Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli.

Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coli B and K-12, have been used to study the assembly and the topology of the protein. This paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies. A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows us to propose a rough model including 2 antigenic sites. The second part is focused on the assembly of the OmpF protein in the outer membrane. Various forms, precursor, unassembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process. The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope. The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly.     

A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli

Inducible hybrid genes encoding two large domains, a periplasmic domain consisting of the PhoS sequence and an outer membrane domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesized hybrid polypeptides are correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivity toward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogold labelling on ultrathin cryosections. Insertion of a hydrophobic sequence between PhoS and OmpF improves the trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimeric conformation which is recognized by specific antibodies. Moreover, the deletion of the C-terminal region impairs the functional insertion of the OmpF domain; this indicates that the last membrane-spanning region of OmpF is necessary for the correct folding and orientation of the protein in the outer membrane.     

The receptor of bacteriophage lambda: evidence for a biosynthesis dependent on lipid synthesis

Inhibition of lipid synthesis in cerulenin-treated cells or in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase after glycerol deprivation, results in a marked decrease of insertion of lamB protein into the outer membrane. No lambda receptor was found in any other cell compartment or in the medium under these conditions. The LamB protein synthesis was inhibited by about 70% in the absence of lipid synthesis. The residual 30% protein produced during inhibition of fatty-acid or phospholipid synthesis, was probably incorporated into the outer membrane since no further incorporation was observed after resumption of these syntheses. Besides OmpF and OmpC protein [Bocquet-Pagès, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105-111], at least four other proteins of the outer membrane are also subject to alteration of levels in the absence of lipid synthesis. Under these conditions the uptake of maltose, like the uptake of 5'AMP [Bocquet-Pagès, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105-111], was inhibited as much as 60%. These results are discussed with regard to the biosynthesis and assembly of the outer membrane proteins.     

Dynamics of the exposure of epitopes on OmpF, an outer membrane protein of Escherichia coli.

The OmpF protein is the major outer membrane trimeric porin of Escherichia coli B. The exposure of several cell-surface-exposed epitopes, that are recognized by various monoclonal antibodies directed against the protein, is investigated. Kinetic analyses show that two epitopes (E18 and E19) appear early during the in-vivo assembly on the folded monomer, just after the removal of the signal peptide, and are conserved in the native trimer. The trimerization that immediately follows or occurs in conjunction with the folding of monomers exposes another antigenic site (E21) at the surface of metastable forms. The binding of nascent lipopolysaccharide promotes the conversion of the heat-modifiable intermediate to a stable trimer and ensures the exposure of E20, E1, E3, E4 and E7. Late epitopes, E1, E3, E4 and E7 are only detected in the outer membrane fraction. These results suggest that different steps induce the sequential exposure of native antigenic sites. The detection of these epitopes depends on conformational changes occurring during the OmpF insertion into the outer membrane.     

Modulations in lipid A and phospholipid biosynthesis pathways influence outer membrane protein assembly in Escherichia coli K-12

The assembly defect of a mutant outer membrane protein, OmpF315, can be corrected by suppressor mutations that lower lipopolysaccharide (LPS) levels and indirectly elevate phospholipid levels. One such assembly suppressor mutation, asmB1 , is an allele of lpxC ( envA ) whose product catalyses the first rate-limiting step in the lipid A (LPS) biosynthesis pathway. Besides reducing LPS levels, asmB1 confers sensitivity to MacConkey medium. A mutation, sabA1 , that reverses the MacConkey sensitivity phenotype of asmB1 maps within fabZ (whose product is needed for phospholipid synthesis from a precursor) is also required for lipid A synthesis. In addition to reversing MacConkey sensitivity, the sabA1 mutation reverses the OmpF315 assembly suppression phenotype of asmB1 . These results show that OmpF315 assembly suppression by asmB1 , which is achieved by lowering LPS levels, can be averted by a subsequent aberration in phospholipid synthesis at a point where the biosynthetic pathways for these two lipid molecules split. OmpF315 assembly suppression can also be achieved in an asmB ⁺ background where FabZ expression is increased. The data obtained in this study provide genetic evidence that elevated phospholipid levels and/or phospholipid to LPS ratios are necessary for assembly suppression.     

Role of lipopolysaccharide in assembly of Escherichia coli outer membrane proteins OmpA, OmpC, and OmpF.

Selection was performed for resistance to a phage, Ox2, specific for the Escherichia coli outer membrane protein OmpA, under conditions which excluded recovery of ompA mutants. All mutants analyzed produced normal quantities of OmpA, which was also normally assembled in the outer membrane. They had become essentially resistant to OmpC and OmpF-specific phages and synthesized these outer membrane porins at much reduced rates. The inhibition of synthesis acted at the level of translation. This was due to the presence of lipopolysaccharides (LPS) with defective core oligosaccharides. Cerulenin blocks fatty acid synthesis and therefore that of LPS. It also inhibits synthesis of OmpC and OmpF but not of OmpA (C. Bocquet-Pagès, C. Lazdunski, and A. Lazdunski, Eur. J. Biochem. 118:105-111, 1981). In the presence of the antibiotic, OmpA synthesis and membrane incorporation remained unaffected at a time when OmpC and OmpF synthesis had almost ceased. The similarity of these results with those obtained with the mutants suggests that normal porin synthesis is not only interfered with by production of mutant LPS but also requires de novo synthesis of LPS. Since synthesis and assembly of OmpA into the outer membrane was not affected in the mutants or in the presence of cerulenin, association of this protein with LPS appears to occur with outer membrane-located LPS.     

Overexpression of protease-deficient DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants in a degP background

Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP(S210A) protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP(S210A) was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP(S210A) also fractionated. These results suggested that the DegP(S210A)-mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.     

Export of altered forms of an Escherichia coli K-12 outer membrane protein (OmpA) can inhibit synthesis of unrelated outer membrane proteins

Expression of mutant ompA genes, encoding the 325 residue Escherichia coli outer membrane protein OmpA, caused an inhibition of synthesis of the structurally unrelated outer membrane porins OmpC and OmpF and of wild-type OmpA, but not of the periplasmic beta-lactamase. There was no accumulation of precursors of the target proteins and the inhibitory mechanism operated at the level of translation. So far only alterations around residue 45 of OmpA have been found to affect this phenomenon. Linkers were inserted between the codons for residues 45 and 46. A correlation between size and sequence of the resulting proteins and presence or absence of the inhibitory effect was not found, indicating that the added residues acted indirectly by altering the conformation of other parts of the mutant OmpA. To be effective, the altered polypeptides had to be channelled into the export pathway. Internal deletions in effector proteins, preventing incorporation into the membrane, abolished effector activity. The results suggest the existence of a periplasmic component that binds to OmpA prior to membrane assembly; impaired release of this factor from mutant OmpA proteins may trigger inhibition of translation. The factor could be a See B-type protein, keeping outer membrane proteins in a form compatible with membrane assembly.     

Assembly of the OmpF porin of Escherichia coli B. Immunological and kinetic studies of the integration pathway

The different conformations of the outer membrane protein OmpF of Escherichia coli B were studied with immunological probes. The antigenic determinants recognized by one monoclonal (MoF3) and two polyclonal antibodies were investigated under various conditions of solubilization which modify the association of OmpF with other membrane components, such as lipopolysaccharide. Several polymeric forms of the protein could be detected after extraction at 37 degrees C or 56 degrees C. The monoclonal antibody, which is specific to an exposed region of native OmpF, recognized various trimeric forms in an immunoprecipitation assay. Under the same conditions, the binding of polyclonal antibodies apparently induced strong conformational rearrangements, since the pattern of trimeric forms detected was greatly modified. The conversion of newly synthesized monomers of OmpF to the various trimer forms was investigated using these antibodies. The trimerization occurred rapidly but the appearance of the native conformation of OmpF was delayed. Some additional step was required to expose the MoF3-specific antigenic site at the surface of the trimeric form. These results are discussed in relation to the structure of OmpF and its association with lipopolysaccharide in the outer membrane.     

Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin.

Deep rought mutants, which produce very defective lipopolysaccharides, are unable to export normal levels of porins into the outer membrane. In this study, we showed that lipopolysaccharides from such mutants were also unable to facilitate the trimerization, in vitro, of monomeric OmpF porin secreted by spheroplasts of Escherichia coli B/r. In contrast, lipopolysaccharides containing most or all of the core oligosaccharides were able to facilitate trimerization.     

An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli

To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA. The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form. Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane. Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins. The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF.     

Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins

asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipo-polysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA-mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.     

Targeting of porin to the outer membrane of Escherichia coli. Rate of trimer assembly and identification of a dimer intermediate

Porin, a transmembrane protein in the outer membrane of Escherichia coli, exists in a trimeric structure which is not dissociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 25 degrees C. This unusual stability was utilized in the study of the conformational changes which accompany the targeting of porin to the outer membrane. A delay of 16-44 s between completion of synthesis of a monomer and its assembly into a trimer was found from the ratio of monomers to trimers found in exponentially growing cells. Pulse-chase experiments showed that rapid processing of precursor OmpF molecules was followed by assembly into sodium dodecyl sulfate-resistant oligomers with a half-time of 20 s at 30 degrees C. An intermediate in assembly was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis below 10 degrees C and was identified as a metastable dimer.     

asmB, a suppressor locus for assembly-defective OmpF mutants of Escherichia coli, is allelic to envA (lpxC).

A novel genetic scheme allowed us to isolate extragenic suppressor mutations that restored mutant OmpF assembly. One group of these mutations, termed asmB for assembly suppressor mutation B, permitted mutant OmpF assembly in a non-allele-specific manner. Genetic mapping analyses placed the asmB mutations at the 2-min region of the Escherichia coli K-12 chromosome. Further analyses revealed that the asmB mutations map within the envA (lpxC) gene, which encodes an enzyme needed for the synthesis of the lipid A moiety of lipopolysaccharide (LPS). Nucleotide sequence analysis showed that the asmB mutations caused a change from F-50 to S (F50S substitution) (asmB2 and asmB3) or a G210S substitution (asmB1) in EnvA. Cells bearing the asmB alleles displayed increased sensitivity to various hydrophobic compounds and detergents, suggesting an alteration within the outer membrane. Direct examination (of the LPS showed that its amounts were reduced by the asmB mutations, with asmB1 exerting a greater effect than asmB2 or asmB3. Thus, it appears that the asmB mutations achieve mutant OmpF assembly suppression by reducing LPS levels, which in turn may alter membrane fluidity.     

Molecular analysis of asmA, a locus identified as the suppressor of OmpF assembly mutants of Escherichia coli K-12

We present the molecular characterization of the asmA gene, whose product is involved in the assembly of outer membrane proteins in Escherichia coli K-12. The asmA locus was initially identified as a site for suppressor mutations of an assembly defective OmpF315. Our data suggest that these suppressor mutations either completely abolish or reduce asmA expression and can be complemented in trans by piasmid clones carrying asmA sequences. The recessive nature of asmA suppressor mutations suggests that the functional AsmA protein participates in Inhibiting the assembly of OmpF315 and other mutant OmpFs. As the assembly of wild-type and parental OmpF proteins was not affected by asmA mutations, AsmA must provide an environment refractory only to the assembly of mutant OmpF proteins. However, we cannot completely rule out the possibility that AsmA plays a minor role in the assembly of wild-type and parental OmpF in wild-type cells. The presence of a putative signal sequence within the amino-terminal sequence of AsmA suggests that it is either a periplasmic or an outer membrane protein. This predicted location of AsmA is compatible with its role in the assembly of outer membrane proteins.     

Differential effects of yfgL mutation on Escherichia coli outer membrane proteins and lipopolysaccharide

YfgL together with NlpB, YfiO, and YaeT form a protein complex to facilitate the insertion of proteins into the outer membrane of Escherichia coli. Without YfgL, the levels of OmpA, OmpF, and LamB are significantly reduced, while OmpC levels are slightly reduced. In contrast, the level of TolC significantly increases in a yfgL mutant. When cells are depleted of YaeT or YfiO, levels of all outer membrane proteins examined, including OmpC and TolC, are severely reduced. Thus, while the assembly pathways of various nonlipoprotein outer membrane proteins may vary through the step involving YfgL, all assembly pathways in Escherichia coli converge at the step involving the YaeT/YfiO complex. The negative effect of yfgL mutation on outer membrane proteins may in part be due to elevated sigma E activity, which has been shown to downregulate the synthesis of various outer membrane proteins while upregulating the synthesis of periplasmic chaperones, foldases, and lipopolysaccharide. The data presented here suggest that the yfgL effect on outer membrane proteins also stems from a defective assembly apparatus, leading to aberrant outer membrane protein assembly, except for TolC, which assembles independent of YfgL. Consistent with this view, the simultaneous absence of YfgL and the major periplasmic protease DegP confers a synthetic lethal phenotype, presumably due to the toxic accumulation of unfolded outer membrane proteins. The results support the hypothesis that TolC and major outer membrane proteins compete for the YaeT/YfiO complex, since mutations that adversely affect synthesis or assembly of major outer membrane proteins lead to elevated TolC levels.     

A novel OmpY porin from Yersinia pseudotuberculosis: structure, channel-forming activity and trimer thermal stability

A novel OmpY porin was predicted based on the Yersinia pseudotuberculosis genome analysis. Whereas it has the different genomic annotation such as "outer membrane protein N" (ABS46310.1) in str. IP 31758 or "outer membrane protein C2, porin" (YP_070481.1) in str. IP32953, it might be warranted to rename the OmpN/OmpC2 to OmpY, "outer membrane protein Y", where letter "Y" pertained to Yersinia. Both phylogenetic analysis and genomic localization clearly support that the OmpY porin belongs to a new group of general bacterial porins. The recombinant OmpY protein with its signal sequence was overexpressed in porin-deficient Escherichia coli strain. The mature rOmpY was shown to insert into outer membrane as a trimer. The OmpY porin, isolated from the outer membrane, was studied employing spectroscopic, electrophoretic and bilayer lipid membranes techniques. The far UV CD spectrum of rOmpY was essentially identical to that of Y. pseudotuberculosis OmpF. The near UV CD spectrum of rOmpY was weaker and smoother than that of OmpF. The rOmpY single-channel conductance was 180 ± 20 pS in 0.1 M NaCl and was lower than that of the OmpF porin. As was shown by electrophoretic and bilayer lipid membrane experiments, the rOmpY trimers were less thermostable than the OmpF trimers. The porins differed in the trimer-monomer transition temperature by about 20°C. The three-dimensional structural models of the Y. pseudotuberculosis OmpY and OmpF trimers were generated and the intra- and intermonomeric interactions stabilizing the porins were investigated. The difference in the thermal stability of OmpY and OmpF trimers was established to correlate with the difference in intermonomeric polar contacts.     

Effect of various mild surfactants on the reassembly of an oligomeric integral membrane protein OmpF porin

Reassembly of OmpF porin from its denatured monomer into the sodium dodecyl sulfate-resistant species was investigated by using 27 kinds of mild surfactants. Polyethyleneoxide-type surfactants with a hydrophilic-lipophilic balance value of 10.8–14.6 induced the trimerization of denatured OmpF porin. Dimerization and trimerization were induced by non-polyethyleneoxide-type mild surfactants that are generally used for membrane protein solubilization. The dependence of surfactant concentrations on reassembly was estimated to obtain a minimal concentration required for the reassembly of the protein. Extensive reassembly (85% yield) into dimer (a putative assembly intermediate) was observed at a protein concentration of 0.05 mg/ml in 7 mg/ml n-octyl--d-glucopyranoside and 1 mg/ml sodium dodecyl sulfate. This condition will be useful for the studies of the dimer and dimerization of OmpF porin. The role of mixed micelle system on the protein renaturation was discussed.