title: A triple mutant of Escherichia coli lacking secondary acyl chains on lipid A.
abstract: All possible combinations of insertion mutations in the three genes encoding the acyl carrier protein-dependent late acyltransferases of lipid A biosynthesis, designated lpxL(htrB), lpxM(msbB), and lpxP, were generated in Escherichia coli K12 W3110. Mutants defective in lpxM synthesize penta-acylated lipid A molecules and grow normally. Strains lacking lpxP fail to incorporate palmitoleate into their lipid A at 12 degrees C but make normal amounts of hexa-acylated lipid A and are viable. Although lpxL mutants and lpxL lpxM double mutants grow slowly on minimal medium at all temperatures, they do not grow on nutrient broth above 32 degrees C. Such mutants retain the ability to synthesize some penta- and hexa-acylated lipid A molecules because of limited induction of lpxP at 30 degrees C but not above 32 degrees C. MKV15, an E. coli lpxL lpxM lpxP triple mutant, likewise grows slowly on minimal medium at all temperatures but not on nutrient broth at any temperature. MKV15 synthesizes a lipid A molecule containing only the four primary (R)-3-hydroxymyristoyl chains. The outer membrane localization and content of lipid A are nearly normal in MKV15, as is the glycerophospholipid and membrane protein composition. However, the rate at which the tetra-acylated lipid A of MKV15 is exported to the outer membrane is reduced compared with wild type. The integrity of the outer membrane of MKV15 is compromised, as judged by antibiotic hypersensitivity, and MKV15 undergoes lysis following centrifugation. MKV15 may prove useful as a host strain for expressing late acyltransferase genes from other Gram-negative bacteria, facilitating the re-engineering of lipid A structure in living cells and the design of novel vaccines.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Mara K
authors.initials: MK
authors.lastname: Vorachek-Warren
authors.forename: Suzanne
authors.initials: S
authors.lastname: Ramirez
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D000217:Acyltransferases / Q000235:genetics; D001426:Bacterial Proteins; D002240:Carbohydrate Sequence; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008050:Lipid A / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008050:Lipid A; D000217:Acyltransferases; C492237:LpxL protein, E coli; C118473:LpxL protein, bacteria; C097895:MsbB protein, E coli; C118452:lpxP protein, E coli