Schuch Raymond, Fischetti Vincent A, Nelson Daniel C
Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, NY, USA.
Methods Mol Biol. 2009;502:307-19. doi: 10.1007/978-1-60327-565-1_18.
Lysins are phage-encoded, peptidoglycan (cell wall) hydrolases that accumulate in the bacterial cytoplasm during a lytic infection cycle. Late during infection, the lysins undergo holin-mediated translocation across the inner membrane into the peptidoglycan matrix where they cleave cell wall covalent bonds required for wall stability and allow bacterial lysis and progeny phage release. This potent hydrolytic activity is now the foundation of a powerful genetic-based screening process for the identification and analysis of phage lysin proteins. Here, we describe a method for identifying a lysin, PlyG, from a bacteriophage that specifically infects the Gram-positive organism Bacillus anthracis; however, the techniques described can be adapted to clone, express, and analyze lysins from any phage infecting Gram-positive bacteria or possibly even Gram-negative bacteria.
溶素是噬菌体编码的肽聚糖(细胞壁)水解酶,在裂解感染周期中积累于细菌细胞质中。在感染后期,溶素通过孔蛋白介导跨内膜转运至肽聚糖基质,在那里它们切割细胞壁稳定所需的共价键,导致细菌裂解和子代噬菌体释放。这种强大的水解活性现已成为基于遗传学的强大筛选过程的基础,用于鉴定和分析噬菌体溶素蛋白。在此,我们描述了一种从专门感染革兰氏阳性菌炭疽芽孢杆菌的噬菌体中鉴定溶素PlyG的方法;然而,所描述的技术可用于克隆、表达和分析来自任何感染革兰氏阳性菌甚至可能是革兰氏阴性菌的噬菌体的溶素。