Unveiling the regulatory network controlling natural transformation in lactococci.

Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we revealed that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium.

Competence shut-off by intracellular pheromone degradation in salivarius streptococci.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation.

In the process of natural transformation bacteria import extracellular DNA molecules for integration into their genome. One strand of the incoming DNA molecule is degraded, whereas the remaining strand is transported across the cytoplasmic membrane. The DNA transport channel is provided by the protein ComEC. Many ComEC proteins have an extracellular C-terminal domain (CTD) with homology to the metallo-ß-lactamase fold. Here we show that this CTD binds Mn2+ ions and exhibits Mn2+ -dependent phosphodiesterase and nuclease activities. Inactivation of the enzymatic activity of the CTD severely inhibits natural transformation in Bacillus subtilis. These data suggest that the ComEC CTD is a nuclease responsible for degrading the nontransforming DNA strand during natural transformation and that this process is important for efficient DNA import.

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans.

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.

Competence inhibition by the XrpA peptide encoded within the comX gene of Streptococcus mutans.

Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first described negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ΔxrpA strain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.

Pneumococcal Competence Coordination Relies on a Cell-Contact Sensing Mechanism.

Bacteria have evolved various inducible genetic programs to face many types of stress that challenge their growth and survival. Competence is one such program. It enables genetic transformation, a major horizontal gene transfer process. Competence development in liquid cultures of Streptococcus pneumoniae is synchronized within the whole cell population. This collective behavior is known to depend on an exported signaling Competence Stimulating Peptide (CSP), whose action generates a positive feedback loop. However, it is unclear how this CSP-dependent population switch is coordinated. By monitoring spontaneous competence development in real time during growth of four distinct pneumococcal lineages, we have found that competence shift in the population relies on a self-activated cell fraction that arises via a growth time-dependent mechanism. We demonstrate that CSP remains bound to cells during this event, and conclude that the rate of competence development corresponds to the propagation of competence by contact between activated and quiescent cells. We validated this two-step cell-contact sensing mechanism by measuring competence development during co-cultivation of strains with altered capacity to produce or respond to CSP. Finally, we found that the membrane protein ComD retains the CSP, limiting its free diffusion in the medium. We propose that competence initiator cells originate stochastically in response to stress, to form a distinct subpopulation that then transmits the CSP by cell-cell contact.

A Novel Feedback Loop That Controls Bimodal Expression of Genetic Competence.

Gene expression can be highly heterogeneous in isogenic cell populations. An extreme type of heterogeneity is the so-called bistable or bimodal expression, whereby a cell can differentiate into two alternative expression states. Stochastic fluctuations of protein levels, also referred to as noise, provide the necessary source of heterogeneity that must be amplified by specific genetic circuits in order to obtain a bimodal response. A classical model of bimodal differentiation is the activation of genetic competence in Bacillus subtilis. The competence transcription factor ComK activates transcription of its own gene, and an intricate regulatory network controls the switch to competence and ensures its reversibility. However, it is noise in ComK expression that determines which cells activate the ComK autostimulatory loop and become competent for genetic transformation. Despite its important role in bimodal gene expression, noise remains difficult to investigate due to its inherent stochastic nature. We adapted an artificial autostimulatory loop that bypasses all known ComK regulators to screen for possible factors that affect noise. This led to the identification of a novel protein Kre (YkyB) that controls the bimodal regulation of ComK. Interestingly, Kre appears to modulate the induction of ComK by affecting the stability of comK mRNA. The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK. The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed. Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

The Alternative Sigma Factor SigX Controls Bacteriocin Synthesis and Competence, the Two Quorum Sensing Regulated Traits in Streptococcus mutans.

Two small quorum sensing (QS) peptides regulate competence in S. mutans in a cell density dependent manner: XIP (sigX inducing peptide) and CSP (competence stimulating peptide). Depending on the environmental conditions isogenic S. mutans cells can split into a competent and non-competent subpopulation. The origin of this population heterogeneity has not been experimentally determined and it is unknown how the two QS systems are connected. We developed a toolbox of single and dual fluorescent reporter strains and systematically knocked out key genes of the competence signaling cascade in the reporter strain backgrounds. By following signal propagation on the single cell level we discovered that the master regulator of competence, the alternative sigma factor SigX, directly controls expression of the response regulator for bacteriocin synthesis ComE. Consequently, a SigX binding motif (cin-box) was identified in the promoter region of comE. Overexpressing the genetic components involved in competence development demonstrated that ComRS represents the origin of bimodality and determines the modality of the downstream regulators SigX and ComE. Moreover these analysis showed that there is no direct regulatory link between the two QS signaling cascades. Competence is induced through a hierarchical XIP signaling cascade, which has no regulatory input from the CSP cascade. CSP exclusively regulates bacteriocin synthesis. We suggest renaming it mutacin inducing peptide (MIP). Finally, using phosphomimetic comE mutants we show that unimodal bacteriocin production is controlled posttranslationally, thus solving the puzzling observation that in complex media competence is observed in a subpopulation only, while at the same time all cells produce bacteriocins. The control of both bacteriocin synthesis and competence through the alternative sigma-factor SigX suggests that S. mutans increases its genetic repertoire via QS controlled predation on neighboring species in its natural habitat.

K+ modulates genetic competence and the stress regulon of Streptococcus mutans.

Potassium (K+) is the most abundant cation in dental plaque fluid. Previously, we reported the link between K+ transport via Trk2 in Streptococcus mutans and its two critical virulence attributes: acid tolerance and surface adhesion. Herein, we build further on the intimate link between K+ levels and S. mutans biology. High (>25 mM) versus low (≤5 mM) K+ concentrations in the growth medium affected conformational epitopes of cell surface-localized adhesin P1. At low K+, the expression of stress response elements gcrR and codY, cell-adhesion-associated genes such as spaP and metabolism-associated genes such as bglP was induced at stationary phase (P<0.05), suggesting that K+-mediated regulation is growth phase-dependent and stress-sensitive. Production of the newly discovered secretory protein encoded by SMU_63c was strongly dependent on the availability of K+ and growth phase. This protein is a newly discovered regulator of genetic competence and biofilm cell density. Thus, the influence of K+ on DNA transformation efficiency was also examined. Compared with 25 mM K+ concentration, the presence of low K+ reduced the transformation frequency by 100-fold. Genetic transformation was abolished in a strain lacking a Trk2 system under all K+ concentrations tested. Consistent with these findings, repression of competence-associated genes, comS and comX, was observed under low environmental K+ conditions and in the strain lacking Trk2. Taken together, these results highlight a pivotal role for environmental K+ as a regulatory cation that modulates stress responses and genetic transformation in S. mutans.

Overexpression of the fratricide immunity protein ComM leads to growth inhibition and morphological abnormalities in Streptococcus pneumoniae.

The important human pathogen Streptococcus pneumoniae is a naturally transformable species. When developing the competent state, it expresses proteins involved in DNA uptake, DNA processing and homologous recombination. In addition to the proteins required for the transformation process, competent pneumococci express proteins involved in a predatory DNA acquisition mechanism termed fratricide. This is a mechanism by which the competent pneumococci secrete a muralytic fratricin termed CbpD, which lyses susceptible sister cells or closely related streptococcal species. The released DNA can then be taken up by the competent pneumococci and integrated into their genomes. To avoid committing suicide, competent pneumococci produce an integral membrane protein, ComM, which protects them against CbpD by an unknown mechanism. In the present study, we show that overexpression of ComM results in growth inhibition and development of severe morphological abnormalities, such as cell elongation, misplacement of the septum and inhibition of septal cross-wall synthesis. The toxic effect of ComM is tolerated during competence because it is not allowed to accumulate in the competent cells. We provide evidence that an intra-membrane protease called RseP is involved in the process of controlling the ComM levels, since △rseP mutants produce higher amounts of ComM compared to wild-type cells. The data presented here indicate that ComM mediates immunity against CbpD by a mechanism that is detrimental to the pneumococcus if exaggerated.

Secretion of a pneumococcal type II secretion system pilus correlates with DNA uptake during transformation.

Streptococcus pneumoniae is a major human pathogen that successfully adapts to the host environment via an efficient uptake system for free DNA liberated from other organisms in the upper respiratory tract, facilitating immune evasion and drug resistance. Although the initial signaling events leading to pneumococcal competence for DNA transformation and the fate of DNA when it has been taken up have been extensively studied, the actual mechanism by which DNA in the environment may traverse the thick capsular and cell wall layers remains unknown. Here we visualize that induction of competence results in the formation of a native morphologically distinct pilus structure on the bacterial surface. This plaited pilus is encoded by the competence (com)G locus, and, after assembly, it is rapidly released into the surrounding medium. Heterologous pneumococcal pilus expression in Escherichia coli was obtained by replacing the pulE-K putative pilin genes of the Klebsiella oxytoca type II secretion system with the complete comG locus. In the pneumococcus, the coordinated secretion of pili from the cells correlates to DNA transformation. A model for DNA transformation is proposed whereby pilus assembly "drills" a channel across the thick cell wall that becomes transiently open by secretion of the pilus, providing the entry port for exogenous DNA to gain access to DNA receptors associated with the cytoplasmic membrane.

Deletion analysis of Streptococcus pneumoniae late competence genes distinguishes virulence determinants that are dependent or independent of competence induction.

The competence regulon of Streptococcus pneumoniae (pneumococcus) is crucial for genetic transformation. During competence development, the alternative sigma factor ComX is activated, which in turn, initiates transcription of 80 'late' competence genes. Interestingly, only 16 late genes are essential for genetic transformation. We hypothesized that these late genes that are dispensable for competence are beneficial to pneumococcal fitness during infection. These late genes were systematically deleted, and the resulting mutants were examined for their fitness during mouse models of bacteremia and acute pneumonia. Among these, 14 late genes were important for fitness in mice. Significantly, deletion of some late genes attenuated pneumococcal fitness to the same level in both wild-type and ComX-null genetic backgrounds, suggesting that the constitutive baseline expression of these genes was important for bacterial fitness. In contrast, some mutants were attenuated only in the wild-type genetic background but not in the ComX-null background, suggesting that specific expression of these genes during competence state contributed to pneumococcal fitness. Increased virulence during competence state was partially caused by the induction of allolytic enzymes that enhanced pneumolysin release. These results distinguish the role of basal expression versus competence induction in virulence functions encoded by ComX-regulated late competence genes.

Serum Albumin and Ca2+ Are Natural Competence Inducers in the Human Pathogen Acinetobacter baumannii.

The increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition. Acinetobacter baumannii is a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca(2+)or albumin. We show that comEA and pilQ are involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence in A. baumannii Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen.

The Thermus thermophilus†comEA/comEC operon is associated with DNA binding and regulation of the DNA translocator and type IV pili.

Natural transformation systems and type IV pili are linked in many naturally competent bacteria. In the Gram-negative bacterium Thermus thermophilus, a leading model organism for studies of DNA transporters in thermophilic bacteria, seven competence proteins play a dual role in both systems, whereas two competence genes, comEA and comEC, are suggested to represent unique DNA translocator proteins. Here we show that the T. thermophilus ComEA protein binds dsDNA and is anchored in the inner membrane. comEA is co-transcribed with the flanking comEC gene, and transcription of this operon is upregulated by nutrient limitation and low temperature. To our surprise, a comEC mutant was impaired in piliation. We followed this observation and uncovered that the impaired piliation of the comEC mutant is due to a transcriptional downregulation of pilA4 and the pilN both playing a dual role in piliation and natural competence. Moreover, the comEC mutation resulted in a dramatic decrease in mRNA levels of the pseudopilin gene pilA1, which is unique for the DNA transporter. We conclude that ComEC modulates transcriptional regulation of type IV pili and DNA translocator components thereby mediating a response to extracellular parameters.

Programmed protection of foreign DNA from restriction allows pathogenicity island exchange during pneumococcal transformation.

In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of S. pneumoniae through programmed protection and incorporation of foreign DNA.

Premethylation of foreign DNA improves integrative transformation efficiency in Synechocystis sp. strain PCC 6803.

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5' untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.

Spo0A~P imposes a temporal gate for the bimodal expression of competence in Bacillus subtilis.

ComK transcriptionally controls competence for the uptake of transforming DNA in Bacillus subtilis. Only 10%-20% of the cells in a clonal population are randomly selected for competence. Because ComK activates its own promoter, cells exceeding a threshold amount of ComK trigger a positive feedback loop, transitioning to the competence ON state. The transition rate increases to a maximum during the approach to stationary phase and then decreases, with most cells remaining OFF. The average basal rate of comK transcription increases transiently, defining a window of opportunity for transitions and accounting for the heterogeneity of competent populations. We show that as the concentration of the response regulator Spo0A∼P increases during the entry to stationary phase it first induces comK promoter activity and then represses it by direct binding. Spo0A∼P activates by antagonizing the repressor, Rok. This amplifies an inherent increase in basal level comK promoter activity that takes place during the approach to stationary phase and is a general feature of core promoters, serving to couple the probability of competence transitions to growth rate. Competence transitions are thus regulated by growth rate and temporally controlled by the complex mechanisms that govern the formation of Spo0A∼P. On the level of individual cells, the fate-determining noise for competence is intrinsic to the comK promoter. This overall mechanism has been stochastically simulated and shown to be plausible. Thus, a deterministic mechanism modulates an inherently stochastic process.

The regulatory network of natural competence and transformation of Vibrio cholerae.

The human pathogen Vibrio cholerae is an aquatic bacterium frequently encountered in rivers, lakes, estuaries, and coastal regions. Within these environmental reservoirs, the bacterium is often found associated with zooplankton and more specifically with their chitinous exoskeleton. Upon growth on such chitinous surfaces, V. cholerae initiates a developmental program termed "natural competence for genetic transformation." Natural competence for transformation is a mode of horizontal gene transfer in bacteria and contributes to the maintenance and evolution of bacterial genomes. In this study, we investigated competence gene expression within this organism at the single cell level. We provide evidence that under homogeneous inducing conditions the majority of the cells express competence genes. A more heterogeneous expression pattern was observable on chitin surfaces. We hypothesize that this was the case due to the heterogeneity around the chitin surface, which might vary extensively with respect to chitin degradation products and autoinducers; these molecules contribute to competence induction based on carbon catabolite repression and quorum-sensing pathways, respectively. Therefore, we investigated the contribution of these two signaling pathways to natural competence in detail using natural transformation assays, transcriptional reporter fusions, quantitative RT-PCR, and immunological detection of protein levels using Western blot analysis. The results illustrate that all tested competence genes are dependent on the transformation regulator TfoX. Furthermore, intracellular cAMP levels play a major role in natural transformation. Finally, we demonstrate that only a minority of genes involved in natural transformation are regulated in a quorum-sensing-dependent manner and that these genes determine the fate of the surrounding DNA. We conclude with a model of the regulatory circuit of chitin-induced natural competence in V. cholerae.

Control of natural transformation in salivarius Streptococci through specific degradation of σX by the MecA-ClpCP protease complex.

Competence for natural DNA transformation is a tightly controlled developmental process in streptococci. In mutans and salivarius species, the abundance of the central competence regulator σ(X) is regulated at two levels: transcriptional, by the ComRS signaling system via the σ(X)/ComX/SigX-inducing peptide (XIP), and posttranscriptional, by the adaptor protein MecA and its associated Clp ATPase, ClpC. In this study, we further investigated the mechanism and function of the MecA-ClpC control system in the salivarius species Streptococcus thermophilus. Using in vitro approaches, we showed that MecA specifically interacts with both σ(X) and ClpC, suggesting the formation of a ternary σ(X)-MecA-ClpC complex. Moreover, we demonstrated that MecA ultimately targets σ(X) for its degradation by the ClpCP protease in an ATP-dependent manner. We also identify a short sequence (18 amino acids) in the N-terminal domain of σ(X) as essential for the interaction with MecA and subsequent σ(X) degradation. Finally, increased transformability of a MecA-deficient strain in the presence of subinducing XIP concentrations suggests that the MecA-ClpCP proteolytic complex acts as an additional locking device to prevent competence under inappropriate conditions. A model of the interplay between ComRS and MecA-ClpCP in the control of σ(X) activity is proposed.

Regulatory elements involved in the expression of competence genes in naturally transformable Vibrio cholerae.

BACKGROUND: The human pathogen Vibrio cholerae normally enters the developmental program of natural competence for transformation after colonizing chitinous surfaces. Natural competence is regulated by at least three pathways in this organism: chitin sensing/degradation, quorum sensing and carbon catabolite repression (CCR). The cyclic adenosine monophosphate (cAMP) receptor protein CRP, which is the global regulator of CCR, binds to regulatory DNA elements called CRP sites when in complex with cAMP. Previous studies in Haemophilus influenzae suggested that the CRP protein binds competence-specific CRP-S sites under competence-inducing conditions, most likely in concert with the master regulator of transformation Sxy/TfoX. RESULTS: In this study, we investigated the regulation of the competence genes qstR and comEA as an example of the complex process that controls competence gene activation in V. cholerae. We identified previously unrecognized putative CRP-S sites upstream of both genes. Deletion of these motifs significantly impaired natural transformability. Moreover, site-directed mutagenesis of these sites resulted in altered gene expression. This altered gene expression also correlated directly with protein levels, bacterial capacity for DNA uptake, and natural transformability. CONCLUSIONS: Based on the data provided in this study we suggest that the identified sites are important for the expression of the competence genes qstR and comEA and therefore for natural transformability of V. cholerae even though the motifs might not reflect bona fide CRP-S sites.