Mapping the twin‐arginine protein translocation network of B acillus subtilis

Bacteria employ twin‐arginine translocation ( T at) pathways for the transport of folded proteins to extracytoplasmic destinations. In recent years, most studies on bacterial T at pathways addressed the membrane‐bound T at A ( B ) C subunits of the T at translocase, and the specific interactions between this translocase and its substrate proteins. In contrast, relatively few studies investigated possible coactors in the T at A ( B ) C ‐dependent protein translocation process. The present studies were aimed at identifying interaction partners of the T at pathway of B acillus subtilis , which is a paradigm for studies on protein secretion by G ram‐positive bacteria. Specifically, 36 interaction partners of the T at A and T at C subunits were identified by rigorous application of the yeast two‐hybrid ( Y 2 H ) approach. Our Y 2 H analyses revealed that the three T at A isoforms of B . subtilis can form homo‐ and heterodimers. Subsequently, the secretion of the T at substrates Y wb N and P ho D was tested in mutant strains lacking genes for the T at AC interaction partners identified in our genome‐wide Y 2 H screens. Our results show that the cell wall‐bound protease W pr A is important for Y wb N secretion, and that the H em AT and C sb C proteins are required for P ho D secretion under phosphate starvation conditions. Taken together, our findings imply that the B acillus T at pathway is embedded in an intricate protein–protein interaction network.