Six David A, Lambert Bliss, Raetz Christian R H, Doerrler William T
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Biochim Biophys Acta. 2012 Jul;1821(7):989-93. doi: 10.1016/j.bbalip.2012.04.003. Epub 2012 Apr 14.
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.
我们之前描述了使用Ludox密度梯度法富集在脂多糖输出方面存在缺陷的条件性大肠杆菌msbA突变体（Doerrler WT（2007年），《应用与环境微生物学》73卷；7992 - 7996页）。在此，我们使用这种方法来分离和表征温度敏感型lpxL突变体。LpxL是脂多糖生物合成途径（雷茨途径）中的一种晚期酰基转移酶。对突变体的lpxL基因进行测序发现存在错义突变和无义突变。错义突变包括几个靠近酶活性位点或保守残基的突变（E137K、H132Y、G168D）。这些数据表明，Ludox梯度可用于有效分离在脂多糖生物合成方面存在缺陷的条件性大肠杆菌突变体，并为LpxL的酶促机制提供见解。
