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

The BamA protein of Escherichia coli plays a central role in the assembly of β-barrel outer membrane proteins (OMPs). The C-terminal 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.

Dissection of β-barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli.

BamA of Escherichia coli is an essential component of the hetero-oligomeric machinery that mediates β-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane β-barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1.

Involvement and necessity of the Cpx regulon in the event of aberrant beta-barrel outer membrane protein assembly.

The Cpx and sigma(E) regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the sigma(E) pathway monitors the biogenesis of beta-barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of beta-barrel OMP mis-assembly, by utilizing mutants expressing either a defective beta-barrel OMP assembly machinery (Bam) or assembly defective beta-barrel OMPs. Analysis of specific mRNAs showed that Delta cpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the sigma(E) pathway.

Analysis of YfgL and YaeT interactions through bioinformatics, mutagenesis, and biochemistry.

In Escherichia coli, YaeT, together with four lipoproteins, YfgL, YfiO, NlpB, and SmpA, forms a complex that is essential for beta-barrel outer membrane protein biogenesis. Data suggest that YfgL and YfiO make direct but independent physical contacts with YaeT. Whereas the YaeT-YfiO interaction needs NlpB and SmpA for complex stabilization, the YaeT-YfgL interaction does not.