MecA is a highly conserved adaptor protein encoded by prokaryotes from the Bacillota phylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiont Streptococcus mutans likely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactions in vivo. In addition, we further develop a new application of SLCA to support in vivo measurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as-yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of the MecA regulatory function still has yet to be discovered.IMPORTANCEDespite multiple decades of study, the regulatory mechanism and function of MecA have remained largely a mystery. The current study provides the first detailed roadmap to investigate these functions in other medically significant bacteria. Furthermore, this study developed new genetic approaches to assay prokaryotic protein-protein interactions via the split luciferase complementation assay (SLCA). SLCA technology is commonly employed in eukaryotic genetic research but has not yet been established for studies of bacterial protein-protein interactions. The SLCA protein binding affinity assay described here is a new technological advance exclusive to the current study and has not been reported elsewhere.
Keywords: MecA interactome; Streptococcus mutans; mass spectrometry; protein binding affinity; split luciferase complementation assays.