Autoregulation of theStreptococcus mutansSloR Metalloregulator Is Constitutive and Driven by an Independent Promoter

ABSTRACTStreptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries,S. mutansharbors a 25-kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives in part from transcriptional readthrough of thesloABCoperon, which encodes a Mn2+/Fe2+ABC transport system. Here we describe the details of thesloABCpromoter that drives this transcription as well as those for a novel independent promoter in an intergenic region (IGR) that contributes to downstreamsloRexpression. Reverse transcriptase PCR (RT-PCR) studies support the occurrence ofsloRtranscription that is independent ofsloABCexpression, and the results of 5′ rapid amplification of cDNA ends (5′ RACE) revealed asloRtranscription start site in the IGR, from which the −10 and −35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with a lower affinity than that for SloR binding to thesloABCRpromoter. The function of thesloRpromoter was validated by semiquantitative real-time PCR (qRT-PCR) experiments. Interestingly,sloRexpression was not significantly affected when bacteria were grown in the presence of a high manganese concentration, whereas expression of thesloABCoperon was repressed under these conditions. The results ofin vitrotranscription studies support the occurrence of SloR-mediated transcriptional activation ofsloRand repression ofsloABC. Taken together, these findings implicate SloR as a bifunctional regulator that repressessloABCpromoter activity and encouragessloRtranscription from an independent promoter.IMPORTANCETooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide.S. mutans-induced carious lesions cause functional, physical, and/or esthetic impairment in the vast majority of adults and in 60 to 90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviatingS. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health, which is inextricably linked to oral health. Research focused on theS. mutansSloR metalloregulatory protein can guide the development of novel therapeutics and thus alleviate the burden of dental cavities.

Autoregulation of theS. mutansSloR metalloregulator is constitutive and driven by an independent promoter

Streptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries,S. mutansharbors a 25kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives, in part, from transcriptional readthrough of thesloABCoperon which encodes a Mn2+/Fe2+ABC transport system. Herein, we describe the details of thesloABCpromoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstreamsloRexpression. RT-PCR studies supportsloRtranscription that is independent ofsloABCexpression, and the results of 5′ RACE revealed asloRtranscription start site in the IGR from which the −10 and −35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with lower affinity than SloR binding to thesloABCRpromoter. Function of thesloRpromoter was validated in qRT-PCR experiments. Interestingly,sloRexpression was not significantly impacted when grown in the presence of high manganese, whereas expression of thesloABCoperon was repressed under these conditions. The results ofin vitrotranscription studies support SloR-mediated transcriptional-activation ofsloRand -repression ofsloABC.Taken together, these findings implicate SloR as a bifunctional regulator that repressessloABCpromoter activity and encouragessloRtranscription from an independent promoter.ImportanceTooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide.S. mutans-induced carious lesions cause functional, physical, and/or aesthetic impairment in the vast majority of adults, and in 60-90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviatingS. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health which is inextricably linked to oral health. Research focused on theS. mutansSloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.