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Gram-negative bacteria are defined by an outer membrane (OM) that contributes to envelope integrity and barrier function. Building this bilayer require proper assembly of lipopolysaccharides, proteins, and phospholipids, yet how the balance of these components is achieved is unclear. One system long known for ensuring OM stability is the Tol-Pal complex, which has been implicated in maintaining OM lipid homeostasis. However, assignment of Tol-Pal function has been challenging, owing to its septal localization and associated role(s) during division. Here, we uncouple the function of Tol-Pal in OM lipid homeostasis from its impact on cell division in Escherichia coli , by engineering a chimeric complex that loses septal enrichment. We demonstrate that this peripherally-localized Tol-Pal complex is fully capable of maintaining lipid balance in the OM, thus restoring OM integrity and barrier. Our work establishes the primary function of the Tol-Pal complex in OM lipid homeostasis, independent of its role during division. The Tol-Pal complex is important for multiple cellular processes in Gram-negative bacteria, but its molecular functions remain unclear. Here, Tan & Chng provide experimental evidence supporting the primary function of the complex in outer-membrane lipid homeostasis.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer with outer leaflet lipopolysaccharides and inner leaflet phospholipids (PLs). This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. To maintain this barrier, the OmpC-Mla system removes mislocalized PLs from the OM outer leaflet, and transports them to the inner membrane (IM); in the first step, the OmpC-MlaA complex transfers PLs to the periplasmic chaperone MlaC, but mechanistic details are lacking. Here, we biochemically and structurally characterize the MlaA-MlaC transient complex. We map the interaction surfaces between MlaA and MlaC in Escherichia coli , and show that electrostatic interactions are important for MlaC recruitment to the OM. We further demonstrate that interactions with MlaC modulate conformational states in MlaA. Finally, we solve a 2.9-Å cryo-EM structure of a disulfide-trapped OmpC-MlaA-MlaC complex in nanodiscs, reinforcing the mechanism of MlaC recruitment, and highlighting membrane thinning as a plausible strategy for directing lipids for transport. Our work offers critical insights into retrograde PL transport by the OmpC-Mla system in maintaining OM lipid asymmetry. Maintenance of lipid asymmetry in the bacterial outer membrane (OM) is mediated by the OmpC-Mla system, but mechanistic details remain to be elucidated. Here, the authors show that electrostatic interactions, conformational dynamics, and membrane thinning may facilitate spontaneous retrograde phospholipid transfer at the OM.

Tuberculosis continues to pose a serious threat to global health. Mycobacterium tuberculosis , the causative agent of tuberculosis, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host. Among their many virulence factors, mycobacteria encode Mce systems. Some of these systems are implicated in lipid uptake, but the molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. We show that the Mce1 system in Mycobacterium smegmatis comprises a canonical ATP-binding cassette transporter associated with distinct heterohexameric assemblies of substrate-binding proteins. Furthermore, we establish that the conserved membrane protein Mce1N negatively regulates Mce1 function via a unique mechanism involving blocking transporter assembly. Our work offers a molecular understanding of Mce complexes, sheds light on mycobacterial lipid metabolism and its regulation, and informs future anti-mycobacterial strategies. Mycobacterial Mce systems are putative ABC transporters for lipids. Here, the authors revealed two distinct Mce1 complexes comprising different heterohexameric substrate binding assemblies that are negatively regulated via a novel mechanism.

The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane β-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD β-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the β-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.

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. 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.

Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1:1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM.

Most Gram-negative bacteria contain lipopolysaccharide (LPS), a glucosamine-based phospholipid, in the outer leaflet of the outer membrane (OM). LPS is unique to the bacterial OM and, in most cases, essential for cell viability. Transport of LPS from its site of synthesis to the cell surface requires eight essential proteins, MsbA and LptABCDEFG. Although the key players have been identified, the mechanism of LPS transport and assembly is not clear. The stable LptD/E complex is present at the OM and functions in the final stages of LPS assembly. Here, we have identified the mutant allele lptE6, which causes a two-amino-acid deletion in the lipoprotein LptE that affects its interaction with LptD. Highly specific suppressor mutations were isolated not only in lptD but also in bamA, which encodes the central component of the β-barrel assembly machine. We show that lptE6 and both suppressor mutations affect the assembly of the LptD/E complex and suggest that the lipoprotein LptE interacts with LptD while this protein is being assembled by the β-barrel assembly machine.

The outer membrane of most Gram-negative bacteria is made up of LPS, and in nearly all bacteria that contain LPS it is essential for the life of the organism. The lipid portion of this molecule, lipid A, also known as endotoxin, is a potent activator of the innate immune response. More than 50 genes are required to synthesize LPS and assemble it at the cell surface. Enormous progress has been made in elucidating the structure and biosynthesis of LPS, but until recently the cellular components required for its transport from its site of synthesis in the inner membrane to its final cellular location at the cell surface remained elusive. Here we describe the identification of a protein complex that functions to assemble LPS at the surface of the cell. This complex contains two proteins: Imp, already identified as an essential outer-membrane protein implicated in LPS assembly; and another protein, RIpB, heretofore identified only as a rare lipoprotein. We show that RIpB is also essential for cell viability and that the Imp/RIpB complex is responsible for LPS reaching the outer surface of the outer membrane.

The presence of lipopolysaccharide (LPS) on the cell surface of Gram-negative bacteria is critical for viability. A conserved β-barrel membrane protein LptD (lipopolysaccharide transport protein D) translocates LPS from the periplasm across the outer membrane (OM). In Escherichia coli, this protein contains two disulfide bonds and forms the OM LPS translocon with the lipoprotein LptE. Here, we identified seven in vivo states on the oxidative-folding pathway of LptD. Proper assembly involved a nonfunctional intermediate containing non-native disulfides. Intermediate formation required the oxidase DsbA, and subsequent maturation to the active form with native disulfides was triggered by LptE. Thus, disulfide bond-dependent protein folding of LptD requires the proper assembly of a two-protein complex to promote disulfide bond rearrangement.

The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which, in part, render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. Although the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. Mycobacterial membrane protein Large 3 (MmpL3), an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several antimycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism of action of BM212 as a potent MmpL3 inhibitor, and use it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future antituberculosis drugs.