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.
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