The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics and a synthetic lethal screen we show that lengthening Lpp to the upper limit does not change the spatial constraint, but rather impacts the load-bearing capacity across the outer membrane. Our findings demonstrate E. coli expressing elongated Lpp homeostatically counteracts periplasmic enlargement with a combination of tilting Lpp and reducing Lpp abundance. By genetic screening we identified all of the genes in E. coli that become essential in order to enact this homeostasis, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to enact this homeostasis. We observed increased levels of factors determining cell stiffness, decrease membrane integrity, increase membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling and protein translocation

中文翻译：

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周质稳态调节维持对细胞包膜过程和细胞活力必不可少的空间限制

革兰氏阴性菌的细胞膜由两层围绕着周质和肽聚糖层的膜组成。跨越细胞外壳的分子机器取决于空间约束和跨越细胞外壳和表面的承载力。决定细胞包络空间约束的机制仍未完全定义。在大肠杆菌中，卷曲螺旋脂蛋白 Lpp 有助于外膜和下面的肽聚糖层之间的唯一共价键。使用蛋白质组学、分子动力学和合成致死筛选，我们表明将 Lpp 延长到上限不会改变空间约束，而是会影响外膜的承载能力。我们的发现证明了大肠杆菌表达延长的 Lpp 稳态地抵消了周质增大，同时倾斜 Lpp 和降低 Lpp 丰度。通过基因筛选，我们鉴定了大肠杆菌中的所有基因这对于实现这种稳态变得必不可少，并且通过定量蛋白质组学发现，很少有蛋白质需要在稳态水平上上调或下调才能实现这种稳态。我们观察到决定细胞硬度、降低膜完整性、增加膜囊泡的因素水平增加，并且依赖其他非必需的系链来维持脂质转运和肽聚糖生物合成。此外，这对于理解跨包膜的空间约束如何控制鞭毛驱动的运动、细胞信号传导和蛋白质易位等过程具有重要意义