Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components Anu Daniel,3 Aparna Singh, Lynette J. Crowther, Paula J. Fernandes, Wiebke Schreiber and Michael S. Donnenberg Correspondence Michael S. Donnenberg mdonnenb@umaryland.edu Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, 20 Penn Street, Baltimore, MD 21201, USA Received 20 January 2006 Revised 1 May 2006 Accepted 8 May 2006 Typical enteropathogenic Escherichia coli strains express an established virulence factor belonging to the type IV pili family, called the bundle-forming pilus (BFP). BFP are present on the cell surface as bundled filamentous appendages, and are assembled and retracted by proteins encoded by the bfp operon. These proteins assemble to form a molecular machine. The BFP machine may be conceptually divided into three components: the cytoplasmic membrane (CM) subassembly, which is composed of CM proteins and cytoplasmic nucleotide-binding proteins; the outer membrane (OM) subassembly and the pilus itself. The authors have previously characterized the CM subassembly and the pilus. In this study, a more complete characterization of the OM subassembly was carried out using a combination of biochemical, biophysical and genetic approaches. It is reported that targeting of BfpG to the OM was influenced by the secretin BfpB. BfpG and BfpU interacted with the amino terminus of BfpB. BfpU had a complex cellular distribution pattern and, along with BfpB and BfpG, was part of the OM subassembly. INTRODUCTION It is becoming increasingly clear that many cellular processes are carried out, not by individual proteins functioning in isolation, but by macromolecular complexes and intricate protein networks (Alberts, 1998; Phizicky et al., 2003). Structural and biochemical characterizations of such macro- molecular assemblies are being undertaken to obtain a better understanding of the cellular processes they control. These considerations also apply to proteins and processes involved in bacterial pathogenesis. Type IV pili (Tfp) of Gram-negative bacteria are assembled by one such multi- component molecular machine (Anantha et al., 2000; Ramer et al., 2002). Tfp are filamentous surface appendages that are expressed by many pathogenic bacteria, including Pseudo- monas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella enterica serovar Typhi, Legionella pneumophila, and enteropathogenic (EPEC) and enterotoxigenic Escherichia coli (Hobbs & Mattick, 1993; Strom & Lory, 1993; Zhang et al., 2000; Stone & Abu Kwaik, 1998; Taniguchi et al., 1995; Giro ´n et al., 1991). Tfp play a role in diverse processes, such as cellular adhesion (Lee et al., 1994; Rudel et al., 1995), colonization (Herrington et al., 1988; Tacket et al., 1998), twitching motility (Bradley, 1980; Henrichsen, 1983; Merz et al., 2000; Wall & Kaiser, 1999), biofilm formation (O’Toole & Kolter, 1998), horizontal gene transfer (Seifert et al., 1988; Yoshida et al., 1999) and virulence (Tacket et al., 1998; Herrington et al., 1988; Bieber et al., 1998). Proteins of the Tfp biogenesis machine share extensive sequence similarity to proteins of type II secretion systems (T2SSs) and DNA uptake systems, and have ortho- logues among the proteins required for the assembly of filamentous phages and archaeal flagellae, suggesting that these systems are structurally and mechanistically similar and share an ancient evolutionary link (Chen & Dubnau, 2003; Russel, 1998; Craig et al., 2004; Sandkvist, 2001; Peabody et al., 2003). Tfp are homopolymeric structures composed of the pilin structural protein (Craig et al., 2004). Mature pilin is generated from the pre-pilin precursor, which contains an unusual short positively charged leader peptide, by a pre-pilin peptidase, which also N-methylates the nascent amino terminus (Strom et al., 1993). A number of conserved accessory genes are required for Tfp biogenesis, including those encoding the pre-pilin peptidase, a polytopic cyto- plasmic membrane (CM) protein, pre-pilin-like proteins, nucleotide-binding proteins, and an outer membrane (OM) secretin. Mutational analyses have revealed that these 3Present address: Laboratory of Bacterial Pathogenesis and Immuno- logy, The Rockefeller University, 1230 York Avenue, NY 10021, USA. Abbreviations: AHT, anhydrotetracycline; AI, autoaggregation index; BFP, bundle-forming pilus; CM, cytoplasmic membrane; DSP, dithio- bis[succinimidyl] propionate; EPEC, enteropathogenic Escherichia coli; a-Gal, a-galactosidase; His 6 , hexahistidine; ITC, isothermal titration calorimetry; MBP, maltose-binding protein; b-ME, b-mercaptoethanol; OM, outer membrane; Tfp, type IV pili. 0002-8860 G 2006 SGM Printed in Great Britain 2405 Microbiology (2006), 152, 2405–2420 DOI 10.1099/mic.0.28860-0