Isolation,Genome Determination and Lysis Function Analysis of Phage Kuerle of Erwinia amylovora
[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 75 599 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)(3 550 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.
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