Biosynthesis and modification of bacterial cell surface

为了解细胞壁的生物合成和修饰机制，对枯草芽孢杆菌的多糖去乙酰化酶同源物（ybaN，更名为pdaB）进行了研究。突变体在营养生长期表现出正常的细胞形态。pdaB缺失孢子在孢子形成的早期至中期看似正常，但超过90%的pdaB孢子在孢子形成后24 h（后期）变暗。对产孢sigma因子缺陷突变体和Northern blot的分析表明，pdaB是由o^E RNA聚合酶转录的。培养48小时的孢子电镜显示孢子内部几乎是空的或贝弗利降解。这些结果表明，多糖去乙酰化酶同源物PdaB与其他同源物PdaA（表达发芽缺陷表型）的功能不同。我们还建立了细胞壁裂解酶亚细胞定位的检测方法。目的基因与目的基因3′端3xFLAG肽序列（其产物含有许多天冬氨酸残基）融合。将融合基因整合到枯草芽孢杆菌的染色体DNA中，表达融合基因。用一抗flag抗体和二抗fitc偶联抗体确定融合蛋白的亚细胞定位，并在荧光显微镜下观察。利用这种方法，确定了三种细胞壁水解酶的亚细胞定位：LytE (Cw1F)-3xFLAG和LytF (Cw1E)-3xFLAG定位在细胞分离位点或极点，而LytC (Cw1B)-3xFLAG定位在整个细胞表面。结果表明细胞壁不是均匀的，含有LysM结构域的一组蛋白（LytF和LytE）识别分离位点和两极。该方法对确定革兰氏阳性菌表面蛋白的亚细胞定位非常有用。

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.