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Biosynthesis and modification of bacterial cell surface

细菌细胞表面的生物合成和修饰

基本信息

批准号：
13460037
负责人：
SEKIGUCHI Junichi
金额：
$ 8.45万
依托单位：
Shinshu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13460037/
关键词：
Bacillus subtilis surface proteins polysaccharide deacetylase cell separation cell wall lytic enzyme FLAG protein 細胞壁東海酵素 PdaB YbaN PdaA Flag蛋白質

项目摘要

To know the mechanisms of biosynthesis and modification of cell wall, a polysaccharide deacetylase homologue (ybaN ; renamed pdaB) of Bacillus subtilis was examined. The mutant exhibited normal cell morphology during the vegetative growth phase. The pdaB-deficient spores during the early to middle stages of sporulation seemed to be normal, but more than 90% of pdaB spores became dark at 24 h (late stage) after the onset of sporulation. Analyses of sporulation sigma factor-deficient mutants and Northern blot indicated that pdaB is transcribed with o^E RNA polymerase. Electron microscopy of spores from a 48-h culture revealed that inside of spores was almost empty or Beverly degraded. These results indicate that the function of the polysaccharide deacetylase homologue PdaB is different from that of the other homologue PdaA (expressing germination-deficient phenotype).We also developed a detection method of subcellular localization of cell wall lytic.enzymes. The target gene was fused to the 3xFLAG peptide sequence (its product contains many aspartic acid residues) at the 3'-terminal of the target gene. After integration of the fused gene into B. subtilis chromosomal DNA, the fused gene was expressed. Subcellular localization of the fused protein was determined with primary anti-FLAG antibody and the secondary FITC-conjugated antibody followed by observation with a fluorescence microscope. Using this procedure, subcellular localization of three cell wall hydrolases was determined : LytE (Cw1F)-3xFLAG and LytF (Cw1E)-3xFLAG were localized at a cell separation site or poles, but LytC (Cw1B)-3xFLAG was localized at the entire cell surface. The result indicates that the cell wall is not homogeneous and a group of proteins (LytF and LytE) containing the LysM domain recognizes a separation site and both poles. This procedure is very useful to determine the subcellular localization of general surface proteins from Gram-positive bacteria.

为了解生物合成和细胞壁修饰的机制，研究了枯草芽孢杆菌多糖脱乙酰酶同源物（ybaN ;更名为pdaB）。该突变体在营养生长期表现出正常的细胞形态。在孢子形成的早期至中期阶段，pdaB缺陷的孢子似乎是正常的，但超过90%的pdaB孢子在孢子形成开始后24小时（后期阶段）变暗。对孢子形成σ因子缺陷突变体的分析和北方印迹表明pdaB是用o^E RNA聚合酶转录的。48小时培养的孢子的电子显微镜显示孢子内部几乎是空的或贝弗利降解。这些结果表明，多糖脱乙酰酶同源物PdaB的功能是不同的，从其他同源物PdaA（表达萌发缺陷表型）。我们还开发了一种检测方法的亚细胞定位的细胞壁溶解。将靶基因与3xFLAG肽序列（其产物含有许多天冬氨酸残基）融合在靶基因的3 '末端。融合基因整合到B中后。枯草芽孢杆菌染色体DNA，表达融合基因。融合蛋白的亚细胞定位用抗FLAG一抗和FITC偶联二抗测定，随后用荧光显微镜观察。使用该方法，确定了三种细胞壁水解酶的亚细胞定位：LytE（Cw 1F）-3xFLAG和LytF（Cw 1 E）-3xFLAG定位于细胞分离位点或两极，但LytC（Cw 1B）-3xFLAG定位于整个细胞表面。结果表明，细胞壁是不均匀的，一组含有LysM结构域的蛋白质（LytF和LytE）识别分离位点和两极。该方法对于确定革兰氏阳性菌一般表面蛋白的亚细胞定位非常有用。