Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans

Abstract

The abilities of Streptococcus mutans to form biofilms and to survive acidic pH are regarded as two important virulence determinants in the pathogenesis of dental caries. Environmental stimuli are thought to regulate the expression of several genes associated with virulence factors through the activity of two-component signal transduction systems. Yet, little is known of the involvement of these systems in the physiology and pathogenicity of S. mutans. In this study, we describe a two-component regulatory system and its involvement in biofilm formation and acid resistance in S. mutans. By searching the S. mutans genome database with tblastn with the HK03 and RR03 protein sequences from S. pneumoniae as queries, we identified two genes, designated hk11 and rr11, that encode a putative histidine kinase and its cognate response regulator. To gain insight into their function, a PCR-mediated allelic-exchange mutagenesis strategy was used to create the hk11 (Em(r)) and rr11 (Em(r)) deletion mutants from S. mutans wild-type NG8 named SMHK11 and SMRR11, respectively. The mutants were examined for their growth rates, genetic competence, ability to form biofilms, and resistance to low-pH challenge. The results showed that deletion of hk11 or rr11 resulted in defects in biofilm formation and resistance to acidic pH. Both mutants formed biofilms with reduced biomass (50 to 70% of the density of the parent strain). Scanning electron microscopy revealed that the biofilms formed by the mutants had sponge-like architecture with what appeared to be large gaps that resembled water channel-like structures. The mutant biofilms were composed of longer chains of cells than those of the parent biofilm. Deletion of hk11 also resulted in greatly diminished resistance to low pH, although we did not observe the same effect when rr11 was deleted. Genetic competence was not affected in either mutant. The results suggested that the gene product of hk11 in S. mutans might act as a pH sensor that could cross talk with one or more response regulators. We conclude that the two-component signal transduction system encoded by hk11 and rr11 represents a new regulatory system involved in biofilm formation and acid resistance in S. mutans.

FIG. 1.

The arrangement of the hk/rr11 genetic locus. Neighboring genes were assigned putative functions based on high blast homology scores with genes for the indicated proteins: 1, primosomal replication factor V (AAK34400); 2, methionyl tRNA transferase Fmt (AAK34399); 3, RNA binding protein SunL (rRNA methyltransferase RsmB) (AAK34398); 4, phosphoprotein Ser/Thr phosphatase PppL (AAK34397); 5, Ser/Thr protein kinase PknB (AAK34396); 6, conserved hypothetical protein (AAK34395); 7, peptidyl-prolyl cis-trans isomerase PpiB (AAK75626); 8, polyribonucleotide nucleotidyltransferase (general stress protein GSP13) (AAK34392); 9, pyruvate formate lyase-activating enzyme PflC (AAK74424); 10, transcriptional regulator RdrA (AAK34719); 11, pyruvate formate lyase 2 PflD (AAK34714); 12, transaldolase-like protein MipB (AAK34713); and 13, glycerol dehydrogenase GldA (AAK74432).

FIG. 2.

Growth curves of the parent strain S. mutans NG8 and hk/rr11 mutants SMHK11 and SMRR11 grown in TYEG medium at pH 7.0 and 5.5.

FIG. 3.

Biofilm formation and quantification of S. mutans strains. The graph represents the turbidity of the biofilms as reflected by their absorbance after safranin staining. The mean values ± SDs are presented.

FIG. 4.

Scanning electron micrographs show spatial distribution and architecture of biofilms formed by S. mutans strains.

FIG. 5.

Effect of pH on the growth of S. mutans strains. The photographs were taken after 40 h of incubation at 37°C with 5% CO₂.

FIG. 6.

Inducible ATR was assayed in log-phase cells of the mutants. The mean percentages of survivors ± SDs from three independent experiments are presented.

FIG. 7.

Autoradiograms of 2D gels obtained with parent strain NG8 (A and B) and mutant HK11 (C and D) of S. mutans exposed to pH 7.5 (A and C) and 5.5 (B and D) for 30 min. Solid and open arrowheads denote proteins that had increased or decreased expression, respectively, in both NG8 and SMHK11 at the acid shock. Note the presence of four protein spots indicated with arrows in panel D that were induced in NG8 but not in SMHK11. Numbers at the top of each panel are pHs; numbers at the left are molecular masses in kilodaltons.