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
). 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 (). 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.
Fig. 6. Hypothetical model showing the role of CipB and CipI in S. mutans competence regulatory cascade. This model integrates the primary circuit sensing CSP, the ComDE two-component system, the ComR/ComS circuit, the CSP-inducible CipB bacteriocin, and its (more ...)
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.