Abstract: Escherichia coli O157∶H7 is a common foodborne pathogen that severely threatens public health. Phage, endolysin and holin are new bacteriostatic agents. In this study, phage EC-p9 was identified by random primer for decammeric oligonucleotide, genes of endolysin Lys 9 and holin Hol 9 of the phage were obtained through PCR, properties of the two proteins were predicted through in-silico analysis, and the two proteins were expressed by E.coli. The results indicated that phage EC-p9 was dsDNA phage with a “holin-endolysin” lysis system. Endolysin Lys 9 was a peptidoglycan hydrolase without a transmembrane structure. Holin Hol 9 was a type III holin with only one transmembrane structure. Endolysin Lys 9 and holin Hol 9 were successfully expressed in E.coli BL21 (DE3), with yield of 2.49 and 0.95 g/L, respectively. Only limited amount of holin Hol 9 was obtained due to its toxic effect on E.coli. The results laid a foundation for research of the bacteriostatic ability of endolysin Lys 9 and holin Hol 9, and also for investigation of the lysis system of phage EC-p9.
[1] 樊晓洁. 食源性致病菌大肠杆菌O157∶H7检测方法的研究进展[J].食品安全质量检测学报,2020,11(7):2 144-2 149. FAN X J.Research progress on detection methods of foodborne pathogenic Escherichia coli O157∶H7[J].Journal of Food Safety and Quality,2020,11(7):2 144-2 149. [2] 黄冠南.大肠杆菌O157∶H7在中国东北部地区砂砾梯度土壤中的存活行为及其影响因素[D].吉林:吉林大学,2020. HUANG G L.Survival and the influence factors of Escherichia coli O157∶H7 in soils over a sandy gradient in northeastern China[D].Jilin:Jilin University,2020. [3] SABOURI S,SEPEHRIZADEH Z,AMIRPOUR-ROSTAMI S,et al.A minireview on the in vitro and in vivo experiments with anti-Escherichia coli O157∶H7 phages as potential biocontrol and phage therapy agents[J].International Journal of Food Microbiology,2017,243:52-57. [4] WONG C S,MOONEY J C,BRANDT J R,et al.Risk factors for the hemolytic uremic syndrome in children infected with Escherichia coli O157∶H7:A multivariable analysis[J].Clinical Infectious Diseases,2012,55(1):33-41. [5] 王磊, 曹巍.全球抗生素耐药性现状分析及对策建议[J].军事医学,2017,41(5):329-333. WANG L,CAO W.Current global antimicrobial resistance and countermeasures[J].Military Medical Sciences,2017,41(5):329-333. [6] LU Z,BREIDT F.Escherichia coli O157∶H7 bacteriophage Φ241 isolated from an industrial cucumber fermentation at high acidity and salinity[J].Frontiers in Microbiology,2015,6:67. [7] 胡颖, 李洪军,贺稚非.噬菌体防控食源性致病菌的研究进展[J].食品与发酵工业,2019,45(1):246-251. HU Y,LI H J,HEN Z F.Research progress on bacteriophages for preventing and controlling foodborne pathogens[J].Food and Fermentation Industries,2019,45(1):246-251. [8] LU N,SUN Y,WANG Q,et al.Cloning and characterization of endolysin and holin from Streptomyces avermitilis bacteriophage phiSASD1 as potential novel antibiotic candidates[J].International Journal of Biological Macromolecules,2020,147:980-989. [9] 韩晗, 李雪敏,程博幸,等.dsDNA噬菌体裂解系统作用机制的研究进展[J].畜牧与兽医,2019,51(7):132-139. HAN H,LI X M,CHEN BX,et al.Progress in research on the lysis mechanism of dsDNA bacteriophage[J].Animal Husbandry and Veterinary Medicine,2019,51(7):132-139. [10] WON G,KIM B,LEE J H.A novel method to generate Salmonella typhi Ty21a ghosts exploiting the lambda phage holin-endolysin system[J].Oncotarget,2017,8(29):48 186-48 195. [11] SHANNON R,RADFORD D R,BALAMURUGAN S.Impacts of food matrix on bacteriophage and endolysin antimicrobial efficacy and performance[J].Critical Reviews in Food Science & Nutrition,2020,60(10):1 631-1 640. [12] YU J H,LIM J A,CHANG H J,et al.Characteristics and lytic activity of phage-derived peptidoglycan hydrolase,LysSAP8,as a potent alternative biocontrol agent for Staphylococcus aureus[J].Journal of Microbiology and Biotechnology 2019,29(12):1 916-1 924. [13] SPRINGER K,REUTER S,KNUPFER M,et al.Activity of a holin-endolysin system in the insecticidal pathogenicity island of Yersinia enterocolitica[J].Journal of Bacteriology,2018,200(16).DOI.10.1128/JB.00180-18. [14] MAHENTHIRALINGAM E,CAMPBELL M E,FOSTER J,et al.Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis[J].Journal of Clinical Microbiology,1996,34(5):1 129-1 135. [15] 陈欣, 胡玲萍,应宇斌,等.食源性致病菌交叉适应现象及分子机制的研究进展[J].食品科学,2020,41(23):243-254. CHEN X,HU L P,YIN Y B,et al.A review of cross-adaptation and its molecular mechanism of foodborne pathogens[J].Food Science,2020,41(23):243-254. [16] WON G,HAJAM I A,LEE J H.Improved lysis efficiency and immunogenicity of Salmonella ghosts mediated by co-expression of lambda phage holin-endolysin and X174 gene E[J].Scientific Reports,2017,7:45 139. [17] SONG J, XIA F,JIANG H Y,et al.Identification and characterization of HolGH15:The holin of Staphylococcus aureus bacteriophage GH15[J].Journal of General Virology,2016,97:1 272-1 281. [18] WALMAGH M,BOCZKOWSKA B,GRYMONPREZ B,et al.Characterization of five novel endolysins from Gram-negeative infecting baceriophage[J].Applied Microbiology & Biotechnology,2013,97(10):4 369-4 375. [19] LOOD R,WINER B Y,PELZEK A J,et al.Novel phage lysin capable of killing the multidrug-resistant gram-negative bacterium Acinetobacter baumannii in amouse bacteremia model[J].Antimicrob Agents Chemother,2015,59(4):1 983-1 991. [20] 程梦珺. 粪肠球菌噬菌体EF-P10及其裂解酶生物学特性与实验治疗的研究[D].吉林:吉林大学,2019. CHEN M J.The biological characteristics of Enterococcus faecalis phage EF-P10 and its lysin and therapeutic experiment study of the lysin[D].Jilin:Jilin University,2019. [21] 陈杰. 水稻细菌性褐条病菌噬菌体AP1新穿孔素-裂解素双组分裂解系统的鉴定和功能研究[D].杭州:浙江大学,2019. CHEN J.Research on identification and function ofa new type of holin-endolysin lysis system in Acidovorax oryzae phage AP1[D].Zhejiang:Zhejiang University,2019.