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

Abstract

Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes—two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data.

Outer Membrane Biogenesis

“…These barrel and eL6 altering mutations, essentially confer a gain of function phenotype bypassing the requirement for BamA communication with the periplasmic lipoproteins. One of the mutations, bamA_F494L , was later shown to also be a partial bypass suppressor of bamD (63). Although bamD is still essential, it can be almost completely depleted in bamA_F494L strain without affecting viability (63).…”

“…One of the mutations, bamA_F494L , was later shown to also be a partial bypass suppressor of bamD (63). Although bamD is still essential, it can be almost completely depleted in bamA_F494L strain without affecting viability (63). …”

Folding outer membrane proteins independently of the β-barrel assembly machinery: an assembly pathway for multimeric complexes?

“…Intriguingly, BamA might prove an example of pathway crossover. How BamA reaches the OM is unknown, but both the BAMlipoproteins BamA associates with [52,53] and BamA itself [2] are suggested to help BamA assembly. Further research should establish whether some or all of these OMPs truly are substrates with mixed BAM-and Lol-dependence or whether some observations require revalidation by independent approaches to rule out erroneous interpretations resulting from the complexity of the in vivo experimental systems used.…”

Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family

“…They facilitate the insertion of β‐barrel‐shaped proteins into the OM of Gram‐negative bacteria, mitochondria, their descendent organelles, and probably even in chloroplasts . The best studied prokaryotic Omp85 protein is BamA (β‐barrel assembly machinery protein A) in Escherichia coli , which together with the lipoproteins BamB, C, D, and E facilitate the insertion of other β‐barrel‐shaped OM proteins . Structural analysis of BamA confirmed that the protein consists of five N‐terminal polypeptide transport‐associated (POTRA) domains and the C‐terminal 16‐stranded pore‐forming β‐barrel domain that is characteristic for all Omp85 proteins .…”