Different roles of EIIABMan and EIIGlc in regulation of energy metabolism, biofilm development, and competence in Streptococcus mutans

Abstract

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major carbohydrate transport system in oral streptococci. The mannose-PTS of Streptococcus mutans, which transports mannose and glucose, is involved in carbon catabolite repression (CCR) and regulates the expression of known virulence genes. In this study, we investigated the role of EII(Glc) and EIIAB(Man) in sugar metabolism, gene regulation, biofilm formation, and competence. The results demonstrate that the inactivation of ptsG, encoding a putative EII(Glc), did not lead to major changes in sugar metabolism or affect the phenotypes of interest. However, the loss of EII(Glc) was shown to have a significant impact on the proteome and to affect the expression of a known virulence factor, fructan hydrolase (fruA). JAM1, a mutant strain lacking EIIAB(Man), had an impaired capacity to form biofilms in the presence of glucose and displayed a decreased ability to be transformed with exogenous DNA. Also, the lactose- and cellobiose-PTSs were positively and negatively regulated by EIIAB(Man), respectively. Microarrays were used to investigate the profound phenotypic changes displayed by JAM1, revealing that EIIAB(Man) of S. mutans has a key regulatory role in energy metabolism, possibly by sensing the energy levels of the cells or the carbohydrate availability and, in response, regulating the activity of transcription factors and carbohydrate transporters.

FIG. 1.

Diauxic growth of UA159 and MMC1 in TV medium supplemented with 0.05% glucose and 0.5% inulin. The circles represent the wild-type strain UA159, whereas the squares represent MMC1, the ptsG knockout strain. The results shown represent the means and standard deviations (error bars) of three independent experiments.

FIG. 2.

Lactose-specific (A) and cellobiose-specific (B) PTS activities of UA159 and JAM1 cells grown in glucose. The asterisk represents a statistically significant P value of ≤0.05. The results shown represent the means and standard deviations (error bars) of three independent experiments.

FIG. 3.

CAT specific activity driven by fruA promoter in the wild-type (TW31) and ptsG-minus (JAM 25) backgrounds. Cells were grown in TV supplemented with galactose or galactose plus inulin. Values shown are means ± standard deviations (error bars) from at least three independent experiments. The results are expressed as nanomoles of Cm acetylated per minute per milligram of protein.

FIG. 4.

Biofilm formation (top) and biofilm biomass quantitation (bottom) of S. mutans UA159, MMC1 (ptsG knockout), and JAM1 (manL knockout) in buffered medium supplemented with glucose. The results shown represent the means and standard deviations (error bars) of at least three independent experiments.

FIG. 5.

Number of genes, grouped in functional categories, that are differentially expressed in JAM1 relative to UA159 grown in BHI broth to an optical density of 0.5. The clusters of orthologous group functional categories are as follows: energy, energy metabolism; PTS, phosphoenolpyruvate:sugar phosphotransferase system (also belongs to energy metabolism); hyp, hypothetical protein; ABC, ABC transporters; CI met, central and intermediate metabolism; transport and binding, transport and binding of metals and cations; FA-plipidbio, fatty acid and phospholipid biosynthesis; TC system, two-component systems; regulatory, transcriptional regulators; others, genes that do not belong to any of the categories described above.