Plasmids of Bacillus thuringiensis var. galleriae strain 612

We studied Bacillus thuringiensis var galleriae, strain 612 plasmids. B. thuringiensis cells contain double-stranded plasmid DNA molecules (ranging of about 12% from total DNA content) with buoyant density 1.59 g/cm3. Plasmid DNA content was constant during the exponential and stationary phases of bacterial growth. The plasmid fractions consist of DNA molecules with molecular weights of 5.9 x 10(6), 10.0 x 10(6), and 110.9 x 10(6) daltons (pVD1, pVD2 pVD3, respectively). Endonuclease EcoRI cuts the plasmids pVD2 and pVD3 into two and four fragments, respectivelyy, but pVDI seemed to be resistent to EcoRI treatment. We found that pVD2 and pVD3 plasmids contain a common DNA fragment with the molecular weight of 6.7 x 10(6) dalton as it was shown by restriction analysis. In contrast, the same plasmids contain the common fragment with molecular weight of 7.5 x 10(6) dalton as shown by heteroduplex analysis. Plasmid pVD3 has a transposon-like structure.     

Cloning and comparative characteristics of genes coding two structurally distant delta-endotoxins of Bacillus thuringiensis var. galleriae and kurstaki

Representative total DNA libraries of Bac. thuringiensis var. kurstaki (strain Dipel) and galleriae (strain 11-67) have been constructed on the basis of phasmid vector lambda pSL5. Recombinant phasmid clones, carrying delta-endotoxin-coding genes of both subspecies have been isolated by means of immunoenzyme screening. Restriction mapping and partial nucleotide sequence determination have demonstrated that phasmid lambda pOC2, isolated from var. kurstaki DNA library, contains the complete delta-endotoxin-coding gene, identical to the one, described by Schnepf H.E. et al. J. Biol. Chem. 1985. V. 260. P. 6264. Recombinant phasmids lambda pOC10 and 11 have been shown to contain the full-sized gene, coding delta-endotoxin of Bac. thuringiensis var. galleriae. The protein products of the cloned genes have been characterized by Western-blot analysis and bioassays. The absence of substantial homology of two genes, evidenced by Southern-blot hybridisation, correlates with sufficiently big differences in biological specificity of the corresponding proteins. This is in accordance with our previous data on N-terminal amino acid sequence determination of delta-endotoxins of those subspecies of Bac. thuringiensis.     

Biological characteristics of the variants of Bacillus thuringiensis subsp. galleriae formed during continuous culture

Bacillus thuringiensis subsp. galleriae S. variants formed during continuous cultivation differ from the parent culture in certain properties. In contrast to the parent R form, their growth in the chemostat does not yield virulent mutants which can cause their lysis on solid media. The chemostat S forms are resistant against virulent phage mutants produced when the R variants are grown under the conditions of continuous cultivation and against a virulent phage obtained from the parent culture 69-6 under the action of vancomycin. The R forms are sensitive to these phages. When the S forms are grown under the chemostat conditions, they do not revert to the R forms. The R and S forms do not differ noticeably in the character of their growth, formation of spores and crystals, and biological activity.     

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.     

On the formation of crystal proteins during sporulation in Bacillus thuringiensis var. thuringiensis

The sporulation potential of Bacillus subtilis as a function of position in the cell cycle was determined by transferring cells from growth medium to sporulation medium at various times during growth. Growth was induced by incubating heat-activated spores in rich medium or by diluting stationary phase vegetative cultures with fresh growth medium. The results supported earlier observations that sporulation potential is cell cycle dependent. The rise in sporulation potential was studied by exposing cultures to the inhibitors of cell wall and protein synthesis, vancomycin and chloramphenicol. The delay in the appearance of the peak of sporulation potential caused by these inhibitors compared with the reported lack of effect of nalidixic acid, indicates that the appearance of sporulation potential requires synthesis of a macromolecular component other than deoxyribonucleic acid. The effect of nalidixic acid in preventing the decline of the sporulation potential was compared with the effect of high temperature on a mutant temperature sensitive for the initiation of DNA replication. It was found that prevention of chromosome completion with nalidixic acid maintained a high sporulation potential, whereas prevention of chromosome re-initiation in the temperature sensitive mutant did not affect the decline in sporulation potential as the cells enter stationary phase.Abbreviations NAL Nalidixic acid - HPUra 6-(p-hydroxyphenylazo)-uracil - VAN Vancomycin - CAM Chloramphenicol - BHI Brain heart infusion broth - c.f.u. Colony forming units     

Molecular modeling and characterization of the B. thuringiensis and B. thuringiensis LDC-9 cytolytic proteins

The Cyt toxins are able to lyse a wide range of cell types in vitro, unlike the Cry delta-endotoxins. It exerts its activity by the formation of pores within target cell membranes. The structural information available for Cyt2Aa (PDB id: 1CBY) consists of a single domain in which two outer layers of alpha-helix wrap around a mixed beta-sheet. Beta-barrel was suggested as a possible structure of the pores. Hence, this study seeks to investigate the structural properties of other Cytolytic proteins by predicting the three-dimensional (3D) model using Cyt2Aa as template. The predicted models are expected to be significantly more accurate as all the Cyt proteins showed significant similarity with the template (PDB id: 1CBY). The refined homology models revealed similar secondary structures (alpha-helices and beta-sheets) and tertiary features as Cyt2Aa. The variation in the loop regions of the tertiary structure accounts for the differential toxicity.     

Structure and biological features of the temperate phage of Bacillus thuringiensis var. galleriae 69/9, sensitive to chloroform

A new temperate bacteriophage designated Px1 has been isolated from the culture of Bacillus thuringiensis var. galleriae 69/6 producing enthobacterin. The bacteriophage belongs to morphological group B1 in accordance with the classification by D. Reanney and H. Ackerman. The bacteriophage head has an isometric multifaceted form with 40 nm diameter. The length of its noncontractile transversely lined tail is 130 nm. High sensitivity to chloroform is peculiar of the phage. The lytical specter of the phage Px1 has been studied. The phage is shown to be capable of efficient transduction of plasmids between the bacteria of Bacillus cereus group.     

Biochemical and immunological characterization of the cloned crystal toxin of Bacillus thuringiensis var. israelensis

The protein components of the cloned crystal toxin of Bacillus thuringiensis var. israelensis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Using an antiserum to the solubilized B. thuringiensis var. israelensis crystal protein as a probe, immunological homology between the crystal protein components of B. thuringiensis var. israelensis and those of the recombinant B. megaterium strain VB131 was tested. The results from this study indicate that the crystal inclusion of the recombinant strain contains only the 130 kilodalton protein and not the 68 or the 28 kilodalton proteins of the crystal toxin of B. thuringiensis var. israelensis and that the 130 kilodalton protein is primarily responsible for the mosquitocidal activity of this organism.     

Stabilization and increased production of insecticidal crystal proteins of Bacillus thuringiensis subsp. galleriae in steady- and transient-state continuous cultures

Bacillus thuringiensis subsp. galleriae, grown in continuous cultures, segregated to spontaneous asporogenic variants replacing the wild-type Spo⁺ Cry⁺ strains [Sachidanandham R, Jayaraman K (1993) Appl Microbiol Biotechnol 40:504–507]. Realizing that this was due to specific but unknown nutritional requirements, we undertook further continuous-culture studies to identify growth requirement(s) by pulsing various medium components and growth factors. While carbon, nitrogen and pulses of nutrients exhibited a neutral pulse response, a group of amino acids were shown to improve the stability and volumetric productivity of biomass. The formation of spores and insecticidal crystal proteins was found to be higher with amino acid supplementation. Comparison of carbon-limited steady-state continuous cultures under two different conditions of growth brought forth the stabilizing effects of the amino acid supplementation. Batch experiments carried out with these inputs demonstrated a better carbon utilization, resulting in a higher biomass as well as enhancement of bioinsecticidal activity. Received: 14 May 1996 / Received revision: 9 September 1996 / Accepted: 13 September 1996     

Purification, characterization, and immunological properties of fumarase from Euglena gracilis var. bacillaris.

A rapid three-step procedure utilizing heat treatment, ammonium sulfate fractionation, and affinity chromatography on Matrex gel Orange A purified fumarase (EC 4.2.1.2) 632-fold with an 18% yield from crude extracts of Euglena gracilis var. bacillaris. The apparent molecular weight of the native enzyme was 120,000 as determined by gel filtration on Sephacryl S-300. The preparation was over 95% pure, and the subunit molecular weight was 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. The pH optimum for E. gracilis fumarase was 8.4. The Km values for malate and fumarate were 1.4 and 0.031 mM, respectively. Preparative two-dimensional gel electrophoresis was used to further purify the enzyme for antibody production. On Ouchterlony double-immunodiffusion gels, the antifumarase serum gave a sharp precipitin line against total E. gracilis protein and purified E. gracilis fumarase. It did not cross-react with purified pig heart fumarase. On immunoblots of purified E. gracilis fumarase and crude cell extracts of E. gracilis, the antibody recognized a single polypeptide with a molecular weight of approximately 60,000, indicating that the antibody is monospecific. This polypeptide was found in E. gracilis mitochondria. The antibody cross-reacted with an Escherichia coli protein whose molecular weight was approximately 60,000, the reported molecular weight of the fumA gene product of E. coli, but it failed to cross-react with proteins found in crude mouse cell extracts, Bacillus subtilis extracts, or purified pig heart fumarase.     

Site-specific restrictases from Bacillus thuringiensis var. Kumantoensis]

The efficiency of bacteriophages CP-54 and CP-55 plating on Bacillus thuringiensis var. kumantoensis H18 (Kum) is decreased about 10-fold as compared with the efficiency of plating on Bacillus thuringiensis var. galleriae H5 (Gal). Bacteriophages having propagated for one cycle in Kum cells might be further grown in this strain without growth restriction. Two site-specific restriction enzymes isolated from Bacillus thuringiensis var. kumantoensis were designated BtkI and BtkII. The endonuclease BtkI recognises the same nucleotide sequence CGCG in DNA as recognised by the restriction endonuclease FnuDII; BtkII recognises the same nucleotide sequence GATC as the endonuclease Sau3A.     

Cytotoxicity of two proteins isolated from Bacillus thuringiensis var. israelensis crystals to insect and mammalian cell lines

Abstract Two toxic proteins (proteins A and B) were isolated from Bacillus thuringiensis var. israelensis crystals using ion-exchange chromatography. A cell cytotoxicity assay based on the uptake of [³H]uridine by tissue culture cells following exposure to the toxic proteins, was used to measure the viability of the cells. Proteins A and B were cytotoxic to Aedes aegypti and MA104 cells. Protein B was also cytotoxic to Spodoptera frugiperda cells.     

Formation of spontaneous asporogenic variants of Bacillus thuringiensis subsp. galleriae in continuous cultures

Bacillus thuringiensis subsp. galleriae forms spontaneous asporogenic, crystalliferous variants (Spo^–Cry⁺) especially under continuous culture conditions. These variants gradually replace the wild-type strain (Spo⁺Cry⁺) entirely in the culture. Spo^–Cry⁺ variants form amorphous insecticidal crystalline inclusion bodies, that are difficult to solubilize and less toxic to the caterpillars of Bombyx mori. However, the defective inclusion bodies and their trypsin-digested peptides exhibited similar antigenic profiles to that of native crystals in Western blot analysis. Apparently a block in the formation of spores does not affect the synthesis of the constitutive peptides of the crystals but may interfere with the proper assembly of the crystalline endotoxin. Correspondence to: K. Jayaraman     

A broad-spectrum cytolytic toxin from Bacillus thuringiensis var. kyushuensis.

Bacillus thuringiensis (Bt) var. kyushuensis synthesizes a mosquitocidal crystalline inclusion containing several proteins ranging from 140 to 14 kDa. We have identified a 25 kDa protein protoxin in this inclusion which is not cytolytic, but when activated proteolytically to 23-22 kDa products is cytolytic to mosquito, lepidopteran and mammalian cells, can release entrapped glucose from liposomes and forms cation-selective channels in a planar lipid bilayer. This broad-spectrum cytolytic toxin is related antigenically to the 23 kDa toxin from Bt var. darmstadiensis strain 73-E10-2, but not to the 25 kDa CytA toxin of Bt var. israelensis. The cytolytic activity of these Bt var. kyushuensis toxins, like that of the latter two toxins, can be neutralized by incubation with liposomes containing phospholipids.