Exhaustive Cell Wall Hydrolase Knockout Bacillus subtilis Strain

In order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall - cell wall hydrolases - has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the multiple-knockout strain is a research tool to characterize hydrolases, using it to identify 3 "helper" hydrolases that act in certain stress conditions.

为实现生长增殖，细菌细胞必须同时合成并分解自身细胞壁。介导这两类过程的酶类，是多款临床优质抗生素的作用靶点。此前，我们对负责细胞壁分解的蛋白质——细胞壁水解酶（cell wall hydrolases）——的认知，受限于多数细菌所携带的大量水解酶之间存在的功能冗余性。为解决这一难题，我们在枯草芽孢杆菌（Bacillus subtilis）中鉴定出42种细胞壁水解酶，并构建了缺失其中40种的工程菌株。研究证实，细菌细胞仅依靠单一细胞壁水解酶即可存活；这意味着，若要阐明标准实验室培养条件下枯草芽孢杆菌的生长机制，仅需对数量极少的蛋白质展开研究，大幅简化了该研究课题。此外，我们验证了该多基因敲除菌株可作为表征水解酶功能的研究工具，并借助其鉴定出3种在特定应激条件下发挥作用的"辅助"水解酶。