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.     

Single mutations in a gene for a tail fiber component of an Escherichia coli phage can cause an extension from a protein to a carbohydrate as a receptor

The T-even type Escherichia coli phage Ox2 recognizes the outer membrane protein OmpA as a receptor. This recognition is accomplished by the 266 residue protein 38, which is located at the free ends of the virion's long tail fibers. Host-range mutants had been isolated in three consecutive steps: Ox2----Ox2h5----Ox2h10----Ox2h12, with Ox2h12 recognizing the outer membrane protein OmpC efficiently and having lost some affinity for OmpA. Protein 38 consists, in comparison with these proteins of other phages, of two constant and one contiguous array of four hypervariable regions; the alterations leading to Ox2h12 were all found within the latter area. Starting with Ox2h12, further host-range mutants could be isolated on strains resistant to the respective phage: Ox2h12----h12h1----h12h1.1----h12h1.11----h12 h1.111. It was found that Ox2h12h1.1 (and a derivative of Ox2h10, h10h4) probably uses, instead of OmpA or OmpC, yet another outer membrane protein, designated OmpX. Ox2h12h1.11 was obtained on a strain lacking OmpA, -C and -X. This phage could not grow on a mutant of E. coli B, possessing a lipopolysaccharide (LPS) with a defective core oligosaccharide; Ox2h12h1.111 was obtained from this strain. It turned out that the latter two mutants used LPS as a receptor, most likely via its glucose residues. Selection for resistance to them in E. coli B (ompA+, ompC-, ompX-) yielded exclusively LPS mutants, and in another strain, possessing OmpA, C and X, the majority of resistant mutants were of this type. Isolated LPS inactivated the mutant phages very well and was inactive towards Ox2h12. By recombining the genes of mutant phages into the genome of parental phages it could be shown that the phenotypes were associated with gene 38. All mutant alterations (mostly single amino acid substitutions) were found within the hypervariable regions of protein 38. In particular, a substitution leading to Ox2h12h1.11 (Arg170----Ser) had occurred at the same site that led to Ox2h10 (His170----Arg), which binds to OmpC in addition to OmpA. It is concluded that not only can protein 38 gain the ability to switch from a protein to a carbohydrate as a receptor but can do so using the same domain of the polypeptide.     

Role of outer membrane components in the nonspecific binding ofEscherichia coli to cellulose

The involvement of lipopolysaccharide and outer membrane proteins in the binding ofEscherichia coli to cellulose was investigated. Cellulose binding was assayed in defined strains with or without O-antigenic polysaccharide and in mutants with defects in lipopolysaccharide core synthesis. Binding was also tested in strains lacking major outer membrane proteins. Optimal cellulose binding was exhibited by rough strains and was reduced to various extents in the presence of different O-antigens. Core defects also reduced but did not abolish binding to cellulose. Reduced binding was also found in mutants lacking OmpC protein, but OmpC/OmpA double mutants orompB mutants lacking OmpC and OmpF were not affected. Mutants with reduced cellulose binding were also isolated directly through selection of nonbinding populations after chromatography on cellulose columns. Each of the independent isolates derived fromE. coli K12 with reduced cellulose binding had multiple mutations, with additional phenotypic changes such as phage resistance, increased sensitivity to bile salts, or altered patterns of outer membrane proteins. These results suggest that no single receptor that could be altered by mutation was responsible for the binding ofE. coli to cellulose. Rather, the nonspecific binding of cellulose was more likely to be due to interaction with, or the combined activity of, several integral outer membrane components that could be masked by O-antigen.     

Analysis of structure-function relationships in Escherichia coli K12 outer membrane porins with the aid of ompC-phoE and phoE-ompC hybrid genes

Summary To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene. The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization. Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies. Most of these properties were found to be determined by multiple regions clearly separated in the primary structure. The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface. The locations of these regions are in agreement with a previously proposed model for porin topology.     

Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors.

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.     

Regulatory mutations conferring constitutive synthesis of major outer membrane proteins (OmpC and OmpF) in Escherichia coli.

An ompB strain of Escherichia coli K-12 lacking major outer membrane proteins OmpC and OmpF was used to isolate a pair of mutants that have restored the ability to synthesize either OmpC or OmpF protein. These mutants were found to produce the respective proteins constitutively under the several conditions where the synthesis in the wild-type strain was markedly repressed; namely, in the absence of the ompB gene function, under restrictive medium conditions, or upon lysogenization with phage PA-2. The mutations ompCp1 and ompFp9 responsible for such synthesis were shown to be located in the close vicinity of the corresponding structural genes, ompC and ompF. Moreover, the mutations affect the expression of these genes in a cis-dominant fashion. Taken together with other evidence, it was suggested that ompCp1 and ompFp9 represent regulatory site mutations occurring at the promoter regions of ompC and ompF respectively. Relevance of these findings to the genetic control of outer membrane protein synthesis is discussed.     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

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.     

Assembly-defective OmpC mutants of Escherichia coli K-12.

Novel ompC(Dex) alleles were utilized to isolate mutants defective in OmpC biogenesis. These ompC(Dex) alleles also conferred sensitivity to sodium dodecyl sulfate (SDS), which permitted the isolation of SDS-resistant and OmpC-specific phage-resistant mutants that remained Dex+. Many mutants acquired resistance against these lethal agents by lowering the OmpC level present in the outer membrane. In the majority of these mutants, a defect in the assembly (metastable to stable trimer formation) was responsible for lowering OmpC levels. The assembly defects in various mutant OmpC proteins were caused by single-amino-acid substitutions involving the G-39, G-42, G-223, G-224, Q-240, G-251, and G-282 residues of the mature protein. This assembly defect was correctable by an assembly suppressor allele, asmA3. In addition, we investigated one novel OmpC mutant in which an assembly defect was caused by a disulfide bond formation between two nonnative cysteine residues. The assembly defect was fully corrected in a genetic background in which the cell's ability to form disulfide bonds was compromised. The assembly defect of the two-cysteine OmpC protein was also mended by asmA3, whose suppressive effect was not achieved by preventing disulfide bond formation in the mutant OmpC protein.     

Release of outer membrane fragments from wild-type Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments.

EDTA-induced outer membrane losses from whole cells of wild-type Escherichia coli (O111:B4) and several lipopolysaccharide (LPS) mutants derived from E. coli K-12 D21 were analyzed. EDTA treatment induced losses of LPS (up to 40%), outer membrane proteins OmpA, OmpF/C, and lipoprotein, periplasmic proteins, and phosphatidylethanolamine. The extent of these releases was strain specific. Successively more EDTA was necessary to induce these losses from strains containing LPS with increasing polysaccharide chain length. An additional heat shock immediately following the EDTA treatment had no effect on LPS release, but it decreased the release of outer membrane proteins and reduced the leakage of periplasmic proteins, suggesting that the temporary increase in outer membrane "permeability" caused by Ca2+-EDTA treatment was rapidly reversed by the redistribution of outer membrane components, a process which is favored by a mild heat shock. The fact that the material released from E. coli C600 showed a constant ratio of lipoprotein, OmpA, and phosphatidylethanolamine at all EDTA concentrations tested suggests that the material is lost as specific outer membrane patches. The envelope alterations caused by EDTA did not result in cell lysis.     

Receptor specificity of the Escherichia coli T-even type phage Ox2. Mutational alterations in host range mutants

The T-even type Escherichia coli phage Ox2 uses the outer membrane protein OmpA as a receptor. The protein is recognized with the ends of the virion's long tail fibers. The 266 residue protein 38 is located at this site and acts as an adhesin. Host-range mutants had previously been isolated from Ox2. Mutant Ox2h5 is able to infect cells possessing an altered OmpA protein, which renders the cell resistant to Ox2. Ox2h10 was selected from Ox2h5. This phage recognizes the OmpC protein in addition to the OmpA protein. Ox2h12, which stems from Ox2h10, binds to OmpC with high affinity, but has lost efficient binding to OmpA. The mutational alterations caused in genes 38 are: Asp231----Asn(h5) and His170----Arg(h10). The triple mutant Ox2h12 possesses an insertion of a Gly residue next to Gly121. The three mutants have additionally acquired mutations affecting their base plate, making them "trigger-happy". When protein 38 was compared with the same protein derived from other E. coli phages, it was found to contain two constant and one variable domains, the latter harboring four hypervariable regions flanked by a largely conserved glycine-rich sequence. The h5 and h10 mutations occurred within two hypervariable areas, while the additional Gly residue was present in one of the flanking conserved sequences. On the basis of these results, as well as those obtained from host-range mutants analyzed previously, a model for such adhesins is proposed. Receptor recognition is most likely performed via the hypervariable regions, which may form loops held together in close proximity by the oligoglycine sequences. The latter may achieve this by being part of highly compact omega loops.     

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.     

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.     

Coliphage which requires either the LamB protein or the OmpC protein for adsorption to Escherichia coli K-12.

Either of two different proteins in the outer membrane of Escherichia coli K-12 (LamB and OmpC) can function in the constitution of receptor activity for a newly isolated T-even bacteriophage. This bacteriophage (SSI) differs from other T-even phages which use the OmpC protein as their receptors. The simple procedure used to isolate phage SSI may be suitable for the detection of bacteriophages with novel outer membrane receptor requirements.     

Site-directed mutagenesis studies to probe the role of specific residues in the external loop (L3) of OmpF and OmpC porins in susceptibility of Serratia marcescens to antibiotics

Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF-ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages T2, K3 and of K3 host range mutants

Genes 38, which code for a receptor-recognizing protein present at the tip of the long tail fibers, have been sequenced from phages T2, the T-even-type phage K3 and its host range mutants K3hx, K3h1 and K3h1h. The genes from phages T2 and K3 code for proteins consisting of 262 and 260 amino acid residues, respectively. Fifty amino-terminal and 25 carboxy-terminal residues are highly conserved. The amino-terminal amino acids are most likely involved in binding to the neighboring protein 37. Between residues 116 and 226 of the T2 protein and residues 116 and 223 of the K3 protein, sequences exist that are similar to sequences present in Escherichia coli outer membrane proteins and which serve as phage receptors. Most likely, all of these regions in the latter proteins are exposed on the cell surface and are part of their phage receptor areas. In the phage proteins, these sequences are flanked by stretches rich in glycine, perhaps providing an increased flexibility for the polypeptide at these sites; some "wobble" may be required during the protein 38-receptor interaction. The mutational alterations in the host range mutants were found in gene 38. In the K3hx protein, a duplication of six base-pairs caused the wild-type sequence -Gly163-Lys-Leu-Ile- to be changed to -Gly163-Lys-Leu-Lys-Leu-Ile-. In the K3h1 protein, a glutamic acid residue at position 203 was substituted by a lysine. Both alterations occurred within areas similar to outer membrane proteins. Mutant K3h1h, derived from K3h1, exhibits an extended host range as compared to K3h1. No mutational alteration, in addition to that found in K3h1, was found in g38 nor was the part of gene 37 that encodes the carboxy-terminal moiety of the protein altered. K3h1h may represent a "trigger-happy" phage. The results of this and other work show that the phage-phage receptor systems under study represent a primitive immune system.     

Cysteine-22 and cysteine-38 are not essential for the functions of maltoporin (LamB protein)

Maltoporin in the outer membrane of Escherichia coli contains two cysteine residues, at positions 22 and 38 in the primary sequence. The role of these residues in determining structural stability, and their contributions to the maltoporin binding sites for maltodextrins and bacteriophage lambda, was investigated. Site-directed mutagenesis was used to alter each of these residues to a serine. A double mutant lacking both cysteines was also isolated. None of the substitutions affected maltodextrin binding or the binding of phage lambda, suggesting the variant proteins retain a native binding-site conformation. The mutants were assembled at wild-type levels into the outer membrane as maltoporin trimers but the temperature-stability of the trimer greater than monomer dissociation was slightly reduced in the presence of the Cys 38 substitution. However, it is unlikely that the stability of trimers was due to disulfide linkages between subunits since the native trimers are stable under highly reducing conditions in the presence of SDS; more likely the Cys greater than Ser substitutions slightly perturb intra- or inter-subunit hydrophobic interactions in regions predicted to span across the outer membrane.     

Escherichia coli phage receptors. Minor porins and proteins participating in the specific transport as phage receptors

Except for the main porin proteins OmpC and OmpF there exist the membrane proteins participating in the transport of specific substrates: phosphates, nucleosides, iron, vitamin B12, maltose and maltodextrins, that also play the role of phage receptors. Some phages use as receptors the porins determined by the genes of lambdoid prophages. LamB protein that serves receptor for phage lambda exposes the amino acids sequence on the outer surface of membranes that participates in phage adsorption. The sequence is similar to tetrapeptide of fibronectin responsible for binding with the surface of cellular receptor in eucaryotes.