Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNA
Boonstra, Mirjam
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Chapter 6
Chapter 6
171 Summary
Bacillus subtilis has evolved to be able to adapt quickly to changes in its environment. This versatility in lifestyle makes it a fascinating subject for scientific inquiry. Unlike many other bacteria capable of the same adaptations B. subtilis is easy to cultivate in the lab and easily genetically modified making it an excellent model organism. In this thesis only two of B. subtilis’ many adaptive strategies, i.e. sporulation and natural competence, were studied. Sporulation results in the formation of highly resistant spores allowing survival under harsh conditions. Competence is the state in which the bacterium can take up DNA from its environment. The process of taking up DNA from the environment and its integration into the chromosome or reconstitution as a plasmid is called transformation. Competence is a transient state that the cell can enter and leave, regardless whether DNA has been taken up or not (Süel et al., 2006). Sporulation however is irreversible past the commitment stage and needs to be completed past this point (Narula et al., 2012; Parker et al., 1996). Both phenotypes are a response to nutrient limitation and both require the presence of the phosphorylated version of the master regulator of sporulation Spo0A (Spo0A~P) (Fujita and Losick, 2005; Fujita et al., 2005; Mirouze et al., 2012). By using specific media we can ensure that the cells preferentially enter one of the two phenotypes (Bott and Wilson, 1967; Schaeffer et al., 1965; Spizizen, 1958). Both sporulation and competence have been extensively studied. A more in depth description of these processes is given in the introduction Chapter 1. Much is also known about the process of transformation and the proteins involved in the uptake and the integration of DNA, but there is always more to discover, such as the timing and localization of DNA integration. We therefore focused on some aspects of these processes which were not fully elucidated yet. The results of these investigations are summarised below.
172 Chapter 2, Spo0A and replication
One of the most important adaptations that B. subtilis can undergo is sporulation. Sporulation as a response to nutrient limitation allows the bacterium to survive for extended periods of time in hostile environments until conditions become more favourable, nutrients become available and germination occurs. Sporulation is an energy intensive process, which is irreversible past the commitment stage (Narula et al., 2012; Parker et al., 1996). It is therefore important that the process is tightly regulated. Spo0A is the master regulator of sporulation and the amount of phosphorylated Spo0A (Spo0A~P) determines whether sporulation is initiated or not, with high amounts of Spo0A~P causing entry into sporulation (Fujita and Losick, 2005; Fujita et al., 2005). An important feature of sporulation is the presence of only two chromosomes, one destined for the spore, with the other remaining in the mother cell. Disruption in chromosome copy number can have a negative impact on sporulation, such as the formation of two instead of one spore (Eldar et al., 2009; Murray and Errington, 2008; Veening et al., 2009; Xenopoulos and Piggot, 2011). In Chapter 2 the role of Spo0A was studied in maintaining correct chromosome copy numbers during sporulation.
The two primary regulators of chromosome copy number are SirA (Sporulation Inhibitor of Replication) (Jameson et al., 2014; Rahn-Lee et al., 2009; Wagner et al., 2009) and Sda (Suppressor of DnaAI) (Burkholder et al., 2001; Cunningham and Burkholder, 2009; Veening et al., 2009). More recently a kinA:spo0F gene dosage effect linking sporulation to nutrient status and also effecting chromosome copy number has been found (Narula et al., 2015). The chromosome of B. subtilis contains Spo0A~P binding sites (0A-boxes) in its origin of replication (oriC)(Berka et al., 2002; Hamoen et al., 2002; Ogura et al., 2002). The presence of 0A-boxes within the oriC, along with the fact that the lack of known factors controlling chromosome copy number sirA and sda does not completely disrupt the generation of correct copy number, led to the hypothesis that Spo0A plays a role in maintaining correct chromosomal copy number.
Summary & Discussion
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Mutating the 0A boxes in the oriC region in a WT background as well as strains in which sda, sirA or both were deleted allowed us to determine the effect that binding of Spo0A~P has on chromosome copy number. The chromosome copy number was visualised using a tetO/tetR-mcherry system. By over-expressing kinA and thereby increasing the amount of phosphorylated spo0A we enhanced the effect of Spo0A on chromosome copy number. The effect of Spo0A on replication is particularly pronounced when the levels of Spo0A are rapidly increased by over expression of kinA rather than when the levels are more gradually increased through nutrient limitation. Although binding of Spo0A to the oriC might not be the primary method of regulating chromosome copy number, it is possible that there are situations in which its role becomes more important. In our data the effect of a high level of spo0A, independent of nutrient status, is more pronounced than when the increase of Spo0A occurs more gradually as a result of nutrient depletion. It is not unlikely that there are situations in nature where regulation of chromosome copy number by Spo0A is important, for instance when sporulation is initiated by environmental factors other than nutrient limitation. To conclude we found that Spo0A is able to assist in maintaining correct chromosome copy number during sporulation (Boonstra et al., 2013).
Chapter 3, Transcriptome and proteome during competence
A prominent aspect of competence is that even in the lab under optimal competence stimulating conditions only a subpopulation (5-25%) of the culture becomes competent. The presence of both competent and non-competent cells within in the culture poses a challenge in transcriptomic analyses as it makes it more difficult to detect small differences in changes in expression when comparing to a non-competent culture. In order to overcome the problem of the relatively small sub-population previous research used mutants that caused all the cells becoming competent and/ or using comK deletion strains making the bacteria unable to become competent (Berka et al., 2002; Hamoen et al., 2002; Ogura et al., 2002).
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Although the use of mutants overcame the problem posed by the small subpopulation of competent cells, no significant down regulation within the competent population was found leading to the conclusion that ComK is a transcriptional activator (Berka et al., 2002; Hamoen et al., 2002; Ogura et al., 2002). In Chapter 3 We decided on a different approach by separating the competent and non-competent sub-populations by FACS using a PcomG-gfp indicator strain in which the competent subpopulation expresses gfp (Smits et al., 2005), while fixing the cells in high molarity sodium chloride solution (Brown and Smith, 2009; Nilsson et al., 2014). We took advantage of the highly sensitive RNA-seq technique, which not only allowed us to detect genes with low levels of expression but also non-coding RNAs. Aside from investigating the transcriptome we also determined protein levels in the two sub-populations. Separation of the two subpopulations by FACS allowed us to compare them without the use of competence mutants. Our results were in accordance with previous studies in particular with regard to the main competence regulon. Interestingly we did find several significantly down regulated genes of which all but one have corresponding up-regulated antisense RNAs. Most of the non-coding RNAs found in our transcriptomics analysis contain predicted K-boxes in their promoter region. In contrast the down-regulated genes do not have K-boxes in their promoter region indicating that down-regulation by ComK is primarily indirect and likely occurs through the use of antisense RNAs. We also found that regulation of a few of the proteins required for competence are found to be regulated at a post-transcriptional level; these are MinD, Noc, SbcC and SbcD.
We discovered two genes involved in competence that had not been found in previous studies, i.e. yhfW and yhxC. The function of these genes is not known, but they have previously been found to be highly expressed during sporulation (Nicolas et al., 2012; Steil et al., 2005; Wang et al., 2006) and are regulated by SigF and SigE respectively (Arrieta‐Ortiz et al., 2015; Wang et al., 2006). Both proteins are predicted to be oxidoreductases and YhfW contains a 2Fe-2S Rieske domain. They are conserved among Firmicutes. Deletion of YhfW does not result in a large reduction of competence cells, but instead reduces the expression level of comG.
Summary & Discussion
175
In contrast a lack of YhxC does cause a reduction in the amount of competent cells. Interestingly they not only effect expression of comG but that of the important regulators comK and srfA under competence conditions and spo0A under sporulation conditions (yhfW).
Metabolomic analysis revealed that during competence there is a reduction in compounds involved in the TCA cycle such as fumarate, 2-oxoglutarate, pyruvate and citrate in the yhfW mutant. Levels of L-threonine, 2-oxoglutarate, phenylpyruvate, L-methionine, L-tryptophan, aspartate and L-glutamate are also reduced in the mutant. The changes in TCA cycle components indicate a role of YhfW in this process. The reduction in citrate levels are interesting as citrate levels have been shown to affect the phosphorelay and Spo0A~P levels (Craig et al., 1997; Ireton et al., 1995). Interestingly RNA seq analysis revealed up-regulation of NAD synthesis genes, but metabolomics data did not reveal changes in NAD(H) levels. We also did not find changes in known TCA cycle or aminoacid synthesis genes. Most of the down-regulated genes are unknown genes. From the data it is likely that the lower levels of aminoacids are the result of disruptions in the TCA cycle due to the absence of YhfW.
Chapter 4, Fluorescent labelling of DNA to be able to follow its uptake and fate
Transformation is a fascinating process in which double stranded DNA is bound after which one strand is degraded and the DNA is transported single stranded into the cells (Dubnau and Cirigliano, 1972; Piechowska and Fox, 1971). This transport occurs though a large multi protein complex primarily localised at the pole (Chung and Dubnau, 1998; Hahn et al., 2005; Kaufenstein et al., 2011). Once inside the cell the single stranded DNA is bound and protected by proteins and either integrated in to the chromosome in case of homology with the chromosome or reconstituted as a plasmid if the requirements for autonomous replication are met (Grove et al., 2005; Yadav et al., 2014).. The transport complex and its components have been extensively studied through various techniques such as fusions to fluorescent proteins and electron microscopy. Much is also known about the proteins involved in recombination and plasmid reconstitution.
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Labelling with fluorescent proteins allows visualisation of proteins and their localisation and interactions. Because proteins are expressed with the purpose of transporting and integrating DNA we decided to look for a method of labelling the transported DNA, while still allowing uptake. Fluorescent labelling of DNA was done before, but this method of labelling did not result in uptake of the DNA into the cytoplasm (Stingl et al., 2010). We used a method of covalently labelling both strands of DNA ether by incorporating Fluorescein-dUTP directly through PCR or by incorporation of aminoallyl-dUTP through PCR and subsequent labelling with DyLight650 (Chapter 4). Labelling with these dyes not only allows visualisation of DNA, but the DNA is also taken up in a DNaseI resistant manner.
Transformation with labelled DNA containing an antibiotic resistance cassette and homologous regions results in antibiotic resistant colonies. In previous studies localisation of components of the complex machinery occurs primarily at the pole. In contrast, the labelled DNA is more often localised inside the cell compared to the transport proteins and can be seen to co-localise with the chromosome. We also found that the labelled DNA co-localises with ComFC of the transport complex and the recombinase RecA. RecA also forms the actively searching filamentous form in the presence of labelled DNA. Co-localisation of DNA with specific labelled regions of the chromosome is also found. Because the competence machinery and the recombination proteins are highly conserved among naturally competent bacteria, it is likely that this method of labelling can also be used for studying other bacteria. When choosing a fluorescent dye it is important to take into account not only the size and structure of the dye molecule, but also its charge. DNA is a negatively charged molecule that is transported through the water-filled ComEC channel. The dye should therefore be hydrophilic and have a negative charge in order to prevent electrostatic interactions that are unfavourable to transport.
Chapter5, Following the transformation process in space and time
Labelling of proteins, DNA and the chromosome not only allows for studying co-localisation, but can also give insight into the dynamics of these interactions and the timing of up-take and integration.
Summary & Discussion
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In chapter 5 we use labelling of DNA, and a specific locus on the chromosome to gain more insight into the dynamics of the uptake and integration process. Using labelled DNA containing an antibiotic resistance gene and an IPTG inducible promoter gfp fusion allowed us to determine when expression of the transformed DNA occurs.
By using a parB-gfp/parB-mkate we could visualise a specific locus on the chromosome. We used both conventional timelapse microscopy using cells fixed on a polyacrylamide slide and a microfluidics system. We found that B. subtilis is easily transformed within a microfluidics system. The integrated exogenous DNA was expressed and the cells grew in the presence of antibiotic. We could also see expression of a Pspank-parB-gfp construct and were able to determine that cell-division is not required before expression of integrated exogenous DNA. Expression of gfp first becomes visible 90 minutes after addition of DNA, but the average time before expression is 6hrs 45minutes after addition of DNA and 4.45 minutes after addition of fresh medium. It has previously been found that after dilution in fresh medium in a flask replication and growth are resumed after 2-3 hours (Haijema et al., 2001).
We found that cell division is not required for expression of integrated DNA. It would therefore be particularly interesting to see if replication is required before expression and if mutation repair is involved in delaying expression. Although we could not clearly determine live co-localisation with the specific locus on the chromosome due to dye bleaching and chromosome mobility, we were able to see replacement of the locus. Loss of ParB foci becomes visible 90 minutes after addition of DNA. This is quite a long time considering that transport takes place at a speed of approximately 80 nucleotides per second (Maier et al., 2004) meaning our DNA molecule should be fully internalised after 31 seconds. It is likely therefore that searching for homology by RecA and the recombination process are responsible for the long time before visible replacement. It is also possible that ParB remains bound to the chromosome during the recombination process further delaying disappearance of the foci.
The main limiting technical factor is bleaching of the fluorescent dye. It would therefore be good to find a more photo stable fluorescent dye, which meets the requirements concerning charge and size.
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Further optimisation of the imaging settings may also contribute to better results. Using a system in which components of the transport machinery, replication proteins, a specific locus on the chromosome and labelling of DNA containing a promoter-FP fusion could allow one to follow the entire process from uptake to integration to expression. By also visualising a locus outside the homologous region one could also determine if DNA replication is required before expression.
General Discussion
Both sporulation and competence are important adaptive phenotypes. Both are regulated by Spo0A, which also affects other adaptive phenotypes. In a highly unpredictable environment, where extensive competition for resources takes place, small effects can be the deciding factor between successful propagation and death. It is therefore likely that directly binding of Spo0A to the oriC is more important in nature than in the lab in particular because the effect is more pronounced upon overexpression of kinA during exponential growth. Although competence and sporulation are two very different processes, both have in common that they require phosphorylated Spo0A (Fujita and Losick, 2005; Fujita et al., 2005; Mirouze et al., 2012). It is interesting therefore that we found during our research described in chapter 3 a gene that effects expression levels of Spo0A during competence and sporulation.
At first glance bacteria may seem simple organisms compared to multi-cellular species, but the processes taking place inside the cell are anything but simple. The two genes yhfW and yhxC both have no known function and yet it is likely that they are involved in at least two processes; sporulation and competence. Most research is done in the lab under optimised conditions which has the advantage of reducing the number of variables effecting an experiment and thereby providing a clear picture. However this is rather far removed from the situation in nature in which the high number of variables is a defining aspect of the environment.
Summary & Discussion
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The first genes often discovered are those that show a very clear phenotype in the conditions of the lab and one might say that therefore these genes are more important than the many genes of which no function has yet been found. This is indeed the case for those genes that are absolutely required for survival under any condition. However some of these unknown genes may play very important roles in the natural environment of bacteria. It is therefore important to further study these types of genes.
With respect to yhfW and yhxC, they affect competence and likely also sporulation based on our data and their high expression during this process (Arrieta‐Ortiz et al., 2015; Nicolas et al., 2012; Steil et al., 2005; Wang et al., 2006). As both sporulation and competence are important adaptive phenotypes and the genes are conserved among firmicutes it would be very interesting to determine what their exact function is. yhfW is interesting, as Blast reveals that when firmicutes are removed from the BLAST (100 species cut off), similar proteins are found in archaea and cyanobacteria, but not in other bacterial species such as gram-negatives. This combined with the fact that iron-sulphur cofactors are among the most ancient of co-factors makes it an interesting candidate for further study. It is my opinion that investigations into the many unknown genes in bacteria in particular those conserved among different species are worthwhile, as these are most likely primarily important in their natural environment, rather than the lab. Chapter 2 and chapter 3 of this thesis contain results from pure fundamental research and uses established techniques to gain insight into biological processes. Chapter 4 and chapter 5 are in a large part about developing a novel methodology to study the process of transformation. Both types of research come with their own challenges. When developing a method it becomes important to take into account the physical properties of the system one wishes to study. In our case we found that in choosing a dye for labelling DNA not only are the size and structure of the dye important but also the charge. We also ran into challenges, such as photostability of the dyes. Despite this we managed to obtain intriguing results, such as cell division not being required for expression of integrated DNA. As the limiting factors for these experiments have been identified it should not be too difficult to further optimise the method.
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