An exhaustive multiple knockout approach to understanding cell wall hydrolase function in Bacillus subtilis.
An exhaustive multiple knockout approach to understanding cell wall hydrolase function in Bacillus subtilis.
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DOI：
10.1128/mbio.01760-23
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发表时间：
2023-10-31
期刊：
影响因子：
6.4
通讯作者：
Garner, Ethan C.
中科院分区：
文献类型：
--
作者：
Wilson, Sean A.;Tank, Raveen K. J.;Hobbs, Jamie K.;Foster, Simon J.;Garner, Ethan C.
大多数细菌都被它们的细胞壁所包围，细胞壁包含一个高度交联的肽聚糖保护膜。为了生长，细菌必须不断地重塑他们的墙，插入新的材料，打破旧的束缚。通过细胞壁水解酶进行键的断裂，使细胞壁扩张。单个水解酶的功能被它们的冗余性所阻碍：单个敲除通常不呈现表型。我们使用彻底的多重敲除方法来确定枯草芽孢杆菌生长所需的最小水解酶集合。我们确定了42个候选水解酶。令人惊讶的是，我们能够在一个菌株中去除除两个以外的所有这些基因；这个“∆40”菌株只显示出轻微的生长速度下降，表明40种水解酶中没有一种是生长所必需的。∆40菌株不能明显地摆脱旧墙，这表明营业额对增长并不是必不可少的。∆40菌株中剩余的水解酶是Lyte和CwlO，此前已证明它们是合成致死的。在∆40中可以去除这两种酶中的任何一种，这表明两种水解酶中的任何一种都足以促进细胞生长。环境条件和生化条件的筛选表明，镁离子对Lyte活性有抑制作用，而RLPA样蛋白对Lyte活性有刺激作用。综上所述，这些结果表明，枯草杆菌细胞壁水解酶的唯一基本功能是通过扩大细胞壁来促进细胞生长，而Lyte或CwlO本身就足以实现这一功能。本实验引入∆40菌株作为研究枯草杆菌水解酶活性及其调控的工具。为了生长，细菌细胞必须创造和分解它们的细胞壁。负责这些过程的酶是我们一些最好的抗生素的目标。我们对分解细胞壁的蛋白质--细胞壁水解酶--的理解一直受到许多细菌含有的大量水解酶之间的冗余的限制。为了解决这个问题，我们在枯草芽孢杆菌中鉴定了42个细胞壁水解酶，并创造了一个缺少40个细胞壁水解酶的菌株。我们证明，细胞只需使用一种细胞壁水解酶就可以存活；这意味着要了解枯草杆菌在标准实验室条件下的生长，只需研究非常有限的蛋白质，大大简化了问题。此外，我们还证明了∆40菌株是一种表征水解酶的研究工具，使用它来鉴定在特定胁迫条件下起作用的三种“辅助”水解酶。
Most bacteria are surrounded by their cell wall, containing a highly cross-linked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this “∆40” strain shows only a mild reduction in growth rate, indicating that none of the 40 hydrolases are necessary for growth. The ∆40 strain does not detectably shed old wall, suggesting that turnover is not essential for growth. The remaining hydrolases in the ∆40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be removed in ∆40, indicating that either hydrolase alone is sufficient for cell growth. Screening of environmental conditions and biochemistry revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results suggest that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone are sufficient for this function. These experiments introduce the ∆40 strain as a tool to study hydrolase activity and regulation in B. subtilis. 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 ∆40 strain is a research tool to characterize hydrolases, using it to identify three “helper” hydrolases that act in certain stress conditions.
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影响因子：
3.2
作者：
FAN, DP;BECKMAN, MM;CUNNINGHAM, WP
通讯作者：
CUNNINGHAM, WP
DOI：
10.1099/00221287-137-8-1987
发表时间：
1991-08-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
--
作者：
FOSTER, SJ
通讯作者：
FOSTER, SJ
DOI：
10.1073/pnas.1004304107
发表时间：
2010-11-02
影响因子：
11.1
作者：
Atilano, Magda L.;Pereira, Pedro M.;Filipe, Sergio R.
通讯作者：
Filipe, Sergio R.
DOI：
10.1111/j.1432-1033.1981.tb05342.x
发表时间：
1981-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
BEACHEY, EH;KECK, W;SCHWARZ, U
通讯作者：
SCHWARZ, U
影响因子：
3.6
作者：
Bisicchia, Paola;Noone, David;Devine, Kevin M.
通讯作者：
Devine, Kevin M.