Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Mol Microbiol. 2022 Jun;117(6):1366-1383. doi: 10.1111/mmi.14914. Epub 2022 May 21.
Conjugative elements are widespread in bacteria and include plasmids and integrative and conjugative elements (ICEs). They transfer from donor to recipient cells via an element-encoded type IV secretion system. These elements interact with and utilize host functions for their lifecycles. We sought to identify essential host genes involved in the lifecycle of the integrative and conjugative element ICEBs1 of Bacillus subtilis. We constructed a library of strains for inducible knockdown of essential B. subtilis genes using CRISPR interference. Each strain expressed one guide RNA in ICEBs1. We induced partial interference of essential genes and identified those that caused an acute defect in acquisition of ICEBs1 by recipient cells. This screen revealed that reducing expression of genes needed for synthesis of cell wall teichoic acids caused a decrease in conjugation. Using three different ways to reduce their synthesis, we found that wall teichoic acids were necessary in both donors and recipients for efficient conjugative transfer of ICEBs1. Further, we found that depletion of wall teichoic acids caused cells involved in ICEBs1 conjugation to die, most likely from damage to the cell envelope. Our results indicate that wall teichoic acids help protect against envelope stress caused by active conjugation machines.
结合元件在细菌中广泛存在，包括质粒、整合和接合元件（ICE）。它们通过元件编码的 IV 型分泌系统从供体转移到受体细胞。这些元件与宿主相互作用并利用宿主功能来完成其生命周期。我们试图鉴定与枯草芽孢杆菌整合和接合元件 ICEBs1 生命周期相关的必需宿主基因。我们构建了一个使用 CRISPR 干扰诱导必需枯草芽孢杆菌基因敲低的菌株文库。每个菌株在 ICEBs1 中表达一个向导 RNA。我们诱导必需基因的部分干扰，并鉴定出那些导致受体细胞急性获得 ICEBs1 缺陷的基因。该筛选揭示了降低细胞壁磷壁酸合成所需基因的表达会导致接合减少。我们使用三种不同的方法来降低它们的合成，发现磷壁酸对于 ICEBs1 的有效接合转移在供体和受体中都是必需的。此外，我们发现磷壁酸的耗尽导致参与 ICEBs1 接合的细胞死亡，这很可能是由于细胞包膜受损。我们的结果表明，磷壁酸有助于防止由活性接合机器引起的 envelope stress。
