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Studies on initiation of sporulation of B.cereus

蜡状芽孢杆菌孢子形成的研究

基本信息

批准号：
09660141
负责人：
HATANO Shoji
金额：
$ 1.79万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660141/
关键词：
Bacillus cereus Sporulation IMP dehydrogenase guaB 菌体内GTP

项目摘要

IMP dehydrogenase activity of B.cereus increased with increase in cell growth in YE-EMM, where B.cereus did not sporulate. When B.cereus was cultured in a modified G medium, a sporulation medium, the activity reached the highest level at 6 hr and decreased thereafter. After induction of sporulation by nutritional shift down in 1/100 G medium, the enzyme activity decreased to about 5% of that of the exponentially growing cells at 1 hr of resuspension. The sporulation rate of B.cereus was over 90% in the modi- fied G medium and 1/100 G medium. The sporulation was strongly inhibited by mycophe- nolic acid at I mM when the drug was added at 0 and I hr of resuspension in 1/100 G medium. Intracellular GTP concentration of B.cereus decreased to the 1owest level about i hr of resuspension. Although the GTP increased to about 50% of the exponentially growing cells at 2 hr of resuspension in control cells, the concentration did not increase in the pres- ence of I mM mycophenolic acid.IMP dehydrogenase was purified from a crude extract of B.cereus cells. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE and 225 kDa by gel filtration.B.cereus ts-4 genomic library was constructed with EMBL3 phage vector. By screening the genomic library with B.subtilis guaB probe, clone GR-1 was obatained. The insert of the clone is under sequencing.

蜡样芽胞杆菌的 IMP 脱氢酶活性随着 YE-EMM 中细胞生长的增加而增加，其中蜡样芽胞杆菌不形成孢子。当蜡状芽胞杆菌在改良的G培养基（孢子形成培养基）中培养时，活性在6小时时达到最高水平，此后下降。在 1/100 G 培养基中通过营养转移诱导孢子形成后，酶活性在重悬 1 小时时降至指数生长细胞酶活性的约 5%。改良G培养基和1/100 G培养基中蜡状芽胞杆菌产孢率均在90%以上。当在 1/100 G 培养基中重悬 0 小时和 1 小时时添加药物时，1 mM 麦考酚酸强烈抑制孢子形成。蜡状芽胞杆菌的细胞内GTP浓度在重悬约1小时时降低至最低水平。尽管在对照细胞中再悬浮2小时时GTP增加到指数生长细胞的约50%，但在1mM麦考酚酸存在下浓度没有增加。IMP脱氢酶从蜡状芽孢杆菌细胞的粗提取物中纯化。 SDS-PAGE测得纯化酶的分子量为56 kDa，凝胶过滤测得其分子量为225 kDa。用EMBL3噬菌体载体构建B.cereus ts-4基因组文库。用枯草芽孢杆菌guaB探针筛选基因组文库，获得克隆GR-1。克隆的插入片段正在测序中。