Identification and functional analysis of an ammonium transporter in Streptococcus mutans.

Streptococcus mutans, a Gram-positive bacterium, is considered to be a major etiologic agent of human dental caries and reported to form biofilms known as dental plaque on tooth surfaces. This organism is also known to possess a large number of transport proteins in the cell membrane for export and import of molecules. Nitrogen is an essential nutrient for Gram-positive bacteria, though alternative sources such as ammonium can also be utilized. In order to obtain nitrogen for macromolecular synthesis, nitrogen-containing compounds must be transported into the cell. However, the ammonium transporter in S. mutans remains to be characterized. The present study focused on characterizing the ammonium transporter gene of S. mutans and its operon, while related regulatory genes were also analyzed. The SMU.1658 gene corresponding to nrgA in S. mutans is homologous to the ammonium transporter gene in Bacillus subtilis and SMU.1657, located upstream of the nrgA gene and predicted to be glnB, is a member of the PII protein family. Using a nrgA-deficient mutant strain (NRGD), we examined bacterial growth in the presence of ammonium, calcium chloride, and manganese sulfate. Fluorescent efflux assays were also performed to reveal export molecules associated with the ammonium transporter. The growth rate of NRGD was lower, while its fluorescent intensity was much higher as compared to the parental strain. In addition, confocal laser scanning microscopy revealed that the structure of biofilms formed by NRGD was drastically different than that of the parental strain. Furthermore, transcriptional analysis showed that the nrgA gene was co-transcribed with the glnB gene. These results suggest that the nrgA gene in S. mutans is essential for export of molecules and biofilm formation.

Role of ABC transporter proteins in stress responses of Streptococcus mutans.

Streptococcus mutans possesses a number of ATP-binding cassette (ABC) transporters, which function as multiple sugar metabolism transporters and are promising targets for antimicrobial strategies. In the present study, we performed functional analyses of SMu0836 and SMu0837 products, which are possible ABC transporters. Isogenic mutant strains Δ0836 and Δ0837 were generated by insertional inactivation of SMu0836, and SMu0837, respectively, of strain MT8148 and found to be more sensitive to antibiotics as compared to MT8148. In addition, assays of membrane transport functions using a fluorescent probe showed that export pumps did not function properly in strain Δ0836. Expression of those genes was elevated when strain MT8148 was cultured with a sublethal concentration of tetracycline, as well as when exposed to heat shock, osmotic stress, and oxidative stress conditions. Furthermore, the expressions of other genes including SMu0374, SMu0215, SMu0475, SMu0986, and SMu1051, which encode possible ABC transporters, were also elevated when strain MT8148 was cultured with a sublethal concentration of tetracycline. Together, these results suggest that the products of SMu0836 and SMu0837 are ABC transporters, which may function in stress response.

Regulation of recombination between gtfB/gtfC genes in Streptococcus mutans by recombinase A.

Streptococcus mutans produces 3 types of glucosyltransferases (GTFs), whose cooperative action is essential for cellular adhesion. The recombinase A (RecA) protein is required for homologous recombination. In our previous study, we isolated several strains with a smooth colony morphology and low GTF activity, characteristics speculated to be derived from the GTF fusions. The purpose of the present study was to investigate the mechanism of those fusions. S. mutans strain MT8148 was grown in the presence of recombinant RecA (rRecA) protein, after which smooth colonies were isolated. The biological functions and sequences of the gtfB and gtfC genes of this as well as other clinical strains were determined. The sucrose-dependent adherence rates of those strains were reduced as compared to that of MT8148. Determination of the sequences of the gtfB and gtfC genes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of the recA gene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions of gtfB and gtfC genes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.