Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli

A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R · Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.     

Insecticidal Activity of the Protein Encoded by the cryV Gene of Bacillus thuringiensis kurstaki INA-02

A new host specificity was discovered with the insecticidal protein encoded by the cryV gene. The cryV gene was cloned from the Bacillus thuringiensis kurstaki INA-02 strain, which was selected among a number of B. thuringiensis isolates because of its high activity against Spodoptera litura. Analyses by polymerase chain reaction (PCR) revealed that INA-02 contained the cryIA(a) and cryV genes. Since no Spodoptera activity was observed with B. thuringiensis sotto, which contained only cryIA(a), insecticidal activity of the protein encoded by the cryV gene was investigated with several insect species including S. litura. For bioassay, the cryV gene was highly expressed in an acrystalliferous B. thuringiensis strain, BT51. The CryV protein from BT51 was assayed against larvae of three lepidopteran species, Bombyx mori, S. litura, and Plutella xylostella. The protein was highly active against S. litura and P. xylostella, suggestive that the protein contributes to the unique activity of INA-02.     

Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium

Significant differences in expression of the delta-endotoxin genes cryA1 and cryA2 of Bacillus thuringiensis subsp. kurstaki were observed in B. subtilis and B. megaterium. The cryA1 gene was expressed when present on a high-copy-number (hcn) vector in B. megaterium but not in B. subtilis. The cryA2 gene was expressed in both hosts, but at a higher level in B. megaterium. Expression of the cryA2 gene in B. megaterium was better from a hcn vector than from a low copy number vector. Inhibition of sporulation was observed when the toxin genes were present on hcn plasmids in B. subtilis while no such effect was evident in B. megaterium. In addition, there was a significant reduction in copy numbers in both B. subtilis and B. megaterium when delta-endotoxin genes or a spoVG promoter-containing fragment of DNA were cloned into hcn plasmids.     

Rocket Immunoelectrophoresis of the Entomocidal Parasporal Crystal of Bacillus thuringiensis subsp. kurstaki

Rocket immunoelectrophoresis was used to quantitate the soluble parasporal crystal of Bacillus thuringiensis subsp. kurstaki. The method described is rapid, reliable, specific, and extremely accurate, and it can be used to measure crystal toxin in commercial microbial insecticides that contain a mixture of spores, vegetative cells, and carrier materials.     

苏云金芽孢杆菌菌株YBT1535转cry1C基因菌的构建

利用电脉冲将ｃｒｙ１Ｃ基因转子苏云金孢杆菌野生菌株ＹＢＴ１５３５,筛选得到３个转化子。质粒电泳、ＰＣＲ扩增及Ｓｏｕｔｈｅｒｎ杂交结果均证明,基因ｃｒｙ１Ｃ已转入菌株ＹＢＴ１５３５。生物测定结果表明,３个转化子对甜菜夜蛾的毒力比出发菌ＹＢＴ１５３５均有显著的提高,转化子ＹＢＴ１５３５－１和ＹＢＴ１５３５－３对小菜蛾和棉铃虫的生物活性与出发菌ＹＢＴ１５３５相近,而转子化子ＹＢＴ１５３５－２则有一定幅度     

Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensis subsp. israelensis

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST–20-kDa fusion protein (GST-20) nor the GST–45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST–60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.     

Conjugal Transfer of a Toxin-Coding Megaplasmid from Bacillus thuringiensis subsp. israelensis to Mosquitocidal Strains of Bacillus sphaericus

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on a large plasmid, pBtoxis. In an attempt to combine the best properties of the two bacteria, an erythromycin resistance-marked pBtoxis plasmid was transferred to B. sphaericus by a mating technique. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in a resistant colony of Culex quinquefasciatus, apparently due to the production of Cry11A but not Cry4A or Cry4B. The stability of the plasmid in the B. sphaericus host was moderate during vegetative growth, but segregational instability was observed, which led to substantial rates of plasmid loss during sporulation.     

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki

AIMS: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. METHODS AND RESULTS: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. CONCLUSIONS: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase 'structure-function' relation.     

Physical Mapping of the Bacillus thuringiensis subsp. kurstaki and alesti Chromosomes

Two strains of the well-known insect pathogen and biopesticide, Bacillus thuringiensis (Bt), belonging to subspecies alesti (strain Bt5) and kurstaki (strain Bt213), were chosen for genetic characterization. The two strains belong to different serotypes and are currently classified into different subspecies, although their insecticidal activity is similar. Physical maps were constructed of Bt alesti and Bt kurstaki using Pulsed Field Gel Electrophoreses (PFGE), and the map positions of several genes were determined. The 5.5 Mb combined genetic and physical chromosome maps of the two strains were found to be indistinguishable, and the only differences detected between the strains were of extrachromosomal origin. A cryIA toxin gene probe hybridised to a chromosome fragment and to two extrachromosomal elements in both strains, migrating as 100 kb and 350 kb, respectively. In addition a cry hybridizing extrachromosomal element migrating as 80 kb was present only in Bt alesti. Both strains were also found to contain sequences hybridizing to an enterotoxin (hbla) gene probe. Such sequences were positioned on the 350 kb extrachromosomal element, as well as on the chromosome. Received: 20 April 2001 / Accepted: 29 May 2001     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Intracellular Proteases of Bacillus thuringiensis subsp. kurstaki and a Protease-Deficient Mutant Btk-q

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis (Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum. Received: 4 January 2002 / Accepted: 6 March 2002     

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.     

Intracellular proteases of Bacillus thuringiensis subsp. kurstaki and a protease-deficient mutant Btk-q

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis ( Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum.     

Persistence of Bacillus thuringiensis subsp. kurstaki in Urban Environments following Spraying

Bacillus thuringiensis subsp. kurstaki is applied extensively in North America to control the gypsy moth, Lymantria dispar. Since B. thuringiensis subsp. kurstaki shares many physical and biological properties with Bacillus anthracis, it is a reasonable surrogate for biodefense studies. A key question in biodefense is how long a biothreat agent will persist in the environment. There is some information in the literature on the persistence of Bacillus anthracis in laboratories and historical testing areas and for Bacillus thuringiensis in agricultural settings, but there is no information on the persistence of Bacillus spp. in the type of environment that would be encountered in a city or on a military installation. Since it is not feasible to release B. anthracis in a developed area, the controlled release of B. thuringiensis subsp. kurstaki for pest control was used to gain insight into the potential persistence of Bacillus spp. in outdoor urban environments. Persistence was evaluated in two locations: Fairfax County, VA, and Seattle, WA. Environmental samples were collected from multiple matrices and evaluated for the presence of viable B. thuringiensis subsp. kurstaki at times ranging from less than 1 day to 4 years after spraying. Real-time PCR and culture were used for analysis. B. thuringiensis subsp. kurstaki was found to persist in urban environments for at least 4 years. It was most frequently detected in soils and less frequently detected in wipes, grass, foliage, and water. The collective results indicate that certain species of Bacillus may persist for years following their dispersal in urban environments.     

Inheritance of resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F(1) reciprocal crosses and backcrosses between F(1) larvae and resistant (P(R)) and susceptible (P(S)) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F(1) crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F(1) larvae was slightly higher than that for P(S), suggesting that resistance is partially recessive. The responses of both backcross progeny (F(1) x P(R), F(1) x P(S)) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F(1) x P(R) backcross but decreased the correspondence with the F(1) x P(S) backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.     

Inheritance of Resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F₁ reciprocal crosses and backcrosses between F₁ larvae and resistant (P_R) and susceptible (P_S) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F₁ crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F₁ larvae was slightly higher than that for P_S, suggesting that resistance is partially recessive. The responses of both backcross progeny (F₁ × P_R, F₁ × P_S) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F₁ × P_R backcross but decreased the correspondence with the F₁ × P_S backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.