title: Density gradient enrichment of Escherichia coli lpxL mutants.
abstract: We previously described enrichment of conditional Escherichia coli msbA mutants defective in lipopolysaccharide export using Ludox density gradients (Doerrler WT (2007) Appl Environ Microbiol 73; 7992-7996). Here, we use this approach to isolate and characterize temperature-sensitive lpxL mutants. LpxL is a late acyltransferase of the pathway of lipid A biosynthesis (The Raetz Pathway). Sequencing the lpxL gene from the mutants revealed the presence of both missense and nonsense mutations. The missense mutations include several in close proximity to the enzyme's active site or conserved residues (E137K, H132Y, G168D). These data demonstrate that Ludox gradients can be used to efficiently isolate conditional E. coli mutants with defects in lipopolysaccharide biosynthesis and provide insight into the enzymatic mechanism of LpxL.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Six
authors.forename: Bliss
authors.initials: B
authors.lastname: Lambert
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
authors.forename: William T
authors.initials: WT
authors.lastname: Doerrler
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002499:Centrifugation, Density Gradient; D018389:Codon, Nonsense; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D008050:Lipid A / Q000096:biosynthesis; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D012822:Silicon Dioxide
chemical_list: D018389:Codon, Nonsense; D029968:Escherichia coli Proteins; D008050:Lipid A; C024922:Ludox AM; D012822:Silicon Dioxide; D000217:Acyltransferases; C492237:LpxL protein, E coli