atpA基因缺失对貉源大肠杆菌生物学特性的影响  

Effect of atpA gene deletion on biological characteristics of Escherichia coli LCE-1 from raccoon dogs

作　　者：侯冠欣 马亚娟 王苗 孙欣艺 王利丽 张闫 张春晓 张志强[1] HOU Guan-xin;MA Ya-juan;WANG Miao;SUN Xin-yi;WANG Li-li;ZHANG Yan;ZHANG Chun-xiao;ZHANG Zhi-qiang(Hebei Normal University of Science&Technology,Qinhuangdao 066004,China;Hebei Agricultural University,Baoding 071001,China;Center for Prevention and Control of Animal Epidemic Disease in Wuhan,Wuhan 430000,China)

机构地区：[1]河北科技师范学院,河北秦皇岛066004 [2]河北农业大学,河北保定071001 [3]武汉市动物疫病预防控制中心,湖北武汉430000

出　　处：《中国预防兽医学报》2023年第8期794-800,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：河北省省级科技计划资助(19226629D);河北省高等学校科学技术研究项目(BJ2021055)。

摘　　要：为研究编码ATP合酶α亚基对大肠杆菌生物学特性的影响,本研究利用λ-Red同源重组技术构建貉源致病性大肠杆菌LCE-1(WT)atpA基因缺失菌株LCE-1ΔatpA(KO)和基因回补株LCE-1ΔatpA/pBR322-atpA(RS),并均采用PCR和测序鉴定。在此基础上,进一步分析atpA基因缺失对大肠杆菌的生长特性、生化特性的影响,结果显示,在LB培养基中KO菌株与WT菌株相比生长无明显差异,但在M9培养基中生长明显减缓;KO菌株的部分生化特性也发生了变化,对蔗糖、肌醇的分解利用发生改变,α-葡萄糖苷酶、β葡萄糖醛酸酶、精氨酸双水解酶活性也发生了改变。通过试管及微量结晶紫法分析3株菌生物被膜(BF)的形成能力,结果显示atp基因缺失能够显著降低大肠杆菌BF的形成能力(P<0.01)。对这3株菌进行酸应激、碱应激、热应激以及氧化应激测定这3种菌株对环境变化的应激能力,结果显示atp基因缺失能够显著提高大肠杆菌对酸应激、碱应激、热应激和氧化应激的敏感性。通过K-B纸片法对这3株菌的耐药性进行测定,结果显示KO菌株对多种抗生素的敏感性有所增强。利用KO、WT菌株分别感染小鼠进行致病性试验,结果显示,WT菌株的LD50为4.12×10^(7) cfu/mL,KO菌株的LD50为3.43×10^(8) cfu/mL,KO菌株毒力下降1个数量级。本研究构建了大肠杆菌的atpA基因缺失株,并证实AtpA蛋白在细菌的生化特性以及应对各种应激过程中具有重要作用,是大肠杆菌一个重要的多功能蛋白。To investigate the effect of theαsubunit of ATP synthase on the biological characteristics of Escherichia coli(E.coli),the atpA gene deletion strain LCE-1ΔatpA(KO)was constructed byλ-Red homologous recombination technique based on a pathogenic E.coli LCE-1(WT)from raccoon dog,and the pBR322-atpA was constructed and transformed into the deletion mutant resulting in the complementary strain LCE-1ΔatpA/pBR322-atpA(RS).On this basis,the effects of atpA gene deletion on the growth and biochemical characteristics of E.coli were further analyzed.The results showed that there was no significant difference between the growth of KO strain and WT strain in LB medium,while the growth of the KO strain obviously slowed down in M9 medium,and some biochemical characteristics of the KO strain altered,for instance the decomposition and utilization of sucrose and inositol and the activities ofα-glucosidase,β-Glucuronic acid and arginine dihydrolase.The biofilm formation(BF)ability of the three strains was evaluated by tube test and microwell test,and it showed that the BF ability of the KO strain significantly reduced(P<0.01).Acid stress,alkali stress,heat stress and oxidative stress were tested to evaluate the stress ability of the three strains to environmental changes,the results showed that the sensitivity of the KO strain to acid stress,alkali stress,heat stress and oxidative stress significantly increased.The drug resistance of these three strains was determined by K-B disk method.The results showed that the KO strain were more sensitive to many kinds of antibiotics.The results of pathogenicity test on mice showed that the LD50 of WT strain and KO strain was 4.12×10^(7)cfu/mL and 3.43×10^(8)cfu/mL,respectively,and the virulence of the KO strain decreased by about 10 times compared to the WT strain.In this study,we successfully constructed the aptA gene deletion strain of E.coli,and found that AptA protein is an important multifunctional protein in E.coli,which plays an important role in the biochemical characteristics

关 键 词：貉源致病性大肠杆菌 λ-Red同源重组 atpA基因缺失 

分 类 号：S852.61[农业科学—基础兽医学]