The beta-barrel outer membrane protein assembly complex of Neisseria meningitidis

Abstract

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the beta-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.

FIG. 1.

BamC, BamE, and ComL mutants. (A) Cell envelopes derived from the strains indicated above the panel were separated by SDS-PAGE and immunoblotted with anti-BamC antiserum. The predicted molecular weight of BamC is 39,000; the bands below the 37,000-molecular-weight marker probably represent nonspecific reactivity of the antiserum. (B) Cell lysates of the strains indicated above the panel were separated by SDS-PAGE and immunoblotted with anti-BamE antiserum. HB-1ΔbamE(pBamE) was analyzed after growth in the presence (+) or absence (−) of 0.5 mM IPTG. (C) Growth of HB-1 and HB-1ΔcomL(pComL) in the absence (−) or presence (+) of 0.5 mM IPTG. HB-1ΔcomL(pComL) was pregrown overnight on plates containing 1 μM IPTG. OD₅₅₀, optical density at 550 nm. (D) Cell lysates of HB-1(pBamD) grown in the absence (−) or presence (+) of 0.5 mM IPTG were subjected to SDS-PAGE, blotted, and probed with anti-BamD antiserum. (E) Immunoblot, probed with anti-ComL antiserum, of cell envelopes of HB-1 and HB-1ΔcomL(pComL) grown in the absence (−) or presence (+) of 0.5 mM IPTG. In this case, HB-1ΔcomL(pComL) was pregrown overnight on plates containing 10 μM IPTG. Numbers to the left of gels are molecular weights (in thousands).

FIG. 2.

Role of putative Bam complex components in OMP assembly. (A to D) Cell envelopes were subjected to denaturing (d) or semi-native (n) SDS-PAGE and stained with Coomassie brilliant blue (A, B, C) or blotted and probed with anti-PorA or anti-PorB antibodies (D). Arrows point at unfolded porins. (E) Cell lysates were subjected to denaturing SDS-PAGE and immunoblotting with anti-PilQ antiserum. Only the relevant parts of the blot are shown. The strains are indicated below the panels as follows: 1, HB-1; 2, HB-1ΔbamC; 3, HB-1ΔbamE; 4, HB-1ΔcomL(pComL); 5, HB-1ΔbamE(pBamE); and 6, HB-1Δmlp. The prominent bands seen in lane 4 of panel A at the 28,000-molecular-weight marker likely represent a combination of phase-variable Opa proteins and ComL, which have similar predicted molecular weights. Numbers to the left of gels are molecular weights (in thousands). α, anti; +, present; −, absent.

FIG. 3.

Composition of the HMW SDS-resistant Omp85 complex. (A) Cell envelopes from strain HB-1 were analyzed by denaturing (d) or semi-native (n) SDS-PAGE, blotted, and probed with antisera indicated above the panels. (B) Cell envelopes derived from the strains indicated above the panels were analyzed by denaturing (d) or semi-native (n) SDS-PAGE, blotted, and probed with an anti-Omp85 MAb. HB-1ΔbamE-(pBamE) was grown in the presence (+) or absence (−) of 0.5 mM IPTG as indicated. HB-1ΔcomL(pComL) was pregrown overnight on plates containing 10 μM IPTG and subsequently grown in TSB in the absence or presence of 0.5 mM IPTG. (C to E) Cell envelopes of 1, HB-1; 2, HB-1-Δcl4; 3, HB-1ΔrmpM; and 4, H44/76-Δcl4 were analyzed by semi-native SDS-PAGE and immunoblotting using an anti-Omp85 MAb. (E) Cell envelopes of HB-1 were incubated with (+) or without (−) 0.4 mM β-mercaptoethanol for 30 min prior to loading on the gel. The positions of various conformations of Omp85 are indicated as follows: UM, unfolded monomer; FM, folded monomer. Numbers to the left of gels are molecular weights (in thousands). α, anti.

FIG. 4.

OMP assembly in RmpM mutants. (A to D) Cell envelopes were subjected to denaturing (d), denaturing without reducing agents (d−) or semi-native (n) SDS-PAGE and stained with Coomassie blue (A, B), or blotted and probed with anti-PorA (C) or anti-PorB (D) antibodies. Cell envelopes were prepared from 1, HB-1; 2, HB-1-Δcl4; 3, HB-1ΔrmpM; and 4, HB-1ΔcomL(pComL) grown in the absence of IPTG. Folded porin monomers, dimers, and trimers are indicated by single, double, and triple asterisks, respectively. #, Unfolded monomeric porin. The ComL-depleted cell envelope sample is included as a control to show defective PorA assembly. (E) Cell lysates were subjected to denaturing SDS-PAGE and immunoblotting with anti-PilQ antiserum. Lane numbers refer to strains as indicated for panels A to D. Numbers to the left of gels are molecular weights (in thousands). α, anti.

FIG. 5.

Protease accessibility of Omp85 in Bam complex mutants. Cell envelopes from the strains indicated above the lanes were treated with (+) or without (−) trypsin (tryp.) and subjected to denaturing SDS-PAGE followed by immunoblotting with antiserum against the C-terminal part of N. meningitidis Omp85. Tryptic fragments with apparent molecular weights of approximately 58,000, 48,000, and 40,000 are indicated with I, II, and III, respectively. Where relevant, the absence (−) or presence (+) of IPTG during growth of the strains is indicated. Numbers to the left of gels are molecular weights (in thousands).

FIG. 6.

Omp85-associated proteins in N. meningitidis. (A) Cell envelope extracts of strains expressing wild-type (1) or His-tagged (2) Omp85 were subjected to Ni²⁺-NTA purification. Shown are elution fractions analyzed by denaturing SDS-PAGE and silver staining or immunoblotting, using antibodies against the indicated proteins. The lane indicated with c contains nonextracted cell envelopes of HB-1 as a control to show reactivity of the anti-Imp antiserum. Numbers to the left of gels are molecular weights (in thousands). (B) Cell envelopes containing His-tagged Omp85 (a) and a Ni²⁺-NTA elution fraction derived from them (b) were blotted. The blot was cut horizontally, and the upper part probed with an anti-Omp85 MAb and the lower part with anti-BamE antiserum. Before exposure to film, the blots were pieced together again.