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. 2010 Apr;192(7):1844-52.
doi: 10.1128/JB.01667-09. Epub 2010 Jan 29.

The hdrRM operon of Streptococcus mutans encodes a novel regulatory system for coordinated competence development and bacteriocin production

Toshinori Okinaga  1 , Guoqing Niu, Zhoujie Xie, Fengxia Qi, Justin Merritt
Affiliations

The hdrRM operon of Streptococcus mutans encodes a novel regulatory system for coordinated competence development and bacteriocin production

Toshinori Okinaga et al. J Bacteriol. 2010 Apr.

Abstract

The Streptococcus mutans hdrRM operon encodes a novel two-gene regulatory system induced by high cell density. Previous studies identified hdrM as the only known negative regulator of competence development in S. mutans. In the present study, we demonstrated that the HdrRM system bypasses the prototypical competence gene regulators ComC and ComDE in the transcriptional regulation of the competence-specific sigma factor comX and the late competence genes. Similarly, the HdrRM system can abrogate the requirement for ComE to produce the bacteriocin mutacin IV. To further probe the regulatory mechanism of hdrRM, we created an hdrR overexpression strain and showed that it could reproduce each of the hdrM competence and mutacin phenotypes, indicating that HdrM acts as a negative regulator of HdrR activity. Using a mutacin IV-luciferase reporter, we also demonstrated that the hdrRM system utilizes the same promoter elements recognized by ComE and thus appears to comprise a novel regulatory pathway parallel to ComCDE.

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Figures

FIG. 1.
FIG. 1.
Transformation efficiencies of various hdrM mutants. Transformation efficiency was measured as described in Materials and Methods, and all values were normalized to the wild-type UA140 value (3.14 × 10−6). The strains are identified as follows: WT (wild-type UA140), WT+CSP (UA140 with CSP), ΔM (hdrM mutant), ΔC (comC mutant), ΔC+CSP (comC mutant with CSP), ΔE (comE mutant), ΔX (comX mutant), ΔY (comY mutant), ΔM/C (hdrM comC double mutant), ΔM/C+CSP (hdrM comC double mutant with CSP), ΔM/E (hdrM comE double mutant), ΔM/X (hdrM comX double mutant), and ΔM/Y (hdrM comY double mutant). The results presented here are the average of three independent experiments. Each experiment measured three independent clones for each sample.
FIG. 2.
FIG. 2.
Transcription of competence genes in the hdrM background. Wild-type UA140 and the hdrM mutant were tested for the expression levels of (A) comC, (B) comE, (C) comX, and (D) comY during the early, mid, and late log phases. Data are presented relative to the transcript abundance of the wild-type early log phase sample, which was arbitrarily assigned a value of 1. Black bars represent wild-type UA140, whereas the hdrM mutant is represented by dark gray bars. These results are the average of three independent experiments. Each experiment measured three independent clones for each sample. All experiments measuring transcription via real-time RT-PCR were normalized by using the 16S rRNA gene as a housekeeping control.
FIG. 3.
FIG. 3.
Analysis of mutacin IV (nlmA) production in the hdrM background. The production of mutacin IV was tested by the deferred antagonism assay as described in Materials and Methods. The development of a growth inhibition halo is indicative of the presence of mutacin IV inhibiting the sensitive strain S. sanguinis NY101. S. mutans strains are identified as follows: WT (wild-type UA140), ΔE (comE mutant), ΔM (hdrM mutant), ΔM/E (hdrM comE double mutant), ΔIV (nlmA mutant), and ΔM/E/IV (hdrM comE nlmA triple mutant). This experiment was performed three times with similar results.
FIG. 4.
FIG. 4.
Analysis of the hdrR overexpression strain. (A) The expression of hdrR was measured by real-time RT-PCR during the early, mid, and late log phases, and results are presented relative to the early log phase transcript abundance of the wild type. Wild-type UA140 is represented by black bars, the translational fusion hdrR overexpression strain is represented by light gray bars, and the RBS-negative hdrR overexpression strain is represented by solid white bars. (B) Comparison of the transformation efficiencies of wild-type UA140 (black), the hdrM mutant (dark gray), the translational fusion hdrR overexpression strain (light gray), and the RBS-negative hdrR overexpression strain (white). Values are presented relative to the wild type, which was arbitrarily assigned a value of 1. (C) Comparison of the expression of comX among wild-type UA140 (black), the hdrM mutant (dark gray), the translational fusion hdrR overexpression strain (light gray), and the RBS-negative hdrR overexpression strain (white). Samples were measured by real-time RT-PCR during the early, mid, and late log phases, and results are presented relative to the early log phase transcript abundance of the wild type. The data in panels A to C are the average of three independent experiments. Each experiment measured three independent clones for each sample.
FIG. 5.
FIG. 5.
Analysis of mutacin IV production in the hdrR overexpression strain. The production of mutacin IV was detected with the deferred antagonism assay using S. sanguinis NY101. S. mutans strains are identified as follows: WT (wild-type UA140), ΔE (comE mutant), ROE (translational fusion hdrR overexpression strain), ROE(RBS−)/ΔE (RBS-negative hdrR overexpression strain in the comE background), ROE/ΔE (translational fusion hdrR overexpression strain in the comE background), ΔIV (nlmA mutant), and ROE/ΔE/ΔIV (translational fusion hdrR overexpression strain in the comE nlmA background). This experiment was performed three times with similar results.
FIG. 6.
FIG. 6.
Investigation of the effect of hdrRM operon overexpression upon competence and mutacin IV production. (A) Comparison of transformation efficiencies among wild-type UA140 (black), the hdrR overexpression strain (gray), and the hdrRM overexpression strain (white). (B) Luciferase activities of the nlmA-luc reporter strain in the following backgrounds: WT (wild-type UA140), ROE (hdrR overexpression strain), RMOE (hdrRM overexpression strain), ΔE (comE mutant), ROE/ΔE (hdrR overexpression strain in the comE background), RMOE/ΔE (hdrRM overexpression strain in the comE background), ΔDR [UA140 in nlmA(Δdirect repeat)-luc], ROE/ΔDR [hdrR overexpression strain in nlmA(Δdirect repeat)-luc], and RMOE/ΔDR [hdrRM overexpression strain in nlmA(Δdirect repeat)-luc]. The competence and luciferase assays were normalized to the wild-type UA140 values. All results are the average of three independent experiments. Each experiment measured three independent clones for each sample.
FIG. 7.
FIG. 7.
Model of the hdrRM-regulated competence pathway. HdrM (M) responds to unknown environmental signals which regulate the antagonism of HdrR (R) transcription factor activity. When the environmental conditions are such that HdrM is unable to antagonize HdrR, this will allow HdrR to activate the transcription of comX for natural competence and nlmA for mutacin IV production. This pathway functions in parallel with ComD (D) and ComE (E), which respond to the presence of CSP.
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