Junker F, Rodríguez-Herva J J, Duque E, Ramos-González M I, Llamas M, Ramos J L
Consejo Superior de Investigaciones Cientificas, Estación Experimental del Zaidin, Department of Plant Biochemistry and Molecular and Cellular Biology, Granada, Spain.
Extremophiles. 2001 Apr;5(2):93-9. doi: 10.1007/s007920100176.
Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.
脂多糖(LPS)是革兰氏阴性菌外膜的主要成分,被视为一种防御屏障。为了确定LPS在耐溶剂恶臭假单胞菌DOT-T1E菌株对溶剂的抗性中是否起作用,我们构建了无法合成LPS O抗原侧链的突变体。克隆、测序了编码耐溶剂菌株LPS O抗原侧链合成起始酶的wbpL基因,并在体外使用编码卡那霉素抗性的盒式结构将其敲除,通过定点诱变在体内产生了DOT-T1E菌株的一个名为WbpL0的突变体。将WbpL突变体与野生型菌株在对多种有毒化合物(包括螯合剂、有机酸、去污剂和芳香烃)的耐受性方面进行了比较。结果发现,该突变体对有机酸和芳香烃的耐受性与野生型菌株相同,但对乙二胺四乙酸和脱氧胆酸盐更敏感。