Takamatsu H, Kodama T, Imamura A, Asai K, Kobayashi K, Nakayama T, Ogasawara N, Watabe K
Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
J Bacteriol. 2000 Apr;182(7):1883-8. doi: 10.1128/JB.182.7.1883-1888.2000.
The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T(4)) in wild-type cells and in a gerE36 (GerE(-)) mutant but not in spoIIAC (SigF(-)), spoIIGAB (SigE(-)), spoIIIG (SigG(-)), and spoIVCB (SigK(-)) 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 SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis.
对枯草芽孢杆菌染色体上从abrB到spoVC区域的六个新基因的表达进行了分析,其中一个基因yabG具有在依赖SigK的基因中保守的预测启动子序列。Northern印迹分析表明,在野生型细胞和gerE36(GerE-)突变体中,对数生长停止后4小时(T(4))首次检测到yabG mRNA,而在spoIIAC(SigF-)、spoIIGAB(SigE-)、spoIIIG(SigG-)和spoIVCB(SigK-)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SigK的RNA聚合酶识别的共有序列非常相似。通过插入红霉素抗性基因使yabG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。yabG孢子在L-丙氨酸以及L-天冬酰胺、D-葡萄糖、D-果糖和氯化钾混合物中的萌发情况也与野生型孢子相同。另一方面,yabG孢子的蛋白质制剂包含15 kDa、18 kDa、21 kDa、23 kDa、31 kDa、45 kDa和55 kDa的多肽,在相同条件下,这些多肽在野生型孢子中含量较低或未被提取。我们确定了它们的N端氨基酸序列,发现这些多肽分别是CotT、YeeK、YxeE、CotF、YrbA(31 kDa和45 kDa)和SpoIVA。在荧光显微镜下,芽孢形成细胞中产生的YabG-绿色荧光蛋白融合体的荧光在前芽孢中可检测到,但在母细胞区室中未检测到。这些结果表明,yabG编码一种芽孢形成特异性蛋白,参与枯草芽孢杆菌的外壳蛋白组成。