The role of bitoxibacillin components in the manifestation of its biological activity

New methods were developed and applied to quantitative determination of beta-exotoxin and antibiotic activity of delta-endotoxin with respect to Micrococcus spp. in bitoxibacillin (BTB) and the fermentation broths prepared under industrial conditions. The biosynthesis of beta-exotoxin in the period of its maximum accumulation during the fermentation was estimated. It was shown that the primary biological effect of BTB on insects consisted in the actions of beta-exotoxin and delta-endotoxin. Biological activity of each of the entomocidal components of the entomocidal components of BTB did not practically correlated with the number of viable spores. There was a correlation between the antibiotic activity of crystalline B. thuringiensis subsp. thuringiensis solutions and the insecticidal activity of the entomopathogenic preparations. Determination of beta-exotoxin and antibiotic activity of delta-endotoxin might be used as a complex procedure for testing the quality of BTB. The method for estimating antibiotic activity of the crystal solutions allowed one to assay the biological activity of other preparations based on Bacillus thuringiensis non-synthesizing beta-exotoxin.     

Features of the kinetics of accumulating entomocidal exotoxins of Bacillus thuringiensis subsp. thuringiensis during fermentation stage of bitoxibacillin production

Accumulation of Bacillus thuringiensis subsp. thuringiensis beta-exotoxin (BET) in the course of industrial fermentation (a stage in the production of the entomocidal biopreparation bitoxibacillin) has been studied. It has been demonstrated in model experiments that the decrease in the content of BET in the culture fluid is accounted for by the toxin interaction with an attendant product, the exogenous metabolite (EM). EM has been isolated from the culture fluid and characterized. EM causes alkalization of the medium, exerts entomocidal effects (in Musca domestica) and fails to form salts on treatment with BaCl2. The absorption spectrum of EM is similar to that of BET, showing a maximum at lambda = 259 nm. The light-absorbing chromophore is a pyrimidine or purine base. A method for quantitative determination of both exotoxins (BET and EM) in bacterial preparations has been developed.     

Update on the detection of beta-exotoxin in Bacillus thuringiensis strains by HPLC analysis

AIMS: The current work aimed to study the presence of beta-exotoxin by high-performance liquid chromatography (HPLC) in supernatant fluids from final whole cultures of the 69 type strains and 13 subtypes of Bacillus thuringiensis strains, as well as from some insecticidal strains. METHODS AND RESULTS: Results from HPLC and bioassays with Ephestia kuhniella (Lepidoptera Pyralidae) were compared. Type I beta-exotoxin was only detected in type strains representing serotypes H1, H9 and H10a,10b. Discrepancies between HPLC and bioassays were found in H8a,8b and some insecticidal strains, which suggests the occurrence of another soluble toxin different from type I beta-exotoxin, possibly type II beta-exotoxin. CONCLUSION: This study shows the need to use bioassays to determine the presence of beta-exotoxin activity. However, HPLC is a fast and sensitive technique if only type I beta-exotoxin is to be determined. The occurrence of beta-exotoxin in a type strain does not imply production of this metabolite by other strains belonging to the same serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: These results complete the characterization of type strains belonging to the International Entomopathogenic Bacillus Collection (Institut Pasteur, Paris, France).     

An ABC transporter from Bacillus thuringiensis is essential for beta-exotoxin I production

beta-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce beta-exotoxin I have suggested that there is a strong correlation between high levels of beta-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry(-))(Pig(+)), with no crystal gene, produced considerable amounts of beta-exotoxin I together with a soluble brown melanin pigment. Therefore, beta-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of beta-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry(-))(Pig(+)) mutants. We screened for nonpigmented mutants with impaired beta-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for beta-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for beta-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers beta-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule.     

Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production

beta-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce beta-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and beta-exotoxin production. A specific HPLC assay for type I beta-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of beta-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed beta-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as "common," "seldom," or "rare" beta-exotoxin producers. The serovar-dependent beta-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovar-dependent regulation of plasmid-encoded genes.     

Specific Features of Accumulation Kinetics of Entomocidal Exotoxins of Bacillus thuringiensis Subsp. thuringiensisduring the Fermentation Stage of Bitoxibacillin Production

Accumulation of Bacillus thuringiensis subsp. thuringiensis -exotoxin (BET) in the course of industrial fermentation (a stage in the production of the entomocidal biopreparation bitoxibacillin) has been studied. It has been demonstrated in model experiments that the decrease in the content of BET in the culture fluid is accounted for by the toxin interaction with an attendant product, the exogenous metabolite (EM). EM has been isolated from the culture fluid and characterized. EM causes alkalization of the medium, exerts entomocidal effects (in Musca domestica), and fails to form salts on treatment with BaCl₂. The absorption spectrum of EM is similar to that of BET, showing a maximum at = 259 nm. The light-absorbing chromophore is a pyrimidine or purine base. A method for quantitative determination of both exotoxins (BET and EM) in bacterial preparations has been developed.     

Correspondence of high levels of beta-exotoxin I and the presence of cry1B in Bacillus thuringiensis

Examination of 640 natural isolates of Bacillus thuringiensis showed that the 58 strains (9%) whose supernatants were toxic to Anthonomus grandis (Coleoptera: Curculionidae) produced between 10 and 175 micro g of beta-exotoxin I per ml. We also found that 55 (46%) of a sample of 118 strains whose culture supernatants were not toxic to A. grandis nevertheless produced between 2 and 5 micro g/ml. However, these amounts of beta-exotoxin I were below the threshold for detectable toxicity against this insect species. Secretion of large amounts of beta-exotoxin I was strongly associated with the presence of cry1B and vip2 genes in the 640 natural B. thuringiensis isolates studied. We concluded that strains carrying cry1B and vip2 genes also possess, on the same plasmid, genetic determinants necessary to promote high levels of production of beta-exotoxin I.     

Toxicity of Bacillus thuringiensis beta-exotoxin to three species of fruit flies (Diptera: Tephritidae)

The current study describes toxic effects of the Bacillus thuringiensis beta-exotoxin toward 3rd instars of 3 fruit fly species: Anastrepha ludens (Loew), A. obliqua (Macquart), and A. serpentina (Wiedemann). The beta-exotoxin was highly toxic to all 3 species tested, with LC50 values calculated as 0.641, 0.512, and 0.408 microgram/cm2 of filter paper used to expose the larvae, for A. ludens, A. obliqua, and A. serpentina, respectively. Exposure to beta-exotoxin was associated with an increase in the incidence of deformed pupae. The adult survivors from beta-exotoxin treatments showed no negative effects in terms of their longevity, fecundity, or egg eclosion (fertility). We conclude that the beta-exotoxin may have potential as a control agent for fruit fly pests.     

Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography.

An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.     

Lack of toxicity to adults of the Mexican fruit fly (Diptera: Tephritidae) of beta-exotoxin in Bacillus thuringiensis endotoxin preparations

beta-Exotoxin (thuringiensin) was found in high titers in centrifugation supernatants and acetone/lactose powders produced from centrifugation pellets of strains Guat 1 and HD 2 of Bacillus thuringiensis (Berliner). Diets containing powders of either strain were toxic, diets containing Guat 1 supernatant were not toxic, diets containing HD 2 supernatant were slightly toxic, and diets containing powders or supernatants from uninoculated culturing medium spiked with beta-exotoxin were not toxic. Most mortality occurred within 3 d when flies fed on powders but not until 6-7 d when flies fed on HD 2 supernatant. These results indicated that the primary toxic principals of the powders were endotoxins/spores and that beta-exotoxin alone was not toxic to adult flies at the concentrations found in the supernatants or powders.     

Partial purification and properties of proteases of bitoxibacillin

The paper describes properties of proteases A and B isolated from the biological insecticide bitoxibacillin by sulphate precipitation and Sephadex G-75 gel filtration. Proteases A and B of bitoxibacillin belong to the neutral bacterial proteases. pH optimum was found to be 6.0 and 7.5 for detection of proteolytic activity of protease A and protease B, respectively. Thermal stability of proteases A and B was similar and increased by 25% upon addition of CaCl2. Both proteases were inhibited with EDTA. The molecular weight of proteases A and B was estimated to be 57,000 and 47,000, respectively.     

An in vitro system for testing Bacillus thuringiensis toxins: the lawn assay

A cell assay system was developed that allows Bacillus thuringiensis delta-endotoxins activated at high pH (10.5) to be tested in vitro without causing alkaline injury to target cells. The assay is carried out on a lawn of gel-suspended cells, requires only 1 microliter of sample per dose, and is quantitative, rapid, and sensitive. The threshold dose for toxicity of B. thuringiensis subsp. kurstaki HD-73 with IPRI-CF-1 cells was 24 pg protein. The assay is also very useful for identifying antibodies which inhibit toxicity and for detecting beta-exotoxin.     

Determination of leucine-binding protein content of Escherichia coli cells]

A radioimmunochemical method of determination of leucine-binding protein and a method, based on the selective absorption of the protein and its complex with leucine on DEAE-cellulose, has been developed. The protein content in the E. coli cells at different stage of growth has been determined by the radioimmunochemical and equilibrium dialysis methods. It was shown that the protein content in the cells is practically independent of the growth-phase.     

Structure and synthesis of a lipid-containing bacteriophage. The molecular weight and other physical properties of bacterophage PM2.

Several physical and chemical parameters of bacteriophage PM2 have been measured. The sedimentation constant was determined to be s-20,w=293 S. The buoyant density in sucrose at 20 degrees C was 1.24 g cm+-3 and in CsCl at 25 degrees C was 1.29 g cm-3. The high-speed equilibrium centrifugation method of Yphantis (1964) was used to measure the molecular weight of PM2. The necessary auxiliary parameters were also determined. A value of 0.771 plus or minus 0.005 cm-3 g-1 for the apparent specific volume at constant chemical potential in 1 M sodiium chloride has been obtained by pycnometry; the viral concentration was determined using the absorption coefficient at 260 nm (4.60 plus or minus 0.10 cm-2 mg-1), which in turn was calculated from the phosphorous content of the virus (17.89 plus or minus 0.28 mu-g of P per mg dry weight dry weight of virus). The molecular weight of PM2 determined with these parameters is (44.1 plus or minus 1.2 x 10-6). From the phosphorous content of the virus, the percentage of phosphorous known to be in its DNA (Camerini-Otero and Franklin, 1972), and the molecular weight of the bacteriophage, we have calculated a molecular weight for PM2 DNA of 6.26 x 10-6, which confirms values determined using empirical relationships.     

The measurement of negative charge content in cartilage using a colloid titration technique.

A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 mu eq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.     

稻米赖氨酸快速测定条件的探讨

以氨基酸自动分析仪测定稻米赖氨酸含量为参照标准。在“茚三酮测定赖氨酸含量”(A法)的基础上,对该法中大米蛋白质的提取温度和时间、色温度和时间及茚三酮用量等条件进行了探讨,提出了稻米赖氨酸含量快速测(B法)。提取条件为90℃5分钟,显色条件为90℃20分钟;茚三酮试剂用1 ml。用B法对10个大米样品进行测定,赖氨酸含量与氨基酸分析仪测得的赖含量基本上一致,无显著差异(P>0.05)。     

An isochore map of the human genome based on the Z curve method

The distribution of the G+C content in the human genome has been studied by using a windowless technique derived from the Z curve method. The most important findings presented in this paper are twofold. First, abrupt variations of the G+C content along human chromosome sequences are the main variation patterns of G+C content. It is found that at some sites, the G+C content undergoes abrupt changes from a G+C-rich region to a G+C-poor region alternatively and vice versa. Second, it is shown that long domains with relatively homogeneous G+C content along each chromosome do exist. These domains are thought to be isochores, which usually have sharp boundaries. Consequently, 56 isochores longer than 3 Mb have been identified in chromosomes 1-22, X and Y. Boundaries, size and G+C content of each isochore identified are listed in detail. As an example to demonstrate the power of the method, the boundary between the Classes III and II isochores of the MHC sequence has been determined and found to be at 2,477,936, which is in good agreement with the experimental evidence. A homogeneity index is introduced to measure the homogeneity of G+C content in isochores. We emphasize that the homogeneity of G+C content is relative. The isochores in which the G+C content keeps absolutely constant do not exist. Isochore structures appear to be a basic organization of the human genome. Due to the relevance to many important biological functions, the clarification of isochore structures will provide much insight into the understanding of the human genome.     

Proline for alanine substitutions in the C-peptide helix of ribonuclease A

The effect on overall alpha-helix content of substituting proline for alanine has been determined at 5 positions (1, 2, 4, 5, and 13) of a 13-residue peptide related in sequence to residues 1-13 of ribonuclease A. The helix content falls off rapidly as proline is moved inward, and the proline residue effectively truncates the helix. No helix-stabilizing effect of proline is found at positions 2 or 4 within the first turn of the helix. Proline substitution at either end position (1, 13) has little effect on overall helix content, in agreement with an earlier study of glycine for alanine substitutions. The two end residues of the helix appear to be strongly frayed.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.