Stability of the larvicidal activity of Bacillus thuringiensis subsp. israelensis: amino acid modification and denaturants

The Bacillus thuringiensis subsp. israelensis mosquito larvicidal toxin is not a sulfhydryl-activated toxin. The protein disulfide bonds were cleaved and blocked without loss of toxicity. In contrast, modification of the lysine side chains eliminated toxicity. Additionally, the toxin was resistant to high concentrations of salt (8 M NaBr), organic solvents (40% methanol), denaturants (4 M urea), and neutral detergents (10% Triton X-100). However, it was inactivated by both positively and negatively charged detergents and by guanidine hydrochloride.     

Reconstitution of tetanus neurotoxin from two antigenically active polypeptide fragments.

Whole tetanus toxin indistinguishable from the undissociated toxin (molecular weight 160,000), in terms of molecular weight, electrophoretic behavior, antigenicity, and toxicity, was reconstituted. This was achieved by removal of urea and dithiothreitol (DTT) by dialysis from a mixture of freshly purified fragment α (molecular weight 53,000) and fragment β (molecular weight 107,000) in buffer containing 2 M urea and 1 mM DTT. The highest yield (almost 100%) of reconstituted toxin was obtained when the two isolated fragments were mixed in a molar ratio of 1:1. No toxin was reconstituted from either one of two fragments alone.     

In vitro reconstitution of the Clostridium botulinum type D progenitor toxin.

Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33). The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

Purification and some properties of Clostridium botulinum type AB toxin

Abstract The progenitor toxin of Clostridium botulinum type AB was purified; both large-sized (L) and medium-sized (M) toxins were found. The toxicity of M toxin increased by about 10-fold upon trypsinization; the increase was due mostly to type B toxin and a little to type A toxin. M toxin appeared to consist of one molecule each of toxic and nontoxic components. The activated toxic component was made up of four fragments, A-H- and L-chains and B-H- and L-chains. AB toxin may be a mixture of A and B toxins.     

Study of the receptor for black widow spider neurotoxin. I. Characteristics of membrane-bound and solubilized receptors from the bovine brain

Iodine-125 labelled alpha-latrotoxin from the venom of Central Asia black widow spider Latrodectus mactans tredecimguttatus binds specifically to the bovine brain membrane receptor producing a stable slowly dissociating complex with Kd = 1.6 x 10(-10) M and Bmax = 0.5 pmol/mg protein. Treatment of the complex with alkaline high-salt buffer induces reversible dissociation of the bound toxin. The antitoxin polyclonal antibody does not increase the dissociation rate of the bound toxin. Wheat germ lectin as well as concanavalin A inhibit the toxin binding to the membrane receptor. The receptor is solubilized with ionic and non-ionic detergents, and methods of latrotoxin binding assay are developed. The solubilized receptor is shown to retain high affinity to toxin, its binding activity being stable but critically dependent on the presence of calcium ions. Chromatographic properties of the receptor suggest its glycoprotein nature.     

Oral toxicity to flesh flies of a neurotoxic polypeptide.

An insect selective neurotoxic polypeptide from venom of the scorpion Androctonus australis (AaIT, M(r) 8,000) was shown to cross the midgut of the flesh fly Sarcophaga falculata, using assays of oral toxicity, column chromatography, and microscopic autoradiography of the native and radioiodinated toxin. AaIT induced paralysis of flies within 1-2 h after oral administration, with a lethal dose (LD50) of 10 micrograms/100 mg of body weight. Oral toxicity was about 0.14% of toxicity by injection. Hemolymph collection 70-85 min after feeding flies with [125I]AaIT showed that 5% of ingested radioactivity appeared in hemolymph. Most of this represented degradation products, but included about 0.3% of the chromatographically intact toxin. In contrast, hemolymph of identically treated lepidopterous larvae (Manduca, Helioverpa [= Heliothis]) contained degradation products but no intact toxin. [125I]AaIT was shown to cross the midgut of Sarcophaga through a morphologically distinct segment of the midgut previously shown to be permeable to a cytotoxic, positively charged polypeptide of similar molecular weight. These results suggest that Sarcophaga midgut contains a morphologically and functionally distinct segment that transports small peptides, and that employment of neurotoxic polypeptides for insect control may be feasible. Activity might be greatly improved through modification and metabolic stabilization of active peptides.     

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.     

Localization of a mosquito-larval toxin of Bacillus sphaericus 1593.

Cell-free wall, membrane, and cytoplasmic fractions were prepared from Bacillus sphaericus 1593, which exhibited toxic activity against larvae of the mosquito Culex pipiens var. quinquefasciatus. Breakage of 12- to 14-h cells by sonication or French pressure cell yielded toxic material which could be assayed in a standard mosquito larva bioassay. When sporulating cells of strain 1593 were fractionated, the majority of the toxic activity was localized in the cell wall rather than in the plasma membrane or cytoplasm. The toxin located in the bacterial cell wall was relatively stable, in that activity was unaffected by treatment with trypsin, pronase, CHCl3-CH3OH-water, Triton X-100, 8 M urea (30 min), heat (80 degrees C, 12 min), sonication, refrigeration, lyophilization, or freezing. Activity was destroyed by boiling for 10 min or by 0.01 N NaOH. Only about 1.0% of the activity present in purified cell walls could be recovered by a 2-h extraction with 8 M urea or 3 M guanidine hydrochloride. A comparison of the toxicity of a cell-free cell wall fraction with that of a sample consisting entirely of heat-stable spores indicated that the spore preparation was about 10 times more active.     

Trichostrongylus colubriformis: egg lethality due to Bacillus thuringiensis crystal toxin

A toxin from crystals of Bacillus thuringiensis israelensis was lethal in vitro to eggs of the ruminant nematode Trichostrongylus colubriformis, with an LD50 of 1.8 ng/ml. Larval viability declined after a 2-hr exposure to B. t. israelensis and was dependent on the development period of eggs prior to exposure. Alkaline solubilization suggested that the insecticidal delta-endotoxin of B. t. israelensis was not responsible for nematicidal activity. Filtration of the toxin through 0.2- or 0.45-micron-pore filters revealed that the nematicidal activity was retained on the filter. Toxicity for nematode eggs was decreased by the enzyme inhibitor L-1-tosylamide 2-phenylethylchloromethyl ketone (10(-4) M) or ethylenediaminetetraacetic acid (10(-5) M) and phenylmethylsulfonyl fluoride (10(-6) M). Ethylenediaminetetraacetic acid from 10(-9) to 10(-5) M had no effect on the toxicity while phenylmethylsulfonyl fluoride from 10(-9) to 10(-5) M inhibited toxicity. Fourteen mammalian and microbial enzymes had no significant effect on larval viability while 12 sugars and lipids failed to reduce the toxicity. Addition of 5 mM calcium to the eggs' medium decreased the B. t. israelensis toxicity by 20-fold. The calcium-dependent inhibition of toxicity was reversed by ethylenediaminetetraacetic acid (10(-5) M) and lanthanum chloride (100 microM). The ionophore A-23187 decreased the LD50 by 18-fold to 33.5 ng/ml. Addition of 5 mM calcium chloride to the ionophore and toxin yielded an LD50 of 9.2 ng/ml. Treatment of nematode eggs with B. t. israelensis toxin for 2 or 24 hr had no effect on subsequent binding of selected fluoresceinated lectins to the eggshell.     

Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. This resistance (> 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture or in suspension or whether choleragen was bound at 0 or 37°C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside G_M1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [³H]G_M1, up to 70% of the cell-associated G_M1 was extracted under the mild conditions. When the G_M1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of G_M1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [³H]galactose by immunoadsorption, only labeled G_M1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.     

Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution and toxin binding chain

Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys, Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC50) of protein synthesis occurring at 4 +/- 3.10(-11) M (n = 7) which was much lower than that of DT DT (7.8 +/- 9.8.10(-9) M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC50 = 5.4 +/- 4.9.10(-10) M, n = 7) was higher than that of DT (IC50 = 5 +/- 4.10(-11) M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10(-8) M, although DT was highly toxic (IC50 = 1.8 +/- 1.4.10(-10), n = 3). In the presence of 5 microM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC50 of 1.2 +/- 0.8.10(-9) M (n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.     

The mutation R(423)S in the Bacillus thuringiensis hybrid toxin CryAAC slightly increases toxicity for Mamestra brassicae L

Bacillus thuringiensis Cry1Ac toxin is 100 times less toxic than Cry1C to Mamestra brassicae. An R(423)S mutation abolishes Cry1Ac toxin proteolysis in M. brassicae gut juice but does not increase its toxicity to this insect. The CryAAC hybrid toxin (1Ac/1Ac/1Ca) is toxic to M. brassicae but is susceptible to gut protease digestion at the R(423) residue. Accordingly we have investigated the effect of the R(423)S mutation in CryAAC on its toxicity for M. brassicae and Pieris brassicae. Bioassays demonstrated that the R(423)S mutation slightly increased the toxicity of CryAAC for M. brassicae by having a significantly inhibitory effect on the growth of surviving larvae. The mutant hybrid was still highly toxic to P. brassicae. Features of CryAACR(423)S such as, (1) stability in M. brassicae gut juice and (2) crystal solubility were investigated. Computer simulations suggest that a possible major increase in flexibility in the CryAAC loop beta7/beta8 (G(391)-P(397)) caused by the R(423)S substitution could be a reason for the increase in M. brassicae toxicity.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

铜绿微囊藻毒株M.8641的毒素

M.8641是从武汉东湖分离培养的一株有毒的铜绿微囊藻(Microcystis aeruginosa),它产生两种环状短肽肝毒素。经凝胶过滤及HPLC等过程纯化,Waters Pico Tag系统测定,其主毒素(毒素Ⅰ)的氨基酸组成为:Glu(1),β-Masp(1),Ala(Ⅰ),Arg(2),Mdha(1),FAB-MS和MS/MS测定分子量为m/z1038,元素组成为C_(49)H_(76)O_(12)N_(13)。毒素Ⅱ的氨基酸组成,除一分子Arg为Leu取代外,其余与毒素Ⅰ相同,分子量m/z 994,分子式C_(49)HT_(75)O_(12)N_(10)。毒素Ⅰ和Ⅱ的毒性分别为129和42μg/kg(i.p.),二者在细胞中的含量各为0.12及0.01mg/kg。FLC测定表明M.8641的两种毒素与MCYST-RR和-LR有相同的Rf值。从氨基酸组成、分子量、分子式及毒性等资料分析,亦证明毒素工相当于MCYST-RR,毒素Ⅱ相当于MCYST-LR。     

Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene.

Two different 30-nucleotide regions of the cryIAc insecticidal protoxin gene from Bacillus thuringiensis were randomly mutagenized. One region was within one of seven amphipathic helices believed to be important for the formation of ion channels. There was no loss of toxicity for three test insects by any of 27 mutants, a result similar to that obtained previously for mutations within another such helix. Only mutations within a region encoding the central helix have resulted in a substantial number of mutants with low or no toxicity. A second mutagenized region encodes amino acids which are unique to this toxin and are within one of the loops in a portion of the toxin important for specificity. Among 21 different mutations of these 10 residues, only changes of two adjacent serine residues resulted in decreased toxicity which was greater for Manduca sexta than for Heliothis virescens larvae. These mutant toxins bound poorly to the single M. sexta CryIAc vesicle-binding protein and to several of the multiple H. virescens-binding proteins. The loop containing these serines must be involved in the formation of a specific toxin recognition domain.     

Single amino acid changes in domain II of Bacillus thuringiensis CryIAb delta-endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles.

Deletion of amino acid residues 370 to 375 (D2) and single alanine substitutions between residues 371 and 375 (FNIGI) of lepidopteran-active Bacillus thuringiensis CryIAb delta-endotoxin were constructed by site-directed mutagenesis techniques. All mutants, except that with the I-to-A change at position 373 (I373A), produced delta-endotoxin as CryIAb and were stable upon activation either by Manduca sexta gut enzymes or by trypsin. Mutants D2, F371A, and G374A lost most of the toxicity (400 times less) for M. sexta larvae, whereas N372A and I375A were only 2 times less toxic than CryIAb. The results of homologous and heterologous competition binding assays to M. sexta midgut brush border membrane vesicles (BBMV) revealed that the binding curves for all mutant toxins were similar to those for the wild-type toxin. However, a significant difference in irreversible binding was observed between the toxic (CryIAb, N372A, and I375A) and less-toxic (D2, F371A, and G374A) proteins. Only 20 to 25% of bound, radiolabeled CryIAb, N372A, and I375A toxins was dissociated from BBMV, whereas about 50 to 55% of the less-toxic mutants, D2, F371A, and G374A, was dissociated from their binding sites by the addition of excess nonlabeled ligand. Voltage clamping experiments provided further evidence that the insecticidal property (inhibition of short-circuit current across the M. sexta midgut) was directly correlated to irreversible interaction of the toxin with the BBMV. We have also shown that CryIAb and mutant toxins recognize 210- and 120-kDa peptides in ligand blotting. Our results imply that mutations in residues 370 to 375 of domain II of CrylAb do not affect overall binding but do affect the irreversible association of the toxin to the midgut columnar epithelial cells of M. sexta.     

Bacteria, molds, and toxins in water-damaged building materials.

Microbial toxins and eukaryotic cell toxicity from indoor building materials heavily colonized by fungi and bacteria were analyzed. The dominant colonizers at water-damaged sites of the building were Stachybotrys chartarum (10(3) to 10(5) visible conidia cm-2), Penicillium and Aspergillus species (10(4) CFU mg-1), gram-negative bacteria (10(4) CFU mg-1), and mycobacteria (10(3) CFU mg-1). The mycobacterial isolates were most similar to M. komossense, with 98% similarity of the complete 16S rDNA sequence. Limulus assay of water extracts prepared from a water-damaged gypsum liner revealed high contents of gram-negative endotoxin (17 ng mg-1 of E. coli lipopolysaccharide equivalents) and beta-D-glucan (210 ng mg-1 of curdlan equivalents). High-performance liquid chromatography analysis of the methanol extracts showed that the water-damaged gypsum liner also contained satratoxin (17 ng mg-1). This methanol-extracted substance was 200 times more toxic to rabbit skin and fetus feline lung cells than extract of gypsum liner sampled from a non-water-damaged site. The same extract contained toxin(s) that paralyzed the motility of boar spermatozoa at extremely low concentrations; the 50% effective concentration was 0.3 microgram of dry solids per ml. This toxicity was not explainable by the amount of bacterial endotoxin, beta-D-glucan, or satratoxin present in the same extract. The novel in vitro toxicity test that utilized boar spermatozoa as described in this article is convenient to perform and reproducible and was a useful tool for detecting toxins of microbial origin toward eukaryotic cells not detectable in building materials by the other methods.     

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-2 toxin metabolism by ruminal bacteria and its effect on their growth.

The effect of T-2 toxin on the growth rates of different bacteria was used as a measure of its toxicity. Toxin levels of 10 micrograms/ml did not decrease the growth rate of Selenomonas ruminantium and Anaerovibrio lipolytica, whereas the growth rate of Butyrivibrio fibrisolvens was uninhibited at toxin levels as high as 1 mg/ml. There was, however, a noticeable increase in the growth rate of B. fibrisolvens CE46 and CE51 and S. ruminantium in the presence of low concentrations (10 micrograms/ml) of T-2 toxin, which may indicate the assimilation of the toxin as an energy source by these bacteria. Three tributyrin-hydrolyzing bacterial isolates did not grow at all in the presence of T-2 toxin (10 micrograms/ml). The growth rate of a fourth tributyrin-hydrolyzing bacterial isolate was unaffected. B. fibrisolvens CE51 degraded T-2 toxin to HT-2 toxin (22%), T-2 triol (3%), and neosolaniol (10%), whereas A. lipolytica and S. ruminantium degraded the toxin to HT-2 toxin (22 and 18%, respectively) and T-2 triol (7 and 10%, respectively) only. These results have been explained in terms of the presence of two different toxin-hydrolyzing enzyme systems. Studies with B. fibrisolvens showed the presence of a T-2 toxin-degrading enzyme fraction in a bacterial membrane preparation. This fraction had an approximate molecular weight of 65,000 and showed esterase activity (395.6 mumol of p-nitrophenol formed per min per mg of protein with p-nitrophenylacetate as the substrate.