Sip1Ab gene from a native Bacillus thuringiensis strain QZL38 and its insecticidal activity against Colaphellus bowringi Baly

In this study, we collected 540 soil samples from northeast China and isolated the wild-type strain of Bacillus thuringiensis (Bt) by identifying and cloning 9 Bt strains that expressed the secreted insecticidal protein (Sip) gene. We selected the strain QZL38 for further study. The sip gene was identified from the Bt strain QZL38 using polymerase chain reaction (PCR). We sequenced a 1095-base pair fragment of DNA that encodes 364 amino acid residues of a 41.18?kDa pro-toxin and compared it with the registered Sip1Ab protein amino acid residue sequence. The sequence was submitted to GenBank with the accession no. KP231523, and the gene was named sip1Ab. The Sip1Ab protein expressed in Escherichia coli showed insecticidal activity against Colaphellus bowringi Baly, with an LC50 of 1.051?μg?mL^?1. To identify the active fragment of the Sip1Ab toxin, four pairs of primers with different truncation positions were designed, and the recombinant proteins were expressed in E. coli. The truncated Sip protein expressed in E. coli showed insecticidal activity against C. bowringi Baly. The insecticidal activity of the recombinant proteins against C. bowringi Baly from the Sip1Ab signal peptide after removal of 30 amino acid residues showed an LC50 of 1.078?μg?mL^?1. Sip proteins may play an important role in the prevention and control of the C. bowringi Baly.     

An engineered Bacillus thuringiensis strain with insecticidal activity against Scarabaeidae (Anomala corpulenta) and Chrysomelidae (Leptinotarsa decemlineata and Colaphellus bowringi)

A genetically-engineered Bacillus thuringiensis (Bt) strain, 3A-HBF, with a broad insecticidal spectrum was constructed by introducing the recombinant plasmid pSTK-3A containing cry3Aa7 into the wild-type Bt strain HBF-1 containing the cry8Ca2 gene. The Cry3Aa7 protein produced by strain 3A-HBF was verified by SDS-PAGE and Western blotting. Flat rectangular crystals of Cry3Aa7 protein were observed besides spherical crystals (Cry8Ca2). The plasmid pSTK-3A was stable when strain 3A-HBF was grown in medium without antibiotics. The growth rate of 3A-HBF was not significantly different from that of the recipient strain, HBF-1. Strain 3A-HBF showed toxicity against two families of pests, Scarabaeidae and Chrysomelidae pests, which are susceptible to Cry8Ca (Anomala corpulenta) and Cry3Aa (Leptinotarsa decemlineata and Colaphellus bowringi). The 50% lethal concentrations of 3A-HBF against A. corpulenta, L. decemlineata and C. bowringi were 0.730 × 10⁸ c.f.u./g dry soil, 1.74 μg/ml and 1.15 μg/ml, respectively.     

Ser-Substituted Mutations of Cys Residues in Bacillus thuringiensis Vip3Aa7 Exert a Negative Effect on its Insecticidal Activity

Vegetative insecticidal proteins (VIPs), which were produced by Bacillus thuringiensis during its vegetative growth stage, display a broad insecticidal spectrum to Lepidoptera larvae. Sequence alignment of the Vip3A-type indicates that three cysteine residues were conserved in Vip3A-type proteins. To determine whether these conserved cysteine residues contributed to the insecticidal activity, the three residues were respectively substituted with serine in the Vip3Aa7 protein by site-directed mutagenesis. Bioassays using the third instar larvae of Plutella xylostella showed that the toxicity of C401S and C507S mutants were completely abolished. To find out the inactivity reason of mutants, three mutants and the wild-type Vip3Aa7 were treated with trypsin. The results indicated that the C507S mutant was rapidly cleaved and resulted in decrease of the 62?kDa toxic core fragment. These results indicated that the replacement of the Cys⁵⁰⁷ with a Ser⁵⁰⁷ caused decrease in C507S resistance against trypsin degradation. It is suggesting a possible association between insecticidal activity and trypsin sensitivity of Vip3A proteins. This study serves a guideline for the study of Vip3A protein structure and active mechanism.     

Single concentration tests show synergism among Bacillus thuringiensis subsp. israelensis toxins against the malaria vector mosquito Anophelesalbimanus

Bioassays of insecticidal proteins from Bacillus thuringiensis subsp. israelensis with larvae of the malaria vector mosquito Anophelesalbimanus showed that the cytolytic protein Cyt1Aa was not toxic alone, but it increased the toxicity of the crystalline proteins Cry4Ba and Cry11Aa. Synergism also occurred between Cry4Ba and Cry11Aa toxins. Whereas many previous analyses of synergism have been based on a series of toxin concentrations leading to comparisons between expected and observed values for the concentration killing 50% of insects tested (LC₅₀), we describe and apply a method here that enables testing for synergism based on single concentrations of toxins.     

The influence of female age on male mating preference and reproductive success in cabbage beetle,Colaphellus bowringi

The influence of female age on male mating preference and reproductive success has been studied using a promiscuous cabbage beetle, Colaphellus bowringi Baly （Coleoptera： Chrysomelidae）. In a simultaneous choice test, middle-aged females had significantly greater mating success than young and old females. In single pair trials, when paired with middle-aged virgin males, middle-aged females mated faster, copulated longer, and had greater fecundity and fertility than young or old females, while the longevity of males was not significantly affected by female age. This study on C. bowringi suggests that middle-aged females are more receptive to mating, which can result in the highest male reproductive success.     

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism

Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop β6-αE K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.     

Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution

Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of ⁴³⁹QAAG⁴⁴² or ⁴⁴³AVYT⁴⁴⁶ of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors.     

Synergism and Antagonism between Bacillus thuringiensis Vip3A and Cry1 Proteins in Heliothis virescens,Diatraea saccharalis and Spodoptera frugiperda

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC₅₀, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC₉₀, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC₉₀ level than at the LC₅₀ level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.     

Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC₅₀ of Lip and HD1 were 33.27 and 128.61 μg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.     

Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests

The insecticidal activities and specificities of the Vip3Aa proteins derived from different Bt strains are very different, although the similarities between these proteins are higher than 95%. In this study, we hypothesised that the differences in Vip3Aa11 and Vip3Aa39 C-terminal amino acids determine their differences in insecticidal activity against three Lepidoptera insects. To find the amino acid residues associated with insecticidal activity, nine different amino acid residues of Vip3Aa11 were substituted with the corresponding amino acid residues from Vip3Aa39 by site-directed mutagenesis. The toxicity of each protein was estimated by bioassays, and the results demonstrated that the mutant Y784N lost its insecticidal activity against three insects (Agrotis ipsilon, Helicoverpa armigera, and Spodoptera exigua). The insecticidal activity of S543N, I544L, and S686R against S. exigua increased 5-fold, 2.65-fold, and 8.98-fold, while the toxicity to H. armigera and A. ipsilon slightly decreased compared with that of the Vip3Aa11 protein. These findings indicate that the amino acid residues Ser⁵⁴³, Ile⁵⁴⁴, Thr⁶⁸⁵, Ser⁶⁸⁶, Arg⁷⁰⁴, Ile⁷⁸⁰, and Tyr⁷⁸⁴ may be insecticidal activity-related residues. Additionally, the trypsin activation of the four mutants indicated that all proteins can form a 62-kDa core fragment, except Y784N. A possible association between the insecticidal activity and trypsin sensitivity of Vip3A proteins is suggested.     

Insecticidal activity of spore-free mutants of Bacillus thuringiensis against the Indian meal moth and almond moth

Three oligosporogenic mutants of Bacillus thuringiensis were assayed for toxicity against larvae of the Indian meal moth, Plodia interpunctella, and the almond moth, Ephestia cautella. The results were compared with insecticidal activity obtained from the parent strain (HD-1) and two standard B. thuringiensis formulations (HD-1-S-1971 and HD-1-S-1980) against the same insect species. The toxicity of the sporeless mutant preparations was significantly diminished against the Indian meal moth (10- to 26-fold increase in LC₅₀) but exceeded the toxicity of the standards against the almond moth. The toxicities of the B. thuringiensis preparations toward the Indian meal moth were consistent with the number of spores in the test samples, but spores did not contribute to toxicity to E. cautella larvae. A rationale for basing dosage on soluble protein was demonstrated for use in situations where spores are not a contributing factor in toxicity.     

Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests

A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding a coleopteran-specific insecticidal protein was constructed and introduced into wild Bacillus thuringiensis subsp. aizawai G03, which contained cry1Aa, cry1Ac, cry1Ca, and cry2Ab genes and was highly toxic to lepidopteran insect pests. The genetically engineered strain were named G033A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry3Aa7 gene was expressed normally and produced a 67 kDa protein in G033A, and the flat rectangular crystals of Cry3Aa7 toxin protein was observed under scanning electron microscope. The recombinant plasmid was maintained in bacteria cultured for 180 generations in culture media containing no antibiotics. Synthesis of the Cry3Aa7 toxin conferred high and broad toxicity to the recombinant strain G033A against coleopteran order, elm leaf beetle (Pyrrhalta aenescens) (LC₅₀ 0.35 mg/ml), for which the parental strain G03 was not toxic. Both the parental strain G03 and recombinant strain G033A showed strong insecticidal activity to lepidopteran pests, beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera), respectively. The lethal concentration 50% (LC₅₀) of G033A against S. exigua, P. xylostella, and H. amigera was 4.26, 0.86, and 1.76 μg/ml, respectively.     

Isolation,cloning, and overexpression of vip3Aa gene isolated from a local Bacillus thuringiensis

Vegetative insecticidal protein (Vip) is a newly discovered family of toxin protein isolated from Bacillus thuringiensis (Bt). An 88.5-kDa Vip3Aa protein was secreted by a local strain of the bacterium during the vegetative growth phase. The full length of the coding region ‘2.3 kbp’ of the vip3Aa gene was isolated from plasmid DNA, cloned in pGEM-T vector and finally cloned in pQE-30 expression vector. Nucleotide sequence revealed 98% homology with that of the previously isolated genes. Expression of the vip3Aa in Escherichia coli was carried out and the expressed protein was detected in the concentrated supernatant, not in the pellet. This indicated that vip3Aa is secreted into the culture medium. Expressed protein was purified, blotted, and assayed against the cotton leaf worm Spodoptera littoralis. The LC₅₀ was found to be 142.4 µ/mL while the LC₅₀ was 90 ppm for the wild strain. These results suggest the use of either the isolated Bt strains or the expressed vip3Aa in an integrated pest management program against lepidopteran insect pests.     

Sip1, a Conserved AP-1 Accessory Protein,Is Important for Golgi/Endosome Trafficking in Fission Yeast

We had previously identified the mutant allele of apm1⁺ that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4⁺/sip1⁺, which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1 -deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl⁻ and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking.     

Discovery and characterization of Sip1A: a novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae

Bioassay screening of Bacillus thuringiensis culture supernatants identified strain EG2158 as having larvicidal activity against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Ion-exchange fractionation of the EG2158 culture supernatant resulted in the identification of a protein designated Sip1A (secreted insecticidal protein) of approximately 38 kDa having activity against Colorado potato beetle (CPB). An oligonucleotide probe based on the N-terminal sequence of the purified Sip1A protein was used to isolate the sip1A gene. The sequence of the Sip1A protein, as deduced from the sequence of the cloned sip1A gene, contained 367 residues (41,492 Da). Recombinant B. thuringiensis and Escherichia coli harboring cloned sip1A produced Sip1A protein which had insecticidal activity against larvae of CPB, southern corn rootworm (Diabrotica undecimpunctata howardi), and western corn rootworm (Diabrotica virgifera virgifera).     

Sequence Analysis of Insecticidal Genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC₅₀] of 2 to 6 μg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P_L promoter were shown to have insecticidal activity (LC₅₀ of 112 μg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

Oligomerization of Cry11Aa from Bacillus thuringiensis Has an Important Role in Toxicity against Aedes aegypti

Cry11Aa and Cyt1Aa of Bacillus thuringiensis are active against mosquitoes and show synergism. Cyt1Aa functions as a membrane receptor inducing Cry11Aa oligomerization. Here we characterized Cry11Aa helix α-3 mutants impaired in oligomerization and toxicity against Aedes aegypti, indicating that oligomerization of Cry11Aa is important for toxin action. Cyt1Aa did not recover the insecticidal activity of Cry11Aa mutants.Bacillus thuringiensis subsp. israelensis has been used worldwide for the control of different mosquitoes that are vectors of several human diseases (10, 11). This bacterium produces different toxins that individually show activity against mosquitoes, i.e., Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa (2). The toxicity of Cry11Aa and Cry4 toxins against Aedes aegypti is greatly increased in the presence of sublethal concentrations of Cyt1Aa (14). Also, Cyt1Aa overcomes the resistance of the Culex quinquefasciatus population to Cry11Aa (12, 13). Cyt1Aa synergizes the toxic activity of Cry11Aa by functioning as a Cry11Aa receptor, facilitating the oligomerization of Cry11Aa and its pore formation activity (7, 8). Oligomerization is a complex event that involves interaction with a toxin receptor and further proteolysis of helix α-1 (3). In the case of the Cry1Ab toxin, helix α-3 of domain I contains coiled-coil structures that are important for oligomerization (4). Some point mutations in helix α-3 do not affect interaction with receptors but severely affected oligomerization, influencing pore formation and toxicity against Manduca sexta larvae (4).Since binding with Cyt1Aa facilitates Cry11Aa oligomerization, we hypothesize that Cry11Aa mutants unable to oligomerize would be affected in synergism with Cyt1Aa and in toxicity. In this report, we analyzed the effect of point mutations in helix α-3 of Cry11Aa on oligomerization, synergism with Cyt1Aa, and toxicity against A. aegypti larvae.Helix α-3 of Cry11Aa potentially forms coiled-coil structures, as determined by the program COILS, which calculates the probability that a sequence will adopt a coiled-coil conformation (6). The coiled-coil structures are characterized by heptads of residues (abcdefg), where positions a and d are occupied mostly by apolar residues and g and e by charged residues. Here we mutagenized some residues located at positions g and a of the predicted coiled-coil (Fig. ​(Fig.1).1). Substitutions R90E, E97A, Y98E, V104E, and S105E were produced by site-directed mutagenesis (Quick Change; Stratagene, La Jolla, CA) using the pCG6 plasmid (1) as a template and appropriate mutagenic oligonucleotides. Point mutations were verified by automated DNA sequencing at Instituto de Biotecnología-UNAM and transformed into the acrystalliferous B. thuringiensis 407 strain. B. thuringiensis strains were grown in solid nutrient broth sporulation medium supplemented with 10 μg/ml erythromycin (5). Crystal inclusions were purified as described previously (8) and solubilized in 100 mM NaOH for 1 h at 4°C. After solubilization, the Cry11Aa protoxins were dialyzed for 12 h against 50 mM Na₂CO₃, pH 10.5. The pH was equilibrated at pH 8.6 with equal volumes of 1 M Tris-HCl, pH 8, and protoxins were activated with trypsin (1:50, wt/wt) for 2 h at 25°C. All mutants, with the exception of the V104E mutant, which was not analyzed further, produced crystal inclusions similar to those for the wild-type toxin, composed of a 70-kDa protoxin (Fig. ​(Fig.2A).2A). After trypsin activation, all mutants produced two polypeptides of 32 and 36 kDa, similarly to the Cry11Aa toxin, suggesting that these mutations did not cause a major structural disturbance (Fig. ​(Fig.2B).2B). The Cry11Aa and mutant activated toxins were analyzed by circular dichroism spectroscopy (Fig. ​(Fig.2C).2C). The activated toxins were dialyzed against 10 mM Na₂HPO₄, 50 mM NaF, pH 9, and then purified by anion-exchange chromatography with HiTrap Q-Sepharose (Pharmacia LKB Biotechnology) in the same buffer, using a linear NaF gradient from 50 to 400 mM. The similarities among the curves indicate that the mutant toxins have a structure similar to that of the wild-type toxin.Open in a separate windowFIG. 1.Schematic representation of the coiled-coil structures of the α-3 helices of Cry1Ab and Cry11Aa toxins. The positions of residues a, b, c, d, e, f, and g of the heptads are presented. The mutated residues in both toxins that affected oligomerization and toxicity are shown in boldface type (reference 4 and this work).Open in a separate windowFIG. 2.SDS-PAGE analysis and circular dichroism spectra of Cry11Aa mutant toxins. (A) The Cry11Aa protoxins were solubilized at pH 10.5 and analyzed by SDS-PAGE (15% acrylamide). (B) SDS-PAGE analysis (15% acrylamide) of the activated toxins with trypsin. Both SDS-polyacrylamide gels were stained with Coomassie blue. Lanes 1, Cry11Aa; lanes 2, E97A mutant; lanes 3, Y98E mutant; lanes 4, R90E mutant; lanes 5, S105E mutant. (C) Analysis of the secondary-structure compositions of the mutants and Cry11Aa activated toxins. Circular dichroism spectra were recorded with a Jasco model J-715 spectropolarimeter equipped with a Peltier temperature control supplied by Jasco. Spectra were collected from 190 to 250 nm. Eight replicate spectra were collected for each sample to improve the signal-to-noise ratios. The final purified-protein concentration was 0.3 mg/ml, and spectra were collected in a 0.1-cm-pathlength cell. The secondary-structure prediction was performed using the CDSSTR algorithm (1a, 11a). Solid black line, Cry11Aa; dotted black line, E97A mutant; dashed black line, Y98E mutant; solid gray line, R90E mutant; dotted gray line, S105E mutant; MRE, mean residue ellipticity; [θ], ellipticity.The toxicity of spore/crystal suspensions of Cry11Aa or the individual mutants (75 to 10,000 ng/ml) was analyzed with bioassays against 10 fourth-instar A. aegypti larvae reared at 28°C, 87% humidity, and 12:12 light-dark conditions in 100 ml dechlorinated water, and mortality was scored after 24 h (four independent assays). The Cry11Aa toxin showed a mean lethal concentration of 355 ng/ml, with 95% confidence limits of 265 to 446 (Probit analysis using Polo-PC LeOra Software). In contrast, the R90E, E97A, Y98E, and S105E mutants were severely affected in toxicity against A. aegypti larvae, since no mortality was observed at the highest concentration used (10,000 ng/ml).We then analyzed the oligomerization of Cry11Aa toxins as previously described (8). Small unilamelar vesicles (SUV), composed of egg yolk phosphatidyl choline, cholesterol (Avanti Polar Lipids, Alabaster, AL), and stearylamine (Sigma, St. Louis, MO) at a 10:3:1 proportion, respectively, were used (8). Cyt1Aa was purified from the 4Q7/pWF45 strain (14) grown as described above. Cyt1Aa inclusions were purified by sucrose gradients, solubilized in 50 mM Na₂CO₃, 10 mM dithiothreitol, pH 10.5 (2 h at 30°C), and activated with 1:100 proteinase K (Sigma-Aldrich Co.), wt/wt, for 20 min at 30°C.For oligomerization assays, 2.5 μg soluble Cry11Aa or mutant protoxin was incubated for 2 h at 37°C in a 100-μl final volume of 50 mM Na₂CO₃, pH 10.5, with 200 μM SUV, 1:50 trypsin (wt/wt), and 0.5 μg Cyt1Aa activated toxin. After 2 h of incubation, 1 mM phenylmethylsulfonyl fluoride was added to stop the reaction, and the membrane fraction was separated by centrifugation (1 h at 100,000 × g). The pellet was suspended in the same buffer solution. Oligomeric structures of Cry toxins are highly stable after boiling as well as after urea denaturation (9). The suspension was boiled for 4 min, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8% acrylamide), and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The oligomeric and monomeric structures of Cry11Aa were detected using polyclonal anti-Cry11Aa antibody (1/15,000; 1 h) and a secondary antibody coupled with horseradish peroxidase (Sigma, St. Louis, MO) (1/5,000; 1 h) followed by luminol (ECL; Amersham Pharmacia Biotech) as described by the manufacturers. Figure ​Figure33 shows that only the Cry11Aa wild-type toxin was able to oligomerize, while the mutants were severely impaired in oligomerization.Open in a separate windowFIG. 3.Analysis of Cry11Aa oligomer formation. Soluble Cry11Aa protoxin was activated with trypsin for 2 h at 37°C in the presence of SUV and Cyt1Aa activated toxin. The membrane fraction was separated by ultracentrifugation, and the Cry11Aa protein was analyzed by Western blotting of the membrane pellet with polyclonal anti-Cry11A antibody. The sizes of the proteins were estimated from a molecular prestained plus standard, all blue (Bio-Rad). Lane 1, Cry11Aa; lane 2, R90E mutant; lane 3, Y98E mutant; lane 4, E97A mutant; lane 5, S105E mutant.Finally, the synergistic activity between Cyt1Aa and Cry11Aa was analyzed. A concentration of Cyt1Aa that produced 10% mortality was assayed in the presence of a protein concentration of wild-type Cry11A that produced 20% mortality. Larvae were examined 24 h after treatment, in three repetitions. This particular protein mixture produced a synergism factor of 8. Under these conditions, mortality was more than 80%, due to the synergistic activities of both toxins. Similar experiments were performed with the mutant toxins, using the same concentration of Cyt1Aa toxin and different concentrations (up to 6,000 ng/ml) of the mutant toxins. Cyt1A did not increase the toxicity of the Cry11Aa mutants, since only 10% mortality was observed, even at the highest concentration of the mutant toxins.Previously, helix α-3 of a lepidopteran-specific toxin (Cry1Ab) was subjected to mutagenesis. The R99E and Y107E mutants of the Cry1Ab toxin were severely impaired in oligomerization and toxicity, showing that oligomer formation is a necessary step to kill the larvae (4). The data presented here indicate that oligomer formation is also an essential step in the mechanism of toxicity of the mosquitocidal Cry11Aa toxin and that helix α-3 is involved in this process.     

大猿叶虫四地理种群遗传多样性的RAPD分析

以大猿叶虫Colaphellus bowringi Baly 4个地理种群的基因组DNA为材料, 进行RAPD分析。从80条引物中筛选出11条稳定性好、多态性高的引物进行扩增, 共得到65个扩增位点, 53个多态位点, Nei氏遗传多样性指数为0.1049~0.2061, Shannon多样性指数为0.1641~0.3167。结果表明所分析的大猿叶虫遗传变异很高, 其中江西龙南种群遗传变异最小, 山东泰安种群遗传变异最高。种群间的遗传距离范围为0.0636~0.3200, 其中江西龙南种群和江西修水种群间的遗传距离最小, 哈尔滨种群与江西龙南种群间的遗传距离最大, 种群遗传距离的大小与其相对地理距离的远近吻合。结果提示种群遗传距离的大小与它们生物学上的相似性有关联。     

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369–375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.