Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins'' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein''s pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.During the last few decades, the insecticidal toxins produced by Bacillus thuringiensis have been used increasingly in the forms of formulated sprays and transgenic plants for the highly focused biological control of insect pests (29). At the same time, the mechanism by which these proteins form pores in the apical membrane of midgut epithelial cells of targeted insects has been studied extensively (7, 29). In the case of the three-domain Cry toxins, specificity is mostly attributable to their capacity to bind to certain proteins located on the surface of the intestinal membrane through specific segments of domains II and III, composed mainly of β sheets (16, 27). On the other hand, membrane insertion and pore formation are thought to occur through elements of domain I, composed of a bundle of six amphipathic α-helices surrounding the highly hydrophobic helix α5 (17, 20).Several lines of evidence indicate that helices α4 and α5 play a particularly important role in these processes (3). Spectroscopic studies with synthetic peptides corresponding to domain I helices revealed that α4 and α5 have the greatest propensity for insertion into artificial membranes, although insertion and pore formation were most efficient when α4 and α5 were connected by a segment corresponding to the α4-α5 loop of the toxin (13, 14). A particularly large number of single-site mutations with altered amino acids from these helices, which lead to a strong reduction in the toxicity and pore-forming ability of the toxin, have been characterized (2, 9, 10, 15, 18, 23, 25, 30, 31, 33). Finally, a site-directed chemical modification study has provided strong evidence that α4 lines the lumens of the pores formed by the toxin (23).Recent studies have established that toxin activity is especially sensitive to modifications not only in the charged residues of α4 (31) but in most of its hydrophilic residues (15). Furthermore, the loss of activity of most of these mutants did not result from an altered selectivity or size of the pores but from a reduced pore-forming capacity of the toxin (15, 31). In order to better understand the role of α4 in the mechanism of pore formation, the present study was carried out with a series of previously characterized Cry1Aa mutants in which most of the residues from this helix were replaced by cysteines (15). By subjecting these mutants to circular dichroism (CD), protease sensitivity, pore formation inhibition, and electrophoretic mobility analyses, our data suggest that the mutations in α4 which alter the pore-forming ability of Cry1Aa do so mainly by preventing the proper oligomerization or membrane insertion of the toxin.     

The effect of oxygen on the sporulation, δ-endotoxin synthesis and toxicity of Bacillus thuringiensis H14

Summary The sporulation and toxicity of Bacillus thuringiensis H14 were studied as a function of aeration. The fed-batch cultures carried out in the similar aeration conditions were followed in four different oxygen transfer rates containing 0, 20, 100 and 250 mmol/l/h. The percentage of total cells which had formed refractile spores in these four oxygen transfer rates were 100, 93, 84 and 48%, respectively. The highest rate of sporulation was observed in the absence of oxygen and the mature spores were the only population present under this condition at the end of culture. Sporulation in a large portion of cells failed under saturated oxygenation and either mature spores or vegetative cells were present at the end of culture. In the intermediate conditions, cells in different physiological states could be observed at the end of culture. It was found that the optimal conditions for spore yield and for δ-endotoxin yield were not the same, even though sporulation and δ-endotoxin formation proceed simultaneously during the fermentation process. The 130-kDa δ-endotoxin seemed to be more sensitive to aeration conditions. The higher toxicity against Culex pipiens was obtained under the saturated condition.     

Specific mutations within the α4–α5 loop of the bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry δ-endotoxins suggests that helices α4 and α5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the α4–α5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-β-d-thiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the α4–α5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.     

Stirred tank culture of Bacillus thuringiensis H-14 for production of the mosquitocidal δ-endotoxin: mathematical modelling and scaling-up studies

The effect of culture conditions on -endotoxin production by strain S128 of Bacillus thuringiensis H-14 (locally isolated in Egypt) was investigated using a 10l working volume fermenter. Fermentation medium formulated from the inexpensive locally available soya (as a nitrogen source) and molasses (as a carbon source) was used. Aeration, agitation, pH and initial concentration of molasses were chosen as experimental factors and their influence on toxin yield was investigated using 2⁴ central composite experimental design. The mathematical model obtained revealed that the optimal batch cultivation conditions with respect to agitation, pH, and initial concentration of molasses were 325 rev min^–1, 7.1 and 2.1% (w/v) respectively. The mathematical model obtained indicated that by increasing the aeration rate over 0.89 v/v per minute the productivity could still be increased. A simulated scaling-up study, in which the Simplex method was applied, is also presented. The results of this investigation could be of great help for large-scale production of a cheap mosquito-larvicide in developing countries where mosquito-borne diseases are still a serious health and economic problem.     

Expression of insecticidal activity in Rhizobium containing the δ-endotoxin gene cloned from Bacillus thuringiensis subsp. tenebrionis

Bacillus thuringiensis subsp. tenebrionis produces a 65 kilodalton polypeptide toxin which is lethal to various coleopteran insect larvae. The gene encoding this toxin was cloned in E. coli in the broad host range vector pKT230 and subsequently transferred to Rhizobium leguminosarum by conjugation. Western blot analysis showed that the toxin gene was expressed in the free living state of Rhizobium producing two major polypeptides of 73 and 68 kilodalton in size. The level of expression of the toxin gene in Rhizobium varied from strain to strain. Cell extracts from toxin-producing rhizobia were toxic to larvae of Gasterophysa viridula. Bioassays also showed that the -endotoxin was toxic to larvae of the clover weevil Sitona lepidus. Furthermore, pea (Pisum sativum) and white clover (Trifolium repens) plants suffered less root and nodule damage by Sitona larvae when they were inoculated with Rhizobium strains containing the toxin gene. This suggests that such rhizobia could be useful in the biological control of this important legume pest.Abbreviations B.t.t. Bacillus thuringiensis subsp. tenebrionis - IPTG isopropyl--D-thiogalactoside     

Structural analysis and molecular dynamics simulations of novel δ-endotoxin Cry1Id from Bacillus thuringiensis to pave the way for development of novel fusion proteins against insect pests of crops

The theoretical three-dimensional structure of a novel δ-endotoxin Cry1Id (81 kDa) belonging to Cry1I class, toxic to many of the lepidopteran pests has been investigated through comparative modeling. Molecular dynamics (MD) simulations was carried out to characterize its structural and dynamical features at 10 ns in explicit solvent using the GROMACS version 4.5.4. Finally the simulated model was validated by the SAVES, WHAT IF, MetaMQAP, ProQ, ModFOLD and MolProbity servers. Despite low sequence identity with its structural homologs, Cry1Id not only resembles the previously reported Cry structures but also shares the common five conserved blocks of amino acid residues. Although the domain II of Cry1Id superpose well with its closest structural homolog Cry8Ea1, variation of amino acids and length in the apical loop2 of domain II was observed. In this work, we have hypothesized that the variations in apical loop2 might be the sole factor for providing variable surface accessibility to Cry1Id protein that could be important in receptor recognition. MD simulation showed the proposed endotoxin retains its stable conformation in aqueous solution. The result from this study is expected to aid in the development hybrid Cry proteins and new potent fusion proteins with novel specificities against different insect pests for improved pest management of crop plants.     

Host specificity of the Bacillus thuringiensis δ-endotoxin toward Lepidopteran species: Spodoptera littoralis Bdv. and Pieris brassicae L

Crystals from several strains of Bacillus thuringiensis among the first 10 serotypes were tested for their effectiveness (in terms of LC₅₀ or LD₅₀) in killing Spodoptera littoralis larvae. The δ-endotoxin obtained from the isolates aizawai 7–29 and entomocidus 601 was found to be the most active against S. littoralis; lethal doses were considerably lower than those obtained with the berliner 1715 strain but were nevertheless 10 times higher than that of this last strain against Pieris brassicae. Interestingly, entomocidus crystals were active toward both Lepidopteran species. Protein fractions with molecular mass ranging from 60 to 70 kDa, obtained by dissolving crystals with gut proteases from the two insect species, proved to be active when delivered by intrahemocoel injection. Based on the use of mild detergents such as Triton N-101, sodium cholate, or both, conditions for stabilizing the activity of the solubilized fractions are reported. Under these conditions only, the molecules of toxin thus obtained were as active against S. littoralis larvae as the native crystals, whatever the route of administration and whatever the enzymes used. The same fractions induced different responses in P. brassicae larvae, which manifested a much higher sensitivity to the proteolysis-activated molecules, particularly after intrahemocoel injection, thus suggesting differences between the two insect species as regards the nature of toxic effects. The results clearly illustrate two different patterns in activity spectrum among Lepidopteran species and they also indicate that structural characteristics of the protoxin are predominant factors in determining host specificity.     

Comparative Study of Bacillus thuringiensis Cry1Aa and Cry1Ac δ-Endotoxin Activation, Inactivation and In Situ Histopathological Effect in Ephestia kuehniella (Lepidoptera: Pyralidae)

A comparative study of different steps in the mode of action of the individual Bacillus thuringiensis kurstaki BNS3 Cry1Aa and Cry1Ac δ-endotoxins on E. kuehniella larvae was performed in order to investigate the origin of the difference in the response of this larvae to each of the latter. Proteolytic activation was shown to be one of the main steps impaired in E. kuehniella tolerance to Cry1Aa. The absence of two proteinase activities as well as an altered activity level observed in the case of Cry1Aa would be the consequence of proteinase-mediated tolerance of E. kuehniella to this toxin. In situ binding and histopathological effect analyses allowed concluding that the binding of the toxin to BBMV receptors is the key step in E. kuehniella tolerance to Cry1Aa toxin. The latter was slightly bound to apical membranes of epithelial cells that remained intact, whereas Cry1Ac was tightly bound to completely damaged cells basal membranes.     

Studies on Bacillus thuringiensis H-14 strains isolated in egypt—IV. characterization of fermentation conditions for δ-endotoxin production

Fermentation media formulated from inexpensive locally available ingredients, namely soya meal, Proflo (a partially defatted cooked cottonseed fiour) and molasses, were assessed for growth, sporulation and -endotoxin production, by a mosquito-toxic strain of Bacillus thuringlensis H-14 locally isolated in Egypt, using an 8 I working volume fermenter. The insecticidal activity was assayed against Culex piplens. Good growth and sporulation were obtained with all the tested medium combinations, but a medium composed of 3% (w/v) soya meal and 1% (w/v) molasses was the best for -endotoxin production. The optimal batch cultivation conditions with respect to aeration (0.37 to 0.62 vol/vol min^–1), agitation (200 rev/min), pH (6.5 to 7.5), temperature (30°C) and initial concentration of carbon (1% w/v) and nitrogen (3% w/v) sources are also presented. This investigation shows that these locally available ingredients could be used for the production of low-priced mosquito larvicide in Egypt and other developing countries where these ingredients are avallable.     

Effect of specific mutations in helix α7 of domain I on the stability and crystallization of Cry3A in Bacillus thuringiensis

Insecticidal crystal (Cry) proteins of Bacillus thuringiensis crystallize after synthesis forming large inclusions that stabilize these toxins in the environment after cell lysis until eaten by an insect. Despite the biological importance of crystallization, little is known about the structural elements of Cry molecules that facilitate this process. We identified subdomains that affect Cry3A structure possibly through improper folding by chimeric-scanning mutagenesis, substituting short peptides of a truncated 70-kDa Cry1C molecule that does not crystallize into Cry3A, a wild-type 70-kDa molecule that crystallizes readily. Cry3A consists of three domains that contain five different blocks of conserved amino acids. Domain substitution and mutagenesis within these blocks suggested that the specific structure of block 2, which spans the junction between domains I and II, was important to the relative stability of Cry3A and subsequent crystallization. Amino acid sequences of particular importance to stability in Cry3A block 2 were identified using three substitution mutants, each spanning about a third of this block. One that consisted of Cry1C helix α7 yielded no detectable protein, whereas the other two produced characteristic Cry3A crystals. Specific mutations in this region showed tyrosine 268 was critical to normal stability of Cry3A and subsequent crystallization in that a mutant, 268L, was less stable than wild-type Cry3A and failed to form a characteristic Cry3A crystal. Circular dichroism analysis showed a decrease in this mutant’s α-helicity, indicating the importance of tyrosine 268 to the specific conformation of helix α7 that facilitates stability and normal crystallization.     

Analyses of α-amylase and α-glucosidase in the malaria vector mosquito,Anopheles gambiae,as receptors of Cry11Ba toxin of Bacillus thuringiensis subsp. jegathesan

Bacillus thuringiensis subsp. jegathesan produces Cry11Ba crystal protein with high toxicity to mosquito larvae. The Cry11Ba toxicity is dependent on its receptors on mosquito larval midgut epithelial cells. Previously, a cadherin-like protein (AgCad2), aminopeptidase (AgAPN2) and alkaline phosphatase (AgALP1) were reported to be involved in regulation of Cry11Ba toxicity on Anopheles gambiae larvae. Here, the cDNAs encoding α-amylase (AgAmy1) and α-glucosidase (Agm3) were cloned from A. gambiae larva midgut. Both are glycophosphatidylinositol (GPI) anchored proteins on brush border membranes (BBMV). Immunohistochemistry revealed their localization on different regions of the larval midgut. AgAmy1 and Agm3 bound Cry11Ba with high affinity, 37.6 nM and 21.1 nM respectively. Cry11Ba toxicity against A. gambiae larvae was neutralized by both AgAmy1 and Agm3. The results provide evidence that both AgAmy1 and Agm3 function as receptors of Cry11Ba in A. gambiae.     

Importance of spores,crystals, and δ-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae

Physical methods were used to produce spores containing impurities of 0.02–0.05% crystals and crystals containing impurities of 0.001–0.01% spores from cultures of Bacillus thuringiensis. In Galleria mellonella larvae, these preparations from varieties galleriae, aizawai, and wuhanensis were only moderately active compared to 1:1 mixtures of spores and crystals. Spores of an acrystalliferous aizawai mutant were inactive and did not contain a polypeptide of the same size as the potent M_r 138000 δ-endotoxin present in spores and crystals of all three wild-type strains. Thus, this polypeptide probably contributed to the moderate activity of wild-type spores. Spore impurities in the crystal preparation were killed by γ irradiation without harming the crystals. The crystals without live spores were virtually inactive (LC₅₀s, ca. 10¹⁰ crystals/g insect food). Addition of 10³ spores to 10⁸ crystals/g food (0.001% spores) increased the mortality of larvae from 0 to 36%, and addition of 10⁴ spores (0.01% spores) killed 64% larvae. Thus, the addition of low levels of spores increased the potency of crystals in G. mellonella from virtually zero to moderate levels, suggesting that the live spore impurities in the crystal preparations were responsible for the observed moderate potency of crystals before γ irradiation, a view supported by a reduction of potency of crystal preparations following admixture of streptomycin to the insect food. In contrast to the results with G. mellonella, crystals were ca. 30 times as active as spores in Pieris brassicae larvae. Many authors have found crystals purified by physical methods to be highly active in a range of lepidopterous hosts. The present work indicates that the role of the spore impurities in these species may need further investigation. Absence of live spores of B. thuringiensis may impair the control of some insect species feeding on spore-free products and on microorganisms or plants into which endotoxins have been introduced by genetic manipulation.     

Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis

The calcium-binding residues, Tyr302 and His235, and the sodium-binding residue, Asp194, on the activity of Bacillus licheniformis α-amylase were investigated using site-directed mutagenesis. Tyr302 and His235 were replaced by Asn and Asp, respectively, to produce the mutants Y302N and H235D; Asp194 was replaced by Ala to produce D194A. The mutant amylases were purified to homogeneity; each was ~53?kDa. The specific activity of the D194A was 236?U?mg(-1), lower than the specific activity of the wild-type enzyme by 55%. No significant changes of thermostability, optimum temperature, and optimum pH level were observed in D194A. Mutant amylases with H235D and Y302N significantly improved their specific activity by 43% (754?U?mg(-1)) and 7% (563?U?mg(-1)), respectively, compared with the wild-type enzyme. H235D substitution decreased its optimum pH by approx. 0.5-1 pH unit.     

Effective control of yellow stem borer and rice leaf folder in transgenic rice indica varieties Basmati 370 and M 7 using the novel δ-endotoxin cry2A Bacillus thuringiensis gene

Transgenic rice indica varieties Basmati 370 and M 7 expressing the novel cry2A (Bt) insecticidal gene were generated by particle bombardment. Molecular and biochemical analyses in R0 and R1 populations confirmed stable integration and expression of this novel Bt transgene. We estimated that the gene product was expressed up to 5% of total leaf protein. Insect feeding bioassays demonstrated that the Cry2A protein was effective against the yellow stem borer and the rice leaf folder, two major rice pests in the Indian Subcontinent. This is the first report of the control of major rice pests using this specific Bt gene. The cry2A gene can now be used in combination with other insecticidal genes for pyramiding resistance against insect pests. This will delay, or perhaps in combination with integrated pest management practices, prevent evolution of insect populations resistant to single insecticidal genes.     

Effects of Bacillus thuringiensis δ-endotoxins Cry1Ab and Cry1Ac on the coccinellid beetle,Cheilomenes sexmaculatus (Coleoptera,Coccinellidae) under direct and indirect exposure conditions

Genes encoding cry1Ab and cry1Ac δ-endotoxins from the bacterium, Bacillus thuringiensis (Bt) that have been incorporated in several crops to enhance their resistance to insect pests may possibly influence the activity and abundance of natural enemies of insect pests. The ladybird beetle, Cheilomenes sexmaculatus (L.) might ingest Bt toxins expressed by genetically modified plants by feeding on aphids, early instar larvae of lepidopterans, and other soft bodied insects feeding on transgenic plants. Therefore, we studied the effects of Cry1Ab and Cry1Ac Bt toxins on C. sexmaculatus under direct and indirect exposure conditions. For direct exposure, the neonate C. sexmaculatus larvae were fed either pure 2M sucrose (control) or sucrose solution containing Cry1Ab or Cry1Ac (0.1%), and on alternate days with aphids till pupation. Direct exposure of C. sexmaculatus larvae to Bt toxins resulted in reduced larval survival and adult emergence as compared to the controls, which might be due to long-term direct exposure. However, there were no adverse effects of the Bt toxins on C. sexmaculatus when the larvae were reared on Aphis craccivora Koch fed on different concentrations of Cry1Ab or Cry1Ac in the artificial diet. A significant and positive correlation was observed between the presence of Bt toxins in aphids, and coccinellid larvae and adults (r=0.53** to 0.86**). The results suggested that a direct exposure to Bt toxins expressed in transgenic plants or predation on H. armigera on Bt-transgenic plants will have little effect on the activity and abundance of the ladybird, C. sexmaculatus.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

Single point mutations of aromatic residues in transmembrane helices 5 and -6 differentially affect TRPV4 activation by 4α-PDD and hypotonicity: Implications for the role of the pore region in regulating TRPV4 activity

The importance of the TRPV4 channel for human physiology has been highlighted in recent years with the identification of an increasing number of hereditary diseases associated with mutations of this channel. However, the functional understanding of TRPV4 associated pathologies remains a puzzle due to incomplete understanding of the polymodal regulation of TRPV4 channels and lack of insight into the structure–function relationship of the channel. In this work, we identified a series of highly conserved aromatic residues in transmembrane (TM) helices 5–6 with profound importance for TRPV4 activity. Substituting F617, Y621 or F624 in TM5 with leucine reduced channel sensitivity to the agonist 4α-PDD and heat, yet two of these mutants – F617L and Y621L – showed increased activation in response to cell swelling. In TM6, a Y702L mutation significantly reduced sensitivity to all of the above stimuli. In conclusion, we have identified residues in TM5-6 which differentially affect heat and agonist activation, and we have demonstrated distinct activation pathways for 4α-PDD and osmolarity.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.     

Interactions between the tobacco budworm,Heliothis virescens,and the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki: Relationship between length of exposure to the toxin and survival

Larvae of the tobacco budworm, Heliothis virescens, which were fed ad libitum for 24, 48, and 72 hr on a diet treated with various levels of the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki and then transferred to an untreated diet, showed an unexpected capacity to recover from the effects of the toxin, although, as the length of exposure increased, the capacity decreased. Observations on larvae held to emergence indicated that recovery from the toxin was complete. X-ray studies using Ba²⁺ incorporated into the diet showed that, although the toxin paralyzed the midgut of the treated animals, many animals recovered after the toxin was removed, with food once again passing through the gut.