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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 [(14)C]-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.

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 ABC 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]-labelled 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 towards discovering new antibiotics against Gram-negative pathogens.Significance StatementBiological membranes define cellular boundaries, allow compartmentalization, and represent a prerequisite for life. In Gram-negative bacteria, the outer membrane (OM) prevents entry of toxic substances, conferring intrinsic resistance against many antibiotics. This barrier function requires unequal distribution of lipids across the OM bilayer, yet how such lipid asymmetry is maintained is not well understood. In this study, we established the directionality of lipid transport for a conserved membrane protein complex, and uncovered mechanistic insights into how ATP powers such transport from the OM to the inner membrane. Our work provides fundamental understanding of lipid trafficking within the Gram-negative double-membrane envelope in the context of OM lipid asymmetry, and highlights the importance of targeting lipid transport processes for future antibiotics development.

In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry. DOI: http://dx.doi.org/10.7554/eLife.19042.001, eLife digest Escherichia coli are bacteria that can cause vomiting and diarrhoea in humans and other mammals. Each E. coli cell is surrounded by two membranes, which are each made of two layers of fat molecules known as lipids. The outer membrane prevents the entry of toxic compounds and allows E. coli to withstand damaging agents from outside the cell, such as antibiotics. The outer membrane’s ability to act as an effective barrier depends on an asymmetric, or uneven, distribution of lipid molecules across its two layers. The inside layer is dominated by phospholipids, whereas the outside layer is comprised mainly of lipids with attached sugars. The distribution of the two lipid types is maintained by a molecular machine with components that can be found in both the inner and outer membranes. This machine is thought to remove phospholipids from the outside layer of the outer membrane and transport them back to the inner membrane. A group (or”complex”) of proteins known as MIaFEDB operates as a part of this machine at the inner membrane. MlaFEDB is believed to use energy derived from the breakdown of a molecule called ATP to help ensure that phospholipids removed from the outside layer of the outer membrane are reinserted into the inner membrane. It was proposed that the complex contains four proteins, but it was not clear exactly how these components are arranged. Now, Thong et al. reveal how MlaFEDB is organized and characterize the roles of the individual protein components. The experiments confirm that the MlaFEDB complex is made up of four proteins, including two core components and two support proteins (called MlaB and MlaD). There are six copies of MlaD in the complex. In addition, MlaD has a strong affinity for phospholipids and plays a role in controlling the rate at which energy is harnessed through the breakdown of ATP. Further experiments show that the other support protein MlaB is necessary for both the proper assembly and activity of the complex, likely through its interaction with one of the core components. The next step following on from this work is to directly observe MlaFEDB in action to find out how it uses energy to insert lipids into the inner membrane. In the long term, more information about the structure of the complex would be needed to further understand how it works at the molecular level. DOI: http://dx.doi.org/10.7554/eLife.19042.002