Putative membrane assembly of EtpM-colicin V chimeras Fabien Gérard a,1 , Nathalie Pradel a , Changyun Ye b , Bérengère Ize a,2 , Liang Yi c , Jianguo Xu b , Ross E. Dalbey c , Long-Fei Wu a, * a Laboratoire de Chimie Bactérienne, UPR9043, Institut de Biologie Structurale et Microbiologie, CNRS, 31, chemin Joseph Aiguier 13402 Marseille cedex 20, France b Department of diarrheal diseases, National Institute of Communicable Diseases Prevention and Control, Chinese Center for Disease Control and Prevention, Beijing 102206, PR China c Department of Chemistry and Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA Received 29 January 2004; accepted 5 April 2004 Available online 28 April 2004 Abstract 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 DtatC 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 ofYidC, 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 andYidC-dependent translocation of ColV. © 2004 Elsevier SAS. All rights reserved. Keywords: Type II secretion; Topology; Membrane insertion; Signal peptide; Colicin;YidC 1. Introduction The type II protein secretion pathway delivers various virulence factors into the growth media [1]. It requires a minimum of 12, and possibly 14 proteins which are highly conserved among different bacteria [2,3]. Enterohemor- rhagic E. coli O157:H7 is the predominant causative agent of hemorrhagic colitis in human [4]. A functional type II secre- tion pathway is encoded by 13 plasmid-bearing genes (etpC to etpO) detected in all 30 O157 strains analyzed [5]. Recent studies suggest that the etp-encoded type II secretion system is ubiquitous among the O157:H7 serotype and is found in some enteropathogenic E. coli O55:H7 strains, but absent from other diarrheagenic E. coli [6]. In addition, the type II system of E. coli O157:H7 secretes a metalloprotease, StcE, that specifically cleaves the C1 esterase inhibitor [7]. There- fore, the type II secretion system plays an important role in the pathogenesis of the enterohemorrhagic pathogen. Among the 13 etp genes, etpM codes an integral membrane protein homologous to the GspMs of Erwinia carotovora, Erwinia chrysanthemi or Klebsiella pneumoniae. The topology de- duced from analysis of fusions to alkaline phosphatase, b-lactamase or b-galactosidase shows that GspMs are type II bitopic inner membrane proteins with a large C-terminal domain protruding into the periplasm [2,3]. Interestingly, EtpM contains a twin-arginine motif in front of its signal anchor, suggesting a Tat-dependent membrane insertion. Recently,YidC was identified as a factor specifically used for the insertion of membrane proteins and not for the trans- location of exported proteins [8]. It assists the integration of both Sec-dependent and Sec-independent IMPs [8–10]. In vitro crosslinking studies revealed specific contact ofYidC to the transmembrane (TM) regions of IMPs [11,12]. The TM of the bitopic IMP FtsQ seems to contact first SecY and then YidC [12]. However, the first TM region of the polytopic model IMP Lep could be crosslinked to both SecY and YidC, even when the nascent chains were very short and the first TM was not yet fully exposed outside the ribosome [13]. * Corresponding author. Tel.: +33-4-9116-4157; fax: +33-4-9171-8914. E-mail address: wu@ibsm.cnrs-mrs.fr (L.-F. Wu). 1 Present address: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville FL 32611, USA 2 Present address: Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK Biochimie 86 (2004) 283–286 www.elsevier.com/locate/biochi © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.04.002