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��ANRV322-MI61-11 ARI 6 August 2007 16:59 Biogenesis of the Gram-Negative Bacterial Outer Membrane Martine P. Bos, Viviane Robert, and Jan Tommassen Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands; email: m.bos@uu.nl, vivianerobert@hotmail.com, j.p.m.tommassen@uu.nl Annu. Rev. Microbiol. 2007. 61:191 214 Key Words First published online as a Review in Advance on lipopolysaccharide, outer membrane proteins, lipoproteins, June 7, 2007 phospholipids The Annual Review of Microbiology is online at micro.annualreviews.org Abstract This article s doi: The cell envelope of gram-negative bacteria consists of two mem- 10.1146/annurev.micro.61.080706.093245 branes, the inner and the outer membrane, that are separated by Copyright � 2007 by Annual Reviews. the periplasm. The outer membrane consists of phospholipids, All rights reserved lipopolysaccharides, integral membrane proteins, and lipoproteins. 0066-4227/07/1013-0191$20.00 These components are synthesized in the cytoplasm or at the inner leaflet of the inner membrane and have to be transported across the inner membrane and through the periplasm to assemble eventually in the correct membrane. Recent studies in Neisseria meningitidis and Escherichia coli have led to the identification of several machineries implicated in these transport and assembly processes. 191 by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 other gram-negative bacteria, however, the protein moiety of OM lipoproteins may also Contents extend into the extracellular medium; ex- INTRODUCTION. . . . . . . . . . . . . . . . . 192 amples are the LbpB and TbpB compo- INTEGRAL OUTER MEMBRANE nents of the lactoferrin and transferrin re- PROTEINS . . . . . . . . . . . . . . . . . . . . . 193 ceptor, respectively, of Neisseria meningitidis Passage Across the IM and (70). Through the Periplasm . . . . . . . . 193 The OM functions as a selective barrier Assembly into the OM . . . . . . . . . . . . 194 that protects the bacteria from harmful com- Role of LPS in OMP Biogenesis . . 199 pounds, such as antibiotics, in the environ- Model for OMP Biogenesis . . . . . . . 200 ment. Unlike the IM, the OM is not energized LIPOPROTEINS. . . . . . . . . . . . . . . . . . . 200 by a proton gradient and ATP is not avail- LIPOPOLYSACCHARIDE . . . . . . . . . 202 able in the periplasm. In the absence of read- Structure and Biosynthesis . . . . . . . . 202 ily available energy sources, nutrients usually Transport to the Cell Surface . . . . . 202 pass the OM by passive diffusion via an abun- Models for LPS Transport . . . . . . . . 205 dant class of trimeric OMPs called porins (66). PHOSPHOLIPIDS . . . . . . . . . . . . . . . . . 206 Porins form water-filled channels that allow PERSPECTIVES . . . . . . . . . . . . . . . . . . . 206 the passage of small hydrophilic solutes with molecular weights up to <"600 Da. Never- theless, energy-requiring transport processes in the OM have also been described. Such INTRODUCTION processes are dependent on complex energy- IM: inner The cell envelope of gram-negative bacteria coupling systems, such as the TonB system, membrane consists of two membranes, the inner mem- which couples the proton-motive force of the OM: outer brane (IM) and the outer membrane (OM), IM to receptor-mediated uptake processes in membrane that are separated by the periplasm containing the OM (110). Periplasm: the peptidoglycan layer. The two membranes Whereas the composition, structure, and compartment have an entirely different structure and com- function of the OM have been known for between the inner position. Whereas the IM is a phospholipid decades, its assembly in the absence of en- and outer bilayer, the OM is an asymmetrical bilayer, ergy sources has remained largely enigmatic. membranes consisting of phospholipids and lipopolysac- All the components of the OM are syn- Peptidoglycan: charides (LPS) in the inner and outer leaflet, thesized in the cytoplasm or at the cyto- bacterial cell wall respectively. Additionally, these membranes plasmic face of the IM, and they have to consisting of sugar polymers covalently differ with respect to the structure of the be transported across the IM and through connected via integral membrane proteins. Whereas inte- the periplasm to reach their destination and oligopeptides gral IM proteins typically span the membrane to assemble into the OM. Recently, many Lipopolysaccharide in the form of hydrophobic �-helices, inte- new components involved in these processes (LPS): glycolipid gral OM proteins (OMPs) generally consist have been described, and in this review we constituting the of antiparallel amphipathic �-strands that fold focus on these recent developments. Apart outer layer of the into cylindrical �-barrels with a hydrophilic from studies in the classical model organisms, bacterial outer membrane interior and hydrophobic residues pointing E. coli and Salmonella enterica, much progress outward to face the membrane lipids (49). in this field was reached by studying N. menin- �-barrel: common structure of outer Both membranes also contain lipoproteins, gitidis, and a comparison between these sys- membrane proteins, which are anchored to the membranes via tems reveals important differences in these consisting of an N-terminal N-acyl-diacylglycerylcysteine, fundamental processes. Hence, what is true amphipathic with the protein moiety usually facing the for E. coli is not necessarily true for other antiparallel �-strands periplasm in the case of Escherichia coli. In bacteria. 192 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 (where Ar is an aromatic residue and X is any INTEGRAL OUTER MEMBRANE residue). Transmembrane �-strands of OMPs PROTEINS Lipoprotein: are particularly enriched with such motifs. Passage Across the IM and Through protein attached to Like skp mutants, surA mutants are viable, but the bacterial inner or the Periplasm the combination of an skp and a surA muta- outer membrane via tion results in a synthetically lethal phenotype Integral OMPs are synthesized in the cyto- an N-terminal lipid (75). Therefore, it was suggested that Skp and plasm as precursors with an N-terminal signal moiety SurA are functionally redundant and that they sequence, which is required for translocation Porin: protein that operate in parallel pathways for chaperone ac- forms water-filled across the IM. Two translocation machineries tivity (75). However, this is not the only pos- channels in the outer have been identified: the Sec system for the membrane sible explanation for the synthetic phenotype. translocation of unfolded proteins (23) and Sec system: general We favor the possibility that Skp and SurA act the Tat system, which transports proteins protein export sequentially in the same pathway (Figure 1). folded in the cytoplasm (57). However, to apparatus Skp may act as a holding chaperone, prevent- our knowledge, all OMPs studied to date are Chaperone: protein ing aggregation of nonnative OMPs in the transported via the Sec system, indicating that that guides another periplasm, and SurA may act as a folding chap- they reach the periplasm in an unfolded state. molecule to its erone. The demand for the holding chaper- After transport across the IM, the nascent destination, being one may be limited when the subsequent fold- the folded state or OMPs are accessible to periplasmic chaper- the right cellular ing and assembly steps are efficient. Under ones (30). These chaperones have been stud- compartment those conditions, the OMPs have only little ied extensively, mainly in E. coli. One of chance to aggregate and an skp null mutation these chaperones, Skp (seventeen-kilodalton- protein), immediately interacts with the OMPs as soon as they emerge from the Sec channel (37). The crystal structure of OM this homotrimeric protein resembles a three- Omp85 OMP Porin pronged grasping forceps that could bind a C nonnative OMP between the prongs and pro- YfgL NlpB YfiO tect it and prevent it from aggregating dur- ing passage through the periplasm (52, 109). SurA An skp mutant of E. coli is viable but contains C Periplasm decreased amounts of OMPs (16). Another periplasmic chaperone, SurA, was initially identified as a protein required for survival Skp during the stationary phase (102). Mutants in surA display an OMP assembly defect (56), and specifically the conversion of unfolded IM monomers of OMPs into folded monomers Sec appears affected in such mutants (77). The protein has peptidyl-prolyl cis/trans isomerase Figure 1 (PPIase) activity (56, 62), which nevertheless Model for OMP biogenesis. As soon as an OMP emerges from the Sec appears to be dispensable for the chaperone translocon, it is bound by the chaperone Skp, which may prevent function (5). Unlike most cytoplasmic chaper- aggregation in the periplasm. The C-terminal signature sequence of the OMP functions as a targeting signal and binds the periplasmic domain of ones, SurA is selective and preferentially binds Omp85. Other chaperones and folding catalysts, such as SurA, may act on nonnative OMPs over other proteins (5). By the OMP. After folding, the OMP inserts into the lipid bilayer, possibly in screening peptide libraries, Hennecke et al. between the Omp85 subunits. Oligomerization of certain OMPs, such as (38) demonstrated that SurA binds peptides porins, may occur after insertion. The exact function of the accessory rich in aromatic residues and preferentially lipoproteins YfgL, YfiO, and NlpB is not known. Another accessory lipoprotein recently identified, SmpA (84), is not indicated here. those containing Ar-Ar or Ar-X-Ar motifs " www.annualreviews.org Outer Membrane Biogenesis 193 by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 has only a limited effect. The demand for such a role for DegP in OMP assembly (75). How- a chaperone will increase when the subsequent ever, the role of DegP as a protease, remov- folding and assembly processes are hampered, ing misfolded or unfolded OMPs from the PPIase: e.g., in a surA mutant. Hence, it is not possible periplasm, may be more important than its peptidyl-prolyl to construct the skp surA double mutant. function as a chaperone in this respect (13). cis/trans isomerase Apart from SurA, the periplasm contains �E: alternative three other PPIases, PpiA (also known as sigma factor that Assembly into the OM guides RNA RotA), PpiD, and FkpA. PpiA is by far the polymerase to the most active of them, because its inactivation The insertion of OMPs into the OM has long promoters of genes leads to barely detectable residual PPIase ac- remained enigmatic and has been considered involved in relieving tivity (47). However, a ppiA null mutation had a spontaneous process (98). However, recent periplasmic stress no detectable effect on OMP assembly (47). In work has identified a proteinaceous machin- conditions contrast, a ppiD null mutation led to an overall ery that is essential for this process. reduction in the level and folding of OMPs, and a combination of ppiD and surA mutations Omp85, an essential component of the was lethal (19), suggesting functional redun- OMP assembly machinery. Recently, we dancy. However, PpiD is anchored in the IM, (105) identified a first component required whereas OMP folding is presumably initiated for OMP insertion, i.e., a protein designated after targeting to the OM, with which, indeed, Omp85 in N. meningitidis. Omp85 was found a proportion of the SurA molecules cofrac- to be essential for the viability of the bacteria, tionated (38). Later, it was reported that ppiD and homologues of the omp85 gene were mutants, like ppiA and fkpA mutants and even found in all gram-negative bacteria for which a ppiD ppiA fkpA triple mutant, did not show the genome sequence was available (106), different overall OMP patterns or OM per- suggesting its involvement in an important meability compared with the wild type and process. Moreover, in many of these genomes, that a ppiD surA double mutant could be con- including those of N. meningitidis and E. coli, structed, which had the same phenotype as a the omp85 gene is flanked by the skp gene, surA single mutant (44). Overall, there is little which encodes the periplasmic chaperone in- evidence for a direct role of PpiA, PpiD, or volved in OMP biogenesis, and rseP (formally FkpA in OMP biogenesis. designated yaeL), which encodes a protease Other proteins that may play a role at involved in the �E-dependent stress response the periplasmic stage of OMPs are DsbA and of E. coli that is induced upon accumulation of DsbC, two enzymes involved in the forma- unfolded OMPs in the periplasm (see below). tion and isomerization, respectively, of disul- All these features are consistent with a vital fide bonds. DsbA catalyzed the formation of role of Omp85 in OMP assembly. Upon de- a disulfide bond between two cysteines engi- pletion of Omp85 in a genetically engineered neered in OM porin PhoE at positions not strain, unfolded forms of all OMPs examined, accessible from the periplasm once the porin including porins, a siderophore receptor, an is inserted into the OM. This observation enzyme, and a secretin involved in type IV showed that the disulfide bond was formed pili assembly, accumulated as aggregates in during periplasmic transit and that at least par- the periplasm (105, 106). The role of Omp85 tial folding occurs prior to OM insertion (30). in OMP assembly was confirmed in E. coli, DegP is a protease that degrades unfolded or in which the corresponding gene, designated misfolded proteins in the periplasm but also yaeT, was also an essential gene. Either deple- has chaperone activity, as was shown in vitro tion of Omp85 or growth of a temperature- on the soluble substrates MalS and citrate syn- sensitive mutant at the restrictive temperature thase (87). A combination of surA and degP resulted in severe OMP assembly defects mutations was synthetically lethal, suggesting (27, 112, 114). Even more interestingly, a 194 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 homolog of Omp85 was essential for the the absence of the �E-dependent periplasmic assembly of �-barrel proteins into the outer stress response in N. meningitidis. In E. coli, membrane of mitochondria, a eukaryotic cell this stress response is induced upon accumu- organelle of endosymbiont origin (33, 54, lation of unfolded OMPs in the periplasm (for 69). Apparently, the OMP assembly pathway a review, see Reference 79). Such OMPs are is highly conserved during evolution. sensed by the PDZ domain of the membrane- A difference was observed with respect to bound protease DegS, upon which a prote- the fate of OMPs in Omp85-depleted cells olytic cascade is initiated that involves the of either N. meningitidis or E. coli. Whereas protease domain of DegS and RseP, result- unfolded OMPs accumulated as aggregates ing in the cleavage of the antisigma factor in such cells of N. meningitidis (105), Omp85 RseA and the release of �E in the cytoplasm depletion in E. coli primarily led to severely (Figure 2). The released �E then binds the reduced amounts of detectable OMPs (112, RNA polymerase core enzyme, resulting in 114). The difference is presumably caused by the transcription of the genes for periplasmic Figure 2 OM Periplasmic stress OMP response. Upon accumulation of Stress unfolded OMPs in C the periplasm, a stress response is initiated that starts Periplasm with the recognition of the C termini of the OMPs by the PDZ domain of the protease DegS. This recognition initiates a proteolytic cascade, IM resulting in the release of the alternative sigma factor �E, which binds RNA polymerase and leads �E to enhanced �E Periplasmic transcription of stress response genes encoding RNA periplasmic polymerase chaperones, such as Skp and SurA, and the protease DegP. The �E response also skp sRNAs leads to production surA of sRNAs that degP negatively regulate OMPs OMP expression. " www.annualreviews.org Outer Membrane Biogenesis 195 Protease PDZ RseB RseB Protease by CNRS-multi-site on 10/11/07. For personal use only. RseP RseP RseA DegS DegS RseA Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org PDZ ANRV322-MI61-11 ARI 6 August 2007 16:59 chaperones, such as Skp and SurA, and for resides in the periplasm but is firmly associ- the potent protease DegP. Together these ated with many different integral OMPs via chaperones and protease relieve the periplas- an N-terminal peptide (71). RmpM proba- sRNA: small mic stress. Moreover, the �E response results bly anchors the OM to the underlying pep- regulatory RNA in the production of small regulatory RNAs tidoglycan layer (88), and it has no particular (sRNAs) in Enterobacteriaceae that prevent function in OMP biogenesis. Cross-linking OMP expression at the translational level (31, experiments revealed that the Omp85 ho- 43, 68). Thus, in E. coli, misfolded OMPs molog of E. coli forms a complex with three are rescued or degraded in the periplasm and lipoproteins, YfgL, YfiO, and NlpB (114). A OMP synthesis is inhibited during the stress similar complex was identified upon a pro- period. Although chaperones such as Skp and teomic analysis of OMP complexes resolved SurA are present in N. meningitidis, many es- by blue-native PAGE (93). Copurification ex- sential components of the signal transduction periments using strains with mutations in the pathway are lacking. Whereas E. coli contains genes for various components of the complex three genes for the related proteases DegP, indicated that YfgL and YfiO directly interact DegS, and DegQ (107), only a homologue of with Omp85, whereas NlpB is associated with DegQ can be found in N. meningitidis (en- the complex via YfiO (58). coded by the NMB0532 locus in the N. menin- The yfgL gene was originally identified gitidis strain MC58). Moreover, homologues in a screen for suppressors that restored the of RseA and RseB appear to be absent. Al- OM permeability defect of a partial loss-of- though there is an alternative � factor belong- function mutation in the imp gene (29, 78), ing to the ECF (extracytoplasmic factor) fam- which encodes an OMP involved in LPS ily (to which �E also belongs) encoded on the biogenesis (see below). Although yfgL is not meningococcal chromosome (i.e., NMB2144 an essential gene, null mutations create an in strain MC58), it seems to have an entirely OM permeability defect and result in reduced different function. Its inactivation in the re- amounts of OMPs (67, 78, 114), consistent lated bacterium Neisseria gonorrhoeae did not with a role in OMP assembly. Remarkably, al- affect global gene expression as analyzed by though the yfgL gene is widely disseminated microarray analysis, whereas its overexpres- among gram-negative bacteria, we could not sion affected only very few genes, including identify a homolog in the sequenced genomes those for methionine sulfoxide reductase (35). of N. meningitidis and N. gonorrhoeae. Thus, the �E-dependent periplasmic stress re- The yfiO gene is essential in E. coli (67), sponse appears to be absent in the pathogenic but a mutant with a transposon insertion near Neisseriaceae. As a result, when there is an the 3 end of the gene was viable (114). This OMP assembly defect, OMPs will continue to mutant showed increased OM permeability be synthesized and they will accumulate in the and reduced amounts of OMPs, and a yfiO periplasm, where they will form aggregates. depletion strain showed a phenotype similar to that of the yaeT/omp85 depletion strain of Omp85 is part of a multisubunit complex. E. coli (58). In N. gonorrhoeae, a transposon Sodium dodecyl sulfate polyacrylamide gel insertion in the middle of the yfiO homolog electrophoresis (PAGE) analysis under non- was described (32). This mutant was viable, denaturing conditions revealed that Omp85 showed a reduced cell size, and was transfor- of N. meningitidis is present in a high- mation deficient; therefore, the gene was des- molecular-weight complex (105). The only ignated comL. However, attempts to introduce other protein identified in this complex was other comL truncations into the chromosome the RmpM protein ( J. Geurtsen, R. Voulhoux failed (32), and we have not yet succeeded & J. Tommassen, unpublished results), which to generate a complete comL deletion in the has a peptidoglycan-binding motif and largely chromosome of N. meningitidis (E. Volokhina, 196 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 M.P. Bos & J. Tommassen, unpublished re- FtsQ/DivIB family of IM proteins involved in sults). Hence, also in the Neisseriaceae, YfiO cell division. The bacterial Omp85 homologs (ComL) appears essential for viability and the are unique in having five of these POTRA do- POTRA: N-terminal half of the protein appears suffi- mains, whereas the number of such domains polypeptide cient for partial functionality. The ComL pro- in the other proteins with known functions transport associated tein is covalently linked to the peptidoglycan, is restricted to one or two. Another member both in N. gonorrhoeae and when expressed of the Omp85 superfamily that is widely in E. coli (32). Furthermore, yfgL mutations disseminated among gram-negative bacteria affected peptidoglycan synthesis, possibly by (encoded by the ytfM gene in E. coli and the regulating the activity of lytic transglycosy- NMB2134 locus in N. meningitidis strain lases (29). Thus, both YfgL and YfiO might MC58) contains three POTRA domains. have a role in modulating the peptidoglycan The function of this protein is unknown (92). to facilitate the passage of OMPs through this To study its structure and function in layer. more detail, we produced the Omp85 protein Whereas yfiO, like yfgL and omp85, is of E. coli in inclusion bodies, which were widely disseminated among gram-negative isolated, and refolded the protein into the bacteria, this is less so for nlpB. Null mutants native conformation in vitro (76). The re- in nlpB of E. coli are viable; they show only folded protein formed oligomers, presumably moderate OM permeability defects and no tetramers, similarly as reported for another obvious defects in OMP assembly (67, 114). member of the Omp85 superfamily, i.e., However, because an nlpB surA double knock- HMW1B, the TpsB component of a TPS out mutant has a synthetic lethal phenotype system of Haemophilus influenzae (95). Of (67), it is clear that NlpB also has a direct role note, the interactions between the subunits in in OMP assembly, possibly redundant to that this homo-oligomeric complex are not stable; of the periplasmic chaperone SurA. blue-native PAGE indicated equilibrium between monomeric and oligomeric forms Structure of Omp85. On the basis of (76). Consistently, the hetero-oligomeric the sequence, we (105) have proposed Omp85/YfiO/YfgL/NlpB complex identified that Omp85 consists of two domains, a in vivo appeared to consist of one copy of C-terminal �-barrel embedded in the OM each subunit (93). and an N-terminal domain extending into the periplasm. Support for this model was Interaction of Omp85 with its substrate obtained in proteolytic digestion experi- OMPs. When reconstituted in planar lipid ments, which resulted in the degradation of bilayers, both in vitro refolded Omp85 and the N-terminal domain and left the predicted Omp85 extracted from E. coli cell envelopes membrane-embedded �-barrel domain intact formed narrow ion-conductive channels (76, (76, 91). The periplasmic extension contains 91). This property was used to study the in- repeats of a conserved domain, POTRA teraction between Omp85 and its substrate (polypeptide transport associated), suggested OMPs, emanating from the idea that such an to have chaperone-like qualities (81). Such interaction would affect the channel activity. POTRA domains were further identified in Indeed, nonnative OMPs drastically enhance other members of the Omp85 superfamily, Omp85 channel activities (76), showing that which includes the Omp85 homologs of mi- these substrates interact directly with Omp85. tochondria, the Toc75 OM component of the Furthermore, using mutant OMPs and syn- chloroplast protein import machinery, and thetic oligopeptides in this assay, we demon- the OM-localized TpsB component of the strated that Omp85 specifically recognizes a two-partner secretion (TPS) systems of gram- C-terminal motif in its substrates (76) that was negative bacteria, and in members of the shown to be required for efficient assembly " www.annualreviews.org Outer Membrane Biogenesis 197 by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 of these proteins into the OM in vivo (94). expression was tolerated and allowed for its This C-terminal signature motif, which con- assembly into the OM (21). Similarly, several sists of a phenylalanine (or tryptophan) at the studies reporting the assembly of neisserial C-terminal position, a tyrosine or hydropho- OMPs into the E. coli OM in vivo (113) may bic residue at position 3 from the C terminus, be explained by low expression levels. Pulse- and also hydrophobic residues at positions 5, chase experiments in E. coli overexpressing 7, and 9 from the C terminus, is present in PhoE revealed the existence of two assem- most bacterial OMPs, including porins, re- bly pathways: approximately half of the PhoE ceptors, enzymes, and autotransporters. It is molecules assembled within the 30-s pulse interesting to note that the same signature period into their native conformation in the sequence is recognized by the PDZ domain OM, whereas the other half of the molecules of DegS when unfolded OMPs accumulate followed much slower kinetics and allowed in the periplasm (108), thereby initiating the for the detection of several assembly inter- �E-dependent periplasmic stress response (see mediates (42). In these assays, the mutant above). PhoE lacking the C-terminal phenylalanine In the planar lipid bilayer experiments an followed only the slower kinetic pathway. This OMP of N. meningitidis, in contrast to E. coli study underscores the role of the C-terminal OMPs, did not enhance the activity of the E. signature sequence as a targeting signal and coli Omp85 channels (76). Consistently, high- indicates that there must be alternative, less level expression of neisserial OMPs in E. coli efficient targeting signals in OMPs. Further- is often lethal and leads to their misassembly, more, kinetic partitioning between OM incor- suggesting that the E. coli OMP assembly ma- poration and aggregation of periplasmic in- chinery cannot deal efficiently with neisserial termediates may explain the observation that proteins. Indeed, expression of E. coli omp85 OMPs with defective signature sequences are cannot complement an omp85 mutation in N. still assembled into the OM at low expression meningitidis and vice versa (E. Volokhina, V. levels, when the kinetics of aggregation is low Robert, M.P. Bos & J. Tommassen, unpub- and hence the time span for assembly into the lished observations). Although the C termini OM is elongated (76). of neisserial OMPs do contain the signature What could be the nature of the al- sequence with the features outlined above, ternative targeting sequences in OMPs? Of they are further characterized by the presence note, the C-terminal signature sequence con- of a positively charged residue at the penulti- tributes an amphipathic �-strand to the �- mate position, which could inhibit the inter- barrel in the folded protein. In the absence action with E. coli Omp85. Indeed substitu- of the C-terminal phenylalanine, Omp85 tion of this positively charged residue in the N. may recognize, though less efficiently, in- meningitidis porin PorA drastically improved ternal �-strands, many of which also end PorA assembly into the E. coli OM in vivo (76). with an aromatic residue. In some classes Thus, although the OMP assembly machin- of OMPs, the C-terminal signature motif ery is highly conserved among gram-negative could not be discerned (94). Perhaps, in bacteria, species-specific adaptations seem to these cases also, Omp85 recognizes an in- have occurred during evolution. ternal �-strand. One of the major OMPs of Of note, the C-terminal signature se- E. coli, OmpA, consists of two domains, an quence of OMPs is not absolutely essential N-terminal OM-embedded �-barrel and a C- for assembly into the OM. Thus, whereas terminal periplasmic extension. The signature the high-level expression of a mutant form of motif was found in this case at the end of the�- the E. coli porin PhoE lacking the C-terminal barrel domain (94) and its importance in OM phenylalanine was lethal and resulted in its targeting was demonstrated in a deletion anal- periplasmic aggregation (21, 94), its low-level ysis (48). Although this example demonstrates 198 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 that the targeting motif can be located inter- rough mutants of E. coli and Salmonella ty- nally in the primary structure of an OMP, this phimurium, which contain truncated LPS appears to be a rather exceptional case be- molecules (2, 51), a role of LPS in OMP cause the C-terminal extension of an OMP assembly has been suggested. Consistently, sequence with, for example, a His tag usually the OM porin PhoE of E. coli could be con- results in severe assembly defects (91). verted in vitro into a folded monomeric form Another class of OMPs that lack the C- in the presence of LPS, detergent, and diva- terminal signature motif is constituted by lent cations. This folded monomer appeared TolC and its homologs, which are involved in to be an assembly intermediate because it type I protein secretion and drugs extrusion. could subsequently be converted into its na- In its native trimeric structure, TolC forms a tive trimeric OM-inserted form (22). Also �-barrel in the OM, to which each monomer for other OMPs, LPS-dependent folding in contributes four �-strands, whereas the ma- vitro has been reported (83). However, LPS- jor portion of the protein extends as long independent in vitro folding conditions were �-helices into the periplasm (53). TolC is de- later established for many OMPs, including pendent on Omp85 for its assembly (112); PhoE (42), arguing against a specific role of hence, possibly also in this case, an inter- LPS in OMP folding. Moreover, an lpxA mu- nal �-strand is recognized. In contrast, Wza, tant of N. meningitidis, which is completely an OMP involved in the export of capsu- defective in LPS biosynthesis, appeared to be lar polysaccharides, has an entirely different viable (90), and all OMPs examined, including structure; the membrane-embedded portion porins, were correctly assembled in vivo into of this octameric protein consists of a barrel the LPS-free OM of this mutant (89). Nev- of eight amphipathic �-helices, to which each ertheless, species-specific differences with re- monomer contributes one helix (28). It is con- spect to the LPS dependency of OMP assem- ceivable that this protein assembles entirely bly cannot yet be excluded at this stage. independently of the Omp85 machinery. The The crystal structure of E. coli Skp re- secretins form a class of OMPs involved in di- vealed a putative LPS-binding site (109), con- verse processes, including type II and type III sistent with the earlier description of the pro- protein secretion, type IV pili biogenesis, and tein in Salmonella minnesota as an LPS-binding the extrusion of filamentous phages (7). The protein (34). Thus, LPS may exert its role structure of these large multimeric proteins is in OMP biogenesis via Skp. Furthermore, it not known at atomic resolution, but they ap- was demonstrated in protease-digestion as- pear to be rather poor in �-sheet content (14), says that binding of LPS and phospholipids which could indicate that they also follow a could inversely modulate the structure of Skp different assembly pathway. However, at least in vitro (20). Thus, after binding nonnative one secretin, PilQ of N. meningitidis, depends OMPs at the IM, a conformational change on Omp85 for its assembly (105). Perhaps, the triggered by LPS binding may release the binding of these proteins and other OMPs cargo at the OM. Note that LPS is present that lack the C-terminal signature motif is only in the outer leaflet of the OM. Hence, indirect and requires accessory factors, such Skp should bind to LPS molecules that have as the lipoproteins YfiO, YfgL, and NlpB, or not yet reached their destination. Consis- specific chaperones, such as the pilotins in the tently, OMP biogenesis is heavily affected by case of the secretins (3). cerulinin, a drug that inhibits lipid synthe- sis (8). However, the putative LPS-binding site in E. coli Skp is largely conserved in Role of LPS in OMP Biogenesis N. meningitidis Skp, where OMP biogene- Since the observation that the amounts of sev- sis is independent of LPS. Hence, the role eral OMPs are severely decreased in deep- of this putative LPS-binding site remains to " www.annualreviews.org Outer Membrane Biogenesis 199 by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 be determined in site-directed mutagenesis mains. Binding initiates folding, which results experiments. in the release of Skp (20) and may be assisted We speculate that the role of LPS in OMP by the presumed chaperone activities of the Imp: increased biogenesis is restricted to late stages after POTRA domains and by periplasmic proteins membrane the insertion into the OM, such as the sta- such as SurA and DsbA. Binding of an OMP permeability bilization of porin trimers (55), which pre- also results in a conformational change in the sumably requires some LPS-mediated rear- C-terminal domain of Omp85, which is re- rangements in the cell surface-exposed loops flected by the increased pore activity observed of the proteins. The severely reduced amounts in the planar lipid bilayer experiments (76). of OMPs in deep-rough mutants may be ex- This conformational change allows the OMPs plained by the induction of the �E response to insert into the OM, possibly in between the in such mutants, resulting in the production Omp85 subunits. Dissociation of the Omp85 of sRNAs that inhibit the synthesis of many subunits releases the assembled OMPs into OMPs (see above). Changes in LPS structure the OM, where final conformation changes in induce the �E response in E. coli by a hitherto the cell surface-exposed loops may be induced unknown mechanism (97). At least one OMP upon interaction with LPS. of E. coli, TolC, appears totally unaffected by The specific role of the accessory lipopro- LPS structure (111). Presumably, the expres- teins YfgL, YfiO, and NlpB has not been ad- sion of TolC is unaffected by the sRNAs. The dressed experimentally so far. Of note, no ho- assembly of this lipid-independent OMP is in- mologs of these proteins have been implicated dependent of the accessory component YfgL in the assembly of �-barrel proteins into the of the Omp85 assembly machinery (15). Of mitochondrial OM, suggesting that their role note, a homolog of the yfgL gene is lacking in is less crucial, although YfiO is definitely es- N. meningitidis, in which the assembly of all sential, at least in E. coli (67). These proteins OMPs is independent of LPS. Hence, there may play a role in the recognition of OMPs may be an additional role for LPS in the as- that do not display the C-terminal signature sembly of lipid-dependent OMPs in E. coli, motif, which includes the essential protein and YfgL may play a role specifically in the Imp (increased membrane permeability), in assembly of this class of OMPs. In this re- modulating the peptidoglycan layer, and/or spect, it may indeed be relevant that the yfgL they may function as chaperones in the fold- mutants were initially picked up as suppres- ing of OMPs. YfgL may have a role in coor- sors of a partial loss-of-function imp mutant dinating the assembly of a subclass of OMPs (29), and the imp gene product is specifically that require LPS for their assembly, but such a involved in LPS biogenesis (9) (see below). role is difficult to assess in E. coli, in which as- sembly defects of OMPs are associated with a feedback inhibition on their synthesis via Model for OMP Biogenesis �E-induced sRNAs, whereas N. meningitidis, We propose the following model for OMP which does not display such a feedback inhibi- biogenesis (Figure 1). After their transport tion, does not have a YfgL homolog. The ab- through the Sec translocon, nascent OMPs sence of this feedback inhibition mechanism are immediately bound by Skp, which may as- makes N. meningitidis a favorable organism to sist their release from the IM and prevent their study the role of other assembly factors. aggregation in the periplasm. The Skp/OMP complex is targeted to the Omp85 complex in LIPOPROTEINS the OM, whereby the C-terminal signature motif of the OMPs functions as the primary Bacterial lipoproteins are membrane targeting signal that binds directly to Omp85, attached via an N-terminal N-acyl- presumably to its N-terminal POTRA do- diacylglycerylcysteine. Lipidation and folding 200 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 of these proteins take place after their translo- cation over the IM via the Sec machinery. OM Prior to cleavage of the signal sequence, a ? diacylglycerol group is transferred by the enzyme Lgt from phosphatidylglycerol to LolB C-X the sulfhydryl group of the cysteine that is invariably present at the +1 position relative to the processing site (82). Subsequently, the Periplasm LolA diacylglycerylprolipoprotein is processed by a dedicated signal peptidase, signal peptidase II LolA (24), after which the free �-amino group of Sorting the cysteine is acylated by the enzyme Lnt (36), yielding the mature lipoprotein. Lipoproteins are sorted to the IM or OM LolC LolE IM Sec according to a sorting signal that comprises LolD LolD the amino acids flanking the lipidated cysteine ATP ADP ATP ADP in the mature protein (101). Lipoproteins Figure 3 lacking an IM retention signal, usually an aspartate at the +2 position of the mature Lipoprotein transport through the bacterial cell envelope. After their transport via the Sec system and subsequent lipidation at the N-terminal protein, are transported to the OM by the cysteine (C), lipoproteins bind to the ABC-transporter LolCDE, provided Lol system (Figure 3). The first component they do not possess a Lol-avoidance motif, which usually is an aspartate of the Lol system was identified in an in vitro (D) flanking the N-terminal C (C-D). Lipoproteins with another amino system, whereby lipoproteins were synthe- acid at the +2 position (X) are destined for the OM. Energy from ATP sized in a radioactive form in spheroplasts of hydrolysis by LolD is transferred to LolC and LolE and utilized to open the hydrophobic LolA cavity to accommodate the lipoprotein. When the E. coli (60). The mature forms of the newly LolA-lipoprotein complex interacts with the OM receptor LolB, the synthesized lipoproteins were retained at lipoprotein is transferred to LolB and inserted into the OM. Further the surface of the spheroplasts, from which transport to the outer leaflet of the OM, if applicable, occurs through they could be released upon addition of a unknown mechanisms. periplasmic extract. The active component of the periplasmic extract was identified and decrease in lipoprotein binding affinity. ATP designated LolA. Furthermore, a complex of hydrolysis is then required for transfer of the the three proteins LolC, LolD, and LolE, lipoprotein from LolC/E to the periplasmic which constitutes an ATP-binding cassette chaperone LolA (41). The LolA-lipoprotein (ABC) transporter in the IM, is required complex crosses the periplasm and interacts for lipoprotein transport (65). The integral with an OM receptor, LolB (61). The lipopro- membrane components of this transporter, tein is then transferred to LolB according LolC and LolE, show considerable sequence to the affinity difference between LolA and similarity and may function as a heterodimer. LolB. LolA and LolB are structurally similar; Of note, N. meningitidis contains only one of both contain a hydrophobic cavity. The Spheroplasts: these proteins (encoded by locus NMB1235 LolA cavity is composed mostly of aromatic bacterial cells of in strain MC58), which may form a homod- residues, whereas the cavity in LolB is made which the outer membrane and the imer. The current model (Figure 3) states mostly of leucine and isoleucine residues, peptidoglycan layer that lipoproteins destined for the OM are first which is likely to explain the difference in have been disrupted bound by LolC/E in an ATP-independent lipoprotein-binding affinity (64, 96). by treatment with manner. This binding results in an increase in In E. coli, all known lipoproteins face the EDTA and lysozyme the affinity of LolD for ATP. Subsequently, periplasm, but in other bacteria, most no- ABC: ATP-binding ATP binding to LolD causes a conformational tably in members of the spirochetes (12), cell cassette change in the LolC/E moiety that results in a surface-exposed lipoproteins also are present. " www.annualreviews.org Outer Membrane Biogenesis 201 C-X C-X C-X C-X C-X C-D C-X C-D/X by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 Whether lipoprotein transport over the OM acids at the 2, 3, 2 , and 3 positions and phos- occurs through an extension of the Lol sys- phate groups at the 1 and 4 positions. The tem or by an unrelated transport system primary 3-hydroxy fatty acids may be substi- is presently unclear. The only cell surface- tuted with secondary fatty acids. The lipid A exposed lipoprotein studied in this respect is biosynthetic pathway, also known as the Raetz the starch-disbranching enzyme pullulanase pathway (74), has been characterized in detail, of Klebsiella oxytoca (72). Pullulanase contains mostly in E. coli and Salmonella typhimurium, an aspartate at position +2, indicating that but it appears to be highly conserved among it is not a substrate for the Lol system. In- other gram-negative bacteria (74). Remark- deed the transport of pullulanase to the cell ably, although LPS has so far only been de- surface requires a type II protein secretion tected in gram-negative bacteria, homologs of apparatus. In the absence of such an appa- the genes encoding the lipid A biosynthetic ratus, pullulanase is retained in the IM (72). enzymes have also been found in sequenced Thus, in K. oxytoca, sorting between periplas- plant genomes (74). mically and cell surface-exposed lipoproteins The core oligosaccharide is much more takes place at the IM level. However, this is variable between bacterial species. In addi- not necessarily always the case. N. meningitidis tion, a huge amount of LPS heterogeneity is contains several cell surface-exposed lipopro- created by numerous modifications of both teins, including LbpB and TbpB, but the se- the lipid A part and the core part of LPS quenced genomes do not reveal the presence (73). The modifying enzymes, which are lo- of a type II secretion apparatus (104). Further- calized in different cellular compartments, are more, LbpB and TbpB contain an isoleucine not generally conserved; so different species and a leucine, respectively, at the +2 posi- can express uniquely modified types of LPS tion, suggesting they are substrates for the (103). The O-antigen, if present, is the most Lol system. Thus, these lipoproteins may be variable part of LPS and shows even a high transported to the cell surface via an exten- degree of variability between different strains sion of the Lol system, which remains to be of the same species. identified. Transport to the Cell Surface LIPOPOLYSACCHARIDE In contrast to the understanding of its biosyn- thesis, the mechanism of the transport of LPS Structure and Biosynthesis from its site of synthesis to its final destina- LPS is a complex glycolipid exclusively tion forms a much less complete picture (25). present in the outer leaflet of the OM of gram- It is clear that the lipid A core moiety and negative bacteria. It consists of a hydrophobic the O-antigen subunits, if present, are trans- membrane anchor, lipid A, substituted with ported separately over the IM. The O-antigen an oligosaccharide core region, which in some subunits are transferred over the IM by one bacteria (e.g., in most E. coli strains, but not in of three different routes: the Wzy-, ABC- the laboratory strain E. coli K-12 and also not transporter-, or synthase-dependent pathway in N. meningitidis) is extended with a repeating (74). After polymerization, the subsequent lig- oligosaccharide, the O-antigen. These differ- ation of the O-antigen to the lipid A core ent LPS constituents are synthesized at the cy- moiety at the periplasmic side of the IM is toplasmic leaflet of the IM. The lipid A moi- an incompletely understood process that in- ety of LPS is rather well conserved among volves at least the WaaL ligase (45). gram-negative bacteria. It usually consists of a The translocation of the lipid A core moi- �-1,6-linked d-glucosamine disaccharide car- ety over the IM is mediated by an ABC fam- rying ester- and amide-linked 3-hydroxy fatty ily transporter called MsbA, as inferred from 202 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 the accumulation of LPS in the IM of a depletion strain. Upon depletion of Imp, temperature-sensitive E. coli msbA mutant at newly synthesized LPS was not accessible to the restrictive temperature (116). The LPS LPS-modifying enzymes in the OM, showing accumulated was not modified by periplas- that it did not reach the outer leaflet of the mic enzymes, demonstrating that it was not OM (115). In E. coli, another OM component transported to the periplasmic leaflet of the was identified to play a role in LPS transport, IM (26). More evidence for a role of MsbA i.e., the essential lipoprotein RlpB. Depletion in LPS transport came from a study of an of this lipoprotein resulted in a phenotype msbA mutant of N. meningitidis. The viabil- similar to that expressed upon depletion of ity of LPS-deficient N. meningitidis mutants Imp. Moreover, Imp and RlpB exist in a com- makes this organism well suited for the study plex. Depletion of Imp or RlpB resulted in of LPS biogenesis, because clean knockouts an increased total cellular LPS content (115), of genes involved in this process can be con- indicating that in E. coli, in contrast to N. structed. Indeed, MsbA is a nonessential pro- meningitidis, defective transport does not lead tein in N. meningitidis (99). A neisserial msbA to feedback inhibition on LPS biosynthesis. mutant produced only low amounts of LPS, a Imp and RlpB homologs are widely dis- feature indicative of a defect in LPS transport seminated among gram-negative bacteria, in this species. In N. meningitidis, biosynthe- suggesting that the mechanism of LPS trans- sis and transport of LPS are coupled in such a port is highly conserved. Imp is predicted to way that synthesis is reduced under conditions contain a �-barrel domain embedded in the in which transport is halted (9, 99). OM, with a long N-terminal domain and a The subsequent steps in LPS transport short C-terminal domain extending into the to the exterior of the bacterium have long periplasm (Figure 4). In the conserved do- remained obscure. However, an OM compo- main database at the National Center for nent required for the appearance of LPS at the Biotechnology Information (59), the �-barrel bacterial cell surface was identified recently. domain is recognized as a conserved domain, This component is an OMP known as Imp designated OstA-C, in all Imp homologs. An- or OstA (organic solvent tolerance), because other conserved domain, COG1934, is recog- E. coli strains expressing mutant versions of nized in the N-terminal periplasmic extension this protein showed altered membrane per- of many but not all Imp homologs (Figure 4). meability (1, 80). Imp is an essential protein in Nevertheless, all Imp homologs show se- E. coli. In a conditional imp mutant, correctly quence similarity over the entire length, and folded OMPs accumulate in aberrant mem- also when the COG1934 domain is not branes with an increased density, indicative recognized. of an altered lipid/protein ratio (11). The Additional components putatively in- precise role of Imp was demonstrated in volved in LPS translocation were identified N. meningitidis. Imp was not essential in this by Sperandeo et al. (86), who discovered sev- bacterial species, allowing the construction eral new essential genes in E. coli, some of of an imp deletion mutant. The phenotype which appeared to play a role in cell enve- of this mutant demonstrated a role for Imp lope biogenesis. Depletion of recombinant in LPS biogenesis: It produced less than bacteria for the proteins encoded by two of 10% of wild-type levels of LPS, which were these genes resulted in similar phenotypes as not accessible to LPS-modifying enzymes described for Imp- and RlpB-depleted cells: recombinantly expressed in the OM or added The bacteria exhibited an altered OM density to the extracellular medium (9). Therefore, and an increased cellular LPS content. The Imp appears to function in the transport of genes, which form an operon, were designated LPS over the OM to the cell surface. This lptA and lptB (LPS transport) (85). Unfortu- role of Imp was confirmed in an E. coli imp nately, we cannot use the same designations in " www.annualreviews.org Outer Membrane Biogenesis 203 by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 # OstA_C COG1934 102 Imp 195 OstA_C LptA/ 319 COG1934 NMB0355 YrbK 214 COG3117 1 Nostoc sp. PCC712 COG3117 COG1934 Acidobacteria bacterium COG1934 COG1934 1 COG3117 Ellin345 1 COG3117 Acinetobacter sp. ADP1 OstA_C Figure 4 Conserved protein domains present in LPS transport components. The Conserved Domain Database at the National Center for Biotechnology Information was searched in December 2006 with the proteins shown at the left. The type and number (#) of retrieved architectures are shown. The species expressing the most unusual architectures are indicated at the right. A topology model for Imp, based on manual inspection for putative �-strands, is shown at the top right-hand corner. N. meningitidis, for which the acronym lptA is of Imp (Figure 4), indicative of a common used for a gene encoding an LPS phospho- function. ethanolamine transferase (17). LptB is a 27-kDa protein present in a Also the neisserial homolog of E. coli 140-kDa IM complex; unfortunately, no in- LptA, encoded by the NMB0355 locus in teracting partners were identified (93). The N. meningitidis strain MC58, plays a role in protein possesses the typical features of an LPS transport: In these bacteria, the cor- ABC protein but has no obvious membrane- responding gene is not essential, and its spanning segments. deletion results in severely decreased lev- Thus, the current data suggest the involve- els of LPS (10). The LPS is accessible to ment of a novel ABC transporter in LPS trans- periplasmic LPS-modifying enzymes in this port. LptB is the ABC component of this mutant, indicating that the LptA/NMB0355 transporter, but the cognate integral mem- protein acts at a step after translocation by brane component remains to be identified. MsbA (10). The LptA protein of E. coli was The protein encoded by the yrbK gene, which found in the soluble periplasmic fraction (85), is located immediately upstream of the lptAB but we found the majority of the corre- operon and which is also essential for viability sponding neisserial protein (NMB0355) in in E. coli (86), may be a part of the transporter. the membrane fraction, although it has no The genetic organization of the yrbK-lptA- obvious membrane-spanning segments (M.P. lptB locus is highly conserved among gram- Bos & J. Tommassen, unpublished obser- negative bacteria, and the observation that a vations). LptA largely consists of the con- conserved protein domain present in YrbK served domain COG1934, the same domain is sometimes present in one polypeptide to- found in the N-terminal periplasmic domain gether with conserved domains from Imp or 204 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org COG1934 ANRV322-MI61-11 ARI 6 August 2007 16:59 LptA (Figure 4) is suggestive for a role of through the periplasm in a soluble complex YrbK in LPS transport. However, secondary with a chaperone that shields its hydrophobic structure predictions of YrbK show only one moiety, similar to the Lol system for lipopro- putative transmembrane helix, making it un- tein transport (Figure 3). Indeed, the recent likely that this protein functions as the integral identification of a novel ABC transporter in- membrane component of the ABC trans- volved in LPS transport may suggest sim- porter, as such components of ABC trans- ilarities with the lipoprotein transport sys- porters usually contain multiple transmem- tem. The LptA protein, which is a soluble brane helices (6). periplasmic protein in E. coli (86), may func- tion as the LPS chaperone, as LolA does for lipoproteins. Furthermore, while LolA passes Models for LPS Transport its cargo to the structurally related OM recep- Two models for LPS transport through the tor LolB, LptA may pass the LPS molecules cell envelope have been considered, and to the periplasmic N-terminal domain of Imp, with the current situation, no model can be which shows sequence similarity to LptA. The considered definitively proven or discounted �-barrel of Imp may form a channel for fur- (Figure 5). One possibility is that LPS passes ther transport to the cell surface. Secondary Membrane contact sites Lol system-like OM Imp Imp RlpB RlpB Periplasm LptA LptA YrbK YrbK YrbK IM ?? MsbA MsbA LptB LptB ATP ADP ATP ADP ATP ADP ATP ADP Figure 5 Models for LPS transport through the bacterial cell envelope. LPS is synthesized at the inner leaflet of the IM and transported over the IM by the ABC transporter MsbA. LPS then travels through the periplasm by an unknown mechanism that could resemble the Lol system (right). LptB together with an unknown (?) membrane protein and possibly YrbK may form an ABC transporter that delivers LPS to the periplasmic chaperone LptA. LPS may then be transferred from LptA to the periplasmic domain of Imp, which may have a structure similar to LptA. Alternatively, LPS transport may take place at contact sites between the two membranes (left). YrbK, LptA, LptB, and an unknown transmembrane domain may function in the formation of these contact sites. Finally, Imp and RlpB are required to transfer LPS to the cell surface, perhaps by acting as a flippase complex. " www.annualreviews.org Outer Membrane Biogenesis 205 by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 structure predictions for LptA show many order to reach the OM, they first need to ro- �-strands, possibly forming a soluble beta- tate (flip-flop) over the membrane. It is not barrel, resembling LolA (10, 96). The LptB clear whether a dedicated flippase is neces- protein could be the functional LolD ho- sary for this process. The LPS transporter molog of the LPS transport system. As ex- MsbA was also implicated in phospholipid plained above, the putative LolC/E compo- transport because the conditional E. coli msbA nents of such an LPS transport system remain mutant accumulated both LPS and phospho- to be found. lipids in the IM under restrictive conditions The other model postulates that LPS ac- (116). However, an msbA mutant of N. menin- tually never leaves its membranous environ- gitidis appeared viable and still made a dou- ment and that it is transported at contact ble membrane, showing that at least in this sites between the IM and OM (Figure 5). bacterium MsbA is not required for phospho- The first indication for the existence of such lipid transport (99). Another distant msbA ho- sites, known as zones of adhesion or Bayer molog in N. meningitidis, i.e., the NMB0264 junctions, came from electron microscopy locus in strain MC58, could be disrupted studies (4), although fixation procedures may without causing any obvious phenotype (M.P. have affected the results (46). Later, M�hlradt Bos, unpublished observations). Moreover, et al. (63) reported that newly synthesized various �-helical membrane-spanning pep- LPS appears in patches in the OM, close tides, but curiously not MsbA, induced phos- to the membrane contact sites. Membrane pholipid translocation in synthetic lipid bi- fractionation studies showed the existence layers (50). Thus, flip-flop of phospholipids of a minor fraction, designated OML, that may not require a specific transporter but contains IM, peptidoglycan, and OM. This merely the presence of the typical �-helical membrane fraction, which contained pepti- membrane-spanning segments of some IM doglycan biosynthesis activity, may represent proteins. The next steps in phospholipid bio- membrane contact sites. Pulse-chase exper- genesis, i.e., transport through the periplasm iments combined with fractionation proce- and incorporation into the inner leaflet of the dures showed that newly synthesized LPS OM, remain obscure. Unlike LPS transport, transiently passed through this fraction on its the transport of phospholipids was halted in way to the OM (40). Furthermore, when a spheroplasts, and unlike lipoproteins, newly similar approach was used that led to the iden- synthesized phospholipids could not be re- tification of LolA (see above), newly synthe- leased from the spheroplasts upon addition of sized LPS could not be released from sphero- a periplasmic extract (100). Thus, the trans- plasts upon addition of periplasmic extracts. port mechanism appears different from those Rather, LPS transport from IM to OM con- of LPS and lipoproteins. Any components in- tinued in the spheroplasts, suggesting that this volved in phospholipid transport remain to be process does not involve a soluble periplas- identified. mic component and proceeds via contact sites (100). In this model, the LptA, LptB, and PERSPECTIVES YrbK components may have a role in the for- mation of these contact sites (Figure 5). In the past few years, much progress has been made in the field of OM biogenesis with the identification of many new compo- PHOSPHOLIPIDS nents involved in the process. The field will The major OM phospholipids of E. coli are rapidly move forward, gaining mechanistic phosphatidylethanolamine and phosphatidyl- insights to which structural analysis of the glycerol. Phospholipids are synthesized at the newly identified components by X-ray crystal- cytoplasmic side of the IM (18, 39). Then, in lography will make important contributions. 206 Bos Robert Tommassen � � by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 The major players required for OMP assem- ies have revealed similarities as well as differ- bly have likely been identified. Provided that ences. For example, whereas an LPS transport no energy-coupling system is required and defect in N. meningitidis results in feedback in- that protein folding and partitioning into the hibition of its synthesis, this is not the case in hydrophobic environment of the membrane E. coli. For OMP assembly, the reverse is true: are the driving forces, it may be possible to An OMP assembly defect leads to feedback set up an in vitro system for OMP assembly inhibition in E. coli, but not in N. meningitidis. with purified components. For LPS, a major Such differences make it attractive to study issue remains how it is transported through specific aspects of OM biogenesis in different the periplasm. Studying the binding of LPS organisms. In addition, considering the dif- to the components together with immuno- ferences already observed between these two gold electron microscopy studies to deter- model organisms, it is likely that studies in mine whether these components are associ- other bacteria will uncover new, unanticipated ated with the contact sites between IM and features. OM will help to address these questions. For Further studies in this field will remain lipoproteins, an important issue is how such important, because they will uncover funda- molecules are transported to the cell surface. mental biological processes. In addition, the Research on the transport of phospholipids knowledge gained from these studies may be to the OM has to start more or less from the useful for medical applications: The essential beginning. nature of the bacterial machineries involved Importantly, much progress in the field has and their surface localization make them at- been reached by studies in two model organ- tractive targets for the development of new isms, E. coli and N. meningitidis. These stud- antimicrobial drugs and vaccines. SUMMARY POINTS 1. OMPs and lipoproteins are transported across the IM via the Sec system. 2. Assembly of bacterial outer membrane proteins requires the outer membrane protein Omp85, which is evolutionary conserved and found even in the OM of mitochondria. 3. Omp85 recognizes its substrate OMPs by virtue of their C-terminal signature sequences. 4. Other proteins involved in OMP transport and assembly are the periplasmic chap- erones Skp and SurA, and the OM-associated lipoproteins YfiO, YfgL, NlpB, and SmpA, the function of which remains to be determined. 5. Transport of lipoproteins to the OM depends on the Lol system, which consists of an ABC transporter in the IM, a soluble periplasmic chaperone, and an OM-attached receptor. 6. MsbA is an ABC transporter required for the transport of LPS across the IM. 7. Further transport of LPS to the cell surface requires, at least, the ABC protein LptB, the periplasmic protein LptA, the OM-attached lipoprotein RlpB, and the integral OMP Imp. 8. Nothing is known regarding the transport of phospholipids to the OM. " www.annualreviews.org Outer Membrane Biogenesis 207 by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org ANRV322-MI61-11 ARI 6 August 2007 16:59 DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS The work in our laboratory is supported by grants from the Netherlands Research Council for Earth and Life Sciences (ALW) and the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Organization for Scientific Research (NWO). LITERATURE CITED 1. Abe S, Okutsu T, Nakajima H, Kakuda N, Ohtsu I, et al. 2003. n-Hexane sensitivity of Escherichia coli due to low expression of imp/ostA encoding an 87 kDa minor protein associated with the outer membrane. Microbiology 149:1265 73 2. Ames GFL, Spudich EN, Nikaido H. 1974. Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117:406 16 3. Bayan N, Guilvout I, Pugsley AP. 2006. Secretins take shape. Mol. 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Downloaded from arjournals.annualreviews.org AR322-FM ARI 9 July 2007 9:23 Annual Review of Microbiology Volume 61, 2007 Contents Frontispiece Margarita Salas xiv 40 Years with Bacteriophage �29 Margarita Salas 1 The Last Word: Books as a Statistical Metaphor for Microbial Communities Patrick D. Schloss and Jo Handelsman 23 The Mechanism of Isoniazid Killing: Clarity Through the Scope of Genetics Catherine Vilch�ze and William R. Jacobs, Jr. 35 Development of a Combined Biological and Chemical Process for Production of Industrial Aromatics from Renewable Resources F. Sima Sariaslani 51 The RNA Degradosome of Escherichia coli: An mRNA-Degrading Machine Assembled on RNase E Agamemnon J. Carpousis 71 Protein Secretion in Gram-Negative Bacteria via the Autotransporter Pathway Nathalie Dautin and Harris D. Bernstein 89 Chlorophyll Biosynthesis in Bacteria: The Origins of Structural and Functional Diversity Aline Gomez Maqueo Chew and Donald A. Bryant 113 Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis Rita Tamayo, Jason T. Pratt, and Andrew Camilli 131 Aggresomes and Pericentriolar Sites of Virus Assembly: Cellular Defense or Viral Design? Thomas Wileman 149 As the Worm Turns: The Earthworm Gut as a Transient Habitat for Soil Microbial Biomes Harold L. Drake and Marcus A. Horn 169 vi by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org AR322-FM ARI 9 July 2007 9:23 Biogenesis of the Gram-Negative Bacterial Outer Membrane Martine P. Bos, Viviane Robert, and Jan Tommassen 191 SigB-Dependent General Stress Response in Bacillus subtilis and Related Gram-Positive Bacteria Michael Hecker, Jan Pan�-Farr�, and Uwe V�lker 215 Ecology and Biotechnology of the Genus Shewanella Heidi H. Hau and Jeffrey A. Gralnick 237 Nonhomologous End-Joining in Bacteria: A Microbial Perspective Robert S. Pitcher, Nigel C. Brissett, and Aidan J. Doherty 259 Postgenomic Adventures with Rhodobacter sphaeroides Chris Mackenzie, Jesus M. Eraso, Madhusudan Choudhary, Jung Hyeob Roh, Xiaohua Zeng, Patrice Bruscella, �gnes Pusk�s, and Samuel Kaplan 283 Toward a Hyperstructure Taxonomy Vic Norris, Tanneke den Blaauwen, Roy H. Doi, Rasika M. Harshey, Laurent Janniere, Alfonso Jim�nez-S�nchez, Ding Jun Jin, Petra Anne Levin, Eugenia Mileykovskaya, Abraham Minsky, Gradimir Misevic, Camille Ripoll, Milton Saier, Jr., Kirsten Skarstad, and Michel Thellier 309 Endolithic Microbial Ecosystems Jeffrey J. Walker and Norman R. Pace 331 Nitrogen Regulation in Bacteria and Archaea John A. Leigh and Jeremy A. Dodsworth 349 Microbial Metabolism of Reduced Phosphorus Compounds Andrea K. White and William W. Metcalf 379 Biofilm Formation by Plant-Associated Bacteria Thomas Danhorn and Clay Fuqua 401 Heterotrimeric G Protein Signaling in Filamentous Fungi Liande Li, Sara J. Wright, Svetlana Krystofova, Gyungsoon Park, and Katherine A. Borkovich 423 Comparative Genomics of Protists: New Insights into the Evolution of Eukaryotic Signal Transduction and Gene Regulation Vivek Anantharaman, Lakshminarayan M. Iyer, and L. Aravind 453 Lantibiotics: Peptides of Diverse Structure and Function Joanne M. Willey and Wilfred A. van der Donk 477 The Impact of Genome Analyses on Our Understanding of Ammonia-Oxidizing Bacteria Daniel J. Arp, Patrick S.G. Chain, and Martin G. Klotz 503 Contents vii by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org AR322-FM ARI 9 July 2007 9:23 Morphogenesis in Candida albicans Malcolm Whiteway and Catherine Bachewich 529 Structure, Assembly, and Function of the Spore Surface Layers Adriano O. Henriques and Charles P. Moran, Jr. 555 Cytoskeletal Elements in Bacteria Peter L. Graumann 589 Indexes Cumulative Index of Contributing Authors, Volumes 57 61 619 Cumulative Index of Chapter Titles, Volumes 57 61 622 Errata An online log of corrections to Annual Review of Microbiology articles may be found at http://micro.annualreviews.org/ viii Contents by CNRS-multi-site on 10/11/07. For personal use only. Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

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