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Distribution study of Bacillus thuringiensis strains in Western Siberian soils allowed us to isolate crystal-forming bacteria assigned to a new pathovar of Bacillus thuringiensis ssp. tohokuensis with a toxic effect on mosquito larvae. A description of this bacterial pathovar is presented.
Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp, berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R1, the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue.
In this study, we rapidly identified Bacillus thuringiensis 4.0718 strain that harbored the known cryl and cry2 type genes by a PCR strategy. Three pairs of universal oligonucleotide primers were designed to detect all known cry1, cry2 and cry3 type gene sequences. Then the DNA of the positive strain 4.0718 was probed with a set of specific primers. One feature of this screening method was that each gene was expected to produce a PCR product having a precise molecular weight. PCR products having different sizes probably represented the gene was a potentially novel gene. Differentiations among these genes was determined on the basis of the electrophoresis patterns of PCR products. Finally, five cry1 type genes (cry1 Aa, cry1 Ab, cry1 Ac, cry1 Cb, a novel cry4.5 type genes) and one cry2 Ac type gene had been detected from Bacillus thuringiensis 4.0718 strain.
When the active form of the Bacillus thuringiensis delta-endotoxin Cry1Ca was expressed in E. coli severe growth retardation was observed. The absence of a short peptide from the N-terminus of the protoxin was responsible for this effect. The introduction of a mutation at an amino acid previously reported as being involved in the initial stages of pore formation within the natural insect target partially abolished the growth retardation effect. We suggest that removal of the N-terminal peptide is a necessary step in toxin activation, the presence of this peptide preventing proper interaction of the toxin with the target membrane. Expression of the truncated toxin in Bacillus thuringiensis also prevented the formation of Cry1Ca crystals.
Intraspecies interrelations between representatives of 7 serotypes of Bacillus thuringiensis, as well as antagonistic interrelations between B. thuringiensis and 130 strains of 16 species of Bacillus bacteria were studied. Antagonistic activity was determined for 18 strains, representatives of genus Bacillus, with respect to B. thuringiensis var. tuviensis. Antagonism was the most pronounced between the strains of the alesti serotype and representatives of other serotypes as well as between the strains V. thuringiensis var. tuviensis and most of other bacillus species. Strain B. thuringiensis var. tuviensis is slightly sensitive to the action of toxic metabolites from strains of this and other species of genus Bacillus.
Bacillus thuringiensis subsp. aizawai (HD133) was grown in culture media in which dextrose was a common carbon source and 30 different agricultural products and by-products were tested as the main nitrogen sources. These products included legumes, cereals, animal proteins, leaf proteins, yeasts, oilseeds, tubers, and casamino acid. Of the 30 products tested, cottonseed meal, defatted soy flour, and corn gluten meal were the most efficient substrates for the production of spore-crystal biomass and endotoxin potency. The carbohydrate/nitrogen ratios for these additives ranged from 0.3 to 0.5 and the glutamic acid content of their proteins from 9.2 to 16.0%. There was no close relationship between the estimates of the amounts of endotoxin produced and the potency of the product when fed to bertha armyworm, Mamestra configurata.
Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.
Bacillus thuringiensis was recovered at a relatively high frequency from both running and still fresh waters in natural environments of Kyushu, Japan. Of 107 water samples examined, 53 (49.5%) contained this organism. The frequency of B. thuringiensis colonies was 4.4% among 4414 colonies of the Bacillus cereus/B. thuringiensis group. The density of this bacterium in fresh waters averaged 0.45 cfu/ml. Serologically, B. thuringiensis isolates were assigned to 26 H serotypes. Of these, H14/36 (H serovar israelensis/malaysiensis) was the predominant, followed by the serotypes H3abc (kurstaki), H27 (mexicanensis), H3ad (sumiyoshiensis), and H35 (seoulensis). Of 195 isolates, 52 (26.7%) exhibited larvicidal activity against aquatic Diptera; 21 killed Culex pipiens molestus (Culicidae) only, and 31 were active on both the culicine mosquito and the moth-fly, Clogmia albipunctata (Psychodidae). The Diptera-toxic isolates produced spherical or irregularly pointed parasporal inclusions.
The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought.
Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.
AIMS: To investigate fusion expression between Bacillus thuringiensis crystal protein and a foreign protein, the expression of a fusion protein comprised of Cry1Ac, and enhanced green fluorescent protein (EGFP) in B. thuringiensis Cry(-)B strain was examined. METHODS AND RESULTS: The N-terminal fusion expression of EGFP in Cry1Ac was attempted under the control of the native cry1Ac promoter. The EGFP gene was cloned into pProMu and named pProMu-EGFP. The transformant, ProMu-EGFP/CB produced parasporal inclusions that were of bipyramidal-shaped crystals in size ranging from 200 to 300 nm. The fusion protein was approximately 150 kDa and identified by the immunoblot analysis using a Cry1Ac antibody and also a GFP antibody. The LC(50) of the ProMu-EGFP/CB was twofold higher when compared with that by the ProAc/CB. However, the crystal protein produced by the ProMu-EGFP/CB was effective on Plutella xylostella larvae. CONCLUSIONS: The ProMu-EGFP/CB produced bipyramidal shaped and insecticidal crystals comprising fusion proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the N-terminal fusion expression of EGFP and Cry1Ac, expression and crystallization between the B. thuringiensis crystal protein and a foreign protein were validated.
Bacillus thuringiensis var. kurstaki (B.t.-k) is a microbial pesticide which has been widely used for over 30 years. Its safety for a human population living in sprayed areas has never been tested. Surveillance for human infections caused by B.t.-k among Lane County, Oregon residents was conducted during two seasons of aerial B.t.-k spraying for gypsy moth control. Bacillus isolates from cultures obtained for routine clinical purposes were tested for presence of Bacillus thuringiensis (B.t.). Detailed clinical information was obtained for all B.t.-positive patients. About 80,000 people lived in the first year's spray area, and 40,000 in the second year's area. A total of 55 B.t.-positive cultures were identified. The cultures had been taken from 18 different body sites or fluids. Fifty-two (95 percent) of the B.t. isolates were assessed to be probable contaminants and not the cause of clinical illness. For three patients, B.t. could neither be ruled in nor out as a pathogen. Each of these three B.t.-positive patients had preexisting medical problems. The level of risk for B.t.-k and other existing or future microbial pesticides in immunocompromised hosts deserves further study.
The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pHi) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K+ and Na+ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pHi depends strongly on the activity of a K+/H+ exchanger. In the absence of toxin, removal of K+ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pHi equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pHi. B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H+, K+ and Na+.
Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 micro g of beta-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 micro g/ml. However, these amounts of beta-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of beta-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of beta-exotoxin I.
The cryIVB gene of Bacillus thuringiensis subsp. israelensis (Bti) codes for a 135-kDa insecticidal crystal protein, which is specifically toxic to dipteran larvae. We have identified a transcription start point (tsp) of cryIVB by a primer extension experiment. The promoter sequence alignment, together with the chronology of appearance of the transcript, suggested that cryIVB is transcribed by RNA polymerase containing sigma 35 (E sigma 35). This was confirmed by investigation of cryIVB transcription in several Bacillus subtilis sporulation mutants. Unlike the lepidopteran-specific crystal protein-encoding genes [cryIA(a) and cryIB], transcription of which is regulated by both sigma 35 and sigma 28, cryIVB transcription was controlled only by the sigma 35-dependent promoter at the midsporulation stage.
The growth of Bacillus thuringiensis subsp. thuringiensis strains (BT) 202, 1140, 98 producing bitoxybacillin in the presence of novobiocin, mitomycin C and at high temperature 43 degrees C resulted in obtaining of the mutants for the synthesis of crystalline protein (Cry) and exotoxin (Exo). Analysis of the plasmid content of the mutants has shown the Cry Exo phenotyre to correlate with the loss of 55 MD plasmid in the strain BT202 and the loss of 60MD plasmid in BT1140. The transfer of these plasmids into Bacillus cereus leads to the transfer of endo- and exotoxin production properties. The synthesis of Cry in BT98 is controlled by the 56MD plasmid, while the synthesis of Exo is encoded by the chromosome or plasmid that cannot be eliminated or transferred into other strains. Localization of Cry on the 55 and 56 MD plasmids in BT202 and BT98 is confirmed by the hybridization of the plasmid DNA with the DNA-probe.
A gene from Bacillus thuringiensis subsp. san diego that is responsible for a delta-endotoxin active against Colorado potato beetle and some other Coleoptera was sequenced and shown to have surprising regional homology with both lepidopteran and dipteran active delta-endotoxins from other strains of B. thuringiensis. Unlike the lepidopteran active toxins from B. thuringiensis subsp. kurstaki that exist as approx. 130-kDa protoxins and form bipyramidal crystalline inclusions, the coleopteran toxic protein forms a square-shaped crystal composed of an approx. 65-kDa protein. Comparisons of the gene sequences encoding the active portions of these protoxins indicate conservation of N-terminal hydrophilic and hydrophobic regions, and suggest a distant ancestral origin for these insecticidal proteins.
A rapid identification of Bacillus thuringiensis strains was established by using multiplex polymerase chain reaction (PCR). Primers of high homology specific to regions within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each strain of B. thuringiensis subsp. kurstaki. Differentiation among these strains was made on the basis of the electrophoretic pattern of the PCR products. Known B. thuringiensis subsp. kurstaki strains as well as unidentified strains isolated from insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a two-step PCR containing five primers capable of distinguishing between the strains giving products of 1,500, 858, and 653 bp for the CryIA(a) CryIA(b), and CryIA(c) genes, respectively. The method can be applied to rapidly detect the strains of B. thuringiensis subsp. kurstaki in commercial formulations and in the field.