The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [¹⁴C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.

革兰氏阴性细菌包膜的标志是存在外膜 (OM)。OM是不对称的，包括外叶中的脂多糖（LPS）和内叶中的磷脂（PLs）；这一关键特征赋予渗透屏障功能以抵御外部损害，包括抗生素。为了维持 OM 脂质不对称，OmpC-Mla 系统被认为可以从 OM 中去除异常定位的 PL，并将它们运输到内膜 (IM)。驱动脂质运输系统的关键是 IM 中的 MlaFEDB ATP 结合盒转运蛋白复合物，但包括运输方向性在内的机械细节仍然是个谜。在这里，我们开发了一种敏感的点对点体外脂质转移试验，可以直接跟踪 [ ¹⁴周质伴侣 MlaC 和 MlaFEDB 之间的 C] 标记的 PL 重组为纳米圆盘。我们揭示了 MlaC 以 MlaD 依赖的方式自发地将 PLs 转移到 IM 转运蛋白，这种方式可以通过耦合的 ATP 水解进一步增强。此外，我们表明 MlaD 对于调节 ATP 水解和这种逆行 PL 转移之间的生产耦合很重要。我们进一步证明，自发的 PL 转移也发生从 MlaFEDB 到 MlaC，但这种顺行运动反而被 ATP 水解所消除。我们的工作揭示了一个模型，其中 PL 在 MlaC 和 MlaFEDB 的两个脂质结合位点之间可逆地分配，并且 ATP 结合和/或水解改变了这种平衡，最终通过 OmpC-Mla 系统驱动逆行 PL 运输。