苏云金芽胞杆菌肠毒素基因的PCR检测

采用多重引物PCR进行了 45株苏云金芽胞杆菌、2株蜡状芽胞杆菌和 2株球形芽胞杆菌溶血素BL ,肠毒素T和entS基因的检测 ,结果表明 95 6%苏云金芽胞杆菌含溶血素hblA基因 ,91 1 %含bceT基因 ,93 3%含entS基因。用两种商业化肠毒素检测试剂盒TECRA和RPLA进行所有菌株肠毒素的体外免疫测定 ,大部分苏云金芽胞杆菌和阳性蜡状芽胞杆菌都能产生不同水平的肠毒素活性 ,同hblA基因PCR检测结果基本相符。尽管DBT0 0 7和T2 4 0 0 1含有hblA基因 ,但用TECRA却检测不到肠毒素 ;Dmu39菌株不含肠毒素基因 ,但用TECRA却检测出高的肠毒素活性。苏云金芽胞杆菌BDT2 4 8和球性芽孢杆菌不含肠毒素基因和肠毒素。结果表明昆虫病原菌苏云金芽胞杆菌的安全性有待进一步研究     

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菌株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 1 0 0kD ;测定了该蛋白的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′端所在的 2 9kbXbaI片段和 3 1kbClaIDNA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     

苏云金芽胞杆菌4.0718菌株中杀虫晶体蛋白基因的定位及鉴定

[目的]对苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)4.0718菌株中的杀虫晶体蛋白基因(insecticidal crystal protein gene,简称cry基因)进行定位和鉴定,系统分析高毒力Bt4.0718菌株的杀虫基因背景.[方法]采用脉冲电泳(PFGE)分离Bt 4.0718菌株的基因组DNA,确定该菌株的PFGE图谱和质粒图谱;采用Southern杂交分析该菌株中cry基因的定位,并使用PCR及PCR产物限制性片段长度多态性分析(PCR-RFLP)方法鉴定该菌株染色体和质粒上含有的cry基因类型.[结果]确定了Bt 4.0718菌株的PFGE图谱和质粒图潜,鉴定到Bt4.0718菌株的染色体和质粒上均定位有cry基因,且分别包含crylAa、crylAc、cry2Aa和cry2Ab 4种基因成分.但染色体上含有的cry基因可能不如质粒上含有的cry基因具有完整的开放阅渎框.[结论]首次在Bt 4.0718菌株的染色体上发现有丰富的cry基因,且与质粒上含有的cry基因类型一致.     

ERIC-PCR鉴别苏云金芽胞杆菌与蜡状芽胞杆菌的研究

利用ERIC-PCR技术对苏云金芽孢杆菌(Bt)、蜡状芽孢杆菌(Bc)和对照菌基因组DNA进行扩增,回收、标记BtPCR扩增片段,分别与各菌株的基因组DNA进行斑点杂交和Southern杂交,筛选Bt标识序列。结果显示:Bt各菌株均可扩增得到250bp的特异片段;Bt和Bc均可得到600bp的共有扩增片段;以筛选得到的569bp片段为探针,可特异性地与Bt基因组DNA杂交;ERIC-PCR技术可以在DNA指纹图谱水平区分鉴别Bt与Bc菌,正确反映出两者的亲缘关系。结果表明ERIC-PCR技术在Bt的检测及在Bt与Bc的鉴定中具有较强的实用性。     

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。     

蜡状芽胞杆菌群菌株中部分病原相关因子的检测

对26株蜡状芽胞杆菌群菌株进行了肠毒素基因及其它病原相关因子的检测。PCR结果表明,17株蜡状芽胞杆菌群菌株中含有病原调控因子plcR的同源序列。采用3组溶血肠毒素hbl基因和3组非溶血肠毒素nhe基因特异性引物,分别可从73%的菌株中至少扩增出一个与预期DNA片段大小一致的片段,其中,苏云金芽胞杆菌菌株中溶血素hbl基因和非溶血素nhe基因的阳性检出率为83%。蜡状芽胞杆菌DBt248完全没有溶血活性,而且在溶血素hbl和非溶血素nhe基因的3个亚基以及病原调控因子plcR的PCR检测中均为阴性,有望作为宿主菌用于苏云金芽胞杆菌晶体蛋白的表达和应用。     

四川盆地生态区苏云金芽胞杆菌cry基因的鉴定及新型模式cry基因的克隆

[目的]为了明确四川盆地生态区土壤中苏云金芽胞杆菌cry,基因资源情况,进一步克隆出新型的杀虫晶体蛋白基因.[方法]本研究主要通过菌株晶体形状的光学显微镜及扫描电镜观察、PCR-RFLP技术鉴定cry基因型法、杀虫晶体蛋白的SDS-PAGE分析和菌株生物活性测定等方法对此地区菌株进行研究.[结果]从四川盆地不同生态区采集2650份土壤样品中分离了791株苏云金芽胞杆菌.PCR-RFLP鉴定结果表明:此地区的苏云金芽胞杆菌主要含有cry1,cry2,cry3,cry4/10,cry9,cry30和cry40等7种cry,基因类型;含cry1基因的菌株最丰富,共有21种不同cry1型基因组合;从中发现了新型模式基因,并采用Tail-PCR技术获得了其中3个基因的全长序列,被国际苏云金芽胞杆菌杀虫晶体蛋白基因命名委员会命名为cry54Aa1、cry30Fa1和cry30Ga1.通过生物活性测定,发现对鳞翅目和双翅目害虫具毒力的菌株.未鉴定出基因型的80个菌株的伴胞晶体SDS-PAGE分析表明:这些菌株均有40～130 kDa蛋白表达,极有可能含新型的杀虫蛋白基因.[结论]研究结果充分体现了四川盆地生态区苏云金芽胞杆菌资源的多样性及特殊性,所蕴含的杀虫蛋白基因在农业生产上具有重要意义和应用前景.     

苏云金芽胞杆菌cry2Ad基因的克隆及其表达产物的活性分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)SBT2是我国新分离出的一株野生菌株.扫描电镜显示该菌株产生双锥体形晶体.琼脂糖凝胶电泳发现其质粒图谱含有5个条带.聚丙烯酰胺凝胶电泳显示此菌株产生130 kD晶体蛋白.利用PCR-RFLP法进行杀虫基因类型鉴定,发现其含有cry1Aa、cry1Da、cry1Hb、cry1Jb、cry1Ka 、cry1Ib、基因.Cry2Ad蛋白的活性至今未见研究报道,本研究克隆和测序了该基因.并对其进行了表达.生物活性测定结果表明其表达产物对舞毒蛾(Lymantria dispar)、棉铃虫(Helicoverpa armigera)、亚洲玉米螟(Ostrinia furnacalis)、小菜蛾(Plutella xylostella)有低活性;对大猿叶甲(Colaphellus bowringi)无活性.     

土壤中对甲虫有活性苏云金芽胞杆菌的筛选

近几年来,甲虫已成为十字花科蔬菜的头号害虫。本文采集了湖北省各地区192份土壤样品,分离到苏云金芽胞杆菌(简称Bt)菌株74株,以甲虫代表——黄粉虫为靶标昆虫筛选了15株典型Bt,得到一株对甲虫有活性的Bt L1;生物测定该菌的半致死浓度(LC50)为221.20μg·g-1;聚丙烯酰胺凝胶电泳(SDS-PAGE)显示L1至少具有3个杀虫晶体蛋白;PCR扩增杀虫晶体蛋白基因,测序发现Bt L1中含有cry1Ac,cry1Aa,cry1Ia基因,推测Bt L1中对甲虫活性的杀虫晶体蛋白基因为cry1Ia。     

苏云金芽胞杆菌sigK 基因插入失活突变体的特点及其对cry3A基因启动子活性的影响

摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt) sigK 基因插入失活突变体,分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因,构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体,生长曲线测定表明,突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示,突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达,所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性,对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需,并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Bacillus and relatives in foodborne illness

Species of Bacillus and related genera have long been troublesome to food producers on account of their resistant endospores. These organisms have undergone huge taxonomic changes in the last 30 years, with numbers of genera and species now standing at 56 and over 545, respectively. Despite this expansion, relatively few new species have been isolated from infections, few are associated with food and no important new agents of foodborne illness have been reported. What has changed is our knowledge of the established agents. Bacillus cereus is well known as a cause of food poisoning, and much more is now understood about its toxins and their involvement in infections and intoxications. Also, although B. licheniformis, B. subtilis and B. pumilus have occasionally been isolated from cases of food‐associated illness, their roles were usually uncertain. Much more is now known about the toxins that strains of these species may produce, so that their significances in such episodes are clearer; however, it is still unclear why such cases are so rarely reported. Another important development is the use of aerobic endosporeformers as probiotics, as the potentials of such organisms to cause illness or to be sources of antibiotic resistance need to be borne in mind.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides

Abstract Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis -based insecticides (Bactimos^TM, DiPel^TM, Florbac^TM FC, Foray^TM 48B, Novodor^TM FC, Turex^TM, VecTobac^TM, XenTari^TM). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel^TM the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP^TM which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis .     

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.     

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.     

The Bacillus cereus bceT enterotoxin sequence reappraised

Bacillus cereus is a known opportunistic human pathogen belonging to the B. cereus group. Establishment of the pathogenesis most likely involves several gene products. One of these gene products, a single gene component named bceT, has been cloned and described from B. cereus B-4ac [Agata et al., Microbiology 141 (1995) 983-988]. However, our sequences of the bceT region from 16 B. cereus group strains showed inconsistency with the published bceT sequence. Only part of the bceT sequence had homology to our sequences. This initiated a more thorough investigation of the bceT sequence. Restriction site search and database searches intimated that the cloned bceT was created by an incidental joining of four DNA fragments during ligation. One of these fragments had 93% homology to an open reading frame (ORF 101) located within the pathogenic island of the Bacillus anthracis pXO1 virulence plasmid. We suggest that the reported enterotoxic activity of the original cloned bceT construct could be due to either the fusion gene or the fragment with homology to ORF 101 in pXO1.     

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Abstract Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus . This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis .     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.