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Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2. The addition of Ca24 was required for enzyme stabilization...
Spores of Bacillus thuringiensis contain a toxin active against lepidopterous larvae. This toxin can be solubilized by extraction with reagents which dissolve the protein crystal of B thuringiensis. It is inactivated by crystal-specific antiserum. Spores of Bacillus cereus contain a similar toxin although the specific activity is much lower than the spores of B. thuringiensis. The B. cereus toxin contains a single major polypeptidecomponent. Toxic activity can be solubliized from spores of both species by incubation with gut juices from Pieris brassicae.
In most Bacillus thuringiensis strains, the cry genes are transcribed by RNA polymerases containing sporulation-sigma factors E or K, leading to the formation of an insecticidal crystal within the mother cell along spore development. The kurstaki HD1 strain, a parent of commercial strains, also releases the insecticidal proteins Cry1I and Vip3A in the extracellular medium. vip3A expression is activated by the transcriptional regulator VipR at the onset of the stationary phase. Here, we expanded the VipR regulon in strain HD1 by identifying the VipR-binding box upstream from the cry2Aa, cry2Ab, and cry1Ia genes, and conducting transcription assays. Unexpectedly, a VipR box was located in the promoter of a putative N-acetylmuramoyl-l-alanine amidase (ami) gene upstream from cry1Ac in strain kurstaki HD73, closely related to the HD1 but devoid of vipR. Introduction of vipR in this strain led to the expression of the ami-cry1Ac operon, resulting in an early and increased production of Cry1Ac. We demonstrated that Cry1Ac was also produced in a VipR-dependent manner in an HD73 ∆spo0A mutant. Similarly, an HD1 ∆spo0A strain produces all the insecticidal proteins encoded in its genome, including cry2Ab, previously considered unexpressed. A genomic analysis also revealed the presence of putative VipR-binding sequences in lepidopteran-active strains, upstream from cry genes such as cry1E, cry1F, cry9D, and cry9E. Overall, our results break the dogma on the regulation of cry1A and cry2A genes and provide evidence of sporulation-independent Cry toxin production in biopesticidal Bt strains.IMPORTANCEBacillus thuringiensis is a remarkably efficient entomopathogen due to its ability to produce various insecticidal proteins, such as Cry or Vip. This property has made it a highly effective biopesticide used worldwide. Our work modifies the paradigm of cry1 and cry2 genes being regulated solely by sporulation-specific sigma factors and thus exclusively expressed during this process. Indeed, we demonstrated that the VipR regulator controls the transcription of vip3Aa, cry2Aa, cry2Ab, cry1Ia, and the ami-cry1A operons encoded by a strain closely related to that of commercial biopesticides and specifically turns on their expression from the onset of the stationary phase, leading to the production of insecticidal crystals independently of sporulation. By providing new knowledge on the regulation of insecticidal protein genes, these findings bring new insight for the genetic improvement of Bt strains used as commercial biopesticides.
Strains of Bacillus thuringiensis were shown to hydrolyse various forms of chitin around growing colonies on a solid medium. In conditions of submerged cultivation on a medium containing demineralized crab shells, Bac. thuringiensis var. caucasicus INMI Arm. 837 manifests the chitinolytic activity at the beginning of the stationary growth phase. The activity of chitinase which is of a constitutive nature increases when the bacterium is cultivated at pH 7.2. The maximum rate of hydrolysis of colloid chitin by chitinase prepared from the cultural broth is displayed at pH 8.0 and 60 degrees C.
Centrifugation of Bacillus thuringiensis 351 DNA in CsCl-ethidium bromide density gradient has revealed, besides the main band of chromosomal DNA (p = 1.56 g/cm2), some additional bands with higher density, which usually correspond cicle superhelix DNAs, are revealed. 6 discrete bands are observed under electrophoresis of total fraction of minor DNA bands, which suggests, that the preparation contains cicle DNAs of different size. The treatment with DNAse results in the appearance of 3 bands corresponding to opened cicle DNA forms, their molecular weight being 12-10(6)--4-10(6) daltons. Cicle super-helix and opened cicle DNA forms are found in minor fraction by means of electrone microscopy. Statistical analysis has revealed the presence of at least three types of cicle molecules of different size. The variant 351-10, free of extrachromosomal elements, is isolated after ethidium bromide treatment at high temperature. Possible mechanisms of cicle DNAs formation are discussed. Biological function of DNA extrachromosomes fragments in Bacillus thuringiensis is still obscure.
The exotoxin of Bacillus thuringiensis, which is an inhibitor of RNA synthesis, inhibits Planarian regeneration. Planarians which have been cut twice at the same level are able to regenerate after the second section in the presence of the toxin. This indicates that the first amputation stimulates a synthesis of stable RNAs which thus are available for regeneration at the moment of the second section.
The paper describes Bacillus thuringiensis var. galleriae strains possessing a bacteriocin-like factor (factor K) which inhibits the cultures of a number of serotypes of Bac. thuringiensis. The K+ cultures exhibit no antibacterial effect on other gram-positive and gram-negative microorganisms. The maximal level of factor K is found by the 8--9th hour of cultivation. No correlation has been established between the strucutred elements in the preparations of K+ cultures and the antibacterial activity. Apparently, factor K can be transferred to other strains.
Forty-eight strains of Bacillus thuringiensis and 12 strains of B. cereus were treated with ultraviolet light and mitomycin C. The former agent was the more effective inducer. Bacillus thuringiensis produces at least seven different phage particles with long, non-contractile tails. The frequencies of lysogeny and polylysogeny are 83 and 25% respectively. Morphologically defective phages occur in 25% of strains, whereas five of them produce low molecular-weight bacteriocins. One strain of B. cereus harbors "killer-particles." There is no apparent correlation between the presence of phage-like particles, phage senstivity, and serotypes, biotypes, or the origin of B. thuringiensis strains.
Asporogenic and oligosporogenic mutants of Bacillus thuringiensis can be isolated by streaking the bacteria onto a solid medium, incubating at 42 degrees C, and picking small, raised areas of growth which appear on the streaks after 48 h.
Ultrathin sections of Bacillus thuringiensis var. galleriae infected by a specific phage were studied by electron microscopy. Stages of the phage reproduction within the cell, from adsorption of phage particles to liberation of the phage from the cell, have been revealed. Growth of the phage follows that of DNA-containing phages. The yield of the phage is very high.
During the submerged cultivation of Bacillus thuringiensis var. thuringiensis in 300- and 3000-liter fermentors, lysis occurred at the end of the exponential phase of growth. New vegetative cells were subsequently formed which usually sporulated. At time of lysis, the amount of soluble sugar was 1-12 g/liter, pH value dropped to 5.3-5.8 from the original PH 6.8 and started to rise after all the cells had lysed. The proteolytic activity was low during the lysis and increased as the sporulation commenced.
The parasporal crystalline protoxin of Bacillus thuringiensis contains a single glycoprotein subunit that has a molecular weight of approximately 1.2 X 10(5). The carbohydrate consists of glucose (3.8%) and mannose (1.8%). At alkaline pH, the proendotoxin is apparently solubilized and activated by an autolytic mechanism involving an inherent sulfhydryl protease that renders the protoxin insecticidal. Activation generates protons, degraded polypeptides, sulfhydryl group reactivity, proteolytic activity, and insect toxicity. Chemical modification of the sulfhydryl groups inhibits the proteolytic and insecticidal activities, suggesting that cysteine residues may be present in the active site of the protein.
The culture of Bacillus thuringiensis var. galleriae was shown to be polylysogenic. As has been found earlier, the culture has a phage with the original structure of particles whose tail possesses a special substructure called a "collar". The phage was described in detail as a phage of Bac. thuringiensis 1-97 with a "collar". Further studies of the culture of Bac. thuringiensis var. galleriae 1-97 has shown that it also contains another phage which differs sharply from the first one in the morphology of particles, the spectrum of lytic action, and other properties. Electron microscopy of the cultural fluid of the Bac. thuringiensis var. galleriae cultures has revealed in them the presence of different amounts of the particles of both morphological types found in the strain 1-97. These data suggest that the cultures of Bac. thuringiensis var. galleriae are polylysogenic. The methods for the isolation of the new phage are described.
Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6-7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity. Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this anti-serum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35s]cysteine. It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.
Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.
Comparative characteristics of sporogenous and asporogenous Bacillus thuringiensis strains is carried out. Asporogenous strains are found to differ from wild type strains in a number of criteria, including colony morphology, character of growth on rich and poor media and UV-sensitivity. Sporogenous strains form R colonies, they are more stable and more rare produce variants forming S colonies. S colonies are typical for asporogenous mutants, and under the cultivation in unfavourable conditions (elevated temperature, a shift of pH, a change of an incubation regime) asporogenous strains dissociate with a high frequency into R form. Initial strains, which are multiple auxotrophs, under certain conditions can form "prototrophic" revertants which are unstable when incubated on rich media. Suppressor mutation is supposed to be a possible mechanism of the origination of "prototrophs".
The beta-exotoxin, thuringiensin A, from Bacillus thuringiensis var. thuringiensis has been found to be C-mitotic in the bone marrow cells of Rattus norvegicus. This exotoxin therefore appears to be a potential tool for metaphase arrest in karyological studies. The delta-endotoxin protein has no similar activity.
Plasmid DNAs were isolated from Bacillus thuringiensis and their physico-chemical properties were studied. The DNAs were purified using ultracentrifugation in a CsCl2 density gradient. The molecular weight of these extrachromosomal DNAs determined by electrophoresis in 0.6% agarose gel and by electron microscopy was 6 . 10(6)--10 . 10(6) daltons; a DNA with a molecular weight of 90 . 10(6) daltons was also found. Digestion with restriction endonuclease EcoRI yielded fragments of the plasmid DNAs with cohesive ends; these were further identified by means of electrophoresis in 1% agarose gel.