2006 Fiscal Year Final Research Report Summary
Mechanism of bacterial cell separation and functions of teichoic acids
| Project/Area Number |
16380059
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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 |
Applied microbiology
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| Research Institution | SHINSHU UNIVERSITY |
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Principal Investigator |
SEKIGUCHI Junichi Shinshu University, Interdisciplinary Graduate School of Science and Technology, Professor, 大学院総合工学系研究科, 教授 (80111053)
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| Co-Investigator(Kenkyū-buntansha) |
SHIDA Toshio Shinshu University, Interdisciplinary Graduate School of Science and Technology, Associate Professor, 大学院総合工学系研究科, 助教授 (40162599)
YAMAMOTO Hiroki Shinshu University, Interdisciplinary Graduate School of Science and Technology, Assistant Professor, 大学院総合工学系研究科, 助手 (20262701)
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| Project Period (FY) |
2004 – 2006
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| Keywords | Bacillus subtilis / Immunofluorescence microscopy / Peptidoglycan / Cell wall hydrolases / Phage / Teichoic acid / Cell separation enzyme / LysM domain |
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| Research Abstract |
Bacterial cell wall consists of peptidoglycan as a major component (it contains 50% in Bacillus subtilis cell wall), negative- charged polymers (teichoic acids and lipoteichoic acids) and sometimes teichuronic acids under the phosphate starvation condition. The other components are polypeptides and polysaccharides. In this project, we studied Bacillus subtilis cell wall as a model system and focused on cell wall-associated proteins and teichoic acids. Among cell-wall binding proteins, we found CwlS as a cell wall hydrolase that digests D-Glu-meso-A_2pm linkage in the stem peptide of peptidoglycan. The immunofluorescence microscopy indicated that CwlS was also localized at cell separation site as well as poles. The localization was similar to those of LytE and LytF as previously reported. In vitro analysis suggested that GST-2xLysM fused protein containing the LysM domain of LytF binds poorly with cell walls but efficiently with peptidoglycan. Since this difference may be caused by the
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presence of teichoic acids in cell wall, cell walls from TagO-or TagA-deficient mutants were prepared and used for the substrate of the binding ability of GST-2xLysM. GST-2xLysM bound efficiently with the mutant cell walls. We also determined the localization of LytF-3xFLAG on cells with anti-FLAG antibody (primary) followed by FITC-conjugated antibody (secondary). Fluorescent microscopy revealed the LytF-3xFLAG was localized as spirals in the minor teichoic acid mutants and also in the major teichoic acid mutants. Therefore, presence of major and also minor teichoic acids affected the localization of LysM domain on the cell surface. We also found new cell wall hydrolase (CwlK ; YojL) that cleaves L-alanine-D-glutamic acid linkage in the stem of peptidoglycan. Cell wall lytic activity of h-ΔCwlK was found to be maximum at pH 6.5 under the conditions of 37℃ without NaCl. Moreover, the optimum temperature and NaCl strength for cell wall lytic activity were 37℃C(conditions : 50 mM MOPS-NaOH [pH 6.5] without NaCl) and 0 mM (conditions : 50 mM MOPS-NaOH [pH 6.5] at 37℃), respectively. The h-ΔCwlK protein is a cell wall lytic enzyme exhibiting a specific activity of 1,086 U/mg under the optimum conditions (pH 6.5, 37℃ and 0 M NaCl). We also analyzed the two-component system, YvrGHb. YvrGHb affected the expression of cell surface protein genes : wprA, wapA, and dltA positively and lytA negatively. Less
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