Regulation of ciaXRH operon expression and identification of the CiaR regulon in Streptococcus mutans

Abstract

The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the -10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes.

FIG. 1.

Effects of ciaH and ciaRH mutation and ciaR overexpression (OEciaR) on competence. The transformation efficiency of the wild type (WT) was arbitrarily assigned as 100%. The experiments were repeated 3 times, and the average values are presented.

FIG. 2.

Expression of ciaR in the ciaH mutant as measured by real-time RT-PCR. Template RNA was isolated from wild-type (WT) and ciaH (ΔciaH) mutant cells grown to an OD₆₀₀ of 0.3. The ratio between the relative cDNA abundance of the ciaH mutant and that of the wild type was calculated and expressed as fold change. The experiments were repeated 3 times, and the average values are presented.

FIG. 3.

(A) Alignment of the putative CiaR binding sites of the putative CiaR regulon promoters. Underlined sequences are the direct repeats (DRI and DRII), and bold letters denote the putative −10 sequence. Shaded regions in SMU.139, SMU.239, SMU.739, and ciaX are protected by CiaR as determined in the DNA footprint assay (B to E). Lines above the sequence indicate the −35 position. (B to E) DNA footprints of the promoters of SMU.139 (B), SMU.239 (C), SMU.739 (D), and ciaX (E) under different concentrations of recombinant CiaR protein. The dashed line labeled FP indicates the protected region, the dashed line labeled CS indicates the consensus CiaR binding sequences, and the solid line labeled −10 indicates the putative −10 sequence.

FIG. 4.

Model for mode of regulation of the CiaRH TCS. Arrows indicate positive effect, and blocked lines indicate negative effect. Thick line represents high levels of transcript, and thin line represents low levels of transcript. See the text for a detailed description.