Showing papers on "Bacillus thuringiensis published in 1987"

Transgenic plants protected from insect attack

Abstract: The Gram-positive bacterium Bacillus thuringiensis produces proteins which are specifically toxic to a variety of insect species. Modified genes have been derived from bt2, a toxin gene cloned from one Bacillus strain. Transgenic tobacco plants expressing these genes synthesize insecticidal proteins which protect them from feeding damage by larvae of the tobacco hornworm.

Insect Tolerant Transgenic Tomato Plants

Abstract: The structure of an insect control protein gene from Bacillus thuringiensis var. kurstaki HD–1 was determined, and truncated forms of the gene that express a functional insecticidal protein were generated. Two of these truncated genes were incorporated into a plant expression vector for Agrobacterium–mediated transformation. Transgenic tomato plants containing the chimeric genes express the insect control protein gene. Such expression confers tolerance to lepidopteran larvae on the transgenic tomato plants and their progeny. These engineered tomato plants represent a significant step to increased selectivity, specificity and efficiency in insect control.

Bacillus thuringiensis §-Endotoxin Expressed in Transgenic Nicotiana tabacum Provides Resistance to Lepidopteran Insects

Abstract: The crystal proteins, or §-endotoxins, of Bacillus thuringiensis are specifically lethal to Lepidopteran insects. We utilized a truncated and modified portion of a cloned crystal protein gene to construct a chimeric gene capable of expression in plant cells. Using an Agrobacterium tumefaciens binary vector system, we then transferred the chimeric toxin gene into tobacco (Nicotiana tabacum cv Havana 425) cells and regenerated recombinant plants. One to several copies per cell of the toxin gene are routinely present in the recombinant plants. Hybridization experiments demonstrated that these plants had a new RNA species of the size expected for the truncated toxin mRNA, and a polypeptide having the mobility expected for the truncated toxin was detected by immunoblotting. Significant variation was found in the levels of toxin-specific RNA expression between different recombinants, but the levels of hybridizing RNA in transformants correlated with the level of toxicity demonstrated against Manduca sexta (tobacco hornworm), and other Lepidopteran insects. The recombinant genes were transmitted to progeny and resistance to insects was maintained, thus demonstrating that the introduction of toxin genes into plants may be a practical method of providing protection against certain insect pests.

Selective Process for Efficient Isolation of Soil Bacillus spp.

Abstract: We were able to isolate Bacillus thuringiensis from environmental samples with a background of 109 bacteria per g of soil. Our selection process differed significantly from classical selection methods which permit only the desired organism to grow. In our process, germination of B. thuringiensis spores was selectively inhibited by sodium acetate, while most of the undesired sporeformers germinated. Next, all of the nonsporulated microbes were eliminated by heat treatment at 80°C for 3 min. The surviving spores were then plated on a rich agar medium and allowed to grow until they sporulated. Of random colonies picked from agar, 20 to 96% were crystal-forming Bacillus species. B. thuringiensis and B. sphaericus were routinely selected by this method.

Specificity of Bacillus thuringiensis delta-endotoxin

Abstract: The insecticidal activity of the delta-endotoxins of 14 Bacillus thuringiensis strains belonging to 12 subspecies was determined against Pieris brassicae, Heliothis virescens, and Spodoptera littoralis. Larvae of P. brassicae were highly susceptible to purified crystals of strains of B. thuringiensis subsp. thuringiensis and B. thuringiensis subsp. morrisoni, whereas H. virescens responded best to B. thuringiensis subsp. kenyae and B. thuringiensis subsp. kurstaki. The crystals of the B. thuringiensis subsp. entomocidus strain were the most potent against S. littoralis. It was shown that the solubility of the crystals within the gut of the three insect species is a first important step in the mode of action. Predissolution of the crystals especially enhanced the insecticidal activity against H. virescens. When in vitro-activated toxins were applied, the relative potency range varied greatly from one insect species to another. It can be concluded that at least three factors influence the potency of B. thuringiensis delta-endotoxins: the strain-related origin of the toxin, the degree of solubility of the crystals in the gut juice, and the intrinsic susceptibility of the insect to the toxin.

Bioprocess developments in the production of bioinsecticides by Bacillus thuringiensis.

Abstract: (1987). Bioprocess Developments in the Production of Bioinsecticides by Bacillus Thuringiensis. Critical Reviews in Biotechnology: Vol. 6, No. 1, pp. 87-127.

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

Abstract: The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.

Identification ofSelf-Transmissib le Plasmids inFour Bacillus thuringiensis Subspecies

Abstract: was obtained by theisolation offertile B.anthracis andB.cereustranscipients thatcontained onlypBC16andone ofthese plasmids. pXO14was efficient inmobilizing thetoxin andcapsule plasmids, pXO1andpXO2,respectively, fromB.anthracis transcipients toplasmid-cured B.anthracis orB.cereusrecipients. DNA-DNAhybridization experiments suggested thatDNA homology exists among pXO13,pXO14,andtheB.thuringiensis subsp. thuringiensis conjugative plasmids pXOllandpXO12.Matings performed betweenstrains whicheach contained thesame conjugative plasmid demonstrated reduced efficiency ofpBC16transfer. However, inmany instances whendonorandrecipient strains contained different conjugative plasmids, theefficiency ofpBC16 transfer appeared tobeenhanced. ThereportsbyGonzalez andco-workers (10-12) ofa Bacillus mating systemprompted us toinvestigate whether we could use ittofacilitate genetic analyses ofvarious Bacillus plasmids. Ourlaboratory strains ofBacillus thuringiensis were screened fortheir donorability inmatings withBacillus anthracis andBacillus cereusrecipients. The plasmids presentinthepotential B.thuringiensis donors were cryptic; hence, tomonitor plasmid transfer during conjugation, we introduced theselectable B.cereusplasmid pBC16(2.8 megadaltons), whichencodes tetracycline resistance(3), intoeachstrain byCP-51-mediated transduction (29). Oursurveyshowedthat 6of12B.thuringiensis strains inour collection, representing five subspecies, were effective donors ofpBC16whengrown inmixedculture withB. anthracis orB.cereusrecipients (2,34). Twolarge plasmids, pXOllandpXO12,fromB.thuringiensis subsp. thuringiensis 4042Awere characterized as being abletomediate conjugal transfer ofthemselves andotherplasmids athigh frequencies (2).pXO12was alsofoundto carrygenes involved incrystal toxin production. Thepresent study was undertaken withtheideaofidentifying andcharacterizing theplasmids whichwere responsible fordonoractivities oftheotherstrains whichwere effective inour screening tests: B.thuringiensis subsp. morrisoni, B.thuringiensis subsp. toumanoffi, B.thuringiensis subsp. alesti, andB.thuringiensis subsp. israelensis. Whenmatings were performed withthese strains asdonors, eachstrain was found toharbor aunique large plasmid which was transmitted ata highfrequency torecipients. These plasmids were namedpXO13,pXO14,pXO15,andpXO16. Experiments presented heredemonstrate an association

Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis.

Abstract: A ca. 10-kilobase (kb) HindIII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicells showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region.

Toxicity of Different Serotypes and Toxins of Bacillus thuringiensis to Resistant and Susceptible Indianmeal Moths (Lepidoptera: Pyralidae)

Abstract: Fifty-seven isolates of Bacillus thuringiensis (BT) known to be toxic to larvae of Indianmeal moths, Plodia interpunctella (Hubner), were tested for activity against an Indianmeal moth colony resistant to the HD-l strain of BT. Twenty-one of the isolates, representing five of the eight serotypes tested, were active against the BT-resistant moths. Fifteen of the isolates, representing serotypes 4a,4c ( kenyae ), 6 ( entomocidus ), 7 ( aizawai ), 9 ( tolworthi ), and 10 ( darmstadiensis ), had no significant toxicity toward house flies, Musca domestica L., indicating that their toxicity toward the BT-resistant Indianmeal moths resulted from differences in the structure, composition, or function of the spore-crystal complex and not from exotoxin contamination. Bioassays confirmed that the Indianmeal moths were resistant to spores and crystals but susceptible to β-exotoxin.

Analysis of the molecular basis of insecticidal specificity of Bacillus thuringiensis crystal delta-endotoxin.

Abstract: The mechanism of action and receptor binding of a dual-specificity Bacillus thuringiensis var. aizawai ICl delta-endotoxin was studied using insect cell culture. The native protoxin was labelled with 125I, proteolytically activated and the affinity of the resulting preparations for insect cell-membrane proteins was studied by blotting. The active preparations obtained by various treatments had characteristic specificity associated with unique polypeptides, and showed affinity for different membrane proteins. The lepidopteran-specific preparation (trypsin-treated protoxin containing 58 and 55 kDa polypeptides) bound to two membrane proteins in the lepidopteran cells but none in the dipteran cells. The dipteran-specific preparation (protoxin treated sequentially with trypsin and Aedes aegypti gut proteases, containing a 53 kDa polypeptide) bound to a 90 kDa membrane protein in the dipteran (A. aegypti) cells but bound to none in the lepidopteran cells or Drosophila melanogaster cells. The toxicity of trypsin-activated delta-endotoxin was completely inhibited by preincubation with D-glucose, suggesting a role for this carbohydrate in toxin-receptor interaction. The toxicity was also decreased by osmotic protectants to an extent proportional to their viscometric radius. These results support a proposal that initial interaction of toxin with a unique receptor determines the specificity of the toxin, following which cell death occurs by a mechanism of colloid osmotic lysis.

New strain of Bacillus, a toxin derived thereof and a composition for combating Coleoptera

Abstract: The present invention provides a new strain of micro-organism, Bacillus thuringiensis of pathotype C, a process for obtaining a toxin therefrom and an insecticide for combating Coleoptera containing the new micro-organism or a toxin obtained therefrom.

Novel isolates of bacilus thuringiensis having activity against the alfalfa weevil, hypera brunneipennis

Abstract: Novel Bacillus thuringiensis (B.t.) isolates are disclosed and claimed. These novel B.t. isolates have activity against the Egyptian alfalfa weevil, which is known to cause heavy agricultural losses.

Transformation of vegetative cells of Bacillus thuringiensis by plasmid DNA

Abstract: Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

Safety of the Entomopathogen Bacillus thuringiensis var. israelensis for Mammals

Abstract: Six preparations of Bacillus thuringiensis var. israelensis de Barjac were tested for toxicity and infectivity to mice, rats, and rabbits. Oral and aerosol administration of the microbial insecticide to rats resulted in no deaths. Intraperitoneal injection of one preparation into athymic mice produced substantial mortality (26/42), but a subsequent experiment with a different preparation did not result in mortality. B. t. israelensis persisted in the spleen of mice for as long as 7 wk, but multiplication in mammals was not evident. Colony-forming units of B. t. israelensis seemed to be cleared from the host as inert particles. B. t. israelensis dusts caused minimal ocular irritation and pastes caused severe conjunctival congestion in rabbit eyes. We conclude that B. t. israelensis is not a virulent, invasive mammalian pathogen and that it can be used safely in environments where human exposure is likely to occur.

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture.

Abstract: Summary An insecticidal protein gene from Bacillus thuringiensis var. aizawal was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteloytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resist-ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawal toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran ceil membranes, which may be the receptor for this toxin.

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

Abstract: The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp israelensis and B thuringiensis subsp morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies The alkali-solubilized parasporal bodies of B thuringiensis subsp israelensis caused 50% lysis of human erythrocytes at 114 micrograms/ml, whereas those of B thuringiensis subsp morrisoni caused similar lysis at 184 micrograms/ml Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B thuringiensis subsp israelensis and B thuringiensis subsp morrisoni were 187 and 1198 micrograms/ml, respectively Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B thuringiensis subsp israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B thuringiensis subsp morrisoni However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies These results indicate that the mosquitocidal and hemolytic properties of B thuringiensis subsp israelensis and B thuringiensis subsp morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies The lower hemolytic activity of the B thuringiensis subsp morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and nucleotide sequencing of two insecticidal δ-endotoxin genes from Bacillus thuringiensis var. kurstaki HD-1 DNA

Abstract: The insecticidal δ-endotoxin genes cry-1-1 and cry-1-2 derived from Bacillus thuringiensis var. kurstaki Hd-1 were cloned, and their nucleotide sequences were determined. The nucleotide sequence of cry-1-1 was identical with the one reported by Schnepf et al. except for one base in the coding region. The coding regions of our two cloned toxin genes were not identical, but showed homology. More intensive variations in the sequences were observed in some regions, predominantly in the center of the coding region, 3′-terminal portion and 3′-flanking region. The differences in nucleotide sequences may have been mainly induced by base exchanges and deletions. Both translated products in E. coli were toxic to Bombyx mori larvae, but their insecticidal activities were considerably different.

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Abstract: Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.

The glycoprotein toxin of Bacillus thuringiensis subsp. israelensis indicates a lectinlike receptor in the larval mosquito gut.

Abstract: The mosquito-active protein crystals produced by Bacillus thuringiensis subsp. israelensis contain covalently attached aminosugars which are critical for their larvicidal activity. The 50% lethal concentrations toward Aedes aegypti larvae were increased up to 10-fold by mild periodate treatment, up to 40-fold by forming the protein crystals in the presence of tunicamycin, and up to 7-fold by the presence during the mosquito bioassays of N-acetylglucosamine or its trimer, triacetylchitotriose. Periodate-treated crystals and crystals formed in the presence of tunicamycin had greatly reduced binding capacities for wheat germ agglutinin, an N-acetylglucosamine-specific lectin. These results suggest that the B. thuringiensis subsp. israelensis glycoprotein toxin binds to a lectinlike receptor in the larval mosquito gut. Furthermore, the distinct lectin-binding patterns exhibited by diptera-active versus lepidoptera-active B. thuringiensis crystals suggest that host specificity for the microbial insecticides is determined, in part, by the carbohydrate portion of their glycoprotein crystals.

Transfer of chromosomal genes and plasmids in Bacillus thuringiensis.

Abstract: A low frequency of chromosomal gene transfer from Bacillus thuringiensis to Bacillus cereus was detected by cell mating, with a tryptophan marker being the most frequently transferred gene among four that were tested. The process was resistant to DNase and was not mediated by cell filtrates. Among several B. thuringiensis subspecies tested, transfer was best with a derivative of B. thuringiensis subsp. kurstaki HD1, which lost several plasmids. All of the B. cereus recombinants contained at least one plasmid from the donor B. thuringiensis; frequently, it was a plasmid that encoded a protoxin gene. In matings with B. thuringiensis subsp. kurstaki HD1, a 29-megadalton plasmid that contained a ca. 2.5-kilobase region of homology with the chromosome was always transferred. No detectable transfer of chromosomal genes was found in B. thuringiensis subsp. kurstaki HD1 strains lacking this plasmid, suggesting that there may be chromosome mobilization.