Ardin Arifah Chieko, Fujita Kazuyo, Nagayama Kayoko, Takashima Yukiko, Nomura Ryota, Nakano Kazuhiko, Ooshima Takashi, Matsumoto-Nakano Michiyo
Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
PLoS One. 2014 Sep 17;9(9):e107569. doi: 10.1371/journal.pone.0107569. eCollection 2014.
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.
变形链球菌是一种革兰氏阳性菌，被认为是人类龋齿的主要病原体，据报道它能在牙齿表面形成称为牙菌斑的生物膜。已知这种生物体在细胞膜中拥有大量用于分子进出的转运蛋白。氮是革兰氏阳性菌必需的营养物质，不过也可以利用铵等替代来源。为了获取用于大分子合成的氮，含氮化合物必须被转运到细胞内。然而，变形链球菌中的铵转运蛋白仍有待鉴定。本研究着重于鉴定变形链球菌的铵转运蛋白基因及其操纵子，同时也分析了相关的调控基因。变形链球菌中与枯草芽孢杆菌的nrgA相对应的SMU.1658基因与铵转运蛋白基因同源，位于nrgA基因上游且预测为glnB的SMU.1657是PII蛋白家族的一员。我们使用nrgA缺陷突变株（NRGD），检测了其在铵、氯化钙和硫酸锰存在下的细菌生长情况。还进行了荧光外流试验以揭示与铵转运蛋白相关的输出分子。与亲本菌株相比，NRGD的生长速率较低，而其荧光强度则高得多。此外，共聚焦激光扫描显微镜显示，NRGD形成的生物膜结构与亲本菌株的截然不同。此外，转录分析表明nrgA基因与glnB基因共同转录。这些结果表明，变形链球菌中的nrgA基因对于分子输出和生物膜形成至关重要。
