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Bacterial differentiation: Its application on production of useful substances.

细菌分化：其在有用物质生产中的应用。

基本信息

批准号：
60303024
负责人：
KOBAYASHI Yasuo
金额：
$ 4.54万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Co-operative Research (A)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

1. Control mechanism of bacterial differentiation (Saito, Sadafe, Fujita, Ikeuchi, Kawamura) (1) Regulatory mechanism of sporulation initiation gene spoOA expression in Bacillus subtilis was studied by the use of lacZ-fusion gene and temperature-sensitive spoOA mutant. It was shown that expression of spoOA gene depends on spoOA, OB, OE, OF, and OH genes and spprulation was not initated unless the function of SpoOA protein is derepressed. (2) spoOA gene was cloned and its gene product was overproduced in Escherichia coli and purified. Purified SpoOA protein has DNA-binding activity. (3)div gene which regulates cell division, sporulation, and protein secretion was cloned. (4) Catabolite-repressible gluconate operon was cloned. The whole nucleotide sequence was determined and the mechanism of catabolite repression of B. subtilis was studied.2. Isolation of regulatory factor of gene expression (Beppu, Nakayama) (1) Streptomycin production and sporulation in Streptomyces griseus is positively controlled by A-factor. A-factor was purified and regulatory network surrounding A-factor was studied. (2) Chromosomal proteins of B. subtilis were isolated and purified and sporulation-specific chromosomal proteins were identified.3. Development of usefulvector (Kobayashi, Yamasaki, Yamane, Kudoh, Tanaka) (1) Isolation and analysis of a temperature-dependent promoter revealed that its expressio is controlled by translational-coupling. Stable promoter-probe vector for B. subtilis was constructed. (2) A commom method to amplify a useful gene in B. Subtilis chromosome was developed. (3) Secretion vector carrying -amylase signal peptide was constructed and improvement of its secretion ability was intended. (4) prtR gene which increases protease production in B. natto was identified and cloned. It was shown that PrtR protein is a positive factor which stimulates the transcription of protease gene.

1.细菌分化的调控机制（Saito，Sadafe，Fujita，Ikeuchi，Kawamura）（1）利用lacZ融合基因和温度敏感型spoOA突变体研究了枯草芽孢杆菌产孢起始基因spoOA表达的调控机制。结果表明，spoOA基因的表达依赖于spoOA、OB、OE、OF和OH基因，除非SpoOA蛋白的功能被解除抑制，否则不能启动生殖。(2)克隆了spoOA基因，并在大肠杆菌中过量表达和纯化了其基因产物。纯化的SpoOA蛋白具有DNA结合活性。（3）克隆了调控细胞分裂、产孢和蛋白质分泌的div基因。(4)克隆了代谢产物阻遏型葡萄糖酸操纵子。测定了全基因序列，并对B的代谢抑制机制进行了初步探讨.对枯草芽孢杆菌进行了研究.基因表达调控因子的分离（别府，Nakayama）（1）灰色链霉菌中链霉素的产生和孢子形成受A因子正控制。纯化了A-因子，并研究了A-因子周围的调控网络。(2)B的染色体蛋白。分离纯化了枯草芽孢杆菌的芽孢形成特异性染色体蛋白.（1）分离并分析了一个温度依赖型启动子，发现其表达受偶联调控。B的稳定启动子-探针载体。subtilis构建。(2)一种扩增B中有用基因的常用方法。亚二倍体染色体发达。(3)构建了携带α-淀粉酶信号肽的分泌载体，并对其分泌能力进行了研究. (4)prtR基因，其增加B中的蛋白酶产生。对纳豆进行了鉴定和克隆。结果表明，PrtR蛋白是一种促进蛋白酶基因转录的正性因子。