Investigating Cooperative SloR Binding in Streptococcus mutans

Objectives: Streptococcus mutans is a primary acidogen in human dental plaque and the principle causative agent of tooth decay. A 25kDa SloR metalloregulatory protein regulates over 200 S. mutans genes, many of which contribute to caries formation. The objective of this study is to elucidate the SloR-DNA interface by defining the stoichiometry with which SloR dimers bind to a 72bp SloR Recognition Element (SRE) that precedes the S. mutans sloABC metal ion transport operon. We propose cooperative homodimeric SloR binding to each of three inverted hexameric repeat sequences on this SRE.
Methods: We performed negative staining and single particle electron microscopy (EM) to visualize SloR-DNA binding, and fluorescence anisotropy to define SloR binding affinities (K_d) and Hill coefficients, the latter as a measure of cooperativity. The results of bioinformatic analyses combined with the SloR crystal structure were used to inform site-directed mutagenesis experiments aimed at mapping residues that are important for SloR dimer interactions. Semi-quantitative real-time PCR (qRT-PCR) experiments were also performed to assess the impact of these mutations on SRE binding and sloABC transcription.
Results: Negative staining and EM revealed three SloR binding sites on the SRE. Fluorescence anisotropy revealed a Hill coefficient equal to 1.8, and K_ds that support strongest SloR binding to the central repeat unit (K_d=1.3nM) versus weaker binding to the flanking imperfect repeats (K_d=4.1nM and 6nM). Interestingly, a F187A mutation at the SloR dimer interface abrogated SloR aggregation in vitro, implicating it in cooperative binding. This is consistent with sloA transcription that was de-repressed 3-fold when this mutation was introduced into the S. mutans UA159 chromosome.
Conclusions: The S. mutans SloR metalloregulator binds to three inverted hexameric repeats on the SRE that precedes the sloABC operon, and it does so via cooperative binding that is dependent, in part, on a phenylalanine residue at position #187.