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The oral biofilm organism Streptococcus mutans must face numerous environmental stresses to survive in its natural habitat. Under specific stresses, S. mutans expresses the competence-stimulating peptide (CSP) pheromone known to induce autolysis and facilitate the uptake and incorporation of exogenous DNA, a process called DNA transformation. We have previously demonstrated that the CSP-induced CipB bacteriocin (mutacin V) is a major factor involved in both cellular processes. Our objective in this work was to characterize the role of CipB bacteriocin during DNA transformation. Although other bacteriocin mutants were impaired in their ability to acquire DNA under CSP-induced conditions, the ΔcipB mutant was the only mutant showing a sharp decrease in transformation efficiency. The autolysis function of CipB bacteriocin does not participate in the DNA transformation process, as factors released via lysis of a subpopulation of cells did not contribute to the development of genetic competence in the surviving population. Moreover, CipB does not seem to participate in membrane depolarization to assist passage of DNA. Microarray-based expression profiling showed that under CSP-induced conditions, CipB regulated ~130 genes, among which are the comDE locus and comR and comX genes, encoding critical factors that influence competency development in S. mutans. We also discovered that the CipI protein conferring immunity to CipB-induced autolysis also prevented the transcriptional regulatory activity of CipB. Our data suggest that besides its role in cell lysis, the S. mutans CipB bacteriocin also functions as a peptide regulator for the transcriptional control of the competence regulon.
Natural transformation is a genetically programmed physiological process, and the state of transformable bacteria is termed competence (17). Competency development requires the formation of a multicomponent DNA uptake machinery as well as the activities of several recombination and DNA repair proteins by competent cells (8). DNA transformation contributes to horizontal gene transfer and the acquisition of new traits by bacteria (23). Naturally competent bacteria are found in many bacterial phyla, although the overall number of bacteria known to be naturally competent is relatively small (17). The low-G+C Gram-positive bacteria contain a number of naturally transformable species. The two best studied are the human pathogen Streptococcus pneumoniae and the soil dweller Bacillus subtilis (for a review, see reference 6). In both species, the competence genes are divided into two sets: the early genes involved in regulation of competency and the late genes required for DNA binding, uptake, and recombination.
In genetic transformation of the cariogenic organism Streptococcus mutans, the regulatory loop is modeled after that in S. pneumoniae (27) and is composed of the competence-stimulating peptide (CSP), the ComDE two-component system, and the alternative ComX sigma factor (also named SigX). CSP is synthesized ribosomally as a peptide precursor containing a double-glycine-type leader sequence at its N terminus and depends on a specific ATP-binding cassette transporter (ComAB) for cleavage and export. One genomic locus contains the comC, comD, and comE genes encoding the CSP precursor, a membrane-bound histidine kinase sensor, and a response regulator, respectively. When the extracellular mature CSP reaches a critical concentration, it interacts with ComD, resulting in its autophosphorylation and the subsequent activation of ComE by phosphorylation. The phosphorylated form of ComE regulates transcription by binding to a specific sequence found upstream of the promoter regions of several genes. Among the early genes is comX, encoding the ComX sigma factor required for the transcription of genes possessing a com-box consensus sequence in their promoter region and encoding proteins involved in DNA processing, uptake, and recombination. In contrast to S. pneumoniae, the S. mutans comX gene does not share any cis-acting site with ComE-dependent genes. It has been recently demonstrated that a novel circuit, ComR/ComS, is the proximal regulator of comX in S. mutans (25). ComR is a member of the Rgg family of transcription factors, while ComS belongs to a novel small double-tryptophan-containing peptide family. The ComR/ComS circuit is critical for development of genetic competence in S. mutans since inactivation of comR and/or comS genes completely abolished competency (25). According to Lemme et al. (20), competence development in S. mutans is a bistable system. Using a combination of flow cytometry sorting (ComX-green fluorescent protein [GFP]) and transcriptome analysis of the separated populations, the authors showed quite convincingly that all cells in a clonal population of S. mutans responded to CSP and that a bifurcation into two distinct subpopulations, one developing competence and one undergoing autolysis, was observed.
Several bacteriocin genes are expressed in the whole population of S. mutans through the CSP-ComDE regulatory system (35). One particular gene, SMU.1914, activated by ComE in the presence of CSP encodes mutacin V or CipB bacteriocin (13, 18, 31). CipB belongs to the class II bacteriocins commonly found in streptococci and lactic acid bacteria. Class II bacteriocins are small, heat-stable, unmodified, and hydrophobic antimicrobial peptides of 20 to 60 amino acid residues in length (26). The killing spectrum is rather narrow, limited to species or strains related to the producers (33). A common mechanism of action for class II bacteriocins is the dissipation of proton motive force (PMF) via pore formation in the cytoplasmic membrane of target cells, leading to leakage of cellular solutes and, eventually, cell death (1, 15). Surprisingly, a ΔcipB mutant unable to produce the CipB bacteriocin had a ~ 2-log-fold reduction in transformation efficiency compared to the wild-type (WT) strain under CSP-induced conditions (31). In S. pneumoniae, it was shown that a two-peptide bacteriocin, CibAB, produced by competent cells was required for the release of virulence factors from killed noncompetent cells (12). It was proposed that CibAB functions as a trigger factor for lysis, most probably by inserting into the membrane of noncompetent cells and depleting cellular energy, a plausible fratricidal mechanism (7). Since the development of genetic competence in S. mutans occurs in part via a small peptide bacteriocin, the aim of this study was to characterize the role of CipB bacteriocin during the process of CSP-induced DNA transformation in S. mutans.
S. mutans strains used in this study are listed in Table 1. Deletion mutants were constructed in the S. mutans UA159 wild-type (WT) strain as described previously (19). All strains were grown in Todd-Hewitt yeast extract (THYE) broth and incubated statically at 37°C in air with 5% CO2. When needed, the antibiotic erythromycin (10 μg/ml) or spectinomycin (1 mg/ml) was added to the culture medium. Cell growth was monitored through optical density at 600 nm (OD600). Cell viability was assessed by counting CFU on replica agar plates.
Overnight cultures of WT and its deletion mutants were diluted (1:20) with fresh THYE and incubated at 37°C until an OD600 of 0.1 was reached. To test the effect of CSP on genetic transformation (CSP-induced conditions), synthetic CSP (sCSP; Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, Ontario, Canada) dissolved in sterile deionized water was added at a final concentration of 0.2 μM. Ten micrograms of UA159 genomic DNA (gDNA) containing the spectinomycin resistance marker inserted into the rgp locus of the UA159 strain, as inactivation of this locus has no effect on transformation efficiency (31), was added to the cultures (0.5-ml aliquots), which were grown for a further 2.5 h at 37°C before differential plating. The transformation efficiency was calculated as the percentage of spectinomycin-resistant transformants divided by the total number of recipient cells, which was determined by the number of CFU on antibiotic-free THYE agar plates. All assays were performed in triplicate from three independent experiments. Statistical significance was determined by using a Student t test and a P value of <0.01.
DNA transformation assays were also performed in the presence of the proton motive force (PMF) inhibitor gramicidin (1.0, 2.5, and 25 μg/ml) or carbonyl cyanide m-chlorophenylhydrazone (CCCP; 1.0 and 10 μg/ml). Transformation assays were performed as described above, except that cultures were incubated with PMF inhibitors for 15 min prior to the addition of streptococcal gDNA.
Two different DNA microarray experiments were performed, one to compare the gene expression profiles of WT and the ΔcipB mutant during CSP-induced transformation (0.2 μM sCSP added) and one to compare the gene expression profiles of WT and the ΔcipI mutant during natural transformation (no sCSP or uninduced conditions). Overnight cultures were diluted (1:20) with fresh THYE and incubated at 37°C until an OD600 of 0.1 was reached. Cells were then grown at 37°C for 1 h 45 min in THYE broth in the presence of Δrgp mutant gDNA (10 μg). Cells were processed with the Bio101 Fast Prep System (Qbiogen), and total RNA was extracted using TRIzol reagent (Invitrogen). WT and ΔcipB and ΔcipI mutant cDNAs were labeled with cyanine 3 (Cy3), whereas reference cDNA (WT mid-log phase of growth) was labeled with cyanine 5 (Cy5). An MAUI hybridization chamber was used for hybridization, and slides were scanned using a GenePix scanner at 532 nm for the Cy3 channel and at 635 nm for the Cy5 channel. Scanned images were processed using TIGR Spotfinder software, and the output data were normalized using the MIDAS program and the Biometric Research Branch microarray tool. Statistically significant genes were then identified using a class comparison analysis. A two-sample t test with the parametric P value cutoff set at <0.001 was used for statistical analysis. Microarray assays of four independent RNA isolations were conducted. Results were validated by real-time quantitative PCR (qPCR) using the CFX96 real-time PCR detection system (Bio-Rad). The gyrA gene was used as internal reference. qPCR assays were performed in triplicate with RNA isolated from three independent experiments.
We previously demonstrated that under high levels of sCSP, a subpopulation of S. mutans cells undergoes autolysis due to the action of CipB bacteriocin (31). Interestingly, a ΔcipB mutant unable to produce the CipB bacteriocin, and consequently unable to undergo CSP-induced autolysis, showed no increase in transformation efficiency in the presence of exogenous sCSP, while a ΔcipI mutant deficient in the CipI immunity factor showed high transformation efficiency levels under both conditions (Fig. 1). These results prompted us to hypothesize that the 2-log increase in DNA transformation efficiency observed for the WT strain under CSP-induced conditions could result from extracellular signal(s) released by lysed S. mutans cells triggering competence in the surviving population. To test this hypothesis, transformation assays were performed with washed ΔcipB mutant cells diluted in the cell-free supernatant of an overnight culture of the WT strain cultivated in the presence of high levels of sCSP (conditions known to induce CipB-dependent autolysis). Our results showed that ΔcipB mutant cells grown in the cell-free supernatant of WT cells were transformed at frequencies similar to those obtained using ΔcipB mutant cells diluted into fresh medium (Fig. 2). The fact that the transformation efficiency of ΔcipB mutant could not be restored to the wild-type level suggested that the autolytic activity of the bacteriocin does not contribute to the CSP-induced DNA transformation process through release of specific secondary signals by the subpopulation of lysed S. mutans cells.
As S. mutans is known to produce several bacteriocins (mutacins) (5, 13, 16), we next sought to determine if all CSP-inducible bacteriocins could affect the cell's ability to become competent. Bioinformatic analysis of the UA159 genome using the BAGEL tool, a web server that identifies putative bacteriocin open reading frames (ORFs) in a DNA sequence using knowledge-based bacteriocin databases and motif databases (9), identified 79 bacteriocin candidates in the genome of the UA159 reference strain. Among the 79 bacteriocin candidates, only those presenting the following criteria were selected: (i) leader sequence ending with a double-glycine motif, (ii) conserved domain of the class II bacteriocin family, and (iii) CSP-inducible bacteriocin gene (based on microarray data; see reference 31). Five candidates were finally selected: SMU.150/151 (NlmAB or mutacin IV), SMU.423 (NlmD or mutacin VI), SMU.1902, SMU.1906, and SMU.1914 (CipB or mutacin V). In order to determine if the competence-altered phenotype observed with the ΔcipB mutant was specific to mutacin V, mutant strains deficient in each of these selected bacteriocin genes were constructed and their transformation efficiencies were evaluated under CSP-induced conditions (Fig. 3). The deletions of mutacin VI (ΔnlmD mutant) and SMU.1906 (Δ1906 mutant) reduced the transformation efficiency, the reduction being 9.03-fold and 22.2-fold, respectively. In contrast, deletions of mutacin IV (ΔnlmAB mutant) and SMU.1902 (Δ1902 mutant) had no significant effect on transformation efficiency. The deletion of mutacin V (ΔcipB mutant) reduced the transformation efficiency more than 1,000-fold, corresponding to less than 0.05% of the wild-type value, the greatest reduction under the conditions tested. Hence, mutacin V was selected for further analysis.
Bacteriocins are normally synthesized as intracellular biological inactive forms called prebacteriocins. The prebacteriocins are processed and exported into the extracellular environment through a dedicated ABC transporter (33). In order to test if the secreted form of CipB bacteriocin was necessary to participate in the CSP-induced DNA transformation process, transformation assays were performed using monocultures of a mutant strain deficient in the export system for CipB. We thus created a deletion mutant of the nlmTE locus (14) unable to secrete mutacin V. As shown in Fig. 4, the ΔnlmTE mutant exhibited transformation efficiencies similar to that of the WT, suggesting that the exported form of CipB was not participating in the CSP-induced DNA transformation process. To confirm this result, we also performed transformation assays using cocultures of the WT and ΔcipB mutant strains. If the secreted form of CipB bacteriocin is necessary to trigger competence, we should be able to restore the transformation efficiency of the ΔcipB mutant to WT levels following direct cocultures with both WT and ΔcipB mutant strains. Results showed that cocultivations of WT and ΔcipB strains failed to yield a transformation efficiency higher that 0.05% of the WT value for the ΔcipB mutant (Fig. 4). Consequently, these results strongly support the hypothesis that the intracellular form (cytoplasmic and/or membrane embedded) of CipB bacteriocin, and not the exported form, is necessary to trigger competence under CSP-induced conditions.
Class II bacteriocins present generally a hydrophobic domain that penetrates the target cell membrane. Subsequently, their activity mainly induces dissipation of the proton motive force (PMF) via pore formation (15). In Bacillus subtilis, the PMF functions as a driving force for the DNA uptake during bacterial transformation (24, 36). As the primary structure of CipB contains a predicted helical transmembrane domain, we hypothesized that CipB might be part of the competence induction network by promoting membrane depolarization, thus facilitating DNA uptake. To test this hypothesis, CSP-induced transformation assays were performed in the presence of gramicidin and CCCP, two decoupling agents traditionally used for membrane potential alteration. The results showed that the use of CCCP did not restore the transformation efficiency of the ΔcipB mutant to the WT levels (data not shown). It is thus unlikely that CipB plays a role in DNA transformation by altering the PMF. Interestingly, transformation assays performed in the presence of gramicidin, even at low concentrations, completely failed to yield any transformants for both WT and ΔcipB mutant strains, suggesting that the PMF is necessary to drive DNA uptake in S. mutans cells.
We then reasoned that CipB could participate in the DNA transformation process by regulating the competence genes. Comparison of the gene expression profiles between WT and ΔcipB mutant during CSP-induced transformation (see Materials and Methods for details) suggested that CipB directly and/or indirectly regulated ~130 genes (see Table S1 in the supplemental material), among which are the comDE locus and comR and comX genes (results confirmed by qPCR), encoding critical factors that influence competence development in S. mutans (Table 2). All previously identified ComX-regulated genes encoding late competence proteins and/or proteins involved in DNA-related function were also found strongly downregulated in the ΔcipB mutant arrays (Table 2).
We also confirmed and/or identified a ComX-binding box in the promoter region of the SMU.507, SMU.769, and SMU.2076 genes encoding proteins of unknown functions. Work recently done by Okinaga et al. (29) demonstrated that the SMU.507 gene was dispensable for transformation in S. mutans. Deletion mutants were constructed in SMU.769 and SMU.2076, and the mutants were tested using our transformation assays (CSP-induced and uninduced conditions). As shown in Fig. 5, the deletion of SMU.769 and SMU.2076 had no effect on transformation efficiency under CSP-induced conditions. In contrast, inactivation of both genes dramatically affected natural transformation (no transformants were recovered after 48 h of incubation under uninduced transformation conditions), suggesting that their gene products may play a critical role during natural transformation.
The microarray results also identified genes encoding a putative transcriptional regulator (SMU.168) and two other two-component systems, the hk4-rr4 and hk9-rr9 loci, whose expression was altered in the ΔcipB mutant. In S. mutans and S. pneumoniae, genetic competence is primarily regulated by the ComDE two-component system. To assess the role of SMU.168 and the HK4/RR4 and HK9/RR9 systems, SMU.168 and each of the response regulator genes were inactivated and the mutants were tested for their ability to be transformed. Under uninduced transformation conditions, the Δrr4 mutant showed significantly lower transformation rates than did the WT (~14-fold decrease), whereas the Δrr9 and Δ168 mutants exhibited transformation efficiencies similar to that of the WT (Fig. 5). The deletions of rr4, rr9, and SMU.168 genes did not affect DNA transformation under CSP-induced conditions.
The expression of several genes encoding bacteriocins and GG-motif-containing peptides was found negatively regulated in the ΔcipB arrays. Most of these genes are located in a 13.5-kb bacteriocin-related genomic island. Three (nlmD, cipI, and SMU.1906) of the five gene deletions tested had a significant effect on transformation efficiency (Fig. 1 and and3).3). In contrast, the deletions of nlmAB and SMU.1902 did not affect DNA transformation (Fig. 3).
Based on these results, we can conclude that the CipB bacteriocin also possesses a regulatory role in the activation of three major regulators of S. mutans genetic competence, ComE, ComR, and ComX, hence affecting indirectly the expression of genes essential for the DNA transformation process.
As illustrated in Fig. 1, a ΔcipI mutant showed increased transformation efficiency under uninduced conditions (no sCSP added). These results prompted us to investigate whether the inactivation of cipI could affect the expression of the competence genes. We thus determined the transcription profile of a ΔcipI mutant cultivated under uninduced transformation conditions (see Table S1 in the supplemental material). As expected, we observed upregulation of all competence genes (Table 2). Interestingly, several genes encoding putative autolysins were also found highly expressed in our ΔcipI mutant arrays. Recently, two studies demonstrated that inactivation of SMU.836 encoding a putative peptidoglycan hydrolase strongly affected natural transformation (10, 29). We thus created deletions of SMU.575/574 (lrgAB), SMU.609, and SMU.772 genes and tested the mutant's ability to be transformed. As shown in Fig. 5, the tested autolysin mutants exhibited transformation efficiencies similar to that of the WT under CSP-induced and uninduced conditions. These results suggest that these putative autolysins are not required for the development of competence in S. mutans.
Horizontal gene transfer (HGT) is a dominant force in the evolution of bacteria, enabling them to acquire new characteristics. DNA transformation is one of the three mechanisms of HGT occurring in bacteria (23). During transformation, DNA material can be transferred between different species of bacteria or bacteria belonging to different genera (17). The potential benefits of natural transformation are numerous, including the rapid acquisition of genes and the possibility of producing offspring with recombinant phenotypes, the acquisition of intact DNA strands for repair of DNA damage by recombination, and the acquisition of the nutrients contained in the DNA molecules (17). Natural transformation might also represent a competitive advantage for one organism where a multispecies biofilm is involved. Indeed, biofilm cells are embedded in a self-produced matrix that holds them together (11). This extracellular polymeric substance is composed of polysaccharides, proteins, and extracellular DNA (eDNA). Despite the fact that a role for eDNA in surface attachment and biofilm strengthening has already been demonstrated, we cannot rule out the possibility that eDNA may also be a source of genes in HGT (27). The natural habitat of S. mutans is the dental plaque biofilm, one of the most complex human microbiota (37). In S. mutans, the CSP-ComDE quorum sensing system controlling genetic competence functions optimally when the cells are living in actively growing biofilms (21). The concept that oral biofilm may provide S. mutans with a reservoir of diverse transferable genetic information has dramatic implications when considering the potential for the transfer of antibiotic resistance genes to pathogens that may transiently reside in the oral cavity.
The regulatory cascade controlling the development of genetic competence in S. mutans is still not completely elucidated. Several groups have demonstrated the implication of many effector genes and different regulatory circuits. Work previously done in our labs has demonstrated that the CSP-ComDE quorum sensing system controls various biological processes in S. mutans, such as virulence, genetic transformation, and production of antimicrobial peptides called bacteriocins. Bacteriocins provide producing organisms with an ecological function over their most likely competitors. The bacteriocins produced by S. mutans are called mutacins. Bioinformatic analyses and mutational studies demonstrated that the antimicrobial repertoire of the UA159 reference strain, a clinical strain isolated from a child with active caries, includes mutacin IV, mutacin V, and mutacin VI (13, 16, 18, 35). We recently demonstrated that the S. mutans competence-stimulating peptide is also a stress response pheromone capable of inducing the expression of mutacin V or CipB under stressful conditions (31). While the extracellular form of CipB interfered with the growth of Lactococcus lactis (organism extensively used in the production of cheese) and Streptococcus oralis (oral streptococci frequently isolated from cases of human infective endocarditis), the intracellular accumulation of unprocessed CipB was lethal to the producing cell. Surprisingly, we found that inactivation of CipB bacteriocin strongly reduced the cell's ability to become competent.
In this study, we report the characterization of CipB bacteriocin during the development of genetic competence in S. mutans. Our results confirmed that besides its role in cell lysis, the S. mutans CipB bacteriocin also participates in the transcriptional control of the competence regulon under CSP-induced conditions. Moreover, we discovered that CipI protein that confers immunity to CipB-induced autolysis also prevents the CipB transcriptional regulatory activity. To the best of our knowledge, this is the first study to report a role for a bacteriocin and its cognate immunity factor in transcriptional regulation. CipB and CipI proteins are most probably members of a new class of active molecules that can play a regulatory role by interacting with protein partners and by modulating protein partner activity. In enterobacteria, recent results have converged to highlight the role of small hydrophobic peptides as regulators (4). These small peptides can promote the degradation/stabilization or activation of membrane proteins. In Escherichia coli, a small protein of 47 amino acid residues, MgrB, directly interacts with the sensor kinase PhoQ of the PhoQ/PhoP two-component system. This interaction results in the repression of multiple genes in the PhoQ/PhoP regulon, which could be due to activation of the phosphatase activity of PhoQ and/or to the inhibition of its kinase activity (22). In B. subtilis, the competence-specific transcription factor ComK is sequestered in a ternary complex with ClpC and MecA proteins until the small protein ComS interacts with ClpC, enabling the liberation of ComK. Liberated ComK can then activate transcription of genes required for the development of genetic competence (34). Recently, Mashburn-Warren and coworkers (25) postulated that ComR and a small peptide named XIP formed a complex that functions as a transcriptional activator of S. mutans ComX sigma factor involved in the control of competence-specific genes. Assuming that CipB bacteriocin is also a peptide regulator, we can speculate that the intracellular unprocessed form of CipB could interact with the ComE response regulator, the first master regulator of the S. mutans competence regulon or the sensor kinase ComD, to promote the activity of ComE (due to inhibition of the phosphatase activity of ComD and/or to activation of its kinase activity). It is also possible that CipB directly interacts with ComR, the proximal regulator of the ComX sigma factor, to enhance the binding affinity of ComR to its own promoter region. Further experiments will be required to explore these possibilities.
The results of this study led us to propose a new model of regulation of the S. mutans competence regulatory network, which integrates the CSP-ComDE quorum sensing system, the ComR/ComS circuit, the CSP-inducible CipB bacteriocin, and its immunity factor, CipI (Fig. 6). In this model, the ComDE two-component system is the primary circuit sensing CSP and is responsible for the activation of cipB transcription. Although the exported form of CipB kills competitors, the intracellular form of CipB is necessary to activate the CSP-induced competence pathway through the ComE, ComR, and ComX master competence regulators. Under conditions of low cell density (low CSP levels), S. mutans upregulates expression of CipI immunity protein through the LiaSR two-component system (30). CipI protein then sequesters intracellular CipB (present at low concentrations) and prevents its regulatory function. Then, when the cell density reaches a critical threshold concentration (high CSP levels), the ComDE two-component system strongly activates cipB gene expression (the balance between CipB and CipI is affected). When unsequestered, intracellular CipB can act as a peptide regulator to activate competence gene expression via ComE and/or ComR transcriptional regulators.
Taken together, our results suggest that the S. mutans CipB bacteriocin is a dual-function peptide that combines bacteriolytic activity and transcriptional regulation. As small noncoding RNAs have emerged as important players in diverse aspects of biology, bacterial small proteins and/or peptides may constitute an important novel class of regulatory molecules in prokaryotes. These small molecules might contribute to bacterial fitness by having multiple roles, such as cell killing, modification of the DNA-binding capacity of a transcription factor, protein degradation/stabilization, activation of sensor kinase, and alteration of the specificity of a membrane transporter.
We thank Julie Perry for helpful discussion and technical assistance with the construction of bacteriocin mutants. We are grateful to Stephanie Koyanagi for technical assistance with the autolysin-deficient mutants.
This study was supported by CIHR grant MOP-93555 to C.M.L. and NIH grant R01DE013230 to D.G.C.
†Supplemental material for this article may be found at http://jb.asm.org/.
Published ahead of print on 7 October 2011.