Gram-negative bacteria are surrounded by a cell envelope that comprises an outer membrane (OM) and an inner membrane that, together, delimit the periplasmic space, which contains the peptidoglycan (PG) sacculus. Covalent anchoring of the OM to the PG is crucial for envelope integrity in Escherichia coli. When the OM is not attached to the PG, the OM forms blebs and detaches from the cell. The Braun lipoprotein Lpp¹ covalently attaches OM to the PG but is present in only a small number of γ-proteobacteria; the mechanism of OM–PG attachment in other species is unclear. Here, we report that the OM is attached to PG by covalent cross-links between the N termini of integral OM β-barrel-shaped proteins (OMPs) and the peptide stems of PG in the α-proteobacteria Brucella abortus and Agrobacterium tumefaciens. Cross-linking is catalysed by l,d-transpeptidases and attached OMPs have a conserved alanyl-aspartyl motif at their N terminus. Mutation of the aspartate in this motif prevents OMP cross-linking and results in OM membrane instability. The alanyl-aspartyl motif is conserved in OMPs from Rhizobiales; it is therefore feasible that OMP–PG cross-links are widespread in α-proteobacteria.

革兰氏阴性细菌被一个细胞包膜包围，该细胞包膜包括一个外膜 (OM) 和一个内膜，它们一起界定了包含肽聚糖 (PG) 球囊的周质空间。OM 与 PG 的共价锚定对于大肠杆菌的包膜完整性至关重要。当 OM 未连接到 PG 时，OM 会形成气泡并与细胞分离。Braun 脂蛋白 Lpp ¹将 OM 共价连接到 PG 上，但仅存在于少数 γ-变形菌中；其他物种中 OM-PG 附着的机制尚不清楚。在这里，我们报告了 OM 通过整合 OM β-桶形蛋白 (OMP) 的 N 末端与流产布鲁氏菌中 PG 的肽茎之间的共价交联连接到 PG。和根癌农杆菌。交联由l , d-转肽酶催化，连接的 OMP 在其 N 末端具有保守的丙氨酰-天冬氨酰基序。该基序中天冬氨酸的突变阻止了 OMP 交联并导致 OM 膜不稳定。丙氨酰-天冬氨酰基序在根瘤菌的 OMP 中是保守的；因此，OMP-PG 交联在 α-变形菌中广泛存在是可行的。