Investigating Post-Translational Regulation of the SloR Metalloregulator in Streptococcus mutans

Objectives: Streptococcus mutans is a member of the human oral microbiota and the primary causative agent of dental caries. Work in the Spatafora laboratory focuses on a 25kDa metalloregulatory protein, called SloR, and whether it is subject to post-translational control by the ATP-dependent Clp protease complex. Previous research in our laboratory revealed increased transcription of the sod, tpx and dpr oxidative stress genes in a S. mutans SloR-deficient mutant, and direct SloR binding to the promotor of the S. mutans spxA gene which encodes a transcriptional enhancer of these and other oxidative stress genes. Taken together, we propose that SloR, as a modulator of the oxidative stress response, may be subject to proteolytic degradation by the Clp protease complex when S. mutans is challenged with oxidative stress.
Methods: Semi-quantitative real-time PCR (qRT-PCR) studies were performed to elucidate sloR transcription in the UA159 wild-type strain under conditions of H₂O₂-induced stress. Immunoblotting was used to reveal the stability of the SloR protein, to investigate the putative relationship between the SloR and Clp proteins, and to monitor SloR and ClpE abundance in the presence of a 0.5mM H₂O₂ stressor for up to 60 minutes.
Results: The results of qRT-PCR indicate that intracellular SloR concentrations are likely subject to post-transcriptional mechanisms of control when S. mutans is grown under oxidative stress conditions. Western blots with UA159 whole cell lysates revealed a decrease in SloR after 15 minutes of exposure to H₂O₂, and an increase in ClpE after 30 minutes of exposure to the oxidative stressor; blots with the S. mutans ClpX, ClpE, and ClpP insertion-deletion mutants revealed SloR accumulation.
Conclusions: The S. mutans SloR metalloregulator is subject to post-translational degradation under conditions of oxidative stress.