Fitzmaurice Dylan R, Amador Anthony, Starr Tahj, Hocky Glen M, Rojas Enrique R
Department of Biology, New York University, New York, New York.
Department of Chemistry and Simons Center for Computational Physical Chemistry, New York University, New York, New York.
Biophys J. 2025 Mar 4;124(5):765-777. doi: 10.1016/j.bpj.2025.01.017. Epub 2025 Jan 27.
The outer membrane is the defining structure of Gram-negative bacteria. We previously demonstrated that it is a major load-bearing component of the cell envelope and is therefore critical to the mechanical robustness of the bacterial cell. Here, to determine the key molecules and moieties within the outer membrane that underlie its contribution to cell envelope mechanics, we measured cell-envelope stiffness across several sets of mutants with altered outer-membrane sugar content, protein content, and electric charge. To decouple outer membrane stiffness from total cell envelope stiffness, we developed a novel microfluidics-based "osmotic force-extension" assay. In tandem, we developed a method to increase throughput of microfluidics experiments by performing them on color-coded pools of mutants. We found that truncating the core oligosaccharide, deleting the β-barrel protein OmpA, or deleting lipoprotein outer membrane-cell wall linkers all had the same modest, convergent effect on total cell-envelope stiffness in Escherichia coli. However, these mutations had large, variable effects on the ability of the cell wall to transfer tension to the outer membrane during large hyperosmotic shocks. Surprisingly, altering the electric charge of lipid A had little effect on the mechanical properties of the envelope. Finally, the presence or absence of OmpA determined whether truncating the core oligosaccharide decreased or increased envelope stiffness (respectively), revealing sign epistasis between these components. Based on these data we propose a putative structural model in which the spatial interactions between lipopolysaccharides, β-barrel proteins, and phospholipids coordinately determine cell envelope stiffness.
外膜是革兰氏阴性菌的标志性结构。我们之前证明它是细胞壁的主要承重成分，因此对细菌细胞的机械稳健性至关重要。在此，为了确定外膜中对其细胞壁力学贡献起基础作用的关键分子和部分，我们测量了几组外膜糖含量、蛋白质含量和电荷改变的突变体的细胞壁刚度。为了将外膜刚度与整个细胞壁刚度解耦，我们开发了一种基于微流控的新型“渗透力-伸展”测定法。同时，我们开发了一种方法，通过在颜色编码的突变体库上进行微流控实验来提高实验通量。我们发现，截断核心寡糖、删除β桶蛋白OmpA或删除脂蛋白外膜-细胞壁连接体，对大肠杆菌整个细胞壁刚度都有相同的适度、趋同的影响。然而，这些突变对细胞壁在大的高渗冲击期间将张力传递到外膜的能力有很大的、可变的影响。令人惊讶的是，改变脂质A的电荷对细胞壁的力学性质几乎没有影响。最后，OmpA的存在与否决定了截断核心寡糖是降低还是增加细胞壁刚度（分别对应），揭示了这些成分之间的符号上位性。基于这些数据，我们提出了一个推定的结构模型，其中脂多糖、β桶蛋白和磷脂之间的空间相互作用协同决定细胞壁刚度。
