8), 10% v/v glycerol, 14

0.05% v/v Bromophenol blue) and boiled for 8 min (heated to 110°C in the case of spores from A163). 15

To improve sample quality for iso-electric focusing, samples were treated with a 2D clean up kit 16

(Amsersham, Biosciences). Iso-electric focusing (IEF) was run as described by Luppens et al (2005). 17

Two samples and one pooled standard were applied in a sample cup and run (1 h 200 V, 3.34 h step 18

from 300 V to a 1000 V, 0.5 h step from a 1000 V to 8000 V, 4 h 8000 V) per 18-cm Immobiline 19

DryStrip (pH 3-10). Following IEF, the strips were layered upon a uniform 12.5% v/v SDS 20

polyacrylamide gel and electrophoresis was run at 2 W per gel for 15 h at a constant temperature of 25 21

°C. Labelled proteins were visualized by scanning o n the Typhoon imager. Gel analysis was 22

performed using DeCyder 5.01 software. To exclude spots which were non representative of proteins, 23

filter settings were: Slope < 1.5, area > 200, peak height > 200, volume > 1000. Differences between 24

the two strains were tested with Student’s t-tests on the proteins found to be significantly different in 25

the 1-way-ANOVA analysis. All tests were un-paired; the p-value for significance was set at < 0.01 to 26

compensate for the repeated testing of the same data. The encircled area is magnified in the lower 27

panels of the figure. These panels also give quantitative plots of the encircled area after differential 28

scanning of the spots (see text for further discussion). 29

30

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Figure 4. 1

A typical example of an experiment describing time resolved image analysis of samples of germinating 2

and outgrowing B. subtilis spores of laboratory strain PS832 (Oomes et al., 2009). The spores were 3

inoculated onto 1.5 mm ‘thick’ low melting point agarose strips placed on a standard microscope slide 4

(CML). These strip were made from minimal growth medium according to Hu et al. (1999) buffered 5

with 80mM MOPS (PH 7.4), containing as carbon- and nitrogen-sources 10 mM glucose, 10 mM 6

glutamate and 10 mM NH4Cl, and as germination triggers additionally 1 mM fructose, 1 mM potassium 7

chloride, and 10 mM L-asparagine. The medium was solidified by the addition of 1.5% low melting 8

point agarose (Sigma). The various forms of spores and outgrowing cells that are generally observed 9

were quantified. At each time point microscope views containing 50-100 spores were examined. 10

Experiments were performed multiple times of which a typical result is shown. Microscope counts of 11

spore numbers at the various time points after the onset of germination were done at least in duplicate. 12

The l/w ratio indicates the length/width proportion of the outgrowing cells. When this number exceeded 13

1.5 we considered the specimen under examination to be an outgrowing rod-like cell rather than a 14

phase dark spore. 15

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Figure 1. 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Hyb B.sub01 B.sub02 B.sub03 B.sub04 B.sub05 B.sub06

rB.coa rB.sub rB.sub - Geo01 Geo02 Geo03

Geo04 Geo05 No Amp Geo06 B.ce01 B.ce02 B.coa01

B.coa02 B.lich01 B.lich02 - B.pum01 B.pum02 B. pum03

- - - - - - -

- - - - - - -

- - - rGeo04 rGeo03 rGeo02 rGeo01

16

17

18

19

Geobacillus

B.subtilis

Geobacillus

B.subtilis

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Figure 2. 1

2

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Figure 3. 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

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22

23

B. subtilis 168 B. subtilis A163

pH 4 pH 7 pH 4 pH 7

85 % identity

B. subtilis 168

B. subtilis A163

B. subtilis 168 B. subtilis A163

pH 4 pH 7 pH 4 pH 7

85 % identity

B. subtilis 168

B. subtilis A163

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Figure 4. 1

2

3

0

10

20

30

40

50

60

70

1 3 5 7 9 11

Phase brightsporesPhase darksporesRod-like cellsl/w 1,5%

of t

he to

tal

Time after onset of germination (hours)