摘要
【背景】解淀粉欧文氏菌(Erwiniaamylovora)是仁果类果树火疫病的病原体,对全球苹果和梨的生产构成严重威胁。随着抗生素耐药菌株的出现,火疫病的防治面临巨大的挑战。【目的】分离一种新的裂解解淀粉欧文氏菌的噬菌体,并分析该噬菌体裂解相关基因的功能,为火疫病的防治提供新的选择。【方法】以解淀粉欧文氏菌Ea102为宿主菌,采用双层平板法从流行火疫病的果园土壤中分离噬菌体。通过噬菌斑和透射电镜观察其形态。用PacBio测序技术测定噬菌体基因组,SPAdes组装序列,RAST注释基因组。通过大肠杆菌原核表达系统分析噬菌体Kuerle的裂解机制。【结果】分离纯化出一株裂解解淀粉欧文氏菌的噬菌体,命名为Kuerle。Kuerle由二十面体衣壳的头部和短尾组成,潜伏期约为50 min,裂解量约为240 pfu/cell。噬菌体基因组全长75599 bp,GC含量48.0%,末端有393 bp的重复序列,未发现与溶原调控相关的基因。共预测到85个开放阅读框(open reading frame,ORF),其中33个已知功能蛋白中包含一个由3550 aa组成的巨大病毒颗粒相关的RNA聚合酶(virion-associated RNA polymerase,vRNAP),该vRNAP是Schitoviridae科噬菌体的主要特征之一。病毒粒子和基因结构表明噬菌体Kuerle属于有尾噬菌体类的Schitoviridae科病毒。聚集在DNA晚期表达区的3个裂解相关基因holin、endolysin和spanin组成了“裂解盒”。在大肠杆菌中Kuerle-holin的表达会抑制细胞的生长,而Kuerle-endolysin的表达可裂解细胞。二者共表达时Kuerle-holin会加速Kuerle-endolysin引起的细胞裂解。用叠氮化钠抑制细菌的一般分泌系统(Sec)或Kuerle-endolysin N端信号序列的缺失均会导致endolysin裂解功能丧失。上述结果表明噬菌体Kuerle具有pinholin-SAR endolysin裂解系统。Kuerle-holin(pinholin)使细胞质膜去极化以激活分泌的Kuerle-endolysin(SAR endolysin)降解肽聚糖层。【结论】Kuerle是一株烈性噬菌体并且Kuerle-endolysin抑菌效果显著,为后续火疫病生防试剂的制备提供了理论依据和研究材料。
【Background】Erwinia amylovora,the causal agent of fire blight in pome fruit trees,poses a serious threat to apple and pear production worldwide.With the emergence of antibiotic-resistant strains,control of E.amylovora causing fire blight is a huge challenge.【Objective】The objective of this study is to isolate a new lytic E.amylovora bacteriophage and analyze the functions of the phage lytic-related genes,and to provide a new option for the control of fire blight.【Method】Phage was isolated from fire-blight-endemic orchard soil using the double-layer plaque assay with E.amylovora strain Ea102 as the host.Morphology was observed by phage plagues and transmission electron microscopy.The phage genome was determined by PacBio sequencing,SPAdes assembly,and RAST annotation.The Escherichia coli prokaryotic expression system was used to analyze the lysis mechanism of bacteriophage.【Result】A new E.amylovora bacteriophage,named Kuerle,was isolated and purified.Kuerle consists of an icosahedral capsid head and a short tail,with a latent phase of about 50 min and burst size of about 240 pfu/cell.The phage has a genome length of 75599 bp with 48.0%GC content,and direct terminal repeat of 393 bp.No regulatory genes related to lysogency were identified.A total of 85 open reading frames(ORFs)were predicted,of which 33 known functional proteins contained a giant virion-associated RNA polymerase(vRNAP)(3550 aa).The vRNAP is considered one of the main features of the family Schitoviridae.The morphology of viron and genome structure agree that phage Kuerle belongs to the family Schitoviridae.Three lysis-associated genes,holin,endolysin and spanin clustered in late expression DNA region compose“lysis cassettes”.The expression of Kuerle-holin in E.coli inhibited the growth of bacteria,while the expression of Kuerle-endolysin caused cell lysis.Kuerle-holin accelerated the process of cell lysis caused by Kuerle-endolysin.Inhibition of the general secretion system(Sec)of bacteria with sodium azide or deletion of the N-terminal signaling sequence of Kuerle-endolysin both resulted in the loss of endolysin lysis function.These results suggest that the phage Kuerle has a pinholin-SAR endolysin lysis system.Kuerle-holin(pinholin)depolarizes cytoplasmic membrane to activate the secreted Kuerle-endolysin(SAR endolysin)which degrades cell wall in periplasm.【Conclusion】Kuerle is a virulent bacteriophage and Kuerle-endolysin has significant bacteriostatic effect,which provides theoretical basis and research materials for the preparation of subsequent fire blight control reagents.
作者
陈妞
余成敏
崔百明
任彩霞
杨丽颖
董钰
刘琳
郑银英
CHEN Niu;YU ChengMin;CUI BaiMing;REN CaiXia;YANG LiYing;DONG Yu;LIU Lin;ZHENG YinYing(College of Life Sciences,Shihezi University,Shihezi 832003,Xinjiang)
出处
《中国农业科学》
CAS
CSCD
北大核心
2024年第2期295-305,共11页
Scientia Agricultura Sinica
基金
兵团科技攻关计划(2023AB004-03)。
