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.

Distribution of oral streptococci highly resistant to amoxicillin in dental plaque specimens from Japanese children and adolescents.

Oral streptococci are major pathogens of infective endocarditis. Prophylactic antibiotics are commonly given to subjects with certain kinds of heart disorders when invasive dental treatments are performed, with amoxicillin (AMPC) being widely used for this purpose. However, there is little information regarding AMPC-resistant oral streptococci. Here, a total of 344 dental plaque specimens collected from 253 healthy Japanese children, adolescents and young adults (aged 2-22 years) were diluted and streaked onto culture medium containing high-dose AMPC. The MICs for the isolated strains were evaluated using a macrodilution broth method described by the Clinical and Laboratory Standards Institute. Bacterial DNA was extracted from each strain and the entire sequences of the 16S rRNA gene were compared with those in GenBank to identify the species. The results showed that strains with AMPC MICs >16 µg ml(-1) were isolated from 18 specimens from 14 patients. Analyses of the 16S rRNA gene sequences of these strains identified them as major oral streptococcal species, including Streptococcus oralis and Streptococcus mitis. These findings indicate that oral streptococci with elevated MICs for AMPC exist in certain small populations of healthy children, and highlight the need for further studies to determine risk factors that lead to the appearance of such strains.