The cell envelope of gram-negative bacteria consists of an inner (IM) and an outer membrane (OM) separated by an aqueous compartment, the periplasm, which contains the peptidoglycan layer. The OM is an asymmetric bilayer, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) facing outward (29, 32). The OM is an effective permeability barrier that protects the cells from toxic compounds, such as antibiotics and detergents, thus allowing bacteria to inhabit several different and often hostile environments. LPS is responsible of most of the permeability properties of the OM and consists of the lipid A moiety (a glucosamine-based phospholipid) linked to the short core oligosaccharide and the distal O-antigen polysaccharide chain. The core oligosaccharide can be further divided into an inner core, composed of 3-deoxy-Dmanno-octulosanate (KDO) and heptose, and an outer core, which has a somewhat variable structure. LPS is essential in most gram-negative bacteria, with the notable exception of Neisseria meningitidis (39).The biogenesis of the OM implies that the individual components are transported from the site of synthesis to their final destination outside the IM by crossing both hydrophilic and hydrophobic compartments. The machinery and the energy source that drive this process are not yet understood.The lipid A-core moiety and the O-antigen repeat units are synthesized at the cytoplasmic face of the IM and are separately exported via two independent transport systems, namely, the O-antigen transporter Wzx (13, 17) and the ATP binding cassette (ABC) transporter MsbA that flips the lipid A-core moiety from the inner leaflet to the outer leaflet of the IM (12,28,45). O-antigen repeat units are then polymerized in the periplasm by the Wzy polymerase and ligated to the lipid A-core moiety by the WaaL ligase (reference 29 and references therein). Escherichia coli K-12 LPS is missing the O antigen, as an IS5 insertion disrupts its synthesis (18). Very recently, a modified LPS in which repeating units of colanic acid, a cell surface polysaccharide synthesized by enteric bacteria in the presence of envelope-damaging stresses (42), are ligated to the core oligosaccharide in a WaaL-dependent manner has been described (21).How LPS reaches the OM is less well understood. A protein complex in the OM of E. coli composed of LptD (formerly Imp), an essential ␤-barrel OM protein (6), and LptE (formerly RlpB), an essential OM lipoprotein, has recently been implicated in LPS assembly (43). Depletion of either protein results in similar OM biogenesis defects, including increased LPS levels, abnormal membrane structures, and activation of the OM enzyme PagP (43). These findings indicate that the LptD/LptE complex is responsible for LPS assembly at the outer surface of the OM (43). LptD has also been shown to be

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.The cell envelope of gram-negative bacteria consists of an inner (IM) and an outer (OM) membrane separated by the periplasmic space, which contains the peptidoglycan layer. The OM is an asymmetric bilayer, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) facing outward (28, 32). LPS, a molecule unique to gram-negative bacteria, consists of the lipid A moiety (a glucosamine-based phospholipid) linked to a short-core oligosaccharide and the distal O-antigen polysaccharide chain. The core oligosaccharide can be further divided into the inner core, composed of 3-deoxy-D-mannooctulosanate (KDO) and heptose, and the outer core, with a somewhat variable structure. Escherichia coli K-12 synthesizes a shorter LPS consisting of a lipid A moiety linked to a short-core oligosaccharide but missing the O-antigen chain (28). LPS is essential in most gram-negative bacteria, with the notable exception of Neisseria meningitidis. In E. coli, the minimal part required for viability consists of the KDO 2 lipid IV A moiety (28).LPS is synthesized in the cytoplasmic face of the IM and must traverse the IM and the periplasm to reach its final destination in the outer face of the OM (6, 28). Translocation across the IM requires the ABC transporter MsbA, which mediates the flipping from the inner leaflet (the site of the synthesis) to the outer leaflet of the IM (14, 27, 44). MsbA has also been implicated in phospholipid transport across the IM of E. coli (13,14). Interestingly, MsbA in N. meningitidis is not essential and seems not to be required for phospholipid transport (39). How LPS traverses the periplasm and is inserted into the OM is much less understood.Because of its hydrophobic lipid A moiety, transport of LPS through the aqueous periplasm is thermodynamically un...

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SUMMARY Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6′-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.