Density gradient enrichment of Escherichia coli lpxL mutants

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• 作者：David A. Six^b ; ¹ ; Bliss Lambert^a ; Christian R.H. Raetz^b ; ² ; William T. Doerrler^a ; ^{wdoerr@lsu.edu}
• 关键词：LPS ; lipopolysaccharide ; TLC ; thin layer chromatography ; Amp ; ampicillin ; Kan ; kanamycin
• 刊名：Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids
• 出版年：2012
• 期刊代码：18_13881981
• 类别：bio
• 出版时间：July, 2012
• 卷：1821
• 期：7
• 页码：989-993
• 文件大小：495 K

摘要

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.