An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.     

Streptomyces sp. 173, an insecticidal micro-organism from marine

AIMS: To find new insecticidal antibiotics from marine micro-organisms. METHODS AND RESULTS: Strains isolated from seawater and sea sediments from Beidiahe and Dagang of the east coast of China were screened for their insecticidal qualities. The screening was carried out using bioassay of brine shrimp and the insect pest Helicoverpa armigera. The fermentation, preliminary extraction and isolation of Streptomyces sp.173 were carried out. CONCLUSIONS: In total 331 isolates were examined through bioassay of brine shrimp and 40 isolates (12.08%) showed potential insecticidal activities. Of the 40 isolates, one isolate, designated Streptomyces sp.173, was found to have strong insecticidal activity against both brine shrimp and H. armigera, similar to that of avermectin B1. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated Streptomyces sp.173 has great insecticidal potency. This work indicated that marine micro-organisms could be an important source of insecticidal antibiotics and the improved anti-brine shrimp bioassay is suitable for primary screening.     

类产碱假单胞菌杀虫蛋白的N端鉴定及抗体制备

The insecticidal protein from Pseudomonas pseudoaligenes was and exotoxin which had toxicity on locusts.In order to elucidate its molecular properties an amino acid sequence,the insecticidal protein was purified from the culture supernatant by ultrafiltration,ion\|exchange chromatography and gel filtration,and showed a single band on SDS\|PAGE.Analysis of the purified insecticidal protein dentified N\|terminal sequence of ten amino acid residues.Its polyclonal antibody was also obtained by immunizing rabb…     

Characteristics of insecticidal substances from the entomopathogenic fungus Metarhizium pinghaense 15R against cotton aphid in Korea

Aphids (Hemiptera: Aphididae) are agricultural pests for a wide range of economically important crops worldwide. Entomopathogenic fungi have been studied and developed as a biological agents to control hard to control insect pests like the aphid. In particular, secondary metabolites produced by entomopathogenic fungi are being studied as insecticidal substances. This study evaluated the insecticidal activity and characteristics of insecticidal substances in culture filtrate against aphids to confirm the insecticidal substances produced by Metarhizium pinghaense 15R. After treating cotton aphids with the culture filtrate, strong insecticidal activity (100 % insect mortality) was observed on the third day of treatment, and it was confirmed that insecticidal substances in the culture filtrate had high thermal stability. In addition, the physicochemical properties of insecticidal substances in the culture filtrate were evaluated, and it was determined that the insecticidal substances were hydrophobic, with small molecules of 3 kDa or less. Based on these results, it was suspected that the insecticidal substances in the culture filtrate were destruxins (DTXs), which are representative secondary metabolites of Metarhizium known to have insecticidal activity. Through HPLC (High Performance Liquid Chromatography) analysis, the presence of DTX A and B was found in all active samples, and it was confirmed that the insecticidal substances of M. pinghaense 15R were DTXs. This study provides primary data for developing biochemical crop protection agents using these insecticidal substances, and we suggest the potential of using insecticidal substances of entomopathogenic fungi as biochemical pesticides in Korea.     

Cloning, functional characterization, and mode of action of a novel insecticidal pore-forming toxin, sphaericolysin, produced by Bacillus sphaericus

An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.     

苏云金杆菌杀虫晶体蛋白活性预测的支持向量机模型

藉均匀设计(UD)方法,构建了苏云金杆菌(Bt)杀虫晶体蛋白氨基酸组成特征与其杀虫活性之间关系的支持向量机(SVM)模型。当惩罚系数为0·01、epsilon值为0·2、gamma值为0·05、域值为0·5时,该模型对Bt杀虫晶体蛋白杀虫活性的预测平均准确率达73%。     

对二点委夜蛾高毒力苏云金芽胞杆菌的筛选及其基因型分析

【目的】获得对二点委夜蛾Athetis lepigone(M(o|¨)schler)高毒力的苏云金芽胞杆菌(Bt)菌株,寻找对该虫具有特异杀虫活性的蛋白毒素,探索Bt菌株或其杀虫基因应用于二点委夜蛾防治的可行性。【方法】通过生物测定方法比较了36株苏云金芽胞杆菌和一株恶臭假单胞工程菌PHB-cry1Ab对二点委夜蛾幼虫的杀虫活性,同时利用PCR-RFLP方法对这些菌株的基因型进行了分析。【结果】不同Bt菌株对二点委夜蛾幼虫的杀虫活性差别很大,杀虫活性高的菌株都含有cry1Ac基因。饲毒72 h后含单基因的BtHD-73菌株(cry1Ac)对二点委夜蛾2龄幼虫的毒力(LC_(50)值为188.51μg/g)明显高于含多基因的Bt SC-40菌株(cry1Ac,cry2Ac,cry1I,vip3A)的毒力(LC_(50)值为418.13μg/g)。含有vip3A基因的Bt SC-40和BtHD-13营养期上清液对二点委夜蛾2龄幼虫表现出一定的杀虫活性(72 h死亡率分别达到42.5%和57.4%),而无vip3A基因的Bt HD-73营养期上清液未表现出明显的杀虫活性。【结论】由cry1Ac基因编码的Cry1Ac蛋白对二点委夜蛾幼虫具有特异杀虫活性,Vip3A蛋白对二点委夜蛾幼虫可能也有一定的杀虫活性。     

Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes

Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.     

脱氧鬼臼毒素对粘虫几种代谢酶系的影响

脱氧鬼臼毒素是砂地柏Sabina vulgaris Ant.中的主要杀虫活性成分之一。为探讨其作用机理,以叶碟饲喂法处理粘虫Mythimna separata Walker 4龄幼虫,测定了饲喂处理12h、24 h、36 h、48 h和72 h后试虫的羧酸酯酶（CarE）、酸性磷酸酯酶(ACP)、碱性磷酸酯酶(AKP)、谷胱甘肽S-转移酶（GSTs）和多功能氧化酶(MFO)的活性。结果表明：脱氧鬼臼毒素对粘虫羧酸酯酶的活性无明显影响;对酸性磷酸酯酶具有明显的抑制作用,且随着处理时间的延长,抑制作用增强;对碱性磷酸酯酶的影响较为复杂,表现为先抑制,后有所恢复,再被抑制的变化过程;对谷胱甘肽S-转移酶处理12 h后活性变化不明显,24 h后被抑制,36 h后逐渐激活,与对照差异极显著;对细胞色素P450酶系的O-脱甲基酶活性具有明显的抑制作用。     

苏云金芽孢杆菌鲇泽变种7—29杀虫蛋白质结构基因的...

The regulative region (181bp) and the fifth toxic active domain (217bp) were removed from the insecticidal protein gene of Bacillus thuringiensis var. aizawai 7-29. After the synthesis of the adaptor (15bp) that contains initiation codon (ATG) and the PCR synthesis of the fifth toxic active domain (229bp) that contains stop codon (TAA), were inserted into on 5' truncated and 3' truncated of the coding fod N-terminal peptid's DNA fragment, that to become a modified structural gene. The modified structural gene can be play initiatic translation-function and stop translation-function during translation of insecticidal protein. The insecticidal protein was determined by western blotting, showed the expression of modified structural gene in Escherichia coli JM 103. The bioassay of insecticidal proteins showed the 3' truncated and 5' truncated of insecticidal gene was higher toxic active than the 3' truncated of insecticidal gene in Escherichia coli JM 103.     

Synthesis of Novel (9S)‐Acyloxy Derivatives of Quinidine and Dihydroquinidine as Insecticidal Agents

Endeavor to discover biorational natural products‐based insecticides, two series (30) of novel (9S)‐acyloxy derivatives of quinidine and dihydroquinidine were prepared and assessed for their insecticidal activity against Mythimna separata in vivo by the leaf‐dipping method at 1 mg/mL. Among all the compounds, especially four derivatives exhibited the best insecticidal activity with final mortality rates of 71.4 %, 75.0 %, 71.4 %, and 75.0 %, respectively. Relatively speaking, 9‐hydroxy group is well tolerated, and the results showed that after modification of the hydroxy group with an acyloxy group, the insecticidal activity was significantly increased; the configuration at C8/9 position is important for insecticidal activity, and the (9S)‐configuration is optimal; modification of the out‐ring double bond is acceptable, and hydrogenation of the double bond enhances insecticidal activity. These preliminary results will pave the way for further modification of quinidine in the development of potential new insecticides.     

Cloning, Functional Characterization, and Mode of Action of a Novel Insecticidal Pore-Forming Toxin, Sphaericolysin, Produced by Bacillus sphaericus

An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.     

Cloning, Expression, and Purification of Insecticidal Protein Pr596 from Locust Pathogen Serratia marcescens HR-3

A novel insecticidal protein (Pr596) produced by Serratia marcescens HR-3 was found be a metalloprotease and responsible for insecticidal activity toward locusts. Two pairs of primers were designed to amplify Pr596, a putative open reading frame (ORF) by similarity search and the N-terminal amino-acid sequence of insecticidal protein. The results revealed that the ORF consisted of 1464 nucleotides encoding a protein of 487 amino-acid residues. Pr596 was cloned into expression vector pET32a(+) and was expressed in Escherichia coli BL21 (DE3)/pLysS strain with isopropyl-β-d-thiogalactopyranoside induction. The Pr596 was found to be highly expressed as inclusion bodies by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Pr596 inclusion bodies were isolated and subjected to Ni-NTA His Bind Resins (Pharmacia, Germany). Pr596 purified and refolded was revealed by SDS-PAGE and had proteolytic activity and insecticidal activity. Results suggested that there is a potential to develop this protein to be used as an alternative locus control agent.     

A Cry1Ac Toxin Variant Generated by Directed Evolution has Enhanced Toxicity against Lepidopteran Insects

Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16–β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.     

Structure-function relationships affecting the insecticidal and miticidal activity of sugar esters

Synthetic sugar esters are a relatively new class of insecticidal compounds that are produced by reacting sugars with fatty acids. The objective of this research was to determine how systematic alterations in sugar or fatty acid components of sugar ester compounds influenced their insecticidal properties. Sucrose octanoate, sorbitol octanoate, sorbitol decanoate, sorbitol caproate, xylitol octanoate, xylitol decanoate and xylitol dodecanoate were synthesized and evaluated against a range of arthropod pests. Dosage-mortality studies were conducted on pear psylla (Cacopsylla pyricola Foerster) on pear, tobacco aphid (Myzus nicotianae) Blackman and tobacco hornworm (Manduca sexta [Johannson]) on tobacco, and twospotted spider mite (Tetranychus urticae Koch) on apple in laboratory bioassays. These sugar esters were compared with insecticidal soap (M-Pede, Dow AgroSciences L.L.C., San Diego, CA), to determine how toxicologically similar these materials were against the arthropod pests. Substitutions in either the sugar or fatty acid component led to significant changes in the physical properties and insecticidal activity of these compounds. The sugar esters varied in their solubility in water and in emulsion stability, yet, droplet spread upon pear leaves occurred at low concentrations of 80-160 ppm and was strongly correlated with psylla mortalities (R2 = 0.73). Sequentially altering the sugar or fatty acid components from lower to higher numbers of carbon chains, or whether the sugar was a monosaccharide or disaccharide did not follow a predictable relationship to insecticidal activity. Intuitively, changing the hydrophile from sorbitol (C6) to xylitol (C5) would require a decrease in lipophile chain length to maintain hydrophilic-lipophilic balance (HLB) relationships, yet an increase in lipophile chain length was unexpectedly needed for increasing insecticidal activity. Thus, the HLB of these materials did not correlate with pear psylla mortalities. Initial insect bioassays and dosage-mortality data found significant differences among sugar ester compounds' toxicity to the range of arthropod species. Sucrose octanoate high in monoester content had the highest activity against the range of arthropod pests at low concentrations of 1200-2400 ppm. No single chemical structure for the xylitol or sorbitol esters were optimally effective against the range of arthropods we tested and sorbitol octanoate and xylitol decanoate had the highest insecticidal activity of this group. All of the sugar ester materials produced high T. urticae mortalities on apple at very low concentrations of 400 ppm. Overall, most of the sugar esters that were examined had superior insecticidal activity compared with insecticidal soap. Sugar ester chemistry offers a unique opportunity to design an insecticide or miticide specific to certain arthropod pests which would be valuable in crop integrated pest management (IPM) programs. Sucrose esters are currently used as additives in the food industry which makes them especially attractive as safe and effective insecticides.     

Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles.

A rapid analysis of Bacillus thuringiensis strains predictive of insecticidal activity was established by using polymerase chain reaction (PCR) technology. Primers specific to regions of high homology within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each insecticidal class. Predictions of insecticidal activity were made on the basis of the electrophoretic patterns of the PCR products. Included in the screen were PCR primers specific for cryI, cryIII, and cryIV genes, which are insecticidal for lepidopterans, coleopterans, and dipterans, respectively. Known B. thuringiensis strains as well as unidentified strains isolated from soil and insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a single PCR reaction containing 12 to 20 primers capable of distinguishing between the different insecticidal genes. Insecticidal activity predicted by the PCR screen was found to correspond with the insecticidal activity of insect bioassays. In addition to identifying strains with known insecticidal genes, the PCR screen can identify strains with altered electrophoretic patterns containing potentially novel genes.     

苏云金芽胞杆菌营养期杀虫蛋白基因特性分析

营养期杀虫蛋白是在苏云金芽胞杆菌营养期中发现的一种非晶体状胞外杀虫蛋白 .通过Southern杂交的基因定位实验 ,证实了营养期杀虫蛋白基因并非位于染色体和环型质粒上 ;在 2 0个不同血清型的 2 3种菌株中 ,营养期杀虫蛋白基因的存在率为 5 6 5 % (13 2 3) .同时对随机选择的 3个不同亚种菌株进行基因克隆和序列分析 ,表明其基因具有相当高的同源性 .运用生物信息技术 ,对营养期杀虫蛋白多肽进行了功能结构域比对分析 ,发现这 789多肽蛋白的N端 31至 15 0残基之间存在一种趋化信号传导因子相似序列 ,C端 5 36至 6 6 6残基之间则存在着纤维素结合结构域相似肽 .上述结果提示 ,营养期杀虫蛋白基因在苏云金芽胞杆菌天然菌株中高度保守并较为广泛分布 ,其编码蛋白的特殊结构提示了营养期杀虫蛋白潜在的功能特性 ,为进一步研究营养期杀虫蛋白奠定了基础 .     

昆虫病原线虫共生菌BP品系杀虫毒素基因簇中各基因与杀虫活性的关系

昆虫病原线虫共生菌Xenorhabdus nematophilus BP的多个杀虫毒素基因集中在一起形成一个约40kb的基因簇。为研究这个基因簇中各基因与杀虫活性的关系,对该共生菌粘粒文库中5个粘粒克隆XnBP76、XnBP83、XnBP203、XnBPp378 和XnBP414及XnBP83的3个亚克隆插入DNA片段的基因结构和它们对棉铃虫的杀虫活性进行了比较,结果显示,xptB1, xptC1和xptA2 3个基因或后两者的联合表达产物具有最强的杀虫效果,缺失其中的任何1个或2个会使杀虫活力大幅度地下降或完全消失;而xptD1和xptA1的缺失对毒素基因簇的表达产物的杀虫活力影响很小;杀虫毒素的物理混合没有明显的增效作用。     

Efficacy and nontarget effects of reduced-risk insecticides on Aphis glycines (Hemiptera: Aphididae) and its biological control agent Harmonia axyridis (Coleoptera: Coccinellidae)

The efficacy of three reduced-risk insecticides (pyrethrins, insecticidal soap, and narrow-range mineral oil) was determined for nymphs and adults of the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), an exotic pest of North American soybean, Glycine max (L.) Merr. These insecticides also were evaluated for nontarget effects on one of the aphid's key biological control agents, multicolored Asian lady beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), including first and third instars, pupae, and adults. A Potter Spray Tower was used to conduct direct spray laboratory bioassays. Results indicated that although pyrethrins and narrow-range mineral oil caused 100% mortality to A. glycines nymphs and adults at 72 h posttreatment, insecticidal soap caused equivalent mortality to only the nymphs during the same time period. However, A. glycines adult mortality due to the insecticidal soap (83.3%) was significantly greater than the control. Pyrethrins were highly toxic to first instars of H. axyridis (98% mortality), but they had no effect on third instars, pupae, or adults. Mineral oil and insecticidal soap were moderately lethal to first (48.9 and 40% mortality, respectively) and third (31.9 and 38.8% mortality, respectively) instars of H. axyridis, but they had no effect on pupae and adults. Our results suggest that pyrethrins, insecticidal soap, and narrow-range mineral oil may prove useful for soybean aphid management in organic soybean due to efficacy against the aphid with differential nontarget effects on select stages of H. axyridis. Additional studies will be necessary to elucidate the efficacy of these insecticides under field conditions.     

新型杀虫蛋白VIPs研究进展

简述了芽孢杆菌属 (Bacillus)中的新型活性杀虫蛋白 营养期杀虫蛋白VIPs及其编码基因的发现、特性、作用模式及机制等研究进展 ,并对其杀虫活性和安全因素作了初步评估和预示。