SppR in Streptococcus mutans coordinates stress tolerance response and BrpA expression

SppR is a transcriptional repressor of fructanase FruA and fructose-1-phosphatase SppA. In this study, a frameshift sppR mutant was constructed and analyzed using in vitro biofilm models and stress challenge assays for its role in S. mutans' stress tolerance response and biofilm formation. In addition, luciferase reporter fusion assay and in vitro transcription assay were used to analyze the role of SppR in regulation of brpA. Results showed that sppR deficiency caused no major effect on planktonic growth and cell morphology in regular BHI broth, however negatively impacted growth at low pH and in the presence of oxidative stressor (P < 0.001). Relative to the parent strain, UA159, the sppR mutant also had a reduced survival rate following acid killing (P<0.01) and hydrogen peroxide challenge (P<0.001). Luciferase reporter assays showed that sppR deficiency reduced brpA promoter-reporter activity by >2-fold (P<0.05), while the disruption of a putative SppR-binding site in the promoter reduced its activity by >7-fold (P<0.001). A recombinant SppR (rSppR) retarded the migration of a brpA promoter-containing probe during electrophoresis and enhanced the transcription of brpA in vitro. RNA-seq analysis also showed that deficiency of SppR led to altered expression of >136 genes by >2-fold (P<0.05), including 68 genes up-regulated and 68 genes reduced. Among the down-regulated genes were those belonging to DNA-repair and acid tolerance response. These results suggest that SppR plays an important role in S. mutans physiology including acid and oxidative stress tolerance response and regulation of BrpA expression.