Kodama T, Takamatsu H, Asai K, Kobayashi K, Ogasawara N, Watabe K
Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
J Bacteriol. 1999 Aug;181(15):4584-91. doi: 10.1128/JB.181.15.4584-4591.1999.
The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T(2)) of sporulation in wild-type cells and in spoIIIG (SigG(-)) and spoIVCB (SigK(-)) mutants but not in spoIIAC (SigF(-)) and spoIIGAB (SigE(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T(2) of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yaaH mutant spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T(2) of sporulation and that it is required for the L-alanine-stimulated germination pathway.
对枯草芽孢杆菌染色体上从dnaA到abrB区域的21个新基因的表达进行了分析。其中一个基因yaaH，具有在依赖SigE的基因中保守的预测启动子序列。Northern印迹分析表明，在野生型细胞以及spoIIIG（SigG缺失）和spoIVCB（SigK缺失）突变体中，从孢子形成对数生长停止后2小时（T(2)）开始首次检测到yaaH mRNA，但在spoIIAC（SigF缺失）和spoIIGAB（SigE缺失）突变体中未检测到。通过引物延伸分析确定了转录起始点；-10和-35区域与含SigE的RNA聚合酶识别的共有序列非常相似。由带有yaaH基因预测启动子的质粒编码的YaaH-组氨酸标签融合蛋白在枯草芽孢杆菌转化体的孢子形成T(2)期产生，并从成熟孢子中提取，表明yaaH基因产物是一种孢子蛋白。通过插入红霉素抗性基因使yaaH基因失活并不影响营养生长或孢子对热、氯仿和溶菌酶的抗性。yaaH突变体孢子在L-天冬酰胺、D-葡萄糖、D-果糖和氯化钾混合物中的萌发与野生型孢子几乎相同，但突变体孢子在L-丙氨酸刺激的萌发中存在缺陷。这些结果表明，yaaH是一个新基因，编码一种在孢子形成T(2)期在母细胞区室中产生的孢子蛋白，并且它是L-丙氨酸刺激的萌发途径所必需的。
