Genetic, biochemical, and molecular characterization of the polypeptide transport-associated domain of Escherichia coli BamA

Patricia Workman, Kristina Heide, Nicolas Giuliano, Nanhee Lee, James Mar, Phu Vuong, Drew Bennion, Rajeev Misra

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

The BamA protein of Escherichia coli plays a central role in the assembly of β-barrel outer membrane proteins (OMPs). The Cterminal domain of BamA folds into an integral outer membrane β-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 α2 helix of the POTRA 1 domain (ΔR44), can be overcome by the insertion of alanine 2 residues upstream or downstream from the ΔR44 site. This highlights the importance of the side chain orientation of the α2 helix residues for normal POTRA 1 activity. The ΔR44-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 BamAΔR44 from a low-copy-number plasmid confers a severe drug hypersensitivity phenotype. A spontaneous drug-resistant revertant of BamAΔR44 was found to carry an A18S substitution in the α1 helix of POTRA 1. In the BamAΔR44, 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 α1 helix of the POTRA 1 domain as a suppressor of the folding defect caused by ΔR44 underscores the importance of the helix 1 and 2 regions in BamA folding.

Original languageEnglish (US)
Pages (from-to)3512-3521
Number of pages10
JournalJournal of bacteriology
Volume194
Issue number13
DOIs
StatePublished - Jul 1 2012

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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