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.     

Selection of chitinolytic strains of Bacillus thuringiensis

Three selected strains of Bacillus thuringiensis native to Mexico produced endochitinases, chitobiosidases, and N-acetyl--glucosaminidases in a medium containing colloidal chitin as a main carbon source. Two types of chitinases were clearly identified: endochitinases and chitobiosidases. Chromosomal location of a chitinase gene in B. thuringiensis LBIT-82 was resolved.     

Molecular approaches towards development of novel Bacillus thuringiensis biopesticides

Bacillus thuringiensis (Bt) has been used for control of lepidopteran, dipteran and coleopteran insects for over three decades. Novel Bt strains harbouring new types of insecticidal genes are being discovered worldwide. Recombinant strains with enhanced toxicity and broadened insecticidal spectrum have been constructed. To increase the field persistence of insecticidal crystal proteins (ICPs), alternative modes of their delivery in Pseudomonas sp. and endophytes have been developed. ICPs have been modified by site-directed mutagenesis to improve their insecticidal efficacy. Higher yields of ICPs have been achieved by use of strong expression promoters and other regulatory elements. Gene-disabling of the sporulation-specific protease has led to yield enhancement of ICPs. Interestingly, Bt toxins have been found to act synergistically with some other pesticidal agents. Optimization of fermentation conditions is an essential requirement for cost-effective commercial production of Bt biopesticides. The environmental impact of deployment of genetically engineered biopesticides has been assessed. Recombinant Bt strains that do not carry any non-Bt DNA, endophytes, encapsulation in killed bacteria (such as Pseudomonas) and asporogenous Bt strains are ecologically safe approaches. Efficient resistance management strategies require judicious use of Bt transgenic plants in conjunction with refugia and Bt biopesticides in an Integrated Pest Management (IPM) program. This revised version was published online in November 2006 with corrections to the Cover Date.     

Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR

One hundred and twenty-six strains of Bacillus thuringiensis representing 57 serovars were allocated to 58 genomic types using random amplified polymorphic DNA (RAPD)-PCR patterns. Serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, morrisoni, pakistani, sotto, thuringiensis and tolworthi each encompassed identical or closely related strains. Despite this genomic homogeneity, most of these serovars also included at least one variant strain. Serovars aizawai, canadensis, entomocidus and sotto biotype dendrolimus, on the other hand, were genomically heterogeneous. Of the 57 serovars examined, 31 contained at least one strain with a closely related or identical RAPD pattern to a strain from a different serovar. We conclude that while the species is genomically diverse, the homogeneous serovars represent clonal lineages of successful insect pathogens.     

Expression and characterization of inhA gene from Bacillus thuringiensis 8010

InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.     

Distribution and diversity of Dipteran-specific cry and cyt genes in native Bacillus thuringiensis strains obtained from different ecosystems of Iran

One hundred and twenty-eight Bacillus thuringiensis isolates from fields of different ecological regions of Iran were collected to study the distribution and diversity of Dipteran-specific cry and cyt genes. The percentage of samples with Bt showed significant differences between different regions and also between different fields. The most Bt frequency was observed in the soil samples collected from Caspianic zone (7%) and soils of cotton (17%). Characterization of isolates was based on morphological characteristics of crystals, plasmid profiles and protein band patterns as well as PCR analysis using general and specific primers for 22 different cry and cyt genes encoding proteins active against mosquitoes. Thirty-eight different cry gene profiles were detected in this collection. Several of them were found to be different from all previously published profiles and none of the previous researches reported these numbers of profiles. Strains containing cry2-type genes were the most abundant and represent 57.1% of the isolates. Strains harboring cry24 and cry10 genes were also highly abundant (38.7 and 32.8%, respectively). cry11, cry4, cry17, cry19, cry21, cry29, cyt1, and cry9 genes were less abundant, found in 25.7, 14.3, 11.4, 1.4, 4.3, 1.4, and 10% of the strains, respectively. Among the cry2 gene containing isolates, 37.5% strains harbored cry2Aa, 55% cry2Ab, 2.5% cry2Ac, and 5% other or novel cry2-type genes. Among the cry4 gene containing isolates, 0% strains harbored cry4A, 60% cry4B, and 40% cry4C, cry4D or novel cry4 type genes. Finally, based on crystal morphology, protein patterns and PCR, 21 strains were selected as potentially high Dipteran-active for bioassays. Also our results showed that some of the isolates may harbor minimum a putative novel cry gene.     

Distribution of Bacillus thuringiensis strains in Southern Sweden

Bacillus thuringiensis strains were found to be naturally present in the soils of southern Sweden, being isolated from nine out of 12 sites examined. Forest soil samples were more rich in B. thuringiensis strains than soil samples collected from cultivated areas. A wide diversity of B. thuringiensis strains, representing different biochemical groups, was isolated; samples from Aspö and Fogdö regions showed the highest degree of diversity.R. Landén and M. Bryne are with the Department of Microbiology, Stockholm University, S-10691 Stockholm, Sweden. A. Abdel-Hameed is with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt. A. Abdel-Hameed's present address is the Department of Applied Chemistry and Microbiology, PO Box 27, Viikki, Building B, SF-00014 University of Helsinki, Helsinki, Finland     

Isolation, geographical diversity and insecticidal activity of Bacillus thuringiensis from soils in Spain

Bacillus thuringiensis is a spore-forming bacterium showing the unusual ability to produce endogenous crystals during sporulation that are toxic for some pest insects. This work was performed to study the composition, ecological distribution and insecticidal activity of isolates of this entomopathogenic bacterium from the Spanish territory. Using a standard isolation method, B. thuringiensis was isolated from 115 out of 493 soil samples collected in the Iberian Peninsula and the Canary and Balearic Archipelagos. The percentages of samples with B. thuringiensis were 31.7, 27.6 and 18.5 and the B. thuringiensis index 0.065, 0.067 and 0.11 for the Iberian Peninsula, Canary and Balearic Archipelagos, respectively. The prairies were shown to be the worst source of B. thuringiensis while forests, urban and agricultural habitats showed similar percentages. Strain classification based on H-antigen agglutination showed a great diversity among the Spanish isolates, which were distributed among 24 subspecies, including three new ones andaluciensis, asturiensis and palmanyolensis. We differentiated 65 different protein profiles of spore-crystal mixtures by sulfate-polyacrylamide gel electrophoresis and we selected 109 isolates representative of these profiles to evaluate their insecticidal activity against insects from the Orders Orthoptera, Dictyoptera, Coleoptera, Lepidoptera and Diptera. We found variable percentages of isolates active against Coleoptera and Lepidoptera, one isolate highly active against mosquito larvae and for the first time, three isolates active against cockroaches and locusts.     

Molecular cloning and sequence analysis of the chitinase gene from Bacillus thuringiensis serovar alesti

Endogenous chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. The chitinase gene was cloned from B. thuringiensis serovar alesti strain HD-16, and the deduced 676 amino acid sequence showed a high degree of similarity with other Bacillus chitinases. Additionally, the deduced amino acid sequence showed that the protein contained an amino terminus signal peptide and consisted of a catalytic domain, a fibronectin type III domain and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase or cellulase sequences.     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

AFLP fingerprinting and genotypic characterization of some serovars of Bacillus thuringiensis

Amplified fragment length polymorphism (AFLP)-based fingerprinting of 24 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 13 EcoR1 (+2) and Mse1 (+3) primer combinations for genotypic characterization. A high degree of polymorphism was established among the Bt serovars. A total of 1107 fragments ranging from 30–850 bp were generated out of which 1106 were polymorphic. Discrimination rates of different primer combinations at various band levels (1–5) among different Bt serovars were more than 90%. Cluster analysis revealed very low similarity values, ranging from 7–50%, among the Bt serovars indicating their remarkable genetic diversity. AFLP analysis establishes the molecular relatedness between the serovars and serotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis

Recently, we reported the synthesis of five bacteriocin-like inhibitor substances (Bt-BLIS: morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524) by Mexican strains of Bacillus thuringiensis. Here we show that, collectively, these Bt-BLIS have a moderate to broad spectrum of antibacterial activity, being toxic to clinically significant against Gram-positive and Gram-negative bacteria, including common etiological agents of human diseases, such as strep throat and scarlet fever, septicemia, pneumonia, urinary tract infection, and emetic and gastrointestinal syndromes. Although synthesis of the five Bt-BLIS was independent of the presence of a target inducing bacterium, we demonstrated for the first time that a proteinaceous component(s) secreted by, or liberated by proteolytic cleavage of Bacillus cereus 183 following treatment with proteinase K, enhanced Bt-BLIS synthesis.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

Studies on Bacillus thuringiensis strains isolated from Swedish soils: insect toxicity and production of B. cereus-diarrhoeal-type enterotoxin

At moderate concentration, 23 of 40 strains of Bacillus thuringiensis isolated from Sweden were toxic to Trichoplusia ni and five were toxic to Aedes aegypti. Five of the strains were toxic to Diabrotica undecimpunctata at high concentration, two were toxic to Heliothis virescens at low concentration and five produced thuringiensin (formerly called -exotoxin). No strain was toxic towards the beet armyworm Spodoptera exigua at low concentration. Twenty-three of the strains produced a B. cereus-diarrhoeal-type enterotoxin.A. Abdel-Hameed is with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt. R. Landén is with the Department of Microbiology, Stockholm University, S-10691 Stockholm, Sweden. A. Abdel-Hameed's present address is the Department of Applied Chemistry and Microbiology, P. O. Box 27, Viikki, Building B, FIN-00014 University of Helsinki;     

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% -helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% -sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both -helical and -sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.     

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pH _i ) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K⁺ and Na⁺ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pH _i depends strongly on the activity of a K⁺/H⁺ exchanger. In the absence of toxin, removal of K⁺ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pH _i equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pH _i . B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H⁺, K⁺ and Na⁺.     

Application of RAPD technique to study polymorphism among Bacillus thuringiensis isolates from Jordan

The soil-borne bacterium Bacillus thuringiensis (Bt) is an important biological agent used against human and plant pests and diseases. Seven Jordanian Bt isolates, which have been analysed for toxicity against important pests, were also differentiated through serotyping. In this study, they were analysed at the molecular level using random amplified polymorphic DNA markers. Five more international strains were incorporated in the analysis. The DNA markers used showed high polymorphism among the isolates tested. However, the data did not align completely with earlier serotyping for most isolates. Therefore, it is recommended to engage several analyses (e.g. biochemical and molecular) when classifying newly surveyed Bt isolates in the world.