Proc Natl Acad Sci U S A 103:11754C11759. level of is usually Fluo-3 a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery. IMPORTANCE The asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens hard to treat. In the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which XRCC9 participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine. INTRODUCTION Gram-negative bacteria are surrounded by two lipid bilayers, the inner membrane (IM) and outer membrane (OM), showing distinct composition, structural, and Fluo-3 functional properties (1). The two membranes delimit an aqueous compartment, the periplasm, made up of a thin peptidoglycan layer. The IM is usually a symmetric bilayer made of phospholipids, whereas the OM is an asymmetric membrane composed of glycerophospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet (1). LPS is usually a complex glycolipid assembled at the outer leaflet of the OM, where it forms a permeability barrier that prevents access of many hydrophobic toxic compounds, including antibiotics (2). In (17), LptA from and (18, 19), the LptD-LptE complex from and serovar Typhimurium (12, 20), and the cytoplasmic ABC protein LptB from (10, 21), have been solved. Interestingly, LptA, the periplasmic region of LptC, and the periplasmic N-terminal region of LptD, despite a lack of sequence similarity, share a very comparable -jellyroll fold, made of the juxtaposition of a variable quantity of antiparallel strands. Such a -jellyroll fold (the Lpt fold) appears to be a key element in Fluo-3 driving the assembly of the Lpt machinery. In fact, through these structurally homologous domains, the C terminus of LptC interacts with the N terminus of LptA, and the C terminus of LptA interacts with the N-terminal periplasmic domain name of LptD, thus forming a protein bridge that connects the IM and the OM (12, 16, 20, 22). The assembly of the transenvelope bridge appears to be finely regulated to prevent LPS mistargeting. Proper conversation of LptC with LptB2FG is necessary for LptA recruitment (22). Conversation of the N-terminal domain name of LptD with LptA requires the correct maturation of the LptDE complex that in turns depends on nonconsecutive disulfide bond formation in LptD (16); LptE and the chaperone/protease BepA have been implicated in this process (23). Based on photo-cross-linking experiments, LPS molecules appear to cross the periplasm inside the jellyroll of LptC and LptA (9). ATP hydrolysis by LptB, the cytoplasmic ATPase of the LptB2FG transporter, is required for LPS extraction from your IM Fluo-3 and its transfer to LptA via LptC. Energy does not seem to be required for the assembly of the transenvelope bridge (9). Nevertheless, LptB also plays a role in the IM LptCFG subcomplex assembly, as shown by mutants defective in assembly but proficient in the ATPase activity (10). The molecular role of LptC in LPS transport is still elusive. LptC does not seem to be a functional component of the IM ABC transporter, as its association with the LptB2FG complex does not impact its ATPase activity (8); on the other hand, it appears relevant for proper IM complex assembly (22). Mutational analyses suggest that the conversation of LptC with the IM LptB2FG complex is usually mediated by the N-terminal region of the jellyroll, whereas its transmembrane domain name appears to be dispensable, as a periplasmic soluble version of LptC and a LptC chimera transporting a heterologous transmembrane segment is usually functional and proficient in Lpt complex assembly (22). To gain further insights into the role of LptC in the LPS export pathway, we dissected LptC (mutants and the properties of LptC from (ectopic expression suppresses the lethality of C-terminal deletion of LptC and allows the functioning of hybrid Lpt machinery that carries the highly divergent LptC orthologue from bacterial strains used in this.
