Escherichia coli enterotoxin. Purification and partial characterization.

Enterotoxin, a diarrheagenic protein elaborated by pathogenic Escherichia coli strains has been isolated from the supernatant of fermenter cultures of E. coli strain P263, a porcine enteropathogen. Purification steps involving Bio-Gel agarose A-5m, Sephadex G-75 chromatography, and preparative isotachophoresis were used in the isolation. The resulting product appears to be pure according to immunoelectrophoretic, disc electrophoretic, ultracentrifugal, and immunologic criteria. The entertoxin has an apparent molecular weight of 102,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and its isoelectric point is 6.90. The isolated product is highly active in inducing experimental diarrhea in adult rabbits and piglets. It also elicits, in small dosage, a marked increase in adenylate cyclase activity in broken cell preparations of cat heart tissue. The enterotoxin activity is acid-labile and is destroyed by heating at 65 degrees for 30 min. It is suggested that the heat-stable enterotoxin material is derived from heat-labile enterotoxin by forming a complex with endotoxin or capsular material present in the culture supernatant.     

Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes'' strain)

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca(2+), Zn(2+). It is a dimer with a molecular weight of approx. 70000, and the K(m) value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH(4) in the presence of substrate. The K(m) value for fructose diphosphate is about 20mum (although the Lineweaver-Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.     

Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein.

Type 1 pili of Escherichia coli contain three integral minor proteins with apparent molecular weights (Mr) of 28,000 (28K protein), 16,500, and 14,500 attached to rods composed of Mr-17,000 pilin subunits (Hanson and Brinton, Nature [London] 322:265-268). We describe here an improvement on our earlier method of pilus purification, which gives higher yields and higher purity. Also reported are methods allowing fractionation of intact type 1 pili into rods of pure pilin and free minor proteins, as well as fractionation of the 28K tip adhesion protein from the 16.5K and 14.5K proteins. We have determined the amino acid composition and amino-terminal sequence of the adhesion protein. This sequence shows limited homology with the amino-terminal sequences of several E. coli pilins, including type 1.     

AFA-I, a cloned afimbrial X-type adhesin from a human pyelonephritic Escherichia coli strain. Purification and chemical, functional and serologic characterization

AFA-I, a mannose-resistant, P-independent, X-binding afimbrial Escherichia coli adhesin was purified from a recombinant strain and chemically, functionally and serologically characterized. AFA-I exists on the bacterial surface and free as a macromolecular aggregate in the supernatant of spent culture medium. It is composed of a single, repeating 16-kDa polypeptide subunit. The AFA-I protein amino acid composition is remarkable for the presence of 22% non-polar hydrophobic residues and 2.5-3.0 cysteines per subunit. Since AFA-I travels as a monomer in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under non-reducing conditions, no disulfide bonds exist between subunits and at least one free sulfhydryl per subunit is available. The AFA-I N-terminal amino acid sequence residues 1-24 was unrelated to E. coli fimbrial sequences; however, the N-terminus of AFA-I and GV-12, another E. coli afimbrial protein, was asparagine. HB101 (pIL 14), the AFA-I recombinant strain, agglutinated only human and gorilla erythrocytes, indicating a preference for receptor molecules on the red cells of man and the anthropoid apes. AFA-I did not bind glycophorin A or sialyl glycosides and is therefore distinct from the E. coli X-binding adhesins with M and S specificity. The AFA-I receptor was found to be abundant and diffusely distributed on HeLa tissue culture monolayer cell surfaces by indirect fluorescent microscopy. Anti-AFA-I sera bound AFA-I in Western blots of 4 out of 16 X-binding E. coli urine isolates. They did not bind MS or P pili. AFA-I may be exemplary of an adhesin class significant for the pathogenesis of human urinary tract infections.     

Purification and characterization of adenylate cyclase from Escherichia coli K12

Adenylate cyclase of Escherichia coli K12 has been purified 17,000-fold to near homogeneity from a 5-fold overproducing strain. One major band of Mr = 92,000 and several minor bands are seen on sodium dodecyl sulfate-polyacrylamide electrophoresis of the purest fractions. Identification of the enzyme with the 92,000-Da protein is based on the correlation of this band with activity when highly purified enzyme is eluted from ADP-sepharose columns. The native enzyme has a molecular weight of 95,000 determined by gel filtration, showing that the enzyme is active as a monomer. The purest enzyme has a specific activity of 700 nmol min-1 mg-1, indicating a turnover number of about 100 min-1. Our data indicate that there are only about 15 molecules of the enzyme in wild type cells of E. coli. In crude extracts, over 80% of the activity is soluble after centrifugation at 100,000 x g, indicating the enzyme is soluble or, at most, loosely membrane bound. The enzyme is only moderately stable in crude extracts and becomes more unstable as purification proceeds. Activity is stabilized by ATP, or at -20 degrees C as an ammonium sulfate precipitate or in 50% glycerol. The enzyme has an absolute requirement for divalent cations. Maximum activity with Mg2+ is reached at 30 mM. Mn2+ is a good substitute; Co2+ activates well at low concentrations but becomes inhibitory at high concentrations; and Ca2+ is a potent inhibitor in the presence of Mg2+. The isoelectric point of the enzyme is 6.1, and its pH optimum is 8.5. The enzyme is inhibited by its substrate, with a Km of about 1 mM and a Ki of about 1.5 mM, and is noncompetitively inhibited by PPi, ADP, GTP, and a number of other compounds. The data suggest that dissociation of PPi from the first enzyme-product complex is the rate-limiting step in the reaction. Activation of the enzyme, inferred to occur in vivo, could be produced by a postulated regulatory effector which speeds release of PPi from the enzyme-product complex.     

Purification and characterization of catalase HPII from Escherichia coli K12

Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90,000 and 92,000. Only a single 92,000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532,000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4-11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.     

A single-step isolation of K99 pili from B-44 strain of Escherichia coli

A single-step procedure has been developed to isolate pili from enterotoxigenic Escherichia coli strain B44 of calf origin. Pili, removed from the bacteria by heat shock treatment, were allowed to aggregate at 4 degrees C for 16 h. The precipitated pili, isolated by centrifugation, had typical pili morphology as shown by electron microscopy; ability to bind pig brush border; molecular weight greater than 6 million; and predominance of hydrophobic amino acids. On sodium dodecyl sulfate gel electrophoresis, the subunit pilin migrated as a single polypeptide of molecular weight 17,000.     

Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli

Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.     

Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli

The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His(6)-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ～85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.     

Preparation and characterization of myosin from cultured cells of Escherichia coli (02:K1 30156 strain)

In this study the myosin preparations isolated from E. Coli cell cultures were analysed. The isolation of myosin from fresh cultures resulted in a substantially higher (approximately 10-fold) yield than from stored cells. The coli myosin, despite the two DEAE-cellulose treatments, contained more RNA and P-lipid than the myosin prepared from skeletal muscle. The RNA content can be removed gradually by acetone denaturation and lipid removal followed by subsequent washings. The fresh preparations contained protein-bound alkali-stable P; part of this was released by Cu ions. The coli myosin can be phosphorylated. The phosphate uptake depended on the concentrations of ATP and Mg2+ and on the time of incubation. The alkaline hydrolysate of the phosphorylated, washed lipid-free preparation was resolved into 8 P-containing peaks on ion-exchange chromatography. Of fractions P-Arg, Pi, P-Lys and 2 P-His conformer were identified by means of synthetic compounds, elution pattern and specific reactions. The remaining compounds could not be identified. The most abundant component was P-Arg suggesting that this compound might play an important role in the cytokinetic movements of E. coli.     

Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4

Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida, respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K(m) values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 microm, respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.     

Purification and partial characterization of coxsackievirus B3 2A protease expressed in Escherichia coli

Reported here is the overexpression, purification and partial characterization of recombinant coxsakievirus B3 2A protease (CVB3 2A(pro)) from bacterial cells transformed with a plasmid containing the CVB3 2A(pro) cDNA sequences. The structural investigation showed that the protein contains mostly beta-strand elements and requires Zn(2+) ions as a structural component which appeared to be inhibitory if added exogenously. The purified enzyme activity was optimal at 4 degrees C and had a short half-life at physiological temperature. This feature can be the result of the presence of a high content of beta-structure and also hydrophobic residues in its structure.     

Purification and characterization of P fimbriae from an Escherichia coli strain isolated from a septicemic turkey

Abstract A pap + Escherichia coli isolate from a turkey with colisepticemia expressed P fimbriae with a major subunit of an apparent molecular mass of 18 kDa which reacted with anti-F11 serum. This fimbriae was purified and polyclonal antiserum was produced in rabbits. The N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of F11. On immunoblotting, the antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae, with F11, and with F165₁ fimbriae. Some antigenic determinants on the major subunits of F13, F7₁, and F7₂ fimbriae, with a stronger reaction against F13 fimbriae, were also recognized. The F11 antiserum reacted similarly to the antiserum against avian P fimbriae although cross-reactions against F13, F7₁, and F7₂ fimbriae were equivalent. In a competitive enzyme-linked immunosorbent assay, serological differences were observed between the purified avian P fimbriae and F11. Thus, the avian P fimbriae is closely related but not identical to F11 fimbriae which are associated with E. coli isolated from human urinary tract infection.     

Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin

Escherichia coli (E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc β (1–4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets’ diarrhea.     

Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes'' strain).

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.     

Purification and partial characterization of a cellodextrin glucohydrolase (beta-glucosidase) from Trichoderma reesei strain QM 9414

A beta-glucosidase (E.C. 3.2.1.21) was isolated from the culture filtrate of fungus Trichoderma reesei QM 9414 grown in continuous culture with biomass retention. The crude extracellular enzyme preparation was fractionated by a three-step purification procedure [chromatography on Fractogel HW-55 (S) and Bio-Gel A 0.5 plus final preparative isoelectric focusing] to yield three beta-glucosidases with isoelectric points at pH 8.4, 8.0, and 7.4. Only one enzyme (pi 8.4) met the stringent criterion of being homogeneous according to titration curve analysis. This enzyme was then characterized not to be a glycoprotein, although the native protein contained 35% carbohydrate (as glucose). It was found to have an apparent molar mass of 7 x 10(4) g/mol (SDS-PAGE), exhibited its optimum activity towards cellobiose at pH 4.5 and 70 degrees C (30 min test), and lost less than 3% activity at 50 degrees C over a period of 7 h. The K(M) values towards cellobiose and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.5mM and 0.3mM, respectively. The enzyme hydrolyzed cellodextrins (cellotriose to cellooctaose) by sequentially splitting off glucose units from the nonreducing end of the oligomers. The extent of the observed transfer reactions varied with the initial substrate concentration. No enzyme activity towards microcrystalline cellulose or carboxymethylcellulose could be detected. The classification of the enzyme as beta-glucosidase or exo-beta-1,4-glucan glucohydrolase is discussed with respect to the exhibited hydrolytic activities.     

Purification and further characterization of the second nitrate reductase of Escherichia coli K12

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.