A Novel Lipolytic Enzyme, YcsK (LipC), Located in the Spore Coat of Bacillus subtilis, Is Involved in Spore Germination
A Novel Lipolytic Enzyme, YcsK (LipC), Located in the Spore Coat of Bacillus subtilis, Is Involved in Spore Germination
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DOI：
10.1128/jb.01527-06
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发表时间：
2007-01
影响因子：
3.2
通讯作者：
Atsushi Masayama;Ritsuko Kuwana;H. Takamatsu;H. Hemmi;T. Yoshimura;K. Watabe;R. Moriyama
中科院分区：
文献类型：
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作者：
Atsushi Masayama;Ritsuko Kuwana;H. Takamatsu;H. Hemmi;T. Yoshimura;K. Watabe;R. Moriyama
枯草芽孢杆菌ycsK的氨基酸序列与脂肪分解酶GDSL家族的氨基酸序列相似。北方印迹分析表明，ycsK mRNA在孢子形成后4 h开始表达，并且其转录依赖于SigK和GerE。在孢子形成细胞中产生的YcsK-绿色荧光蛋白融合蛋白的荧光在荧光显微镜下在母细胞中可检测到，但在前孢子隔室中不可检测到，并且融合蛋白定位在依赖于CotE、SafA和SpoVID的发育孢子周围。通过插入红霉素抗性基因使ycsK基因失活并不影响营养生长或孢子对热、溶菌酶或氯仿的抗性。ycsK孢子在L-天冬酰胺、D-葡萄糖、D-果糖和氯化钾的混合物和LB培养基中的萌发也与野生型孢子相同，但突变体孢子在L-丙氨酸刺激的萌发中有缺陷。此外，酶谱分析表明，在大肠杆菌中异源表达的YcsK蛋白显示脂解活性。因此，我们建议将ycsK改名为lipC。这是第一次研究细菌孢子萌发相关的脂肪酶。
ABSTRACT The predicted amino acid sequence of Bacillus subtilis ycsK exhibits similarity to the GDSL family of lipolytic enzymes. Northern blot analysis showed that ycsK mRNA was first detected from 4 h after the onset of sporulation and that transcription of ycsK was dependent on SigK and GerE. The fluorescence of the YcsK-green fluorescent protein fusion protein produced in sporulating cells was detectable in the mother cell but not in the forespore compartment under fluorescence microscopy, and the fusion protein was localized around the developing spores dependent on CotE, SafA, and SpoVID. Inactivation of the ycsK gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, lysozyme, or chloroform. The germination of ycsK spores in a mixture of l-asparagine, d-glucose, d-fructose, and potassium chloride and LB medium was also the same as that of wild-type spores, but the mutant spores were defective in l-alanine-stimulated germination. In addition, zymogram analysis demonstrated that the YcsK protein heterologously expressed in Escherichia coli showed lipolytic activity. We therefore propose that ycsK should be renamed lipC. This is the first study of a bacterial spore germination-related lipase.