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Omp85, an evolutionarily conserved bacterial protein involved in outer-membrane-protein assembly
Research in Microbiology 155 (2004) 129–135 www.elsevier.com/locate/resmic
Mini-review
Omp85, an evolutionarily conserved bacterial protein involved in outer-membrane-protein assembly Romé Voulhoux 1 , Jan Tommassen ∗ Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Received 25 November 2003; accepted 25 November 2003 First published online 27 November 2003
Abstract The insertion of proteins into membranes generally requires the assistance of membrane proteins. A protein, designated Omp85 in Neisseria meningitidis, was shown to be required for the assembly of bacterial outer-membrane proteins. The protein is essential for the viability of the bacteria and is ubiquitous among Gram-negative bacteria. Omp85 depletion results in the accumulation of aggregates of unfolded outer-membrane proteins, and we argue that Omp85 is directly involved in outer-membrane-protein assembly. Omp85 shows sequence similarity with Toc75 of the chloroplast protein-import machinery, suggesting a common evolutionary origin. 2003 Elsevier SAS. All rights reserved. Keywords: Outer membrane proteins; Omp85; Protein transport; Neisseria meningitidis
1. Introduction The cell envelope of Gram-negative bacteria consists of two membranes, the inner membrane, which is a phospholipid bilayer, and the outer membrane, which is an asymmetrical bilayer with phospholipids and lipopolysaccharides (LPS) in the inner and outer monolayer, respectively. The membranes are separated by the peptidoglycancontaining periplasm. Both membranes contain proteins. Whereas integral inner-membrane proteins span the membrane by hydrophobic α-helical segments, outer-membrane proteins (OMPs) present an entirely different structure, the β-barrel. The main structural feature of β-barrel OMPs is their composition of an even number of 8 to 22 membranespanning β-strands with an antiparallel topology, which are connected by alternating long and short loops, forming β-hairpin structures [29]. Already 10 amino acids are sufficient for a β-strand to traverse the membrane bilayer. Hydrophobic residues may face the lipid environment, and amino acids with an intermediate polarity may indicate the * Corresponding author.
E-mail address: [email protected] (J. Tommassen). 1 Present address: Laboratoire d’Ingénierie des Systèmes Macromolécu-
laires, UPR9027, IBSM/CNRS, 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. 0923-2508/$ – see front matter 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2003.11.007
interior of the channel. In the range of the bilayer interface, these secondary structures expose aromatic amino acid residues to the outer environment [21]. The biogenesis of OMPs has been under study already for several decades, but the final step in the pathway, the assembly of the proteins into the outer membrane, has remained enigmatic. Here we will review recent progress that has been made in this field.
2. Does OMP insertion require proteinaceous machinery? The insertion of inner-membrane proteins in vivo generally requires the Sec translocon and/or the integral innermembrane protein YidC [9]. Hence, it could be anticipated that the insertion of OMPs may require proteinaceous machinery as well. However, no component of such machinery was found until recently. Moreover, it has been reported that OmpA and OmpF can spontaneously fold and insert into small vesicles in vitro (for a review, see [25]), suggesting that OMP insertion machinery might be nonexistent. However, the kinetics of folding and insertion of OmpA into the liposomes in vitro was very slow, taking 20–30 min to reach completion at 37 ◦ C [25]. Furthermore, the spontaneous insertion of OMPs into lipid bilayers fails to explain the exclusive insertion of these proteins into the outer membrane
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and not into the inner membrane after their transport via the Sec system into the periplasm. Thus, although OMPs may be able to fold and insert spontaneously into liposomes in vitro, a proteinaceous machinery is expected to be required to confer specificity to and increase the kinetics of the process in vivo. Similarly, whereas some small innermembrane proteins, such as the M13 procoat protein, have been reported to insert spontaneously into liposomes in vitro [10], their insertion into the inner membrane is assisted in vivo [20].
3. Involvement of Omp85 in OMP biogenesis Recently, a component of the putative OMP insertion machinery was identified in N. meningitidis [27] This component is an integral OMP, designated Omp85, and was shown to be essential for the viability of the bacteria. To study the role of Omp85 in OMP biogenesis, the chromosomal copy of the gene was inactivated in a strain carrying a copy of omp85 under lac-promoter control on a plasmid. By omission of the inducer of the lac promoter from the growth medium, the cells were depleted of Omp85. Under those conditions, all OMPs examined accumulated in a nonnative form, probably as aggregates which could be extracted from the cell envelopes with urea, demonstrating that they were not integrally inserted into the outer membrane. The defective OMP insertion in the Omp85-depleted strain was confirmed in immunofluorescence microscopy experiments, which showed a strongly reduced surface labelling with antibodies directed against OMPs. Additionally, a cell division defect was observed, apparently caused by the failure to insert newly synthesized OMPs into the outer membrane. The absence of labelling was particularly apparent in the cell division plane, suggesting that newly synthesized OMPs are preferentially assembled into the outer membrane at this site, where indeed membrane growth is expected to occur. It will be interesting to determine the labelling pattern in an Omp85-depleted strain of a baculiform bacterium, such as E. coli, which also incorporates OMPs in the lateral membranes during the cell elongation phase.
4. The involvement of Omp85 in OMP biogenesis is a direct one The negative effect of Omp85 depletion on OMP assembly could be a direct or an indirect one. Notably, it has been reported that LPS stimulates the folding of OMPs in vitro [6]. Thus, the OMP assembly defect in the Omp85-depleted strain could possibly be a consequence of a defect in LPS transport. Indeed, it was recently reported that Omp85 has a role in the transport of LPS and phospholipids to the outer membrane [11]. In that paper, it was demonstrated that: (i) the neisserial omp85 gene is co-transcribed with several downstream genes, including lpxD and lpxA, which encode
LPS biosynthetic enzymes (see Fig. 1); (ii) OMPs still fractionated with the high-density outer membrane fraction after sucrose-gradient density centrifugation to separate inner and outer membranes of an Omp85-depleted strain, arguing against a role of Omp85 in OMP assembly; (iii) LPS and phospholipids were shown to accumulate in the lower density, inner-membrane fraction of the Omp85-depleted strain. However, although omp85 is indeed located in a gene cluster that includes LPS biosynthesis genes, the gene immediately adjacent to omp85, skp, encodes a chaperone well known to be involved in OMP biogenesis (Fig. 1; see Section 5 for further details). Furthermore, it is well established that misassembled OMPs still fractionate with approximately the same density as the outer membrane in sucrose gradients. Using immunoelectron microscopy, such misassembled OMPs can be detected as aggregates in the periplasm [1,26]. Indeed, Genevrois et al. [11] reported the presence of electron-dense material accumulated in the periplasm of the Omp85-depleted strain, but they failed to reveal the identity of this material. Considering our previous experience with misassembled OMPs [1,26] and the proposed role of Omp85 in OMP biogenesis [27], this material most likely represents aggregates of misassembled OMPs. Finally, the reported inner membrane accumulation of LPS and phospholipids under Omp85 depletion most likely represents an artefact of the fractionation technique as well. The difference in density of the inner and outer membranes is determined to a large extent by their different protein content. Thus, whereas the outer membrane of an msbA mutant, which is enriched in OMPs due to a defect in the transport of LPS and phospholipids, has been reported to become heavier [7], the OMP-devoid outer membrane of the Omp85-depleted strain would be expected to become lighter and, eventually, to fractionate with the inner membranes on sucrose gradients. Alternatively, OMPs may be required for the assembly of the lipidic components into the outer membrane (see below), and the failure to incorporate these OMPs into the outer membrane of an Omp85-depleted strain may lead to the accumulation of these lipids in the inner membrane. Anyhow, it should be noted that the sucrose gradient density centrifugation technique to separate inner and outer membranes, which is well established for bacteria such as Escherichia coli and Salmonella, is notoriously problematic for Neisseria [16]. Apart from the accumulation of misfolded OMPs in an Omp85-depleted strain, a direct role for Omp85 in OMP biogenesis rather than in LPS transport was supported by the demonstration of an interaction between Omp85, immobilized on a nitrocellulose membrane, and a nonnative form of the porin PorA in an overlay assay [27]. In addition, (i) LPS is not essential in N. meningitidis [23], and OMPs are normally assembled in an LPS-deficient strain [22]. Hence, the OMP-assembly defect in the Omp85-depleted strain [27] cannot be the consequence of a defect in LPS biogenesis. (ii) An omp85 homologue is also present in Gram-negative
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Fig. 1. Chromosomal organization of the omp85 locus of N. meningitidis and its homologues in 26 fully sequenced bacteria. For each neisserial Omp85 homologue, the percentage of identity over a certain number of amino acids and the corresponding E-values (in brackets) are indicated. The E-value reports the number of hits expected to be found by chance. If they exist, homologues of omp85-flanking genes in N. meningitidis, i.e., yaeL, skp and LPS biosynthesis genes, are represented in white when they are in the same genetic organization as in N. meningitidis and in black when they are present in another locus on the chromosome. Gene alignments and Blast searches were performed using the PEDANT web site (http://pedant.gsf.de). Abbreviations used and Omp85 homologues gi numbers are: Neisseria meningitidis (MC58) gi_7225401; Nitrosomonas (ATCC 19718) gi_30249672; Ralstonia solanacearum gi_17428427; Pseudomonas syringae pv. tomato (DC3000) gi_28868748; Xanthomonas campestris pv. campestris (ATCC 33913) gi_21230822; Xylella fastidiosa (Temecula1) gi_28198247; Salmonella typhimurium (LT2) gi_16418729; Escherichia coli (K12) gi_1786374; Pseudomonas putida (KT2440) gi_26988331; Yersinia pestis (CO92) gi_15979119; Shigella flexneri (2a_301) gi_24050380; Pseudomonas aeruginosa (PAO1) gi_9949808; Vibrio cholerae (N16961) gi_9656813; Haemophilus influenzae (Rd KW20) gi_1573938; Pasteurella multocida (PM70) gi_12722432; Agrobacterium tumefaciens (C58) gi_15156447; Brucella melitensis (16M) gi_17987113; Rickettsia conorii (M7) gi_15619252; Campylobacter jejuni (NCTC 11168) gi_6967623; Helicobacter pylori (99) gi_4155152; Bacteroides thetaiotaomicron (VPI-5482) gi_29349133; Aquifex aeolicus (VF5) gi_2983730; Chlamydophila pneumoniae (JI38) gi_8978674; Treponema pallidum (Nichols) gi_3322602; Borrelia burgdorferi (B31) gi_2688732; Synechocystis (PCC6803) gi_1652591.
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Fig. 2. Alignment of the two Omp85 homologues found in Pseudomonas putida KT2440 genome. Omp85a (PP1599; gi_26988331) is the Omp85 homologue represented Fig. 1 and Omp85b (PP3373; gi_26990088) is the second, atypical Omp85 homologue found in P. putida. Identical and similar residues are marked by double and single dots, respectively. The numbers at the right indicate the position of the amino acid. The underlined sequence is fully identical in the two Omp85 homologues.
bacteria lacking LPS, such as Treponema pallidum and Borrelia burgdorferi (see Fig. 1 and Section 5), arguing against a primary role of Omp85 in LPS transport. (iii) Omp85 shows sequence similarity to well-known protein transporters (see Section 6), suggesting that Omp85 itself is also a protein transporter, rather than a lipid transporter. All these experimental data and theoretical considerations argue for a direct, rather than an indirect role of Omp85 in OMP assembly. Besides, there is a good candidate for the real LPS transporter in the outer membrane. In a recent study, an OMP differ-
ent from Omp85 and involved in outer-membrane biogenesis was identified in E. coli [2]. Moreover, this OMP, designated Imp or OstA, appeared to be essential for the viability of the bacteria. OMPs assembled correctly into their native conformation upon Imp depletion, but they appeared in a membrane fraction with a higher density in sucrose gradient centrifugation than that of the outer membrane, suggesting an increased protein to lipid ratio. Thus, it seems plausible that Imp/OstA plays a direct role in the insertion of these lipid components into the outer membrane. Indeed, evidence that
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Fig. 3. Topology model of the neisserial Omp85. The Omp85 protein is predicted to consist of two domains, the N-terminal periplasmic domain until residue 483 and the C-terminal β-barrel domain, which is constituted of 12 β-strands in the outer membrane (online supplementary material to Ref. [27]).
this protein is implicated in the transport of LPS to the cell surface was recently obtained in N. meningitidis (M.P. Bos, B. Tefsen, J. Tommassen, manuscript submitted for publication).
5. Genetic organization of the omp85 locus Previously, the presence of a gene encoding an Omp85 family member was noted in all Gram-negative bacterial genomes sequenced at that time [17,27]. We now further confirm Omp85 ubiquity among Gram-negative bacteria by identifying an omp85 homologue in the newly sequenced Gram-negative bacterial genomes (Fig. 1), supporting the important general function of Omp85 in OMP biogenesis. Interestingly, apart from PP1599, which is the Pseudomonas putida omp85 homologue depicted in Fig. 1, a second Omp85 homologue (PP3373) was found in the P. putida genome. When compared with N. meningitidis Omp85, it presents 33% identity over 811 amino acids and an E-value of 1e-138. The two P. putida proteins are highly homologous to each other showing 83% amino acid identity. The differences are located in the signal sequence and in the C-terminal half of the protein (Fig. 2), which is presumed to be the membrane-embedded part of Omp85 according to the topology model proposed previously (Fig. 3). Curiously, when the Omp85 proteins of different bacteria are compared, the membrane-embedded part is most conserved, whereas the N-terminal periplasmic domain is more variable. It would be very interesting to investigate in P. putida whether the two homologues have the same or overlapping functions. In most Gram-negative bacterial genomes, the omp85 gene is flanked by genes designated yaeL and skp in E. coli (Fig. 1). These genes encode proteins also implicated in OMP biogenesis. Skp is a periplasmic chaperone. It
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has been shown to bind selectively to unfolded OMPs in vitro [5], and skp mutants contain reduced amounts of OMPs [3]. YaeL plays a role in the activation of the response to periplasmic stress resulting from the accumulation of misfolded OMPs. The molecular mechanism leading to the activation of the periplasmic stress-response regulator σ E was recently discovered [28]. DegS, the periplasmic stress sensor, becomes activated when its PDZ domain recognizes C-terminal consensus sequences of OMPs [24], when they are improperly exposed. This interaction relieves the inhibition of the neighboring protease domain of DegS, triggering a proteolysis cascade that leads to the degradation of the anti-sigma factor RseA and, consequently, to the σ E -driven expression of periplasmic chaperones. YaeL plays an essential role in this proteolysis cascade by cleaving RseA at the cytoplasmic side of the membrane [14]. It is interesting to note that the second omp85 homologue in P. putida, PP3373, is located next to the cpxA and cpxR genes encoding a two-component regulatory system. In E. coli, this twocomponent system constitutes a second pathway to signal and respond to periplasmic stress [4].
6. Homologues of Omp85 It is noteworthy that a homologue of Omp85, designated Toc75, is also present in chloroplasts, where it functions as a component of the chloroplast protein-import machinery [17]. The presence of Omp85 homologues in Gramnegative bacteria and chloroplasts suggests a common evolutionary origin. It is probable that, after the transfer of the ancestral gene to the plant nucleus early in endosymbiosis, the transporter might have reversed orientation to mediate import of proteins into the stroma of the chloroplast. Toc75 is a component of the multisubunit Toc complex in the chloroplast outer membrane. It is not known if Omp85 functions together with other proteins in the OMP assembly machinery. However, cross-linking experiments [15] and seminative SDS-PAGE [27] indicated that Omp85 is part of a multisubunit complex, and it will be interesting to identify the other components of this complex and to determine whether they are involved in OMP assembly as well. Interestingly, some additional components of the chloroplast protein-import machinery appear to have prokaryotic homologues as well [18]. Two reports [17,30] indicate that the Omp85 family is related to another family of bacterial channel proteins involved in a secretion pathway, designated Two-Partner Secretion (TPS) [13]. This secretion pathway is involved in the transfer of virulence factors across the bacterial outer membrane and requires only a single OMP with the general designation TpsB. The region of sequence similarity with TpsB proteins concerns the second halves of Omp85 and Toc75. This indicates that Omp85, Toc75 and the TpsB might derive from a common ancient channel protein, which probably forms a typical β-barrel pore.
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Fig. 4. Model for the biogenesis of OMPs. The main steps in OMP biogenesis are numbered 1–4. (1) The periplasmic chaperone Skp binds to OMPs directly as they emerge at the periplasmic side from the Sec channel. (2) The OMP crosses the periplasm in association with the chaperone Skp, which may prevent aggregation. (3) The OMP interacts directly, presumably via its C-terminal consensus sequence, with the periplasmic domain of Omp85. (4) The protein inserts in a channel formed by the C-terminal membrane-embedded domain of Omp85, which laterally opens to allow for the stable insertion of the fully folded and assembled OMP into the outer membrane.
7. Model for OMP assembly
Acknowledgement
Although many details remain to be revealed, we propose the following model for OMP assembly into the outer membrane (Fig. 4). The periplasmic chaperone Skp binds to OMPs directly as they emerge at the periplasmic side from the Sec channel [12]. Its role might be to prevent aggregation of the unfolded OMPs. Subsequently, the OMP interacts directly, presumably via its C-terminal consensus sequence [24], with the periplasmic domain of Omp85. Here, folding takes place while the protein is still accessible to periplasmic folding catalysts, such as SurA [19] and DsbA [8]. Subsequently, the protein inserts in a channel formed by the C-terminal membrane-embedded domain of Omp85, which laterally opens to allow for the stable insertion of the OMP into the bilayer of the membrane.
We thank the European community (Grant HPRN-CT2000-00075) for support.
8. Conclusions The first identification of a component of the OMPassembly machinery is a major step forward in understanding this assembly process. Analysis of the structure and the structure–function relationship of Omp85, identification of additional components of the machinery, the interaction of OMPs with the machinery and in vitro reconstitution of the machinery are key issues for future research. Additionally, since OMP assembly is of vital importance for the bacteria, the Omp85 system may be an attractive novel target for the development of new antimicrobials.
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Mini-review
Omp85, an evolutionarily conserved bacterial protein involved in outer-membrane-protein assembly Romé Voulhoux 1 , Jan Tommassen ∗ Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Received 25 November 2003; accepted 25 November 2003 First published online 27 November 2003
Abstract The insertion of proteins into membranes generally requires the assistance of membrane proteins. A protein, designated Omp85 in Neisseria meningitidis, was shown to be required for the assembly of bacterial outer-membrane proteins. The protein is essential for the viability of the bacteria and is ubiquitous among Gram-negative bacteria. Omp85 depletion results in the accumulation of aggregates of unfolded outer-membrane proteins, and we argue that Omp85 is directly involved in outer-membrane-protein assembly. Omp85 shows sequence similarity with Toc75 of the chloroplast protein-import machinery, suggesting a common evolutionary origin. 2003 Elsevier SAS. All rights reserved. Keywords: Outer membrane proteins; Omp85; Protein transport; Neisseria meningitidis
1. Introduction The cell envelope of Gram-negative bacteria consists of two membranes, the inner membrane, which is a phospholipid bilayer, and the outer membrane, which is an asymmetrical bilayer with phospholipids and lipopolysaccharides (LPS) in the inner and outer monolayer, respectively. The membranes are separated by the peptidoglycancontaining periplasm. Both membranes contain proteins. Whereas integral inner-membrane proteins span the membrane by hydrophobic α-helical segments, outer-membrane proteins (OMPs) present an entirely different structure, the β-barrel. The main structural feature of β-barrel OMPs is their composition of an even number of 8 to 22 membranespanning β-strands with an antiparallel topology, which are connected by alternating long and short loops, forming β-hairpin structures [29]. Already 10 amino acids are sufficient for a β-strand to traverse the membrane bilayer. Hydrophobic residues may face the lipid environment, and amino acids with an intermediate polarity may indicate the * Corresponding author.
E-mail address: [email protected] (J. Tommassen). 1 Present address: Laboratoire d’Ingénierie des Systèmes Macromolécu-
laires, UPR9027, IBSM/CNRS, 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. 0923-2508/$ – see front matter 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2003.11.007
interior of the channel. In the range of the bilayer interface, these secondary structures expose aromatic amino acid residues to the outer environment [21]. The biogenesis of OMPs has been under study already for several decades, but the final step in the pathway, the assembly of the proteins into the outer membrane, has remained enigmatic. Here we will review recent progress that has been made in this field.
2. Does OMP insertion require proteinaceous machinery? The insertion of inner-membrane proteins in vivo generally requires the Sec translocon and/or the integral innermembrane protein YidC [9]. Hence, it could be anticipated that the insertion of OMPs may require proteinaceous machinery as well. However, no component of such machinery was found until recently. Moreover, it has been reported that OmpA and OmpF can spontaneously fold and insert into small vesicles in vitro (for a review, see [25]), suggesting that OMP insertion machinery might be nonexistent. However, the kinetics of folding and insertion of OmpA into the liposomes in vitro was very slow, taking 20–30 min to reach completion at 37 ◦ C [25]. Furthermore, the spontaneous insertion of OMPs into lipid bilayers fails to explain the exclusive insertion of these proteins into the outer membrane
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and not into the inner membrane after their transport via the Sec system into the periplasm. Thus, although OMPs may be able to fold and insert spontaneously into liposomes in vitro, a proteinaceous machinery is expected to be required to confer specificity to and increase the kinetics of the process in vivo. Similarly, whereas some small innermembrane proteins, such as the M13 procoat protein, have been reported to insert spontaneously into liposomes in vitro [10], their insertion into the inner membrane is assisted in vivo [20].
3. Involvement of Omp85 in OMP biogenesis Recently, a component of the putative OMP insertion machinery was identified in N. meningitidis [27] This component is an integral OMP, designated Omp85, and was shown to be essential for the viability of the bacteria. To study the role of Omp85 in OMP biogenesis, the chromosomal copy of the gene was inactivated in a strain carrying a copy of omp85 under lac-promoter control on a plasmid. By omission of the inducer of the lac promoter from the growth medium, the cells were depleted of Omp85. Under those conditions, all OMPs examined accumulated in a nonnative form, probably as aggregates which could be extracted from the cell envelopes with urea, demonstrating that they were not integrally inserted into the outer membrane. The defective OMP insertion in the Omp85-depleted strain was confirmed in immunofluorescence microscopy experiments, which showed a strongly reduced surface labelling with antibodies directed against OMPs. Additionally, a cell division defect was observed, apparently caused by the failure to insert newly synthesized OMPs into the outer membrane. The absence of labelling was particularly apparent in the cell division plane, suggesting that newly synthesized OMPs are preferentially assembled into the outer membrane at this site, where indeed membrane growth is expected to occur. It will be interesting to determine the labelling pattern in an Omp85-depleted strain of a baculiform bacterium, such as E. coli, which also incorporates OMPs in the lateral membranes during the cell elongation phase.
4. The involvement of Omp85 in OMP biogenesis is a direct one The negative effect of Omp85 depletion on OMP assembly could be a direct or an indirect one. Notably, it has been reported that LPS stimulates the folding of OMPs in vitro [6]. Thus, the OMP assembly defect in the Omp85-depleted strain could possibly be a consequence of a defect in LPS transport. Indeed, it was recently reported that Omp85 has a role in the transport of LPS and phospholipids to the outer membrane [11]. In that paper, it was demonstrated that: (i) the neisserial omp85 gene is co-transcribed with several downstream genes, including lpxD and lpxA, which encode
LPS biosynthetic enzymes (see Fig. 1); (ii) OMPs still fractionated with the high-density outer membrane fraction after sucrose-gradient density centrifugation to separate inner and outer membranes of an Omp85-depleted strain, arguing against a role of Omp85 in OMP assembly; (iii) LPS and phospholipids were shown to accumulate in the lower density, inner-membrane fraction of the Omp85-depleted strain. However, although omp85 is indeed located in a gene cluster that includes LPS biosynthesis genes, the gene immediately adjacent to omp85, skp, encodes a chaperone well known to be involved in OMP biogenesis (Fig. 1; see Section 5 for further details). Furthermore, it is well established that misassembled OMPs still fractionate with approximately the same density as the outer membrane in sucrose gradients. Using immunoelectron microscopy, such misassembled OMPs can be detected as aggregates in the periplasm [1,26]. Indeed, Genevrois et al. [11] reported the presence of electron-dense material accumulated in the periplasm of the Omp85-depleted strain, but they failed to reveal the identity of this material. Considering our previous experience with misassembled OMPs [1,26] and the proposed role of Omp85 in OMP biogenesis [27], this material most likely represents aggregates of misassembled OMPs. Finally, the reported inner membrane accumulation of LPS and phospholipids under Omp85 depletion most likely represents an artefact of the fractionation technique as well. The difference in density of the inner and outer membranes is determined to a large extent by their different protein content. Thus, whereas the outer membrane of an msbA mutant, which is enriched in OMPs due to a defect in the transport of LPS and phospholipids, has been reported to become heavier [7], the OMP-devoid outer membrane of the Omp85-depleted strain would be expected to become lighter and, eventually, to fractionate with the inner membranes on sucrose gradients. Alternatively, OMPs may be required for the assembly of the lipidic components into the outer membrane (see below), and the failure to incorporate these OMPs into the outer membrane of an Omp85-depleted strain may lead to the accumulation of these lipids in the inner membrane. Anyhow, it should be noted that the sucrose gradient density centrifugation technique to separate inner and outer membranes, which is well established for bacteria such as Escherichia coli and Salmonella, is notoriously problematic for Neisseria [16]. Apart from the accumulation of misfolded OMPs in an Omp85-depleted strain, a direct role for Omp85 in OMP biogenesis rather than in LPS transport was supported by the demonstration of an interaction between Omp85, immobilized on a nitrocellulose membrane, and a nonnative form of the porin PorA in an overlay assay [27]. In addition, (i) LPS is not essential in N. meningitidis [23], and OMPs are normally assembled in an LPS-deficient strain [22]. Hence, the OMP-assembly defect in the Omp85-depleted strain [27] cannot be the consequence of a defect in LPS biogenesis. (ii) An omp85 homologue is also present in Gram-negative
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Fig. 1. Chromosomal organization of the omp85 locus of N. meningitidis and its homologues in 26 fully sequenced bacteria. For each neisserial Omp85 homologue, the percentage of identity over a certain number of amino acids and the corresponding E-values (in brackets) are indicated. The E-value reports the number of hits expected to be found by chance. If they exist, homologues of omp85-flanking genes in N. meningitidis, i.e., yaeL, skp and LPS biosynthesis genes, are represented in white when they are in the same genetic organization as in N. meningitidis and in black when they are present in another locus on the chromosome. Gene alignments and Blast searches were performed using the PEDANT web site (http://pedant.gsf.de). Abbreviations used and Omp85 homologues gi numbers are: Neisseria meningitidis (MC58) gi_7225401; Nitrosomonas (ATCC 19718) gi_30249672; Ralstonia solanacearum gi_17428427; Pseudomonas syringae pv. tomato (DC3000) gi_28868748; Xanthomonas campestris pv. campestris (ATCC 33913) gi_21230822; Xylella fastidiosa (Temecula1) gi_28198247; Salmonella typhimurium (LT2) gi_16418729; Escherichia coli (K12) gi_1786374; Pseudomonas putida (KT2440) gi_26988331; Yersinia pestis (CO92) gi_15979119; Shigella flexneri (2a_301) gi_24050380; Pseudomonas aeruginosa (PAO1) gi_9949808; Vibrio cholerae (N16961) gi_9656813; Haemophilus influenzae (Rd KW20) gi_1573938; Pasteurella multocida (PM70) gi_12722432; Agrobacterium tumefaciens (C58) gi_15156447; Brucella melitensis (16M) gi_17987113; Rickettsia conorii (M7) gi_15619252; Campylobacter jejuni (NCTC 11168) gi_6967623; Helicobacter pylori (99) gi_4155152; Bacteroides thetaiotaomicron (VPI-5482) gi_29349133; Aquifex aeolicus (VF5) gi_2983730; Chlamydophila pneumoniae (JI38) gi_8978674; Treponema pallidum (Nichols) gi_3322602; Borrelia burgdorferi (B31) gi_2688732; Synechocystis (PCC6803) gi_1652591.
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Fig. 2. Alignment of the two Omp85 homologues found in Pseudomonas putida KT2440 genome. Omp85a (PP1599; gi_26988331) is the Omp85 homologue represented Fig. 1 and Omp85b (PP3373; gi_26990088) is the second, atypical Omp85 homologue found in P. putida. Identical and similar residues are marked by double and single dots, respectively. The numbers at the right indicate the position of the amino acid. The underlined sequence is fully identical in the two Omp85 homologues.
bacteria lacking LPS, such as Treponema pallidum and Borrelia burgdorferi (see Fig. 1 and Section 5), arguing against a primary role of Omp85 in LPS transport. (iii) Omp85 shows sequence similarity to well-known protein transporters (see Section 6), suggesting that Omp85 itself is also a protein transporter, rather than a lipid transporter. All these experimental data and theoretical considerations argue for a direct, rather than an indirect role of Omp85 in OMP assembly. Besides, there is a good candidate for the real LPS transporter in the outer membrane. In a recent study, an OMP differ-
ent from Omp85 and involved in outer-membrane biogenesis was identified in E. coli [2]. Moreover, this OMP, designated Imp or OstA, appeared to be essential for the viability of the bacteria. OMPs assembled correctly into their native conformation upon Imp depletion, but they appeared in a membrane fraction with a higher density in sucrose gradient centrifugation than that of the outer membrane, suggesting an increased protein to lipid ratio. Thus, it seems plausible that Imp/OstA plays a direct role in the insertion of these lipid components into the outer membrane. Indeed, evidence that
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Fig. 3. Topology model of the neisserial Omp85. The Omp85 protein is predicted to consist of two domains, the N-terminal periplasmic domain until residue 483 and the C-terminal β-barrel domain, which is constituted of 12 β-strands in the outer membrane (online supplementary material to Ref. [27]).
this protein is implicated in the transport of LPS to the cell surface was recently obtained in N. meningitidis (M.P. Bos, B. Tefsen, J. Tommassen, manuscript submitted for publication).
5. Genetic organization of the omp85 locus Previously, the presence of a gene encoding an Omp85 family member was noted in all Gram-negative bacterial genomes sequenced at that time [17,27]. We now further confirm Omp85 ubiquity among Gram-negative bacteria by identifying an omp85 homologue in the newly sequenced Gram-negative bacterial genomes (Fig. 1), supporting the important general function of Omp85 in OMP biogenesis. Interestingly, apart from PP1599, which is the Pseudomonas putida omp85 homologue depicted in Fig. 1, a second Omp85 homologue (PP3373) was found in the P. putida genome. When compared with N. meningitidis Omp85, it presents 33% identity over 811 amino acids and an E-value of 1e-138. The two P. putida proteins are highly homologous to each other showing 83% amino acid identity. The differences are located in the signal sequence and in the C-terminal half of the protein (Fig. 2), which is presumed to be the membrane-embedded part of Omp85 according to the topology model proposed previously (Fig. 3). Curiously, when the Omp85 proteins of different bacteria are compared, the membrane-embedded part is most conserved, whereas the N-terminal periplasmic domain is more variable. It would be very interesting to investigate in P. putida whether the two homologues have the same or overlapping functions. In most Gram-negative bacterial genomes, the omp85 gene is flanked by genes designated yaeL and skp in E. coli (Fig. 1). These genes encode proteins also implicated in OMP biogenesis. Skp is a periplasmic chaperone. It
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has been shown to bind selectively to unfolded OMPs in vitro [5], and skp mutants contain reduced amounts of OMPs [3]. YaeL plays a role in the activation of the response to periplasmic stress resulting from the accumulation of misfolded OMPs. The molecular mechanism leading to the activation of the periplasmic stress-response regulator σ E was recently discovered [28]. DegS, the periplasmic stress sensor, becomes activated when its PDZ domain recognizes C-terminal consensus sequences of OMPs [24], when they are improperly exposed. This interaction relieves the inhibition of the neighboring protease domain of DegS, triggering a proteolysis cascade that leads to the degradation of the anti-sigma factor RseA and, consequently, to the σ E -driven expression of periplasmic chaperones. YaeL plays an essential role in this proteolysis cascade by cleaving RseA at the cytoplasmic side of the membrane [14]. It is interesting to note that the second omp85 homologue in P. putida, PP3373, is located next to the cpxA and cpxR genes encoding a two-component regulatory system. In E. coli, this twocomponent system constitutes a second pathway to signal and respond to periplasmic stress [4].
6. Homologues of Omp85 It is noteworthy that a homologue of Omp85, designated Toc75, is also present in chloroplasts, where it functions as a component of the chloroplast protein-import machinery [17]. The presence of Omp85 homologues in Gramnegative bacteria and chloroplasts suggests a common evolutionary origin. It is probable that, after the transfer of the ancestral gene to the plant nucleus early in endosymbiosis, the transporter might have reversed orientation to mediate import of proteins into the stroma of the chloroplast. Toc75 is a component of the multisubunit Toc complex in the chloroplast outer membrane. It is not known if Omp85 functions together with other proteins in the OMP assembly machinery. However, cross-linking experiments [15] and seminative SDS-PAGE [27] indicated that Omp85 is part of a multisubunit complex, and it will be interesting to identify the other components of this complex and to determine whether they are involved in OMP assembly as well. Interestingly, some additional components of the chloroplast protein-import machinery appear to have prokaryotic homologues as well [18]. Two reports [17,30] indicate that the Omp85 family is related to another family of bacterial channel proteins involved in a secretion pathway, designated Two-Partner Secretion (TPS) [13]. This secretion pathway is involved in the transfer of virulence factors across the bacterial outer membrane and requires only a single OMP with the general designation TpsB. The region of sequence similarity with TpsB proteins concerns the second halves of Omp85 and Toc75. This indicates that Omp85, Toc75 and the TpsB might derive from a common ancient channel protein, which probably forms a typical β-barrel pore.
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Fig. 4. Model for the biogenesis of OMPs. The main steps in OMP biogenesis are numbered 1–4. (1) The periplasmic chaperone Skp binds to OMPs directly as they emerge at the periplasmic side from the Sec channel. (2) The OMP crosses the periplasm in association with the chaperone Skp, which may prevent aggregation. (3) The OMP interacts directly, presumably via its C-terminal consensus sequence, with the periplasmic domain of Omp85. (4) The protein inserts in a channel formed by the C-terminal membrane-embedded domain of Omp85, which laterally opens to allow for the stable insertion of the fully folded and assembled OMP into the outer membrane.
7. Model for OMP assembly
Acknowledgement
Although many details remain to be revealed, we propose the following model for OMP assembly into the outer membrane (Fig. 4). The periplasmic chaperone Skp binds to OMPs directly as they emerge at the periplasmic side from the Sec channel [12]. Its role might be to prevent aggregation of the unfolded OMPs. Subsequently, the OMP interacts directly, presumably via its C-terminal consensus sequence [24], with the periplasmic domain of Omp85. Here, folding takes place while the protein is still accessible to periplasmic folding catalysts, such as SurA [19] and DsbA [8]. Subsequently, the protein inserts in a channel formed by the C-terminal membrane-embedded domain of Omp85, which laterally opens to allow for the stable insertion of the OMP into the bilayer of the membrane.
We thank the European community (Grant HPRN-CT2000-00075) for support.
8. Conclusions The first identification of a component of the OMPassembly machinery is a major step forward in understanding this assembly process. Analysis of the structure and the structure–function relationship of Omp85, identification of additional components of the machinery, the interaction of OMPs with the machinery and in vitro reconstitution of the machinery are key issues for future research. Additionally, since OMP assembly is of vital importance for the bacteria, the Omp85 system may be an attractive novel target for the development of new antimicrobials.
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