Role of HtrA in growth and competence of Streptococcus mutans UA159

Abstract

We report here that HtrA plays a role in controlling growth and competence development for genetic transformation in Streptococcus mutans. Disruption of the gene for HtrA resulted in slow growth at 37 degrees C, reduced thermal tolerance at 42 degrees C, and altered sucrose-dependent biofilm formation on polystyrene surfaces. The htrA mutant also displayed a significantly reduced ability to undergo genetic transformation. A direct association between HtrA and genetic competence was demonstrated by the increased expression of the htrA gene upon exposure to competence-stimulating peptide. The induction of htrA gradually reached a maximum at around 20 min, suggesting that HtrA may be involved in a late competence response. Complementation of the htrA mutation in a single copy on the chromosome of the mutant could rescue the defective growth phenotypes but not transformability, apparently because a second gene, spo0J, immediately downstream of htrA, also affects transformation. The htrA and spo0J genes were shown to be both individually transcribed and cotranscribed and probably have a functional connection in competence development. HtrA regulation appears to be finely tuned in S. mutans, since strains containing multiple copies of htrA exhibited abnormal growth phenotypes. Collectively, the results reveal HtrA to be an integral component of the regulatory network connecting cellular growth, stress tolerance, biofilm formation, and competence development and reveal a novel role for the spo0J gene in genetic transformation.

FIG. 1.

Schematic diagram of the htrA locus and construction of two htrA mutant strains. (A) Gene assignments and gene numbers above the schematic diagram are based on the genomic sequence information available for S. mutans UA159. Arrows indicate the directions of transcription. The numbers inside the schematic diagram and between open reading frames indicate the sizes of the open reading frames and intergenic regions in base pairs, respectively. The grey boxes indicate the consensus binding sites for DnaA (DnaA box). A putative transcriptional terminator is shown immediately downstream of the htrA gene. (B) The htrA gene was mutated either by allelic replacement of the whole gene encoding HtrA by a cassette containing the polar erythromycin resistance cassette (SAB2) or by in vitro insertion of a transposon (EZ::TN) containing the nonpolar kanamycin resistance gene (SAB2-13). See the text for more details.

FIG. 2.

Transcriptional analysis in the htrA-spo0J locus of S. mutans UA159. (A) Northern blot analysis. Total RNA (10 μg) from UA159 strain was separated in a 0.9% formaldehyde gel, transferred to a nylon membrane, and hybridized to a probe specific for the htrA gene. (B) Following reverse transcription with reverse primer spo0J-RV, PCR amplification was performed with a primer set of htrA2-FW and spo0J-RV. The PCR products were run on Tris-acetate-EDTA gel. Lane M, size marker; lane 1, RT-PCR product; lane 2, negative control of RT; lane 3, positive control of PCR from chromosomal DNA of UA159. (C) Real-time PCR. For measuring the htrA (1) and htrA-spo0J (2) mRNAs, total RNA from UA159 was used for RT with htrA2-RV and spo0J-RV, respectively. For measuring the spo0J mRNA, total RNA from SAB2 was used for RT with the spo0J-RV primer. See the text for more details. Data represent means ± standard deviations which were from two separate experiments. Arrows indicate the relative amounts of mRNA.

FIG. 3.

Growth phenotypes of S. mutans UA159 (wild type) and two htrA-disrupted mutants, SAB2 (polar) and SAB2-13 (nonpolar). (A) Growth curves grown in BHI broth at 37°C (top) and 42°C (bottom). The data shown are from a single experiment representative of three independent experiments. (B) Adhesive films formed on the bottom of the glass test tubes. After being grown in BHI at 37°C for 1 day, the culture broths were removed.

FIG. 4.

Biofilm formation of S. mutans UA159 (wild type) and SAB2 in BM medium supplemented with glucose (BM-glucose) or sucrose (BM-sucrose) at a final concentration of 20 mM. Biofilm was assayed on polystyrene microtiter plates, stained by using crystal violet (A) and quantified by adding ethanol/acetone mix (B). See the text for more details. Data are representative of no fewer than three separate experiments. The error bars represent standard deviations.

FIG. 5.

Effects of complementation with htrA on growth phenotypes. The complemented strain (SAB2C) was constructed by integrating the entire htrA gene into the genome of the mutant strain (SAB2) in a single copy. SAB2C behaved almost identically like the parent strain in terms of growth rate (A), the formation of adhesive films on the bottom of glass test tubes (B), and biofilm formation on polystyrene microtiter plates in BM medium supplemented with sucrose (C). The data shown were obtained from at least three independent experiments. See the text for more details.

FIG. 6.

Transformability of htrA mutants compared to UA159. The S. mutans strains used were UA159 (wild type), SAB2(polar htrA mutant), SAB2-13 (nonpolar htrA mutant), SAB3 (polar spo0J mutant), SAB2C (htrA-complemented strain of SAB2), SAB3C (spo0J-complemented strain), and SAB2Cspo0J (htrA-spo0J-complemented strain of SAB2). Percent transformability was determined by the ratio of the number of transformants and that of the total viable recipients, multiplied by 100. The data shown are means ± standard deviations (error bars) of at least three independent experiments. The transformation was performed in 200-μl culture with or without CSP (5 nmol). See the text for more details.