The outer membrane of Gram-negative bacteria is essential for their survival in harsh environments and provides intrinsic resistance to many antibiotics. This membrane is remarkable; it is a highly asymmetric lipid bilayer. The inner leaflet of the outer membrane contains phospholipids, whereas the fatty acyl chains attached to lipopolysaccharide (LPS) comprise the hydrophobic portion of the outer leaflet. This lipid asymmetry, and in particular the exclusion of phospholipids from the outer leaflet, is key to creating an almost impenetrable barrier to hydrophobic molecules that can otherwise pass through phospholipid bilayers. It has long been known that these lipids are not made in the outer membrane. It is now believed that conserved multisubunit protein machines extract these lipids after their synthesis is completed at the inner membrane and transport them to the outer membrane. A longstanding question is how the cell builds and maintains this asymmetric lipid bilayer in coordination with the assembly of the other components of the cell envelope. This Review describes the transenvelope lipid transport systems that have been identified to participate in outer-membrane biogenesis: LPS transport via the Lpt machine, and phospholipid transport via the Mla pathway and several recently proposed transporters.

Actinorhizal plants form a symbiotic association with the nitrogen-fixing actinobacteria Frankia. These plants have important economic and ecological benefits including land reclamation, soil stabilization, and reforestation. Recently, many non-Frankia actinobacteria have been isolated from actinorhizal root nodules suggesting that they might contribute to nodulation. Two Nocardia strains, BMG51109 and BMG111209, were isolated from Casuarina glauca nodules, and they induced root nodule-like structures in original host plant promoting seedling growth. The formed root nodule-like structures lacked a nodular root at the apex, were not capable of reducing nitrogen and had their cortical cells occupied with rod-shaped Nocardiae cells. Both Nocardia strains induced root hair deformation on the host plant. BMG111209 strain induced the expression of the ProCgNin:Gus gene, a plant gene involved in the early steps of the infection process and nodulation development. Nocardia strain BMG51109 produced three types of auxins (Indole-3-acetic acid [IAA], Indole-3-Byturic Acid [IBA] and Phenyl Acetic Acid [PAA]), while Nocardia BMG111209 only produced IAA. Analysis of the Nocardia genomes identified several important predicted biosynthetic gene clusters for plant phytohormones, secondary metabolites, and novel natural products. Co-infection studies showed that Nocardia strain BMG51109 plays a role as a "helper bacteria" promoting an earlier onset of nodulation. This study raises many questions on the ecological significance and functionality of Nocardia bacteria in actinorhizal symbioses.

ATP-binding cassette (ABC) transporters constitute a large family of proteins present in all domains of life. They are powered by dynamic ATPases that harness energy from binding and hydrolyzing ATP through a cycle that involves the closing and reopening of their two ATP-binding domains. The LptB2FGC exporter is an essential ABC transporter that assembles lipopolysaccharides (LPS) on the surface of Gram-negative bacteria to form a permeability barrier against many antibiotics. LptB2FGC extracts newly synthesized LPS molecules from the inner membrane and powers their transport across the periplasm and through the outer membrane. How LptB2FGC functions remains poorly understood. Here, we show that the C-terminal domain of the dimeric LptB ATPase is essential for LPS transport in Escherichia coli. Specific changes in the C-terminal domain of LptB cause LPS transport defects that can be repaired by intragenic suppressors altering the ATP-binding domains. Surprisingly, we found that each of two lethal changes in the ATP-binding and C-terminal domains of LptB, when present in combined form, suppressed the defects associated with the other to restore LPS transport to wild-type levels both in vivo and in vitro. We present biochemical evidence explaining the effect that each of these mutations has on LptB function and how the observed cosuppression results from the opposing lethal effects these changes have on the dimerization state of the LptB ATPase. We therefore propose that these sites modulate the closing and reopening of the LptB dimer, providing insight into how the LptB2FGC transporter cycles to export LPS to the cell surface and how to inhibit this essential envelope biogenesis process. IMPORTANCE Gram-negative bacteria are naturally resistant to many antibiotics because their surface is covered by the glycolipid LPS. Newly synthesized LPS is transported across the cell envelope by the multiprotein Lpt machinery, which includes LptB2FGC, an unusual ABC transporter that extracts LPS from the inner membrane. Like in other ABC transporters, the LptB2FGC transport cycle is driven by the cyclical conformational changes that a cytoplasmic, dimeric ATPase, LptB, undergoes when binding and hydrolyzing ATP. How these conformational changes are controlled in ABC transporters is poorly understood. Here, we identified two lethal changes in LptB that, when combined, remarkably restore wild-type transport function. Biochemical studies revealed that the two changes affect different steps in the transport cycle, having opposing, lethal effects on LptB’s dimerization cycle. Our work provides mechanistic details about the LptB2FGC extractor that could be used to develop Lpt inhibitors that would overcome the innate antibiotic resistance of Gram-negative bacteria.

Lipopolysaccharides (LPS) are essential envelope components in many Gram‐negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP‐binding cassette (ABC) transporter LptB2FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS‐binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP‐driven movements of LptB and those of the substrate‐binding cavity in LptFG are bi‐directionally coordinated through the rigid‐body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.

The cell surface of the Gram-negative cell envelope contains lipopolysaccharide (LPS) molecules, which form a permeability barrier against hydrophobic antibiotics. The LPS transport (Lpt) machine composed of LptB2FGCADE forms a proteinaceous trans-envelope bridge that allows for the rapid and specific transport of newly synthesized LPS from the inner membrane (IM) to the outer membrane (OM). This transport is powered from the IM by the ATP-binding cassette transporter LptB2FGC. The ATP-driven cycling between closed- and open-dimer states of the ATPase LptB2 is coupled to the extraction of LPS by the transmembrane domains LptFG. However, the mechanism by which LPS moves from a substrate-binding cavity formed by LptFG at the IM to the first component of the periplasmic bridge, the periplasmic β-jellyroll domain of LptF, is poorly understood. To better understand how LptB2FGC functions in Escherichia coli, we searched for suppressors of a defective LptB variant. We found that defects in LptB2 can be suppressed by both structural modifications to the core oligosaccharide of LPS and changes in various regions of LptFG, including a periplasmic loop in LptF that connects the substrate-binding cavity in LptFG to the periplasmic β-jellyroll domain of LptF. These novel suppressors suggest that interactions between the core oligosaccharide of LPS and periplasmic regions in the transporter influence the rate of LPS extraction by LptB2FGC. Together, our genetic data reveal a path for the bi-directional coupling between LptB2 and LptFG that extends from the cytoplasm to the entrance to the periplasmic bridge of the transporter. IMPORTANCE Gram-negative bacteria are intrinsically resistant to many antibiotics due to the presence of lipopolysaccharide (LPS) at their cell surface. LPS is transported from its site of synthesis at the inner membrane to the outer membrane by the Lpt machine. Lpt proteins form a transporter that spans the entire envelope and is thought to function similarly to a PEZ candy dispenser. This trans-envelope machine is powered by the cytoplasmic LptB ATPase through a poorly understood mechanism. Using genetic analyses in Escherichia coli, we found that LPS transport involves long-ranging bi-directional coupling across cellular compartments between cytoplasmic LptB and periplasmic regions of the Lpt transporter. This knowledge could be exploited in developing antimicrobials that overcome the permeability barrier imposed by LPS.