SppR in Streptococcus mutans Plays an Important Role in Stress Tolerance Response and BrpA Expression

ABSTRACT: SppR (formerly GlcR) in S. mutans is a transcriptional repressor of fructanase FruA and fructose-6-phopshatase SppA. In this study, a markerless frameshift sppR mutant was constructed and analyzed by acid killing and hydrogen peroxide challenge assays for its role in S. mutans’ stress tolerance response. In addition, luciferase reporter fusion assay, along with electrophoretic mobility shift assay and in vitro transcription assay were used to analyze the role of SppR in expression of brpA, which encodes a multi-functional surface associated protein. Results showed that sppR deficiency has no major effect on its growth and cell morphology when growing regular BHI broth, but significantly reduced the ability of the deficient mutant to grow in low pH and especially in the presence of methyl viologen, a commonly used oxidative stressor at 25 µM (P<0.001). Relative to the parent strain, UA159, the sppR mutant also had a reduced survival rate, following acid killing at pH 2.8, as compared to the wild-type (P<0.01), and such defects appeared to be related adaptive acid tolerance response. Hydrogen peroxide challenge assay at 58 mM also showed the sppR deficiency led to reduction of survival rate of >3-logs after 60 minutes (P<0.001). However, no significant differences in biofilm formation were measured between the sppR mutant and its parent strain. Luciferase reporter fusion assays showed that sppR deficiency led to reduction of reporter activity by >2-fold (P<0.05) when the reporter was under the intact brpA promoter and by >7-fold (P<0.001) when a putative SppR-binding site was deleted or mutated. EMSA assay showed that inclusion of recombinant SppR (rSppR) resulted in electrophoretic shift of the brpA promoter probes. In vitro transcription assay also showed that rSppR enhanced the transcription of brpA. Transcriptomics (RNA-seq) analysis of an allelic exchange mutant revealed that deficiency of SppR led to altered expression of >136 genes by >2-fold (P<0.05), including 68 genes up-expressed and 68 genes reduced. Among the up-regulated genes were those acid and oxidative stress tolerance responses, which include those for malolactic enzyme, DNA process protein DprA, class I SAM-dependent methyltransferase SMT, and mutanobactin biosynthesis and transport. These results suggest that SppR in S. mutans plays an important role in S. mutans physiology including acid and oxidative stress tolerance response and brpA expression.

ORGANISM(S): Streptococcus mutans

PROVIDER: GSE328150 | GEO | 2026/08/19

REPOSITORIES: GEO