Use of colicin-based genetic tools for studying bacterial protein transport A. Filloux a, *, R. Voulhoux a , B. Ize b , F. Gérard b , G. Ball a , L.F. Wu b a Laboratoire d’Ingénierie des Systèmes Macromoléculaires, UPR9027, Institut de Biologie Structurale et Microbiologie/Centre National de la Recherche Scientifique, 31, chemin Joseph-Aiguier, 13402 Marseille cedex 20, France b Laboratoire de Chimie Bactérienne, UPR9043, Institut de Biologie Structurale et Microbiologie/Centre National de la Recherche Scientifique, 31, chemin Joseph-Aiguier, 13402 Marseille cedex 20, France Received 1 March 2002; accepted 7 June 2002 Abstract Transport of proteins across the envelope of Gram-negative bacteria is a very challenging domain of investigation, which involves membrane-embedded proteinaceous complexes at which specific targeting occurs. These transporters (translocon or secreton) have been studied both with genetics and biochemistry. In this review we report recent developments that should help to identify novel interactions that exist within these complexes, and to decipher the signals that specifically direct transported proteins to the cognate system. These developments are exclusively based on the re-routing of colicins to these molecular machineries. The re-routing induces a lethal situation in the case of efficient or inefficient transport, depending on the system, thus creating a genetic tool for selection of mutations that correct or generate a transport default. © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Tat; Xcp; Bacterial cell envelope; Signal peptide 1. Introduction Colicins are bacterial toxins, plasmid-encoded [1], that kill Escherichia coli and related strains. The strains that produce colicins are normally protected against their action due to the co-synthesis of a so-called immunity protein [2]. The transport of colicins is generally not mediated by the use of typical prokaryotic export signals. However, the secretion of colicins generally involves a protein called “lysis protein” [3] whose production results in the appari- tion of several signs of membrane permeability alteration. The level of expression of lysis protein and colicin should be optimal for maximising colicin production before the suicidal process become effective. The colicin structural gene, the immunity gene and the gene required for colicin export are found on a single region of the Col plasmid. Synthesis of most colicins is controlled by the “SOS” regulatory circuit normally dedicated to the control of genes involved in the repair of damaged DNA [4]. The molecular mechanisms that allow colicin gene ex- pression, colicin release into the medium, uptake into the target cell, and the mechanism of action that results in the killing of the infected cells have been extensively studied [5]. This knowledge contributed to the development of colicin-based genetic tools that have been efficiently used in the study of protein transport in bacteria. In this review, we will briefly introduce the salient feature of bacterial protein transport network, and then demonstrate the power of the colicin-based genetic tools for dissecting various aspects of this network. 2. Bacterial protein transport network Proteins are synthesised in the bacterial cytosol from which a certain number should be re-directed to the extra- cytoplasmic compartments. In Gram-negative bacteria, the cytoplasm is surrounded by a cell envelope, which is made * Corresponding author. Tel.: +33-4-91-16-41-27; fax: +33-4-91-71-21-24. E-mail address: filloux@ibsm.cnrs-mrs.fr (A. Filloux). Biochimie 84 (2002) 489–497 © 2002 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 3 0 0 - 9 0 8 4 ( 0 2 ) 0 1 4 1 2 - 8