假单胞菌ZS1鼠李糖基转移酶migA和wapR的缺失对细胞壁结构及鼠李糖脂合成的影响  

Loss of Rhamnoyl Transferase migA and wapR Affects Cell Wall Structure and Rhamnolipid Synthesis in Pseudomonas aeruginosa ZS1

作　　者：张增平 金扬帆 马昀灏 杨支力 刘建华 ZHANG Zengping;JIN Yangfan;MA Yunhao(School of Marine Science and Technology of Zhejiang Ocean University,Zhoushan 316022,China)

机构地区：[1]浙江海洋大学海洋科学与技术学院,浙江舟山316022

出　　处：《浙江海洋大学学报（自然科学版）》2023年第4期352-359,共8页Journal of Zhejiang Ocean University：Natural Science

基　　金：国家自然科学基金面上项目(31571392)。

摘　　要：假单胞菌ZS1是本实验室在前期工作中从油污中分离出的1株高产鼠李糖脂菌株。本实验拟探究阻断脂多糖O-抗原添加鼠李糖基对假单胞菌ZS1鼠李糖脂合成的影响。通过两步法敲除技术,构建了假单胞菌ZS1的wapR和migA缺失突变菌株。发酵结果表明wapR缺失突变株在培养过程中鼠李糖脂合成量显著增加,而migA缺失突变株则只有少量的提升。SDS-PAGE银染结果表明,wapR缺失突变菌株的脂多糖的组分出现显著变化,O-抗原部分几乎完全消失。而migA缺失突变菌株脂多糖组成无显著变化。通过对突变株和野生株在不同抗生素的梯度浓度培养,发现wapR缺失突变菌株对四环素浓度敏感性从125μg·mL^(-1)降低至31.2μg·mL^(-1),而migA缺失突变菌株对四环素的敏感性与野生株相仿,说明假单胞菌脂多糖的缺失能够提高细菌对抗生素的敏感性,从而降低病原菌的危害性。Pseudomonas aeruginosa ZS1 is a high-yielding rhamnolipid strain isolated from oil stains in our laboratory in the previous work.This experiment intends to explore the effect of blocking lipopolysaccharide O-antigen adding rhamnosyl group on the synthesis of rhamnolipid in P.aeruginosa ZS1.We constructed a wapR and migA deletion mutant strain of P.aeruginosa ZS1 by a two-step resistance-free marker gene residue knockout technique.Fermentation results showed that rhamnolipid synthesis was significantly increased in the wapR mutant during the culture process,while only a small increase in the migA deletion mutant.At the same time,the results of SDS-PAGE analysis showed that the structure of lipopolysaccharide in wapR deletion mutant strains changed significantly,and the O-antigen part almost completely disappeared.By culturing the mutant strain and the wild strain under different antibiotic gradient concentrations,it was found that the wapR deletion mutant strain decreased from the highest resistance concentration to tetracycline from 125μg·mL^(-1)to 31.2μg·mL^(-1),while the migA deletion mutant strain was similar to the wild strain,indicating that the destruction of the lipopolysaccharide structure of Gram-negative bacteria can help improve the sensitivity of bacteria to antibiotics and reduce the harm of pathogenic bacteria.

关 键 词：铜绿假单胞菌 鼠李糖脂 脂多糖 O-抗原 基因敲除 

分 类 号：TQ925.6[轻工技术与工程—发酵工程]