Identification and functional analysis of an ammonium transporter in Streptococcus mutans.
Identification and functional analysis of an ammonium transporter in Streptococcus mutans.
复制标题
变形链球菌中铵转运蛋白的鉴定和功能分析。
DOI：
10.1371/journal.pone.0107569
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Matsumoto-Nakano M
中科院分区：
文献类型：
--
作者：
Ardin AC;Fujita K;Nagayama K;Takashima Y;Nomura R;Nakano K;Ooshima T;Matsumoto-Nakano M
变形链球菌是一种革兰氏阳性细菌，被认为是人类龋齿的主要病原体，据报道可在牙齿表面形成称为牙菌斑的生物膜。众所周知，这种生物体在细胞膜上拥有大量用于分子输出和输入的转运蛋白。氮是革兰氏阳性菌的必需营养素，但也可以使用铵等替代来源。为了获得用于大分子合成的氮，必须将含氮化合物运输到细胞内。然而，变形链球菌中的铵转运蛋白仍有待表征。本研究重点分析了变形链球菌的铵转运蛋白基因及其操纵子，同时对相关调控基因进行了分析。与变形链球菌中的 nrgA 相对应的 SMU.1658 基因与枯草芽孢杆菌中的铵转运蛋白基因同源，而位于 nrgA 基因上游并预测为 glnB 的 SMU.1657 是 PII 蛋白家族的成员。使用 nrgA 缺陷突变株 (NRGD)，我们检查了铵、氯化钙和硫酸锰存在下的细菌生长。还进行了荧光流出测定，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，但其荧光强度却高得多。此外，共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生物膜结构有很大不同。此外，转录分析表明nrgA基因与glnB基因共转录。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。
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.
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