Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica, from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis. Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.     

Involvement of a transmissible plasmid in heat-stable exotoxin and delta-endotoxin production inBacillus thuringiensis subspeciesdarmstadiensis

A strain ofBacillus thuringiensis subsp.darmstadiensis (serotype 10), which produces heat-stable exotoxin and delta-endotoxin (Exo⁺Cry⁺), was used for curing and conjugation-like transformation experiments. After treatment with sodium dodecyl sulfate, nine independent mutants that lacked exotoxin productivity (Exo^–) were obtained. Agarose gel electrophoresis showed that all Exo^– strains had lost a plasmid, whose size was 62 megadaltons (Mdal). WhenB. thuringiensis was mated with a streptomycin-resistant (Str^r)B. cereus strain, five Exo⁺Str^r transformants that had acquired the 62-Mdal plasmid were isolated. Furthermore, the Cry⁺ phenotype was consistently associated with the Exo⁺ phenotype. These results indicate that a transmissible plasmid is involved in production of both heat-stable exotoxin and delta-endotoxin.     

Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery

Vibrio anguillarum was isolated as a pathogen in the commercial culture of oyster spat at Pigeon Point, Calif. A water-soluble, heat-stable exotoxin extracted from cultures of the vibrio inhibited larval swimming and contributed to larval mortality. Although the vibrio was insensitive to penicillin in standard plate testing, this antibiotic proved useful in preventing mass larval mortalities in the hatchery.     

Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery.

Vibrio anguillarum was isolated as a pathogen in the commercial culture of oyster spat at Pigeon Point, Calif. A water-soluble, heat-stable exotoxin extracted from cultures of the vibrio inhibited larval swimming and contributed to larval mortality. Although the vibrio was insensitive to penicillin in standard plate testing, this antibiotic proved useful in preventing mass larval mortalities in the hatchery.     

Properties of the isoforms of α-amylase from kilned and unkilned malted sorghum (Sorghum bicolor)

We have previously reported the presence of a relatively heat-stable α-amylase with a low K_m for starch in kilned malted sorghum. In order to establish the industrially useful and more efficient isoforms, we have separated this α-amylase into different isoforms using both cation and anion-exchange chromatographies. Unkilned malted α-amylase crude was separated into three different isoforms (a1, a2 and a3) whereas kilned samples were separated into two (a1 and a2). Apparently one isoform (a3) was lost during kilning due to heat lability. a1 isoform which appears to have a neutral pI and constitute about 60% of the total α-amylases protein that were induced during germination, have the lowest K_m for starch. They are more generally stable than other isoforms at all the temperatures studied. These isoforms lost only 10% activity at 80 °C for 30 min and still had some residual activity at 100 °C incubation for 30 min. a1 isoform could therefore be adapted for industrial starch conversion processes which are carried out within this range of gelatinizing temperatures because of its properties.     

Persistence of Bacillus thuringiensis in foundation beeswax and beecomb in beehives for the control of Galleria mellonella

Beecomb was protected against wax moth attack by impregnating foundation beeswax with Bacillus thuringiensis. Bakthane, a serotype I product, gave good protection of broodcomb for 2 yr at 0.5% in wax and partial protection for 6 yr at 2%. The active component was exotoxin present at about 0.9% in Bakthane in a very insoluble state. No harm to the bees was detected, even when a partially purified preparation of exotoxin was used, but further tests on the leaching of pure exotoxin from comb into honey are required before the latter can be regarded as a practical method of wax moth control. The action of exotoxin on Galleria mellonella larvae was slower than that of the spore-crystal complex and less than that of exotoxin on some Diptera. Spores and crystals of serotype V were × 500 as potent in G. mellonella as those of serotype I and exotoxin on honey-rich artificial food in laboratory assays, but their activity deteriorated in the first hive trials with treated foundation wax. Prehive deterioration was due both to Teepol and soap in the liquid lubricating the wax mill rollers and to moist storage of the foundation. This deterioration was prevented by the use of Triton X-100 as lubricant, by drying the newly impregnated foundation wax and by storing it in dry conditions. This resulted in good protection of comb for one hive season by 1% of serotype V Thuricide in foundation wax. However, protection after two seasons varied, making the use of serotype V spores and crystals uneconomical for commercial practice, although safe for bee and man.     

Identification and characterization of a heat-labile type I glutamine synthetase fromStreptomyces cinnamonensis

Streptomycetes have two distinct glutamine synthetases (GS): a heat-stable dodecameric GSI and a heat-labile octameric GSII. A heat-inactivated GS activity was detected in crude extracts ofStreptomyces cinnamonensis cells grown with nitrate or glutamate as the nitrogen source. The purified enzyme obtained from crude extracts of the nitrate-grown cells after affinity and anion-exchange chromatography was also heat-labile; it was inactivated by 80 % when incubated at 50 °C for 1 h. However, the enzyme has properties typical of GSI and similar with those of the heat-stable GSI purified fromS. aureofaciens: It is composed of twelve subunits, each ofM 55 kDa, and has a native molar mass of 625 kDa and an isoelectric point at pH 4.2. In addition, its activity is regulated by reversible adenylylation. Mg²⁺ and NaCl but not Mn²⁺ protected the purified enzyme from thermal inactivation, and both NaCl and Mn²⁺ or Mg²⁺ stabilized its activity at 4–8 °C. As compared with GSI fromS. aureofaciens, theS. cinnamonensis enzyme was cleaved more extensively during SDS-PAGE, was less sensitive to feedback inhibitors, and similarly affected by divalent cations. TheK _m values were 12.5 mmol/L forl-glutamate, 0.1 for NH ₄ ⁺ , 1.25 for ATP, 18.5 forl-glutamine, 3.3 for hydroxylamine and 0.087 for ADP. To our best knowledge, this is the first report of a heatlabile GSI from any source.     

Investigation of the heterogeneity of xyloglucans from the cell walls of apple

Cell-wall material from parenchymatous tissues of apple was sequentially extracted with 50mm NaOH at 1°, m KOH at 1° and 20°, and 4m KOH at 20°, to leave a residue of α-cellulose. From the 4m KOH-soluble fraction, a crude xyloglucan was isolated by anion-exchange chromatography, and further resolved into seven xyloglucans by borate anion-exchange chromatography. The relative amounts of the xyloglucans, in order of elution, were 2.7:1.3:29.7:1.0:3.2:1.2:10.3. The structural features of five of the xyloglucans were determined by methylation analysis. These results show that apple xyloglucans exhibit heterogeneity.     

Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A is representative of a class of enzymes, the monoADP-ribosyl, which catalyze the covalent transfer of an ADP-ribose moiety of NAD⁺ to a target substrate. Availability of the three-dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD⁺ binding site of exotoxin A: the binding of NAD⁺ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 Å, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP-ribosyl transferase activity.     

Schistosoma japonicum: identification and characterization of neutrophil chemotactic factors from egg antigen

Two neutrophil chemotactic factors were identified in soluble egg antigen preparations of Schistosoma japonicum. The higher-molecular-weight neutrophil chemotactic factor was not separable from eosinophil chemotactic factor by means of gel filtration, anion-exchange chromatography, isoelectric focusing, or affinity chromatography; this neutrophil chemotactic factor is apparently identical to the higher-molecular-weight eosinophil chemotactic factor which we purified previously from the soluble egg antigen. The chemotactic activity of the eosinophil chemotactic factor for neutrophils was stable to periodate oxidation but was notably affected by heating or Pronase digestion, suggesting that the determinant for neutrophil chemotaxis exists on the peptide moiety of the eosinophil chemotactic factor. The lower-molecular-weight neutrophil chemotactic factor was separable from the higher-molecular-weight eosinophil chemotactic factor by gel filtration or anion-exchange chromatography. This neutrophil chemotactic factor was rather hydrophobic and heat-stable, but was sensitive to Pronase or carboxypeptidase A digestion. These results suggest that the receptors on the surfaces of neutrophils and eosinophils for those chemoattractants would be different from each other. We suppose that neutrophil chemotactic factors and eosinophil chemotactic factors from the eggs are responsible for neutrophil and eosinophil accumulation around the eggs in schistosomiasis japonica.     

Characterization of Competitive Inhibitors for the Transferase Activity of Pseudomonas aeruginosa Exotoxin A

A series of small, nonpolar compounds were tested for their ability to inhibit the ADP-ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A. The IC 50 values for the compounds tested ranged from 87 nM to 484 μM for NAP and CMP12, respectively. It was demonstrated that NAP was a competitive inhibitor of the ADPRT reaction for the NAD + substrate with a K i of 45 ± 5 nM, which was in good agreement with the dissociation constant determined independently (K D =56 ± 6 nM). The IC 50 value for NAP was 87 ± 12 nM, which strongly correlated with the K i and K D values. Furthermore, NAP was shown to noncovalently associate with the exotoxin A active site using exhaustive dialysis, NMR, and electrospray mass spectrometry. Finally, a computer molecular model using the X-ray structure of the substrate-bound toxin was generated with NAP bound to the active site of exotoxin A at the nicotinamide-binding site. This model is consistent with the X-ray structure of the catalytic domain of poly-ADP-ribose polymerase complexed with 4-amino-naphthalimide (Compound 4) that was included in this study.     

An alternative oven method combined with different detergent strengths in the analysis of neutral detergent fibre

Two methods for preparing neutral detergent fibre (NDF) – the standard method of Van Soest, P.J., Robertson, G.B., Lewis, B.A., `Symposium: Carbohydrate methodology, metabolism, and nutritional implications in dairy cattle.' J. Dairy Sci. 74 3583–3597 (1991), and an alternative 16 h oven method at 90°C. Neutral detergent solution (100%) was compared with solutions diluted to 50, 25 and 0% of normal strength. Sodium sulfite and a heat-stable amylase were used in all treatments. Forty-four samples consisting of 11 dried and ensiled forages, three straws, nine starchy concentrates, 10 high-protein concentrate feeds, three samples of tropical browse leaves and eight manures from both ruminants and non-ruminants were used. The 0% treatment was the only treatment giving significantly higher NDF values compared to the others (p<0.05). Sensitivity to detergent strength seemed to be sample-specific in the high protein group as meat meal and maize gluten had higher NDF values at reduced detergent strengths. We recommend using the oven method and a detergent strength of 25% of the current standard in combination with sodium sulfite for low protein feeds. For starchy feeds a heat-stable amylase should be used and high lipid samples should be acetone-extracted before isolating NDF. For high protein feed samples only the standard method can yet be recommended. The oven method is labour-saving and more convenient and arguments for reduced detergent strength suggest lower cost and reduced waste water pollution.     

Mapping a novel heat-stable resistance to Meloidogyne in Lycopersicon peruvianum

 The root-knot nematode heat-stable resistance locus from L. peruvianum LA2157 was mapped on chromosome 6. All wild tomato LA2157 entries and the LA2157 S₁ progeny tested were resistant to Mi-avirulent Meloidogyne spp. isolates at 32°C, indicating that the self-compatible accession is homozygous for heat-stable nematode resistance. The novel resistance locus was mapped on a RFLP linkage map; this map was based on a segregating F₂ population obtained from the interspecific F₁ between L. esculentum cv ‘Solentos’ and L. peruvianum LA2157. The inheritance of the heat-stable resistance was evaluated in 100 F₃ lines derived from one F₁ interspecific hybrid. The genotype of the resistance locus of the individual F₂ plants was based on the phenotypic classification of their F₃ lines, and the data were used to map the resistance locus on the arm of chromosome 6 with the closest linkage to TG178. The position of the novel heat-stable resistance of LA2157 was localized in the resistance genes’ cluster close to the location of gene Mi-1. Cuttings of the F₃ lines expressed resistance to Mi-1-avirulent M. incognita and M. javanica biotypes at 25°C and at 32°C (a temperature at which Mi-1 resistance is not expressed). There was no difference in the segregating population for expression of heat-unstable resistance and heat-stable resistance to Mi-1-avirulent Meloidogyne spp. However, LA2157 and cuttings of the above F₃ lines were susceptible to a Mi-1-virulent M. incognita isolate at 30°C and to a M. hapla isolate at 25°C. Received: 6 July 1998 / Accepted: 28 July 1998     

Purification of the endogenous glucocorticoid receptor stabilizing factor

A ubiquitous, low molecular weight, heat-stable component of cytosol stabilizes the glucocorticoid receptor in its untransformed state in association with hsp90. This heat-stable factor mimics molybdate in its effects on receptor function, and it has the heat stability, charge, and chelation properties of a metal oxyanion [Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., & Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817]. In this paper, we describe the further purification of the endogenous factor from rat liver cytosol by anion-exchange HPLC (Ion-110) after prepurification by molecular sieving, cation absorption, and charcoal absorption. Elution of the factor with an isocratic gradient of ammonium bicarbonate results in recovery of all of the bioactivity in a single peak which coelutes with inorganic phosphate and contains all of the endogenous molybdenum. The bioactivity can be separated from inorganic phosphate by chromatography of the partially purified endogenous factor on a metal-chelating column of Chelex-100. The chelating procedure results in complete loss of bioactivity with recovery of 98% of the inorganic phosphate in both the column drop-through and a subsequent 1 M NaCl wash. The factor preparation purified through the Ion-110 HPLC step inhibits temperature-mediated dissociation of the immunopurified glucocorticoid receptor-hsp90 complex, but it is considerably more effective at stabilizing the unpurified receptor-hsp90 complex in a Chelex-treated cytosol system that has been depleted of metal components. These observations support the proposal that an endogenous metal can stabilize the binding of hsp90 to the receptor but it is likely that other cytosolic components that are not present in the immunopurified complex must contribute to the stability of the soluble protein-protein complex in cytosol.     

Purification of Protein Disulfide Isomerase from a Thermophilic Fungus

A protein disulfide isomerase (PDI) was purified to homogeneity from the thermophilic fungus Humicola insolens by a rapid three-step procedure, anion-exchange chromatography, concanavalin A-affinity chromatography, and reverse phase high performance liquid chromatography. Forty-oneμg of PDI was obtained from 100 g of wet mycelium. Concanavalin A-Sepharose chromatography is available for purification of the fungal PDI, indicating that the enzyme is also glycosylated like the yeast PDI. The fungal PDI exists as a dimer (2x60kDa), has a pI of 3.5, and is fairly heat-stable. The amino acid composition of the PDI is similar to those of yeast and bovine liver PDI, and the high content of acidic amino acid residues agrees with the lower acidic pI.     

Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.

The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.     

Sample preparation for beta-exotoxin determination in Bacillus thuringiensis cultures by reversed-phase high-performance liquid chromatography.

Beta-exotoxin is a nucleotide analogue produced by the entomopathogenic bacterium Bacillus thuringiensis. We have defined two new HPLC procedures for quantification of this exotoxin in culture supernatants of B. thuringiensis grown in poor or rich medium. The sample is prepared either by precipitation in solvent or by solid-phase extraction. Solvent precipitation is achieved treating the sample with acetone and acetonitrile. Solid-phase extraction is performed with a C18 and an anion-exchange cartridge. Reversed-phase HPLC with gradient elution of the prepared samples gives a limit of quantitation of 2 microg/ml for samples prepared by solvent precipitation and of 0.3 microg/ml for samples prepared by solid-phase extraction.     

Streptomyces griseus aminopeptidase is a calcium-activated zinc metalloprotein. Purification and properties of the enzyme

A heat-stable aminopeptidase with an N-terminal Ala-Pro-Asp-Ile-Pro-Leu sequence has been purified from Streptomyces griseus by heat treatment followed by gel-exclusion and anion-exchange chromatographic procedures. The enzyme is a monomeric zinc metalloenzyme showing an apparent molecular mass of 33 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 21 kDa by gel filtration on Superose 12. Calcium ions bind to the enzyme, pKCa 4.5, and activate it about sixfold when the substrate is leucine-4-nitroanilide (0.4 mM in 50 mM Tris/HCl pH 8.0, 25 degrees C). Binding of Ca2+ also contributes to the thermal stability of the protein. This aminopeptidase may be useful for two-stage assays of bacterial and mammalian metalloendopeptidases; it may also serve in studies of proteolytic enzyme activation by calcium ions.     

Infectious allergy. XIV. Histochemical changes in redox enzymes in cells of the small intestine smooth muscle during sensitization with diphtheria exotoxin]

Administration to guinea pigs of 1/Dlm of diphtheria exotoxin twice at an interval of 1 and 24 hours, or once in a dose of 25/100 Dlm caused changes in the activity of the redox enzymes. The character of their changes in the animals to which the whole dose of the exotoxin was administered once or repeatedly was directly reverse: in the first case the activity of the enzymes was uniformly increased, and in the second--ununiformly depressed; besides, there was a marked disturbance of coordination between the enzymes. An increased sensitivity of the infectious allergy type following injection of low doses of diphtheria exotoxin was not accompanied by immunity formation. Specific antibodies against diphtheria exotoxin formed only in the organism of animals to which 1/100 Dlm of diphtheria toxin was injected once a day for 15 days, but in this case as well no immunity against diphtheria exotoxin was observed.     

The superantigenic activity of streptococcal pyrogenic exotoxin B is independent of the protease activity

The nature of the mitogenic activity of pyrogenic streptococcal exotoxin B, also known as streptococcal cysteine protease, has been debated in the literature. Streptococcal exotoxin B has been shown to cleave interleukin-1beta precursor and create biologically active interleukin-1beta, a major cytokine mediating inflammation and shock. This activity could mimic the mitogenicity and cytokine release induced by superantigens in lymphocyte stimulating experiments. In this study, the protease activity of streptococcal exotoxin B was irreversibly inhibited by covalent binding of a tripeptide and the superantigenic properties of streptococcal exotoxin B were found not to be influenced by this inactivation. Native as well as protease-inactivated streptococcal exotoxin B was shown to stimulate T-cell proliferation without a need of metabolically active antigen presenting cells. Furthermore, streptococcal exotoxin B-induced T-cell proliferation was shown to require HLA-DQ since addition of HLA-DQ monoclonal antibodies totally inhibited the mitogenic activity of streptococcal exotoxin B, indicating that streptococcal exotoxin B, as other superantigens, makes direct contact with the T-cell receptor via HLA class II. The aim of this study was to characterize the relationship between the proteolytic and superantigenic properties of streptococcal exotoxin B.