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PubMed2005

The clonal structure of Bacillus thuringiensis isolates from north-east Poland does not correlate with their cry gene diversity.

The genetic relationship among 103 natural Bacillus thuringiensis isolates was investigated on the basis of polymerase chain reaction amplification of their specific crystal (cry) protein type genes and chromosomal DNA profiling by pulsed-field gel electrophoresis (PFGE). The strains were recovered from the intestines of small wild rodents and insectivores from the Biebrza National Park and the Lomza Landscape Park of the Narew River Valley in north-east Poland. The percentage of B. thuringiensis strains harbouring genes coding for toxins active against Lepidoptera (cry1, cry2, cry9) was very high (64%) compared with that of Diptera-specific strains (cry4, 14%). No strain with cry genes coding for proteins directed against coleopteran larvae and nematodes was found. After digestion with NotI and AscI, only nine PFGE pulsotypes were observed among all isolates, indicating a clonal structure for the B. thuringiensis population from NE Poland. Interestingly, no correlation was observed between the DNA pulsotype strains and their crystal gene content and diversity. These results therefore emphasize the importance of cry gene horizontal transfer occurring among natural isolates of B. thuringiensis.

Animals↗
PubMed1982

[Differences in primary structures of delta-endotoxins produced by various serotypes of Bacillus thuringiensis].

The primary structures of delta-endotoxins (crystal-forming proteins) produced by two serotypes of Bacillus thuringiensis--V (var. galleriae) and III (var. alesti) were compared. These proteins differ by the specificity of their action on Lepidoptera larvae as well as by their molecular weights. To evaluate the homology of primary structures the tryptic hydrolysates of both endotoxins were fractionated by ion-exchange and thin-layer cellulose chromatography with a subsequent amino acid determination in the hydrolysates of thus obtained the fraction. Only 10%, of the fractions gained from the tryptic hydrolysates of the two delta-endotoxins had similar amino acid composition. In control experiments on comparison of two hydrolysates of the same delta-endotoxins the percentage of fractions with similar amino acid composition was as high as 50%, which reflects the tendency of this approach to overestimate the extent of the differences between the two sequences. Hence the delta-endotoxins produced by two serotypes of Bacillus thuringiensis, being clearly homologous, reveal substantial differences in their amino acid sequences, which are dispersed along the whole polypeptide chain. These striking differences in the primary structures are indicative of an unusually high rate of their evolution, which may be of functional importance for B. thuringiensis serotypes adaptation to different ecological niches.

Amino Acid Sequence↗
PubMed2003

Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9.

AIMS: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. METHODS AND RESULTS: Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121 degrees C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12.4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. CONCLUSIONS: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry.

Animals↗
PubMed1997

Inheritance of Resistance to the Bacillus thuringiensis Toxin Cry1C in the Diamondback Moth.

Laboratory selection increased resistance to the Bacillus thuringiensis toxin Cry1C in a strain of diamondback moth (Plutella xylostella). The selected strain was derived from a field population that had evolved high levels of resistance to Bacillus thuringiensis subsp. kurstaki and moderate resistance to Cry1C. Relative to the responses of a susceptible strain of diamondback moth, the resistance to Cry1C of the selected strain increased to 62-fold after six generations of selection. The realized heritability of resistance was 0.10. Analysis of F(inf1) hybrid progeny from reciprocal crosses between the selected strain and a susceptible strain showed that resistance to Cry1C was autosomally inherited. The dominance of resistance to Cry1C depended on the concentration; inheritance was increasingly dominant as the concentration decreased. Responses of progeny from single-pair families showed that resistance to Cry1C and resistance to Cry1Ab were inherited independently, which enhances opportunities for managing resistance. However, compared with projections based on previously reported recessive inheritance of resistance to Cry1A toxins, the potentially dominant inheritance of resistance to Cry1C observed here could accelerate evolution of resistance.

Journal Article↗
PubMed2005

Differentiated pattern of protein composition of crystalline inclusions of newly isolated Bacillus thuringiensis strains from Silesia in Poland.

Protein profiles of crystal delta-endotoxins were determined in twenty nine Bacillus thuringiensis strains-soil and phylloplane isolates--from Poland. Electrophoretic analysis revealed quantatively and qualitatively different patterns of delta-endotoxin crystal preparations of these B. thuringiensis strains. The crystalline parasporal inclusions of B. thuringiensis isolates were composed of two, three, four or five proteins. Molecular weights of these polypeptides varied from 23.4 kDa to 142 kDa. There is lack of correlation between serovars of B. thuringiensis strains, the morphology of crystals and the number and size of proteins in parasporal inclusions.

Bacillus thuringiensis↗
PubMed1994

Characterization of the type strain of Bacillus thuringiensis subsp. cameroun serotype H32.

The strain 273 B, the type strain of a H serotype of Bacillus thuringiensis not yet characterized: B. thuringiensis subsp. cameroun, serotype H32, was isolated from soil samples collected in Cameroon. This strain produces cuboidal parasporal bodies composed of two major proteins of 53 kDa and 35 kDa. N-terminal sequences of the major proteins share no homology with published sequences. Only the 35 kDa protein is susceptible to digestion by trypsin. A complex array of 9 plasmids was revealed.

Amino Acid Sequence↗
PubMed1997

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.

Animals↗
PubMed1997

Simultaneous production of the 34-kDa and 40-kDa proteins from Bacillus thuringiensis subsp. thompsoni is required for the formation of inclusion bodies.

Cooperation of two crystal proteins from Bacillus thuringiensis subsp. thompsoni. strain HnC was shown to be essential for the formation of inclusion bodies. Expression of the operon containing the 34-kDa and 40-kDa protein genes from HnC in a B. thuringiensis crystal minus strain resulted in the formation of inclusion bodies identical to those from strain HnC. Interruption of one of the genes in the operon led to the lack of inclusion body and to low production of the remaining protein. Absence of inclusion body and low rate of protein production were also observed when both genes were simultaneously expressed but on different vectors. To show a cooperative effect in the formation of the inclusion body, both proteins must be produced from the same transcript.

Bacillus thuringiensis↗
PubMed1987

Cloning and nucleotide sequence of different iso-IS231 elements and their structural association with the Tn4430 transposon in Bacillus thuringiensis.

A family of five repetitive sequences (RS) has been isolated from a plasmid DNA library of Bacillus thuringiensis strain berliner 1715. In a previous paper [Mahillon et al., EMBO J. 4(1985)3895-3899] one of these was shown to harbor all the features of an IS element (IS231). Further nucleotide sequence analysis revealed that two other RS, flanking the delta-endotoxin gene, are actually variants of IS231. Comparison of the nucleotide sequences surrounding the iso-IS231 elements showed a unique structural association between some of these elements and the transposon Tn4430. Although these IS231 elements have transposed into Tn4430, both these IS231 s and the transposon Tn4430 remain structurally intact.

Bacillus thuringiensis↗
PubMed1995

Immunohistochemical detection of binding of CryIA crystal proteins of Bacillus thuringiensis in highly resistant strains of Plutella xylostella (L.) from Hawaii.

We detected binding of insecticidal crystal proteins from Bacillus thuringiensis in one susceptible strain and six resistant strains of diamondback moth, Plutella xylostella, from Hawaii. Immunohistochemical tests with tissue sections from larval midguts showed specific binding of CryIA(a), CryIA(b), and CryIA(c) to brush border membranes. CryIE, which is not toxic to P. xylostella, did not bind to midgut tissues. Larvae from one of the resistant strains ingested extremely high concentrations of a commercial formulation containing the three CryIA proteins without suffering midgut cell damage or mortality. This same resistant strain had previously been found to have greatly reduced binding of radioactively-labeled CryIA(c) to vesicles prepared from brush border membranes. The finding that binding as detected in immunohistochemical tests was not sufficient for toxicity suggests that low levels of binding can occur without harmful effects; the tests did not reflect in vivo binding, or post-binding factors contribute to resistance. Comparison with resistant strains from Florida and the Philippines, which did not bind CryIA(b) in previously reported immunohistochemical tests, suggests that more than one mechanism of resistance to B. thuringiensis may occur within a single species.

Animals↗
PubMed1995

Domain organization of Bacillus thuringiensis CryIIIA delta-endotoxin studied by denaturation in guanidine hydrochloride solutions and limited proteolysis.

Denaturation of Bacillus thuringiensis CryIIIA delta-endotoxin--an insecticidal protein, active against Coleoptera larvae--in concentrated guanidine hydrochloride solutions was pursued by fluorescence and circular dichroism spectroscopy and limited proteolysis. It was found that the protein consists of two fragments that differ by their stability to denaturation by guanidine hydrochloride at pH 3. The less stable fragment corresponds to the N-terminal alpha-helical domain limited by Leu-279; the more stable one starts with Ile-280, contains about 330 amino acid residues, and corresponds to the molecule C-terminal moiety that consist of its two beta-structural domains forming a superdomain.

Amino Acid Sequence↗
PubMed1997

CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus.

Cry proteins produced by Bacillus thuringiensis are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito, Culex quinquefasciatus, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of B. thuringiensis. Resistance at the LC95 level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the LC95 level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.

Animals↗
PubMed1985

Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein.

The insecticidal crystal protein (ICP) gene, icp, from a 68-kb plasmid derived from Bacillus thuringiensis subsp. sotto was cloned in Escherichia coli. The icp expression in E. coli cells was confirmed by both immunological and insect-toxicity assays of the cell extract. The entire icp gene resides in the 6.6-kb PstI fragment, which codes for a 144-kDal peptide identical to the intact ICP, as determined by its size and reaction with anti-ICP antibody. Deletion analysis further revealed that the 2.8-kb region within the 6.6-kb PstI fragment codes for ICP. Analysis of the nucleotide sequence indicated that a peptide of 934 amino acid residues truncated at the C-terminal end is encoded by this 2.8-kb fragment. A unique feature of this truncated ICP is the abundance of cysteine and lysine residues within its C-terminal region.

Bacillus thuringiensis↗
PubMed1999

Characterization of Bacillus thuringiensis serovar bolivia (serotype H63), a novel serovar isolated from the Bolivian high valleys.

The type strain Bacillus thuringiensis var. bolivia (serotype H63), isolated from the Bolivian high valleys, has been characterized at different levels. Its parasporal crystal has an unusual shape and it is composed of a protein of 155 kDa which shows two bands of 75 and 80 kDa after activation. Analysis by PCR shows the presence of cry1 genes, and amplification with specific primers gave products for cry1 E, cry1 D, cry4 A and cry4 B with sizes different to those expected. Immunoblotting tests showed positive reaction for Cry1 E, Cry3 A, Cry4 A and Cry11 A crystal proteins. The plasmid pattern revealed two large and two small plasmids. Toxicity tests were performed against 14 insects and a slight toxicity was found against Plutella xylotella and Trichoplusia ni.

Animals↗
PubMed2003

Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing toxin genes from Bacillus thuringiensis subsp. israelensis.

Genes encoding the mosquito larvicidal toxins Cry4Aa, Cry11Aa, Cyt1Aa and the regulatory P20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under control of two strong promoters P(psbA) and P(A1). The clone pRVE4-ADRC displayed toxicity against fourth-instar larvae of Aedes aegypti, the highest ever achieved in cyanobacteria. It was about 2.5-fold more toxic than the respective clone without cyt1Aa [Wu et al., Appl. Environ. Microbiol. 63 (1997) 4971-4975]. Cyt1Aa synergized the combination of Crys by about five-fold. Consistently, the lethal times exerted by pRVE4-ADRC were also reduced (it killed exposed larvae more quickly). This clone may become a useful biological control agent which reduces the probability of resistance development in the target organisms [Wirth et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10536-10540].

Aedes↗
PubMed1990

Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins.

To investigate the biochemical basis of the differences in the insecticidal spectrum of Bacillus thuringiensis insecticidal crystal proteins (ICPs), we performed membrane binding and toxicity assays with three different ICPs and three lepidopteran species. The three ICPs have different toxicity patterns in the three selected target species. Binding studies with these 125I-labeled ICPs revealed high-affinity saturable binding to brush border membrane vesicles of the sensitive species. ICPs with no toxicity against a given species did not bind saturably to vesicles of that species. Together with previous data that showed a correlation between toxicity and ICP binding, our data support the statement that differences in midgut ICP receptors are a major determinant of differences in the insecticidal spectrum of the entire lepidopteran-specific ICP family. Receptor site heterogeneity in the insect midgut occurs frequently and results in sensitivity to more than one type of ICP.

Animals↗
PubMed2000

Characterization of a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders.

We report here the first Bacillus thuringiensis (Bt) toxin which is toxic to insects from three insect orders (Diptera, Coleoptera, and Lepidoptera). An oligonucleotide probe based on the delta-endotoxin N-terminal sequence was used to detect the gene. A 23-kb BamHI fragment containing the intact gene was identified and cloned from Bt strain YBT-226 plasmid DNA into the vector pBluescript II. Through a series of DNA manipulations the size of this fragment was reduced and the gene sequenced. The deduced amino acid sequence gave a predicted molecular mass of 137 kDa and was identical to a cry1Ba protein from Bt subsp. thuringiensis HD-2, which is now designated as Cry1Ba1 under a new classification scheme. This protein also showed 81.6% similarity with the Cry1B protein (Cry1Bb1) from Bt strain EG 5847. When the YBT-226 cry1Ba1 gene was expressed in an acrystalliferous Bt subsp. israelensis strain it produced irregular bipyramidal crystals during sporulation, which reacted specifically with anti-Cry1Ba antiserum. Bioassays using these crystals after purification resulted in significant mortality at low to moderate concentrations to larvae of the house fly (Musca domestica, Diptera), cottonwood leaf beetle (Chrysomela scripta, Coleoptera), and tobacco hornworm (Manduca sexta, Lepidoptera). This broad-spectrum toxicity was not dependent on presolubilization. In assays with insect cell lines not derived from midgut cells, the soluble toxin killed CH1t (Manduca sexta cells) but was inactive against CF1 (Choristoneura fumiferana cells), Aa(s) (Aedes aegypti), and C2 (Culex quinquefasciatus) mosquito cells.

Bacillus thuringiensis↗