Resolving the native conformation of Escherichia coli OmpA

The native conformation of the 325-residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow-pore, two-domain structure has vied with a large-pore, single-domain structure. Our recent studies show that Ser163 and Ser167 of the N-terminal domain (1-170) are modified in the cytoplasm by covalent attachment of oligo-(R)-3-hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N-terminal domain to be incorporated into planar lipid bilayers as narrow pores (≈ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M-OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I-OmpA). Chemical and Western blot analysis and 1H-NMR showed that segment 264-325 in M-OmpA, but not in I-OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA. Planar lipid bilayer studies indicated that narrow pores formed by M-OmpA undergo a temperature-induced transition into stable large pores (≈ 450 pS, 1 M KCl, 22 °C) [energy of activation (Ea) = 33.2 kcal·mol(-1)], but this transition does not occur with I-OmpA or with M-OmpA that has been exposed to disulfide bond-reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB-modification, disulfide bond formation and temperature in folding OmpA into its native large-pore configuration.     

Pore-forming activity of OmpA protein of Escherichia coli.

Escherichia coli outer membrane protein OmpA was purified to homogeneity, as a monomer, from a K12 derivative deficient in both OmpF and OmpC porins. When proteoliposomes reconstituted from the purified OmpA, phospholipids, and lithium dodecyl sulfate were tested for permeability to small molecules by osmotic swelling, it was found that OmpA produced apparently nonspecific diffusion channels that allowed the penetration of various solutes. The pore-forming activity was destroyed by the heat denaturation of the OmpA protein, and the use of an OmpA-deficient mutant showed that the activity was not caused by copurifying contaminants. The size of the OmpA channel, estimated by comparison of diffusion rates of solutes of different sizes, was rather similar to that of E. coli OmpF and OmpC porins, i.e. about 1 nm in diameter. The rate of penetration of L-arabinose caused by a given amount of OmpA protein, however, was about a hundredfold lower than the rate produced by the same amount of E. coli OmpF porin. The addition of large amounts of lithium dodecyl sulfate to the reconstitution mixture increased the permeability through the OmpA channel, apparently by facilitating the correct insertion of OmpA into the bilayer.     

Evidence that TraT interacts with OmpA of Escherichia coli

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells. TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F-factors become poor recipients in conjugation. To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed.     

An alternative topological model for Escherichia coli OmpA.

The current topological model for the Escherichia coli outer membrane protein OmpA predicts eight N-terminal transmembrane segments followed by a long periplasmic tail. Several recent reports have raised serious doubts about the accuracy of this prediction. An alternative OmpA model has been constructed using (1) computer-aided predictions developed specifically to predict topology of bacterial outer membrane porins, (2) the results of two reports that identified sequence homologies between OmpA and other peptidoglycan-associated proteins, and (3) biochemical, immunochemical, and genetic topological data on proteins of the OmpA family provided by numerous previous studies. The new model not only agrees with the varied experimental data concerning OmpA but also provides an improved understanding of the relationship between the structure and the multifunctional role of OmpA in the bacterial outer membrane.     

The function of ubiquinone in Escherichia coli

1. The function of ubiquinone in Escherichia coli was studied by using whole cells and membrane preparations of normal E. coli and of a mutant lacking ubiquinone. 2. The mutant lacking ubiquinone, strain AN59 (Ubi(-)), when grown under aerobic conditions, gave an anaerobic type of growth yield and produced large quantities of lactic acid, indicating that ubiquinone plays a vital role in electron transport. 3. NADH and lactate oxidase activities in membranes from strain AN59 (Ubi(-)) were greatly impaired and activity was restored by the addition of ubiquinone (Q-1). 4. Comparison of the percentage reduction of flavin, cytochrome b(1) and cytochrome a(2) in the aerobic steady state in membranes from the normal strain (AN62) and strain AN59 (Ubi(-)) and the effect of respiratory inhibitors on these percentages in membranes from strain AN62 suggest that ubiquinone functions at more than one site in the electron-transport chain. 5. Membranes from strain AN62, in the absence of substrate, showed an electron-spin-resonance signal attributed to ubisemiquinone. The amount of reduced ubiquinone (50%) found after rapid solvent extraction is consistent with the existence of ubiquinone in membranes as a stabilized ubisemiquinone. 6. The effects of piericidin A on membranes from strain AN62 suggest that this inhibitor acts at the ubiquinone sites: thus inhibition of electron transport is reversed by ubiquinone (Q-1); the aerobic steady-state oxidation-reduction levels of flavins and cytochrome b(1) in the presence of the inhibitor are raised to values approximating those found in the membranes of strain AN59 (Ubi(-)); the inhibitor rapidly eliminates the electron-spin-resonance signal attributed to ubisemiquinone and allows slow oxidation of endogenous ubiquinol in the absence of substrate and prevents reduction of ubiquinone in the presence of substrate. It is concluded that piericidin A separates ubiquinone from the remainder of the electron-transport chain. 7. A scheme is proposed in which ubisemiquinone, complexed to an electron carrier, functions in at least two positions in the electron-transport sequence.     

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.     

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.     

Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA beta-barrel from those of OmpX. Overall, the present data demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.     

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.     

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.     

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.     

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.     

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.     

Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.     

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.     

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.     

Complement-mediated inhibition of function in complement-resistant Escherichia coli

C-mediated inhibition of function in C-sensitive strains of Escherichia coli involves the assembly of the membrane attack complex (MAC) on the outer membrane with subsequent inhibition of inner membrane function. The inhibition of inner membrane function is critical for effective cell killing as damage to the outer membrane alone is insufficient to kill a cell in the absence of serum lysozyme. Studies on the measurement of oxygen consumption for cells under complement attack showed that C-sensitive cells were inhibited by assembly of the MAC, and that this represents damage to some component of the respiring inner membrane. Mechanisms of cellular resistance to C attack could include 1) inhibition of the assembly of the MAC, 2) inhibition of effective activation of the assembled MAC, or 3) reversal of the inhibitory effects of the MAC. Demonstration of a transient C-mediated inhibition of inner membrane function in C-resistant cells implies that the latter case should be considered as one possible component of cellular resistance to C attack.