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 ß-barrel outer membrane proteins (OMPs). The C-terminal domain of BamA folds into an integral outer membrane ß-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 BamA(ΔR44) from a low-copy-number plasmid confers a severe drug hypersensitivity phenotype. A spontaneous drug-resistant revertant of BamA(ΔR44) was found to carry an A18S substitution in the α1 helix of POTRA 1. In the BamA(ΔR44, 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.

Dissection of ß-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 ß-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane ß-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 ß-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site-specific cross-linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most ß-barrel OMPs except for TolC, which folds into a unique soluble α-helical barrel and an OM-anchored ß-barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans-membrane ß-barrel domain of BamA.

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. The synthetic conditional lethal phenotype of Delta cpxR in mutant Bam or beta-barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant beta-barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly-defective beta-barrel OMP species. Together, these results showed that both the Cpx and sigma(E) regulons are required to reduce envelope stress caused by aberrant beta-barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression.

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. Using bioinformatics, genetics, and biochemical approaches, we have identified three residues, L173, L175, and R176, in the mature YfgL protein that are critical for both function and interactions with YaeT. A single substitution at any of these sites produces no phenotypic defect, but two or three simultaneous alterations produce mild or yfgL-null phenotypes, respectively. Interestingly, biochemical data show that all YfgL variants, including those with single substitutions, have weakened in vivo YaeT-YfgL interaction. These defects are not due to mislocalization or low steady-state levels of YfgL. Cysteine-directed cross-linking data show that the region encompassing L173, L175, and R176 makes direct contact with YaeT. Using the same genetic and biochemical strategies, it was found that altering residues D227 and D229 in another region of YfgL from E221 to D229 resulted in defective YaeT bindings. In contrast, mutational analysis of conserved residues V319 to H328 of YfgL shows that they are important for YfgL biogenesis but not YfgL-YaeT interactions. The five YfgL mutants defective in YaeT associations and the yfgL background were used to show that SurA binds to YaeT (or another complex member) without going through YfgL.