Abstract: Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely.
Abstract: Bacterial nucleoid associated proteins play a variety of roles in genome maintenance and dynamics. Their involvement in genome packaging, DNA replication and transcription are well documented but it is still unclear whether they play any specific roles in genome repair. We discovered that untwisting of the DNA double helix by bacterial non-specific DNA binding proteins stimulates the activity of a repair endonuclease of the Nth/MutY family involved in abasic site removal during base excision repair. The essential Bacillus subtilis primosomal gene dnaD, coding for a protein with DNA-untwisting activity, is in the same operon with nth and the promoter activity of this operon is transiently stimulated by H(2)O(2). Consequently, dnaD mRNA levels persist high upon treatment with H(2)O(2) compared to the reduced mRNA levels of the other essential primosomal genes dnaB and dnaI, suggesting that DnaD may play an important role in DNA repair in addition to its essential role in replication initiation. Homologous Nth repair endonucleases are found in nearly all organisms, including humans. Our data have wider implications for DNA repair as they suggest that genome associated proteins that alter the superhelicity of the DNA indirectly facilitate base excision repair mediated by repair endonucleases of the Nth/MutY family.
Abstract: Much of our knowledge of the initiation of DNA replication comes from studies in the gram-negative model organism Escherichia coli. However, the location and structure of the origin of replication within the E. coli genome and the identification and study of the proteins which constitute the E. coli initiation complex suggest that it might not be as universal as once thought. The archetypal low-G+C-content gram-positive Firmicutes initiate DNA replication via a unique primosomal machinery, quite distinct from that seen in E. coli, and an examination of oriC in the Firmicutes species Bacillus subtilis indicates that it might provide a better model for the ancestral bacterial origin of replication. Therefore, the study of replication initiation in organisms other than E. coli, such as B. subtilis, will greatly advance our knowledge and understanding of these processes as a whole. In this minireview, we highlight the structure-function relationships of the Firmicutes primosomal proteins, discuss the significance of their oriC architecture, and present a model for replication initiation at oriC.
Abstract: In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis, sometimes resulting in colectomy or death. The main virulence factors of C. difficile are toxin A and toxin B. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC). The important role of TcdR as a sigma factor for toxin expression is undisputed, whereas the role of TcdC as an anti-sigma factor, inhibiting toxin expression, is currently the subject of debate. To clarify the role of TcdC in toxin expression, we generated an isogenic ClosTron-based mutant of tcdC in Clostridium difficile strain 630Δ Erm (CT::tcdC) and determined the transcription levels of the PaLoc genes and the expression levels of the toxins in the wild type strain and the tcdC mutant strain. We found only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and total toxin levels did not significantly differ either. These results suggest that in C. difficile 630Δerm TcdC is not a major regulator of toxin expression under the conditions tested.
Abstract: The initiation of DNA replication requires the binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. DnaA also binds to many sites around the chromosome, outside oriC, and acts as a transcription factor at several of these. In low-G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase at oriC, and this depends on DnaA. DnaD and DnaB also are required to load the replicative helicase outside oriC during replication restart, independently of DnaA. Using chromatin immunoprecipitation, we found that DnaD and DnaB, but not the replicative helicase, are associated with many of the chromosomal regions bound by DnaA in Bacillus subtilis. This association was dependent on DnaA, and the order of recruitment was the same as that at oriC, but it was independent of a functional oriC and suggests that DnaD and DnaB do not require open complex formation for the stable association with DNA. These secondary binding regions for DnaA could be serving as a reservoir for excess DnaA, DnaD, and DnaB to help properly regulate replication initiation and perhaps are analogous to the proposed function of the datA locus in Escherichia coli. Alternatively, DnaD and DnaB might modulate the activity of DnaA at the secondary binding regions. All three of these proteins are widely conserved and likely have similar functions in a range of organisms.
Abstract: The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature-sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI-mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes.
Abstract: The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.
Abstract: DnaD and DnaB are essential DNA-replication-initiation proteins in low-G+C content Gram-positive bacteria. Here we use sensitive Hidden Markov Model-based techniques to show that the DnaB and DnaD proteins share a common structure that is evident across all their structural domains, termed DDBH1 and DDBH2 (DnaD DnaB Homology 1 and 2). Despite strong sequence divergence, many of the DNA-binding and oligomerization properties of these domains have been conserved. Although eluding simple sequence comparisons, the DDBH2 domains share the only strong sequence motif; an extremely highly conserved YxxxIxxxW sequence that contributes to DNA binding. Sequence alignments of DnaD alone fail to identify another key part of the DNA-binding module, since it includes a poorly conserved sequence, a solvent-exposed and somewhat unstable helix and a mobile segment. We show by NMR, in vitro mutagenesis and in vivo complementation experiments that the DNA-binding module of Bacillus subtilis DnaD comprises the YxxxIxxxW motif, the unstable helix and a portion of the mobile region, the latter two being essential for viability. These structural insights lead us to a re-evaluation of the oligomerization and DNA-binding properties of the DnaD and DnaB proteins.
Abstract: Natural competence for genetic transformation, i.e. the ability to take up DNA and stably integrate it in the genome, has so far only been observed in the bacterial kingdom (both in gram-negative and gram-positive species) and may contribute to survival under adverse growth conditions. Bacillus subtilis, the model organism for the Bacillus genus, possesses a well-characterized competence machinery. Phylogenetic analysis of several genome sequences of different Bacillus species reveals the presence of many, but not all genes potentially involved in competence and its regulation. The recent demonstration of functional DNA uptake by B. cereus supports the significance of our genome analyses and shows that the ability for functional DNA uptake might be widespread among Bacilli.
Abstract: Clonal populations of microbial cells often show a high degree of phenotypic variability under homogeneous conditions. Stochastic fluctuations in the cellular components that determine cellular states can cause two distinct subpopulations, a property called bistability. Phenotypic heterogeneity can be readily obtained by interlinking multiple gene regulatory pathways, effectively resulting in a genetic logic-AND gate. Although switching between states can occur within the cells' lifetime, cells can also pass their cellular state over to the next generation by a mechanism known as epigenetic inheritance and thus perpetuate the phenotypic state. Importantly, heterogeneous populations can demonstrate increased fitness compared with homogeneous populations. This suggests that microbial cells employ bet-hedging strategies to maximize survival. Here, we discuss the possible roles of interlinked bistable networks, epigenetic inheritance, and bet-hedging in bacteria.
Abstract: The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region. Each K-box consists of two AT-boxes with the consensus sequence AAAA-(N)(5)-TTTT, which are separated by a flexible spacer resulting in either two, three, or four helical turns between the starting nucleotides of the repeating AT-box units. In this study, the effects of potential determinants of ComK regulation in K-boxes were investigated by testing ComK's transcription activation and DNA-binding affinity on altered K-boxes with mutations either in the spacer between the AT-boxes or in the consensus sequence of the AT-boxes. The most striking result demonstrates the importance of the second thymine base in the AT-boxes. Mutation of this T into a guanine resulted in a threefold reduction in transcription activation and DNA binding by ComK. Transcription activation, as well as DNA binding, was almost completely abolished when both AT-boxes contained a T(2)-to-G mutation. This result was corroborated by in silico analyses demonstrating that a combination of mutations at the T(2) positions of both AT-boxes is not found among any ComK-activated K-boxes, indicating that at least one consensus T(2) position is required to maintain a functional K-box. The results suggest an important structural role for T(2) in ComK binding, probably by its specific position in the minor groove of the DNA.
Abstract: Competence for genetic transformation in the bacterium Bacillus subtilis is a bistable differentiation process governed by the minor groove DNA binding protein ComK. No detectable comK transcription occurs in the absence of an intact comK gene, indicating that ComK has auto-activating properties. ComK auto-stimulation, which is dependent on ComK binding to the comK promoter, is a critical step in competence development, ensuring quick and high-level expression of the late-competence genes. Auto-stimulation is also essential for the bistable expression pattern of competence. Here, we demonstrate that ComK acts as an activator at its own promoter by antagonizing the action of two repressors, Rok and CodY. Importantly, antirepression occurs without preventing binding of the repressing proteins, suggesting that ComK and the repressors might bind at distinct surfaces of the DNA helix. DegU, a DNA binding protein known to increase the affinity of ComK for its own promoter, potentiates the antirepression activity of ComK. We postulate that antirepression is primarily achieved through modulation of DNA topology. Although to our knowledge ComK is the only DNA binding protein shown to act in this novel fashion, other minor groove binding proteins may act similarly.
Abstract: Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ alpha-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example, citM, ylxF, yloA, ykoJ, and several genes of the flgB operon. However, high-affinity CssR binding was observed only for htrA, htrB, and, possibly, citM. In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (sigmaD-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the alpha-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the hag promoter, a well-established reporter for sigmaD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of alpha-amylase overproduction.
Abstract: In bacterial differentiation, mechanisms have evolved to limit cells to a single developmental pathway. The establishment of genetic competence in Bacillus subtilis is controlled by a complex regulatory circuit that is highly interconnected with the developmental pathway for spore formation, and the two pathways appear to be mutually exclusive. Here we show by in vitro and in vivo analyses that a member of the Rap family of proteins, RapH, is activated directly by the late competence transcription factor ComK, and is capable of inhibiting both competence and sporulation. Importantly, RapH is the first member of the Rap family that demonstrates dual specificity, by dephosphorylating the Spo0F-P response regulator and inhibiting the DNA-binding activity of ComA. The protein thus acts at the stage where competence is well initiated, and prevents initiation of sporulation in competent cells as well as contributing to the escape from the competent state. A deletion of rapH induces both differentiation pathways and interferes with their temporal separation. Together, these results indicate that RapH is an integral part of a multifactorial regulatory circuit affecting the cell's decision between distinct developmental pathways.
Abstract: To survive in rapidly changing environmental conditions, bacteria have evolved a diverse set of regulatory pathways that govern various adaptive responses. Recent research has reinforced the notion that bacteria use feedback-based circuitry to generate population heterogeneity in natural situations. Using artificial gene networks, it has been shown that a relatively simple 'wiring' of a bacterial genetic system can generate two or more stable subpopulations within an overall genetically homogeneous population. This review discusses the ubiquity of these processes throughout nature, as well as the presumed molecular mechanisms responsible for the heterogeneity observed in a selection of bacterial species.
Abstract: Understanding the basis for the heterogeneous (or bistable) expression patterns of competence development and sporulation in Bacillus subtilis.
Abstract: Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in Bacillus subtilis (T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of B. subtilis, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of lacZ fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of rok into the B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens group by horizontal transmission.
Abstract: In Bacillus subtilis competence for genetic transformation develops only in a subpopulation of cells in an isogenic culture. The molecular mechanisms underlying this phenotypic heterogeneity are unknown. In this study, we stepwise simplify the signal transduction cascade leading to competence, yielding a strain devoid of all regulatory inputs for this process that have been identified so far. We demonstrate that auto-stimulation of ComK, the master regulator for competence development, is essential and in itself can be sufficient to generate a bistable expression pattern. We argue that transcriptional regulation determines the threshold of ComK to initiate the auto-stimulatory response, and that the basal level of ComK (in a wild-type strain governed by MecA-mediated proteolytic control) determines the fraction of cells that reach this threshold, and thus develop competence.
Abstract: Thioredoxins are important thiol-reactive proteins. Most knowledge about this class of proteins is derived from proteome studies, and little is known about the global transcriptional response of cells to various thioredoxin levels. In Bacillus subtilis, thioredoxin A is encoded by trxA and is essential for viability. In this study, we report the effects of minimal induction of a strain carrying an IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible trxA gene (ItrxA) on transcription levels, as determined by DNA macroarrays. The effective depletion of thioredoxin A leads to the induction of genes involved in the oxidative stress response (but not those dependent on PerR), phage-related functions, and sulfur utilization. Also, several stationary-phase processes, such as sporulation and competence, are affected. The majority of these phenotypes are rescued by a higher induction level of ItrxA, leading to an approximately wild-type level of thioredoxin A protein. A comparison with other studies shows that the effects of thioredoxin depletion are distinct from, but show some similarity to, oxidative stress and disulfide stress. Some of the transcriptional effects may be linked to thioredoxin-interacting proteins. Finally, thioredoxin-linked processes appear to be conserved between prokaryotes and eukaryotes.
Abstract: Genome2D is a Windows-based software tool for visualization of bacterial transcriptome and customized datasets on linear chromosome maps constructed from annotated genome sequences. Genome2D facilitates the analysis of transcriptome data by using different color ranges to depict differences in gene-expression levels on a genome map. Such output format enables visual inspection of the transcriptome data, and will quickly reveal transcriptional units, without prior knowledge of expression level cutoff values. The compiled version of Genome2D is freely available for academic or non-profit use from http://molgen.biol.rug.nl/molgen/research/molgensoftware.php.
Abstract: The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities. Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis. By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved. Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part of ComGA of B. subtilis overcomes the slow translation initiation that is provoked by the eukaryotic codon bias present in the original cfp and yfp genes. Using these new vectors, we demonstrate that, within an isogenic population of sporulating B. subtilis cells, expression of the abrB and spoIIA genes is distinct in individual cells.
Abstract: Generally, the presence of a consensus sequence in the promoter of a gene is taken as indication for regulation by the transcription factor that binds to this sequence. In light of the recent developments in genome research, we were interested to what extent this supposition is valid. We examined the relationship between the presence of a binding site for ComK, the competence transcription factor of Bacillus subtilis, and actual transcriptional activation by ComK. Bacillus subtilis contains 1062 putative ComK-binding sites (K-boxes) in its genome. We employed DNA macroarrays to identify ComK-activated genes, and found that the presence of a K-box is an unreliable predictor for regulation. Only approximately 8% of the genes containing a K-box in the putative promoter region are regulated by ComK. The predictive value of a K-box could be improved by taking into consideration the degree of deviation from the K-box consensus sequence, the presence of extra ComK-binding motifs and the positions of RNA polymerase-binding sites. Finally, many of the ComK-activated genes show no apparent function related to the competence process. Based on our findings, we propose that the ComK-dependent activation of several genes might serve no biological purpose and can be considered 'evolutionary noise'.
