球形芽孢杆菌和苏云金芽孢杆菌以色列亚种杀蚊毒素间的协同作用

本研究测定了分别表达苏云金芽孢杆菌Cry4Aa、Cry4Ba、Cry11Aa、Cyt1Aa和球形芽孢杆菌二元毒素Bin的转化菌株Bt B60 1、Bt B611、Bt B640、Bt U 30和Bt CW 3全发酵培养物两两或两两以上不同组合对抗性库蚊的毒力 ,分析了杀蚊毒素间的协同作用。结果表明 ,Bin和Cry4Aa、Bin和Cry 4Ba间有明显的协同作用 ,此外 ,Cry4Aa和Cry4Ba、Cry4Aa和Cry11Aa、Cyt1Aa和Cry4Aa之间也有明显的协同作用     

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)     

苏云金芽孢杆菌以色列亚种cyt1 Aa基因在球形芽孢杆菌中表达

将编码cyt1Aa基因和 p2 0蛋白基因的DNA片段分别克隆连接于两个不同的穿梭载体 pBU 4和pMK 3上 ,构建了重组质粒 pBA 30和 pMA 6,通过电击法 ,将重组质粒分别转化 B .s野生株2 2 97,获得了转化菌株Bs 97 30和Bs 97 6。SDS PAGE和Westernblot分析证实了cyt1Aa基因在转化菌株Bs 97 30中获得了表达 ,而在转化菌株Bs 97 6中未检测到cyt1Aa基因表达的蛋白。转化菌株Bs 97 30中 ,cyt1Aa基因与B .s二元毒素基因同步于菌体生长的对数期起始表达 ,并持续至芽孢形成。生测结果表明 ,转化菌株Bs 97 30中cyt1Aa基因的表达并未明显增强其对敏感和抗性致倦库蚊幼虫的毒力。其原因可能是弱毒性的 cyt1Aa蛋白在转化菌株中的表达量不高。     

Development of mutants of the mosquitocidal bacterium Bacillus thuringiensis subspecies morrisoni (PG-14) toxic to lepidopterous or dipterous insects

The parasporal body of the mosquitocidal isolate (PG-14) of Bacillus thuringiensis subsp. morrisoni (BTM) contains five major proteins with molecular masses of, respectively, 27.3, 65, 128, 135, and 144 kDa. Proteins corresponding in mass to the first four of these also occur in the mosquitocidal strain, B. thuringiensis subsp. israelensis (BTI), and it is thought therefore that the mosquitocidal activity of both strains is due to these four proteins. In other studies it has been shown that each of these proteins exhibits from moderate to high toxicity to mosquitoes, though the specific toxicity of the 144 kDa protein in PG-14 to mosquitoes remains unknown. In the present study, two parasporal body mutants (M146 and M242) of PG-14 were developed growing the wild-type strain at 42 degrees C. The parasporal body of M146 contained less of the 65-kDa protein and was less toxic (LC50 = 108 ng/ml) to mosquitoes than the wild-type strain (LC50 = 8.3 ng/ml). The parasporal body of M242 consisted of a bipyramidal crystal composed of a 144-kDa protein that was not toxic to the mosquito, Aedes aegypti, but exhibited substantial toxicity (LC50 = 2.5 micrograms/ml) to the lepidopteran. Trichoplusia ni. Because the parasporal bodies of BTI and BTM PG-14 are similar in mosquitocidal toxicity on a weight basis, the latter results suggest the 144-kDa protein, though not mosquitocidal alone, can contribute to mosquitocidal, activity when in the presence of other mosquitocidal proteins.     

苏云金杆菌以色列亚种几丁质酶基因的克隆及序列分析

从苏云金芽孢杆菌以色列亚种（Bacillus thuringiensis subsp israelensis)中提取基因组DNA,通过合成1对特异性引物,用touchdown PCR的方法扩增几丁质酶ichi基因序列（GenBank登录号：AF526379）。ichi序列全长为2570bp,含有1个2067bp的开放阅读框（ORF）,编码688个氨基酸,推测分子量为75.79kDa,等电点pI=5.90的几丁质酶前体。序列和结构比较分析表明：Ichi氨基酸序列与蜡状芽孢杆菌（Bacillus cereus)28-9几丁质酶CW、蜡状芽孢杆菌CH几丁质酶B及苏云金芽孢杆菌墨西哥亚种几丁质酶的同源性分别为97.24%、97.18%、97.63%,而与苏云金芽孢杆菌巴基斯坦亚种的同源性只有63.07%。Ichi编码区由分泌信号肽（46AA）、催化区（105AA)、粘蛋白Ⅲ型同源区（74AA）及几丁质结合区（40AA）组成。     

Bacterial (Bacillus thuringiensis subsp. thuringiensis and B. thuringiensis subsp. kurstaki) Entomocidal Preparations Decrease Chlorophyll Accumulation in Larch Needles

Chlorophyll a plus b content and absorption spectra of the homogenates from the cotyledonary leaves of 30-day-old seedlings of two larch species, Larix gmelinii (Rupr.) Rupr. and L. sibirica Ldb. were studied. The seedlings were grown on Perlite containing aqueous solutions of entomocidal biopreparations isolated from Bacillus thuringiensis subsp. thuringiensis (bitoxybacillin) and B. thuringiensis subsp. kurstaki (lepidocide) at various final concentrations (2, 6, and 12 g/l). Changes in the form of chlorophyll absorption spectra induced by biopreparations were established. A marked inhibition of pigment accumulation in the needles dependent on the biopreparation concentration was noted. At a low concentration (2 g/l), the biopreparations virtually did not affect the chlorophyll content; an increase in their concentrations resulted in a decrease in chlorophyll content in leaves by 20% (at 6 g/l) and 40% (at 12 g/l). It is concluded that bitoxybacillin and lepidocide inhibited the chlorophyll accumulation in larch needles to a similar extent.     

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

Aims: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. Methods and Results: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. Conclusions: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. Significance and Impact of the Study: Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.     

Transfer and expression of the cryIVB and cryIVD genes of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus 2297

Abstract The genes encoding the CryIVB and CryIVD crystal polypeptides of B. thuringiensis subsp. israelensis were cloned indepently on a stable shuttle vector, and transfered into B. sphaericus 2297. Recombinant cells expressed the B. thuringiensis toxins during sporulation and were shown to be toxic to Aedes aegypti fourth instar larvae, whereas the parental strain was not.     

Assessment of the toxicity of the cloned 27 kDa toxin of Bacillus thuringiensis var. morrisoni strain PG14

Abstract Expression of the cloned 27 kDa toxin gene from Bacillus thuringiensis strain PG14 in Bacillus subtilis facilitated the evaluation of the in vivo and in vitro toxicities of the pure toxin. This toxin is known to differ from the 27 kDa δ-endotoxin of var. israelensis by a single amino acid. Comparison of toxicity values with data obtained from similarly produced 27 kDa toxin from var. israelensis suggested that the amino acid difference has no detectable effect on the potency of the toxin under the assay conditions employed.     

苏云金芽孢杆菌的一个新亚种(Btsubsp.sinensis)

198 9年自云南昆明市石林的红棕壤中分离到数株苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎ ｓｉｓ,Ｂｔ)菌株[1] ,对其中的一株ＹＫ30 0 4进行了生物学特性、杀虫特性研究及分类鉴定。1　材料与方法1.1　供鉴定的Ｂｔ菌株由云南昆明市石林的红棕壤中分离的苏云金芽孢杆菌ＹＫ30 0 4菌株。1.2　标准Ｂｔ菌株血清型Ｈ1 Ｈ4 1、Ｈ4 4 Ｈ55及Ｈ57 Ｈ69标准Ｂｔ菌株由法国巴斯德研究院ＤｒＬｅｃａｄｅｔ提供 ,其余为本实验室保存。1.3　生物测定用昆虫小菜蛾 (Ｐｌｕｔｅｌｌａｘｙｌｏｓｔｅｌｌａ) 3龄幼虫 ;斜纹夜盗蛾 (Ｐｒ…     

Susceptibility of Field Populations of Anopheles sinensis (Diptera: Culicidae) to Bacillus thuringiensis subsp. israelensis

The susceptibility of Anopheles sinensis to Bacillus thuringiensis subsp. israelensis (Bti) was evaluated through exposure to varying concentrations of Bti. Mosquitoes were collected from five field populations of southern and central China (Zhongshan, Hengxian, Wuchang, Xinzhou, and Ruichang) and compared with a standard laboratory strain. The LC₅₀ values were 146.4, 50.4, 57.9, 50.0, 41.6 and 24.6 ng/mL, respectively. Differences in susceptibility to Bti ranging from 1.7 to 5.9-fold compared with the laboratory strain. The Zhongshan strain had the greatest resistance. At low doses, Bti displayed residual effects. Peak mortality occurred from the first to the fourth day. Exposure to low doses increased the duration of larval development and decreased the duration of pupal development, indicating that low doses of Bti have a sublethal effect on their hosts.     

Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line

A cloned CryIVB toxin was purified from a cured strain of Bacillus thuringiensis (BT) containing the cryIVB gene on the recombinant plasmid Cam135. Solubilized protoxin was treated with Aedes gut extract or trypsin for varying times and tested for toxicity in vitro on three dipteran and one lepidopteran cell line. Treatment with the Aedes extract but not trypsin, produced an active toxin which lysed only Aedes aegypti cells out of those tested. This activation was time-dependent reaching a maximum after 6 h. Both the Aedes extract-treated and trypsin-treated toxin killed A. aegypti larvae, but this toxicity declined rapidly with increasing time of exposure to the proteolytic preparations.     

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.     

A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria

A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus has been constructed. The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter. The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated. Cry4B expression was highest compared to other toxins. Although the level of toxin expression was low compared with E. coli expressing single toxins, the recombinant E. coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae. This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.     

苏云金芽孢杆菌的筛选和初步鉴定

采集四川温江昆虫孳生地的土壤样本94份,利用醋酸钠-抗生素法分离、筛选,共获得苏云金芽孢杆菌9株。镜检可观察到大菱形、小菱形、方形、圆形等四种主要形态的伴胞晶体;采用cryⅠ、cryⅡ、cryⅢ、cryⅤ基因的通用对9株Bt分离菌进行的PCR检测结果表明：9株菌全部含cryⅠ和cryⅤ基因,2株菌含cryⅡ基因,5株菌含cryⅢ基因,而且各菌株均包含2—3个基因型。利用这些菌株对菜青虫进行了室内和室外生物毒力测定,达到了较好的杀虫效果。     

Utilisation of response surface methodology to optimise the culture medium for Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis israelensis (Bti) is a sporulating Gram-positive bacterium that produces protein crystals with insecticide activity against Diptera. The aim of the present work was to optimise the culture medium for this bacterium, based on mathematical and statistical concepts (factorial designs and response surface methodology). The variables studied were carbon and nitrogen source concentrations. The main response analysed was toxicity, evaluated by means of bioassay with Aedes aegypti. The nutrient sources were first selected and then optimised. Ground Bombyx mori pupae, ammonium sulphate and glucose were the most suitable sources of organic nitrogen, inorganic nitrogen and organic carbon, respectively. The toxicity of optimised medium (LC₅₀ = 0.703 ppm, v/v) was higher than that the medium used as reference (LC₅₀ = 3.01 ppm, v/v), which is commonly used in the laboratory culture of Bti. Besides, the optimised medium showed a cost 7.36 times less than that of an alternative medium, based on soybean flour and sugarcane molasses. Factorial design and response surface methodology were effective methods for culture medium optimisation. The results will contribute to the development of local production and utilisation of agroindustrial waste locally.     

Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing toxin genes from Bacillus thuringiensis subsp. israelensis

Genes encoding the mosquito larvicidal toxins Cry4Aa, Cry11Aa, Cyt1Aa and the regulatory P20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under control of two strong promoters P(psbA) and P(A1). The clone pRVE4-ADRC displayed toxicity against fourth-instar larvae of Aedes aegypti, the highest ever achieved in cyanobacteria. It was about 2.5-fold more toxic than the respective clone without cyt1Aa [Wu et al., Appl. Environ. Microbiol. 63 (1997) 4971-4975]. Cyt1Aa synergized the combination of Crys by about five-fold. Consistently, the lethal times exerted by pRVE4-ADRC were also reduced (it killed exposed larvae more quickly). This clone may become a useful biological control agent which reduces the probability of resistance development in the target organisms [Wirth et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10536-10540].