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

ABSTRACT: 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.

SUBMITTER: Ardin AC

PROVIDER: S-EPMC4167856 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Project description:BackgroundStreptococcus mutans is the primary etiological agent of human dental caries. It can metabolize a wide variety of carbohydrates and produce large amounts of organic acids that cause enamel demineralization. Phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays an important role in carbohydrates uptake of S. mutans. The ptxA and ptxB genes in S. mutans encode putative enzyme IIA and enzyme IIB of the L-ascorbate-specific PTS. The aim of this study was to analyze the function of these proteins and understand the transcriptional regulatory mechanism.ResultsptxA (-), ptxB (-), as well as ptxA (-) , ptxB (-) double-deletion mutants all had more extended lag phase and lower growth yield than wild-type strain UA159 when grown in the medium using L-ascorbate as the sole carbon source. Acid production and acid killing assays showed that the absence of the ptxA and ptxB genes resulted in a reduction in the capacity for acidogenesis, and all three mutant strains did not survive an acid shock. According to biofilm and extracellular polysaccharides (EPS) formation analysis, all the mutant strains formed much less prolific biofilms with small amounts of EPS than wild-type UA159 when using L-ascorbate as the sole carbon source. Moreover, PCR analysis and quantitative real-time PCR revealed that sgaT, ptxA, ptxB, SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon. The transcription levels of these genes were all elevated in the presence of L-ascorbate, and the expression of ptxA gene decreased significantly once ptxB gene was knockout.ConclusionsThe ptxA and ptxB genes are involved in the growth, aciduricity, acidogenesis, and formation of biofilms and EPS of S. mutans when L-ascorbate is the sole carbon source. In addition, the expression of ptxA is regulated by ptxB. ptxA, ptxB, and the upstream gene sgaT, the downstream genes SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon, and L-ascorbate is a potential inducer of the operon.

Project description:Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, noncoding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA SmsR4. Our data demonstrate that SmsR4 regulates the enzyme IIA (EIIA) component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria. IMPORTANCE Small RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.

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

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