Characterization of DNA binding sites of the ComE response regulator from Streptococcus mutans

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.     

Identification and Characterization of ComE and ComF, Two Novel Pilin-Like Competence Factors Involved in Natural Transformation of Acinetobacter sp. Strain BD413

Although the high level of competence for natural transformation of Acinetobacter sp. strain BD413 has been the subject of numerous studies, only two competence genes, comC and comP, have been identified to date. By chromosomal walking analysis we found two overlapping open reading frames, designated comE and comF, starting 61 bp downstream of comC. comE and comF are expressed as stable proteins in Escherichia coli, thus proving that they are indeed coding regions, but expression was successful only with 5′-deleted genes. ComE and ComF are similar to pilins and pilin-like components. Both genes were mutated, and the phenotypes of the mutants were analyzed. Natural transformation in comF mutants is 1,000-fold reduced, whereas comE mutants exhibit 10-fold-reduced transformation frequencies. This is clear evidence that comE and comF are involved in natural transformation. However, ComE and ComF are specific for DNA translocation, since comE and comF defects affected neither piliation nor lipase secretion. These results suggest that the type IV pili, the general protein secretion pathway, and the DNA translocation machinery in Acinetobacter sp. strain BD413 are evolutionary related but functionally distinct systems.     

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.     

Peptide pheromone induced cell death of Streptococcus mutans

Streptococcus mutans is considered one of the main causative agents of human dental caries. Cell-cell communication through two-component signal transduction systems (TCSTS) plays an important role in the pathogenesis of S. mutans. One of the S. mutans TCSTS, ComDE, controls both competence development and biofilm formation. In this study, we showed that addition of exogenous competence-stimulating peptide (CSP) beyond the levels necessary for competence inhibited the growth of S. mutans in a ComDE-dependent manner. We also demonstrated that further increases of CSP stopped S. mutans cell division leading to cell death. Use of CSP as a possible therapeutic agent is discussed.