| Autor: |
Haswell JR; Middlebury College, Department of Biology, Middlebury, Vermont, USA., Pruitt BW, Cornacchione LP, Coe CL, Smith EG, Spatafora GA |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of bacteriology [J Bacteriol] 2013 Jan; Vol. 195 (1), pp. 126-34. Date of Electronic Publication: 2012 Oct 26. |
| DOI: |
10.1128/JB.01648-12 |
| Abstrakt: |
Streptococcus mutans is a commensal member of the healthy plaque biofilm and the primary causative agent of dental caries. The present study is an investigation of SloR, a 25-kDa metalloregulatory protein that modulates genes responsible for S. mutans-induced cariogenesis. Previous studies of SloR homologues in other bacterial pathogens have identified three domains critical to repressor functionality: an N-terminal DNA-binding domain, a central dimerization domain, and a C-terminal FeoA (previously SH3-like) domain. We used site-directed mutagenesis to identify critical amino acid residues within each of these domains of the SloR protein. Select residues were targeted for mutagenesis, and nonconservative amino acid substitutions were introduced by overlap extension PCR. Furthermore, three C-terminally truncated SloR variants were generated using conventional PCR. The repressor functionality and DNA-binding ability of each variant was assessed using CAT reporter gene assays, real-time semiquantitative reverse transcriptase (qRT)-PCR, and electrophoretic mobility shift assays. We identified 12 residues within SloR that cause significant derepression of sloABC promoter activity (P < 0.05) compared to the results for wild-type SloR. Derepression was particularly noteworthy in metal ion-binding site 1 mutants, consistent with the site's importance in gene repression by SloR. In addition, a hyperactive SloR(E169A/Q170A) mutant was identified as having significantly heightened repression of sloABC promoter activity, and experiments with C-terminal deletion mutants support involvement of the FeoA domain in SloR-mediated gene repression. Given these results, we describe the functional domains of the S. mutans SloR protein and propose that the hyperactive mutant could serve as a target for rational drug design aimed at repressing SloR-mediated virulence gene expression. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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