Outer Membrane Proteins OmpC Research Articles

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes.

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2×10 7 plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.

Purification of Integral Outer-Membrane Protein OmpC, a Surface Antigen from Salmonella typhi for Structure–Function Studies: A Method Applicable to Enterobacterial Major Outer-Membrane Protein

Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure–function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins.

The porin OmpC ofSalmonella typhimuriummediates adherence to macrophages

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.

Crystallization of the immunodominant outer membrane protein OmpC; the first protein crystals from Salmonella typhi, a human pathogen

OmpC, a surface antigen of Salmonella typhi was crystallized after several attempts, using PEG 3350. Well shaped hexagonal crystals were grown from vapor diffusion method using octyl glucoside and C 12E 9 as detergents. Crystals are sensitive to X-ray and diffract weakly up to 7 Å. Porin isoforms, due to the bound lipopolysaccharides, could be the cause for poor diffraction. Crystal quality depends largely on the purification method, and in case of LPS contamination, the genetic background of the bacteria. Crystallization and initial data collection suggest optimum conditions and the method of choice for OmpC crystallization.

Characterization of a Phage Specific to Hemorrhagic Escherichia coli O157:H7 and Disclosure of Variations in Host Outer Membrane Protein OmpC

Phage AR1, previously known to infect Escherichia coli O157:H7 with high specificity, was further characterized for its genetic properties. The phage DNA sequences including capsid genes and a putative α-glucosyltransferase gene (α-gt) have been deduced. These sequences are conservative but not identical to those of T4 phage. However, a nonessential gene, SegD, organized within the capsid gene cluster of T4 is missing in the corresponding region of AR1 genome, and this characteristic has not been observed among T-even related phages. The difference between AR1 and T4 was further exemplified by their distinct host ranges. Strains of E. coli O157:H7 collected from different sources were permissive to AR1 but resistant to T4 that normally infects K-12 strains of E. coli through contact with the outer membrane protein OmpC. Thus, the O157:H7 strains must have a varied OmpC. Indeed, the OmpC sequence of O157:H7 strains was proved to differ from that of K-12 strains by a total of 15 amino acid substitutions and two gaps (a five-residue deletion and a four-residue insertion). The OmpC molecules are relatively conserved across the gram-negative bacteria, and this is the first time OmpC divergence has been found within the same E. coli species. Since OmpC is located in the outer membrane and its expression is regulated by environmental conditions, alteration of the structure in pathogenic O157:H7 strains may have biological significance.

Characterization of a phage specific to hemorrhagic Escherichia coli O157:H7 and disclosure of variations in host outer membrane protein ompC.

Phage AR1, previously known to infectEscherichia coli O157:H7 with high specificity, was further characterized for its genetic properties. The phage DNA sequences including capsid genes and a putative α-glucosyltransferase gene(α-gt) have been deduced. These sequences are conservative but not identical to those of T4 phage. However, a nonessential gene,SegD, organized within the capsid gene cluster of T4 is missing in the corresponding region of AR1 genome, and this characteristic has not been observed among T-even related phages. The difference between AR1 and T4 was further exemplified by their distinct host ranges. Strains ofE. coli O157:H7 collected from different sources were permissive to AR1 but resistant to T4 that normally infects K-12 strains ofE. coli through contact with the outer membrane protein OmpC. Thus, the O157:H7 strains must have a varied OmpC. Indeed, the OmpC sequence of O157:H7 strains was proved to differ from that of K-12 strains by a total of 15 amino acid substitutions and two gaps (a five-residue deletion and a four-residue insertion). The OmpC molecules are relatively conserved across the gram-negative bacteria, and this is the first time OmpC divergence has been found within the sameE. coli species. Since OmpC is located in the outer membrane and its expression is regulated by environmental conditions, alteration of the structure in pathogenic O157:H7 strains may have biological significance.

Construction of CTB fusion proteins for screening of monoclonal antibodies against Salmonella typhi OmpC peptide loops

Mice were immunized with resin-bound peptides whose sequences have been proposed to be part of exposed loops in Salmonella typhi outer membrane protein OmpC. To screen hybridomas for monoclonal antibodies against those epitopes, we designed fusion proteins where the candidate peptide sequence was attached to the amino end of cholera toxin B-subunit (CTB). The constructed fusion proteins allowed the efficient selection of positive clones by GM1-ELISA. Selected antibodies recognized purified OmpC and whole Salmonella bacteria. This suggests a native structure of our genetically attached peptides in agreement with immunological properties reported for previous CTB recombinant fusion proteins. In a more general context, CTB hybrids could be used to screen for antibodies towards immunogenic epitopes in other systems. This might turn out to be particularly useful when producing antibodies against peptide sequences in microorganisms whose handling is difficult or that pose inherent health risks.

Construction of CTB fusion proteins for screening of monoclonal antibodies against Salmonella typhi OmpC peptide loops.

Mice were immunized with resin-bound peptides whose sequences have been proposed to be part of exposed loops in Salmonella typhi outer membrane protein OmpC. To screen hybridomas for monoclonal antibodies against those epitopes, we designed fusion proteins where the candidate peptide sequence was attached to the amino end of cholera toxin B-subunit (CTB). The constructed fusion proteins allowed the efficient selection of positive clones by GM1-ELISA. Selected antibodies recognized purified OmpC and whole Salmonella bacteria. This suggests a native structure of our genetically attached peptides in agreement with immunological properties reported for previous CTB recombinant fusion proteins. In a more general context, CTB hybrids could be used to screen for antibodies towards immunogenic epitopes in other systems. This might turn out to be particularly useful when producing antibodies against peptide sequences in microorganisms whose handling is difficult or that pose inherent health risks.

Crystallization and X-ray Studies of the DNA-binding Domain of OmpR Protein, a Positive Regulator Involved in Activation of Osmoregulatory Genes in Escherichia coli

The OmpR protein of Escherichia coli is a positive regulator involved in the activation of expression of ompC and ompF genes encoding the major outer membrane protein OmpC and OmpF, respectively. The C-terminal half domain of OmpR (OmpR-C), which is responsible for DNA-binding, has been crystallized using the hanging drop vapour diffusion method. X-ray studies show that the crystals belong to the trigonal space group P3 121 (or P3 221) with a = b = 60·4 Å, c = 58·8 Å and γ = 120°. The asymmetric unit contains one molecule. The crystals diffract to at least 3 Å resolution and are suitable for X-ray structure analysis.

Formation of active heterologous nitrate reductases between nitrate reductases A and Z of Escherichia coli.

Two nitrate reductases, NRA and NRZ, are present in Escherichia coli. These isoenzymes have the same alpha beta gamma, subunits composition and have similar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcloning the different genes and expressing them from separate transcriptional units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantly but independently can meet efficiently and associate to form active enzymes, and (ii) that the alpha subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yield an active membrane-bound heterologous enzyme. One such heterologous enzyme, alpha A beta Z gamma Z, has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heterologous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.

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 0x2 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 → h12h1·111 → h12h1·111. 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.

Isolation and characterization of extragenic suppressor mutants of thetolA-876 periplasmic-leaky allele inEscherichia coliK-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.

Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.

Receptor-recognizing proteins of T-even type bacteriophages: The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb

Escherichia coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors by means of a C-terminal region of protein 37; a dimer of this polypeptide (1026 residues in T4) is located at the distal part of the long tail fibers. Virions of the T2 family use protein 38 (which is attached to the free end of protein 37) for this purpose. The corresponding areas of genes 37 belonging to TuIa and TuIb were cloned and sequenced. Comparison of the deduced protein primary structures, including those of T4 and lambda Stf (Stf most likely representing a subunit of the side tail fibers of phage lambda) showed that an area of 70 to 100 residues is characterized by very variable sequences, while the sequences of the adjacent 43 to 44 C-terminal residues as well as those upstream from the variable region are highly homologous. The variable regions are flanked and interrupted seven or eight times by the motif His-x-His-y, with x and y most often being Ser or Thr; furthermore, the locations of these repeated tetrapeptides are conserved. Using hybrid phages obtained by recombination of one phage with cloned fragments of gene 37 of another, it could be shown that the area of this gene encoding receptor specificity includes the variable area. The situation is analogous to the known receptor-recognizing region of proteins 38 belonging to the T2-type family, except that the repeating sequence is of a different nature. In T4, receptor specificity is coded for by 382 base-pairs of the 3'-end of the gene, starting exactly at the variable area. It was found that T4 can use the outer membrane protein OmpC or lipopolysaccharide as receptors with the same efficiency, and it is proposed that the 70 residues of the variable part of the protein serve to bind to both ligands.

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.

Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, stimulates the transcription of the ompF and ompC genes in Escherichia coli

The OmpR of Escherichia coli is a positive regulator specific for the ompF and ompC genes, which encode outer membrane proteins OmpF and OmpC, respectively. The EnvZ protein is a protein kinase which specifically phosphorylates the OmpR protein. In this study, the results of in vitro transcription experiments revealed that the phosphorylation of the OmpR protein by the EnvZ protein stimulates the transcription of both the ompF and ompC genes.

A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4

The distal part of the long tail fiber of Escherichia coli bacteriophage T4 consists of a dimer of protein 37. Dimerization requires the catalytic action of protein 38, which is encoded by T4 and is not present in the virion. It had previously been shown that gene tfa of the otherwise entirely unrelated phage lambda can functionally replace gene 38. Open reading frame (ORF) 314, which encodes a protein that exhibits homology to a COOH-terminal area of protein 37, is located immediately upstream of tfa. The gene was cloned and expressed in E. coli. An antiserum against the corresponding polypeptide showed that it was present in phage lambda. The serum also reacted with the long tail fibers of phage T4 near their free ends. An area of the gene encoding a COOH-terminal region of ORF 314 was recombined, together with tfa, into the genome of T4, thus replacing gene 38 and a part of gene 37 that codes for a COOH-terminal part of protein 37. Such T4-lambda hybrids, unlike T4, required the presence of outer membrane protein OmpC for infection of E. coli B. An ompC missense mutant of E. coli K-12, which was still sensitive to T4, was resistant to these hybrids. We conclude that the ORF 314 protein represents a subunit of the side tail fibers of phage lambda which probably recognize the OmpC protein. ORF 314 was designated stf (side tail fiber). The results also offer an explanation for the very unusual fact that, despite identical genomic organizations, T4 and T2 produce totally different proteins 38. An ancestor of T4 from the T2 lineage may have picked up tfa and stf from a lambdoid phase, thus possibly demonstrating horizontal gene transfer between unrelated phage species.

Location of phosphorylation site and DNA-binding site of a positive regulator, OmpR, involved in activation of the osmoregulatory genes of Escherichia coli

The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N- and C-terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N-terminal portion contains a site involved in phosphorylation by an OmpR-specific protein kinase EnvZ, whereas the C-terminal part possesses a DNA-binding site for the ompC and ompF promoters.

Location of DNA-binding segment of a positive regulator, OmpR, involved in activation of the ompF and ompC genes of Escherichia coli

OmpR protein is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. OmpR protein is considered to have a two-domain structure. In the present study we isolated a C-terminal fragment of the OmpR molecule, which bound to a specific promoter sequence of the ompF gene. We conclude that the C-terminal portion of the OmpR protein contains a DNA-binding site.