title: Replacement of lipopolysaccharide with free lipid A molecules in Escherichia coli mutants lacking all core sugars.
abstract: Escherichia coli mutants deficient in 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by overexpression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetraacylated precursor lipid IV(A) replacing lipopolysaccharide [Meredith, T. C., et al. (2006) ACS Chem. Biol. 1, 33-42]. Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IV(A) reaches the outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexaacylated lipid A, we overexpressed the lauroyl- or the myristoyltransferase of lipid A biosynthesis, encoded by lpxL and lpxM, respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IV(A). Overexpression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 degrees C without the need for MsbA overproduction. These strains accumulated penta- and hexaacylated free lipid A containing a secondary laurate chain or a laurate and a myristate chain, respectively. Deletion of kdtA in strains overexpressing LpxM accumulated pentaacylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 degrees C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexaacylated lipid A, which is optimal for the MsbA flippase.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: C Michael
authors.initials: CM
authors.lastname: Reynolds
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics; D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002241:Carbohydrates / Q000172:deficiency* / Q000235:genetics*; D048168:Escherichia coli K12 / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008050:Lipid A / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D014166:Transferases / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002241:Carbohydrates; D029968:Escherichia coli Proteins; D008050:Lipid A; D008070:Lipopolysaccharides; C079726:MsbA protein, Bacteria; D014166:Transferases; D000217:Acyltransferases; C492237:LpxL protein, E coli; C019917:3-deoxy-D-manno-octulosonate transferase