Genetic, Biochemical, and Molecular Characterization of the Polypeptide Transport-Associated Domain of Escherichia coli BamA

Abstract

The BamA protein of Escherichia coli plays a central role in the assembly of ␤-barrel outer membrane proteins (OMPs). The Cterminal domain of BamA folds into an integral outer membrane ␤-barrel, and the N terminus forms a periplasmic polypeptide transport-associated (POTRA) domain for OMP reception and assembly. We show here that BamA misfolding, caused by the deletion of the R44 residue from the ␣2 helix of the POTRA 1 domain (⌬R44), can be overcome by the insertion of alanine 2 residues upstream or downstream from the ⌬R44 site. This highlights the importance of the side chain orientation of the ␣2 helix residues for normal POTRA 1 activity. The ⌬R44-mediated POTRA folding defect and its correction by the insertion of alanine were further demonstrated by using a construct expressing just the soluble POTRA domain. Besides misfolding, the expression of BamA ⌬R44 from a low-copy-number plasmid confers a severe drug hypersensitivity phenotype. A spontaneous drug-resistant revertant of BamA ⌬R44 was found to carry an A18S substitution in the ␣1 helix of POTRA 1. In the BamA ⌬R44, A18S background, OMP biogenesis improved dramatically, and this correlated with improved BamA folding, BamA-SurA interactions, and LptD (lipopolysaccharide transporter) biogenesis. The presence of the A18S substitution in the wild-type BamA protein did not affect the activity of BamA. The discovery of the A18S substitution in the ␣1 helix of the POTRA 1 domain as a suppressor of the folding defect caused by ⌬R44 underscores the importance of the helix 1 and 2 regions in BamA folding.

Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of -Barrel Outer Membrane Proteins, Including That of BamA Itself

“…If a mutant BamA protein fails to correctly assemble itself or other ␤-barrel OMPs, including the lipopolysaccharide (LPS) transporter LptD, then cells expressing the mutant BamA protein may have a compromised outer membrane permeability barrier resulting in vancomycin and rifampin sensitivity. Consistent with this view, we have recently reported a BamA POTRA domain mutant with increased sensitivity toward vancomycin and rifampin (36). Because cells expressing BamA AAA or BamA ⌬RGF grow best on glycerol minimal medium at 30°C (Fig.…”

“…The folding status of BamA was assessed by conducting the heat modifiability test, in which the correctly folded form of BamA is distinguished from the unfolded (denatured) form based on the faster mobility of the former on an SDS-polyacrylamide gel (16,28,29,36). As expected, wild-type BamA was correctly folded, as no slowly migrating (denatured) species was present in the unheated envelope sample (Fig.…”

“…The POTRA domain is thought to interact with substrate OMPs via ␤-augmentation and initiate their folding into ␤-barrels (16,17,23). In Escherichia coli, the POTRA domain is also the site of interactions with other lipoprotein BAM members (37) and the periplasmic chaperone SurA (3,36). Deletion of POTRA domains 1 and 2 of E. coli BamA is possible; cells expressing a BamA variant with POTRA domain 2 deleted grow normally, while those with a deletion of POTRA domain 1 grow poorly and have reduced OMP levels (3,16).…”

In Vivo Roles of BamA, BamB and BamD in the Biogenesis of BamA, a Core Protein of the β-Barrel Assembly Machine of Escherichia coli

“…The soluble N-terminal end of BamA folds into five polypeptide-transport-associated (POTRA) domains [23] whose structure revealed possible interactions with substrate polypeptides via β-augmentation [24-26]. Mutational and pull-down assays have also revealed that the POTRA domains of BamA are critical for interactions with the Bam lipoproteins [24] and a periplasmic chaperone, SurA [27,28]. Thus, the POTRA domains appear to serve as the initial staging ground for substrate interaction and folding.…”

Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression

“…The Cterminal domain of BamA forms a transmembrane 16-stranded β-barrel core, while the Nterminal region of BamA forms five soluble periplasmic polypeptide-transport-associated domains (POTRA) (Jansen et al, 2015b, Kim et al, 2007, Gatzeva-Topalova et al, 2010, Gatzeva-Topalova et al, 2008. These POTRA domains contribute to BamA stability and directly interact with BamBD within the periplasm, nascent polypeptides and the periplasmic chaperone SurA (Bennion et al, 2010, Kim et al, 2007, Workman et al, 2012, Dong et al, 2012a. The lipoproteins BamC and BamE interact directly with BamD (Kim et al, 2011a, Malinverni et al, 2006, Wu et al, 2005, Stenberg et al, 2005.…”