Characterization of irvR, a novel regulator of the irvA-dependent pathway required for genetic competence and dextran-dependent aggregation in Streptococcus mutans

Abstract

Previous studies identified irvA as a normally repressed but highly inducible transcription regulator capable of repressing mutacin I gene expression in Streptococcus mutans. In this study, we aimed to identify and characterize the regulator(s) responsible for repressing the expression of irvA. An uncharacterized open reading frame (SMU.1398) located immediately adjacent to irvA and annotated as a putative transcription repressor was identified as a likely candidate. The results of mutation studies confirmed that the expression of irvA was greatly increased in the SMU.1398 background. Mutation of SMU.1398 ("irvR") abolished genetic competence and reduced the expression of the late competence genes/operons comEA, comY, and dprA without affecting the expression of the known competence regulators comC, comED, or comX. In addition, irvR was found to be a potent negative regulator of dextran-dependent aggregation (DDAG) and gbpC expression. Each of these irvR mutant phenotypes could be rescued with a double mutation of irvA or complemented by introducing a wild-type copy of irvR on a shuttle vector. These data indicate that the repression of irvA is critically dependent upon irvR and that irvA repression is essential for the development of genetic competence and the proper control of DDAG in S. mutans.

FIG. 1.

Expression of irvA in various irvR (SMU.1398) backgrounds. The relative expression levels of irvA were determined by real-time RT-PCR and compared between the wild-type strain UA159 (arbitrarily assigned as 1), the irvR mutant (GN01R), the irvR mutant strain harboring an empty shuttle vector (GN01Rs), and the complemented mutant strain (GN01Rc). Data are shown as the averages of the results of three independent experiments. All real-time RT-PCR values were normalized according to the abundance of 16S rRNA in each sample. Error bars show standard deviations.

FIG. 2.

Results of transformation efficiency assays in irvR mutant strains. The transformation efficiency values are presented relative to the wild-type value (6.0 × 10⁻⁷), which was arbitrarily assigned as 100% and compared to the values for the irvR mutant (GN01R), the mutant strain harboring an empty shuttle vector (GN01Rs), the complemented mutant strain (GN01Rc), and the irvR irvA double mutant (GN01RA). Data are presented as the averages of the results of three independent experiments performed in triplicate. Error bars show standard deviations.

FIG. 3.

Results of real-time RT-PCR analysis of late competence genes. The expression levels of late competence genes are presented relative to that of the wild type (UA159), which was arbitrarily assigned a value of 1. (A) Expression level of comEA in the irvR mutant strain GN01R compared to the levels in the wild-type strain UA159 (P < 0.001) and the irvR irvA double-mutant strain GN01RA (P < 0.01). (B) Expression level of comYA in strain GN01R compared to the levels in the wild type (P < 0.0001) and GN01RA (P < 0.0001). (C) Expression level of dprA in strain GN01R compared to the levels in the wild type (P < 0.0001) and GN01RA (P < 0.01) All data are presented as the averages of the results of five independent experiments. Statistical analysis was performed by using the two-tailed Student's t test. Error bars show standard deviations.

FIG. 4.

Results of DDAG assays in various irvR backgrounds. (A) Dextran T2000 was added to the wild-type strain UA159, the irvR mutant (GN01R), the irvR mutant strain harboring an empty shuttle vector (GN01Rs), the complemented irvR mutant (GN01Rc), and the irvR irvA double mutant (GN01RA). (B) The same experiment was performed using the wild-type strain UA159, the irvR mutant (GN01R), the gbpC mutant (LZ02C), and the gbpC irvR double mutant (GN01RC). Shown here are representative results seen after approximately 1 to 2 min of gentle agitation. These experiments were performed three times with similar results.

FIG. 5.

Results of real-time RT-PCR analysis of gbpC. The expression level of gbpC in the wild type (arbitrarily assigned as 1) is compared to the levels in the irvR (GN01R) and irvR irvA (GN01RA) mutant backgrounds. The data are the averages of the results of three independent experiments. Statistical analysis was performed by using the two-tailed Student's t test. Error bars show standard deviations.