Regulation of the competence pathway as a novel role associated with a streptococcal bacteriocin

Abstract

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.

Fig. 1.

DNA transformation assays performed under uninduced (no added sCSP) and CSP-induced (exogenous sCSP added) conditions. Overnight cultures of S. mutans UA159 wild-type (WT) and its deletion mutants (ΔcipB, ΔcipI, and ΔcipB ΔcipI) were diluted in fresh medium and grown to early log phase prior to the addition of UA159 gDNA carrying an antibiotic resistance marker, in the absence and presence of sCSP (0.2 μM). Cells were grown for a further 2.5 h before differential plating. The transformation efficiency was expressed as the percentage of spectinomycin-resistant transformants divided by the total number of recipient cells. The mutant unable to produce CipB bacteriocin (ΔcipB) showed no increase in transformation efficiency under CSP-induced conditions, whereas the ΔcipI mutant unable to produce the immunity factor (mutant with increased lysis potential; see reference 31) showed an increase in transformation efficiency in the absence of added sCSP. The cells of the double mutant were transformed at frequencies similar to those obtained using the ΔcipB mutant under both transformation conditions, suggesting that CipB is the major component.

Fig. 2.

Transformation efficiency of UA159 wild-type (WT) and ΔcipB mutant cells diluted in fresh medium and in the cell-free supernatant of WT cells. Overnight cultures were diluted in fresh THYE medium or in the cell-free supernatant of the WT strain cultivated in the presence of high sCSP levels (2 μM). When the early log phase was reached, UA159 gDNA and sCSP (0.2 μM) were added to the cultures, which were grown for a further 2.5 h before differential plating. The transformation efficiency was expressed as the percentage of spectinomycin-resistant transformants divided by the total number of recipient cells.

Fig. 3.

Transformation efficiency of UA159 wild-type (WT) strain and its bacteriocin-deficient mutants. CSP-induced transformation assays were performed as described above (see Fig. 1 legend for details).

Fig. 4.

Transformation efficiency of UA159 wild-type strain (WT) and ΔcipB and ΔnlmTE mutants. Experiments were performed using monocultures (WT and ΔcipB and ΔnlmTE mutants) and cocultures (WT/ΔcipB) under CSP-induced conditions (see Fig. 1 legend for details). For the cocultivation experiments, equal quantities of WT and ΔcipB mutant cells were mixed. CSP-induced DNA transformation assays were then performed as described for monocultures.

Fig. 5.

Transformation efficiency of UA159 wild-type (WT) strain and its deficient mutants under CSP-induced (sCSP) and uninduced (no sCSP) transformation conditions. The transformation assays were performed as described above (see Fig. 1 legend for details).

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 immunity factor, CipI. At low cell density (low CSP levels), the major competence system remains inactive and other systems, such as CiaHR (3, 32), HtrA (2), and HdrMR (28, 29), induce a basal level of genetic competence. Both cipB and cipI genes are dependent on the ComE regulator for their transcription. However, the expression of cipI shows an additional level of density-dependent control via the LiaSR two-component system (30). CipI then sequesters CipBⁱⁿ present at low concentrations and prevents its regulatory function. When the cell density reaches a critical threshold concentration (high CSP levels), the ComDE two-component system activates cipB gene expression, affecting the balance between CipB and CipI proteins. Unsequestered CipBⁱⁿ can act as a peptide regulator to activate competence gene expression via the ComE and/or ComR regulators (see text for additional details). CipBⁱⁿ, unprocessed intracellular form of CipB; CipB^out, mature extracellular (outside) form of CipB. Solid arrows indicate processing steps or transcriptional regulation that has been previously demonstrated. Dashed arrows indicate hypothetical links.