Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Reduction of resistance of Culex pipiens larvae to the binary toxin from Bacillus sphaericus by coexpression of cry4Ba from Bacillus thuringiensis subsp. israelensis with the binary toxin gene

The cry4Ba gene from Bacillus thuringiensis subsp. israelensis and the binary toxin gene from B. sphaericus C3-41 were cloned together into a shuttle vector and expressed in an acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. Transformed strain Bt-BW611, expressing both Cry4Ba protein and binary toxin protein, was more than 40-fold more toxic to Culex pipiens larvae resistant to B. sphaericus than the transformed strains expressing Cry4Ba protein or binary toxin protein independently. This result showed that the coexpression of cry4Ba of B. thuringiensis subsp. israelensis with B. sphaericus binary toxin gene partly suppressed more than 10,000-fold resistance of C. pipiens larvae to the binary toxin. It was suggested that production of Cry4Ba protein and binary toxin protein interacted synergistically, thereby increasing their mosquito-larvicidal toxicity.     

Binding and activity ofBacillus thuringiensis delta-endotoxin to invertebrate cells

Fluorescein isothiocyanate was used as a label to detect delta-endotoxin ofBacillus thuringiensis subsp.thuringiensis andisraelensis in binding studies with different in vitro cell systems. Protoxin of the subspeciesthuringiensis could be labelled directly whereas the activated toxin had to be traced indirectly with labelled antibodies. Both protoxin and activated toxin bound to primary midgut cell cultures ofPieris brassicae larvae as well as to cells of an established culture ofDrosophila melanogaster. No binding with either toxin form could be observed with hemocytes ofP. brassicae. Biological activity as shown by the trypan blue viability assay was obtained only with the activated toxin against the midgut cells. Toxin of the subspeciesisraelensis reacted very unspecifically. Binding followed by rapid destruction was obtained with all the tested cultures.Abbreviation FITC fluorescein isothiocyanate     

Transfer of plasmids and chromosomal genes amongst subspecies ofBacillus thuringiensis

Summary The plasmids pBC16 and pC194 fromBacilus thuringiensis subsp.israelensis strains A084-16-194 were transferred to 25 subspecies ofB. thuringiensis by a conjugation-like process using broth mating technique. The frequencies of transfer varied considerably between different mating pairs, ranging from 1.1×10^–9 to 9.8×10^–5. Additionally, chromosomal transfer could also be demonstrated in tenB. thuringiensis subspecies with very low frequencies (4.3×10^–9 to 3.7×10^–7). The intersubspecies matings within a group of eight subspecies strains gave higher frequencies of transfer than the matings between the subspecies. Furthermore, the results indicated that the capability to transfer plasmids among these various subspecies did not depend on the presence of large plasmid.     

Development of low-cost media for the culture of mosquito larvicides, Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis

Mosquito larvicides like Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis have been widely and effectively used in mosquito control programs, but the industrial production of these bacilli is expensive. Here we have attempted to develop three cost-effective media, based on cheap sources, potato, common sugar and bengalgram. Growth and production of the insecticidal proteins from these bacteria were satisfactory. Bioassay studies with different mosquito larvae showed considerable toxicity. Therefore the investigation suggests that potato-based culture media are more economical for the industrial production of B. sphaericus and B. thuringiensis serovar. israelensis.     

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.     

Characterization of a New Bacillus thuringiensis Strain That Is Toxic to Coleoptera

A new strain of Bacillus thuringiensis 2-7 was found to belong to the serotype H8. Cells of this strain contained irregular and flat crystalline inclusions and two large plasmids. The gene responsible for crystal formation is most likely located on the large plasmid greater than 105 MDa in size. Comparison of the cry gene of B. thuringiensis 2-7 and the cryIIIA gene of B. thuringiensis subsp. tenebrionis showed that their nucleotide sequences are identical.     

Specificity of cultured insect tissue cells for bioassay of entomocidal protein fromBacillus thuringiensis

Summary Cultured tissue cells from lepidopteran and dipteran sources displayed an order-specific response to entomocidal protein from crystals ofBacillus thuringiensis. Protein isolated from crystals ofB. thuringiensis subsp.kurstaki was effective against cells of the spruce budworm (Choristoneura fumiferana) and the tobacco hornworm (Manduca sexta), but was inactive against both mosquito cell lines tested (Aedes aegypti andAnopheles gambiae). Conversely, protein from inclusion bodies ofB. thuringiensis subsp.israelensis was fully active only against the mosquito cell lines but displayed reduced (four- to seven-fold) toxicity for the lepidopteran cell lines. One exception to this pattern of specificity was observed with aPlodia interpunctella cell line, which failed to respond to either crystal protein preparation. The moth toxin was stable at 4° C for months, whereas the mosquito toxin was susceptible to proteolytic degradation and was unstable for periods longer than 2 wk.     

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。     

Transfer of plasmids pBC 16 and pC 194 into Bacillus thuringiensis subsp. israelensis

A lysozyme sensitive strain of B. thuringiensis (strain O 016) was isolated and shown to be effectively transformed with plasmids pC 194 and pHV 33 using the protoplast transformation technique. The plasmid pC 194 from one successful transformant, strain O 016–194, was subsequently transferred to B. thuringiensis subsp. israelensis by a “conjugation-like” process. The plasmid pBC 16 from B. cereus could also be transferred to B. thuringiensis subsp. israelensis with high frequency using the conjugation-like process. Further, both plasmids, pC 194 and pBC 16, were transferred between strains of B. thuringiensis subsp. israelensis to yield transcipient strains that harbored and expressed properties of both plasmids. This work constitutes effective gene transfer system in B. thuringiensis subsp. israelensis.     

Assessment of protoxin composition of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zujiao Fu and Yunjun Sun contributed equally to this work.     

Response of the Carrot Weevil,Listronotus oregonensis(Coleoptera: Curculionidae), to Strains ofBacillus thuringiensis

Mortality and frass production bioassays were used to investigate the toxicity of seven strains ofBacillus thuringiensisagainst the adult carrot weevil,Listronotus oregonensis(Le Conte). A semi-artificial diet of carrot foliage with 4% agar was selected to maximize feeding by the insects.Bacillus thuringiensissubsp.tenebrionis(Krieg, Huger, Langenbruch, and Schnetter) (BTT) and two unidentifiedB. thuringiensisstrains, A30 and A429, gave the lowest LC₅₀values. The frass bioassay supported the conclusions of the mortality assay. Mortality of adults continued after their removal from the insecticidal medium, with the highest mortality being caused by strains A429 and BTT. Survivors from the frass bioassay, initially exposed to strains A30, A429, and BTT, did not resume normal levels of feeding after their removal from the insecticidal medium.     

Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group

Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12 095 bp), pFR12.5 (12 459 bp) and pFR55 (55 712 bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study of the molecular basis of the conjugative process in Gram positive bacteria, particularly due to the similarity with known conjugation systems. It is also a contribution to the expansion of the non-pathogenic B. cereus plasmid gene pool.     

Expression of the mosquitocidal cryIVB gene under the control of different promoters in Bacillus sphaericus 2362 and acrystalliferous Bacillus thuringiensis subsp. israelensis c4Q2-72

The 3.7 kb XbaI fragment harbouring the cryIVB gene which encoded a 130 kDa mosquitocidal toxin protein from Bacillus thuringiensis subsp. israelensis (B.t.i.) was placed downstream to the cat-86 gene promoter (P _cat-86, spore stage specific expression) or bgaB gene promoter (P _bgaB , vegetative stage specific expression). The constructs were subcloned into pBC16 to obtain pBTC3 and pBTC6, respectively. Both plasmids and the other construct, pBTC1 were successfully transferred into B. thuringiensis subsp. israelensis c4Q2-72 and B. sphaericus 2362. Western blot analysis showed that P _bgaB in front of P _cryIVB could enable cells to produce a 130 kDa protein from the vegetative stage (4 h) whereas those with P _cat-86 could not. The positive detection of 130 kDa crystal protein during the vegetative stage (4 h) by Western blot analysis indicated the vegetative-stage-specific expression of P _bgaB , while the 130 kDa crystal protein produced from cryIVB gene under control of P _cat-86 was detected only at 48 h. The strong activity of P _bgaB , together with P _cryIVB within pBTC6 in both bacterial hosts was also shown by the toxicity assay against Aedes aegypti larvae (B.t.i. c4Q2-72, 5.6 ± 3.6 × 10² c.f.u./ml; B. sphaericus 2362, 5.4 ± 2.5 × 10² c.f.u./ml) which were 100-fold and 10-fold more toxic to such larvae when compared with pBTC3 (P _cat-86 together with P _cryIVB ) and pBTC1 (contained only its self promoter) in the same bacterial host strains, respectively. The plasmid pBTC6 is not stable in either Bacillus host.     

Efficient screening and breeding of Bacillus thuringiensis subsp. kurstaki for high toxicity against Spodoptera exigua and Heliothis armigera

Spodoptera exigua is one of the most renowned agricultural pest insects and relatively insensitive to Bacillus thuringiensis subsp. kurstaki strains which are widely used commercial products to control lepidopterans such as Heliothis armigera. In the current study, we have developed a new and efficient approach to screen and breed a B. thuringiensis subsp. kurstaki strain exhibiting high toxicity against S. exigua while retaining its high toxicity against H. armigera. UV and diethyl sulfate methods were used for mutagenesis, followed by an agar plug plate diffusion assay for preliminary screening of Zwittermicin A over-producing mutants, from which we obtained a mutant strain, designated here as B. thuringiensis subsp. kurstaki D1-23, with high toxicity against S. exigua. The toxicity of D1-23 against S. exigua and H. armigera was improved by 115.4 and 25.9%, respectively, compared to its parental commercial strain BMB005.     

Protection from UV-B Damage of Mosquito Larvicidal Toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">israelensis</Emphasis> Expressed in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed δ-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent. Received: 20 November 2001 / Accepted: 31 December 2001     

Comparative Sensitivity to UV-B Radiation of Two Bacillus thuringiensis Subspecies and Other Bacillus sp.

Susceptibility of Bacillus thuringiensis spores and toxins to the UV-B range (280–330 nm) of the solar spectrum reaching Earth's surface may be responsible for its inactivation and low persistence in nature. Spores of the mosquito larvicidal B. thuringiensis subsp. israelensis were significantly more resistant to UV-B than spores of the lepidopteran-active subsp. kurstaki. Spores of subsp. israelensis were as resistant to UV-B as spores of B. subtilis and more resistant than spores of the closely related B. cereus and another mosquito larvicidal species B. sphaericus. Sensitivity of B. thuringiensis subsp. israelensis spores to UV-B radiation depended upon their culture age; 24-h cultures, approaching maximal larvicidal activity, were still sensitive. Maximal resistance to UV-B was achieved only at 48 h. Received: 13 December 2000/Accepted: 19 January 2001     

Serotyping of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolates,their distribution in different Jordanian habitats and pathogenicity in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The investigation of Bacillus thuringiensisin 40 different samples collected from 12 different Jordanian habitats involved the isolation of 80 Bacillus thuringiensis isolates. Out of these isolates, 47 were pathogenic to the third instar larvae of Drosophila melanogaster. The highest viable count of Bacillus thuringiensis was estimated among soil samples contaminated with decomposed animal bodies (14.25 × 10⁷ c.f.u./g), and the lowest viable count was obtained from soils contaminated with engine oil (0.17 × 10⁷c.f.u./g). Serotyping of the 80 isolates against 55 antisera indicated the presence of 13 serotypes, 12 were identical or cross-reacted withaizawai, higo,israelensis, kenyae, kumamotoensis, kurstaki, malaysiensis, morrisoni, pakistani,sooncheon,tohokuensis, andthuringiensis, whereas the remaining one reacted negatively with the 55 tested antisera indicating the presence of an unknown serotype. Israelensis was the dominant serotype among all the samples except those from decomposed animal and olive-cultivated soils. The pathogenic isolates were found to be in 11 of the 13 serotypes. Spherical parasporal crystals were the most common and toxic crystal types.     

Construction of an <Emphasis Type="Italic">Escherichia coli</Emphasis> to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> shuttle vector for large DNA fragments

Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research.