F187 and Y167 aid cooperative SloR-DNA binding in Streptococcus mutans

Objectives: Streptococcus mutans is among the most acidogenic constituents of the caries microbiome and the primary causative agent of dental caries in dysbiotic plaque. S. mutans harbors a 25-kDa protein, called SloR, that plays a central role in metal ion homeostasis by regulating the expression of genes whose products encode essential metal ion transport and virulence. Work currently ongoing in our laboratories implicates a “secondary” manganese binding pocket in SloR that may be essential for SloR binding to DNA and repression of downstream sloABC transcription. Informed by the SloR crystal structure, we propose that a phenylalanine at amino acid position 187 (F) and a tyrosine at position 167 (Y) interact with one another across dimers to instigate cooperative SloR-DNA binding and appropriate transcription of the sloABC operon. The results of previous fluorescence anisotropy studies support cooperative binding of SloR homodimers to the sloABC promoter region with a Hill coefficient of 1.8. In the present study, we set out to determine whether F187 and T167 are important mediators of the SloR-DNA interaction and downstream gene transcription.
Methods: We used an overlap-extension PCR approach to introduce alanine residues (A) at positions 187 and 167 that localize to the C-terminal FeoA domain of the SloR protein and contribute to one of three manganese binding pockets. Expression profiling experiments (qRT-PCR) were then conducted with S. mutans transformants expressing the SloR mutant variants in trans to reveal their potential impact on sloABC gene transcription.
Results: The results of qRT-PCR indicate that non-conservative amino acid substitutions at positions 187 (F187A) and 167 (Y167A) compromise the ability of SloR to bind DNA and repress sloABC transcription. Conclusion: These findings support a central role for F187 and Y167 in SloR-DNA binding and regulation of the sloABC metal ion transport operon, and represent putative targets for the development of an anti-caries therapeutic.
Conclusions: These findings support a central role for F187 and Y167 in SloR-DNA binding and regulation of the sloABC metal ion transport operon, and represent putative targets for the development of an anti-caries therapeutic.