Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNA
Boonstra, Mirjam
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Boonstra, M. (2017). Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNA. Rijksuniversiteit Groningen.
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Bacillus subtilis: sporulation,
competence and the ability to
take up fluorescently labelled
DNA
Mirjam Boonstra
The research presented in this thesis was carried out in the department of Moleclar Genetics, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Faculty of Science and Engineering, University of Groningen, the Netherlands. The research done by M. Boonstra was funded by the Simon Stevin Meester price awarded to O.P. Kuipers by Stichting Technische Wetenschappen (STW), 13344.
Printing of this thesis was financially supported by the Graduate School of Sciences, University of Groningen.
Cover design by M. Boonstra Printed by Ipskamp Printing
© Mirjam Boonstra, 2017, Lelystad, the Netherlands
ISBN (printed version): 978-94-034-0231-4 ISBN (electronic version): 978-94-034-0230-7
Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNA
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op vrijdag 8 december 2017 om 16.15 uur
door
Mirjam Boonstra geboren op 28 juli 1983
Promotores
Prof. dr. O.P. Kuipers Prof. dr. J.W. Veening
Beoordelingscommissie
Prof. dr. J.M. van Dijl Prof. dr. L.W. Hamoen Prof. dr. I. Mandič-Mulec
Chapter 1: General Introduction 9-43
Chapter 2: Spo0A regulates chromosome copy number
during sporulation by directly binding to the origin of replication in B. subtilis
47-75
Chapter 3: Analyses of the competent and non-competent
subpopulations of B. subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence
79-117
Chapter 4: Following the fate of incoming DNA during
natural transformation of Bacillus subtilis with fluorescently labelled DNA
121-145
Chapter 5: Visualizing and tracking down the uptake and
integration of labelled DNA during competence of
Bacillus subtilis by time-lapse microscopy
149-168
Chapter 6: Summary & Discussion 171-183
Chapter 7: Nederlanse Samenvatting voor niet
wetenschappers
Chapter 1
The different adaptive phenotypes of Bacillus subtilis
Bacillus subtilis is a Gram positive soil bacterium. It grows on dead organic
matter in the soil but also on the roots of plants and can be beneficial to plant growth (Cazorla et al., 2007; Siala et al., 1974; Vilain et al., 2006). B.
subtilis is capable of many different adaptive phenotypes including the
formation of spores, which are highly resistant against harsh environmental conditions. The abundance of B. subtilis on plant material and its ability to form spores has led to research into the fate of ingested B.
subtilis spores in the gastro-intestinal tract of different animals. It has been
discovered that B. subtilis can complete its entire life cycle from spore to growth to sporulation within the gastrointestinal (GI) tract (Leser et al., 2008; Tam et al., 2006).
B. subtilis plays an important role in the soil ecology and its effect on plant
growth makes it of relevance to agriculture. The recent discoveries of its ability to not only survive the GI tract, but to grow there makes it an interesting subject for studies into its effect on the intestinal microbiota and the possible effects on health. B. subtilis has many different lifestyles. When nutrients are abundant Bacillus grows vegetatively, but once nutrients become limited it can enter different adaptive states such as motility, swarming, biofilm formation, competence, cannibalism, production of antimicrobials, and sporulation (Fig. 1).
These many adaptive states combined with easy genetic modification methods make B. subtilis an interesting model organism. It is therefore extensively studied as a model for bacterial development and investigations into the cell cycle. One of the first responses to nutrient limitation is motility, by which the bacteria can move to new locations with more nutrients. Active motility in B. subtilis is driven by flagella (Matsuura et al., 1977) (Fig. 2). Flagella-driven motility plays a role in two distinct motility phenotypes; swimming and swarming (Henrichsen, 1972). Swimming is exhibited by individual cells in liquid, whereas swarming is a multicellular phenotype on solid surfaces that requires the production of the lipopeptide biosurfactant surfactin (Angelini et al., 2009; Calvio et al., 2005; Henrichsen, 1972; Kearns, 2010).
Interestingly, surfactin does not only assist in swarming motility, it is also a powerful antimicrobial and antiviral agent (Peypoux et al., 1999). It lyses cells by the permeabillisation of membranes (Carrillo et al., 2003). Another fascinating aspect of surfactin production is that it is linked to natural competence during which cells can take up exogenous DNA from the environment (Fig. 3). Within, but out of frame with the surfactin synthetase gene srfAB, lies the comS gene. The expression of srfA and
comS is regulated by quorum sensing and a two component regulatory
system, which will be discussed in more detail later.
B. subtilis can also produce a wide variety of other antimicrobials that not
only can be used to kill competing bacteria, but also play a role in the development of adaptive phenotypes by functioning as pheromones (Stein, 2005).
Fig. 1. Schematic representation of several adaptive phenotypes of Bacillus subtilis
Bacillus subtilis is capable of several adaptive phenotypes in response to
changes in its environment such as nutrient limitation. Swimming and swarming motility allow for movement towards nutrients. Secretion of degradative enzymes degrades proteins in the environment which can then be taken up as nutrients. Competence enables the acquisition of new traits. Production of specific toxins to kill sibling B.subtilis cells releases nutrients. Sporulation results in highly resistant spores that can survive harsh conditions and can germinate and resume vegetative growth when conditions are more favourable.
Aside from its ability to kill competing bacteria of a different species B.
subtilis can also engage in cannibalism. Cannibalism is a phenotype in
which one subpopulation actively kills another subpopulation to release nutrients. During cannibalism cells expressing the master regulator of sporulation spo0A kill cells that do not express spo0A by producing the toxins Skf and Sdp. B. subtilis uses it as a method to delay sporulation and enhance biofilm formation (González-Pastor, 2011; González-Pastor et al., 2003; Lopez et al., 2009) (Fig 4).
Within the biofilm different phenotypically distinct subpopulations are present (Vlamakis et al., 2008). Biofilms are an incredibly important part of the bacterial lifestyle. As bacteria can form biofilms on virtually any surface and because it appears to be a near universal trait it has important implications for health, industry and water purification (Hall-Stoodley and Stoodley, 2009; Hall-Stoodley et al., 2004; Singh et al., 2006). For reviews on biofilm formation see Abee et al., 2011; López et al., 2010; Vlamakis et al., 2013).
Fig. 3. Competent B. subtilis 168 (Pxyl
-comK-PcomG-gfp) with fluorescently labelled DNA
Fluorescence microscopy image of a hyper competent B. subtilis strain expressing gfp when competent. The blue cells are the competent gfp expressing cells, the red foci are fluorescently labelled DNA which binds only to the competent cells.
Fig. 2. B. subtilis 168 with flagella. Transmission electron microscopy picture of B. subtilis 168 with flagella.
As a last resort strategy to nutrient limitation B. subtilis can produce spores, which are highly resistant to harsh environmental conditions and which protect the bacterial DNA until conditions become more favourable making the spores germinate. Sporulation will be discussed in more detail later.
These many phenotypes of B. subtilis and its ecological and agricultural significance make it a very interesting subject of study. Aside from its use in fundamental science B. subtilis also has industrial relevance as it is used for the production of enzymes such as those used in laundry detergents. Its ability to sporulate and grow on dead organic matter causes serious problems for the food industry, as the highly resistant spores can survive most sterilisation methods. These spores can later germinate and cause food spoilage.
Bacteria are often seen as simple organisms compared to eukaryotes. However B. subtilis and many other bacteria have multiple adaptive phenotypes and regulation of these phenotypes is far from simple. Bacteria play crucial roles within ecosystems and in the case of B. subtilis especially within soil and plant ecosystems. It is important to understand its role within agricultural systems, and its recently discovered abillity to survive within the GI tract of animals opens new avenues of investigation into animal health. In this thesis we focus on only two of the many phenotypes of B. subtilis, i.e. sporulation and competence.
Fig. 4. Cannibalism. In a sub-population of cells the presence of phosphorylated Spo0A~P results in the production of the toxins Skf (sporulation killing factor) and Sdp (sporulation delay factor). Cells not expressing Spo0A are sensitive to the toxins and lyse resulting in release of nutrients which are taken up by the spo0A expressing cells resulting in delayed sporulation.
Competence
The benefits of competence
Competence is the state in which bacteria become capable of transformation during which exogenous DNA is taken up from the environment. It is one of the methods by which horizontal gene transfer can occur. Horizontal gene transfer in bacteria can increase genetic diversity and possibly lead to an increase in fitness. There are three main ways in which horizontal gene transfer in bacteria can occur, transformation, transduction and conjugation. Transduction is the transfer of DNA from one bacterium to another via bacteriophages. During conjugation a plasmid or conjugative transposon is transferred from a donor bacterium to a recipient bacterium. In this thesis only competence and transformation will be discussed further. Competence is primarily a response to nutrient limitation and is also regulated through quorum sensing.
Only a fraction of the bacteria within the population become competent. under competence-stimulating lab conditions, 5-25% of the B. subtilis 168 population becomes competent.
Fig. 5. Biofilm of B. subtilis NCIB 3610 comI - on msgg agar medium. The bacteria form a colony with a three dimensional structure.
In the wild ancestral strain NCIB3610 the competent subpopulation is much lower and DNA uptake can be further inhibited by the transmembrane protein ComI which is encoded on the pBS32 plasmid (Konkol et al., 2013).
There are three hypotheses regarding the evolutionary benefit of taking up exogenous DNA. Using DNA as a nutrient, DNA repair and increasing genetic diversity. In two naturally competent bacteria, i.e. B. subtilis and
Haemophilus influenzae, DNA damage did not induce competence
indicating that at least in these bacteria it is not directly a response to damaged DNA needing repair (Redfield, 1993). For some bacteria there is evidence for the nutrient hypothesis of competence. For instance in H.
influenzae competence is inhibited by the availability of nucleic acid
precursors AMP and GMP (Macfadyen et al., 2001). Despite this it is generally accepted that the role of competence in B. subtilis is the acquisition of new genes; for reviews see (Dubnau, 1999; Levin and Bergstrom, 2000; Redfield, 2001).
Although there is a large amount of DNA present in the environment of the bacteria, only a very small fraction is likely to have sufficient homology for integration. Even if the transformed DNA is successfully integrated into the genome the gene(s) encoded may not benefit fitness at all. It is therefore a rather risky strategy for increasing fitness, in particular because cell division and replication are also halted. Although competence may be used as a way to utilise DNA as a nutrient source in some bacteria this does not match with the halting of DNA replication and cell division during competence in B. subtilis, which also has other systems dedicated to the uptake of nucleosides and nucleotides (Beaman et al., 1983; Fučik et al., 1974; Kloudová and Fučik, 1974; Saxild et al., 1996). It is of course possible that repair of DNA damage or use as a nutrient are secondary benefits of DNA uptake. The ability of B. subtilis to take up exogenous DNA is a fascinating aspect of its evolution. It allows the acquisition of traits that are unlikely to arise through mutation alone.
Initiation of competence
Competence is an extensively controlled phenotype (Fig. 7) with regulation occurring on a transcriptional and protein level and even on the level of mRNA (Gamba et al., 2015; Hamoen et al., 2003a; Maamar et al., 2007). It is a transient state that the cells can exit from, in order to resume vegetative growth (Hahn et al., 1995).
Competence in B. subtilis is controlled by the master regulator of competence ComK (van Sinderen et al., 1995). Under nutrient rich conditions the expression of comK is repressed. In particular abundance of amino acids results in repression of comK by CodY (Serror and Sonenshein, 1996). Expression of comK is also inhibited by the transition state regulator AbrB and Rok, which also represses its own expression and whose gene is repressed in turn by ComK. (Hahn et al., 1996; Hamoen et al., 2003b; Hoa et al., 2002; Maamar and Dubnau, 2005). AbrB controls the expression of many stationary state genes. Repression of comK by AbrB can be relieved by phosphorylated Spo0A (Spo0A~P) which inhibits expression of abrB by binding to its promoter region (Perego et al., 1988; Strauch et al., 1989). Spo0A is involved in the regulation of multiple phenotypes of B. subtilis with specific levels of Spo0A~P determining which phenotype is entered (Fujita et al., 2005; Mirouze et al., 2012). Expression of comK is also stimulated by unphosphorylated DegU that in its phosphorylated form is required for the production of degradative enzymes (Dahl et al., 1992; Hamoen et al., 2000; Ogura and Tanaka, 1996). Aside from inhibiting the expression of rok, ComK also enhances its own expression via a positive feedback loop (Maamar and Dubnau, 2005; Smits et al., 2005).
A decrease in amino acid levels and subsequent relieve of repression by CodY and increased levels of Spo0A~P are not the only paths to initiation of the competence state, it is also controlled by quorum sensing. This sensing involves the two extracellular peptides, i.e. ComX and Competence and Sporulation Factor (CSF) also known as phosphatase (RapC) regulator PhrC. ComX interacts with the two component system ComP-ComA leading to activation of the srfA operon and production of ComS which releases ComK from the MecA/ClpC/ClpP degradation complex (Hamoen et al., 1995; Nakano et al., 1991).
Competence and Sporulation Factor (CSF) inhibits the inhibitor of ComA; RapC (Anne Shank and Kolter, 2011; Lazazzera et al., 1999; Weinrauch et al., 1991). Recently a new factor named Kre (comK-repressor previously known as YkyB) affecting comK mRNA stability has been discovered (Gamba et al., 2015). When sufficient levels of ComK are present the cells can enter competence. ComK is a transcriptional regulator forming a tetramer of two dimers, which recognizes ComK binding sites in the promoter region. The K-box consensus site consists of 2 binding boxes containing the sequence A4N5T4 separated by 8, 18 or 31 base pairs, which
contains no more than 2 deviations from the consensus per binding site (Hamoen et al., 1998, 2002).
During competence approximately 150 genes are differentially expressed; this thesis). Many of these genes are not directly involved in competence or the uptake and integration of DNA which is why this state is also called the K-state. The core ComK regulon consists of genes that are involved in competence. Among these genes are those involved in the formation of the DNA transport system, homologous recombination, cell division and regulation of competence. Aside from ComK regulated expression of genes there is also differential expression of non-coding RNAs during competence ((Nicolas et al., 2012); this thesis).
Much is already known about the gene regulation by ComK, but some questions still remain. ComK has been found to be an almost exclusive transcriptional activator with no significantly down-regulated genes found in previous transcriptomics studies. The recently discovered Kre is so far the only gene found that may be directly inhibited by ComK as its expression is repressed in competent cells and it contains several potential ComK binding sites (Gamba et al., 2015). Little is known yet about the role of non-coding RNAs in competence and possible direct regulation of ncRNAs by ComK. The difference in protein levels between the competent and non-competent subpopulations also has not been studied before. In chapter three of this thesis we attempt to answer these remaining questions by separating the competent and non-competent subpopulations by FACS and by using RNA-seq instead of microarray and ESI-MS to determine differential protein levels.
Inhibition of cell division and replication
During competence DNA replication and cell division are halted to facilitate homologous recombination. When competent cells are transferred to fresh medium they are delayed in resuming cell division. Several proteins are involved in the inhibition of cell division and replication during competence. The operon containing the inhibitor of cell division MinCD has been found differentially expressed during competence (Berka et al., 2002). MinCD is a regulator of cell division, since it inhibits polar cell division through DivIVA mediated localisation at the cell pole (Bramkamp et al., 2008; Levin et al., 1992; Marston and Errington, 1999; Marston et al., 1998).
An important protein in inhibition of cell division during competence is ComGA. ComGA is a multifunctional traffic ATPase that also plays a role in the binding and uptake of DNA (Chung and Dubnau, 1998).
Fig. 7. Schematic overview of some of the known regulators of competence.
Intermediate levels of Spo0A~P stimulate competence whereas high levels repress competence by binding to the comK promoter region. ComK is released from the Clp/MecA degradative complex by ComS. ComK stimulates its own expression and inhibits expression of therepressor of competence rok.
ComGA has been found to inhibit Z-ring formation, cell elongation and nucleoid division during competence (Haijema et al., 2001). When comGA is deleted, competent cells transferred to fresh medium form Z-rings and elongate, but they are still delayed in cell division (Haijema et al., 2001). The fact that cell division remains inhibited in ComGA deficient cells means that there is another competence-dependent inhibitor of division involved. Briley et al. identified Maf as a protein that inhibits cell division downstream of ComGA (Briley et al., 2011). maf lies in an operon that is differentially expressed during competence (Berka et al., 2002; Hamoen et al., 2002b; Ogura et al., 2002b). Maf inhibits septum formation and interacts with ComGA, DivIVA and FtsW and in the absence of both ComGA and Maf the delay in cell division of competent cells is no longer present (Briley et al., 2011; Butler et al., 1993). The inhibition of cell elongation during escape from competence by ComGA is a result of its interaction with MreB (Mirouze et al., 2015).
Another operon containing cell division proteins, which is up-regulated during competence is the thdF operon. The last gene in the operon, noc, has been found to play an important role in the coordination of cell division with chromosome segregation (Adams et al., 2015; Sievers et al., 2002). Interestingly, although the genes upstream of noc in the operon have been found up-regulated, noc itself has not been found differentially expressed in the previous transcriptomics studies. In chapter three of this thesis we show that although noc is not up-regulated on the RNA level the amount of Noc protein is increased in competent cells.
Also up-regulated during competence are parA and parB (soj and spo0J). ParA regulates initiation of DNA replication by interaction with the initiator of DNA replication DnaA. ParB in turn can inhibit dimerisation of ParA switching it from an activator of DNA replication to an inhibitor (Murray and Errington, 2008; Scholefield et al., 2011).
Inhibition of cell division and DNA replication is an important aspect of competence. During transformation DNA is taken up and integrated into the chromosome. It takes time to successfully integrate this DNA through homologous recombination and to repair the site of integration. The delay in division and replication is likely necessary to allow repair to occur as premature replication and division could result in deleterious mutations and a subsequent decrease in fitness.
Many of the genes found differentially expressed during competence are involved in metabolism or are of unknown function. In Chapter 3 we describe two genes of unknown function that are involved in competence. Recently, there has been an increase in research into the effect of metabolic genes on cell division and replication. We have discovered that the large antisense RNA covering the overflow metabolism gene pta is over-expressed in competent subpopulations. Mutants of pta have been found to rescue cell-division in a replication-defective mutant of Escherichia coli (Maciąg-Dorszyńska et al., 2012). Within the same operon as pta also lies
lipL, which we find down-regulated in the competent sub-population. LipL
is essential in the production of the enzyme co-factor lipoic acid (Christensen et al., 2011; Martin et al., 2011). One of the most important enzyme complexes requiring lipoic acid is pyruvate dehydrogenase that recently has been found to be important for Z-ring formation and may play a role in linking nutrient availability with cell division (Monahan et al., 2014). It is not unlikely that the role of some of the metabolic and unknown genes differentially expressed during competence goes beyond adjusting to nutrient limitation to a more direct role in competence.
The uptake and integration of DNA
During transformation exogenous DNA is taken up from the environment and integrated into the genome if there is sufficient homology to the genome of the recipient. If there is no homology, but the DNA is capable of autonomous replication it can be reconstituted as a plasmid (G J Stewart and Carlson, 1986; Viret et al., 1991). DNA is transported into the cell by the competence machinery, which primarily localises at the cell pole. The majority of the cells have only one uptake machinery, although localisation at both poles has also been observed (Hahn et al., 2005; Kaufenstein et al., 2011). DNA is bound double stranded to the competence machinery but transported single stranded (Dubnau and Cirigliano, 1972; Piechowska and Fox, 1971). In contrast to other bacteria B. subtilis does not require a specific sequence in the donor DNA for transport (Dubnau, 1999). The transport system is primarily formed by the proteins encoded in the comG,
Other proteins such as BdbC and BdbD, which are important for the formation of disulfite bonds in ComGC and ComEC, play a role in the assembly of the machinery (Chung and Dubnau, 1998; Hahn et al., 1993; Londoño-Vallejo and Dubnau, 1993; Meima et al., 2002). The competence pseudopilus is formed by the ComG proteins and facilitates the transport of the DNA through the cell wall (Chung and Dubnau, 1998; Chung et al., 1998). DNA is then bound to ComEA and the nuclease NucA generates double stranded breaks (Hahn et al., 1993; Inamine and Dubnau, 1995; Provvedi et al., 2001; Takeno et al., 2012). An as yet unknown protein degrades one strand of the DNA and single-stranded DNA is transported across the membrane through the channel ComEC, assisted by the ATPase ComFA (Draskovic and Dubnau, 2005; Takeno et al., 2011). B. subtilis does not show a preference for the polarity of ssDNA as it transports both 3'to 5' and 5' to 3’ ssDNA (Vagner et al., 1990).
Once the ssDNA enters the cytosol several general and competence-specific proteins are involved in the process of protection, recombination and DNA repair. (for a review see Kidane et al., 2012). In most cases only one uptake site is present and when there is more than one present, the recombinase RecA appears to preferentially localise at one of the machineries, indicating that in the majority of cases only one uptake site is active at a time (Kaufenstein et al., 2011). Upon entrance the ssDNA is bound to SsbA and SsbB. SsbA is an essential protein and SsbB is competence specific, they play a role in the protection of the ssDNA (Grove et al., 2005; Yadav et al., 2012). DprA facilitates the loading of RecA on the ssDNA and overcomes the inhibition by the SSB proteins (Baitin et al., 2008; Yadav et al., 2014). RecA recombines homologous DNA into the chromosome and its activity is modulated by RecF, RecU and RadA (Carrasco et al., 2004, 2005; Kidane et al., 2004). Integration and DNA repair are further assisted by rescuers such as AddAB and RecQ (Bernstein et al., 2010; Lenhart et al., 2012; Sanchez et al., 2006; Wigley, 2013). The addAB operon also contains two other genes, i.e. the exonucleases sbcC and sbcD that have only been found to be differentially expressed in one replicate of the Ogura study; chapter three of this thesis will discuss these genes in more detail.
Many of the studies into the DNA uptake machinery involves fluorescent labelling of the various protein components. This allows insight into the interactions and localisation of the proteins.
In this thesis we also developed a method for studying the interaction between the components of the competence machinery and the DNA. By using fluorescently labelled DNA it is possible to directly investigate the interaction of DNA with the competence machinery. Labelling DNA with homologous flanking regions also allows for following the entire process from the uptake to integration. In chapters four and five these techniques will be discussed in more detail.
Sporulation
The ability of bacteria to form spores is of interest not only to microbiologists but also to industry and food producers. Spores are metabolically dormant and are capable of withstanding harsh environmental conditions such as radiation, extreme temperatures and desiccation. Spores can survive these conditions for long periods of time up to millions of years (Cano and Borucki, 1995; Vreeland et al., 2000). Sporulation can be a very useful survival strategy for bacteria as it allows their genetic material to survive in conditions where the vegetative cells would die. When conditions become favourable, the spores can germinate and resume vegetative growth. This resistance of spores to harsh treatments make them problematic for food production because the treatments used for killing vegetative cells do not kill the spores. The surviving spores can later grow out and cause food spoilage in the case of B.
subtilis or food poisoning and/or infection in the case of pathogenic spore
formers. Although sporulation is primarily a response to nutrient limitation, a small number of cells initiate sporulation during conditions promoting vegetative growth.
The presence of multiple differential states of bacteria, independent of nutrient levels and genetic variation, is called phenotypic heterogeneity. By having sub-populations with different phenotypes bacteria may increase the chances of survival in changing environments (Ackermann, 2015). Figure 8 gives an overview of the sporulation process.
Initiation of sporulation
Sporulation is a reaction to starvation. Depletion of multiple nutrients such as carbon, nitrogen and phosphorous can be the trigger for sporulation (Schaeffer et al., 1965). Sporulation is initiated when sufficient levels of phosphorylated Spo0A (Spo0A~P) are present (Fujita and Losick, 2005). The main factor linking nutrient status to sporulation is CodY. CodY senses intracellular GTP levels and when concentrations of GTP are high it represses the expression of stationary state genes. It has been shown to bind to the promoter of the master regulator of sporulation spo0A (Molle et al., 2003a; Ratnayake-Lecamwasam et al., 2001). Another represssor of
spo0A is SinR, a DNA-binding protein essential for motility and
competence (Mandic-Mulec et al., 1995). Aside from nutrient limitation high cell density is also a factor in sporulation initiation (Grossman and Losick, 1988; Hofmeister et al., 1995).
Fig. 8. Schematic overview of sporulation
Sporulation is initiated when sufficiently high levels of Spo0A~P are present. The cell enters asymmetric division after which the forespore is engulfed by the mother cell. the spore-coat and cortex are formed. When the endospore is mature the mother cell lyses and the spore is released. When sufficient nutrients are once again present in the environment, the spore germinates and resumes vegetative growth.
The presence of the before-mentioned sporulating cells in non-sporulation stimulating conditions is the result of stochastic variation. Variation in phosphate flux results in different levels of Spo0A~P, which in some cases will be high enough to allow the cell to enter sporulation (Ackermann, 2015; Maughan and Nicholson, 2004; Veening et al., 2005, 2006). Expression of spo0A is positively regulated by the sigma factor SigH (Predich et al., 1992). The activation of Spo0A through phosphorylation relies on a phosphorelay system.
The phosphorelay consists of five kinases (KinA, KinB, KinC, KinD and KinE) and the phosophorelay proteins Spo0B and Spo0F (Burbulys et al., 1991; Jiang et al., 2000). As mentioned before Spo0A~P not only regulates sporulation but also other developmental processes such as competence. (Fujita and Losick, 2005; Fujita et al., 2005; Schultz et al., 2009). Upon phosphorylation Spo0A dimerises and becomes able to bind to DNA and regulate transcription (Spo0A~P). High levels of phosphorylated Spo0A result in entry into sporulation and lower levels can initiate cannibalism, biofilm formation or competence (González-Pastor, 2011; Hamon and Lazazzera, 2001; Mirouze et al., 2012). KinA in particular is important for initiation of sporulation. Increased levels of KinA result in higher levels of Spo0A~P and over-expression of kinA during exponential growth results in initiation of sporulation (Fujita and Losick, 2005).
Recently, an important role for DNA replication in the initiation of sporulation has been found. Narula et al. found that the localisation on the chromosome of spo0F (near the oriC) and kinA are responsible for increase of Spo0A after completion of replication. Spo0F can inhibit KinA thereby lowering the level of Spo0A~P during replication. When replication is completed the ratio returns to one and the level of SPo0A~P shoots up. Spo0A~P activates expression of Spo0F which inhibits KinA resulting in a negative feedback loop (Narula et al., 2015). This feedback loop appears to be a key factor in controlling Spo0A~P dynamics. It plays a crucial role in linking nutrient status and development of sporulation, as during nutrient-rich conditions new rounds of replication begin before the previous one has ended (Narula et al., 2015; Wang and Levin, 2009). Spo0A~P directly regulates the transcription of over 100 genes and is involved in the indirect regulation of more than 500 genes (Fawcett et al., 2000; Molle et al., 2003b).
Direct regulation by Spo0A~P is depended on the presence of specific regions called Spo0A boxes or 0A boxes. The consensus sequence for the 0A boxes is 5’-TGTCGAA-3’ (Molle et al., 2003; Strauch et al., 1990). The amount of Spo0A~P determines which genes are expressed as the affinity of Spo0A~P to the promoter region determines the level needed to initiate transcription. The sinI-sinR operon has multiple 0A boxes with different affinity levels in its promoter region. Binding to the lower-affinity 0A boxes results in transcription of the sinI-sinR operon and promotes the formation of biofilms and matrix production. When sufficiently high levels of Spo0A~P are present, the lower affinity 0A boxes will be bound and the expression of sinI-sinR will be repressed (Fujita and Losick, 2005; Fujita et al., 2005; Kearns et al., 2005; Tan and Ramamurthi, 2014). The sporulation specific genes contain low affinity boxes in the region and therefore require higher Spo0A~P levels. High levels of Spo0A also negatively regulate the expression of genes involved in cell-division and those involved in other developmental processes such as competence. Among the early sporulation specific genes directly activated by Spo0A are
racA, spoIIA, spoIIE and spoIIG (Fujita et al., 2005). Aside from Spo0A
SigH also regulates the transcription of sporulation specific genes (Britton et al., 2002).
Asymmetric division and chromosome copy number
After sporulation is initiated the cell will engage in asymmetric cell division. During vegetative growth the septum is located mid-cell and the cell divides into two cells of a similar size. Sporulation however evolved such that the cell is divided into a larger mother cell and a smaller forespore by placing the septum nearer to the pole. As with symmetric division asymmetric division requires the formation of a Z-ring by FtsZ. During symmetric division the localisation of the peripheral membrane protein DivIVA at both sites of the septum assures assembly of the Z-ring at mid-cell. DivVIA assembles into rings and recruits the MinCD complex which prevents assembly of the Z-ring at the pole (Eswaramoorthy et al., 2011; Tan and Ramamurthi, 2014). The asymmetric localisation of the Z ring during sporulation requires SpoIIE and increased levels of FtsZ, which is regulated by Spo0A and SigH.
SpoIIE and FtsZ localise at both poles, but at only one pole a septum is formed (Ben-Yehuda and Losick, 2002; Khvorova et al., 1998). Another factor important for localisation of FtsZ at the pole is RefZ, which destabilises the Z ring at mid cell and stimulates Z ring formation at the poles. RefZ binds to TetR sites on the chromosome indicating that the organization of the chromosome plays a role in the the switch from symmetric to asymmetric division (Miller et al., 2016; Wagner-Herman et al., 2012). A protein called RacA Binds to GC rich inverted repeats near the origin of replication and through its interaction with DivIVA it anchors the chromosomes to the cell poles (Ben-Yehuda et al., 2005; Tan and Ramamurthi, 2014).
During asymmetric division only two copies of the chromosome are present one in the mother cell and one in the forespore. Disruptions in chromosome copy number result in less efficient sporulation (Eldar et al., 2009; Murray and Errington, 2008; Veening et al., 2009; Xenopoulos and Piggot, 2011). Several factors have been found to control generation of the correct chromosome copy number. Suppressor of DnaAI (Sda) can bind to KinA and KinB and prevent their phosphorylation resulting in reduced levels of Spo0A~P. The level of Sda increases during initiation of replication by transcriptional activation of DnaA. Degradation of Sda by ClpXP result in a decrease of Sda and a relief of the inhibition of the kinase allowing a window in which Spo0A~P levels can increase sufficiently for cells to enter sporulation. The cells that do not enter sporulation during this narrow window enter a new replication cycle. Sda thus prevents initiation of sporulation in replicating cells (Burkholder et al., 2001; Cunningham and Burkholder, 2009; Rowland et al., 2004; Ruvolo et al., 2006; Veening et al., 2009; Whitten et al., 2007). Another protein Sporulation Inhibitor of Replication A (sirA) prevents initiation of replication in cells committed to sporulation. Spo0A~P directly activates transcription of SirA. SirA interacts with the initiator of replication DnaA and displaces it from the origin of replication. In this way it contributes to the maintenance of only two copies of the chromosome during sporulation (Rahn-Lee et al., 2009; Wagner et al., 2009).
Although deletion of these two factors results in aberrations of chromosome copy number during sporulation there are still cells in which chromosomal diploidy is maintained (Veening et al., 2009).
In chapter two of this thesis we will show that Spo0A~P can directly inhibit DNA replication by binding to 0A boxes in the origin of replication. The before-mentioned gene dosage effect of kinA:spo0F is another factor which plays an important role in preventing defects such as incorrect copy number during sporulation (Narula et al., 2015). The chromosomes during the initial stages of asymmetric division are localized away from the poles, and therefore only part of the chromosome destined for the spore is at the correct position (Burton et al., 2007; Wu and Errington, 1994, 1998). The DNA translocase SpoIIIE transports the rest of the chromosome into the forespore (Burton et al., 2007; Wu and Errington, 1994, 1998).
Compartment specific gene expression
An important aspect of sporulation is differential gene expression between the mother cell and the forespore. During sporulation different sigma factors are active in the mother cell and the forespore. Upon completion of asymmetric division, SigF becomes active in the forespore. Although SigF is also present in the mother cell it is inactivated by the anti-sigma factor SpoIIAB. In the forespore however inhibition of SigF by SpoIIAB is relieved by SpoIIE, which dephosphorylates SpoIIAA (Duncan et al., 1995; King et al., 1999). During asymmetric division SpoIIE becomes enriched in the forespore ensuring that SigF is only active here (Dworkin and Losick, 2001; Guberman et al., 2008; Wu et al., 1998). The exact mechanism of SpoIIE enrichment in the forespore is not yet know, but its interaction with FtsZ may play a role (Lucet, 2000).
Another factor causing the increased level of active SigF in the forespore is the localization of the spoIIAB operon on the chromosome. Because it is located closer to the terminal part of the chromosome it is one of the last genes to be transported into the forespore causing it to go without a copy of SpoIIAB for about 10 minutes. This, combined with the relative instability of SpoIIAB results in lower SpoIIAB levels in the forespore (Dworkin and Losick, 2001; Errington, 2003; Lewis et al., 1996; Pan et al., 2001). Upon activation of SigF in the forespore, SigE becomes active in the mother cell. SigE is expressed in its inactive form and activation of sigE in the mother cell requires the expression of the SigF-regulated forespore-specific SpoIIR.
SpoIIR is transported to the interseptal space and activates the membrane protein and protease SpoIIGA . SpoIIGA in turn converts SigE to its active form in the mother cell (Hofmeister et al., 1995; Imamura et al., 2008; Peters and Haldenwang, 1991). SpoIIR localization is dependent on SpoIIGA which is present in both the mother cell side of the membrane and the forespore membrane, but at higher levels in the mother cell septum. SpoIIR activation requires de novo fatty acid synthesis and cleavage by a signal peptidase. Fatty acid modification of SpoIIR may play a role in its localisation by concentrating it in a specific domain of the membrane in which both SpoIIGA and the signal peptidase are present (Diez et al., 2012).
Forespore engulfment
Once asymmetric division has been completed and differential expression is established a process called engulfment is started. Engulfment is a phagocytosis like process in which the septum peptidoglycan is degraded and the septal membranes curve around the forespore cytosol. Although most of the peptidoglycan is degraded a thin layer remains, possibly serving as a template for subsequent process of engulfment and cortex formation (Tocheva et al., 2013). The degradation of septum peptidoglycan involves a degradation machinery composed of SpoIID, SpoIIM and SpoIIP (Chastanet and Losick, 2007; Eichenberger et al., 2001; Gutierrez et al., 2010). SpoIIB localises the DMP machinery to the centre of the septum where degradation starts. The DMP machinery not only degrades the petidoglycan it also moves the membrane to surround the forespore cytoplasm (Abanes-De Mello et al., 2002; Aung et al., 2007; Chastanet and Losick, 2007). The synthesis of new peptidoglycan also plays an important role in the engulfment process (Meyer et al., 2010). SpoIIB is not the only protein capable of localising the DMP complex. In its absence the SpoIIQ and SpoIIIAH complex can localise the DMP machinery via SPoIVFA (Fredlund et al., 2013). SpoIIQ-SpoIIIAH is also required for engulfment in the absence of peptidoglycan (Aung et al., 2007; Broder and Pogliano, 2006). The SpoIIQ-SpoIIAH complex acts as a ratchet during engulfment preventing backward membrane movement (Broder and Pogliano, 2006).
There are thus two mechanisms, DMP-mediated and SpoIIQ-SpoIIIAH by which engulfment occurs resulting in a robust process. The current model of forespore membrane fission involves a protein called FisB that interacts with cardiolipin. At the end of engulfment a membrane tube forms at the cell pole, which due to its high negative curvature becomes enriched in cardiolipins. This tube is pinched off through polymerization of FisB or interaction of FisB with cardiolipin (Doan et al., 2013).
Formation of the coat and cortex
Upon completion of engulfment the forespore is released from the pole and SigG is activated in the forespore compartment. Interestingly the activation of SigG is dependent on SigE which is only active in the mother cell. The metabolic activity of the forespore is reduced once engulfment is completed. A TypeIII secretion system is formed by the genes encoded in the SpoIIIA operon which is under the control of SigE, and SpoIIQ which is controlled by SigF. This 'feeding tube' allows transport of molecules from the mother cell to the forespore. This feeding tube is required for the activation of SigG, but it is not yet known how it activates SigG (Camp and Losick, 2009; Doan et al., 2009; Meisner et al., 2008). The activation of SigG in the forespore leads to activation of SigK in the mother cell which like SigE is expressed in its inactive from. The intermembrane protease SpoIVFB is responsible for the activation of SigK in the mothercell (Lu et al., 1995; Rudner et al., 1999; Yu and Kroos, 2000). Before activation of SigG, SpoIVFB is present in an inactive form in a complex with SpoIVFA and BofA (Resnekov and Losick, 1998). It is activated by SigG-regulated SpoIVB, which cleaves SpoIVFA. SPoIVFA can also be cleaved by CtpB which is activated by SPoIVB (Campo and Rudner, 2006; Zhou and Kroos, 2005).
Once SigG and SigK have been activated the formation of the protective layers of the spore begins. In the mature spore of B. subtilis most of the water is replaced by Ca2+ -dipicolinic acid. The core is surrounded by the
inner spore membrane, the germ cell wall, cortex the outer spore membrane and the coat (Aronson and Fitz-James, 1976; Warth et al., 1963). The coat itself is contains four layers (McKenney et al., 2010).
For an excellent review on the B. subtilis spore coat see (McKenney et al., 2013). An important aspect of spore maturation after engulfment is the localization of proteins to the outer surface of the forespore. One of the first proteins to localise to the outer part of the spore is SpoVM. SpoVM is produced by the mother cell and differentiates between the mother cell membrane and the forespore membrane by the convex curvature of the forespore membrane (Levin et al., 1993; Ooij and Losick, 2003; Ramamurthi et al., 2009; Wasnik et al., 2015). SpoVM is crucial for the initiation of spore coat formation. SpoVM serves as an anchor for SpoIVA which is a structural component of the spore coat (Ramamurthi et al., 2006).
Other components of the basement layer of the spore coat are deposited on top of SpoIVA and SpoVM, which thereby act as a scaffold for spore coat assembly (McKenney et al., 2010). Not only are these two proteins involved in the assembly of the coat, they also play a role in the formation of the cortex (Levin et al., 1993). A fascinating recently discovered property of spore formation is the existence of a quality control system. This system involves a protein called CmpA which is produced by the mother cell (Schmalisch et al., 2010). It is an adaptor protein for the degradation of SpoIVA by ClpX. It represses formation of the cortex until spore coat formation is successfully initiated (Ebmeier et al., 2012). Recently it has been found that defects in the spore envelop cause programmed cell death in a CmpA dependent manner (Tan et al., 2015). Spore coat formation is primarily an activity of the mother cell as the proteins assemble on the outer layer of the spore membrane. The SpoIIIAH-SpoIIQ complex, of which SpoIIQ is a forespore protein, has also been found to have a possible coordinating function in coat assembly (McKenney and Eichenberger, 2012). The spore cortex lies between the inner and outer spore membrane. It is made up of the spore germ wall which is located adjacent to the inner membrane and has a structure similar to the vegetative cell wall (Tipper and Linnett, 1976). The peptidoglycan layer of the cortex however is different from that of vegetative cells. It has a lower level of transpeptidation between peptidoglycan chains and contains muramic lactam (Popham and Setlow, 1993; Warth and Strominger, 1969). There are different degrees of cross linking in the cortex, with the level increasing
The presence of muramic acid is required for germination (Popham et al., 1996). For detailed reviews on sporulation see (Errington, 2003; Higgins and Dworkin, 2012; McKenney et al., 2013; Tan and Ramamurthi, 2014).
Outline of this thesis
The main aims of the thesis were to increase insights into competence and the mechanisms by which replication is controlled during sporulation. We also aimed to develop a new method for following DNA uptake during transformation.
Chapter 1 is the introduction where the various developmental strategies
of B .subtilis are discussed and where the competence and sporulation processes and their regulation are discussed in more detail.
Chapter 2 describes the role of the master regulator of sporulation Spo0A
in maintaining two copies of the chromosome during sporulation.
In chapter 3 We investigated the competent and non-competent subpopulations during competence using RNA-seq and ESI-MS. Two new genes involved in competence were discovered and one was investigated further using metabolomics methods and RNA-seq. Also the involvement of some small RNAs was demonstrated.
In chapter 4 we aimed to develop a method for studying interactions of DNA with components of the competence machinery using fluorescently labelled DNA.
Chapter 5 describes the use of fluorescently labelled DNA in combination
with microfluidics and time lapse microscopy in studying transformation.
Chapter 6 contains a summary of the results and discusses future
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