Bacterial differentiation: Its application on production of useful substances.

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的SpoA突变体，研究了芽胞形成起始基因SpoA在枯草芽孢杆菌中表达的调控机制。结果表明，SPOA基因的表达依赖于SPOA、OB、OE、OF和OH基因，除非SpoOA蛋白的功能被解压，否则不会引发发芽。(2)克隆了SpoA基因，并在大肠杆菌中进行了高效表达和纯化。纯化的SpoOA蛋白具有DNA结合活性。(3)克隆了调控细胞分裂、产孢量和蛋白质分泌的div基因。(4)克隆了分解代谢抑制型葡萄糖酸操纵子。结果：1.测定了枯草芽孢杆菌的全核苷酸序列，并对其分解代谢抑制机制进行了研究。基因表达调控因子的分离(BepPu，Nakayama)(1)灰色链霉菌产孢量和产孢量受A因子正调控。纯化了A因子，并研究了A因子周围的调控网络。(2)分离纯化枯草杆菌染色体蛋白，鉴定出产孢子特异的染色体蛋白。(1)温度依赖性启动子的分离和分析表明其表达受翻译偶联控制。构建了枯草芽孢杆菌稳定的启动子-探针载体。(2)建立了一种在枯草杆菌染色体上扩增有用基因的通用方法。(3)构建了携带淀粉酶信号肽的分泌载体，以期提高其分泌能力。(4)鉴定并克隆了提高纳豆产酶活性的prtR基因。结果表明PrtR蛋白是一种正向刺激蛋白水解酶基因转录的因子。

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.