Spontaneous occurrence of heat-stable somatic antigen variants in Bacillus thuringiensis

Heat-stable somatic antigen (HSSA) variants were isolated from Bacillus thuringiensis strain T84A1-A by antiserum-mediated selection. Substantial differences in HSSAs were clearly shown between variants and the parent strain by HSSA agglutination tests, Ouchterlony tests and precipitin-halo-formation (PHF) tests of extracellular HSSAs, although a 'one way'cross-reaction was evident. The frequency of HSSA variants in the population was < 10^-4, as determined by PHF tests on antiserum-agar plates. Heat-stable somatic antigen variants showed no alteration in flagellar antigenic structure and in 30 phenotypic characteristics. The insecticidal activity of HSSA variants was on the level with that of the parent strain, when tested with larvae of three lepidopterous species.     

Isolation of a non-insecticidal Bacillus thuringiensis strain belonging to serotype H8a8b

Bacillus thuringiensis strains non-toxic to Lepidoptera, Bombyx mori and Diptera, Culex pipiens pallens larvae were isolated from Korean soil samples during an investigation of B. thuringiensis isolates highly toxic to insect pests. One of these isolates, NTB-88, produces parasporal inclusions about 138 kDa in size and is non-toxic to 19 insect species of three orders, Lepidoptera, Diptera and Coleoptera, even though it is highly susceptible to tryptic cleavage. Study of flagellar (H) antibodies of 33 B. thuringiensis strains revealed that NTB-88 has an H antigen identical with that of subsp. morrisoni (serotype 8a8b). Comparison of parasporal inclusion proteins and plasmid DNA patterns of strain NTB-88 with B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. morrisoni PG-14 showed that the isolate is a novel non-insecticidal B. thuringiensis strain belonging to serotype 8a8b.     

苏芸金杆菌血清型I中存在鞭毛抗原亚因子

The strain G was isolated from a dead army worm (Leucania separata Walker) in corn field, at Hebei. It has the typical morphological characteristics of Bacillus thuringiensis. The serological analysis showed that the flagellar suspensions of the strain G was agglutinated only by the antisera against the H1 antigen and not by the antisera against the other 22 known H-antigens. The agglutination titre was 1:6,400. The flagellar suspension of the serotype 1, subsp. thuringiensis, was agglutinated by the antiserum against the strain G, with a titre of 1:12,800. However, the cross-saturation of antisera showed that there were different fractions in the H1 antigen. The H antiserum against subsp. thuringiensis still agglutinated with subsp. thuringiensis, having a titre of 1:6,400, even if it was fully saturated by strain G. When it was saturated by subsp. thuringiensis the H antiserum of strain G did not agglutinate by himself. Therefore, the H1 antigen (subsp. thuringiensis) must be divided into subfactors H1alb and strain G antigen is H1a. On the basis of this study, we propose the strain G represents the serotype H1a under the name of Bacillus thuringiensis subsp. hebeiensis.     

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.     

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.     

The solubility of inclusion proteins from Bacillus thuringiensis is dependent upon protoxin composition and is a factor in toxicity to insects.

Bacillus thuringiensis subsp. aizawai HD133 is one of several strains particularly effective against Plodia interpunctella selected for resistance to B. thuringiensis subsp. kurstaki HD1 (Dipel). B. thuringiensis subsp. aizawai HD133 produces inclusions containing three protoxins, CryIA(b), CryIC, and CryID, and the CryIC protoxin has been shown to be active on resistant P. interpunctella as well as on Spodoptera larvae. The CryIA(b) protoxin is very similar to the major one in B. thuringiensis subsp. kurstaki HD1, and as expected, this protoxin was inactive on resistant P. interpunctella. A derivative of B. thuringiensis subsp. aizawai HD133 which had been cured of a 68-kb plasmid containing the cryIA(b) gene produced inclusions comprising only the CryIC and CryID protoxins. Surprisingly, these inclusions were much less toxic for resistant P. interpunctella and two other Lepidoptera than those produced by the parental strain, whereas the soluble protoxins from these strains were equally effective. In contrast, inclusions from the two strains were about as active as soluble protoxins for Spodoptera frugiperda larvae, so toxicity differences between inclusions may be due to the solubilizing conditions within particular larval guts. Consistent with this hypothesis, it was found that a higher pH was required to solubilize protoxins from inclusions from the plasmid-cured strain than from B. thuringiensis subsp. aizawai HD133, a difference which is probably attributable to the absence of the CryIA(b) protoxin in the former. The interactions of structurally related protoxins within an inclusion are probably important for solubility and are thus another factor in the effectiveness of B. thuringiensis isolates for particular insect larvae.     

Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography.

An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.     

球形芽孢杆菌C3—41二元毒素基因在苏云金芽孢杆菌以色列亚种中的？…

球形芽孢杆菌Ｃ３－４１是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于２３６２菌株,Ｓｏｕｔｈｅｒｎ杂交证明Ｃ３－４１总ＤＮＡ中３．５ＫｂＨｉｎｄＩＩＩ片段上带有４１．９和５１．４ｋＤ二元毒素基因。     

球形芽孢杆菌C3-41二元毒素基因在苏云金芽孢杆菌以色列亚种中的克隆和表达

球形芽孢杆菌C3-41是我国分离的一株对蚊幼虫有毒杀作用的高毒力菌株,对库蚊、按蚊幼虫的毒性高于2362菌株,Southern杂交证明C\-3\|41总DNA中35Kb HindIII片段上带有419和514kD二元毒素基因,该片段由3479个核苷酸组成,核苷酸序列同2362菌株的二元毒素基因序列完全相同。含二元毒素基因的重组质粒pCW\|1和pCW\|2能在大肠杆菌中表达产生二元毒蛋白,但表达量低,重组子杀蚊毒性低。无晶体型苏云金芽孢杆菌以色列亚种重组子在其芽孢形成中能产生以晶体形式存在的二元毒素蛋白,其全发酵液和纯化晶体蛋白的杀蚊活性与C\-3\|41相近。     

Characterization of melanin produced by a wild-type strain of Bacillus thuringiensis

Bt L-7601 is a UV resistant wild-type strain, which belongs to Bacillus thuringiensis subsp. dendrolimus serotype H4a4b. It was isolated from nature, and produced a dark brown pigment during the exponential phase of growth. Bt L-7601 had the ability to produce pigment in a general nutrition-abundant medium, which had no L-tyrosine. The pigment was identified as melanin based on chemical testing, its light absorbance, and FT-IR analysis. Bt L-7601 has a strong resistance to UV light. After 30 min irradiation its survival rate was 17 times higher than that of the strain B. thuringiensis subsp. colmeri 15A3, which had no pigment. Results of the bioassays of residual insecticidal activity of Bt formulation with and without pigment produced by Bt L-7601 against larvae of Helicoverpa armigera and Spodoptera exigua after exposure to UV irradiation showed that the pigment is an excellent UV protective agent for the insecticidal proteins.     

Isolation of Bacillus thuringiensis subsp. israelensis from diseased field-collected larvae of the saturniid moth, Hylesia metabus, in French Guiana

Abstract An isolate of Bacillus thuringiensis subsp. israelensis (BTI) was obtained from field-collected larvae of the saturniid moth, Hylesia metabus . This isolate was shown to belong to serotype H 14, and to produce a spherical parasporal body identical to that of the type strain of BTI. With an LC₅₀ of 0.764 μg cm⁻², this isolate was more toxic to H. metabus than the HD-1 strain of B. thuringiensis subsp. kurstaki (HD-1). These results demonstrate that BTI can be active against lepidopterous insects, a wider spectrum of activity than previously realized.     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌被鉴定为苏云金芽胞杆菌,鞭毛血清型H2,幕虫亚种;产生卵圆形伴胞晶体,伴胞晶体蛋白为100kD;测定了该蛋白 N－末端序列,该序列与炭疽芽胞杆菌的细胞表面S－层蛋白具92－93％相似性,根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱,分别克隆了该基因5’和3’端所在2．9kb XbaI片段和3．1kb Cla I DNA片段,彼此间具0．6kb重叠,通过拼接获得含完整ctc基因的克隆,含该基因的大肠杆菌与表达S－层蛋白的大肠杆菌具相似生长特征,初步表明CTC菌株的伴胞晶体由细胞表面S－层蛋白组成,苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体,由于S－层是细胞表面的结构成分,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑。     

Sulfonate-sulfur utilization involves a portion of the assimilatory sulfate reduction pathway in Escherichia coli

Abstract The strain 273 B, the type strain of a H serotype of Bacillus thuringiensis not yet characterized: B. thuringiensis subsp. cameroun , serotype H32, was isolated from soil samples collected in Cameroon. This strain produces cuboidal parasporal bodies composed of two major proteins of 53 kDa and 35 kDa. N-terminal sequences of the major proteins share no homology with published sequences. Only the 35 kDa protein is susceptible to digestion by trypsin. A complex array of 9 plasmids was revealed.     

Characterization and pathogenic evaluation of Bacillus thuringiensis and Bacillus sphaericus isolates from Argentinean soils

Eighty soil samples of different origin (from urban, agricultural, forested and horticultural areas) which had not previously been treated with bioinsecticides, were collected and examined to investigate the presence of Bacillus thuringiensis and B. sphaericus. From a total of 1473 bacterial isolates examined by differential staining techniques and growth on nutrient agar with the addition of penicillin and streptomycin, 31 (2.1%) strains of Bacillus sphaericus and 25 (1.6%) strains of Bacillus thuringiensis were isolated. These strains were tested for their pathogenicity against Diptera (Culex quinquefasciatus) and Lepidoptera (Anticarsia gemmatalis and Spodoptera frugiperda). Seven strains of Bacillus thuringiensis subspecies kurstaki were found to be pathogenic to Spodoptera frugiperda and twenty-two strains showed a pathological effect against Anticarsia gemmatalis. None of the strains of Bacillus thuringiensis nor the Bacillus sphaericus investigated, showed pathogenic activity against Culex quinquefasciatus. The strains of Bacillus thuringiensis were characterized serologically as belonging to six serotypes (darmstadiensis, entomocidus, kurstaki, muju, sotto and xianguangiensis). One strain seemed to be a new serotype. The electrophoretic profiles of the strains of Bacillus thruringiensis showed bands of 130 kDa similar to those found in strains pathogenic against Lepidoptera. Some physicochemical characteristics were also studied in the soil samples, in order to relate them to the presence or absence of these Bacillus species.     

Insecticidal toxins from Bacillus thuringiensis subsp. kenyae: gene cloning and characterization and comparison with B. thuringiensis subsp. kurstaki CryIA(c) toxins

Genes encoding insecticidal crystal proteins were cloned from three strains of Bacillus thuringiensis subsp. kenyae and two strains of B. thuringiensis subsp. kurstaki. Characterization of the B. thuringiensis subsp. kenyae toxin genes showed that they are most closely related to cryIA(c) from B. thuringiensis subsp. kurstaki. The cloned genes were introduced into Bacillus host strains, and the spectra of insecticidal activities of each Cry protein were determined for six pest lepidopteran insects. CryIA(c) proteins from B. thuringiensis subsp. kenyae are as active as CryIA(c) proteins from B. thuringiensis subsp. kurstaki against Trichoplusia ni, Lymantria dispar, Heliothis zea, and H. virescens but are significantly less active against Plutella xylostella and, in some cases, Ostrinia nubilalis. The sequence of a cryIA(c) gene from B. thuringiensis subsp. kenyae was determined (GenBank M35524) and compared with that of cryIA(c) from B. thuringiensis subsp. kurstaki. The two genes are more than 99% identical and show seven amino acid differences among the predicted sequences of 1,177 amino acids.     

土壤来源的五个苏云金芽孢杆菌新亚种的鉴定

从中国土壤中分离的大量苏云金芽孢杆菌菌株中鉴定出H42、H43、H56、H60及H62等5种新H血清型,并进行了形态、培养特征、生化反应、晶体蛋白质成分及毒力特性等项检测鉴定,鉴定了5个苏云金芽孢杆菌新亚种： Bacillus thuringiensis subsp. jinghongiensis (H42), B.thuringiensis subsp. guiyangiensis (H43),B.thuringiensis subsp. rongseni(H56),B.thuringiensis subsp. pingluonsis(H60)及B.thuringiensis subsp. zhaodongensis(H62) 。毒力生物测定证明5个新亚种的代表菌株对棉铃虫(Heliothis armigera)\,小菜蛾(Plutella xylostella)\,柳蓝叶甲(Plagiodera versicolora)幼虫均无毒力。H42、H43、H56、H60对埃及伊蚊(Aedes aegypti)\,斑须按蚊(Anopheles stephensi)及尖音库蚊(Culex pipiens)亦均无毒;H62对埃及伊蚊无毒,但对尖音库蚊与斑须按蚊有低毒。     

Identification of Flagellar (H) antigenci subfactors in Bacillus thuringiensis H serotypes 10, 18, and 24 isolated in Japan

A total of 95 Bacillus thuringiensis strains isolated from Japan and belonging to H serotypes 10, 18 and 24, were examined for their H antigenic subfactors. Of 84 H serotype 10 isolates, 83 were identified as the H serotype 10a: 10b (serovar darmstadiensis ) and only one isolate was assigned to the II serotype 10a: 10c (serovar londrina ). Among five isolates belonging to the H serotype 18, three were allocated to the H serotype 18a: 18b (serovar kumamotoensis ), while two isolates did not react to antisera against the two known H antigenic subfactors, 18b and 18c. All of the six H serotype 24 isolates were assigned to the H serotype 24a: 24b (serovar neoleonensis ).     

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.     

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.     

Formation of Crystalline delta-Endotoxin or Poly-beta-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of delta-endotoxin on M medium, which contained 330 mug of potassium per ml, but not on ST and ST-a media, each of which contained only 11 mug of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-beta-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH(2)PO(4) (3.7 mM) led to the formation of crystalline delta-endotoxin. The replacement of KH(2)PO(4) with equimolar amounts of KCl, KNO(3), and potassium acetate or an equivalent amount of K(2)SO(4) had a similar effect, whereas the addition of an equimolar amount of NaH(2)PO(4) or NH(4)H(2)PO(4) did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced delta-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-beta-hydroxybutyric acid granules on the media containing 相似文献