Genome-wide screens: novel mechanisms in colicin import and cytotoxicity.

Protein import and export across cellular membranes involves an assembly of integral membrane polypeptides that provide a conduit for protein transfer across the hydrophobic membrane. Colicin import into bacteria is a model for the study of protein import across bacterial membranes. Colicins are produced by E. coli in response to stresses such as nutrient depletion and overcrowding, and kill closely related sibling cells that contain the set of receptors and trans-envelope import proteins, but do not contain the respective immunity protein. Based on known components of the translocation network utilized for cell entry, colicins have been divided into two groups. Group A utilizes the Tol network consisting of genes tolA, tolB, tolQ, tolR and pal (Nagel de Zwaig and Luria, 1967; Davies and Reeves, 1975a; Lazzaroni et al., 2002). Group B employs the Ton network comprised of tonB, exbB and exbD (Davies and Reeves, 1975b; Braun et al., 2002). Loss of the colicin receptors in the outer membrane, proteins in the translocation pathway, or of the immunity protein, are the only known mechanisms of colicin resistance or tolerance. The first step in colicin import is binding to its outer membrane primary receptor. The protein component of this primary receptor is known to be BtuB for colicin A, E1, E2, E3 and E7, OmpF for colicin N, and FepA for colicins B and D. Subsequent to binding to the primary binding steps, nuclease E colicins, for which the import process has been studied extensively (Di Masi et al., 1973; Mock and Pugsley, 1982; Benedetti et al., 1989; Bouveret et al., 1997; Garinot-Schneider et al., 1997; Kurisu et al., 2003; Housden et al., 2005; Duche et al., 2006; Loftus et al., 2006; Duche, 2007; Sharma et al., 2007; Yamashita et al., 2008), utilize a second receptor/translocator to accomplish translocation across the outer membrane. This secondary receptor/translocator is OmpF for colicin A, TolC for colicin E1 and OmpF/OmpC for colicins E2, E3 and E7. No secondary receptor/translocator has yet been identified for colicin N and the group B colicins. In addition to the primary receptor and secondary receptor/translocator several other proteins have been identified that are responsible for colicin import across the double membrane of the target cell. A total of ten genes, btuB, iutA, ompF, ompC, tolC, tolQRAB and tsx have been identified to play a role in the uptake of group A colicins, while the corresponding number for group B colicins is nine ((cir, exbBD, fepA, fhuA, lepB, tonB, tsx (Cascales et al., 2007) and the recently identified fkpA (Hullmann et al., 2008)). This number is considerably smaller than the number of proteins showed to be involved in protein transport across the mitochondrial double membrane. In the mitochondrial system, virtually all the pre-proteins traverse the outer membrane through the initial entry gate, the TOM (translocase of the outer membrane) complex that consists of seven polypeptides. The full translocation of the pre-proteins across the inner membrane into the mitochondrial matrix requires 10 more polypeptides (Bohnert et al., 2007; Bolender et al., 2008). Thus, on the one hand, it might be expected that additional components, proteins or lipids as yet unidentified, could be involved in colicin uptake across the E. coli double membrane. On the other, it is possible that some gene(s) previously proposed to be required for colicin cytotoxicity are not essential. The “Keio Collection” (www.EcoliHub.org/GenoBase) can be used to clarify this situation. This collection, a single-gene knockout library of the entire E. coli genome (Datsenko and Wanner, 2000; Baba et al., 2006), consists of 3985 strains, in duplicate, inoculated in 96-well plates, each well consisting of a distinct single-gene knockout mutant. In the present study, the collection was screened for the sensitivity of each knockout strain to eight colicins, A, B, D, E1, E2, E3, E7 and N, allowing for the identification of knockout strains that are able to grow in the presence of colicin. These colicin ‘tolerant’ or ‘resistant’ single gene knockout strains were further analyzed by complementation with the respective genes from the “ASKA” orfeome library, which restored the colicin sensitivity to the knockout strain. Since only the genes non-essential for cellular viability are represented in the Keio collection, proteins that are required by colicin but which are also essential for cell growth (e. g. lepB for colD) will not be scored in the present genomic screening. It is noted that while a ‘positive hit’ implies that the particular gene is important for the colicin cytotoxicity, a ‘negative hit’, where a gene deletion does not affect colicin activity, is equally significant. A ‘negative hit’ implies that, barring a redundancy in the gene function, the gene is not critical for colicin cytotoxicity. Through this study we have been able to define the requirement of each of the 3985 genes in the Keio collection for colicin cytotoxicity.