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.
Zezhang T. Wen, Bo Yang, L. Guillot et al.· Frontiers in Cellular and In...· 0 citations
ABSTRACT This study used a functional genomics approach to explore the role of a xenobiotic response element (XRE)-type regulator (SMU.405c) in Streptococcus mutans physiology, including the expression of biofilm regulatory protein BrpA. Results showed that deletional mutation of xre significantly reduced the ability of the deficient mutant to grow in the presence of methyl viologen, a commonly used oxidative stressor (P < 0.001). When challenged in a hydrogen peroxide killing assay, the survival rate of the ∆xre mutant was >2-log less than the parent strain after 60 min (P < 0.001). Luciferase reporter fusion assays showed that xre deficiency had no significant effect on luciferase expression when it was under the control of the intact brpA promoter, but the reporter activity increased by >6-fold (P < 0.001) when the reporter gene was fused to a brpA promoter derivative with deletion of a putative XRE-binding box. Electrophoretic mobility shift assay (EMSA) showed that recombinant XRE interacted with the brpA promoter, resulting in an electrophoretic shift of the promoter probes. In vitro transcription assay also showed that inclusion of XRE caused transcription to fall off, significantly reducing full-length brpA transcripts. RNA-seq analysis revealed that deficiency of XRE led to altered expression of >102 genes by >2-fold (P < 0.05), including 28 with increased expression, and 74 with decreased expression. Among the down-regulated were genes for DNA repair and oxidative stress tolerance response. These results suggest that XRE (SMU.405c) in S. mutans plays an important role in brpA expression and oxidative stress tolerance response. IMPORTANCE Streptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response. Streptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response.
Zezhang T. Wen, K. Ellepola, L. Guillot et al.· Microbiology spectrum· 1 citation
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