The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane

Abstract

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.

Fig. 1. (A) Schematic representation of the lpx locus of different Gram-negative bacteria. Arrows of the same shadings represent homologous genes. (B) Results of RT–PCR experiments. Top: transcriptional organization of the neisserial lpx locus showing the regions amplified by the primer pairs 1–9. Bottom: agarose gel of the RT–PCR amplification products. For each primer pair, three lanes are shown [a, negative control using RNA as template (no RT); b, positive control using genomic DNA as template; c, RT–PCR]. The cdsA-lpxA transcript is indicated with grey arrows.

Fig. 2. Omp85 is essential for viability of N.meningitidis. Construction of a meningococcal strain in which Omp85 expression is under the control of the tac promoter. (A) Chromosomal arrangement of the H44/76 wild-type and CMomp85 strains. Transcriptional terminators are indicated by black triangles. Details of cloning are explained in Materials and methods. (B) Western blot on whole-cell extracts of the H44/76 CMomp85 strain grown without (–) and with (+) 0.05 mM IPTG, and the H44/76 wild-type strain as control, at different time points of the first 7 h of growth. The antibody used is a rabbit polyclonal Omp85 antiserum. (C) Growth curves of the lacI^q-Ptac-regulated omp85 strain in the absence (black diamonds) and presence of 0.05 mM IPTG (grey squares). In this panel, two growth curves are shown: in (a), colonies were picked up from IPTG-containing plates, washed and grown for 7 h. After 4 h of growth without IPTG, bacteria were diluted into new fresh medium without IPTG and grown further for 6 h. This is shown in (b).

Fig. 3. Electron micrographs of ultrathin sections of H44/76 wild-type and CMomp85 grown in the presence and absence of 0.05 mM IPTG. (A) Overview and (B–D) high magnification of the cell envelope of the different strains. Bacteria were fixed in a Karnovsky solution and contrasted according to standard procedures (A and B), or they were fixed in a combined glutaraldehyde and OsO₄ solution to retain lipids and contrasted to standard procedures (C), and fixed in PFA, followed directly by embedding without contrasting (D1/2). Sections in (D1) were not post-stained and all the contrast was imparted by the embedding medium. A cytochemical reaction for the presence of PAS-positive material on ultrathin sections is illustrated in (D2). Depletion of Omp85 leads to accumulation of electron-dense amorphous material (small arrows in A, white asterisk in B) and to vesicular structures (large arrow in B) in the periplasmic space. This amorphous material is not lipophilic or cytochemically stained for polysaccharides (white asterisk in C and D2). The small arrows in (C) mark the osmiophilic granules outside the OM. The black asterisks in (D1) and (D2) denote a layer of PAS-positive material outside the cells, presumably the capsular polysaccharide. Arrowheads in (B–D) mark the peptidoglycan layer. The bars represent 200 µm in (A) and 100 µm in (B–D).

Fig. 4. Analysis of fractions 1–12, collected after isopycnic sucrose gradient centrifugation, to separate the membranes of the H44/76 CMomp85 strain grown for 6 h in the presence and absence of 0.05 mM IPTG. (A) LDH activity per 5 µg of proteins as a marker for the IM. (B) SDS–PAGE analysis of the fractions. The positions of molecular weight standard proteins are indicated on the left in kDa; the positions of the porins PorA and PorB are indicated on the right. The black arrow shows the 35 kDa band. (C) Western blot using PorA (MN5C11G) and Opa (15-1-P5.5 + αD2) monoclonal antibodies as markers for the OM.

Fig. 5. Omp85 is required for LPS export to the OM. Silver-stained Tricine-SDS–polyacrylamide gel showing the LPS pattern of proteinase K-treated fractions containing 5 µg of protein of the H44/76 CMomp85 strain grown in the presence (top) and absence (bottom) of 0.05 mM IPTG.

Fig. 6. Omp85 is required for PL export to the OM. The H44/76 CMomp85 strain was grown in the presence of [1,2-¹⁴C]acetic acid for 6 h in the presence and absence of 0.05 mM IPTG. (A) Collected fractions 1–12 were analysed by western blot using PorA monoclonal antibody as a marker for the OM. (B) Relative percentage of the c.p.m. value in each fraction [(c.p.m. value FX/total c.p.m. value) × 100]. (C) TLC separation of PLs. PLs were extracted using the method of Bligh and Dyer (1959) and separated on TLC plates as explained in Materials and methods.

Fig. 7. Secondary structure predictions for Omp85 in N.meningitidis and its homologues in other Gram-negative bacteria (N.gonorrhoeae, S.enterica, E.coli, H.influenzae, P.multocida and B.abortus). Multiple sequence alignments were performed using the ClustalW program. Shown is the N.meningitidis Omp85 sequence. Black and grey squares represent identical and similar residues in the alignment, respectively. Predicted β-strands and α-helices are represented by grey arrows and white rectangles, respectively.