Functional Analysis of Streptococcus mutans Glutamine Transporters in Biofilm Formation

Abstract

Gram-positive Streptococcus mutans, a major pathogenic bacterium for human caries known to play an important role in biofilm formation, is exposed to drastic environment fluctuations, such as temperature and pH changes, nutrient availability, and invasion by foreign substances. This organism possesses a large number of transport proteins in the cell membrane for export and import of molecules, and its membrane transport function allows it to survive stress, as well as create conditions conductive for its growth and development. In addition, transport of nitrogen sources plays a major role in biofilm formation by S. mutans. Nitrogen is an essential nutrient for growth of Gram-positive bacteria and taken up into bacterial cells in various forms, with the nonessential amino acid glutamine, synthesized from ammonium, known to be a nitrogen source for biosynthesis of a variety of compounds. The present study focused on characterizing the glutamine membrane transporter (GlnP) with molecular biological methods.

Using S. mutans MT8148 as the parental strain, GlnP-deficient (GEMR) and complementary (compGEMR) strains were generated by molecular methods. In the logarithmic growth phase, the growth rate of MT8148 and comp-GEMR strains was decreased whereas that of the GEMR strain was not affected by glutamine. Furthermore, to confirm the relevance of membrane transport of glutamine in S. mutans, that was analyzed using fluorescent probes, which showed that fluorescence polarization of the GEMR strain was significantly decreased in a concentration-dependent manner. In addition, the structure of biofilm formed by the GEMR strains was coarser as compared to those associated with the MT8148 and comp-GEMR strains.

The present results indicate an important role for GlnP in regard to bacterial growth, membrane transport of glutamine, biofilm formation, and maintenance of biofilm structure.