Modulation of covR expression in Streptococcus mutans UA159.
The biofilm-forming Streptococcus mutans is a gram-positive bacterium that resides in the human oral cavity and is considered to be the primary etiological agent in the formation of dental caries. The global response regulator CovR, which lacks a cognate sensor kinase, is essential for the pathogenesis and biofilm formation of this bacterium, but it is not clear how covR expression is regulated in S. mutans. In this communication, we present the results of our studies examining various factors that regulate the expression of covR in S. mutans UA159. The results of Southern hybridization and PCR analysis indicated that CovR is an orphan response regulator in various isolates of S. mutans. The transcriptional start site for covR was found to be 221 base pairs upstream of the ATG start codon, and site-directed mutagenesis of the upstream TATAAT box confirmed our findings. The expression of covR is growth phase dependent, with maximal expression observed during exponential-growth phase. While changes to the growth temperature did not significantly affect the expression of covR, increasing the pH or the concentration of Mg(2+) in the growth medium leads to an increase in covR expression. The results of semiquantitative reverse transcriptase PCR analysis and in vivo transcriptional-fusion reporter assays indicated that CovR autoregulates its own expression; this was verified by the results of electrophoretic mobility shift assays and DNase I protection assays, which demonstrated direct binding of CovR to the promoter region. Apparently, regulation by Mg(2+) and the autoregulation of covR are not linked. A detailed analysis of the regulation of CovR may lead to a better understanding of the pathogenesis of S. mutans, as well as providing further insight into the prevention of dental caries.
Chong
Patrick
P
Department of Microbiology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Drake
Laura
L
Biswas
Indranil
I
D001426:Bacterial Proteins / Q000235:genetics*; D015139:Blotting, Southern; D018387:Codon, Initiator / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013295:Streptococcus mutans / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic / Q000187:drug effects
D001426:Bacterial Proteins; D018387:Codon, Initiator; D008274:Magnesium