Thuringiensis Subspecies Research Articles

D-cycloserine or similar physiochemical compounds may be uniquely suited for use inBacillus anthracisspore decontamination strategies

As observed in the aftermath of the anthrax attacks of 2001, decontamination and remediation of a site contaminated by the accidental or intentional release of Bacillus anthracis spores is difficult, costly and potentially damaging to the environment. The identification of novel strategies that neutralize the threat of spores while minimizing environmental damage remains a high priority. We investigated the efficacy of d-cycloserine (DCS), an antibiotic and inhibitor of the spore-associated enzyme (alanine racemase) responsible for converting l-alanine to d-alanine, as a spore germination enhancer and antimicrobial agent. We characterized the impact of DCS exposure on both germinating spores and vegetative cells of fully virulent B.anthracis by evaluating spore germination kinetics, determining the minimum inhibitory concentrations (MICs) required to affect growth of the bacteria and performing macrophage viability assays. DCS enhanced germination induced by l-alanine and also efficiently killed the newly germinated spores. Furthermore, DCS proved nontoxic to macrophages at concentrations that provided protection from the killing effects of spores. Similar tests were conducted with Bacillus thuringiensis (subspecies kurstaki and Al Hakam) to determine its potential as a possible surrogate for B.anthracis field trials. Bacillus thuringiensis spores responded in a similar manner to B.anthracis spores when exposed to DCS. These results further support that DCS augments the germination response of spores in the presence of l-alanine but also reveal that DCS is bactericidal towards germinating spores. DCS (or similar compounds) may be uniquely suited for use as part of decontamination strategies by augmenting the induction of spore germination and then rendering the germinated spores nonviable.

English

Bacillus thuringiensis (Bt) is a delta-endotoxins producing bacterium that inhibits the digestive process of many insect orders such as; lepidopteran, dipteran and coleopteran. A comparative study between sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and DNA hybridization was performed on 25 chosen different strains of B. thuringiensis. Based on patterns from these molecular tools, the 25 isolates where grouped based on insect killing capability. Soil samples from Tennessee were collected and analyzed for the presence of B. thuringiensis.Fifty six (56) of more than one hundred isolates collected were initially processed using the sodium acetate and a crystal protein staining procedure. Proteinaceous and genomic profiles were gathered for 25 isolates which were named Bt1 thru Bt25. DNA was extracted, quantified, amplified, cloned and sequenced. Alignment studies were performed on the sequenced products. This sequencing data helped decipher which of the Bt isolates belonged to the resembling parent category. The spore/crystal mixtures produced during sporulation were harvested by centrifugation at 10,000 g at 4°C. The spores and crystals were then separated using a discontinuous sucrose gradient with ultracentrifugation. The separation was confirmed by polarized microscopy. The crystal proteins were quantified and then separated using sodium dodecyl sulphate-polyacrylamide gel electrophresis (SDS-PAGE). A correlation was achieved between genomic and proteomic profiles which directly helped in grouping the 25 isolates. The compilation of data suggest that 14 of the 25 isolates resembledBacillus thuringiensis subspecies kurstaki, four of the 25 isolates resembled B. thuringiensis subspecies israelensis while just one of the 25 isolates resembled B. thuringiensis subspecies tenebrionis. The overwhelming majority of the Bt isolates collected in our study resembled B. thuringiensis subspecies kurstaki. This can bedue to the over use of genetically modified Bt corn in the late 1980’s which was subsequently dispersed into the environment.  Key words: Bacillus thuringiensis, Nashville Tennessee area, bacterium, SDS-PAGE

Evidence of Formosan Subterranean Termite Group Size and Associated Bacteria in the Suppression of Entomopathogenic Bacteria, Bacillus thuringiensis subspecies israelensis and thuringiensis

Abstract The Formosan subterranean termite, Coptotermes formosanus Shiraki, was studied for its ability to suppress two entomopathogenic bacteria, Bacillus thuringiensis subspecies israelensis (Bti) and thuringiensis (Btt). Different group sizes (50, 25, 10, and no termites [control]) of C. formosanus were placed on well-grown Bti or Btt agar plates. On day 1, the diameters of Bti and Btt colonies in the three treatments containing termites were significantly smaller than in the controls. The diameters of Bti and Btt colonies in the 50-termite treatment were significantly smaller than in the 10-termite treatment. However, neither was significantly different from the 25-termite treatment. This group sized dependent suppression was even more distinct on day 2. On day 5, inhibitory zones were observed in all three treatments containing termites where Bti or Btt colonies originally grew. The Bti and Btt cells from these inhibitory zones regenerated on new plates after transfer from 25- and 10-termite treatments as did the controls, but no regeneration was observed after transfer from 50-termite treatment. Results show that the presence of C. formosanus can suppress the growth of Bti and Btt and the suppression effect enhanced with increased of group size. Moreover, antagonistic tests show that natural bacteria carried by termites play a role in the suppression of Bti and Btt.

Induction of L-form Bacteria from Bacillus thuringiensis

Induction of L-form bacteria from Bacillus thuringiensis was investigated with the long-term view of their use as targeted biological control agents within plants. L-forms were successfully induced from B. thuringiensis subspecies israelensis 36-3 by preparing pour plates of exponential cultures in L-phase medium (LPM) containing μg ml-1 cephalosporin C. The induced L-forms were then subcultured and maintained in L-phase medium supplemented with inactivated mycoplasma screened 5% (v/v) horse serum (5HS) with a combination of 225 μg ml-1 cephalosporin C and 0.6 mg ml-1 penicillin G, using the push block method. Induced L-forms showed typical features with pleiomorphic cells that contained granular particles and small vacuoles but no parasporal bodies. These L-forms were capable of growing on solidified media but failed to grow in liquid media. Ceylon Journal of Science (Bio. Sci.) 41 (2): 137-144, 2012 DOI: http://dx.doi.org/10.4038/cjsbs.v41i2.5383

Toxicity of local Malaysian Bacillus thuringiensis subspecies Kurstaki against Plutella xylostella

The toxicity effect of Bacillus thuringiensis against Plutella xylostella is well established. However, effective B. thuringiensis strain especially local isolate is not well tested. In this study local strain B. thuringiensis subspecies kurstaki, SN5 was assessed for its effectiveness against P. xylostella 3rd instar larvae. Other factors such as spore germination, spore coat, L-alanine-adenosine (LAA) and streptomycin were evaluated with their possible effects on the toxicity of B. thuringiensis cry protein. The result of the study showed that SN5 spore exhibit higher toxicity than the commercial strain, HD-1. L-Alanine-adenosine not only improves rate of spore germination but also synergy effect of spore-crystal mix by increasing toxicity of the mixture. These results demonstrating the potential of local isolate in managing P. xylostella and its potential effect can be increase by adding LAA. Key words: Bacillus thuringiensis, cry-like protein, L-alanine-adenosine, Plutella xylostella, streptomycin, toxicity.

Activity of a Brazilian Strain of Bacillus thuringiensis israelensis Against the Cotton Boll Weevil Anthonomus grandis Boheman (Coleoptera: Tenebrionidae)

A Brazilian Bacillus thuringiensis subspecies israelensis, toxic to Diptera, including mosquitoes, was found also to show toxicity to the coleopteran boll weevil Anthonomus grandis Boheman at an equivalent level to that of the standard coleopteran-active B. thuringiensis subspecies tenebrionis T08017. Recombinant B. thuringiensis strains expressing the individual Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa toxins from this strain were assessed to evaluate their potential contribution to the activity against A. grandis, either alone or in combination. Whilst individual toxins produced mortality, none was sufficiently potent to allow calculation of LC50 values. Combinations of toxins were unable to attain the same potency as the parental B. thuringiensis subsp. israelensis, suggesting a major role for other factors produced by this strain.

Evaluation of organically acceptable insecticides as stand‐alone treatments and in rotation for managing yellowmargined leaf beetle, Microtheca ochroloma (Coleoptera: Chrysomelidae), in organic crucifer production

Yellowmargined leaf beetle, Microtheca ochroloma, is the most damaging pest of organic crucifer crops in the southern United States. Experiments were conducted over four growing seasons (2007-2010) in Alabama to evaluate some botanical and microbial insecticides approved by the Organic Materials Review Institute (OMRI) against M. ochroloma in organically grown crucifer crops. Insecticides evaluated included PyGanic(®) (pyrethrum), Aza-Direct(®) (azadiractin), Entrust(®) (spinosad), Mycotrol O(®) (Beauveria bassiana strain GHA) and NOFLY(®) [Isaria ( = Paecilomyces) fumosoroseus strain FE 9901]. Two experimental organic formulations, Tick-Ex (Metarhizium anisopliae strain F52) and MBI-203 (Chromobacterium subtsugae), and one non-OMRI-listed formulation, Novodor(®) (Bacillus thuringiensis subspecies tenebrionis), were also evaluated. The insecticides were applied as stand-alone treatments at recommended field rates on a weekly schedule. In 2010, some of the treatments were also evaluated in rotation/alternation with Entrust(®) . Insecticide efficacy was determined by comparing densities of M. ochroloma larvae and adults and crop damage ratings in treated versus untreated turnip plots. Entrust(®) consistently performed well in suppressing M. ochroloma adults, larvae and crop damage. PyGanic(®) was the second best treatment. PyGanic(®) or NOFLY(™) can be applied in rotation with Entrust(®) for effective management of M. ochroloma. Entrust(®) applied weekly or in alternation with PyGanic(®) or NOFLY(™) provided acceptable control of M. ochroloma in organic crucifer production.

Enhanced Toxicity of Bacillus thuringiensis Subspecies kurstaki and aizawai to Black Cutworm Larvae (Lepidoptera: Noctuidae) With Bacillus sp. NFD2 and Pseudomonas sp. FNFD1

Bacillus thuringiensis subspecies kurstaki and aizawai are important control agents for lepidopteran pests. Bioassays were designed to test B. t. kurstaki and aizawai against second- and-fourth instar black cutworm larvae with and without Bacillus sp. NFD2 and Pseudomonas sp. FNFD1 bacteria. B. thuringiensis subsp. aizawai (XenTari) was more toxic to both second- and fourth-instar black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), larvae than B. t. kurstaki (DiPel) at 7 d after treatment (DAT). When DiPel was combined with NFD2 or FNFD1 versus second instars, the LC50s were 5.0X and 4.7X lower, respectively, than with DiPel alone. DiPel combined with both NFD2 and FNFD1 versus second instars resulted in an LC50 value 7.7X lower than with DiPel alone. When XenTari was combined with NFD2 or FNFD1 versus second instars, the LC50s were 5.2X and 3.8X lower, respectively, than with XenTari alone. XenTari combined with both NFD2 and FNFD1 versus second instars resulted in an LC50 9.7X lower than with XenTari alone. When DiPel was combined with NFD2 or FNFD1 versus fourth instars, the LC50s were 4.4X and 3.4X lower, respectively, than with DiPel alone. DiPel combined with both NFD2 and FNFD1 versus fourth instars resulted in an LC50 5.0X lower than with DiPel alone. When XenTari was combined with NFD2 or FNFD1 versus fourth instars, the LC50s were 5.7X and 3.3X lower, respectively, than with XenTari alone. XenTari combined with both NFD2 and FNFD1 versus fourth instars resulted in an LC50 6.7X lower than with XenTari alone.

Combined molecular dynamics and continuum solvent studies of the pre-pore Cry4Aa trimer suggest its stability in solution and how it may form pore

Cry4Aa toxin is one of the highly specific mosquito-larvicidal proteins produced by the bacterium Bacillus thuringiensis subspecies israelensis. It is thought to form pores in the larval midgut membrane that cause membrane leakage and subsequent insect death. Therefore, Cry4Aa and other Cry toxins have been used as efficient and safe bacterial insecticides to control the disease-carrying mosquitoes such as Aedes, Anopheles, and Culex. However, we still do not clearly understand how Cry toxins kill mosquito-larvae at molecular details. Recent electron crystallographic images of Cry4Ba toxin, another toxin closely related to Cry4Aa toxin, have suggested that the protein forms trimer in aqueous solution and in lipid monolayer. Moreover, the unit cell of X-ray crystal structure of Cry4Ba toxin has been shown to be trimeric. In this study, we constructed the first full-atom structural model of Cry4Aa trimer using the trimeric unit cell structure of Cry4Ba toxin as a template and then used the methods of molecular dynamics (MD) and molecular mechanics combined with Poisson-Boltzmann and surface area (MM-PBSA) to show that the trimeric structure of Cry4Aa toxin is stable in 150 mM KCl solution on 10 ns timescale. The results reveal that Cry4Aa toxins use polar amino acid residues on α-helices 3, 4, and 6 to form trimer and suggest that the proteins form trimer to reduce their non-polar interactions with surrounding water. Based on the obtained trimeric structure of Cry4Aa toxins, we propose that pore formation of Cry toxins may involve a 90°-hairpin rotation during the insertion of their three α4-α5 hairpins into the membrane. This process may be mediated by water and ions.PACS Codes: 87.15.ap, 87.15.bk, 87.14.ep

Spatial and temporal changes in genetic structure of greenhouse and field populations of cabbage looper,Trichoplusia ni

Trichoplusia ni is a subtropical moth that migrates annually from southern California to southern British Columbia, Canada where it invades vegetable greenhouses and field crops. The heated greenhouse environment has altered the natural extinction-recolonization dynamics of T. ni populations, and allows year-round persistence in some locations. In addition, the extensive use of the biopesticide, Bacillus thuringiensis subspecies kurstaki (Bt) in some greenhouses has selected for resistance. Here we investigated the genetic structure of T. ni populations in British Columbia greenhouses and in field populations in California and British Columbia using amplified fragment length polymorphisms (AFLP) as related to patterns of Bt resistance. The majority of British Columbia field populations were similar to the California field populations, the potential source of migrants. However populations in two geographic areas with high concentrations of greenhouses showed local genetic differentiation. Some of these populations experienced severe bottlenecks over-winter and following Bt sprays. Greenhouse populations showed a pattern of isolation by distance and a strong positive relationship between genetic differentiation and levels of Bt resistance. These patterns indicate that greenhouses that sometimes support year-round populations of T. ni and the ensuing strong bottlenecking effects following winter cleanups and Bt application cause genetic differentiation of T. ni populations. Long distance migrants to field populations contribute to genetic homogeneity of these.

Specific Targeting to Murine Myeloma Cells of Cyt1Aa Toxin from Bacillus thuringiensis Subspecies israelensis

Multiple myeloma is currently an incurable cancer of plasma B cells often characterized by overproduction of abnormally high quantities of a patient-specific, clonotypic immunoglobulin "M-protein." The M-protein is expressed on the cell membrane and secreted into the blood. We previously showed that ligand-toxin conjugates (LTC) incorporating the ribosome-inactivating Ricin-A toxin were very effective in specific cytolysis of the anti-ligand antibody-bearing target cells used as models for multiple myeloma. Here, we report on the incorporation of the membrane-disruptive Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis into LTCs targeted to murine myeloma cells. Proteolytically activated Cyt1Aa was conjugated chemically or genetically through either its amino or carboxyl termini to the major peptidic epitope VHFFKNIVTPRTP (p87-99) of the myelin basic protein. The recombinant fusion-encoding genes were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis through the shuttle vector pHT315. Both chemically conjugated and genetically fused LTCs were toxic to anti-myelin basic protein-expressing murine hybridoma cells, but the recombinant conjugates were more active. LTCs comprising the Cyt1Aa toxin might be useful anticancer agents. As a membrane-acting toxin, Cyt1Aa is not likely to induce development of resistant cell lines.

Synergistic interaction between sublethal doses of Bacillus thuringiensis and Campoletis chlorideae in managing Helicoverpa armigera

Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) is an important solitary larval endoparasitoid of the tomato fruit borer Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in India. The interaction between Bacillus thuringiensis subspecies kurstaki (Btk) HD-1 and C. chlorideae was studied under laboratory condition to explore their compatibility in managing H. armigera. The results had indicated that the growth and development of H. armigera was affected in a dose-dependent manner upon feeding on sublethal doses of Btk HD-1-treated diets. There were no larval survivors in lethal doses of Btk HD-1 (LC70 and LC90). The growth and survival of the parasitoid were normal when the host larvae were fed with sublethal doses or subjected to short time exposure to lethal doses of Btk HD-1. However, the parasitoid offsprings developed slowly and pupal as well as adult period, adult weight and adult emergence rate were reduced significantly if the parasitoid was developing inside a severely Bt intoxicated host larvae. There were no evident differences in longevity of parasitoid adults that were fed on honey solution containing different concentrations of Btk HD-1 as compared to adults fed only on honey solution. This indicates no direct adverse effect of Btk HD-1 on C. chlorideae. Further, the gravid female parasitoid did not discriminate Btk HD-1 intoxicated and normal H. armigera larvae for oviposition. The result implies that spore crystal formulation of Btk HD-1 can be effectively used in a synergistic manner along with existing natural or prereleased population of C. chlorideae in organic farming or as components in biointensive IPM module for managing H. armigera.

Bacillus thuringiensis proteases: Production and role in growth, sporulation and synergism

Bacillus thuringiensis (Bt) subspecies produces metalloproteases and serine alkaline proteases (endogenous) which affect sporulation and entomotoxicity against different insect orders. The production of Bt proteases is investigated in conventional medium and alternative substrates with future repercussions on Bt formulations and larval mortality. Relationship between protease activity and total cell count during Bt fermentation has been discussed while protease activity as a potential indicator of entomotoxicity has also been explored. In general, the proteases influence entomotoxicity in two divergent ways—processing of inactive protoxins to active toxin fractions (by endogenous Bt as well as exogenous larval midgut proteases) and degradation of protoxins to fragments which sometimes lack insecticidal activity (usually by Bt proteases). In fact, the function of endogenous (intra and extracellular) proteases is ambiguous and has been raising serious questions on their role in larval mortality. The review explores various schools of thoughts (traditional as well as advanced) to solve the enigma of protease interactions with crystal toxins at different levels (sporulation and insecticidal action).

Antimicrobial activity of different proteins and their fragments from Bacillus thuringiensis parasporal crystals against clostridia and archaea

Proteins of parasporal crystals (Cry proteins) from entomopathogenic bacterium Bacillus thuringiensis (subspecies kurstaki, galleriae, tenebrionis) as well as some fragments of these proteins, obtained by limited proteolysis, are capable of antimicrobial action against anaerobic bacteria and archaea— Clostridium butyricum, Clostridium acetobutylicum and Methanosarcina barkeri. The MICs are 45–150 μg/mL. Electron microscopy showed that lysis of M. barkeri cells in the presence of 49 kDa fragment of Cry3Aa toxin is generally similar to the bacterial cell lysis, which has been previously detected in the presence of Cry11A, Cry1Ab and other Cry proteins. The Cry1D-like toxin from crystals of B. thuringiensis subsp. galleriae has been put forward as an example of the supposition that cell wall and some of its components like teichoic acid and N-acetylgalactosamine have possible influence on Cry toxins, enhancing their antimicrobial activity. The possible ecological role of the antimicrobial activity of Cry proteins is also discussed.

Characterization of Bacillus thuringiensis isolates toxic to cotton boll weevil (Anthonomus grandis)

The cotton boll weevil (Anthonomus grandis) is the major cotton pest in the Americas. One of the alternatives for its control is the utilization of Bacillus thuringiensis (Bt), an entomopathogenic bacterium characterized by its production of insecticidal crystal proteins. Embrapa Genetic Resources and Biotechnology has a collection of Bacilli in which different isolates of Bt are stored. A method for rearing and maintenance of Anthonomus grandis on artificial diet in the laboratory was developed, and a robust larval bioassay protocol was established for the selection of B. thuringiensis isolates toxic to boll weevil. After preliminary bioassays performed with 215 isolates, 5 were selected that demonstrated a good level of toxicity and these were analyzed in more detail. The most toxic were S601 and S1806 presenting LC50 (lethal concentration to kill 50% if the larvae) of 0.14 mg/ml and 0.30 mg/ml, respectively. S601 showed an LC50 value that was half that of the standard B. thuringiensis subspecies tenebrionis and S1806 demonstrated similar values of LC50 to the standard. S601 contained the cry1B gene and S1806 had the cry4A, cry4B, cry10, cry11, cyt1 and cyt2 genes like B. thuringiensis subspecies israelensis.

Detection and phylogenic analysis of one anthrax virulence plasmid pXO1 conservative open reading frame ubiquitous presented within Bacillus cereus group strains

The presence of one of the anthrax virulence plasmid pXO1 conserved fragments was analyzed in 24 Bacillus cereus and B. thuringiensis strains, including 6 B. thuringiensis subspecies, by polymerase chain reactions. Twelve out of 24 strains showed PCR-positive for an ORF101 homologous sequence. Two pXO1-ORF101-like fragments from a B. cereus B-4ac and a commercial B. thuringiensis kurstaki HD1 were cloned, sequenced and expressed in Escherichia coli. Toxicity assays revealed that the product encoded by the pXO1-ORF101-like fragment had no impact on either Vero cells or Chinese Hamster Ovary cells, suggesting that this fragment probably not contribute to enterotoxic activity. Sequence alignment of the pXO1-ORF101 from three Bacillus anthracis and ORF101-like fragments from other 12 B. cereus group isolates indicated high identity (more than 90%) and the presence of subgroup- and strain-specific SNPs among these fragments.

EVALUATION OF SEVERAL REDUCED-RISK INSECTICIDES IN COMBINATION WITH AN ACTION THRESHOLD FOR MANAGING LEPIDOPTERAN PESTS OF COLE CROPS IN ALABAMA

Several reduced-risk insecticides were evaluated for management of three lepidopteran cole crop pests, Plutella xylostella (L.), Pieris rapae (L.), and Trichoplusia ni (Hübner) in central Alabama in 2004 (spring and fall plantings) and 2005 (spring only). The following formulated sprays were evaluated: Dipel® (Bacillus thuringiensis subspecies kursatki), XenTari® (B. thuringiensis subspecies aizawai), Dipel XenTari (a premixed test formulation consisting of both subspecies of B. thuringiensis), Entrust® (a formulation of spinosad for use in organic crop production), and Novaluron (insect growth regulator). Variations in the populations of the three pest species were recorded from season to season with pest pressure being generally higher in both spring seasons than in the fall season. While moderate to high populations of P. xylostella and P. rapae were recorded in all three seasons, T. ni was detected only in spring 2005. An action threshold of 0.5 cabbage looper equivalents (CLE) per plant was used to determine the need for insecticide applications. Insecticide efficacy was determined by comparing densities of larvae and immatures (larvae pupae) of each pest species, crop damage ratings, densities of key non-target arthropods, and number of insecticide applications in plots treated with each material versus untreated control plots. All five reduced-risk insecticide formulations were effective in reducing infestations of the three lepidopteran pests and in providing marketable cabbage and collards in Alabama. Among the treatments, Entrust® consistently produced the lowest mean damage ratings with the minimum number of applications per season. No significant effects of insecticide treatments were recorded in the numbers of spiders and lady beetles found per plant. The results also suggest that the 0.5 CLE action threshold can be used to produce marketable cabbage and collards in Alabama with only minimal applications of reduced-risk insecticides.

Transfer and expression of the mosquitocidal plasmid pBtoxis in Bacillus cereus group strains

The toxicity of Bacillus thuringiensis subsp. israelensis to dipteran larvae (mosquitoes and black flies) depends on the presence of the pBtoxis plasmid. In this paper, two antibiotic resistance tagged pBtoxis were transferred by conjugation to other Bacillus cereus group strains. Among 15 potential recipients, only a lepidopteran active B. thuringiensis subspecies kurstaki and a B. cereus strain received the plasmid pBtoxis with a low transfer rate of about 10 −8 transconjugants/recipient. The resulting B. thuringiensis subspecies kurstaki transconjugant was active to both lepidopteran and dipteran targets and the B. cereus transconjugant was active against dipteran insects. Phase contrast microscopy showed that the B. cereus transconjugants could produce only round crystalline inclusion bodies while B. thuringiensis subspecies kurstaki transconjugant could produce both round and bipyramidal crystals during sporulation. SDS–PAGE revealed that all the major mosquitocidal proteins from pBtoxis could express in the two transconjugants, including Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa and Cyt1Aa. However, none of the experiment showed any indications of mobilising abilities of pBtoxis. The limited number of strains, which could receive and maintain pBtoxis using a conjugational helper plasmid, indicates a very narrow host range of the B. thuringiensis subsp. israelensis pBtoxis plasmid.

Susceptibility of Archaea to the Antibiotic Effect of the Parasporal Inclusion Proteins from Different Bacillus thuringiensis subspecies

The proteins of parasporal inclusions from three Bacillus thuringiensis subspecies (kurstaki, amagiensis, and monterrey) inhibited growth of methanogenic archaea of two species belonging to two genera, Methanobrevibacter arboriphilus and Methanosarcina barkeri. The minimal inhibitory concentrations of these proteins were 20 to 50 micrograms/ml. Lysozyme exhibited similar bactericidal effect on archaea. The perspective of comparative studies on the effect of polyfunctional proteins on bacteria and archaea is discussed.

Expression of the Bacillus thuringiensis Cyt2Aa1 toxin in Pichia pastoris using a synthetic gene construct.

The nucleotide sequence data corresponding to the syncyt2Aa1 open reading frame was deposited with the EMBL/GenBank Nucleotide Sequence Databases under the accession number AF398463. Bacillus thuringiensis delta-endotoxins are membrane-active, pore-forming proteins with highly specific insecticidal activities. In addition to a well-established role in the biological control of a wide variety of crop pests and disease vectors, these toxins also have great potential for the development of anti-tumour agents called immunotoxins (ITs), chimaeric molecules consisting of a cell-binding ligand coupled to a toxin or its subunits. The ultimate goal of our study was the recombinant production of such ITs based on the Cyt2Aa1 toxin from B. thuringiensis subspecies kyushuensis. We explored the use of Pichia pastoris for recombinant IT production because earlier attempts in our laboratory using the Escherichia coli expression system or various chemical conjugation strategies yielded only low levels of functional product. However, our initial attempts were not successful because the A+T-rich bacterial cyt2Aa1 gene contained fortuitous polyadenylation sites, causing premature transcription termination in this yeast. Accordingly, we designed and constructed a synthetic cyt2Aa1 gene (syncyt2Aa1) optimized for heterologous expression in P. pastoris. This was achieved by increasing the overall G+C content of the bacterial cyt2Aa1 while changing its codon usage to that preferred by the methylotrophic yeast. Here we describe in detail the design, synthesis and requisite PCR repair of syncyt2Aa1, then present analyses of recombinant Cyt2Aa1 expression in P. pastoris using this synthetic gene. Following the results presented in this paper, the syncyt2Aa1 gene was also successfully used for the recombinant production of a Cyt2Aa1-based IT in the same expression host.