Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel β-strands, forming an amphiphilic β connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of β-strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed β-barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

Export and sorting of theEscherichia coli outer membrane protein OmpA

Results of studies, mostly using the outer membrane, 325 residue protein OmpA, are reviewed which concern its translocation across the plasma membrane and incorporation into the outer membrane ofEscherichia coli. For translocation, neither a unique export signal, acting in a positive fashion within the mature part of the precursor, nor a unique conformation of the precursor is required. Rather, the mature part of a secretory protein has to be export-compatible. Export-incompatibility can be caused by a stretch of 16 (but not 8 or 12) hydrophobic residues, too low a size of the polypeptide (smaller than 75 residue precursors), net positive charge at the N-terminus, or lack of a turn potential at the same site. It is not yet clear whether binding sites for chaperonins (SecB, trigger factor, GroEL) within OmpA are importantin vivo. The mechanism of sorting of outer membrane proteins is not yet understood. The membrane part of OmpA, encompassing residues 1 to about 170, it thought to traverse the membrane eight times in antiparallel -sheet conformation. At least the structure of the last -strand (residues 160–170) is of crucial importance for membrane assembly. It must be amphiphilic or hydrophobic, these properties must extend over at least nine residues, and it must not contain a proline residue at or near its center. Membrane incorporation of OmpA involves a conformational change of the protein and it could be that the last -strand initiates folding and assembly in the outer membrane.     

Bacteriophage receptor area of outer membrane protein OmpA of Escherichia coli K-12.

A number of T-even-like bacteriophages use the outer membrane protein OmpA of Escherichia coli as a receptor. We had previously analyzed a series of ompA mutants which are resistant to such phages and which still produce the OmpA protein (R. Morona, M. Klose, and U. Henning, J. Bacteriol. 159:570-578, 1984). Mutational alterations were found near or at residues 70, 110 and 154. Based on these and other results a model was proposed showing the amino-terminal half of the 325-residue protein crossing the outer membrane repeatedly and being cell surface exposed near residues 25, 70, 110, and 154. We characterized, by DNA sequence analysis, an additional 14 independently isolated phage-resistant ompA mutants which still synthesize the protein. Six of the mutants had alterations identical to the ones described before. The other eight mutants possessed seven new alterations: Ile-24----Asn, Gly-28----Val, deletion of Glu-68, Gly-70----Cys, Ser-108----Phe, Ser-108----Pro, and Gly-154----Asp (two isolates). Only the latter alteration resulted in a conjugation-deficient phenotype. The substitutions at Ile-24 and Gly-28 confirmed the expectation that this area of the protein also participates in its phage receptor region. It is unlikely that still other such sites of the protein are involved in the binding of phage, and it appears that the phage receptor area of the protein has now been characterized completely.     

Energetics and intermediates of the assembly of Protein OmpA into the outer membrane of Escherichia coli

OmpA is a major protein of the outer membrane of Escherichia coli. It is made as a larger precursor, pro-OmpA, which requires a membrane potential for processing. We now show that pro-OmpA accumulates in the cytoplasm of cells treated with carbonyl cyanide m-chlorophenylhydrazone, an uncouple which lowers the membrane potential. Upon restoration of the potential, this pro-OmpA is secreted, processed, and assembled into the outer membrane. Pro-OmpA made in vitro is also recovered with the postribosomal supernatant. It is efficiently processed to OmpA by liposomes which have bacterial leader peptidase that is exclusively internally oriented. These experiments show that: (i) the insertion of pro-OmpA into the plasma membrane is not coupled to its synthesis; (ii) insertion is promoted by the transmembrane electrochemical potential; (iii) pro-OmpA can cross a bilayer spontaneously; and (iv) pro-OmpA is processed by the same leader peptidase which converts M13 procoat to coat.     

Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli K-12. A model, in which this protein crosses the membrane eight times in an antiparallel beta-sheet conformation and in which regions around amino acids 25, 70, 110 and 154 are exposed at the cell surface, had been proposed. Linkers were inserted into the ompA gene with the result that OmpA proteins, carrying non-OmpA sequences between residues 153 and 154 or 160 and 162, were synthesized. Intact cells possessing these proteins were treated with proteases. Insertion of 15 residues between residues 153 and 154 made the protein sensitive to proteinase K and the sizes of the two cleavage products were those expected following proteolysis at the area of the insertion. Addition of at least 17 residues between residues 160 and 162 left the protein completely refractory to protease action. Thus, the former area is cell surface exposed while the latter area appears not to be. The insertions did not cause a decrease in the concentration of the hybrid proteins as compared to that of the OmpA protein, and in neither case was synthesis of the protein deleterious to cell growth. It is suggested that this method may serve to carry peptides of practical interest to the cell surface and that it can be used to probe surface-located regions of other membrane proteins.     

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.     

Proteomic analysis of Escherichia coli biofilms reveals the overexpression of the outer membrane protein OmpA

Bacterial colonisation and biofilm formation on the surface of urinary catheters is a common cause of nosocomial infection, and as such is a major impediment to their long-term use. Understanding the mechanisms of biofilm formation on urinary catheters is critical to their control and will aid the future development of materials used in their manufacture. In this report we have used proteomic analysis coupled with immunoassays to show that the major outer membrane protein (OmpA) of Escherichia coli is overexpressed during biofilm formation. A series of synthetic hydrogels being developed for potential use as catheter coatings were used as the substrata and OmpA expression was increased in biofilms on all these surfaces, as well as being a feature of both a laboratory and a clinical strain of E. coli. Up-regulation of OmpA may, therefore, be a common feature of E. coli biofilms. These findings present OmpA as a potential target for biofilm inhibition and may contribute to the rational design of biofilm inhibiting hydrogel coatings for urinary catheters.     

Pseudomonas aeruginosa outer membrane protein F: structural role and relationship to the Escherichia coli OmpA protein.

A Pseudomonas aeruginosa outer membrane protein F-deficient omega-insertion mutant strain H636, in contrast to its protein F-sufficient parent strain H103, was unable to grow on unsupplemented Proteose Peptone no. 2 broth (Difco Laboratories, Detroit, Mich.). Addition of high concentrations of NaCl, KCl, glucose, sucrose, or potassium succinate permitted growth of strain H636 at rates approaching those of the parent strain H103. Strain H636 cells were 33% shorter and had a 46% smaller cross-sectional area than did the parent strain growing at similar rates on the same medium. These properties of the oprF::omega mutant were analogous to those previously observed for Escherichia coli ompA mutants in an lpp (Braun lipoprotein-deficient) mutant background. Therefore, we compared P. aeruginosa protein F and the E. coli OmpA protein. In addition to many similarities previously described, sequence alignment demonstrated substantial amino acid sequence homology throughout the carboxy-terminal 168 to 180 amino acids of the two proteins. Consistent with this observation, polyclonal antiserum specific for OmpA reacted on Western blots (immunoblots) with protein F. Expression of protein F from the cloned oprF gene in an E. coli ompA lpp double mutant resulted in a 1.7-fold increase in cell length and a 2.1-fold increase in cross-sectional area compared with values for the same mutant containing only the plasmid vector onto which the oprF gene had been cloned. These results favor a structural role for P. aeruginosa protein F and suggest that it is strongly related to the E. coli OmpA protein.     

Lipid fluidity-dependent biosynthesis and assembly of the outer membrane proteins of E. coli.

The reduction of the membrane lipids of E. coli to a nonfluid state resulted in the accumulation in the cell envelope of a high molecular weight precursor of the protoIG protein, a major outer membrane protein. The protoIG protein was as sensitive to trypsin as the mature toIG protein assembled in the outer membrane. In contrast to the toIG protein, however, the accumulated protoIG protein was easily released from the envelope fraction by both sodium lauryl sarcosinate extraction and sonication. This indicated that the precursor protein was loosely associated with the cell membrane. When a fluid lipid state was restored, the protoIG protein was processed to the mature form which was then correctly assembled in the outer membrane. These results suggest that the protoIG protein produced under nonfluid lipid conditions was properly translocated across the cytoplasmic membrane, but could not be assembled in the outer membrane due either to the reversible inhibition of the processing of the ProtoIG to the toIG protein or to the lack of interaction with a specific outer membrane component(s). Reduced lipid fluidity also caused various alterations in the biosynthesis and assembly of other membrane proteins. In addition to the toIG protein, a large number of new proteins were accumulated in the membrane. Alternatively, the matrix protein as well as the promatrix protein were not detected in the cell envelope. On the other hand, the lipoprotein was normally produced, processed, modified and assembled in the outer membrane. These results indicate that the outer membrane proteins are synthesized and assembled according to several different mechanisms, on which the physical state of the membrane has various effects.     

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.     

Conjugation-deficient mutants of Escherichia coli distinguish classes of functions of the outer membrane OmpA protein

Summary Sixty-two E. coli mutants, selected as being deficient as recipients in F factor conjugation, are altered either in the amount or function of the outer membrane OmpA protein or in lipopolysaccharide structure. These two components may function together in conjugation, since the residual conjugation activity of a mutant lacking OmpA protein was unaffected by the additional presence of a lipopolysaccharide defect. Sixty of the strains carried mutations mapping to ompA, and these could be divided into classes depending on the amount of OmpA protein in their membranes. Representatives of these classes of mutant alleles failed to complement in diploids, indicating that they all affect the ompA structural gene and nearby sequences needed for its expression. The properties of these classes distinguish three groups of OmpA protein functions: 1) the structural function in the outer membrane in providing resistance to chelating agents and the hydrophobic antibiotic novobiocin, 2) the receptor functions in phage Tull^* and K3 infection, and 3) the functions of binding cells together during conjugation, facilitating the uptake of receptorbound colicin K or L, and allowing phage Ox2 to infect. Different cellular amounts or sites in OmpA protein are thus required for these three groups of functions.     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel -strands, forming an amphiphilic connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of -strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed -barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K-12.

We studied whether information required for export is present within the mature form of the Escherichia coli 325-residue outer membrane protein OmpA. We had previously analyzed overlapping internal deletions in the ompA gene, and the results allowed us to conclude that if such information exists it must be present repeatedly within the membrane part of the protein encompassing amino acid residues 1 to 177 (R. Freudl, H. Schwarz, M. Klose, N. R. Movva, and U. Henning, EMBO J. 4:3593-3598, 1985). A deletion which removed the codons for amino acid residues 1 to 229 of the OmpA protein was constructed. In this construct the signal sequence was fused to the periplasmic part of the protein. The resulting protein, designated Pro-OmpA delta 1-229, was processed, and the mature 95-residue protein accumulated in the periplasm. Hence, information required for export does not exist within the OmpA protein.     

The influence of amino substitutions within the mature part of an Escherichia coli outer membrane protein (OmpA) on assembly of the polypeptide into its membrane

The membrane part of the 325-residue outer membrane protein OmpA of Escherichia coli encompasses residues 1-177. This part is thought to cross the membrane eight times in antiparallel beta-strands, forming four loops of an amphipathic beta-barrel. With the aim of gaining some insight into the mechanism of sorting, i.e. the way the protein recognizes and assembles into its membrane, a set of point mutants in the ompA gene has been generated. Selection for toxicity of ompA expression following mutagenesis with sodium bisulfite yielded genes with multiple base pair substitutions, the majority of which resulted in amino acid substitutions in the membrane moiety of the protein. None of the altered proteins was blocked in membrane incorporation. A proline residue exists at or near each of the presumed turns at the inner side of the outer membrane. Using oligonucleotide-directed mutagenesis, each of them was replaced by a leucine residue which is thought to be a turn blocking residue. None of these proteins had lost the ability to be incorporated into the membrane. Apparently, leucine residues are tolerated at turns in this protein. To interfere with the formation of antiparallel beta-strands, four double mutants were prepared: ompA-ON3 (Ala11----Pro, Leu13----Pro), -ON4 (Ala11----Asp, Leu13----Pro), -ON5 (Gly160----Val, Leu162----Arg), and -ON6 (Leu164----Pro, Val166----Asp). The former three proteins and even quadruple mutants consisting of a combination of ompA-ON2 or -ON4 with -ON5 were not defective in membrane assembly. In contrast, the OmpA-ON6 protein was translocated across the plasma membrane but could not be incorporated into the outer membrane. It is concluded that at least one rather small area of the polypeptide is of crucial importance for the assembly of OmpA into the outer membrane.     

Conformation of Escherichia coli outer membrane protein OmpA produced in Bacillus subtilis: Influence of lipopolysaccharide

Abstract The conformation of the outer membrane protein OmpA of Escherichia coli produced in Bacillus subtilis and solubilized in Sarkosyl was studied by measuring its ability to bind OmpA-specific phage K3 and to inhibit F-mediated conjugation. The partially purified protein was inactive in both these assays. Refolding of the protein in the presence of lipopolysaccharide resulted in preparations with full phage-binding and conjugation-inhibiting capacity, indicating the formation of surface-exposed loops of OmpA of native conformation. The finding is of importance for the potential use of outer membrane proteins of Gram-negative bacteria as vaccines.     

Refolding of an integral membrane protein. OmpA of Escherichia coli

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.