不同培养时期的大肠杆菌和枯草芽孢杆菌间发生的跨属自然遗传转化

携带穿梭质粒的大肠杆菌与作为受体的枯草芽孢杆菌分别培养至不同生长阶段混合均匀后静置40min,涂布选择性平板,37℃培养30h后得到一定数目的转化子,DNaseⅠ敏感实验证实质粒是通过自然遗传转化而非其它形式发生转移。实验发现大肠杆菌可以在特定生长时期向胞外分泌DNA,并且在对数期具有最高的提供质粒的能力,而生长后期的细胞因为体系中DNase量的增加转化频率下降。进一步的研究发现枯草芽孢杆菌在营养丰富的LB培养基中也具有与基本培养基中相当的转化能力,并且在对数生长前期具有较高的转化频率。     

枯草芽孢杆菌感受态研究新进展

在枯草芽孢杆菌中,感受态的形成受到一种二元信号转导系统的调节,这种系统对胞外的感受态信息素浓度作出感应而激活晚期感受态基因的表达。各种晚期感受态蛋白分别负责外源DNA的吸附、吸收和内源化,它们共同构成了DNA的运输系统。初步探讨了枯草芽孢杆菌感受态调节在细胞生长和进化中的意义。     

适用于野生型枯草芽孢杆菌转化有机溶剂方法的建立和优化

用经典的spizizen方法将穿梭质粒pLJ导入Bacillus subtilis168、Bacillus subtilisWB600、Baci／／ussubtihsW-B800中,均能达到70多个转化子／μgDNA的较高转化效率,但用此方法将该质粒导入由本实验室筛选并保存的野生型BacillussubtilisNx-2中,不能获得转化子。本研究对spizizen转化方法进行了改进。通过添加不同浓度的吐温-80,DMSO、丙酮、甲苯、乙醇等有机溶剂,均能获得转化子。同时,本研究对转化条件进行了优化,分别选取吐温-80、DMSO、甲苯等3种效果较好的添加剂设计正交实验。试验结果表明,在添加4％吐温-80、3％DMSO、1％甲苯的条件下得到了34个转化子／斗gDNA的较高转化效率。还研究了质粒与感受态共培养时间和感受态稀释倍数对转化的影响,当质粒与感受态共培养时间为3h,感受态稀释5倍时．转化效率较高。     

枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究

为便于枯草芽孢杆菌工业化生产应用,对Spizizen创立的枯草芽孢杆菌DNA转化方法进行改进.用GMI和GMII溶液处理枯草芽孢杆菌野生型菌株BS501a、营养缺陷型突变株DBl342和非营养缺陷型突变株WB800,用改进的方法制备感受态细胞,用7.5kb质粒pSBPTQ进行转化,并研究RNA、酵母粉、水解酪蛋白、培养方法对枯草芽孢杆菌质粒转化的影响.结果表明,该方法适用于不同基因型枯草芽孢杆菌的质粒转化,营养缺陷型突变株DBl342的转化率为750 CFU/μg/DNA,非营养缺陷型突变株WB800转化率为1 070 CFU/xg DNA,野生型菌株BS501a转化率为270 CFU/μg/DNA.根据影响转化效率的因素,推测在该方法中,枯草芽孢杆菌质粒转化原理:一定生物量的枯草芽孢杆菌在外界营养条件和钙、镁离子作用下,细胞壁和细胞膜形成缺陷,使外源DNA转入枯草芽孢杆菌细胞内.     

枯草芽孢杆菌噬菌体载体的构建

以φ0105DI：It为原始株构建的重组噬菌体φ105S35和φ10 5S36具有自主侵染能力和溶源化特征。其基因组内插入的lkb片段上的cat,基因赋予二者所在宿主以氯霉素抗性,在两株噬菌体中插入位点相同,即原φ105DI ：It的smal酶切片段D、E之间,但插入片段在二者中的定向相反。与cat基因同时引入的单一BamHI和Xbal位点提供了外源DNA的插入位置。重组噬菌体DNA可高效转染枯草芽孢杆菌原生质体。因此φ105S35和币φ105S36可作为枯草芽孢杆随系统的载体而被利用。     

枯草芽孢杆菌bgIS基因在酵母染色体上整合

将枯草杆菌β－１,３－１,４－葡聚糖酶基因（ｂｇＩＳ）２．７ｋｂＥｃｏＲＩ片段和酵母染色体ｒＤＮＡ片段克隆到整合型不含酵母自主复制序列（Ａｕｔｏｎｏｍｕｓｌｙ ｒｅｐｌｉｃａｔｉｎｇ ｓｅｑｕｅｎｃｅ）的大肠杆菌／酵母菌穿梭质粒ＹＩＰ５上,构建成ＹＩＰ５－ｂｇＩＳ－ｒＤＮＡ的杂种质粒ＰＣＺＨ,转化Ｓ．ｃｅｒｅｖｉｓｉａｅ并得到表达。稳定性测定表明,Ｙ３３（ＰＣＺＨ１０１）和Ｙ３３（ＰＣＺＨ１０４）     

遗传工程微生物细胞间发生的自然遗传转化

将两株具有不同遗传标记的枯草芽孢杆菌在基本培养基中分别培养至对数生长后期后进行短时间混合静置培养 ,经选择平板筛选、DNaseI敏感性试验、质粒检测和产蛋白酶活性检测 ,发现两菌株之间可通过自然遗传转化进行染色体DNA和质粒DNA的交换。研究结果表明 ,自然遗传转化可在细胞间进行 ,这对揭示微生物群居的自然环境中可能存在的细胞间的DNA转移 ,以及正确评估遗传工程微生物(GEMs)的安全使用具有重要意义。     

蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段,该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达,核黄素产量从39．5mg／L增加到61．7mg/L.     

THE POTENTIAL FOR GENETIC EXCHANGE BY TRANSFORMATION WITHIN A NATURAL POPULATION OF BACILLUS SUBTILIS

We have investigated the potential for genetic exchange by transformation within a Mojave Desert population of Bacillus subtilis. Almost all strains surveyed were competent for transformation, and the strains varied over almost three orders of magnitude in their levels of competence. This high degree of variation suggests that natural selection toward an optimal level of competence is, at most, very weak in this population. Six of 24 competent strains showed sexual isolation from laboratory strain 168 (i.e., heterogamic transformation was reduced). Direct crosses between selected pairs of Mojave strains indicated sexual isolation within the Mojave population. Levels of sexual isolation observed within this population of B. subtilis were much less than those previously observed for transformation between named Bacillus species. Sexual isolation between 168 and Mojave strains, and among Mojave strains, was due to differences in restriction-modification systems and to DNA sequence divergence.     

RECOMBINATION AND MIGRATION RATES IN NATURAL POPULATIONS OF BACILLUS SUBTILIS AND BACILLUS MOJAVENSIS

We have investigated the rates of recombination and migration in native populations of two closely related, naturally competent Bacillus species. Native soil isolates of Bacillus subtilis and Bacillus mojavensis were obtained from three continents and, within North America, from populations at a range of geographical distances from one another. The rate of recombination within populations of each species was estimated from restriction-site data for three genes. Recombination was shown to occur within each species at about the same rate as neutral mutation, whatever the geographical scale or phylogenetic scale over which strains were sampled. The rate of migration between populations was estimated by a cladistic analysis and was shown to be high (i.e., Nm > 1), even among populations on different continents. The level of migration within each species is sufficient to prevent neutral geographical divergence within species.     

影响枯草杆菌原生质体转化的因素

原生质体转化是将外源基因导入细菌的主要方法之一,其转化频率受制于多种因素。本研究以激BacillussubtilisDB104为宿主菌,以枯草杆菌的高表达型质粒pNQ122为外源DNA,研究了原生质体再生率、原生质体浓度和用于制备原生质体的细胞生长期对转化频率的影响,获得了在该系统中实现高频率转化的条件。该转化条件使外源基因在多个B.subtilis菌株中的转化成为可能,并使从B.subtilis中筛选中性蛋白酶基因获得成功。     

芽孢杆菌原生质体的形成,再生及种间融合的研究

原生质体融合技术不仅能使遗传基因高频率重组,而且可以集双亲优良遗传性状为一体.自Schaeffer等人成功地进行了微生物原生质体融合以来,这项技术便广为人们所接受.枯草芽孢杆菌分泌的抗菌蛋白能抑制多种植物病原菌的生长,苏云金芽孢杆菌生成的伴孢晶体蛋白可毒杀植物害虫.利用原生质体融合技术将这两种芽孢杆菌抑菌杀虫的遗传特性融为一体,选育出防治植物病虫害的新一代生防菌株成为可能,有关这方面的研究国内外尚未见报道.本文报道了抗菌蛋白产生菌TG26-10和晶体蛋白产生菌AS1.904-17原生质体的形成,再生及种间融合的影响因素,为进一步筛选目的融合子提供基础.     

枯草芽孢杆菌BF7658噬菌体的研究

从一些工厂分离到6株感染枯草芽孢杆菌BF7658的噬菌体,对它们的形态学和生物学特性以及DNA和结构蛋白进行了比较,电镜观察指出,所有噬菌体的头部外形都呈现椭园形,但其大小和尾部长度有所不同,它们的宿主范围较窄,用限制酶分析,噬菌体DNA分子量在29.9kb和98kb之间,根据解链温度计算出噬菌体DNA的G+C含量在45.1和60.2mol％之间,6株噬菌体的蛋白经SDS-聚丙烯酰胺凝胶电泳测定呈现四条主带和五条次带。     

FINE-SCALE GENETIC AND PHENOTYPIC STRUCTURE IN NATURAL POPULATIONS OF BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS: IMPLICATIONS FOR BACTERIAL EVOLUTION AND SPECIATION

The genetic and phenotypic structure of sympatric populations of wild bacteria traditionally identified as Bacillus subtilis and B. licheniformis was analyzed. Small soil samples were taken from a single, tiny site in the Sonoran Desert of Arizona, USA, to provide a true population analysis, in contrast to many analyses of genetic structure using bacterial strain collections of widely heterogeneous origin. Genetic analyses of isolates used multilocus enzyme electrophoresis, mismatches in restriction fragment length polymorphism, and variants from Southern hybridization with B. subtilis DNA probes. Phenotypic analyses of isolates used the API test system for detection of growth and acid production on specific carbon sources. The two species were distinct both phenotypically and genetically, despite their known potential for genetic exchange in laboratory experiments. Genic and genotypic diversity were high in both species, and only 16% of observed allozyme variants might possibly be common to both species. Hence, there is probably modest genetic exchange, if any, between the species in nature. Clear hierarchies of population-genetic structure were found for both species. Different types of genetic data yield concordant population structures for B. subtilis. For both species, two-locus and multilocus statistical analyses of linkage demonstrated modest to strong disequilibrium at the species level but truly panmictic subunits within each species. The evidence for extensive genetic recombination within these fine-scale subdivisions is unequivocal, indicating that the sexuality of these bacteria can be well expressed in nature. The relation of these results to processes of bacterial evolution and speciation is discussed.     

腺嘌呤对枯草杆菌GMI—971发酵生产肌苷的影响

培养基中腺嘌呤含量对腺嘌呤缺陷型的枯草杆菌GMI－971发酵产肌苷有显著影响。在拟定条件下,腺嘌呤浓度在150mg／L时摇瓶肌苷产量最高。对不同来源的四种酵母粉分别添加腺嘌呤,当其含量增加至l50ml／L时,肌苷产量也最高。     

枯草芽孢杆菌分子克隆系统受体的改造

实验证明枯草芽孢杆菌(Bacillus subtilis)BR151和MI112中的pUB110质粒,在液体中传代要比固体斜面传代稳定。通过NTG诱变获得了对pUB110稳定的受体BSG.来自大肠杆菌的穿梭质粒在BSG菌中转化频率较亲本高。小的外源DNA片段插入pBE2后在BSG菌中转管传代30次是稳定的。     

枯草芽孢杆菌中怀植酸酶的纯化和酶学性质

从土壤中分离到了产中性植酸酶的枯草芽孢杆菌菌株并对所产植酸酶进行了分离纯化,此中性植酸酶的反应最适pH为7．5,最适温度为55度,在37度下以植酸钠为底物的Km值为0．19mmol/L,植酸酶活性依赖Ca^2 的存在,酶蛋白的分子量大小约为45kD,纯酶蛋白N端序列为Lys-His-Lys-Leu-Ser-Asp-Pro-Tyr-His-Phe-Thr。