The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane
2003; Springer Nature; Volume: 22; Issue: 8 Linguagem: Inglês
10.1093/emboj/cdg174
ISSN1460-2075
Autores Tópico(s)Bacterial Infections and Vaccines
ResumoArticle15 April 2003free access The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane Stéphanie Genevrois Corresponding Author Stéphanie Genevrois Research Unit in Molecular Biology (URBM), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur, Belgium Search for more papers by this author Liana Steeghs Liana Steeghs Laboratory of Vaccine Research (LVR) Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands Search for more papers by this author Paul Roholl Paul Roholl Laboratory of Pathology and Immunobiology (LPI), National Institute of Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands Search for more papers by this author Jean-Jacques Letesson Jean-Jacques Letesson Research Unit in Molecular Biology (URBM), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur, Belgium Search for more papers by this author Peter van der Ley Peter van der Ley Laboratory of Vaccine Research (LVR) Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands Search for more papers by this author Stéphanie Genevrois Corresponding Author Stéphanie Genevrois Research Unit in Molecular Biology (URBM), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur, Belgium Search for more papers by this author Liana Steeghs Liana Steeghs Laboratory of Vaccine Research (LVR) Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands Search for more papers by this author Paul Roholl Paul Roholl Laboratory of Pathology and Immunobiology (LPI), National Institute of Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands Search for more papers by this author Jean-Jacques Letesson Jean-Jacques Letesson Research Unit in Molecular Biology (URBM), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur, Belgium Search for more papers by this author Peter van der Ley Peter van der Ley Laboratory of Vaccine Research (LVR) Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands Search for more papers by this author Author Information Stéphanie Genevrois 1, Liana Steeghs2, Paul Roholl3, Jean-Jacques Letesson1 and Peter van der Ley2 1Research Unit in Molecular Biology (URBM), University of Namur (FUNDP), Rue de Bruxelles, 61, 5000 Namur, Belgium 2Laboratory of Vaccine Research (LVR) Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands 3Laboratory of Pathology and Immunobiology (LPI), National Institute of Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, PO Box 1, 3720 BA, Bilthoven, The Netherlands ‡S.Genevrois and L.Steeghs contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1780-1789https://doi.org/10.1093/emboj/cdg174 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The envelope of Gram-negative bacteria consists of an inner membrane (IM), the peptidoglycan layer and an outer membrane (OM) (Nikaido, 1996). The latter is an asymmetrical bilayer with phospholipids (PLs) in the inner leaflet and lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) in the outer leaflet. LPS has a cell surface-exposed oligosaccharide chain, which in many organisms is linked further to a longer O antigen chain composed of repeating sugar subunits (Raetz and Whitfield, 2002). LPS and PL biosynthesis takes place in both the cytoplasm and IM of the bacterial cell. The enzymes responsible for the lipid A biosynthesis pathway have been well characterized in Escherichia coli (Raetz and Whitfield, 2002) and are targets for the design of novel antibacterial agents (Onishi et al., 1996; McMurry et al., 1998). During the past 20 years, both genetic and biochemical studies have led to the description of LPS composition, biosynthesis and regulation. The formation of LPS is a complex process involving the synthesis of activated precursors in the cytoplasm, followed by the assembly of the lipid A core at the IM (Raetz and Whitfield, 2002). In E.coli, the lipid A biosynthetic pathway has been elucidated entirely. Conditionally lethal mutants have been used to isolate and characterize genes involved in the early steps of lipid A biosynthesis. These genes (lpxA, lpxC and lpxD) were shown to be essential for viability (Beall and Lutkenhaus, 1987; Galloway and Raetz, 1990; Kelly et al., 1993). However, in Neisseria meningitidis, it is possible to make a knockout mutant of lpxA, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis (Galloway and Raetz, 1990), while maintaining cell viability (Steeghs et al., 1998). This mutant still possesses an OM in which the PL composition is altered in comparison with the wild-type strain (Steeghs et al., 2001). In E.coli, the lpxA gene belongs to a largely conserved cluster of genes, present in almost all Gram-negative bacteria for which genome sequences are available. Figure 1A shows the chromosomal arrangement of this locus in several Gram-negative bacteria. In addition to the lipid A biosynthesis genes lpxA, lpxD and lpxB, this cluster also includes fabZ and cdsA required for fatty acid and phospholipid synthesis, respectively (Raetz and Dowhan, 1990; Mohan et al., 1994), as well as yaeM and yaeL which are known to be involved in membrane biogenesis in E.coli: yaeM encodes 1-deoxy-D-xylulose 5-phosphate reductoisomerase involved in the synthesis of isoprenoids (Takahashi et al., 1998), which have been characterized in many diverse organisms and have been shown to serve as pigments, defensive agents, constituents of membranes or components of signal transduction networks (Sacchettini and Poulter, 1997). yaeL encodes the essential EcfE IM protease, which is involved in regulation of the heat shock response in E.coli (Dartigalongue et al., 2001). EcfE modulates the level of expression of both RpoH and RpoE σ factors, which control protein folding and degradation in the cytoplasm and extracytoplasm, respectively. Moreover, RpoE regulates lipid biogenesis in response to environmental stress (Raivio and Silhavy, 2001). The yaeT gene, encoding a putative outer membrane protein (OMP) of unknown function, is always found upstream of lpxD, in most cases associated with hlpA, which encodes a periplasmic chaperone (Chen and Henning, 1996; Missiakas et al., 1996). The presence of a gene encoding a highly conserved OMP in a cluster dedicated to LPS and PL biosynthesis suggests a possible involvement in lipid transport. Figure 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. Download figure Download PowerPoint The way in which completed LPS and PL are translocated to the cell surface represents the major unresolved question in their biosynthesis. Recently, a clue to the mechanism of lipid A core and PL transport within the IM has emerged from the discovery that a mutation in msbA, encoding an essential ABC transporter, results in a defect in lipid export (Doerrler et al., 2001). The MsbA protein is thought to function as an IM flippase for PL and lipid A core transport to the periplasm. To complete the LPS biosynthesis, lipid A core is attached further to the O-polysaccharide chain once both molecules are in the periplasm. The three currently known pathways for O-polysaccharide biosynthesis are distinguished by their export mechanisms (Raetz and Whitfield, 2002). The pathways are called Wzy-dependent, ABC transporter-dependent and synthase-dependent. The first two processes are widespread, while the last one has limited distribution in O-polysaccharide biosynthesis. Despite the export differences, the initiation reactions and ligation to lipid A core are similar. The molecular mechanism underlying the transport of the completed LPS molecules to the OM is still unknown. As for LPS, the mechanisms of PL transport from the IM to OM are unknown. Several hypotheses have been proposed, such as PL and LPS translocation through membrane adhesion zones (Bayer's junctions) (Bayer, 1968), but it is still not clear whether or not these structures are physiologically relevant (Kellenberger, 1990). Another possibility is that specific proteins may serve as a shuttle for lipid transfer to the OM, but this has never been demonstrated. Neisseria meningitidis is, to date, the only Gram-negative bacterium in which a completely LPS-deficient but still viable mutant can be obtained (Steeghs et al., 1998). This makes it a uniquely suitable organism for the study of LPS and PL transport. In the present study, we have investigated the involvement of Omp85, the neisserial YaeT homologue, in the export of lipids to the OM. We first showed that omp85, the gene encoding Omp85, is part of a transcriptional unit that starts with cdsA and ends with lpxA. We further demonstrated that Omp85 is essential for the viability of N.meningitidis and is required for LPS and PL transport in the OM. We also showed that Omp85 is not directly involved in the transport of integral OMPs such as PorA and Opa, though the depletion of Omp85 leads to increased amounts of their degradation products in the OM. Results The omp85 gene is transcriptionally linked to genes involved in lipid A, fatty acid and phospholipid biosynthesis The lpx locus is highly conserved among different Gram-negative bacteria (Figure 1A), containing genes involved in lipid A, fatty acid and PL biosynthesis together with genes implicated in membrane biogenesis. In order to determine if omp85 is co-transcribed with these genes in N.meningitidis, RT–PCR assays were performed (Figure 1B). A negative control, consisting of DNase-treated RNA, and a positive control, consisting of genomic DNA, were included in the experiment. Using cDNA as template, we could amplify overlapping regions between genes of the cluster, from cdsA to lpxA. This result demonstrates that omp85 is part of a transcriptional unit that contains eight genes, starting with cdsA and ending with lpxA, which could reflect the fact that they encode proteins or enzymes involved in a common pathway. Omp85 is essential for viability of N.meningitidis To test directly the involvement of Omp85 in LPS and PL transport, we first attempted to make an omp85 knockout mutant in N.meningitidis wild-type strain H44/76 and its LPS-deficient lpxA mutant by allelic replacement with constructs containing either a kanamycin or chloramphenicol marker in omp85, but without any success. Moreover, we tried to delete omp85 in the HA3003 strain in which LPS expression could be switched on or off through a tac promoter-controlled lpxA gene (Steeghs et al., 2001). Again, no double crossover recombination events leading to the inactivation of the omp85 gene could be obtained, whether HA3003 expressed LPS or not, indicating an essential role for Omp85 in the viability of both the wild-type and LPS-deficient mutant. To study the function of Omp85 in spite of this limitation, we next constructed a meningococcal strain in which Omp85 expression is regulated from the tac promoter (Figure 2A). The lacIq-Ptac-regulated omp85 DNA fragment was first introduced at the rmpM locus. Secondly, the omp85 gene was deleted at its own locus using the kanamycin cassette, which does not contain a transcriptional terminator. The latter insertion did not affect expression of downstream lpx genes in the resulting omp85 conditional mutant (termed CMomp85), as was checked by RT–PCR (results not shown). We could only select kanamycin-resistant clones in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG), again demonstrating that omp85 expression is essential for bacterial survival. Using this mutant, the expression of Omp85 could be regulated by the presence or absence of IPTG in the medium. Depletion of Omp85 was checked every hour of growth by western blot on whole-cell extracts using a rabbit polyclonal antiserum directed against Omp85 (Figure 2B). After 3 h of growth without IPTG, depletion of Omp85 became visible, and was apparently complete after 5 h of growth. Upon induction with IPTG, omp85 is transcribed and cells are viable (Figure 2C). When no IPTG was added in the medium, cell growth was affected. The effect of depletion of Omp85 on bacterial growth was more pronounced when, after 4 h of incubation without IPTG, the culture was diluted further into fresh medium without IPTG (see Figure 2C, panel b). This clearly demonstrates that Omp85 is essential for the viability of N.meningitidis. Figure 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 lacIq-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). Download figure Download PowerPoint Analysis of the ultrastructure of the cell envelope upon depletion of Omp85 The ultrastructure of the H44/76 CMomp85 strain grown in the presence and absence of IPTG, and its parental strain was examined by transmission electron microscopy (Figure 3). We did not observe any ultrastructural difference in the cell envelope between the CMomp85 strain grown with IPTG and the wild-type strain. In contrast, cells of H44/76 CMomp85 grown without IPTG were more heterogeneous in size, and a significant proportion of cells showed signs of lysis (Figure 3A and B). A clear accumulation of both electron-dense material and vesicular structures in the periplasm was observed for these latter cells, suggesting that the depletion of Omp85 leads to a defect in transport of some component(s) that can no longer reach the OM. We further analysed ultrastructurally whether this accumulating material consists of lipids or polysaccharides. Therefore, bacteria were fixed in glutaraldehyde/osmium tetroxide solution to retain lipids and in a paraformaldehyde (PFA) solution to perform the periodic acid–Schift (PAS) reaction. H44/76 wild-type and IPTG-stimulated CMomp85 bacteria showed small lipophilic granules at the outside of the OM, whereas CMomp85 bacteria grown without IPTG did not show these lipophilic granules. The accumulating material in these cells was also not reactive with osmium and is therefore considered as not lipophilic (Figure 3C). A cytochemical reaction for polysaccharides revealed many granules in a broad band directly outside these cells, presumably representing the capsule. No reaction was seen within the periplasmic space (Figure 3D). These findings suggest that the accumulating electron-dense material in the periplasm is of a proteinaceous nature. Figure 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). Download figure Download PowerPoint IM and OM separation of the H44/76 CMomp85 strain The cell envelope composition of the H44/76 CMomp85 strain, grown for 4 and 6 h in the presence and absence of IPTG, was examined by IM and OM separation through isopycnic sucrose gradient centrifugation (Steeghs et al., 2001). We will only present the data on the collected fractions after 6 h of growth. Gradient fractions of 1 ml were collected, protein quantified and analysed for the presence of lactate dehydrogenase (LDH) activity as a marker for the IM (Osborn et al., 1972), and for the presence of PorA and Opa as markers for the OM (Figure 4). LDH activity peaked in fractions 3 and 4 when the H44/76 CMomp85 strain was grown without IPTG (Figure 4A). In contrast, in the presence of IPTG, LDH activity was higher and peaked in fractions 3–6. In both cases, no LDH activity was detected in fractions 8–12, which are therefore regarded as pure OM fractions. We analysed the protein content on each collected fraction by SDS–PAGE followed by Coomassie Blue staining (Figure 4B). The protein profile for these fractions is the same for bacteria grown in the presence or absence of IPTG, with the exception of a band (marked with a black arrow) at a molecular weight of 35 kDa that disappears when bacteria are not induced with IPTG. We performed a western blot experiment using monoclonal antibodies directed against PorA and Opa, two integral OMPs (Figure 4C). PorA and Opa proteins are visible in almost all the fractions, but with strong enrichment of these proteins in the OM fractions. This was observed previously using the same procedures for the IM and OM separation of the wild-type strain and the LPS-deficient mutant (Steeghs et al., 2001). The Opa protein remains present in the OM fractions of the strain grown without IPTG, suggesting that the 35 kDa band that disappears upon depletion of Omp85 does not correspond to Opa. Moreover, depletion of Omp85 did not lead to accumulation of PorA and Opa in the IM fractions, indicating that Omp85 is not directly involved in the transport of these proteins to the OM. The only effect is a somewhat increased amount of PorA and Opa degradation products in the OM fractions. Degradation of OMPs has also been reported in the LPS-deficient mutant (Steeghs et al., 2001). Figure 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. Download figure Download PowerPoint Omp85 is required for LPS transport to the OM To study the effect of Omp85 depletion on LPS localization, the LPS content of each proteinase K-treated fraction was analysed by Tricine-SDS–PAGE followed by silver staining (Lesse et al., 1990) (Figure 5). When Omp85 was present, LPS was observed mainly in the OM fractions. In contrast, when Omp85 was depleted, LPS mostly disappeared from the OM fractions and instead accumulated in the IM fractions. Moreover, a two band pattern was observed, suggesting that depletion of Omp85 in addition leads to the relative increase in a modified form of LPS with slightly lower gel mobility. After 4 h of growth without IPTG, the same result was observed, though the effect of depletion of Omp85 on LPS accumulation in IM fractions was less marked, suggesting that LPS accumulation in the IM occurs directly and progressively upon depletion of Omp85. Figure 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. Download figure Download PowerPoint Omp85 is required for PL transport to the OM The results shown in Figure 5 indicate a role for Omp85 in LPS export. However, as a knockout omp85 mutant could not be isolated in the LPS-deficient mutant, Omp85 might also be required for another function in membrane biogenesis. Therefore, we examined the effect of Omp85 depletion on PL transport to the OM (Figure 6). Bacteria were grown for 6 h as previously described, but in the presence of [1,2-14C]acetic acid. Bacterial cell fractionation was performed by another method, based on the lysis of lysozyme–EDTA spheroblasts (Osborn et al., 1972). IMs and OMs were separated on a sucrose gradient. Each fraction was analysed for the presence of PorA as a marker for the OM (Figure 6A). Compared with the previous membrane separation, we observed a slightly different distribution of OM fractions in this experiment: PorA is distributed in fractions 4–10. This might be explained by the use of lysozyme, which degrades the peptidoglycan layer, leading to a shift of OM to the lower density fractions of the gradient. This has been observed previously in the sucrose gradient fractionation of membranes from Salmonella typhimurium using the same procedures (Osborn et al., 1972). The N.meningitidis OM localizes in the same density region of the gradient (38–43% sucrose) as the 'H' band representing the S.typhimurium OM. PLs were extracted and analysed for their c.p.m. value as shown in Figure 6B. We calculated the relative percentage of each PL extract compared with the sum of the c.p.m. of all extracts. The distribution of PLs can also be visualized after their separation on thin-layer chromatography (TLC) as shown in Figure 6C. Compared with the strain grown with IPTG, PLs are decreased in the OM (fractions 6–10) and instead accumulate in the IM (particularly in fraction 2) after depletion of Omp85 (−IPTG). These results demonstrate that Omp85 is also required for PL transport to the OM. Moreover, this might explain why no omp85 knockout mutant could be isolated in the LPS-deficient mutant. Figure 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. Download figure Download PowerPoint Discussion In the present study, we have investigated the potential involvement of Omp85 in transport of lipids to the OM of N.meningitidis. In contrast to most Gram-negative bacteria, N.meningitidis is viable without LPS, making it a uniquely suitable organism to study lipid transport. The omp85 gene has a conserved location in all Gram-negative bacterial genomes sequenced to date. We demonstrated that, in N.meningitidis, omp85 is part of a transcriptional unit that starts with cdsA and ends with lpxA. The association of omp85 with genes encoding proteins involved in LPS and PL biosynthesis and cell envelope assembly suggested a common involvement in lipid biosynthesis and membrane biogenesis pathways. The omp85 gene is essential for survival of N.meningitidis wild-type and its LPS-deficient mutant. We constructed a derivative strain of H44/76, designated H44/76 CMomp85, which carries an IPTG-inducible omp85 gene on the chromosome, allowing us to study the function of Omp85 after its gradual depletion. The analysis of the ultrastructure of the bacterial envelope by electron microscopy revealed that together with depletion of Omp85, an electron-dense amorphous material, as well as vesicular structures are accumulating between the IM and OM. Different staining techniques were used to demonstrate that the electron-dense material probably consists of protein. The vesicular structures are reminiscent of the IM reduplications seen in the E.coli msbA mutant (Doerrler et al., 2001), and suggest a block in lipid export. IM and OM separation of the CMomp85 strain, grown in the presence and absence of IPTG, demonstrated that Omp85 is required for LPS transport to the OM. Analysis of the LPS content in the IM and OM revealed a more pronounced two-band pattern in the mutant, indicating that depletion of Omp85 leads to some modification of LPS. This could be explained by increased sialylation of LPS in the mutant. In Neisseria, sialylation is catalysed by an α-2,3-sialyltransferase (Lst) that monosialylates the terminal galactose of LOS (for lipooligosaccharide) by using 5′-cytidine monophospho-N-acetylneuraminic acid as donor (Mandrell et al., 1990). Recently, it has been shown that Lst is a surface-exposed OMP with its active site possibly facing the periplasm (Shell et al., 2002). In the CMomp85 strain grown without IPTG, LPS is accumulating in a compartment where it could be subjected to increased sialylation by Lst. To check whether the accumulating upper band in the non-induced cells corresponds to intensive sialylation of LPS, these samples were subjected to neuraminidase treatment. The upper band is disappearing following this treatment, indicating that this band corresponds to a sialylated form of LPS (results not shown). Transport of the integral OMPs PorA and Opa to the OM is not impaired when Omp85 is depleted, though an increased amount of PorA and Opa degradation products is observed in the OM fractions. Various studies have shown that in E.coli, LPS is required for the correct assembly of OMP (de Cock and Tommassen, 1996). In contrast, in N.meningitidis, the trimerization of porins is not affected in the LPS-deficient mutant (Steeghs et al., 2001), though some degradation products of these OMPs were observed. We have shown that in the absence of Omp85, LPS accumulates in the IM; this is probably toxic for the bacteria and would explain why an omp85 knockout mutant could not be isolated. The isolation of an omp85 knockout mutant in the LPS-deficient mutant would have demonstrated that Omp85 is only involved in LPS transport to the OM. Using different constructs containing either a kanamycin or chloramphenicol marker inserted into omp85, it was not possible to isolate such a mutant, suggesting that Omp85 might have an additional function. We therefore checked if depletion of Omp85 also has an effect on PL transport to the OM. This turned out to be the case, explaining why we could not isolate a double omp85-lpxA mutant. Consistently, homologues of Omp85 have also been found in the genomes of Treponema pallidum and Borrelia burgdorferi (Fraser et al., 1997, 1998), which are spirochetes that do not contain LPS in their OM (Hardy and Levin, 1983). In these genomes,
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