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Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Forster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven [alpha] helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the [alpha]3-[alpha]4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time course of the donor fluorescence. We found that Cry1Aa is present on both membrane leaflets. doi/ 10.1085/jgp.200910347

Bacillus thuringiensis subsp, israelensis produces a potent mosquitocidal protein, Cry4A. We have identified a 15-bp catabolite responsive element (cre), overlapping the -35 element of the cry4A promoter. Changing a guanine to adenine at position -49 in the promoter abolished glucose catabolite repression of cry4A and enhanced promoter activity two- to threefold. This cis regulatory element is essential for controlled toxin synthesis, vital to evolutionary success of B. thuringiensis subsp, israelensis.

Conjugation, mobilization, and retromobilization are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in gram-negative species, where retromobilization, the capture of DNA from a recipient by a donor cell, was shown to result from two successive steps: the transfer of the conjugative plasmid from the donor to the recipient followed by the retrotransfer of the mobilizable plasmid to the donor. This successive model was established for gram-negative bacteria but was lacking experimental data from the gram-positive counterparts. In the present work, the mobilization and retromobilization abilities of the conjugative plasmid pXOl6 from Bacillus thuringiensis subsp, israelensis were studied using the mobilizable plasmids pUB110 and pE194 and the 'nonmobilizable' element pC194 lacking the mob and oriT features (all from Staphylococcus aureus). Experimental data suggested a successive model, since different retromobilization frequencies were observed between the small plasmids. More importantly, retromobilization was shown to be delayed by 50 and 150 min for pUB110 and pE194, respectively, compared to pXO16 conjugation. Natural liquid foods (cow milk, soy milk, and rice milk) were used to evaluate the putative ecological impact of these transfers. In cow and soy milk, conjugation, mobilization, and retromobilization were shown to occur at frequencies of 8.0 x [10.sup.-1], 1.0 x [10.sup.-2], and 1.2 x [10.sup.-4] transconjugants per recipient, respectively. These data are comparable to those obtained with LB medium and about 10-fold lower than in the case of rice milk. Taken together, these results emphasize the potential role of plasmid capture played by B. thuringiensis in natural environments.

Swarming is a social phenomenon that enables motile bacteria to move co-ordinately over solid surfaces. The molecular basis regulating this process is not completely known and may vary among species. Insertional mutagenesis of a swarming-proficient Bacillus thuringiensis strain was performed, by use of the transposon mini-Tn 10, to identify novel genetic determinants of swarming that are dispensable for flagellation, swimming motility, chemotaxis and active growth. Among the 67 non-swarming mutants obtained, six were selected that showed no defect in flagellar assembly and function, chemotaxis or growth rate. Sequence analysis of DNA flanking the transposon insertion led to the identification of previously uncharacterized genes that are involved in the development of swarming colonies by B. thuringiensis and that are highly conserved in all members of the Bacillus cereus sensu lato group. These genes encode non-flagellar proteins with putative activity as sarcosine oxidase, catalase-2, amino acid permease, ATP-binding cassette transporter, dGTP triphosphohydrolase and acetyltransferase. Functional analysis of two of the isolated mutants demonstrated that swarming differentiation depends on the intracellular levels of the osmoprotectant glycine betaine and on the quantity of synthesized phenazine secondary metabolites. The finding that proteins involved in diverse physiological processes have a role in swarming motility underlines the complexity of the molecular mechanisms governing this behaviour in B. thuringiensis.

Cyt1 Aa is a [delta]-endotoxin protein that is produced by Bacillus thuringiensis subsp, israelensis. It is a membrane pore-forming toxin that is lethal to insect larvae and is broadly cytolytic to vertebrate as well as invertebrate cells. Cyt1 Aa is produced as a protoxin of 27 kDa. Proteolytic activation results in a reduction of the molecular mass to approximately 23-24 kDa and a threefold increase in activity. In this research, Cyt1 Aa crystals were purified from B. thuringiensis IPS78/11 harbouring the expression vector pHT-cyAp20. The activity of the activated form of Cyt1 Aa (23-24 kDa) was examined on a pathogenic strain of the Gram-negative Escherichia coil and the Gram-positive species Staphylococcus aureus. The Cyt1 Aa minimal inhibitory concentration for E. coil and S. aureus was 1.25 and 5 [micro]g [ml.sup.-1], respectively. Cyt1Aa was found to be bactericidal for E. coli, whereas it was bacteriostatic for S. aureus. Furthermore, Cyt1 Aa increased the lethal effect when acting in combination with antibiotics. The association of Cyt1 Aa with cells of these two bacteria was demonstrated by Western blot analysis using antibodies against the whole [delta]-endotoxin crystal. Scanning electron microscopy displayed damage to Cyt1 Aa-treated cells. Ion imbalance due to damage of the cell walls and membranes was confirmed by X-ray microanalysis. These experiments show that Cyt1 Aa has an antibacterial effect on pathogenic species and demonstrate, apparently for the first time, that exogenous Cyt1 Aa has a bactericidal effect upon Gram-negative bacteria.

The impact of genetically modified trees on soil nitrogen-fixing bacterial communities is investigated. It is concluded in the absence of evidence that the presence of the Bacillus thuringiensis cry1Ab gene effects diazotroph communities.

A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 [mm.sup.2]. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 [+ or -] 31 (n = 11), 2742 [+ or -] 38 (n = 3), 3053 [+ or -] 19 (n = 2), 2777 [+ or -] 26 (n = 2), 2953 [+ or -] 24 (n = 2), and 3105 [+ or -] 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 [min.sup.-1]. A hindrance factor (6) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x [10.sup.5] BT + BC spores/mL, suggesting that [alpha] is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.

The infectivity of Bacillus thuringiensis Cry proteins for the pest Lobesia botrana on grape plants is analyzed using Cry1, Cry2 and Cry9 group proteins. The proteins belonging to Cry9 group were found to be the most toxic.

We report the generation of transgenic soybean [Glycine max (L.) Merr.] via Agrobacterium-mediated gene transfers of a cry1A gene (tic107) from Bacillus thuringiensis (Bt) that exhibits a high degree of resistance against the lepidopteran pests Pseudoplusia includens (Walker) (soybean looper), Helicoverpa zea (Boddie) (soybean podworm), and Anticarsia gemmatalis Hubner (velvetbean caterpillar). Three transgenic soybean lines (862, 726, and 781) were evaluated for expression, molecular composition, efficacy against target pests, and agronomic characteristics. We have designed and tested an expression cassette that consistently and reproducibly generates transgenic soybeans that accumulate the tic107 protein at levels as high as 6.12 [micro]g [mg.sup.-1] of total extractable protein. Expression levels of this magnitude of a Bt insecticidal protein have never been reported in soybean. Previous reports have indicated expression levels 100-fold lower in the highest-expressing lines. In addition, the phenotypes of these high-expressing lines were indistinguishable from their negative segregant and the transformation parent (Asgrow var. 'A3237'). Insect bioassay data demonstrate complete protection against soybean looper, soybean podworm, and velvetbean caterpillar when negative controls exhibited defoliation as high as 98%. Unlike previous reports of transgenic soybeans, we report here highly efficacious, single-copy, and normal phenotypes of transgenic soybean plants containing the highly expressed cry1A gene.

Bacillus thuringiensis is the most widely applied biological insecticide and is used to manage insects that affect forestry and agriculture and transmit human and animal pathogens. This ubiquitous spore-forming bacterium kills insect larvae largely through the action of insecticidal crystal proteins and is commonly deployed as a direct bacterial spray. Moreover, plants engineered with the cry genes encoding the B. thuringiensis crystal proteins are the most widely cultivated transgenic crops. For decades, the mechanism of insect killing has been assumed to be toxin-mediated lysis of the gut epithelial cells, which leads to starvation, or B. thuringiensis septicemia. Here, we report that B. thuringiensis does not kill larvae of the gypsy moth in the absence of indigenous midgut bacteria. Elimination of the gut microbial community by oral administration of antibiotics abolished B. thuringiensis insecticidal activity, and reestablishment of an Enterobacter sp. that normally resides in the midgut microbial community restored B. thuringiensis-mediated killing. Escherichia coil engineered to produce the B. thuringiensis insecticidal toxin killed gypsy moth larvae irrespective of the presence of other bacteria in the midgut. However, when the engineered E. coli was heat-killed and then fed to the larvae, the larvae did not die in the absence of the indigenous midgut bacteria. E. coli and the Enterobactersp. achieved high populations in hemolymph, in contrast to B. thuringiensis, which appeared to die in hemolymph. Our results demonstrate that B. thuringiensis-induced mortality depends on enteric bacteria. insect-microbe interactions | microbe-microbe interactions | synergy | Lepidoptera | pore-forming toxin

Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use. anthelminthic | Cry5B | nematode

Germination is the process by which a dormant spore returns to its vegetative state when exposed to suitable conditions. We report on the real-time detection of kinetic germination and heterogeneity of single Bacillus thuringiensis spores in an aqueous solution by monitoring the calcium dipicolinate (CaDPA) biomarker with laser tweezers Raman spectroscopy (LTRS). A single B. thuringiensis spore was optically trapped in a focused laser beam, and its Raman spectra were recorded sequentially in time after exposure to a nutrient-rich medium, so that the CaDPA amount inside the trapped spore was monitored during the dynamic germination process. The CaDPA content in an individual spore was observed to remain almost constant in the first period and then decrease very rapidly due to its release into the medium (within ~2 min). The time-to-germination ([t.sub.germ]), defined as the time required for the CaDPA band intensity to decrease to the midpoint from its initial value, was found to be stochastic for individual spores with a typical value of ~30 min under the experimental conditions. The distribution of the time-to-germination was measured from a time lapse measurement of a population of spores. The results demonstrated that LTRS can be used to noninvasively detect the kinetic germination process at the single-cell level and explore cellular heterogeneity.

The hypothesis that Heliothis virescens cadherin-like protein (HevCaLP) is a functional receptor for Cry1A and Cry1F toxins is tested using Drosophila Schneider 2 (S2) cells and a fluorescence-based cytotoxicity assay is presented. The results proved that HevCaLP is a functional Cry1A but not Cry1Fa receptor.

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation that are extensively used in the field for control of mosquito populations. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in resistant colony of culex quinquefasclatus, apparently due to the production of Cry11A but not Cry4A or Cry4B.

Byline: C. Sengonca (1), B. Liu (2), Y. J. Zhu (1) Keywords: GCSC-BtA; Bacillus thuringiensis; Abamectin; Insecticidal activity; Antifeedant Abstract: Insecticidal activity and antifeedant effect of a new type biocide GCSC-BtA and its two reactants Bacillus thuringiensis (B.t.) crystal and Abamectin against third instar larvae of Plutella xylostella L. (Lep., Plutellidae) were investigated using bioassays. GCSC-BtA showed significantly higher toxicity to P. xylostella with LC.sub.50 of 0.021 mg/ml than B.t. crystal with 0.060 mg/ml and Abamectin with 0.139 mg/ml in leaf-dip bioassay. Net leaf-dip test gave L[C.sub.50]s of 0.073, 0.071 and 0.670 mg/ml for GCSC-BtA, B.t. crystal and Abamectin, respectively. The significant differences of the biocide toxicities between leaf-dip and net leaf-dip methods showed the potential of net leaf-dip method for separating ingestion and contact poisons. Variation in toxicity of the biocide and two reactants in net leaf-dip method and in mortality in topical application bioassay showed that GCSC-BtA possessed a combination of strong stomach as well as contact poison property higher than B.t. or Abamectin. GCSC-BtA indicated no fumigant and systemic toxicities, which the two reactants lacked. In the antifeedant bioassay, GCSC-BtA acted as a strong antifeedant to the larvae of P. xylostella. Leaf consumption by the larvae after GCSC-BtA treatment was 0.04--0.60 mm.sup.2 with a concentration range of 0.04--4.00 mg/ml, which was lower than 0.26--1.02 and 0.04--6.47 mm.sup.2 for B.t. crystal and Abamectin, respectively. DC.sub.50 (concentration resulting in a deterrence index of 50%) value for GCSC-BtA was 0.089 mg/ml, which was also significantly lower than 0.275 and 0.736 mg/ml for B.t. crystal and Abamectin, respectively. Author Affiliation: (1) Department of Entomology and Plant Protection, Institute of Phytopathology, University of Bonn, Nussallee 9, 53115, Bonn, Germany (2) Biotechnology Center, Fujian Academy of Agricultural Sciences, 350003, Fuzhou, Fujian, PR China Article History: Registration Date: 28/11/2005 Received Date: 29/04/2005 Accepted Date: 28/11/2005 Online Date: 14/12/2005 Article note: Communicated by Andreas Vilcinskas

Bacillus thuringiensis subsp, israelensis produces crystal proteins, Cry (4Aa, 4Ba, 10Aa, and 11Aa) and Cyt (1Aa and 2Ba) proteins,toxic to mosquito vectors of human diseases. Cyt1Aa overcomes insect resistance to Cry11Aa and Cry4 toxins and synergizes the toxicity of these toxins. However, the molecular mechanism of synergism remains unsolved. Here, we provide evidence that Cyt1Aa functions as a receptor of Cry11Aa. Sequential-binding analysis of Cyt1Aa and Cry11Aa revealed that Cyt1Aa binding to Aedes aegypti brush border membrane vesicles enhanced the binding of biotinylated-Cry11Aa. The Cyt1Aa- and Cry11Aa-binding epitopes were mapped by means of the yeast two-hybrid system, peptide arrays, and heterologous competition assays with synthetic peptides. Two exposed regions in Cyt1Aa, loop [beta]6-[alpha]E and part of [beta]7, bind Cry11Aa. On the other side, Cry11Aa binds Cyt1Aa proteins by means of domain II-loop [alpha]8 and [beta]-4, which are also involved in midgut receptor interaction. Characterization of single-point mutations in Cry11Aa and Cyt1Aa revealed key Cry11Aa (S259 and E266) and Cyt1Aa (K198, E204 and K225) residues involved in the interaction of both proteins and in synergism. Additionally, a Cyt1Aa loop [beta]6-[alpha]E mutant (K198A) with enhanced synergism to Cry11Aa was isolated. Data provided here strongly indicates that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium because it produces a toxin and also its functional receptor, promoting toxin binding to the target membrane and causing toxicity. receptor interaction | binding epitopes | insect resistance | mode of action

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests and thus can reduce reliance on insecticides. Widespread planting of such Bt crops increased concerns that their usefulness would be cut short by rapid evolution of resistance to Bt toxins by pests. Pink bollworm (Pectinophora gossypiella) is a major pest that has experienced intense selection for resistance to Bt cotton in Arizona since 1997. We monitored pink bollworm resistance to Bt toxin for 8 years with laboratory bioassays of strains derived annually from 10-17 cotton fields statewide. Bio-assay results show no net increase from 1997 to 2004 in the mean frequency of pink bollworm resistance to Bt toxin. A synthesis of experimental and modeling results suggests that this delay in resistance can be explained by refuges of cotton without Bt toxin, recessive inheritance of resistance, incomplete resistance, and fitness costs associated with resistance. Bacillus thuringiensis | resistance management | transgenic crops

Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtxl. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3' end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca.

The three-dimensional structure of the N-acyl-L-homoserine lactone hydrolase (AHL lactonase) from Bacillus thuringiensis has been determined, by using single-wavelength anomalous dispersion (SAD) phasing, to 1.6-[Angstrom] resolution. AHLs are produced by many Gram-negative bacteria as signaling molecules used in quorum-sensing pathways that indirectly sense cell density and regulate communal behavior. Because of their importance in pathogenicity, quorum-sensing pathways have been suggested as potential targets for the development of novel therapeutics. Quorum-sensing can be disrupted by enzymes evolved to degrade these lactones, such as AHL lactonases. These enzymes are members of the metallo-[beta]-lactamase superfamily and contain two zinc ions in their active sites. The zinc ions are coordinated to a number of ligands, including a single oxygen of a bridging carboxylate and a bridging water/hydroxide ion, thought to be the nucleophile that hydrolyzes the AHLs to ring-opened products, which can no longer act as quorum signals. bridged bimetallo center | lactonase | single-wavelength anomalous dispersion

The Cry family Bacillus thuringiensis insecticidal and nematicidal proteins constitute a valuable source of environmentally benign compounds for the control of insect pests and disease agents. Model invertebrates such as Caenorhabditis elegans and Drosophila melanogaster, as well as advances in insect genomics is used to accelerate efforts to clone Cry toxin resistance genes and come to a detailed and broad understanding of Cry toxin resistance.

The insecticidal protoxin from Bacillus thuringiensis is shown to be a major component of the spore coat. A novel surface display system is developed using Bacillus thuringiensis spores in which the N-terminal portion of the protoxin is replaced with a heterologous protein.

The male reproductive costs in pink bollworm resistant to an insecticidal protein of Bacillus thuringiensis (Bt) produced by transgenic cotton, using contrasts between two pairs of related susceptible and resistant strains were examined. It is found that the reduced first-male paternity in resistant males might involve reduced sperm precedence caused by mutations in a cadherin gene linked with resistance to Bt cotton.

A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, is developed for simultaneous determination of microbial diversity and abundance within a complex community. The results indicate that the real-time-t-RFLP assay can be a powerful molecular tool for learning about microbial communities in various engineered systems and natural habitats.

The current hypothesis on the mode of action of the cytolytic delta-endotoxin Cyt 1 A from Bacillus thuringiensis is that the toxin self-assembles into well-defined cation-selective channels or pores, which result in colloidosmotic lysis of the cell. But, a new hypothesis suggests that Cyt 1 A aggregates on the membrane surface and acts in a detergent-like manner, since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes.

The permeabilizing behavior of the native binary toxin and its two individual components expressed as recombinant proteins was studied using calcein efflux determination in liposomes and by ion channel activity measurements in planar lipid bilayers (PLBs).

The two new classes of crystal proteins Bacillus thuringiensis strain EG4851 that comprise a binary toxin active against western corn rootworm (WCR) are described. The antirootworm proteins produced by strain EG4851 represent a new binary toxin and the genes encoding these proteins may become an important component of a sustainable resistance management strategy against this insect pest.

The identification and characterization of two genes, yqgB and yqfZ, of unknown function are reported. The findings suggest that the yqgB and ygfZ genes encode adaptive factors that may act in synergy, enabling the bacteria to cope with the physical environment in vivo, facilitating colonization of the host.

[beta]-Exotoxin I is an insecticidal nucleotide analogue secreted by various Bacillus thuringiensis strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated sigW-ecfX-ecfY, that is essential for [beta]-exotoxin I production in B. thuringiensis subsp, thuringiensis strain 407-1. In this strain, the disruption of the sigW cluster resulted in nontoxic culture supernatants. sigW encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from Bacillus anthracis strain Ames and Bacillus cereus strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from Clostridium perfringens and SigW and SigX from Bacillus subtilis, respectively. EcfX, encoded by the gene following sigW, significantly repressed the expression of sigW when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable cry plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1([Cry.sup.-])([Pig.sup.+]), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of sigW indicated that this gene was expressed during the stationary phase and only in the 407-1 ([Cry.sup.-])([Pig.sup.+]) mutant. This suggests that in the wild type-407([Cry.sup.+]) strain, [beta]-exotoxin I was produced from determinants located on a cry gene-bearing plasmid and that sigW is able to induce [beta]-exotoxin I production in B. thuringiensis in the absence of cry gene-bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that [beta]-exotoxin I production in B. thuringiensis can be restored or induced via an alternative pathway that requires sigW expression.

The use of Bacillus thuringiensis (Bt) endotoxins to control insect vectors of human diseases and agricultural pests is threatened by the possible evolution of resistance in major pest species. In addition to high levels of resistance produced by receptor insensitivity (5, 16, 17), several cases of tolerance to low to medium levels of toxin have been reported in laboratory colonies of lepidopteran species (3, 18). Because the molecular basis of some of these cases of tolerance to the toxin are not known, we explored alternative mechanisms. Here, we present evidence that tolerance to a Bt formulation in a laboratory colony of the flour moth Ephestia kuehniella can be induced by preexposure to a low concentration of the Bt formulation and that the tolerance correlates with an elevated immune response, The data also indicate that both immune induction and Bt tolerance can be transmitted to offspring by a maternal effect and that their magnitudes are determined by more than one gene. coagulation | endotoxin tolerante | immune induction | maternal effect | melanization

The Cry1A toxin of Bacillus thuringiensis interacts with more toxin-binding proteins when it is denatured. These toxin-binding proteins make the insect resistant to the toxin.

Three experiments were conducted to evaluate the impact of the Bacillus thuringiensis (Bt)-11 transformation event in two parental corn hybrids differing in date of maturity on beef and dairy cattle performance. Sixteen lactating Holstein dairy cows in replicated 4 x 4 Latin squares were assigned to four diets in a 2 x 2 factorial arrangement: Bt vs non-Bt trait and early- vs late-maturing corn hybrids. The diets contained 40% of the test corn silage plus 28% corn grain from the same corn hybrid (DM basis). There was no effect of the Bt trait on efficiency of milk production, ruminal pH, acetate:propionate ratio, or in situ digestion kinetics of NDF. The early-maturing corn hybrids resulted in greater total VFA concentrations in the rumen and efficiency of 4% fat-corrected milk production than the later-maturing hybrids (P < 0.05). Sixty-seven steer calves were used in a 70-d corn residue grazing trial for the late-maturing corn hybrids only. Daily BW gain of steers was similar for those grazing Bt and non-Bt corn residues, and the steers exhibited no grazing preference between Bt and non-Bt corn residue. One hundred twenty-eight steer calves were assigned to four silage-based growing diets in a 2 x 2 factorial arrangement: Bt vs non-Bt trait and early- vs late-maturing corn hybrids. The diets contained 90% corn silage and 10% supplement (DM basis). The DMI was higher for steers fed Bt compared with non-Bt hybrids (P < 0.02). An interaction (P < 0.03) was observed for feed efficiency between hybrid genotype and incorporation of the Bt trait. Feed efficiency was greater (P < 0.05) for steers fed the later-maturing non-Bt hybrid compared with the later-maturing Bt hybrid; however, feed efficiency was similar between steers fed early-maturing Bt and non-Bt corn silages. Steers fed the early-maturing hybrid gained 11% faster and were 7% more efficient compared with those fed the late-maturing hybrid. These latter results agree with the dairy experiment in which the early-maturing hybrid resulted in 5% greater efficiency of milk production than the later-maturing corn hybrid. In all experiments, incorporation of the Bt trait into corn had no consistent effect on cattle performance. In addition, background genetics of the corn hybrids appeared to have a more consistent impact on performance than did presence of the Bt trait. Key Words: Beef Cattle, Dairy Cattle, Maize, Maize Byproducts, Maize Silage, Bacillus thuringiensis

Research reveals that glycosylphosphatidylinositol- (GPI-) anchor cleavage by phosphatidylinositol-specific phospholipase of Bacillus thuringiensis is influenced by membrane fluidity and packing and low phase transition temperature as shown by the GPI-anchored coenzyme ecto-5'-nucleotidase reconstituted lipid bilayer vesicles.

Domain III of the Bacillus thuringiensis Cry1Ac toxin maintains the structure of the protein and also participates in the formation of the ion channel inside the midgut of insects. Arginine residues at positions 525, 527, 529, and 531 appear to be important for this purpose.

Researchers describe a vegetative insecticidal protein (VIP) from Bacillus thuringiensis that is toxic to several lepidopteran pests. The amino acid sequence differs slightly from other bacterial VIPs and it may have a different mode of action as well. VIPS are produced during the vegetative growth phase of the bacterium.

The Cry1Ja and Cry1A toxins of Bacillus thuringiensis share binding sites in six moth and butterfly species from different families. This means that the insects might develop cross-resistance to the two toxins.

Residue Asn135 in alpha-helix 4 of the Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins is necessary for pore formation and toxin oligomerization in the gut epithelium of Manduca sexta. However, a mutant protein created by substituting Gln for Asp still binds to the epithelium.

A 20-kDa helper protein in Bacillus thuringiensis enhances the production of Cry1Ac toxin. The P20 protein increases toxin yield more than two-fold, produces larger crystals, and Cry1Ac is more toxic against Heliothis armigera larvae when synthesized in the presence of the helper protein.

Research demonstrates that the binding of Cry3A delta-endotoxin to liposomes does not cause structural changes in domain I as revealed by unchanged denaturation activation energy. Furthermore, removal of 4-terminal alpha-helices and C-terminal 56 amino acids does not interfere in the interaction of the toxin with liposomes.

The diamondback moth has two genes that confer resistance to the Bacillus thuringiensis Cry toxins. One is dominant and one is partially recessive. Cross-resistance among insects from these two lines also differs.

Differences in packaging of the delta-endotoxins of Bacillus thuringiensis may account for the fact that the Cry1Da1 endotoxin is present in much smaller amounts in inclusions than the other endotoxins. The inclusions are ingested by susceptible insect larva and the protoxins are metabolized to toxins.

Strains of the diamondback moth Plutella xylostella have different sensitivities to the Cry toxins of Bacillus thuringiensis depending on what part of the world they come from. Strains from England, Hawaii, and the Netherlands had different sensitivities but in all three, Cry1Ab and Cry1Ac were the most toxic and Cry1Da the least toxic.

Some insecticidal crystal toxins from Bacillus thuringiensis are less effective in inducing pores in membranes at a high pH. This was the conclusion of researchers who tested the Cry1Ac, Cry1C, and Cry1E toxins.

Combining Bacillus sphaericus with the Cyt1Ab1 or Cyt2Ba1 cytotoxins from Bacillus thuringiensis can overcome a mosquito's resistance to B. sphaericus alone. This combination suppressed resistance of Culex quinquefasciatus to B. sphaericus from greater than 17,000- to 2-fold.

There is very little cross-resistance in the diamondback moth to the different insecticidal crystal proteins of Bacillus thuringiensis. Therefore, different Cry proteins could be used in rotation and in different combinations to prevent the moth from becoming resistant to them. The Cry9Ca protein is more potent than some other Cry proteins and may be especially useful.

Researchers show that the P20 protein from Bacillus thuringiensis interacts with the Cry11A protein, making it more toxic to mosquito larvae. This was confirmed in Escherichia coli and Pseudomonas putida that had been genetically engineered to produce both proteins.

Insects possess varying degrees of resistance to the Bacillus thuringiensis Cry1Ab and Cry1Ac toxins. The resistance appears to be mediated by a protease.

Alpha helix 7 of the Cry1Ab delta endotoxin appears to be necessary for its structural stability, its ion channel activity, and its insecticidal activity. The seven alpha helices are part of domain I.

The Cry1Ab, Cry1Ac, and Cry1F endotoxins of Bacillus thuringiensis bind to the brush border of the European corn borer via isoforms of aminopeptidase and cadherin. Commercial corn has been genetically modified with the Cry1Ab endotoxin to control insect pests.

Some insects may be resistant to the Cry1Ac delta-endotoxin of Bacillus thuringiensis because they contain intestinal proteases that completely degrade the toxin. Some degree of proteolysis is necessary to activate the toxin inside the insect, but too much can inactivate the toxin.