Identification and Characterization of SdbA, a Novel Thiol-Disulfide Oxidoreductase in Streptococcus gordonii

Abstract

Disulfide bonds are important for the stability of certain extracellular proteins, including bacterial virulence factors. Thiol disulfide oxidoreductases (TDORs) catalyze disulfide bond formation. Although these enzymes have been well characterized in some organisms, little is known about how disulfide bonds are formed in Gram-positive bacteria, particularly among facultative anaerobes such as streptococci. An analysis of the Streptococcus gordonii sequenced genome found five putative TDORs. These TDORs were systematically investigated for their affect on autolysis, extracellular DNA release, biofilm formation, bacteriocin production, and genetic competence. This revealed a single TDOR with a pleiotropic phenotype that we named Streptococcus disulfide bond protein A (SdbA). SdbA was demonstrated to catalyze disulfide bond formation, and using an in silico approach, we identified the major autolysin AtlS as a natural substrate. The low homology of SdbA to known TDORs prompted us to investigate its catalytic mechanism. TDORs catalyze disulfide bonds using a CXXC motif. Typically, the N-terminal cysteine interacts with substrates, while the C-terminal cysteine is buried, and both are essential for activity. We show that SdbA functions differently, and that mutants with a single, buried C-terminal cysteine of the CXXC motif can complement a ΔsdbA mutant, and restore disulfide bond formation to recombinant and natural substrates. These results distinguish SdbA from previously described TDORs. Finally, we found that mutation of SdbA generated an activating signal for the CiaRH two-component system. CiaRH is involved in stress response and affects virulence in pathogenic streptococci. Our results suggest that CiaRH can sense disulfide bond formation in S. gordonii. Activation of CiaRH repressed a second signaling system, ComDE, and eliminated bacteriocin production. Together, CiaRH and ComDE regulate >100 genes. By modulating the activity of CiaRH, SdbA can influence a broad spectrum of bacterial physiological processes that are not directly related to disulfide bond formation. In summary, this study establishes SdbA as a novel TDOR that catalyzes disulfide bond formation in S. gordonii and is important for the normal physiology of the cell.