Method: Reporter gene fusions to the nlmD promoter and Real-Time PCR were used to monitor nlmD expression in S. mutans UA159 and its derivatives growing in different carbohydrate sources, with or without aeration. Electrophoretic mobility shift assays (EMSA) were performed to assess whether particular transcriptional regulators could bind directly to the promoter region of nlmD.
Result: In ΔccpA, ΔvicK, and ΔcomDE strains of S. mutans, the level of expression of nlmD was decreased compared to the parental strain in aerated cultures. In contrast, all three mutants displayed higher nlmD expression than in the wild-type background in anaerobic conditions. Expression of nlmD was increased >10-fold over the wild-type stain in a ΔccpA mutant growing anaerobically in glucose, but was lower than the wild-type strain in glucose with aeration. Purified CcpA protein was shown to bind directly to the nlmD promoter region. Growth in galactose or sorbitol, which poorly elicit catabolite repression, lead to dramatic decreases in expression of nlmD under aerated or anaerobic conditions.
Conclusion: CcpA was a dominant regulator of nlmD expression in response to carbohydrate and aeration, but two-component systems also contribute to the response to aeration. Expression of nlmD was remarkably sensitive to carbohydrate source in a way that could not be attributed exclusively to CcpA binding or catabolite repression. Collectively, these results reveal new complexities in the molecular pathways integrating oxygen- and carbohydrate-dependent gene expression in S. mutans.
Keywords: Gene expression, Genetics, Microbiology, Molecular biology and Oral biology
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