Studies on initiation of sporulation of B.cereus

在YE-EMM中，蜡样芽孢杆菌的IMP脱氢酶活性随着细胞生长的增加而增加，蜡样芽孢杆菌不产孢。当蜡样芽孢杆菌在改良的G培养基（一种产孢培养基）中培养时，其活性在6小时达到最高水平，此后逐渐下降。在1/100 G培养基中通过营养转移诱导产孢后，再悬浮1小时，酶活性降至指数生长细胞的5%左右。蜡样芽孢杆菌在改良G培养基和1/ 100g培养基上的产孢率均在90%以上。在1/100 G培养基中，再悬0、1 hr时，加药1 mM时，分枝霉酸对产孢有较强的抑制作用。蜡样芽孢杆菌细胞内GTP浓度在重悬1小时左右降至最低水平。在对照细胞中，虽然GTP在重悬2小时时增加到指数生长细胞的50%左右，但在I mM霉酚酸的存在下，GTP的浓度没有增加。IMP脱氢酶是从蜡样芽孢杆菌细胞粗提物中纯化得到的。经SDS-PAGE和凝胶过滤，纯化酶的分子量分别为56 kDa和225 kDa。利用EMBL3噬菌体载体构建蜡样芽孢杆菌ts-4基因组文库。利用枯草芽孢杆菌guaB探针筛选基因组文库，获得克隆GR-1。克隆的插入物正在测序中。

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.