The conserved Tol-Pal trans-envelope complex is important for outer membrane (OM) stability and cell division in Gram-negative bacteria. It is proposed to mediate OM constriction during cell division via cell wall tethering. Yet, recent studies suggest the complex has additional roles in OM lipid homeostasis and septal wall separation. How Tol-Pal facilitates all these processes is unclear. To gain insights into its function(s), we applied transposon-insertion sequencing, and report here a detailed network of genetic interactions with the tol-pal locus in Escherichia coli. We found one positive and > 20 negative strong interactions based on fitness. Disrupting osmoregulated-periplasmic glucan biosynthesis restores fitness and OM barrier function, but not proper division, in tol-pal mutants. In contrast, deleting genes involved in OM homeostasis and cell wall remodeling causes synthetic growth defects in strains lacking Tol-Pal, especially exacerbating OM barrier and/or division phenotypes. Notably, the ΔtolA mutant having additional defects in OM protein assembly (ΔbamB) exhibited severe division phenotypes, even when single mutants divided normally; this highlights the possibility for OM phenotypes to indirectly impact cell division. Overall, our work underscores the intricate nature of Tol-Pal function, and reinforces its key roles in cell wall-OM tethering, cell wall remodeling, and in particular, OM homeostasis.

中文翻译：

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遗传相互作用图突出了 Tol-Pal 复合体的关键作用

保守的 Tol-Pal 反式包膜复合物对于革兰氏阴性菌的外膜 (OM) 稳定性和细胞分裂很重要。建议通过细胞壁束缚来介导细胞分裂过程中的 OM 收缩。然而，最近的研究表明，该复合物在 OM 脂质稳态和间隔壁分离中具有额外的作用。Tol-Pal 如何促进所有这些过程尚不清楚。为了深入了解其功能，我们应用了转座子插入测序，并在此报告了与大肠杆菌中tol-pal基因座的遗传相互作用的详细网络。我们发现了一种基于适应度的积极和> 20 种消极的强相互作用。在tol-pal突变体。相反，删除参与 OM 稳态和细胞壁重塑的基因会导致缺乏 Tol-Pal 的菌株的合成生长缺陷，特别是加剧 OM 屏障和/或分裂表型。值得注意的是，在 OM 蛋白组装（Δ bamB ）中具有额外缺陷的Δ tolA突变体表现出严重的分裂表型，即使单个突变体正常分裂也是如此。这突出了 OM 表型间接影响细胞分裂的可能性。总体而言，我们的工作强调了 Tol-Pal 功能的复杂性，并加强了其在细胞壁-OM 束缚、细胞壁重塑，特别是 OM 稳态中的关键作用。