Molecular characterization of Bacillus thuringiensis strains from Argentina

Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.     

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.     

Kinetic Properties of the Channels Formed by the Bacillus thuringiensis Insecticidal Crystal Protein Cry1C in the Plasma Membrane of Sf9 Cells

Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K⁺ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K⁺. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K⁺ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K⁺ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line. Received: 4 October 1999/Revised: 21 December 1999     

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.     

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.     

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.     

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.     

Functional expression of rat renal Na/Pi-cotransport (NaPi-2) in Sf9 cells by the baculovirus system

The recently cloned Na/P _i -cotransport system NaPi-2 is an apical membrane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further functional/structural studies, this transport system has been expressed in Sf9 insect cells using a recombinant baculovirus. Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/P _i -cotransport up to 20- to 50-fold over noninfected Sf9 cells. Transport of phosphate in infected cells was highly dependent on sodium, exhibited a K _m for P _i of 0.114 mm and an apparent K _m for Na of 63 mm (Hill coefficient of approximately 3) and was stimulated by high external pH. Infected cells expressed a polypeptide of 65 kDa representing a nonglycosylated form of the 85 kDa mature NaPi-2 transporter as present in proximal tubular brush-border membranes. By confocal microscopy expression of NaPi-2 protein was observed in the plasma membrane, yet submembranous accumulation of NaPi-2 protein could not be excluded. This demonstrates that the rat proximal tubular Na/P _i -cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics similar to that in isolated brush-border membranes. The 6His tagging will permit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.We would like to thank W. Scherle and M. Lötscher (Institute of Anatomy) for their generous help using the confocal microscope and Ch. Gasser for the art work. Financial support by the Swiss National Fonds [Grant No. 32-30785.91 (to H.M.) and 32-28664.90 (to J.B.)] and by Stiftung für wissenschaftliche Forschung an der Universitát Zürich is greatly acknowledged.     

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

Summary Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25–35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.The assistance of A. Mazza and G.A.R. Mealing is gratefully acknowledged. The trypsin-activated, HPLC-purified CryIC toxin isolated from B. thuringiensis var. entomocidus crystal was a kind gift from M. Pusztai, Institute for Biological Sciences, NRC, Ottawa.     

On the Adaptive Nature of the Dissociation Process in Bacillus thuringiensis

The process of dissociation into variants that differ in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40°C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

Starvation‐induced autophagy in Spodoptera frugiperda Sf9 ovarian cells

Autophagy controls insect development and can be targeted for pest control in agriculture. In the present study, starvation‐induced autophagy is investigated in the insect species Spodoptera frugiperda. Bioinformatics analysis and a search of the EST database (http://bioweb.ensam.inra.fr/spodobase) identifies a putative ATG8 gene of S. frugiperda. To generate a biomarker of autophagosome, the DNA sequence encoding the open reading frame of this gene is amplified and cloned into a pIEX‐4‐mCherry‐EGFP‐SfATG8 recombinant vector. Sf9 cells are then transfected with this expression vector and starved in phosphate‐buffered saline solution for 4 h to induce autophagy, which is examined by LysoTracker staining (Life Technologies, Grand Island, New York), western blotting and fluorescence microscopy. The results obtained show that starvation stimulates lipidation of SfATG8‐PE and the formation of autophagosomes, providing a foundation for further research with respect to autophagy in insects.     

Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food

Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.     

Persistence ofBacillus thuringiensis andBacillus cereus in soil supplemented with grass or manure

Summary Persistance of inocula ofBacillus thuringiensis spores, parasporal crystals, andBacillus cereus spores in soil supplemented with dried-grass or partly composted, dried-chicken manure (100 mg supplement per 900 mg soil,^–0.01 MPa water availability, 25°C) were monitored over a period of up to 64 days by dilution plating and bioassay with larvae ofPieris brassicae. The inoculantB. thuringiensis population increased 22 x in level in grass-supplemented soil, but declined in manure-supplemented soil to 0.22 x the original level. TheB. cereus inocula declined in both soil treatments to approximately 0.1 x the original level. Insecticidal activity of theB. thuringiensis parasporal crystal decreased exponentially in grass and manuresupplemented soils, with half-lives of approximately 9.5 and 8.5 days respectively.     

Functional expression of full length Limulus Factor C in stably transformed Sf9 cells

Limulus Factor C is a potent antagonist of endotoxin from Gram-negative bacteria. A fusion construct containing full length Factor C has been cloned intoSpodoptera frugiperda Sf9 cell. Stable Sf9 cell transfectants were obtained using Zeocin selection for 2 weeks. The recombinant Factor C (rFC) was secreted into the culture medium at 9 mg l^–1. Both the crude and partially purified rFC were able to detect lipid A at 10 pg ml^–1 in an ELISA-based lipid A binding assay.     

An index for determining the culture time of inoculum in cultivation of Bacillus thuringiensis

The intensity of motility of Bacillus thuringiensis was used to determine the optimal culture time of the inoculum being used for thuringiensin production. Thuringiensin production with an inoculum taken at its maximum motility was about 3 times higher than that with an inoculum taken in the late exponential phase.     

An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells

It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells. A key aspect of this work was to reduce the amount of glutamine in the cell culture medium while maintaining sufficient energy yielding metabolites for vigorous growth by supplementing with glucose and algae-derived amino acids. We present an analysis of cell growth and protein production in Sf9 insect cells expressing secreted Thy1-GFP fusion construct. We also demonstrate isotopic enrichment of the Thy-1 protein backbone with ¹⁵N and carbohydrates with ¹³C by NMR spectroscopy.Electronic supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.