Insecticidal activity of Bacillus laterosporus

Strains of Bacillus laterosporus demonstrated pathogenic activity for second-instar larvae of the mosquito, Culex quinquefasciatus, but failed to demonstrate detectable pathogenicity against larvae of the cabbage looper, Trichoplusia ni. Of 29 strains of the bacterium screened, 16 displayed pathogenicity for mosquito larvae. One of the most pathogenic strains, NRS 590, also demonstrated pathogenic activity for larvae of the mosquito, Aedes aegypti, and for larvae of the black fly, Simulium vittatum. The pathogenicity for Culex larvae was associated with the cell mass rather than with the culture supernatant. A suspension of ultraviolet irradiation-killed cells demonstrated no loss in pathogenic activity, an indication that the pathogenicity is toxin mediated. The toxic substance produced by NRS 590 was found to be resistant to heating at 96°C for 10 min. The toxin was not associated with the heat-resistant, bacterial endospore or with the associated paraspore since a suspension consisting primarily of spores was not toxic to mosquito larvae. Toxic activity in stationary phase cells of NRS 590 was associated with the cell's particulate fraction rather than with the soluble fraction.     

Mosquitocidal activity of the CryIC delta-endotoxin from Bacillus thuringiensis subsp. aizawai.

The cloned 135-kDa CryIC delta-endotoxin from Bacillus thuringiensis is a lepidopteran-active toxin, displaying high activity in vivo against Spodoptera litoralis and Spodoptera frugiperda larvae and in vitro against the S. frugiperda Sf9 cell line. Here, we report that the CryIC delta-endotoxin cloned from B. thuringienesis subsp. aizawai HD-229 and expressed in an acrystalliferous B. thuringiensis strain is also toxic to Aedes aegypti, Anophles gambiae, and Culex quinquefasciatus mosquito larvae. Furthermore, when solubilized and proteolytically activated by insect gut extracts, CryIC is cytotoxic to cell lines derived from the first two of these dipteran insects. This activity was not observed for two other lepidopteran-active delta-endotoxins, CryIA(a) and CryIA(c). However, in contrast to the case with a lepidopteran and dipteran delta-endotoxin cloned from B. thuringiensis subsp. aizawai IC1 (M.Z. Haider, B. H. Knowles, and D. J. Ellar, Eur. J. Biochem. 156:531-540, 1986), no differences in the in vitro specificity or processing of CryIC were found when it was activated by lepidopteran or dipteran gut extract. The recombinant CryIC delta-endotoxin expressed in Escherichia coli was also toxic to A. aegypti larvae. By contrast, a second cryIC gene cloned from B. thuringiensis subsp. aizawai 7.29 (V. Sanchis, D. Lereclus, G. Menou, J. Chaufaux, S. Guo, and M. M. Lecadet, Mol. Microbiol. 3:229-238, 1989) was nontoxic. DNA sequencing showed that the two genes were identical. However, CryIC from B. thuringiensis subsp. aizawai 7.29 had been cloned with a truncated C terminus, and when it was compared with the full-length CryIC delta-endotoxin, it was found to be insoluble under alkaline reducing conditions. These results show that CryIC from B. thuringiensis subsp. aizawai is a dually active delta-endotoxin.     

Parasporal bodies of Bacillus laterosporus sporangia.

Intact colonies of Bacillus laterosporus examined by thin-section transmission electron microscopy revealed sporangia in various stages of development and degeneration as the endospores matured. The sporangia formed a surface layer of hexagonally arranged subunits. The variety of parasporal bodies raised questions of developmental and ecologic utility.     

Insecticidal Activity of Bacillus laterosporus

The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The larvicidal activity of B. laterosporus was associated with spores and crystalline inclusions. Purified B. laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.     

高效杀蚊苏云金芽孢杆菌BRC-LLP29的发酵优化

苏云金芽孢杆菌BRC-LLP29为新型高效杀蚊菌株,应用快速有效的数学统计方法对其杀蚊毒力的发酵培养进行优化.通过单因素筛选确定最佳碳源为葡萄糖、麦芽糖、可溶性淀粉,氮源为typetone、大豆蛋白胨、干酪素;最佳金属离子为Mg²⁺、Al³⁺.采用二水平Placlkett-Burman设计对影响毒力的8因素进行显著性筛选,获得培养基成分中3个重要影响因子:葡萄糖、干酪素和Al₂（SO₄）₃;运用爬坡路径法对这3种因子进行试验,获得3种重要因子的最适浓度范围;通过响应面分析法得到3个重要因子的交互作用和最佳条件,确定BRC-LLP29菌株最佳毒力水平的发酵培养基为:葡萄糖19.8g/L、干酪素28.4g/L、Al₂（SO₄）₃1.2g/L、MgSO₄ 2g/L、K₂HPO₄ 3g/L、CaCO₃ 0.5g/L,优化后毒力水平达到致死率61.11%,与响应面数学模型的预测值只有5.91%的误差.发酵条件优化结果表明:发酵温度为31°,发酵初始pH为7.0,摇瓶装量为40mL/250mL三角瓶,每瓶的接种量为3.5%,发酵72h,对致倦库蚊最终致死率达到最高为83.33%.     

侧孢短芽胞杆菌产生线虫侵染性蛋白酶的优化

采用单因素试验确定侧孢短芽胞杆菌G4产线虫侵染性蛋白酶的最佳碳氮源,通过Placket-Burman设计筛选影响蛋白酶活力的主效因子,最陡坡试验和Box-Behnken设计获得主效因子的最佳水平,建立线虫侵染性蛋白酶的最佳生产体系:葡萄糖9.78 g/L、牛肉膏16.65 g/L、磷酸氢二钾0.75 g/L、可溶性淀粉12.5 g/L、氯化钠0.75 g/L、硫酸镁0.5 g/L、初始pH值自然、装液量50 mL,37℃摇瓶培养32 h,蛋白酶活力可达12 379.41 U/mL,较优化前的2 476.3 U/mL提高了4倍。     

Conjugal Transfer of a Toxin-Coding Megaplasmid from Bacillus thuringiensis subsp. israelensis to Mosquitocidal Strains of Bacillus sphaericus

Both Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis produce mosquitocidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on a large plasmid, pBtoxis. In an attempt to combine the best properties of the two bacteria, an erythromycin resistance-marked pBtoxis plasmid was transferred to B. sphaericus by a mating technique. The resulting transconjugant bacteria were significantly more toxic to Aedes aegypti mosquitoes and were able to overcome resistance to B. sphaericus in a resistant colony of Culex quinquefasciatus, apparently due to the production of Cry11A but not Cry4A or Cry4B. The stability of the plasmid in the B. sphaericus host was moderate during vegetative growth, but segregational instability was observed, which led to substantial rates of plasmid loss during sporulation.     

Mosquitocidal activity of penfluridol as juvenile hormone antagonist

Juvenile hormone antagonists (JHANs) are known to interfere with the formation of juvenile hormone (JH) receptor complex. JHANs might be effective for control of target pests in larval stages at which stages high level of endogenous JH titer is maintained. In order to identify novel insecticidal compounds, 2352 compounds were surveyed on their JHAN activities using the yeast-two hybrid system. Among 53 compounds with JHAN activities, penfluridol showed high level of insecticidal activity against larvae of Aedes albopictus. JHAN activity was increased in proportion to the concentration of penfluridol. Larvicidal activity of penfluridol was 1.3–2.0 folds higher than that of pyriproxyfen. These results suggested that penfluridol could be useful for control of mosquito larvae.     

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. The mode of action of the enzyme.

The acid-soluble products of exhaustive digestion of native DNA with Bacillus laterosporus DNase consist of 6.5% of mononucleotides and 93.5% of oligonucleotides with an average chain length of 3.2. The results of viscometric studies and inactivation of transforming DNA indicate the existence of acid-insoluble intermediates and the selective degradation of the population of substrate molecules rather than a random nucleolytic action. Furthermore, sucrose density gradient analysis of partially digested DNA showed that the initial DNA added as a substrate disappeared progressively during the reaction, being replaced by much more slowly sedimenting acid-insoluble materials, which were eventually degraded into acid-soluble end products during the reaction; products intermediate in size between these two components were not detectable. Studies with DNA labeled at the 3'-terminus indicate that Bacillus laterosporus DNase does not attack DNA from 3'-hydroxyl ends to yeild acid-soluble or acid-insoluble materials in a random manner. The results presented in this paper indicate that the nature of the attack of B. laterosporus nuclease is similar to that previously proposed for Micrococcus luteus DNase. The possibility of the sequential release of acid-insoluble intermediate fragments as well as acid-soluble products from the terminal portion of DNA by the enzyme is discussed.     

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme.

A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.     

侧孢芽孢杆菌质粒提取方法的探究

以能够降解有机磷农药的两株侧孢芽孢杆菌BL-21和BL-22为研究对象,分别采用碱裂解法、试剂盒提取法和SDS法对侧孢芽孢杆菌BL-21和BL-22的质粒进行提取,并通过凝胶电泳和紫外分光光度法对提取结果进行分析,试验结果证明,适合侧孢芽孢杆菌BL-21和BL-22的质粒提取方法是SDS裂解法,该方法提取的质粒大小为10kb,且该方法提取的结果稳定,质粒的产量和质量均符合分子生物学实验的要求。     

Characteristics of extracellular proteases produced by Bacillus laterosporus and Flavobacterium sp. isolated from gelatinfactory effluents

Forty bacterial isolates from the effluents of a gelatin factory (Jabalpur, India) were screened for protease activity and the two most potent producers were identified as Bacillus laterosporus and a Flavobacterium sp. The enzymes of both isolates were optimal at pH 8 and 60°C, with maximum activity after 90 min. The enzyme activity of B. laterosporus was suppressed by Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ ions but was enhanced by Ba²⁺ and Ca²⁺. That of Flavobacterium sp. was suppressed by Mg²⁺ and Mn²⁺ ions but enhanced by Ba²⁺, Ca²⁺ and Fe²⁺. The enzyme activity of the former was strongly inhibited by KCN, whereas that of the latter was only slightly inhibited by 8-hydroxyquinoline.     

Two pH optima of adenosine 5'-triphosphate dependent deoxyribonuclease from Bacillus laterosporus

The various catalytic activities of the ATP-dependent deoxyribonuclease (DNase) of Bacillus laterosporus have pH optima at 6.3 and 8.3. Although the pH profile of ATP-dependent DNase activity on duplex DNA is bell shaped with a maximum at about pH 8.3, ATP-dependent DNAse activity on single-stranded DNA has optima at pH 6.3 and 8.3. ATPase activities dependent on double-stranded and single-stranded DNA have a high bell-shaped peak with a maximum at pH 6.3 with a low and broad shoulder at about pH 8.3. ATP-independent DNase activity also has optima at pH 6.3 and 8.3. The ratio of the amount of ATP hydrolyzed per number of cleaved phosphodiester bonds in DNA increases with decrease in the pH value of the reaction. The ratios obtained at pH 8.3 and 6.3 were respectively about 3 and 22 with duplex DNA as substrate and 5 and 17 with single-stranded DNA as substrate. Formation of a single-stranded region of 15000-20000 nucleotides, which is linked to duplex DNA and about half of which has 3'-hydroxyl termini, was observed at about pH 6.3, but not at above pH 7.5. Furthermore, the optimum concentrations of divalent cations for the activity producing the single-stranded region and the activity hydrolyzing ATP were identical (3 mM Mn2+ or 5 mM Mg2+). Thus the two activities are closely related. These results indicate that the enzyme has two different modes of action on duplex DNA which are modulated by the pH.     

An adenosine triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Improved purification, subunit structure and substrate specificity

The ATP-dependent deoxyribonuclease from Bacillus laterosporus has been purified to near homogeneity by a procedure involving ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex A-25 chromatography and DNA-cellulose affinity chromatography. The purified enzyme has a molecular weight of 210,000 +/- 8,000 as determined by sucrose gradient sedimentation. It is composed of two nonidentical polypeptide chains with close molecular weights of around 110,000. The substrate preference of the pure enzyme is essentially identical with the previous result obtained with the partially purified enzyme preparation (Anai, M., Mihara, T., Yamanaka, M., Shibata, T., & Takagi, Y. (1975) J. Biochem. 78, 105-114). Thus, the enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of ATP. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of ATP. Furthermore, no endonuclease activity is observed on covalently closed circular duplex DNA and open circular duplex DNA.     

Morphological and Chemical Studies of the Spores and Parasporal Bodies of Bacillus laterosporus

Spores of Bacillus laterosporus were studied to determine the chemical and morphological nature of their basophilic canoe-shaped parasporal bodies. An unusually high phosphorus content of these spores compared to other Bacillus species appeared to be associated with the parasporal body. Preparations of these "canoes" still attached to the spore coats were indeed high in phosphorus, but also in nitrogen. They were free of lipide-soluble and nucleic acid phosphorus and stained for protein. Some 50 per cent of the total nitrogen, but only 6 to 10 per cent of the total P were liberated by extraction with alkali-thioglycollate (pH 11.5) or alkali alone (pH 12.2–12.5). Proteinaceous material was recovered from these alkaline extracts and electron microscopy indicated that there had been a marked loss of "canoe" substance. Extraction with acid, removed some 80 per cent of the phosphorus associated with the "canoes" as orthophosphate. Chromatographic analyses for amino acids indicated some 14 ninhydrin-positive spots in the canoe-coat preparations whereas the whole spores contained at least 16.     

Morphological and chemical studies of the spores and parasporal bodies of Bacillus laterosporus

Spores of Bacillus laterosporus were studied to determine the chemical and morphological nature of their basophilic canoe-shaped parasporal bodies. An unusually high phosphorus content of these spores compared to other Bacillus species appeared to be associated with the parasporal body. Preparations of these "canoes" still attached to the spore coats were indeed high in phosphorus, but also in nitrogen. They were free of lipide-soluble and nucleic acid phosphorus and stained for protein. Some 50 per cent of the total nitrogen, but only 6 to 10 per cent of the total P were liberated by extraction with alkali-thioglycollate (pH 11.5) or alkali alone (pH 12.2-12.5). Proteinaceous material was recovered from these alkaline extracts and electron microscopy indicated that there had been a marked loss of "canoe" substance. Extraction with acid, removed some 80 per cent of the phosphorus associated with the "canoes" as orthophosphate. Chromatographic analyses for amino acids indicated some 14 ninhydrin-positive spots in the canoe-coat preparations whereas the whole spores contained at least 16.     

几株侧孢芽孢杆菌解磷能力的研究

菌株BL-11、BL-12、BL-2l、BL-22是自行分离得到的4株具有解磷能力的细菌,经鉴定为侧孢芽孢杆菌(Bacillus laterosporus)。实验分别以Ca3(PO4)2、氧化乐果和水胺硫磷为唯一磷源,接种4株菌,在30℃、180r／min条件培养4d后,以钼蓝比色法测上清液中的水溶性磷含量。在以Ca3(PO4)2为唯一磷源的培养液体中菌株BL-11、BL-12的解磷能力分别为10．9l％和7．34％,均高于菌株BL-21、BL-22;而在以水胺硫磷为唯一磷源的培养液中,菌株BL-21、BL-22的解磷能力显著好于菌株BL-11、BL-12,解磷效率分别为58．98％和75．50％;而在以氧化乐果为唯一磷源的培养液中,菌株BL-21、BL-22的解磷效率分别为32．66％和29．10％,均高于菌株BL-11、BL-12。     

Mosquitocidal activity of anthraquinones isolated from symbiotic bacteria Photorhabdus of entomopathogenic nematode

Two anthraquinones were isolated from the symbiotic bacteria Photorhabdus temperata of entomopathogenic nematodes Heterorhabditis spp. by repeated column chromatography. They were abundantly present in the culture medium and identified as 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone and 3-methoxychrysazine by spectral analysis. The isolated anthraquinones were highly lethal to larvae of Culex pipiens pallens. Our results suggest that anthraquinones might be useful as biopesticides for the biological control of mosquitoes.     

侧孢芽孢杆菌Bl13对番茄早疫病防治效果及机制

以对番茄早疫病原菌有良好拮抗效果的侧孢芽孢杆菌Bl13为研究对象,采用盆栽试验,通过测定番茄株高、茎粗、番茄早疫病病情指数、叶片内防御酶活性以及根区土壤微生物多样性、微生物群落结构组成等指标,探究侧孢芽孢杆菌Bl13防治番茄早疫病的效果及机制。结果表明: 接种Bl13可显著降低番茄早疫病的病情指数,提高叶片内多酚氧化酶(PPO)、过氧化物酶(POD)等防御酶活性,降低病害对植物地上部分及根系生长发育的影响。同时,改善番茄根区土壤微生物群落结构,使芽孢杆菌属、假单胞菌属等常见有益菌属相对丰度显著提高,油壶菌属、血赤壳属相对丰度显著降低。侧孢芽孢杆菌Bl13可通过提高番茄叶片内防御酶活性并增加根区中有益微生物的数量来增强植物对番茄早疫病的抗性,从而实现对番茄早疫病的防治。     

Permeabilization of Model Lipid Membranes by Bacillus sphaericus Mosquitocidal Binary Toxin and its Individual Components

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of ≈100–200 pS with long open times and a high open probability. Larger channels (≥400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (≤100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity. Received: 29 March 2001/Revised: 20 July 2001