Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

• Published in: Journal of Bacteriology Vol. 195; no. 5; pp. 1100 - 1108
• Main Authors: Villa, Riccardo, Martorana, Alessandra M, Okuda, Suguru, Gourlay, Louise J, Nardini, Marco, Sperandeo, Paola, Dehò, Gianni, Bolognesi, Martino, Kahne, Daniel, Polissi, Alessandra
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01-03-2013
• Subjects: ABC transporters; ATP-Binding Cassette Transporters - metabolism; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial proteins; Bacteriology; Binding sites; Biochemistry; Biological Transport; Carrier Proteins - metabolism; E coli; Escherichia coli; Escherichia coli - cytology; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Gram-negative bacteria; Lipids; lipopolysaccharides; Lipopolysaccharides - metabolism; Membrane Proteins - metabolism; Membranes; mutants; Mutation; Permeability; Protein Structure, Tertiary; toxicity
• ISSN: 0021-9193; 1098-5530; 1098-5530; 1067-8832
• DOI: 10.1128/JB.02057-12

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.