Expression of BrpA in Streptococcus mutans is Regulated by FNR-box Mediated Repression

Abstract

Our previous studies showed that brpA in S. mutans, which encodes a member of the LytR-CpsA-Psr family of proteins, can be cotranscribed with brpB upstream as a bicistronic operon, while the intergenic region also has strong promoter activity. To elucidate how brpA expression is regulated, the promoter regions were analyzed using PCR-based deletions and site-directed mutagenesis and a promoterless luciferase gene as a reporter. Allelic exchange mutagenesis was also used to examine genes encoding putative trans-acting factors, and the impact of such mutations on brpA expression was analyzed by reporter assays. Multiple elements in the short brpA promoter (nucleotide -1 to -344 relative to start cordon ATG) were shown to have a major impact on brpA expression, including an FNR-box, for putative binding site of an FNR-type of transcriptional regulator. When compared to the intact brpA promoter, mutations of the highly conserved nucleotides in FNR-box from TTGATgtttAcCtt to TTACAgaaaGtTac resulted in 1362-fold increases of luciferase activity (P<0.001), indicative of the FNR-box mediated repression as a major mechanism in regulation of brpA expression. When luciferase reporter was fused to the upstream brpBA promoter (nt -784 to -1144), luciferase activity was decreased by 4.5-fold (P<0.001) in the brpA mutant, TW14D, and by 67.7-fold (P<0.001) in the brpB mutant, JB409, as compared to the wild-type, UA159. However, no such effects were observed when the reporter gene was fused to the short brpA promoter and its derivatives. These results also suggest that brpA expression in S. mutans is auto-regulated through the upstream brpBA promoter.

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