Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans.

Streptococcus mutans, the primary aetiological agent of dental caries, possesses an YjeE-like protein that is encoded by locus SMU.409, herein designated brpB. In this study, a BrpB-deficient mutant, JB409, and a double mutant deficient of BrpB and BrpA (a paralogue of the LytR-CpsA-Psr family of cell wall-associated proteins), JB819, were constructed and characterized using function assays and microscopy analysis. Both JB409 and JB819 displayed extended lag phases and drastically slowed growth rates during growth in brain heart infusion medium as compared to the wild-type, UA159. Relative to UA159, JB409 and JB819 were more than 60- and 10-fold more susceptible to acid killing at pH 2.8, and more than 1 and 2 logs more susceptible to hydrogen peroxide, respectively. Complementation of the deficient mutants with a wild-type copy of the respective gene(s) partly restored the acid and oxidative stress responses to a level similar to the wild-type. As compared to UA159, biofilm formation by JB409 and JB819 was drastically reduced (P<0.001), especially during growth in medium containing sucrose. Under a scanning electron microscope, JB409 had significantly more giant cells with an elongated, rod-like morphology, and JB819 formed marble-like super cells with apparent defects in cell division. As revealed by transmission electron microscopy analysis, BrpB deficiency in both JB409 and JB819 resulted in the development of low electron density patches and formation of a loose nucleoid structure. Taken together, these results suggest that BrpB likely functions together with BrpA in regulating cell envelope biogenesis/homeostasis in Strep. mutans. Further studies are under way to elucidate the mechanism that underlies the BrpA- and BrpB-mediated regulation.

The redox-sensing regulator Rex modulates central carbon metabolism, stress tolerance response and biofilm formation by Streptococcus mutans.

The Rex repressor has been implicated in regulation of central carbon and energy metabolism in gram-positive bacteria. We have previously shown that Streptococcus mutans, the primary causative agent of dental caries, alters its transcriptome upon Rex-deficiency and renders S. mutans to have increased susceptibility to oxidative stress, aberrations in glucan production, and poor biofilm formation. In this study, we showed that rex in S. mutans is co-transcribed as an operon with downstream guaA, encoding a putative glutamine amidotransferase. Electrophoretic mobility shift assays showed that recombinant Rex bound promoters of target genes avidly and specifically, including those down-regulated in response to Rex-deficiency, and that the ability of recombinant Rex to bind to selected promoters was modulated by NADH and NAD(+). Results suggest that Rex in S. mutans can function as an activator in response to intracellular NADH/NAD(+) level, although the exact binding site for activator Rex remains unclear. Consistent with a role in oxidative stress tolerance, hydrogen peroxide challenge assays showed that the Rex-deficient mutant, TW239, and the Rex/GuaA double mutant, JB314, were more susceptible to hydrogen peroxide killing than the wildtype, UA159. Relative to UA159, JB314 displayed major defects in biofilm formation, with a decrease of more than 50-fold in biomass after 48-hours. Collectively, these results further suggest that Rex in S. mutans regulates fermentation pathways, oxidative stress tolerance, and biofilm formation in response to intracellular NADH/NAD(+) level. Current effort is being directed to further investigation of the role of GuaA in S. mutans cellular physiology.