Autoregulation of the Streptococcus mutans SloR Metalloregulator Is Constitutive and Driven by an Independent Promoter

Abstract

, 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, harbors 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 the operon which encodes a Mn/Fe ABC transport system. Herein, we describe the details of the promoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstream expression. RT-PCR studies support transcription that is independent of expression, and the results of 5' RACE revealed a transcription 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 the promoter. Function of the promoter was validated in qRT-PCR experiments. Interestingly, expression was not significantly impacted when grown in the presence of high manganese, whereas expression of the operon was repressed under these conditions. The results of transcription studies support SloR-mediated transcriptional-activation of and -repression of Taken together, these findings implicate SloR as a bifunctional regulator that represses promoter activity and encourages transcription from an independent promoter. Tooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide. -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 alleviating-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 the SloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.

The S. mutans mntE gene encodes a manganese efflux transporter

“…In S. mutans, the sloABCR operon plays a crucial role in metal ion uptake across the bacterial cell membrane. The operon encodes a SloABC transport system for Mn 2+ /Fe 2+ that is regulated by a 25 kDa SloR metalloregulatory protein expressed immediately downstream (Merchant & Spatafora, 2014; Monette et al, 2018). Specifically, the sloABC genes encode an ATP‐binding cassette (ABC) transport system that enables the acquisition of Mn 2+ and Fe 2+ via a membrane spanning SloC lipoprotein (Haswell et al, 2013).…”

“…In response, S . mutans downregulates the expression of its sloABC gene products so as to prevent the over‐accumulation of metal ions which could have cytotoxic effects, owing to the generation of reactive oxygen species (ROS) (Crepps et al, 2016; Monette et al, 2018). During periods of fasting, however, the bacterium upregulates the expression of the transmembrane SloC lipoprotein along with its accompanying ATP‐binding ( sloA ) and ‐hydrolyzing ( sloB ) proteins, all of which are pivotal for metal ion uptake.…”

Manganese uptake, mediated by SloABC and MntH, is essential for the fitness ofStreptococcus mutans

“…Here, our results confirm that SloR contributes to the regulation of mntH , though the results of both RNAseq and qRT-PCR analyses indicate that SloR repression of the sloABC operon is tighter than it is for the mntH gene. Such robust sloABC repression by SloR can be explained by our previous characterization of cooperative, homodimeric binding between SloR and each of three hexameric repeats that overlap the sloABC promoter [46]. Whether SloR binding at the mntH locus is cooperative, and whether the SloR binding sites overlap with the mntH promoter remains to be determined.…”

“…Final comparisons between the control and Mn-depleted conditions were performed with Degust (http://degust.erc.monash.edu/ ) , with a False Discovery Rate (FDR) of 0.05, and a 2-fold change cutoff. Quantifications of mntH and sloA mRNA were obtained by quantitative real time PCR (qRT-PCR) using gene-specific primers (Table 4) on triplicate samples of the S. mutans UA159 and GMS584 (Δ sloR ) strains grown to mid-logarithmic phase (OD 600 of 0.5) according to established protocols [46]. Student’s t -test was applied to the analysis of the qRT-PCR results.…”

“…EMSAs were performed according to established protocols [46]. Briefly, primers were designed to amplify the promoter regions of the S. mutans sloABC and mntH genes (Table 4).…”