构建大肠埃希菌pgaABCD基因缺失株和互补株的生物被膜形成能力  

Construction of pga ABCD gene deletion and complementation strains and properties of biofilm formation ability for Escherichia coli

作　　者：宫海燕[1] 程倩 赵智龙 施洋[3] Gong Haiyan;Cheng Qian;Zhao Zhilong;Shi Yang(Department of Laboratory Medicine,the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830000,Xinjiang Uygur Autonomous Region,China;National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Xinjiang Medical University,Urumqi 830000,Xinjiang Uygur Autonomous Region,China)

机构地区：[1]新疆医科大学第五附属医院检验科,新疆维吾尔自治区乌鲁木齐市830000 [2]中国疾病预防控制中心传染病预防控制所,北京市102206 [3]新疆医科大学,新疆维吾尔自治区乌鲁木齐市830000

出　　处：《中国组织工程研究》2022年第17期2738-2743,共6页Chinese Journal of Tissue Engineering Research

基　　金：新疆维吾尔自治区卫生健康青年医学科技人才专项科研项目(WJWY-201916),项目负责人:宫海燕;新疆维吾尔自治区创新环境(人才、基地)建设专项—天山青年计划项目(青年博士科技人才项目,2019Q081),项目负责人:宫海燕。

摘　　要：背景:大肠埃希菌形成生物被膜引发难治性的慢性感染,严重威胁了人类的健康。pgaABCD操纵子是生物被膜形成的重要调节基因之一。目的:构建大肠埃希菌缺失菌株MG1655/Δpga,并分别回补pgaA、pgaB、pgaC、pgaD,构建互补株Δpga/pgaA、Δpga/pgaB、Δpga/pgaC、Δpga/pgaD,观察缺失株、互补株的生物被膜形成能力、胞外多糖含量及菌株形态学特征,分析pga基因对生物被膜的调节作用。方法:在Lambda Red重组系统基础上,将pKD46辅助质粒转化入MG1655感受态细胞,获得MG1655/pKD46单克隆。pgaABCD打靶片段转入感受态细胞MG1655/pKD46,利用辅助质粒pCP20构建缺失株MG1655/ΔG16。以质粒pBR322作为回补载体,构建pBR322-pgaA、pgaB、pgaC、pgaD四个质粒,通过电转化的方式分别转入MG1655/ΔG16,获得相应的互补株。应用刚果红平板、结晶紫半定量法、透射电镜检测构建菌株生物被膜形成能力特性的变化,苯酚-硫酸法检测其胞外多糖的含量。结果与结论:(1)缺失株生物被膜形成能力和胞外多糖含量显著降低(P<0.05),有细胞溃烂溶解现象,切片发现菌内空泡变性;(2)提示大肠埃希菌pgaABCD基因的缺失会影响生物被膜的特性,且pgaA、pgaB、pgaC、pgaD均参与生物被膜的形成,但不是唯一调控基因。BACKGROUND:Escherichia coli can cause difficult refractory infections,which seriously threaten human health,due to the formation of“biofilms.”The pgaABCD opero is one of the important regulatory genes for biofilm formation.OBJECTIVE:To construct gene deletion strain MG1655/Δpgaof Escherichia coli,construct the complementation strains ofΔpga/pgaA,Δpga/pgaB,Δpga/pgaC,Δpga/pgaD by complementing pgaA,pgaB,pgaC and pgaD,respectively,observe the biofilm formation ability,and exopolysaccharide and morphological characteristics of deletion and complementation strains,and analyze the effects of gene pgaon the regulation and characteristics of biofilm.METHODS:Based on the Lambda Red recombination system,helper plasmid p KD46 was transferred into competent cells MG1655 to obtain MG1655/p KD46 monoclones.Targeting fragment of pgaABCD gene was transformed to competent cells MG1655/p KD46 to construct gene deleted mutant of MG1655/Δpga.Using plasmid pBR322 as vector,pBR322-pgaA,pBR322-pgaB,pBR322-pgaC,and pBR322-pgaD were constructed and transformed into MG1655/Δpgato obtain the corresponding complementation strains.Congo red-broth agar plate,crystal violet semi-quantitative method,and transmissionelectronmicroscopy were utilized to detect property changes of biofilm formation capacity of construction strains.At the same time,extracellular polysaccharide was detected using phenol-sulfuric acid method.RESULTS AND CONCLUSION:The biofilm formation capacity and extracellular polysaccharide of deletion strains were significantly decreased(P<0.05).Some cells were damaged and collapsed.The section showed vacuolardegeneration in bacteria.To conclude,deletion of gene pgaABCD can affect the biofilm characteristics for Escherichia coli,and the genes pgaA,pgaB,pgaC and pgaD,but not the only regulatory gene,are involved in the biofilm formation.

关 键 词：大肠埃希菌 pgaABCD 生物被膜 胞外多糖 缺失株 互补株 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]