Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)¹. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins^2,3. In Escherichia coli, Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability⁴; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present⁵. We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

革兰氏阴性菌的细胞包膜包括外膜 (OM)、肽聚糖 (PG) 层和内膜 (IM) ¹。OM 和 PG 是承重、选择性渗透的结构，通过 IM 和 OM 蛋白^2,3之间的合作相互作用来稳定。在大肠杆菌中，布劳恩脂蛋白 (Lpp) 是 OM 和 PG 之间唯一的共价链，对细胞包膜稳定性至关重要⁴；然而，大多数其他革兰氏阴性菌缺乏 Lpp，因此假设存在 OM 稳定的替代机制⁵。我们使用 PG 的糖蛋白组学分析表明，β-桶 OM 蛋白在几种革兰氏阴性菌中与 PG 共价连接，包括Coxiella burnetii、Agrobacterium tumefaciens和Legionella pneumophila。在C. burnetii中，我们发现 OM 蛋白和 PG 之间发生四种不同类型的共价连接，β-桶 OM 蛋白 BbpA 的束缚在静止期变得最丰富，脂蛋白 LimB 的束缚在整个细胞周期中相似。使用遗传方法，我们证明 BbpA 的细胞周期依赖性束缚部分依赖于发育调节的 L、D-转肽酶 (Ldt)。我们利用我们的研究结果提出了一个革兰氏阴性细胞包膜稳定模型，其中包括细胞周期控制和 Ldts 在将表面蛋白共价连接到 PG 中的扩展作用。