Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence

Abstract

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB'-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB'-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.

FIG. 1.

Induction of nlmAB′-lacZ expression by CSP. An overnight culture of S. mutans strain OMZ1008 was diluted 100-fold in fresh medium and grown aerobically at 37°C. After 4 h of growth, the culture was split and CSP was added to a final concentration of 0.5 μg/ml to one portion of the culture. The optical density at 600 nm (closed symbols) and β-galactosidase activity (open symbols) were measured throughout growth. Circles: the culture did not receive CSP; squares, the culture received CSP. The figure shows the results of a representative experiment.

FIG. 2.

Influence of the amount of CSP on expression of nlmAB′-lacZ. An overnight culture of S. mutans strain OMZ1008 was diluted 100-fold in fresh medium and grown aerobically at 37°C. After 4 h of growth, the culture was divided in several aliquots. Each aliquot of the culture received a different amount of CSP and was allowed to grow for another 2 hours. The activity of β-galactosidase was then measured in each fraction. The figure shows the results of three independent experiments (indicated by circles, diamonds, and crosses).

FIG. 3.

Clustal W sequence alignment of the leader peptide sequences of putative type II bacteriocins and CSP. Asterisk, identical residue; colon, conserved residue; period, semiconserved residue.

FIG. 4.

Genetic organization of loci harboring the bsm, imm, and competence-related genes. Genes shown by black arrows and arrowheads encode putative bacteriocins, whereas genes associated with competence and bacteriocin production are shown by shaded arrows. The scale above the maps is in base pairs. Numbers above or below the genes correspond with the numbering by Ajdic et al. (1). Incomplete genes are denoted by a prime. Vertical arrows indicate the position of the conserved direct repeat involved in ComCDE-dependent expression.

FIG. 5.

Sequence alignment of upstream regions from four genes encoding putative bacteriocins and one gene encoding a putative bacteriocin immunity protein. The direct repeat elements and the extended −10 promoter regions are underlined. The consensus sequence is shown above the sequences (nucleotides present in each of the sequences are in uppercase, and nucleotides occurring in four of the sequences are in lowercase), whereas the general consensus binding site proposed for TCSTSs of the AlgR/AgrA/LytR family (22) is given below the sequences in boldface. The start of the bsmA transcript is indicated by an arrow. The distance in nucleotides between the end of the conserved region and the start codon is shown.

FIG. 6.

Mutations in the direct repeat of the nlmAB promoter region and their effect on activity of β-galactosidase expressed from a transcriptional nlmAB′-lacZ fusion. Direct repeats are underlined. Residues that were mutated are shown in lowercase and boldface. In S. mutans strain OMZ1037, both repeats and the region in between the repeats were deleted. Activity of β-galactosidase was measured in late-exponential-phase cultures 3 h after addition of 100 ng/ml of CSP.