| Project/Area Number |
13460037
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Shinshu University |
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Principal Investigator |
SEKIGUCHI Junichi Shinshu University, Faculty of Textile Science and Technology, Professor, 繊維学部, 教授 (80111053)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Tsutomu Tokyo University of Agriculture and Technology, Faculty of Agriculture, Associate Professor, 農学研究科, 助教授 (70215812)
YAMAMOTO Hiroki Shinshu University, Faculty of Textile Science and Technology, Research Associate, 繊維学部, 助手 (20262701)
SHIDA Toshio Shinshu University, Faculty of Textile Science and Technology, Associate Professor, 繊維学部, 助教授 (40162599)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2001: ¥7,800,000 (Direct Cost: ¥7,800,000)
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| Keywords | 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 |
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| Research Abstract |
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.
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