Mechanism of bacterial cell separation and functions of teichoic acids

在磷酸盐饥饿条件下，细菌细胞壁主要由肽聚糖(在枯草芽孢杆菌细胞壁中占50%)、带负电荷的聚合物(磷壁酸和脂磷壁酸)以及偶尔的磷壁酸组成。其他成分是多肽和多糖。在本项目中，我们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，它能消化肽聚糖干肽中的D-Glu-Meso-A_2 Pm键。免疫荧光显微镜显示，CwlS也定位于细胞分离部位和极点。其定位与先前报道的Lyte和LytF相似。体外分析表明，含有LytF LysM结构域的GST-2xLysM融合蛋白与细胞壁结合较差，但与肽聚糖结合效果较好。因为这种差异可能是由…引起的从Tago或TagA缺乏突变体的细胞壁中制备了更多的磷壁酸，并将其用作GST-2xLysM结合能力的底物。GST-2xLysM能与突变体细胞壁有效结合。我们还测定了LytF-3xFLAG在抗FLAG抗体(一次)和FITC偶联抗体(二次抗体)细胞上的定位。荧光显微镜显示LytF-3xFLAG在次要的磷壁酸突变体和主要的磷壁酸突变体中以螺旋形式定位。因此，主要的和次要的磷壁酸的存在影响了LysM结构域在细胞表面的定位。我们还发现了一种新的细胞壁水解酶(CwlK；YojL)，它能在肽聚糖的茎中裂解L-丙氨酸-D-谷氨酸键。H-ΔCwlK在37℃的无盐条件下，细胞壁裂解活性在pH 6.5时达到最大。细胞壁裂解的最适温度为37℃C(条件：50 mM MOPS-NaOH[pH 6.5]，无盐)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，在最适条件下(pH 6.5，37℃，0 M NaC l)比活力为1,086U/mg。我们还分析了二组分体系YvrGHb。YvrGHb对细胞表面蛋白基因的表达有正向影响：wprA、wapA和dltA，对lytA有负向影响。较少

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