Mechanism of bacterial cell separation and functions of teichoic acids

细菌细胞分离机制及磷壁酸的功能

• 批准号: 16380059
• 负责人: SEKIGUCHI Junichi
• 金额: $ 9.98万
• 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: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16380059/
• 关键词: Bacillus subtilis; Immunofluorescence microscopy; Peptidoglycan; Cell wall hydrolases; Phage; Teichoic acid; Cell separation enzyme; LysM domain; 細胞壁; FLAGタグ

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 … More 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

在磷酸盐饥饿条件下，细菌细胞壁由作为主要组分的肽聚糖（其在枯草芽孢杆菌细胞壁中含有50%）、带负电荷的聚合物（磷壁酸和脂磷壁酸）和有时磷壁糖醛酸组成。其他成分是多肽和多糖。本研究以枯草芽孢杆菌细胞壁为模型系统，重点研究了细胞壁相关蛋白和磷壁酸。在细胞壁结合蛋白中，我们发现CwlS是一种细胞壁水解酶，可以消化肽聚糖茎肽中的D-Glu-meso-A_2 pm键。免疫荧光显微镜观察表明，CwlS也定位于细胞分离部位和两极。该定位与先前报道的LytE和LytF的定位相似。体外分析表明，含有LytF的LysM结构域的GST-2xLysM融合蛋白与细胞壁结合较差，但与肽聚糖结合有效。由于这种差异可能是由 ...更多信息 在细胞壁中存在磷壁酸的情况下，制备来自TagO-或TagA-缺陷突变体的细胞壁，并用于GST-2xLysM结合能力的底物。GST-2xLysM与突变体细胞壁有效结合。我们还用抗FLAG抗体（一抗）和随后的FITC偶联抗体（二抗）测定了LytF-3xFLAG在细胞上的定位。荧光显微镜显示LytF-3xFLAG在次要磷壁酸突变体和主要磷壁酸突变体中定位为螺旋。因此，主要和次要磷壁酸的存在影响LysM结构域在细胞表面上的定位。我们还发现了新的细胞壁水解酶（CwlK ; YojL），其切割肽聚糖茎中的L-丙氨酸-D-谷氨酸键。h-ΔCwlK的细胞壁溶解活性在pH 6.5、37℃、无NaCl条件下达到最大值。细胞壁裂解活性的最适温度和NaCl浓度分别为37℃（条件：50 mM MOPS-NaOH [pH 6.5]，无NaCl）和0 mM（条件：50 mM MOPS-NaOH [pH 6.5]，37℃）。h-ΔCwlK蛋白是一种细胞壁裂解酶，在最适条件（pH 6.5，37℃和0 M NaCl）下表现出1，086 U/mg的比活性。我们还分析了双组分系统YvrGHb。YvrGHb影响细胞表面蛋白基因的表达：wprA、wapA和dltA呈阳性，lytA呈阴性。少

项目成果

A new D,L-Endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis

• DOI: 10.1128/jb.00188-06
• 发表时间: 2006-08-01
• 期刊: JOURNAL OF BACTERIOLOGY
• 影响因子: 3.2
• 作者: Fukushima, Tatsuya;Afkham, Anahita;Sekiguchi, Junichi
• 通讯作者: Sekiguchi, Junichi

生物工学ハンドブック

生物技术手册

• 发表时间: 2005
• 作者: 高橋恭子;羅智靖;日本生物工学会編
• 通讯作者: 日本生物工学会編

Solution structure of the peptidoglycan binding domain of B. subtilis cell wall lyticenzyme CwlC : Characterization of the sporulation-related repeats by NMR

枯草芽孢杆菌细胞壁溶解酶 CwlC 的肽聚糖结合域的溶液结构：通过 NMR 表征孢子形成相关的重复序列

• 发表时间: 2005
• 期刊: Biochemistry 44・30
• 作者: Numata;T.;Fukai;S.;Ikeuchi;Y;Suzuki;T.;Nureki;O.;Y.Tanaka;M.Mishima;Y.Tanaka;K.Furuita;Toshitatsu Kobayashi;Emiko Iida;Yuki Sudo;Kazuhiko Nakatani;Masaki Mishima
• 通讯作者: Masaki Mishima

Solution structure of the peptidoglycan binding domain of B. subtilis cell wall lytic enzyme, CwIC : Characterization of the sporulation-related repeats by NMR

枯草芽孢杆菌细胞壁裂解酶的肽聚糖结合域的溶液结构，CwIC：通过 NMR 表征孢子形成相关的重复序列

• 发表时间: 2005
• 期刊: Biochemistry 44
• 作者: Mishima;M.
• 通讯作者: M.

バイオテクノロジーの基本技術

生物技术基础技术

• 发表时间: 2006
• 作者: Mishima;M.;T.Shida;K.Yabuki;K.Kato;J.Sekiguchi;C.Kojima;関口 順一
• 通讯作者: 関口 順一