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。     

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.     

基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用

枯草芽孢杆菌（Bacillus subtilis）作为食品级安全菌株,因其具有理化特征清晰、培养发酵方便等特点,广泛应用于异源蛋白质的高效表达以及高附加值物质的合成。传统的B. subtilis遗传转化方法存在操作流程繁琐、效率低等缺点,因此,开发方便高效的遗传转化系统具有重要意义。转录因子ComK被证实能调控B. subtilis感受态的形成,并在B. subtilis高效转化中有重要作用。构建1个含有木糖诱导启动子P_xyl调控comK表达的穿梭质粒pUBC01?P_xyl?comK的菌株B. subtilis K1,经木糖诱导条件优化后,质粒pHY300?p43?egfp的转化效率达到4.8×10³ CFU·μg^-1。此外,质粒pUBC01?P_xyl?comK可在无胁迫条件下连续培养及消除。木糖诱导感受态体系及质粒消除极大地提高了芽孢杆菌基因编辑和菌株改造的便捷性,同时增强了菌株尤其是生产菌株的性状稳定性。     

枯草芽孢杆菌基因修饰生产核黄素

【目的】研究枯草芽孢杆菌核黄素合成途径、木糖代谢相关基因修饰对核黄素合成的影响。【方法】单独过表达或共同过表达核黄素操纵子中的基因、过表达木糖代谢相关基因构建相应的重组菌株。通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量,表征各个基因修饰的效应。采用摇瓶和5 L罐发酵,考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响。【结果】ribA基因单独过表达,使核黄素产量提高99%,但生物量降低30%,出现细胞自溶现象。ribA-ribH基因共表达,使核黄素产量提高280%,并且无细胞自溶和生物量下降现象。1.5%蔗糖与6.5%木糖作为碳源,5 L发酵罐发酵70 h,核黄素产量达到3.6 g/L,与8%蔗糖为碳源的发酵相比,核黄素产量提高80%。木糖代谢相关基因过表达,均明显降低核黄素产量。【结论】与ribA基因单独过表达相比,ribA-ribH基因共表达可有效避免细胞自溶现象,并能进一步提高核黄素产量。蔗糖与木糖共代谢,能够改善前体物供给,有利于提高核黄素产量。     

细菌感受态细胞摄取和分泌DNA的相关性研究I.含有质粒的枯草芽孢杆菌自然感受态缺陷突变株的诱变和分离

以携带ｐＵＢ１１０质粒的枯草芽孢杆菌ＢＲ１５１菌株为出发菌株,利用亚硝基胍作诱变剂,直接在ＬＢ平板上进行诱变。从２９４９个菌落中筛选出一个转化频率低于出发菌株２～３个数量级的突变株,并对其营养缺陷型、ＵＶ的敏感性及Ｋｍ抗性和质粒进行了检测,确证其转化能力降低为自然感受态缺陷而非营养缺陷型的改变或重组缺陷所致     

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）     

地衣芽孢杆菌感受态细胞的形成及高效电转化

芽孢杆菌在营养缺乏的饥饿状态下,细胞易产生感受态因子,处于生长芽孢时期的芽孢杆菌更容易产生感受态。基于此原则利用芽孢杆菌极限营养培养基通过体外处理诱导使地衣芽孢杆菌产生感受态性能,同时调整参数,建立了感受态细胞对质粒pAPR的高效电转化方法。当质粒DNA浓度为1．5μg/ml、转化时电压为1750V的时候,可以得到261个转化子,经鉴定均为阳性克隆子。而常规电转化的最高仅为20个转化子。为以芽孢杆菌为宿主进行高效电转化提供了模型,也为建立适合工业应用的分泌型表达载体的构建打下了一定基础。     

产嘌呤核苷的枯草杆菌prsA基因在大肠杆菌中的表达

为了克隆产嘌呤核苷的枯草杆菌prsA基因。用PCR扩增的方法,从产肌苷的枯草杆菌Bacillus subtilis JSIM-1019中克隆出一个长1kb长的DNA片段,经功能检测,证明正向插入片段与大肠杆菌的磷酸核糖焦磷酸营养缺陷特性（PREP-）能够营养互补。含有该重组质粒的PRPP缺陷大肠杆菌JSIM—DH-27在基本培养基上的能够生长。     

Characterization of high hydrostatic pressure-injured Bacillus subtilis cells*

High hydrostatic pressure (HHP) affects various cellular processes. Using a sporulation-deficient Bacillus subtilis strain, we characterized the properties of vegetative cells subjected to HHP. When stationary-phase cells were exposed to 250 MPa of HHP for 10 min at 25 °C, approximately 50% of cells were viable, although they exhibited a prolonged growth lag. The HHP-injured cells autolyzed in the presence of NaCl or KCl (at concentrations ≥100 mM). Superoxide dismutase slightly protected the viability of HHP-treated cells, whereas vegetative catalases had no effect. Thus, unlike HHP-injured Escherichia coli, oxidative stress only slightly affected vegetative B. subtilis subjected to HHP.     

A Bacillus subtilis 168 mutant with increased xylose uptake can utilize xylose as sole carbon source

Abstract Bacillus subtilis 168 is unable to effectively utilize xylose as sole carbon source. We demonstrate here that this strain cannot actively transport xylose into the cell. After leaving B. subtilis 168 for a few days on minimal plates with xylose as sole carbon source large colonies arise with a frequency of 1 × 10⁻⁶/cell. These mutants grow well on xylose and efficiently take up that sugar. This new property is not inducible by xylose, indicating that the mutation is neither in the xyl nor in the xyn operon.     

Production of diphtheria toxin CRM228 in B. subtilis

The gene coding for a nontoxic diphtheria toxin (DT), tox228, was isolated from lysogenic Corynebacterium diphtheriae and cloned into pBR322. A mature form of the tox228 gene, lacking its signal sequence, was expressed in Bacillus subtilis using a B. amyloliquefaciens alpha-amylase secretion vector. To test the possibility of producing partially deleted DT molecules, which could be used for cell-directed toxin conjugates, a truncated form lacking 151 amino acids from the C-terminus of the DT was generated by oligonucleotide mutagenesis. Both the truncated and intact DT were efficiently secreted into the culture medium. During prolonged cultivation, the truncated form was less stable than the intact DT molecule.     

In vitro complementation of Bacillus subtilis and Escherichia coli. 2-oxoglutarate dehydrogenase complex mutants and genetic mapping of B. subtilis citK and citM mutations

Abstract The 2-oxoglutarate dehydrogenase complex of the tricarboxylic acid cycle (TCA) consists of multiple copies of 3 different subenzymes; E₁, E₂ and E₃. The E₃ subenzyme is also a component of the pyruvate dehydrogenase complex. Bacillus subtilis 2-oxoglutarate dehydrogenase mutants were studied. The mutants defective in E₁, E₂ and E₃ subenzyme activity, respectively, could be separated into 3 groups by biochemical complementation analyses. The groups correspond to the citK, citM and citL genes. A B. subtilis subenzyme defect, probably E₁, could be complemented with the corresponding Escherichia coli wild-type subenzyme and vice versa. Mutations in citK and citM are closely linked. The gene order kauA——citK-citM was determined from 3-factor transformation crosses. It is concluded that the gene organization and the subenzyme structure of the 2-oxoglutarate dehydrogenase complex are similar in B. subtilis and E. coli .     

枯草芽孢杆菌代谢工程改造的策略与工具

作为一种食品安全级的典型工业模式微生物,枯草芽孢杆菌Bacillus subtilis由于具有非致病性、胞外分泌蛋白能力强以及无明显的密码子偏爱性等特点,现已被广泛应用于代谢工程领域。近年来,随着分子生物学和基因工程技术等的迅速发展,多种研究策略和工具被用于构建枯草芽孢杆菌底盘细胞进行生物制品的高效合成。文中从启动子工程、基因编辑、基因回路、辅因子工程以及途径酶组装等方面介绍枯草芽孢杆菌在代谢工程领域的研究历程,并总结其在生物制品生产中的相关应用,最后对其未来的研究方向进行展望。     

枯草芽孢杆菌渗透压调节基因proB的克隆和表达

用PCR扩增的方法从耐盐的枯草杆菌中克隆出一个13kb长的DNA片段,经功能检测,证明正向插入片段与大肠杆菌的脯氨酸营养缺陷特性(proB-)能够营养互补。含有该重组质粒的大肠杆菌DH5α在基本培养基上的耐盐能力从2%提高至4%。通过引物步行法测定了该插入片段的核苷酸序列。利用DNAsis软件进行序列分析发现,该片段第122～1235bp核苷酸编码一个由370个氨基酸组成的蛋白质分子,其上游存在非典型的-10区,典型的-35区和核糖体结合位点,起始密码子处有最佳翻译起始效率的侧翼核苷酸序列。将其与Genebank中的已知基因的序列和编码的氨基酸序列进行同源性比较,结果表明该片段与枯草杆菌168的核苷酸序列、氨基酸序列的同源性分别为81%和90%。证明该基因确实是一个proB基因。通过与三十个不同种属微芽生物proB基因的氨基酸序列比较,发现该蛋白存在有可能与形成酶的活性中心和三维结构有密切关系的几个绝对保守的区域。     

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

Aims: To optimize the transformation conditions and improve the transformation efficiency of Bacillus subtilis WB800 and DB104. Methods and Results: Trehalose, which could decrease the damage of electric shock to the cells, was added to the electroporation medium containing sorbitol and mannitol. The factors affecting the transformation efficiency, such as the growth phase of bacteria, cell concentration, electric field strength and plasmid variety, were examined and improved. The new method increased the transformation efficiency of B. subtilis by nearly 100‐fold compared with the conventional one. Conclusions: With the optimized method, the transformation efficiency came up to 3·64 × 10⁵ transformants μg^?1 DNA for WB800, and 2·10 × 10⁵ transformants μg^?1 DNA for DB104. Significance and Impact of the Study: This improvement in transformation efficiency will be largely attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for directed evolution and transformation of wild‐type B. subtilis strains.     

Effect of the clay minerals montmorillonite and kaolinite on the genetic transformation of competentBacillus subtilis cells

The effect of the clay minerals montmorillonite and kaolinite on the transformation of competentBacillus subtilis cells with chromosomal DNA was studied. Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation. The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles.