Enterotoxigenicity and cytotoxicity of Bacillus thuringiensis strains and development of a process for Cry1Ac production

Bacillus thuringiensis is indistinguishable from Bacillus cereus except for the production of insecticidal crystal proteins (ICPs). B. thuringiensis strains may show enterotoxin profiles and toxin levels similar to those of B. cereus strains isolated from food-poisoning cases. It is important for the food industry and farmers to consider that with the application of B. thuringiensis strains to crops, their spores may be introduced into the human food chain. In this study, 59 B. thuringiensis strains were assayed for their hemolysin BL (HBL) using a BCET-RPLA kit and their cytotoxicity to Chinese hamster ovary (CHO) cells. The enterotoxin titer was as high as that of B. cereus diarrheal-type strain ATCC 49064. In an attempt to obtain a food safety strain for bioinsecticide use, in this study, a 3.5-kb cry1Ac DNA fragment was amplified with PCR from the total DNA of B. thuringiensis subsp. kurstaki CCRC 11502 and cloned into the Bacillus expression vector pHY300PLK. The alpha-amylase promoter, amyE, was then introduced into the promoter region and, afterward, the recombinant plasmid pHYe1Ac35 was introduced into a non-enterotoxigenic and non-cytotoxic B. thuringiensis subsp. kurstaki Tt14 strain. The transformant, without any detectable enterotoxigenicity or cytotoxicity, produced Cry1Ac toxin properly, and its insecticidal activity against Trichoplusia ni larvae was found to be satisfactory.     

天津滨海盐碱土地区城市绿地土壤微生物特性研究

对天津滨海盐碱土地区城市绿地土壤微生物特性研究表明不同绿地植物配置模式下0～40cm土壤微生物数量差异极显著,防护林>灌+草>乔木>乔+草>草坪>滨海盐土,0～20cm土层微生物数量是20cm～40cm的3～4倍。在细菌、放线菌和真菌三大类群中,细菌占绝对优势,其次是放线菌和真菌。滨海盐土微生物的季节变化不大,防护林土壤中微生物总数最多时出现在秋季,乔木、乔+草、灌+草、草坪绿地土壤中微生物数量最多时出现在夏季或秋季。细菌、放线菌数量与土壤全盐含量呈显著负相关,细菌的数量与土壤有机质含量呈极显著相关,真菌数量与土壤养分相关不显著.芽孢杆菌的11个种在不同土壤条件和植物配置模式下的生态分布是不同的,最常见的是蜡质芽孢杆菌、拟霉芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌和蜂房芽孢杆菌,放线菌主要是链霉属,包括链霉菌属的10大类群,分布最广的类群是灰褐类群、金色类群和青色类群,真菌分布有11个属。在不同植物配置下芽孢杆菌、放线菌和真菌的组成、优势种的组合也存在显著差异。     

黑龙江凉水自然保护区苏云金芽孢杆菌的收集与鉴定

黑龙江凉水自然保护区是我国现有保存下来的较大片原始红松林基地之一,总面积6394hm^2,森林覆盖率95％以上。本研究从小兴安岭凉水自然保护区采集土样782份,采用醋酸钠培养基结合高温方法筛选土壤中的芽孢杆菌,通过光学显微镜观察鉴定产生伴胞晶体的苏云金芽孢杆菌（Bacillus thuringiensis．Bt）。总计分离得到芽孢杆菌和苏云金芽孢杆菌分别为1735株和33株,Bt菌株的分离率和出菌率分别为1．90％和4．22％。利用SDS-PAGE和PCR—RFLP方法对筛选获得的成分离株进行了杀虫晶体蛋白和基因型分析,结果表明14株产菱形伴胞的＆菌株,SDS-PAGE电泳分析芽孢后期产生分子量大小130kD蛋白带,PCR-RFLP分析初步鉴定为crylAc基因,其它产圆形或其它不定形晶体蛋白的＆分离菌中,芽孢后期主要蛋白大小为20～150kD不等,PCR—RFLP方法鉴定结合PCR片段测序分析这些菌株含有新型cry4、cry39和cry40基因等。本研究是“中国助资源收鉴与利用”项目组成部分之一,凉水自然保护区苏云金芽孢杆菌的收集与鉴定目的是要对整个东北地区成资源分布和多样性作一个初步评估,实验结果表明东北森林地区具有丰富多样的威菌株和杀虫基因资源。     

Use of fatty acid profiles to identify food-borne bacterial pathogens and aerobic endospore-forming bacilli

Capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of various food-borne microbial pathogens and to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen bacteria, representing eight genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species were used to compare the extracted fatty acid methyl esters (FAMEs). Endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. A data set for each bacterial agent was prepared using fatty acid profiles from five replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for Bacillus anthracis and Bacillus cereus show that there are two branched chain fatty acids, iso 17:1 omega10c and 17:1 anteiso, which are unique in these species. Iso 17:1 omega10c is present in B. cereus vegetative cells and spores but is not observed in B. anthracis. The 17:1 anteiso fatty acid is present in B. anthracis cells but not in B. cereus cells. Fatty acids 16:0 2OH and 17:0 iso 3OH are present in B. anthracis and B. cereus spores but not in the vegetative cells. In summary, analysis of FAMEs from bacteria and spores can provide a sensitive procedure for the identification of food-borne pathogens.     

Size-dependent lipid content in human milk fat globules

Human milk fat globules (HMFGs) are considered to constitute a triglyceride-rich source of fat and energy. However, milk contains lipid particles at different sizes ranging from tens of micrometers to less than 1 microm. In particular, the physical, chemical, and biological properties of submicron sized particles are poorly described. Individual HMFGs were analyzed using laser trapping confocal Raman spectroscopy, and their chemical signature was obtained and compared to 1, 5, and 10 microm globules. Significant differences in both lipid composition and relative lipid content were found between the classes of particles with different diameters. A strong Raman peak at 1742 cm(-1) corresponding to the triacylglycerol core was detected in the 5 and 10 microm diameter globules, whereas in the smaller HMFGs no detectable peak was found. In addition, the submicron particles produced Raman signals consistent with large quantities of unsaturated fatty acids. Moreover, cis and trans isomers of unsaturated fatty acids were found to be unequally distributed between large and small milk fat globules. Interestingly, trans unsaturated fatty acids were found only in 1 and 5 microm globules although more prominent in the 5 microm diameter range. This is the first evidence for size related differential lipid composition of various diameter classes of HMFGs. The results suggest that the milk fat globule size distribution determines milk lipid composition. In addition, large portions of the HMFGs are secreted into milk conspicuously not for fat delivery. Thus, small HMFGs may offer novel metabolic and nutritional functions.     

蜡样芽孢杆菌ATCC14579毒力基因plcR缺失株的构建及其一般特性

蜡样芽孢杆菌是土壤中的优势菌,具有作为益生菌的潜在能力,同时它也是条件致病菌,能引起食物中毒等。蜡样芽孢杆菌的多种毒力因子受到多效性调控子（pleiotropic regulator,plcR）的调控,在其条件致病性作用中起着重要作用。真养产碱杆菌JMP34（Alcaligenes eutrophus）质粒上的2,4-二氯苯氧乙酸（2,4-dichlorophenoxyacetic acid,2,4-D）单加氧酶（tfdA）基因可以降解2,4-D。本研究利用同源重组技术使tfdA基因置换掉蜡样芽孢杆菌的plcR基因,构建了蜡样芽孢杆菌ATCC14579突变株B.cereus△plcRΩtf-dA,并对其毒性、一般生理生化特性进行分析。研究结果表明,突变株B.cereus△plcRΩtfdA的毒性显著减弱;生理生化实验结果显示突变株与野生株并没有明显区别,且突变株并没有表现出tfdA酶活性。所有的结果表明plcR控制着蜡样芽孢杆菌ATCC14579的致病性,同时剔除plcR并不破坏其酶系统。本研究为今后构建蜡样芽孢杆菌工程菌提供了新的思路和依据。     

芽孢杆菌TS01的ARDRA评估和遗传分析

利用自主分离的芽孢杆菌菌株TS01和15种芽孢杆菌(地衣芽孢杆菌,枯草芽孢杆菌,短小芽孢杆菌,巨大芽孢杆菌,凝结芽孢杆菌,蜡状芽孢杆菌,迟缓芽孢杆菌,苏云金芽孢杆菌,嗜热脂肪芽孢杆菌,解淀粉芽孢杆菌,环状芽孢杆菌,球形芽孢杆菌,侧孢短芽孢杆菌,多粘类芽孢杆菌,泛酸枝芽孢杆菌)模式菌种进行ARDRA分析。采用16S rDNA通用引物16S-27和16S-1525进行PCR扩增,16S rDNA扩增片段经六种限制性酶（Alu I、Taq I、Mse I、Bst UI、Hha I和Tsp509 I）酶切电泳,获得了TS01菌株的特征性ARDRA指纹图谱。ARDRA图谱通过GelcomparⅡ软件进行聚类分析(UPGMA),结果表明菌株TS01和地衣芽孢杆菌处于同一分支,亲缘关系最近。ARDRA分析鉴定结果与实验室前期菌株TS01形态、生化鉴定和16S rDNA序列分析结果一致,TS01是一株地衣芽孢杆菌菌株,从而证明ARDRA技术在菌种水平上对芽孢杆菌TS01进行鉴别具有可靠性。     

Stimulation of nitrogen fixation in soddy-podzolic soils with fungi

Stimulation of nitrogen fixation in soddy-podzolic soils is related to the hydrolytic activity of fungi decomposing plant polymers. It was found that the rate of nitrogen fixation upon the simultaneous inoculation of the strains of nitrogen-fixing bacteria Bacillus cereus var. mycoides and the cellulolytic fungus Trichoderma asperellum into a sterile soil enriched with cellulose or Jerusalem artichoke residues is two to four times higher than upon the inoculation of the strains of Bacillus cereus var. mycoides L1 only. The increase in the nitrogen fixation depended on the resistance of the substrates added into the soil to fungal hydrolysis. The biomass of the fungi decomposing plant polymers increased by two–four times. The nitrogen-fixing activity of the soil decreased when the growth of the fungi was inhibited with cycloheximide, which attested to a close correlation between the intensity of the nitrogen fixation and the decomposition of the plant polymers by fungi. The introduction of an antifungal antibiotic, together with starch or with plant residues, significantly (by 60–90%) decreased the rate of nitrogen fixation in the soll.     

苏云金芽胞杆菌类ABC transporter基因zwa-FEG能提高对双效菌素的抗性

双效菌素（Zwittermicin A,ZwA）是一种氨基多元醇类新型广谱抗生素,最初是从蜡状芽胞杆菌(Bacillus cereus) UW85中发现的,zmaR为它的抗性基因。在本实验室已测得的苏云金芽胞杆菌（Bacillus thuringiensis）YBT-1520菌株全基因组序列中有ZwA合成基因簇完整序列,分析发现在其末端存在一个类似ABC transporter的序列（命名为zwa-FEG）。将zwa-EFG在大肠杆菌（Escherichia coli）中表达,大肠杆菌能获得对ZwA的抗性;将zwa-EFG转移到产ZwA的菌株UW85中,能使其ZwA产量显著提高。推测zwa-EFG是一种ZwA专一性的ABC transporter,能将ZwA分泌到胞外,从而增强ZwA的合成和对ZwA的抗性。     

苏云金芽胞杆菌类ABC transporter基因zwa-FEG能提高双效菌素的抗性

双效菌素(zwittermicin A,ZwA)是一种氨基多元醇类新型广谱抗生素,zmaR为其抗性基因。在苏云金芽胞杆菌(Bacillus thuringiensis)YBT-1520菌株ZwA合成基因簇末端存在一个类似ABC transporter的序列(命名为zwa-FEG)。将zwa-FEG基因在大肠杆菌(Escherichia coli)中表达,大肠杆菌能获得ZwA的抗性;将zwa-FEG转移到产ZwA的蜡状芽胞杆菌(Bacillus cereus)菌株UW85中,能使其ZwA产量显著提高。推测zwa-FEG是一种ZwA专一性的ABC transporter,它能将ZwA分泌到胞外,从而增强ZwA的合成和ZwA的抗性。     

蜡样芽胞杆菌905铜锌超氧化物歧化酶基因的克隆及原核表达

蜡样芽孢杆菌905（Bacillus cereus 905）是从小麦体内分离获得的一株植物有益内生细菌。为从氧自由基毒性角度分析该细菌在植物体内的定殖机制,本研究利用PCR方法得到了B. cereus 905的CuZn-SOD基因全长（sodC）。该基因由537 bp组成,编码179个氨基酸残基。构建表达载体pET-22b-sodC,转化Escherichia coli BL21(DE3),IPTG诱导表达结果表明：该基因表达出的蛋白表现出SOD功能,化学抑制法验证其为CuZn-SOD。     

低分子量有机配体对黏粒矿物吸附苏云金芽孢杆菌的影响

土壤是微生物的理想栖息地,土壤微生物群落组成复杂,数量巨大。1g土壤中可能栖息着上百亿个微生物,其中细菌可达1010个[1]。约80%～90%的土壤微生物寄居于土壤固相表面,如黏土矿物、金属氧化物或有机质表面[2]。细菌与矿物间的相互作用在土壤污染物转化与降解、团聚体形成[3]、矿物风化[4-6]及病原菌运移[7-8]等诸多过程起着关键作用。     

H5N1型禽流感病毒血凝素HA1蛋白在苏云金芽胞杆菌细胞表面的展示及其免疫原性

利用苏云金芽胞杆菌S-层蛋白CTC表面展示系统研究在细胞表面展示H5N1型禽流感病毒HA1蛋白的可行性及其免疫原性，为研制既安全有效又能常温长期保藏和运输的禽流感口服基因工程疫苗奠定基础。用部分ha1基因（ha1p）代替S-层蛋白ctc基因中部且位于表面锚定序列slh下游的片段，构建了融合基因ctc-ha1p和 csa-ctc-ha1p；利用电脉冲转化法将含融合基因的重组质粒转入苏云金芽胞杆菌无质粒突变株BMB171中，获得了重组菌株BCCH（含ctc-ha1p，并导入一个载有协助细胞表面展示的csaAB操纵子的质粒）和CH（含csa-ctc-ha1p）。通过血凝和血凝抑制试验以及小鼠免疫学实验证实，2个重组菌株均成功地在细胞表面展示了重组HA1蛋白并具有一定的特异性和免疫原性，其中，CH的效果强于BCCH，说明csa-ctc-ha1p这种融合基因的构建方式更胜一筹。研究结果表明，苏云金芽胞杆菌S-层蛋白CTC表面展示系统可用来研制禽流感口服疫苗。     

N-acyl-homoserine lactones from Enterobacter sakazakii (Cronobacter spp.) and their degradation by Bacillus cereus enzymes

A chemical study of acyl-homoserine lactones (acyl-HSLs) produced by Enterobacter sakazakii resulted in the identification of three molecules: (S)-N-heptanoyl-HSL, (S)-N-dodecanoyl-HSL and (S)-N-tetradecanoyl-HSL. Mixed cultures of E. sakazakii and Bacillus cereus depleted E. sakazakii acyl-HSLs, suggesting acyl-HSL degradation by B. cereus hydrolases (hydrolysis of the lactone or amide moiety). The expression of B. cereus acyl-HSL lactonase and acyl-homoserine acylase was confirmed by monitoring the biotransformation of (S)-N-dodecanoyl-HSL into (S)-N-dodecanoyl-homoserine, dodecanoic acid and homoserine in the presence of B. cereus whole cells, using electrospray-mass spectrometry (ESI-MS).     

Microbial glucosylation of thaxtomin A, a partial detoxification

Thaxtomin A (1) and B (2), the two major phytotoxins associated with the common scab of potato disease, were transformed into C-14 linked beta-glucosides (3) and (4), respectively, when individually incubated with cultures of Bacillus mycoides in oatmeal broth at 26 degrees C. These biotransformation products when assayed on aseptically produced potato minitubers proved to be much less phytotoxic than the parent compounds.     

广西大王岭和大明山自然保护区苏云金芽孢杆菌收集与鉴定

从广西大王岭和大明山两个自然保护区共采集到土样264份，共分离出597株芽孢杆菌，通过光学和电子显微镜检观察，16株分离株观察到伴胞晶体蛋白，初步确定为苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt），出菌率为6．06％。在16株肪分离株中，有4株在芽孢形成过程中能产菱形晶体蛋白，其余12株能产圆形和其他形状的晶体蛋白。利用PCR—RFLP方法和SDS．PAGE方法对16株助分离菌进行了蛋白和基因型的鉴定，结果表明，16株分离株中含有4株crylAc基因，表达约130kD的晶体蛋白，其中含有cry30基因和cry40基因的菌株分别1株和3株，表达大约75kD的晶体蛋白；另外8株戤菌株表达蛋白大小不一，其基因型尚不能确定，有待进一步分析。生物测定表明，产菱形晶体含有crylAc基因的4株励分离株对鳞翅目小菜夜蛾幼虫有很强的毒杀活性，而其它分离株对小菜夜蛾没有毒杀活性。     

Enumeration and confirmation of Bacillus cereus in foods: collaborative study

A collaborative study was conducted in 15 laboratories to evaluate 2 different techniques for enumerating Bacillus cereus in foods. A direct plating technique using mannitol-egg yolk-poly-myxin agar and a most probable number (MPN) technique using trypticase-soy-polymyxin broth were compared for the enumeration of high and low populations of B. cereus in mashed potatoes. The collaborative results showed that the overall mean recovery obtained with the low population level was essentially the same by both techniques. However, the overall mean recovery was significantly higher by the direct plating technique at the high population level. A statistical evaluation of the data also showed that the direct plating technique had better repeatability and reproducibility than did the MPN technique at both the high and low population levels. These results suggest that the MPN technique is suitable for examining foods containing low populations of B. cereus, but that the direct plating technique is preferable for foods that contain a high population of this organism. The confirmatory technique used in the proposed method is reliable for presumptive identification of isolates as B. cereus. The method has been adopted as official first action.     

苏云金芽胞杆菌Bt9875杀虫晶体蛋白可诱导Caspase和Bcl-2家族参与调控HL-60细胞凋亡

研究Caspase家族与Bcl-2家族参与调控苏云金芽胞杆菌(Bacillus thuringiensis,Bt)Bt9875杀虫晶体蛋白对人急性髓细胞性白血病细胞HL-60的影响.本实验采用MTT法检测了杀虫晶体蛋白诱导HL-60细胞凋亡后的Caspase家族的活性和Caspase凋亡酶抑制剂对杀虫晶体蛋白诱导HL-60细胞凋亡的影响;采用Western blot检测了杀虫晶体蛋白诱导多聚ADP-核糖聚合酶(PARP)降解、Bcl-2/Bax调控和细胞色素C的释放.研究结果表明,杀虫晶体蛋白作用HL-60细胞后,激活了Caspase-3、Caspase-8和Caspase-9,在48 h内Caspase家族抑制剂(Z-VAD-FMK)、Caspase-3抑制剂(z-DEVD-FMK)和Caspase-9抑制剂(Z-LEHD-FMK)均可显著抑制杀虫晶体蛋白诱导的细胞凋亡;杀虫晶体蛋白可明显上调促凋亡蛋白Bax的表达,同时下调抗凋亡蛋白BCl-2的表达,并观察到胞浆中细胞色素C的释放.初步证明了Bt9875杀虫晶体蛋白诱导的HL-60细胞凋亡是由Caspase家族和Bcl-2家族共同调控的,线粒体途径在诱导细胞凋亡过程中起着重要作用.     

苏云金杆菌天门变种晶体蛋白基因的克隆和表达

用改进的Birnboim法检测了苏云金杆菌9个变种的质粒组成,其中我国分离到的天门变种有12条质粒带,用Southern杂交法证明,天门变种的δ-内毒素基因位于1个约60MD的质粒上。该质粒经BglⅡ酶切后与Bam HI酶切的pBR322载体连接,转化大肠杆菌HB101。通过菌落原位放射免疫反应和Southern杂交方法,筛选带有δ-内毒素基因并能在大肠杆菌中表达的转化体。对玉米螟幼虫进行生物测定表明,阳性克隆pTCC65及pTCC18有较强的抑制幼虫生长的作用。     

几株固氮芽孢杆菌的分离与鉴定

摘要： 从小麦（Triticum aestivum）、玉米（Zea mays）、黑麦草（Lolium sp.）和柳树（Salix sp.）的根际土壤中分离得到能在无氮培养基上生长的29株芽孢杆菌（Bacilli），通过固氮酶活的测定以及固氮酶结构基因 nifH 的PCR扩增得到7株固氮芽孢杆菌。对这7株固氮菌株进行了生理生化性状测定、16S rDNA序列分析(GenBank accession No. AY373358, AY373360,~AY373364和AY376876)、G+C mol%含量的测定及DNA-DNA杂交实验，结果表明，其中5株菌属于芽孢杆菌，另外2株菌属于类芽孢杆菌。在这7株被鉴定的菌株中，菌株T1被鉴定为Bacillus cereus；菌株G1、C4和C5被鉴定为B. megaterium；菌株W5的生理生化性状、16S rDNA和G+C mol%与Bacillus marisflavi 相近；G2的生理生化性状和16S rDNA 与Paenibacillus polymyxa 接近，但在基于16S rDNA的系统发育树中却与P. durus 聚在最小的分支内；T7可能是Paenibacillus属中的一个新种。