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In Streptococcus pneumoniae, competence and bacteriocin genes are controlled by two two-component systems, ComED and BlpRH, respectively. In Streptococcus mutans, both functions are controlled by the ComED system. Recent studies in S. mutans revealed a potential ComE binding site characterized by two 11 bp direct repeats shared by each of the bacteriocin genes responsive to the competence-stimulating peptide (CSP). Interestingly, this sequence was not found in the upstream region of the CSP structural gene comC. Since comC is suggested to be part of a CSP-responsive and ComE-dependent autoregulatory loop, it was of interest to determine how it was possible that the ComED system could simultaneously regulate bacteriocin expression and natural competence. Using the intergenic region IGS1499, shared by the CSP-responsive bacteriocin nlmC and comC, it was demonstrated that both genes are likely to be regulated by a bifunctional ComE. In a comE null mutant, comC gene expression was increased similarly to a fully induced wild-type. In contrast, nlmC gene expression was nearly abolished. Deletion of ComD exerted a similar effect on both genes to that observed with the comE null mutation. Electrophoretic mobility shift assays (EMSAs) with purified ComE revealed specific shift patterns dependent on the presence of one or both direct repeats in the nlmC–comC promoter region. The two direct repeats were also required for the promoter activity of both nlmC and comC. These results suggest that gene regulation of comC in S. mutans is fundamentally different from that reported for S. pneumoniae, which implicates a unique regulatory mechanism that allows the coordination of bacteriocin production with competence development.

In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

DNA has recently been described as a major structural component of the extracellular matrix in biofilms. In streptococci, the competence-stimulating peptide (CSP) cell-to-cell signal is involved in competence for genetic transformation, biofilm formation, and autolysis. Among the genes regulated in response to the CSP are those involved in binding and uptake of extracellular DNA. We show in this study that a functional DNA binding-uptake system is involved in biofilm formation. A comGB mutant of Streptococcus mutans deficient in DNA binding and uptake, but unaffected in signaling, showed reduced biofilm formation. During growth in the presence of DNase I, biofilm was reduced in the wild type to levels similar to those found with the comGB mutant, suggesting that DNA plays an important role in the wild-type biofilm formation. We also showed that growth in the presence of synthetic CSP promoted significant release of DNA, with similar levels in the wild type and in the comGB mutant. The importance of the DNA binding-uptake system in biofilm formation points to possible novel targets to fight infections.

Previous studies identified irvA as a normally repressed but highly inducible transcription regulator capable of repressing mutacin I gene expression in Streptococcus mutans. In this study, we aimed to identify and characterize the regulator(s) responsible for repressing the expression of irvA. An uncharacterized open reading frame (SMU.1398) located immediately adjacent to irvA and annotated as a putative transcription repressor was identified as a likely candidate. The results of mutation studies confirmed that the expression of irvA was greatly increased in the SMU.1398 background. Mutation of SMU.1398 (“irvR”) abolished genetic competence and reduced the expression of the late competence genes/operons comEA, comY, and dprA without affecting the expression of the known competence regulators comC, comED, or comX. In addition, irvR was found to be a potent negative regulator of dextran-dependent aggregation (DDAG) and gbpC expression. Each of these irvR mutant phenotypes could be rescued with a double mutation of irvA or complemented by introducing a wild-type copy of irvR on a shuttle vector. These data indicate that the repression of irvA is critically dependent upon irvR and that irvA repression is essential for the development of genetic competence and the proper control of DDAG in S. mutans.

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.

The com operon of naturally transformable streptococcal species contains three genes, comC, comD, and comE, involved in the regulation of competence. The comC gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The comD and comE genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the comD-encoded protein being a receptor for CSP. Here we report on the genetic variability of comC and comD within Streptococcus pneumoniae isolates. Comparative analysis of sequence variations of comC and comD shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many S. pneumoniae strains in the laboratory, there is a clear correlation between the presence of a particular comC allele and the cognate comD allele. These findings effectively rule out the possibility that the presence of noncognate comC and comD alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional com pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of S. mitis and S. oralis. The CSPs from these isolates are much more closely related to those from S. pneumoniae than to most CSPs previously reported from S. mitis and S. oralis, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of S. pneumoniae that are under strong selective pressure.

ComA is a response regulator protein of Bacillus subtilis which is required for the transcription of several genes which are involved in late-growth expression and in responses to environmental stress. Among these genes are degQ, gsiA, and srfA. The last is an operon needed for the development of genetic competence, surfactin production, and normal sporulation. We show here that partially purified ComA protein, isolated from an overproducing Escherichia coli strain, is phosphorylated in vitro by incubation with acetyl phosphate and that ComA could bind specifically to a DNA fragment containing the promoter of srfA and associated sequences. The binding affinity is enhanced when ComA is phosphorylated. DNase I protection analysis identified two protected sites located upstream from the srfA promoter. The presence of DNase I-hypersensitive bonds induced by ComA binding which are located between the protected sequences is consistent with a model for ComA action involving the bending of DNA.

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Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (σX), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the σX protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of σX. As σX is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of σX depends on ComW. The clpP comW double mutant accumulated an amount of σX higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for σX synthesis, it acts both in stabilization of σX and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of σX, but only the N terminus is important for stimulating its activity.

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm “lifestyle” for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.

Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using Streptococcus mutans strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of comE or comD had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competence-stimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of comD, comX, and comYA, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of comC, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal comC expression. This study reveals that S. mutans integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of com genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and com genes in different S. mutans isolates and between S. mutans and Streptococcus pneumoniae, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.

Natural genetic transformation in Streptococcus pneumoniae entails transcriptional activation of at least two sets of genes. One set of genes, activated by the competence-specific response regulator ComE, is involved in initiating competence, whereas a second set is activated by the competence-specific alternative sigma factor ComX and functions in DNA uptake and recombination. Here we report an initial characterization of CoiA, a ComX-dependent gene product that is induced during competence and is required for transformation. CoiA is widely conserved among gram-positive bacteria, and in streptococci, the entire coiA locus composed of four genes is conserved. By use of immunoblot assay, we show that, similar to its message, CoiA protein is transient, appearing at 10 min and largely disappearing by 30 min post-competence induction. Using complementation analysis, we establish that coiA is the only gene of this induced locus needed for transformability. We find no indication of CoiA having a role in regulating competence. Finally, using 32P- and 3H-labeled donor DNA, we demonstrate that a coiA mutant can internalize normal amounts of donor DNA compared to the wild-type strain but is unable to process it into viable transformants, suggesting a role for CoiA after DNA uptake, either in DNA processing or recombination.

Streptococcus mutans develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun. 27:444, 1980). We confirmed a previous report that in this medium comS mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS11-17) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that comS+ (but not comS mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a comS mutation at levels equivalent to 1 μM synthetic ComS11-17.

Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 × 104-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.

Streptococcus pneumoniae secretes two different peptide pheromones used for intercellular communication. These peptides, which have completely unrelated primary structures, activate two separate signal transduction pathways, ComABCDE and BlpABCSRH, which regulate natural genetic transformation and bacteriocin production, respectively. Each signal transduction pathway contains a response regulator (ComE and BlpR, respectively) that activates transcription of target genes by binding to similar, but not identical, imperfect direct repeat motifs. In general the direct repeat binding sites are specific for one or the other of the two response regulators, ensuring that competence development and bacteriocin production are regulated separately. However, in the present study we show that the rate of transcription of an operon, encoding an ABC transporter of unknown function, can be stimulated by both peptide pheromones. We also show that this cross-induction is due to a hybrid direct repeat motif that can respond to both ComE and BlpR. To our knowledge this kind of convergent gene regulation by two separate two-component regulatory systems has not been described before in bacteria.

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.

Author Summary

Streptococcus pneumoniae is a major cause of pneumonia, ear infection and meningitis. Antibiotic resistance among S. pneumoniae isolates is increasingly a major clinical problem. The acquisition of antibiotic resistance genes in S. pneumoniae is controlled by a peptide pheromone called competence-stimulating peptide (CSP). CSP binds to a receptor called ComD, which in turn activates its cognate transcription factor ComE to initiate DNA uptake and integration into the S. pneumoniae genome. CSP-ComD/E also regulates the expression of virulence factors required for infection. In this study, multiple synthetic analogues of CSP pheromone were examined for their ability to inhibit acquisition of exogenous DNA, and to control infection by S. pneumoniae in mice. Two of these analogues, CSP1-E1A and CSP2-E1A, competitively inhibit the ability of S. pneumoniae to acquire the streptomycin resistance rpsL gene and the capsule gene cap3A during mouse models of acute pneumonia and bacteremia. CSP1-E1A also reduces mouse mortality during lung infection by S. pneumoniae. This is the first demonstration of the use of CSP analogues to attenuate virulence and to inhibit acquisition of an antibiotic resistance gene in S. pneumoniae. Because the CSP-ComD/E system is conserved among many pathogenic bacteria, CSP analogues may be applicable to reduce the spread of antibiotic resistance genes and to treat infections.

In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor σX, which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.

The Streptococcus mutans hdrRM operon encodes a novel two-gene regulatory system induced by high cell density. Previous studies identified hdrM as the only known negative regulator of competence development in S. mutans. In the present study, we demonstrated that the HdrRM system bypasses the prototypical competence gene regulators ComC and ComDE in the transcriptional regulation of the competence-specific sigma factor comX and the late competence genes. Similarly, the HdrRM system can abrogate the requirement for ComE to produce the bacteriocin mutacin IV. To further probe the regulatory mechanism of hdrRM, we created an hdrR overexpression strain and showed that it could reproduce each of the hdrM competence and mutacin phenotypes, indicating that HdrM acts as a negative regulator of HdrR activity. Using a mutacin IV-luciferase reporter, we also demonstrated that the hdrRM system utilizes the same promoter elements recognized by ComE and thus appears to comprise a novel regulatory pathway parallel to ComCDE.

In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress-tolerance response, and caries production. We have previously shown that CovR acts as a transcriptional repressor by binding to the upstream promoter regions of its target genes. Here, we report that in vivo, CovR activates the transcription of SMU.1882, which encodes a small peptide containing a double-glycine motif. We also show that SMU.1882 is transcriptionally linked to comA that encodes a putative ABC transporter protein. Several genes from man gene clusters that encode mannose phosphotranferase system flank SMU.1882 -comA genes. Genomic comparison with other streptococci indicates that SMU.1882 is uniquely present in S. mutans, while the man operon is conserved among all streptococci, suggesting that a genetic rearrangement might have taken place at this locus. With the use of a transcriptional reporter system and semi-quantitative RT-PCR, we demonstrated the transcriptional regulation of SMU.1882 by CovR. In vitro gel shift and DNase I foot-printing analyses with purified CovR suggest that CovR binds to a large region surrounding the -10 region of the P1882. Using this information and comparing with other CovR regulated promoters, we have developed a putative consensus binding sequence for CovR. Although CovR binds to P1882, in vitro experiments using purified S. mutans RpoD, E. coli RNA polymerase, and CovR did not activate transcription from this promoter. Thus, we speculate that in vivo, CovR may interfere with the binding of a repressor or requires a cofactor.

Many streptococcal species belonging to the mitis and anginosus phylogenetic groups are known to be naturally competent for genetic transformation. Induction of the competent state in these bacteria is regulated by a quorum-sensing mechanism consisting of a secreted peptide pheromone encoded by comC and a two-component regulatory system encoded by comDE. Here we report that a natural isolate of a mitis group streptococcus (Atu-4) is competent for genetic transformation even though it has lost the gene encoding the competence pheromone. In contrast to other strains, induction of competence in Atu-4 is not regulated by cell density, since highly diluted cultures of this strain are still competent. Interestingly, competence in the Atu-4 strain is lost if the gene encoding the response regulator ComE is disrupted, demonstrating that this component of the quorum-sensing apparatus is still needed for competence development. These results indicate that mutations in ComD or ComE have resulted in a gain-of-function phenotype that allows competence without a competence pheromone. A highly similar strain lacking comC was isolated independently from another individual, suggesting that strains with this phenotype are able to survive in nature in competition with wild-type strains.

Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species.

The Spx protein of Bacillus subtilis exerts both positive and negative transcriptional control in response to oxidative stress by interacting with the C-terminal domain of the RNA polymerase (RNAP) alpha subunit (αCTD). Thus, transcription of the srf operon at the onset of competence development, which requires the ComA response regulator of the ComPA signal transduction system, is repressed by Spx-αCTD interaction. Previous genetic and structural analyses have determined that an Spx-binding surface resides in and around the α1 region of αCTD. Alanine-scanning mutagenesis of B. subtilis αCTD uncovered residue positions required for Spx function and ComA-dependent srf transcriptional activation. Analysis of srf-lacZ fusion expression, DNase I footprinting, and solid-phase promoter retention experiments indicate that Spx interferes with ComA-αCTD interaction and that residues Y263, C265, and K267 of the α1 region lie within overlapping ComA- and Spx-binding sites for αCTD interaction. Evidence is also presented that oxidized Spx, while enhancing interference of activator-RNAP interaction, is not essential for negative control.

A novel fibronectin-binding protein from Pasteurella multocida (PM1665) that binds to the fibronectin type III9-10 modules via two helix-hairpin-helix motifs has recently been described [1]. This protein shares homology with competence-related DNA-binding and uptake proteins (ComEA and ComE) from Gram-positive and Gram-negative bacteria. Here, we show that recombinant PM1665 (now designated ComE1) also binds to DNA through the same helix-hairpin-helix motifs required for fibronectin-binding. This binding to DNA is non sequence-specific and is confined to double-stranded DNA. We have cloned and expressed ComE1 proteins from five members of the Pasteurellaceae in order to further investigate the function(s) of these proteins. When expressed as recombinant GST-fusion proteins, all of the homologues bound both to fibronectin and to double-stranded DNA. Inactivation of the gene encoding the ComE1 homologue in Actinobacillus pleuropneumoniae indicates major roles for these proteins in at least two processes: natural transformation, and binding of bacteria to fibronectin.

Oxygen controls competence development in Streptococcus pneumoniae. Oxygen signaling involves the two-component signal transduction systems CiaRH and ComDE and the competence-stimulating peptide encoded by comC and processed by ComAB. We found that NADH oxidase (Nox) was required for optimal competence. Transcriptional analysis and genetic dissection showed that Nox was involved in post-transcriptional activation of the response regulator ComE and in the transcriptional control of ciaRH and comCDE. Thus, in S. pneumoniae, Nox, with O2 as its secondary substrate, is part of the O2-signaling pathway.

To facilitate the study of pneumococcal genes that are essential for viability or normal cell growth, we sought to develop a tightly regulated, titratable gene depletion system that interferes minimally with normal cellular functions. A possible candidate for such a system is the recently discovered signal transduction pathway regulating competence for natural transformation in Streptococcus thermophilus. This pathway, which is unrelated to the ComCDE pathway used for competence regulation in Streptococcus pneumoniae, has not been fully elucidated, but it is known to include a short unmodified signaling peptide, ComS*, an oligopeptide transport system, Ami, and a transcriptional activator, ComR. The transcriptional activator is thought to bind to an inverted repeat sequence termed the ECom box. We introduced the ComR protein and the ECom box into the genome of S. pneumoniae R6 and demonstrated that addition of synthetic ComS* peptide induced the transcription of a luciferase gene inserted downstream of the ECom box. To determine whether the ComRS system could be used for gene depletion studies, the licD1 gene was inserted behind the chromosomally located ECom box promoter by using the Janus cassette. Then, the native versions of licD1 and licD2 were deleted, and the resulting mutant was recovered in the presence of ComS*. Cultivation of the licD1 licD2 double mutant in the absence of ComS* gradually affected its ability to grow and propagate, demonstrating that the ComRS system functions as intended. In the present study, the ComRS system was developed for use in S. pneumoniae. In principle, however, it should work equally well in many other Gram-positive species.

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type α-subunits and with C-terminal deletion mutants of the α-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.