Some of the experimental conditions we tested are stressful, and in these cases, BamE function becomes essential for the cell
Some of the experimental conditions we tested are stressful, and in these cases, BamE function becomes essential for the cell. with proteinase K, BamA is degraded beyond detection. Taken together, our findings suggest that BamE modulates the conformation of BamA, likely through its interactions with CP 375 BamD. == INTRODUCTION == The outer membrane (OM) is an essential organelle of Gram-negative bacteria such asEscherichia coli(26). A key function of the OM is to serve as a barrier preventing access of toxic molecules from the extracellular environment into the bacterial cell (4,5,8,26). Unlike the inner membrane (IM), the OM is an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet (5,26). LPS repels hydrophobic compounds, including some antibiotics, and is a well-characterized antagonist of the human innate immune system. The OM also contains many proteins, including integral outer membrane beta-barrel proteins CP 375 (OMPs) and lipoproteins which are tethered to the OM by amino-terminal lipid moieties (28). OMPs are involved in a variety of physiological processes, including nutrient acquisition and efflux of small molecules such as antibiotics (18,20,21). OMPs are assembled by a machine localized in the OM called the Bam complex (10,14,26). InE. coli, this multisubunit complex is composed of the OMP BamA and four associated OM lipoproteins, BamBCDE (13,21). Depletion of either BamA or BamD results in severe OMP assembly defects, and both proteins are essential for growth (16,19,30). In contrast, null mutations inbamB,bamC, orbamEcause minor OMP assembly defects (27,30). Genetic and biochemical studies have begun to show how the subunits of the Bam complex interact with one another (13,21). For example, it is now known that BamCDE form a stable subcomplex that interacts with BamA and that the BamA-BamCDE interaction occurs independently of the interaction between BamA and BamB (12,27).In vitroreconstitution studies CP 375 have demonstrated that to achieve optimal assembly of OmpT, all five members of the Bam complex (and the periplasmic chaperone SurA) must be present in the reaction mixture (11,12). However, the full extent of the interactions between Bam complex members and their contribution to OMP assembly remains poorly defined. Very little is known about the underlying mechanism by which the Bam complex folds and inserts OMPs into the OM. In this report, we describe efforts to better define the role of the BamCDE subcomplex during OMP assembly. In particular, we wanted to understand the function of BamC and BamE and their Rabbit Polyclonal to KITH_VZV7 relationship to BamA and BamD. Biochemical data show that BamC and BamE share a function in that both are important for stabilizing the interaction between BamA and BamD. By comparing the phenotypes conferred by and the genetic interactions observed between bamCand bamEand genes for other proteins involved in OMP biogenesis and by exploiting the accessibility of BamA to externally added protease, we have uncovered an additional novel function for BamE: BamE functions to control CP 375 the conformation of BamA, likely through its interactions with BamD. == MATERIALS AND METHODS == == Bacterial strains, plasmids, and growth conditions. == All strains and plasmids used in this study are listed inTable 1. ThebamC::kanandbamE::kanalleles, obtained from the Keio collection (2), were cured as described previously to generate the bamCand bamEalleles. The BamD overexpression plasmid contains a mutation in pBamD (27) that increases plasmid copy number and was isolated in a screen forbamDmutants (N. W. Rigel and T. J. Silhavy, unpublished). Cultures were grown in Luria-Bertani (LB) medium at 30C or 37C as indicated. Where appropriate, antibiotics were included in the growth medium at the following concentrations: ampicillin, 125 g/ml; kanamycin, 25 g/ml; tetracycline, 25 g/ml; and chloramphenicol, 20 g/ml. == Table 1. == Strains and plasmids used in this study == Growth curves. == Cultures of each strain were grown overnight in LB broth at 30C. The next day, strains were diluted.