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.     

Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera:Noctuidae)

AIMS: To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains. METHODS AND RESULTS: The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet. CONCLUSIONS: It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.     

Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan

A total of 1700 Japanese strains of Bacillus thuringiensis, belonging to at least 47 H serogroups, were examined for insecticidal activity against larvae of the diamondback moth, Plutella xylostella. The high-level toxicity was associated with 612 isolates (36.0%). Of these, 608 isolates (99.3%) fell into 13 H serogroups belonging to the low-numbered H serotypes, H1-H10. Conversely, most isolates belonging to the high-numbered serotypes (>H10) had little or no larvicidal activity; only one isolate of the serovar japonensis H23 was active. P xylostella larvae were susceptible to 89.8% of the serovar morrisoni H8a:8b strains and 85.7% of galleriae H5a:5b strains. High values of 60-80% were also obtained in six serovars (thuringiensis H1, alesti H3a:3c, kurstaki H3a:3b:3c, kenyae H4a:4c, aizawai H7, and tolworhi H9), while relatively low values of <60% in two other common serovars, sotto H4a:4b and darmstadiensis H10a:10b. Five selected isolates, belonging to H serovars other than kurstaki and aizawai, were 10-60 times less toxic than the reference strain HD-1 (serovar kurstaki). Parasporal inclusion proteins of these strains were immunologically unrelated to those of the strain HD-1 and the aizawai type strain.     

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.     

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.     

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.     

Naturally occurring Bacillus thuringiensis in Indonesia

S. HASTOWO, B.W. LAY AND M. OHBA. 1992. A total of 135 strains of Bacillus thuringiensis were isolated from soils of sericultural and natural environments of various regions in Indonesia: Sumatra, Java, Bali, Timor, Sulawesi and Kalimantan. Serologically, 63 strains were assigned to flagella (H) serotypes 3abc, 3ade, 4ac, 5ac, 6ab, 8ab, 9, 11, 17, 20ac, and 24, indicating a varied flora of B. thuringiensis in Indonesia. Of these, the serotype 3ade predominated, followed by the serotypes 3abc and 6ab. The other 72 strains (53·3%) were not sero-reactive against any of the H antisera to B. thuringiensis H serotypes 1–24. In toxicity tests, 34 strains belonging to serotypes 3abc, 3ade, 4ac, and 8ab showed larvicidal activity to the silkworm, Bombyx mori, while 74·8% did not exhibit insecticidal activity against larvae of B. mori and the mosquitoes, Aedes aegypti and Culex quinquefasciatus. Morphotypes of parasporal inclusions were not correlated with insecticidal activities.     

PCR-based identification of Bacillus thuringiensis pesticidal crystal genes

The polymerase chain reaction (PCR) is a molecular tool widely used to characterize the insecticidal bacterium Bacillus thuringiensis. This technique can be used to amplify specific DNA fragments and thus to determine the presence or absence of a target gene. The identification of B. thuringiensis toxin genes by PCR can partially predict the insecticidal activity of a given strain. PCR has proven to be a rapid and reliable method and it has largely substituted bioassays in preliminary classification of B. thuringiensis collections. In this work, we compare the largest B. thuringiensis PCR-based screenings, and we review the natural occurrence of cry genes among native strains. We also discuss the use of PCR for the identification of novel cry genes, as well as the potential of novel technologies for the characterization of B. thuringiensis strains.     

Cytotoxic Bacillus spp. belonging to the B. cereus and B. subtilis groups in Norwegian surface waters

AIMS: To investigate the presence and numbers of Bacillus spp. spores in surface waters and examine isolates belonging to the B. cereus and B. subtilis groups for cytotoxicity, and to discuss the presence of cytotoxic Bacillus spp. in surface water as hazard identification in a risk assessment approach in the food industry. METHODS AND RESULTS: Samples from eight different rivers with variable degree of faecal pollution, and two drinking water sources, were heat shocked and examined for the presence of Bacillus spp. spores using membrane filtration followed by cultivation on bovine blood agar plates. Bacillus spp. was present in all samples. The numbers varied from 15 to 1400 CFU 100 ml(-1). Pure cultures of 86 Bacillus spp. isolates representing all sampling sites were characterized using colony morphology, atmospheric requirements, spore and sporangium morphology, and API 50 CHB and API 20E. Bacillus spp. representing the B. cereus and B. subtilis groups were isolated from all samples. Twenty-one isolates belonging to the B. cereus and B. subtilis groups, representing eight samples, were screened for cytotoxicity. Nine strains of B. cereus and five strains belonging to the B. subtilis group were cytotoxic. CONCLUSIONS: The presence of cytotoxic Bacillus spp. in surface water represents a possible source for food contamination. Filtration and chlorination of surface water, the most common drinking water treatment in Norway, do not remove Bacillus spores efficiently. This was confirmed by isolation of spores from tap water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of food with water containing low numbers of Bacillus spores implies a risk for bacterial growth in foods. Consequently, high numbers of Bacillus spp. may occur after growth in some products. High numbers of cytotoxic Bacillus spp. in foods may represent a risk for food poisoning.     

Association of PCR and feeding bioassays as a large-scale method to screen tropical Bacillus thuringiensis isolates for a cry constitution with higher insecticidal effect against Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

AIMS: To verify whether the presence of any of the cry1C, 1D, 1E and 1F genes could be associated with high toxicity against fall armyworm. METHODS AND RESULTS: A sample of 60 strains from a large collection of tropical Bacillus thuringiensis (B.t.) isolates was subjected to feeding bioassays and gene-specific PCR. Positive amplification of cry-specific fragments, so confirmed by sequencing, revealed that cry1C was ubiquitous and distributed among high and low mortality classes, cry1D was underrepresented and showed no clear association to high toxicity, and cry1F was not detected. The presence of cry1E significantly correlated to high levels of insecticidal activity, as estimated by linear regression analysis. CONCLUSION: The PCR amplification of cry1E-specific fragments alone appears to be sufficient to identify B.t. strains with high mortality levels against tropical armyworm. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach presented is promising as a simple and efficient method for first-tier, marker-assisted screening of environment-specific B.t. germplasm effective in controlling a single target pest.     

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.     

A screening for the presence of four different crystal protein gene types in 25 Bacillus thuringiensis strains

25 different strains of Bacillus thuringiensis, belonging to 18 different serotypes were screened by Southern hybridization analysis for the presence of nucleotide sequences of four different gene types coding for crystal proteins showing different insecticidal spectra. Many but not all strains showed sequences homologous to any of the probes used. Homology with sequences of the gene type present in strain kurstaki HD1 occurred in all positive strains, whereas the other gene types were much less abundant. Sequences homologous to those of gene type VI, coding for a Spodoptera-specific crystal protein, appeared only in strains of serotypes aizawai and entomocidus.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Bacillus cereus produces several nonproteinaceous insecticidal exotoxins

Bacillus cereus is mainly known as a human food-borne opportunistic pathogen. Here, we used biological assays and HPLC to investigate the ability of B. cereus to produce insecticidal exotoxins during the stationary growth phase. None of the 575 B. cereus strains screened produced detectable levels of β-exotoxin I, a small, heat-stable insecticidal nucleotide analogue. However, six out of a subset of 270 B. cereus strains produced several small, nonproteinaceous insecticidal exotoxins different from β-exotoxin I. Thus, B. cereus can secrete a large array of proteinaceous and nonproteinaceous toxins acting on insects and mammals.     

Identification and characterization of the new bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59)

Two new Bacillus thuringiensis strains have been classified by the H antigen of the cells and differentiated by their morphological, biochemical and molecular characteristics. The flagellar agglutination showed that both strains bore specific H antigens which allowed their classification as the new serotypes H57 and H59. The serovar names proposed for the type strains characterized in this work are B. thuringiensis ser. pirenaica, for the H serotype 57, and B. thuringiensis ser. iberica, for the H serotype 59. Further characterization of these strains, by means of SDS-PAGE, Western inmunodetection, plasmid profile and cry -gene identification by polymerase chain reaction, confirmed the originality of the two novel serotypes. Toxicity tests carried out against several insect species, belonging to the orders Lepidoptera, Diptera and Coleoptera, showed no detectable insecticidal activity for either of the B. thuringiensis strains.     

Combined analysis of supernatant-based feeding bioassays and PCR as a first-tier screening strategy for Vip -derived activities in Bacillus thuringiensis strains effective against tropical fall armyworm

AIMS: To assess whether feeding bioassays using culture-supernatant proteins could be combined with PCR into a first-tier screening strategy for Vip3A-like genes efficient against tropical Spodoptera frugiperda. METHODS AND RESULTS: Out of 12 Bacillus thuringiensis strains studied, the total protein concentrated from the culture supernatant of only the strain HD125 yielded a significantly increased armyworm mortality and an intense band of the predicted size for VIP3A protein in SDS-PAGE. However, PCR and sequencing data indicated Vip-like genes are ubiquitous in tropical B. thuringiensis isolates. Interestingly, the HD125 strain was also the only one displaying a single-band amplification pattern and the highest sequence identity to the reported Vip3A(a) gene. CONCLUSIONS: Results suggest the insecticidal effectiveness of putative VIPs in B. thuringiensis isolates can be preliminarily estimated by the use of supernatant-derived total protein in feeding experiments, though only in a limited manner. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple and cost-effective first-tier screening strategy for VIP-derived activities in B. thuringiensis collections can be developed by combining PCR and feeding bioassays. Moreover, the employed primers showed to be useful as a tool for strains differentiation at DNA level, and for characterization and isolation of Vip-like genes in tropical B. thuringiensis germplasm.     

Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae)

A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.     

Identification of Flagellar (H) antigenci subfactors in Bacillus thuringiensis H serotypes 10, 18, and 24 isolated in Japan

A total of 95 Bacillus thuringiensis strains isolated from Japan and belonging to H serotypes 10, 18 and 24, were examined for their H antigenic subfactors. Of 84 H serotype 10 isolates, 83 were identified as the H serotype 10a: 10b (serovar darmstadiensis ) and only one isolate was assigned to the II serotype 10a: 10c (serovar londrina ). Among five isolates belonging to the H serotype 18, three were allocated to the H serotype 18a: 18b (serovar kumamotoensis ), while two isolates did not react to antisera against the two known H antigenic subfactors, 18b and 18c. All of the six H serotype 24 isolates were assigned to the H serotype 24a: 24b (serovar neoleonensis ).