Putative membrane assembly of EtpM-colicin V chimeras

EtpM of the enterohemorrhagic E. coli O157:H7 is a bitopic membrane protein of the type II protein secretion apparatus. There is a twin-arginine (RR) motif in front of its signal anchor, suggesting a Tat-dependent membrane targeting of EtpM. By exploiting the periplasmic bactericidal activity of colicin V (ColV), we constructed EtpM-ColV fusions and studied the EtpM-mediated translocation of ColV. The wild type strain and the DeltatatC mutant were killed by the expressed fusions and were fully protected from the killing effect by the ColV-specific immunity protein. In contrast, cold-inactivation of YidC, which is generally required for integral membrane protein assembly, significantly attenuated the killing effect in the cold-sensitive yidC mutant. These results confirmed the predicted N(in)-C(out) EtpM topology, and suggests an EtpM-mediated, Tat-independent and YidC-dependent translocation of ColV.