枯草芽孢杆菌在琼脂平板上进行的自然遗传转化

本文对发生在琼脂平板上的枯草芽孢杆菌自然遗传转化进行了初步的研究。结果表明,在相同条件下,该菌在琼脂平板上的自然转化率明显高于传统液体转化,并且转化反应对ＤＮａｓｅ 的抗性增强,通常被认为不能建立感受态的ＬＢ 培养物当涂布到平板上后也很快具有了自然转化的能力,说明在固相物表面进行的转化过程与传统的液体法存在一定的差异。在琼脂平板上,也能观察到具不同遗传标记的菌株间进行的细胞间自然转化。     

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。     

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.     

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。 Abstract：The culture fluids of two genetically distinct Bacillus subtilis strains were mixed and coincubated for a short time after they reached post-exponentially growth phase in minimal media.The steadily bidirectional gene transfer involving chromosomal DNA and plasmid DNA by natural genetic transformation between these two strains has been demonstrated by the methods of selective medium screening,DNaseI sensitivity test,plasmid detection and the detection of the capability of producing protease.This result indicates that natural genetic transformation occurs not only between“naked”DNA and cells but also between cells.This conclusion is significant in the assessment of both the possibility of intercelluar DNA transfer in natural habitats of microorganisms and the risk of the application of genetically engineered microorganisms (GEMs).     

嗜碱性芽孢杆菌PB92碱性蛋白酶分别在大肠杆菌和枯草芽孢杆菌中表达

从Bacillus alcalophillus PB92中扩增出碱性蛋白酶基因Mapr,Mapr分别插入到大肠杆菌载体pET-22b( )和枯草芽孢杆菌载体pWB980中构建成重组分泌型表达载体pET22b( )-Mapr、pWB980-Mapr。碱性蛋白酶基因分别在大肠杆菌宿主BL21和枯草芽孢杆菌DB104中得到表达。SDS-PAGE分析,重组蛋白酶的分子量为28kD。在大肠杆菌,所得酶活为231U/ml,而在枯草芽孢杆菌,其酶活为1563U/ml。大概是由于碱性蛋白酶在枯草芽孢杆菌折叠成熟机制与大肠杆菌的不同造成的。     

枯草芽孢杆菌整合载体研究进展

芽孢杆菌质粒经常在复制时出现不稳定的单链(ssDNA)形式,从而导致质粒载体的丢失,而采用整合载体将克隆基因整合到宿主染色体,是克服枯草杆菌质粒不稳定性的一个有效途径。本综述了芽孢杆菌整合载体的研究历程、整合机理、整合类型及其应用和前景。     

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。     

杀虫防病基因工程枯草芽孢杆菌的构建

分别以枯草芽孢杆菌大肠杆菌穿梭质粒pHB201和pRP22为载体,通过感受态转化方法,将Bt-HD-1杀虫蛋白基因cry1Ac导入了水稻纹枯病生防菌株枯草芽孢杆菌B916。工程菌株质粒酶切电泳分析、Southern印迹分析和杀虫生物活性测定结果证实了cry1Ac基因的导入及其在B916中的有效表达。抑菌测定证明工程菌株保持了原野生型菌株良好的抑菌活性。质粒稳定性分析表明以载体pRP22构建的工程菌株Bs2249具有良好的稳定性,而以载体pBH201构建的工程菌株Bs2014则不稳定。此外,实验还证实Bt基因的导入与表达对B916的生长没有不良影响。     

PVA-biocatalyst with entrapped viable Bacillus subtilis cells

Bacillus subtilis cells were entrapped in polyvinyl alcohol (PVA)-cryogel beads without decay in their viability and capability of secretion of proteolytic enzymes (metalloproteinase and subtilisin). Conditions for preparation of the PVA-biocatalyst with suitable stability and viability of B. subtilis cells were optimized. Diffusion of various compounds into the cryogel (sliced beads) has been monitored on-line using image analysis system. Optimal working conditions and kinetic constants for hydrolysis of proteins catalyzed by the PVA-biocatalyst containing whole B. subtilis cells were estimated. The PVA-biocatalyst was applied in the hydrolysis of casein. The productivity of the biocatalyst (expressed as an amount of liberated aromatic amino acids) reached a maximal level of 12 mg g⁻¹ h⁻¹. Composition of mixture of peptides was dependent on pH, concentrations of Na⁺ and glucose, and in the reaction milieu. Protein hydrolysates of desired composition can be obtained using B. subtilis viable cells immobilized in PVA-gel. Incubation of the immobilized cells in a nutrient medium with casein successfully regenerated proteolytic activity of the biocatalyst.     

Cloning and expression of a Bacillus cellulase gene in Escherichia coli

A Bacillus cellulase gene coding for carboxymethylcellulase (CMCase) has been cloned in Escherichia coli using pBR 322 as a vector. The gene was expressed independently of its orientation in the cloning vector showing enzyme activity 40 times greater than that produced by the original Bacillus species. The high production of CMCase in E. coli by the foreign gene did not impede growth of the host cells and the E. coli produced CMCase responded to various pH values and temperatures in the same way as that produced by the gene donor cells.     

枯草芽胞杆菌孢子表面展示外源蛋白的研究

枯草芽胞杆菌孢子表面展示技术是最近十几年新兴的一种外源蛋白固定方法,已在酶学、疫苗学、靶向药物制备、金属污染治理等领域获得了广泛应用。以孢子衣壳蛋白为载体蛋白,已经成功地把许多抗原、酶和其他蛋白展示在孢子外表面。枯草芽胞杆菌孢子衣壳由多种衣壳蛋白组成,但可用做载体蛋白的并不多,且它们的特性不同。综合介绍了枯草芽胞杆菌孢子表面展示外源蛋白这种新型技术的具体机理,及其在国内外各领域应用的研究进展。     

Mutational analysis of the nucleoid-associated protein HBsu of Bacillus subtilis

The essential nucleoid-associated protein HBsu of Bacillus subtilis comprises 92 residues, 20% of which are basic amino acids. To investigate the role of the residues located within the DNA-binding arm, the arginine residues R58 and R61 were changed to leucine, while lysine residues K80 and K86 were replaced by alanine. All altered proteins exhibited a reduction in DNA binding capacity, ranging from 10% to 30% of HBsu wild type DNA-binding ability. To investigate the physiological effect of these mutations in B. subtilis, the indigenous hbs gene was replaced by the mutated genes. B. subtilis strain PK20, which carries the HBsu mutation R58L which exhibits the lowest DNA binding ability in vitro, showed the strongest retardation of growth compared to the wild type. Furthermore, PK20 cells displayed an increased rate of cell lysis, diminished sporulation efficiency and a reduced level of negatively supercoiled DNA. These observations suggest that the DNA binding ability of HBsu DNA is important for growth and differentiation and influences DNA topology. Received: 27 July 1998 / Accepted: 22 September 1998     

Functional relationship between Escherichia coli RNase E and the CafA protein

We analyzed the functional relationship between the Escherichia coli RNase E and the CafA protein, which show extensive sequence similarity. The temperature-sensitive growth of the RNase E mutant strain ams1 was partially suppressed by multicopy plasmids bearing the cafA gene. Introduction of a cafA::cat mutation enhanced the temperature sensitivity of the ams1 mutant. These results suggest that there is a functional homology between these two proteins. Received: 17 May 1996 / Accepted: 1 October 1996     

Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: Use of AUA as a restart codon

Summary An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the -galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active -galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the -galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.     

Plasmid DNA in a groundwater aquifer microcosm -adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23°C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg²⁺ can be replaced by Na⁺, K⁺ or NH₄ The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.     

Direct selection of recombinant plasmids in Bacillus subtilis

A system is described which permits the direct, positive selection of recombinant plasmids in Bacillus subtilis. This system relies on the plasmid pBD214 which confers chloramphenicol (Cm) resistance and carries a thy gene, and on BD393, a highly competent B. subtilis thy A thy B host. Thy⁻ strains are resistant to trimethoprim (Tmp), and Thy⁺ strains are sensitive. Inactivation of the pBD214 thy determinant by insertion of a DNA fragment permits selection of Cm^r Tmp^r clones, all of which carry recombinant plasmids. This insertional inactivation can be accomplished using the unique EcoRl, Bell, Pvull, or EcoRV sites, all of which are located within the thy gene on pBD214. Some properties of this selective system are described, and its uses for molecular cloning are discussed