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PubMed1991

Compatibility of Bacillus thuringiensis var. Israelensis and Bacillus sphaericus with the fungal pathogen Lagenidium giganteum (Oomycetes: Lagenidiales).

Larvae of Culex quinquefasciatus were exposed to infection by Lagenidium giganteum and various concentrations of B.t.i. or B. sphaericus. The resulting larval mortalities, percentages of infected dead larvae and percentages of larval body regions containing the fungus were compared. Overall, the effectiveness of Lagenidum giganteum against the larvae was not significantly affected by the presence of B.t.i. or B. sphaericus, and the fungal and bacterial agents were compatible. In experiments using 3-day-old larvae, the extent of growth of the fungus in the infected larvae and the percentage of the larvae infected were related to the concentration of B.t.i. in the range of 0.057-0.456 ITU/ml tested but were not related to the concentration of B. sphaericus in the range of 0.6-4.8 x 10(4) spores/ml. With larvae of various ages treated with a low concentration of B.t.i. (0.114 ITU/ml), exposure to the fungus increased the mortality rate in early but not late instars. After single and multiple applications of B.t.i. and B. sphaericus in the presence of the fungus, followed by drying and reflooding, the fungus persisted and reinfected larvae while the B. sphaericus persisted but the B.t.i. did not.

Animals↗
PubMed1968

Isolation and characterization of three autolytic enzymes associated with sporulation of Bacillus thuringiensis var. thuringiensis.

Cells of Bacillus thuringiensis containing refractile spores autolyzed readily when suspended in buffer. The autolysate contained enzymes which lysed vegetative cell walls of the organism. Three enzymes were isolated from the autolysate, and each was purified approximately 30-fold. One enzyme, most active near pH 4.0, was found to be an N-acetylmuramidase. The other two enzymes exhibited pH optima at 8.5. One was stimulated by cobalt ions and the other was not. The cobalt-stimulated enzyme was shown to be an N-acetylmuramyl-l-alanine amidase. The cobalt insensitive enzyme exhibited both N-acetylmuramyl-l-alanine amidase and endopeptidase activity. The amidase activity may reflect incomplete separation of the cobalt-stimulated enzyme. The endopeptidase cleaved the peptide bond between l-alanine d-glutamic acid. A cell wall lytic endopeptidase with this specificity has not been previously reported. All three enzymes were extremely limited in the range of bacterial cell walls which they attacked. Except for cell walls of Micrococcus lysodeikticus, which were lysed by the muramidase, only cell walls of members of the genus Bacillus were attacked.

Alanine↗
PubMed1989

Expression of a cloned Bacillus thuringiensis delta-endotoxin gene in Bacillus subtilis.

The entire coding region of the Bacillus thurigiensis HD73 crystal protein gene was subcloned from plasmid pJWK20 into the integration vector pUG2-15. This plasmid expresses chloramphenicol resistance when integrated into the Bacillus subtilis chromosome in the outH locus near the recE region. The correct molecular organization of the integrated plasmid was verified by hybridization to Southern blots of chromosomal DNA digests. Production of the toxic crystal protein was monitored at different time points during the life cycle of B. subtilis. Toxicity assays against Anagasta (Ephestia) larvae, direct electron microscopy crystal detection, and immunoblotting assays proved that the expression of the gene in B. subtilis is time regulated and restricted mainly to the sporulation stage. RNase protection experiments defined the transcription initiation start point and the transcription timing. All tests were made in a strain containing one to three copies of the integrated plasmid and in a strain subjected to an amplification regimen.

Animals↗
PubMed1991

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.

Amino Acid Sequence↗
PubMed1989

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.

Animals↗
PubMed1994

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis.

NB176 is a Bacillus thuringiensis mutant derived by gamma-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by gamma-irradiation. Integration of additional copies of the cryIIIA gene into the native 90 MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.

Amino Acid Sequence↗
PubMed1991

Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.

Bacillus thuringiensis↗
PubMed1990

[Expression of delta-endotoxin gene from Bacillus thuringiensis var. berliner 1715 in Escherichia coli and Bacillus megaterium cells].

Effects of the structure of plasmids carrying the cloned delta-endotoxin gene (tox) ot Bacillus thuringiensis and of the culture media on the expression of the gene have been studied. The DNA region located upstream from the crystal protein gene promoter inhibited the expression of the tox gene in Escherichia coli cells, but enhanced the expression in Bacillus megaterium cells grown in LB medium. The upstream DNA region did not affect the tox gene expression when Bacillus megaterium cells were grown in SSM medium.

Bacillus megaterium↗
PubMed2006

Molecular characterization of a DNA fragment harboring the replicon of pBMB165 from Bacillus thuringiensis subsp. tenebrionis.

BACKGROUND: Bacillus thuringiensis belongs to the Bacillus cereus sensu lato group of Gram-positive and spore-forming bacteria. Most isolates of B. thuringiensis can bear many endogenous plasmids, and the number and size of these plasmids can vary widely among strains or subspecies. As far as we know, the replicon of the plasmid pBMB165 is the first instance of a plasmid replicon being isolated from subsp. tenebrionis and characterized. RESULTS: A 20 kb DNA fragment containing a plasmid replicon was isolated from B. thuringiensis subsp. tenebrionis YBT-1765 and characterized. By Southern blot analysis, this replicon region was determined to be located on pBMB165, the largest detected plasmid (about 82 kb) of strain YBT-1765. Deletion analysis revealed that a replication initiation protein (Rep165), an origin of replication (ori165) and an iteron region were required for replication. In addition, two overlapping ORFs (orf6 and orf10) were found to be involved in stability control of plasmid. Sequence comparison showed that the replicon of pBMB165 was homologous to the pAMbeta1 family replicons, indicating that the pBMB165 replicon belongs to this family. The presence of five transposable elements or remnants thereof in close proximity to and within the replicon control region led us to speculate that genetic exchange and recombination are potentially responsible for the divergence among the replicons of this plasmid family. CONCLUSION: The replication and stability features of the pBMB165 from B. thuringiensis subsp. tenebrionis YBT-1765 were identified. Of particular interest is the homology and divergence shared between the pBMB165 replicon and other pAMbeta1 family replicons.

Bacillus thuringiensis↗
PubMed1987

[Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis].

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.

Bacillus thuringiensis↗
PubMed1990

Antibacteriophage action on the larvicidal activity of Bacillus thuringiensis H-14 and Bacillus sphaericus against Culex pipiens.

An organically polluted mosquito breeding water was tested for the presence of bacteriophages which could inhibit the larvicidal activity of B. thuringiensis H-14 and B. sphaericus. More than one bacteriophage were isolated which could inhibit the tested bacteria. The sensitivity of the two bacterial species to 12 antibiotics was tested. Two of them, showed no antibacterial action, were selected and considered as antivirus agents in the bacteriophage assays; namely, Amoxycillin and Co-Trimoxazole. Results indicated their antivirus activity as the addition of the antibiotics to the isolated phages could permit normal bacterial growth as well as persistence of larvicidal activity against Culex pipiens larvae.

Animals↗
PubMed2005

[Co-expression of crystal protein gene cry26Aa and cry28Aa has an ability to form parasporal crystal inside exosporium in Bacillus thuringiensis subsp. finitimus].

Parasporal crystal in Bacillus thuringiensis subsp. finitimus T02 forms within the exosporium and remains attached to the spore after mother cell lysis, which leads to spore-crystal connection. According to the crystal protein gene sequence in B. thuringiensis subsp. finitimus, cry26 and cry28 were cloned from strain T02 by PCR amplification and transformed into the crystal negative Bacillus thuringiensis strain BMB171 by shuttle vectors, alone or in combination together. Crystal can be observed inside of exosporium when co-expression of these two genes. But crystal mainly formed outside of exosporium when they expressed alone. This suggests interaction between of the two crystal proteins may be involved in enclosure.

Bacillus thuringiensis↗
PubMed1992

Purification and crystallization of insecticidal delta-endotoxin CryIIIB2 from Bacillus thuringiensis.

CryIIIB2, an insecticidal protein from Bacillus thuringiensis has been crystallized from 0.6 M NaBr and HEPES buffer at pH 7.0 and X-ray diffraction data collected on a native crystal to 2.4 A. The insecticidal protein was obtained from a Bacillus thuringiensis (Bt) strain EG7231. Crystals of the endotoxin are orthorhombic, space group C2221, with unit cell dimensions of a = 122.44, b = 131.81, and c = 105.37 A. A unit cell contains one molecule of the 67,000 Da endotoxin per asymmetric unit.

Bacillus thuringiensis↗
PubMed2006

Bacteriocin-like inhibitor substances produced by Mexican strains of Bacillus thuringiensis.

Bacteriocins are antimicrobial peptides synthesized and secreted by bacteria and could potentially be used as natural food preservatives. Here, we report the production of bacteriocin-like inhibitor substances (Bt-BLIS) by five Mexican strains of Bacillus thuringiensis. Bacillus thuringiensis subsp. morrisoni (LBIT 269), B. thuringiensis subsp. kurstaki (LBIT 287), B. thuringiensis subsp kenyae (LBIT 404), B. thuringiensis subsp. entomocidus (LBIT 420) and B. thuringiensis subsp. tolworthi (LBIT 524) produced proteinaceous Bt-BLIS with high levels of activity against Bacillus cereus and other gram-positive bacteria. Although none was active against the gram-negative bacteria, Escherichia coli, Shigella species and Pseudomonas aeruginosa, the five Bt-BLIS demonstrated antimicrobial activity against Vibrio cholerae, the etiologic agent of cholera. Biochemical and biophysical studies demonstrated that the five Bt-BLIS could be categorized into two groups, those produced by LBIT 269 and 287 (Group A) and LBIT 404, 420, 524 (Group B), based on relative time of peptide synthesis, distinctive bacterial target specificity and stability in a wide range of temperatures and pH. Because of their stability and bactericidal activities against B. cereus and V. cholerae agents of emetic, diarrheal and lethal syndromes in humans, these Bt-BLIS could potentially be used as biodegradable preservatives in the food industry.

Bacillus cereus↗
PubMed2003

Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering.

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC(50)], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC(50) congruent with 114 ng/ml) and C. pipiens (LC(50) 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.

Aedes↗
PubMed1993

Identification of two expressed flagellin genes in the insect pathogen Bacillus thuringiensis subsp. alesti.

Flagellin from Bacillus thuringiensis subspecies alesti strain Bt75 was isolated both from the culture medium and from flagella. Two protein forms with molecular masses close to 32 kDa were obtained from flagella; one form was identical to the flagellin purified from the culture medium. The N-terminal amino acid sequences were identical for both forms. Two genes coding for flagellin have been identified in B. thuringiensis subsp. alesti. The flaB gene was cloned and sequenced in its entire length. The clone containing the flaA gene was incomplete. Both genes were expressed in the mid-exponential growth phase. The flaB gene was flanked by long (355 bp) direct repeats protruding into the coding region in both the N- and C-terminal parts of the gene. DNA sequences related to the flaB gene were found in most other B. thuringiensis subspecies, and in two of them, subsp. kurstaki and subsp. entomocidus, such sequences were present in multiple copies.

Amino Acid Sequence↗
PubMed1986

Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli.

The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A.

Antigens, Bacterial↗