Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain

Abstract

beta-Barrel proteins are present in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The central component of their assembly machinery is called Omp85 in bacteria. Omp85 is predicted to consist of an integral membrane domain and an amino-terminal periplasmic extension containing five polypeptide-transport-associated (POTRA) domains. We have addressed the function of these domains by creating POTRA domain deletions in Omp85 of Neisseria meningitidis. Four POTRA domains could be deleted with only slight defects in Omp85 function. Only the most carboxy-terminal POTRA domain was essential, as was the membrane domain. Thus, similar to the mitochondrial Omp85 homologue, the functional core of bacterial Omp85 consists of its membrane domain and a single POTRA domain, that is, POTRA5.

Figure 1

Domain organization of Omp85 and constructed Omp85 variants. POTRA domains were defined according to Sánchez-Pulido et al (2003) and the membrane domain according to Voulhoux et al (2003). The numbers at the top indicate the positions of the amino- and carboxy-terminal amino acids of each domain in the Omp85 sequence. Protein designations are shown on the left. OMPLA, outer membrane phospholipase A; POTRA, polypeptide-transport-associated.

Figure 2

Analysis of Omp85 mutants. Wild type (WT) refers to strain HB-1. Molecular-weight markers are indicated on the left side of the blots. (A) Immunoblots containing cell lysates from bacteria expressing functional Omp85 variants were probed with an Omp85 carboxy-terminal antiserum. Loadings were equalized on the basis of optical density of the cultures. (B) Growth curves of bacteria expressing functional Omp85 variants in tryptic soy broth plus IPTG. (C) Cell lysates of bacteria expressing Omp85 variants that did not allow disruption of the chromosomal omp85 gene were probed with Omp85 amino-terminal antiserum. Protein designations are as indicated in Fig 1. (D) Cell envelopes of bacteria expressing Δ5 were treated at room temperature with 50 μg/ml trypsin for the time periods indicated below the lanes, subjected to denaturing SDS–PAGE, blotted and probed with Omp85 N-terminal antiserum (top panel), RmpM antibody (middle panel) or stained with Coomassie brilliant blue (lower panel). IPTG, isopropyl-β-D-thiogalactopyranoside; OD, optical density; Omp, outer membrane protein; PorA, porin A; POTRA, polypeptide-transport-associated; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.

Figure 3

Assessment of outer membrane protein folding and assembly in POTRA deletion mutants. Cell envelopes were denatured by boiling in SDS (d) or left on ice (n). The number of deleted POTRA domains is indicated below the lanes. Molecular-weight markers are indicated next to the blots. (A) Coomassie brilliant blue-stained gel. The positions of the Omp85 proteins are indicated on the gel with crosses. (B,C) Immunoblots of similar samples as shown in (A) were probed with antibodies against PorA (B) or NspA (C). (D) Cells were grown under iron limitation and the blot was probed with antibodies against LbpA. The positions of folded monomer (fLbpa), denatured monomer (dLbpa) and folded oligomers (foLbpa) of LbpA are indicated. (E) Blot containing only native samples probed with Omp85 amino-terminal antiserum. The arrowhead indicates folded monomers of intact Omp85. The lanes are from one single blot, but different exposure time durations are shown (separated by the black lines) to optimize visualization of Omp85 reactivity for all samples. Omp, outer membrane protein; PorA, porin A; POTRA, polypeptide-transport-associated.