Biosynthesis and modification of bacterial cell surface

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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.