Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (2): 295-305.doi: 10.3864/j.issn.0578-1752.2024.02.006

• PLANT PROTECTION • Previous Articles     Next Articles

Isolation, Genome Determination and Lysis Function Analysis of Phage Kuerle of Erwinia amylovora

CHEN Niu(), YU ChengMin(), CUI BaiMing, REN CaiXia, YANG LiYing, DONG Yu, LIU Lin, ZHENG YinYing()   

  1. College of Life Sciences, Shihezi University, Shihezi 832003, Xinjiang
  • Received:2023-09-08 Accepted:2023-11-11 Online:2024-01-16 Published:2024-01-19
  • Contact: ZHENG YinYing

RichHTML

10

PDF

96

Abstract:

【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.

Key words: Erwinia amylovora, phage isolation, genome analysis, lysis system, holin, endolysin

Table 1

Primers for testing"

引物名称
Primer name		 引物序列
Sequence of primers (5′-3′)		 对应质粒名称
Name of the corresponding plasmid
Hol-1F GGCATATGgctcgcaaagtgaagttagta (Nde I) pET-holin
Hol-249R GGGAATTCctactcactggcatcgttact (EcoR I)
Lys-1F GGCATATGccagtgagtagcactactagg (Nde I) pET-endolysin
Lys-609R GGGAATTCtcatttagcatcttcctggc (EcoR I)
Hol-1F GGCATATGgctcgcaaagtgaagttagta (Nde I) pET-endolysin-holin
Lyt-609R GGGAATTCtcatttagcatcttcctggc (EcoR I)
LysΔSP2-F GGCATATGgagggtggatattctaatga (Nde I) pET-endolysinΔsp
LysΔSP2-R GGGAATTCtcatttagcatcttcctggc (EcoR I)
LysΔSP1-F GGGAATTCggagggtggatattctaatga (EcoR I) pET-endolysinΔsp-holin
LysΔSP549-R GGAAGCTTCtcatttagcatcttcctggc (Hind III)

Fig. 1

Phage plagues, particle morphology of Kuerle"

Fig. 2

The one-step growth curves of Kuerle in Ea102"

Table 2

Host range of phage Kuerle"

菌株 <BOLD>S</BOLD>train 裂解谱 Lytic spectrum
巨大芽孢杆菌KD7 B. megaterium KD7 －
巨大芽孢杆菌KB17 B. megaterium KB17 －
巨大芽孢杆菌KB3 B. megaterium KB3 －
萎缩芽孢杆菌B4 B. atrophaeus B4 －
萎缩芽孢杆菌KB14 B. atrophaeus KB14 －
萎缩芽孢杆菌B8 B. atrophaeus B8 －
萎缩芽孢杆菌KB16 B. atrophaeus KB16 －
解淀粉欧文氏菌Ea102 E. amylovora Ea102 +
解淀粉欧文氏菌Ea915 E. amylovora Ea915 +

Fig. 3

Comparison of the genomes of phage Kuerle and Enterobacteria phage N4 (accession number: NC_008720) Arrows indicate predicted ORFs, red vertical lines indicate tRNAs, and gene similarity profiles between phages are shown as percentages in grey chromatograms"

Fig. 4

Structural analysis of phage Kuerle lysis genes"

Fig. 5

Phylogenetic tree of lysis-associated proteins A: Kuerle-endolysin; B: Kuerle-holin"

Fig. 6

Kuerle-endolysin enters periplasmic space via the Sec pathway"

[1]
MOLINA L, REZZONICO F, DEFAGO G, DUFFY B. Autoinduction in Erwinia amylovora: Evidence of an acyl-homoserine lactone signal in the fire blight pathogen. Journal of Bacteriology, 2005, 187(9): 3206-3213.

doi: 10.1128/JB.187.9.3206-3213.2005
[2]
ZHAO Y, TIAN Y, WANG L, GENG G, ZHAO W, HU B, ZHAO Y. Fire blight disease, a fast-approaching threat to apple and pear production in China. Journal of Integrative Agriculture, 2019, 18(4): 815-820.

doi: 10.1016/S2095-3119(18)62033-7
[3]
MANSFIELD J, GENIN S, MAGORI S, CITOVSKY V, SRIARIYANUM M, RONALD P, DOW M, VERDIER V, BEER S V, MACHADO M A, TOTH I, SALMOND G, FOSTER G D. Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular Plant Pathology, 2012, 13(6): 614-629.

doi: 10.1111/j.1364-3703.2012.00804.x pmid: 22672649
[4]
中华人民共和国农业农村部公告第351号. 中华人民共和国农业农村部公报, 2020(12): 96-98.
Ministry of Agriculture and Rural Affairs of the People’s Republic of China Announcement No.351. Gazette of the Ministry of Agriculture and Rural Affairs of the People’s Republic of China, 2020(12): 96-98. (in Chinese)
[5]
王俊, 高建诚, 巴音克西克, 木也沙·买买提, 张军恒, 田艳丽, 胡白石. 利用电加热自动消毒修枝剪阻断梨火疫病田间传播. 植物检疫, 2022, 36(2): 25-28.
WANG J, GAO J C, BAYINKEXIKE, MUYASSAR·MAMAT, ZHANG J H, TIAN Y L, HU B S. Blocking field spread of fire blight by electric heating automatic disinfection pruning scissors. Plant Quarantine, 2022, 36(2): 25-28. (in Chinese)
[6]
王俊, 高建诚, 巴音克西克, 吴莉莉. 利用蜜蜂传播生防菌防治梨火疫病. 植物检疫, 2022, 36(1): 9-12.
WANG J, GAO J C, BAYINKEXIKE, WU L L. Dispersal of biocontrol bacterium by honey bee for control of pear fire blight. Plant Quarantine, 2022, 36(1): 9-12. (in Chinese)
[7]
韩丽丽, 荆珺, 王杰花, 陈卫民. 杏树梨火疫病在中国首次发生. 植物检疫, 2022, 36(6): 46-49.
HAN L L, JING J, WANG J H, CHEN W M. First report of pear fire blight on apricot caused by Erwinia amylovora in China. Plant Quarantine, 2022, 36(6): 46-49. (in Chinese)
[8]
MCMANUS P S, STOCKWELL V O, SUNDIN G W, JONES A L. Antibiotic use in plant agriculture. Annual Review of Phytopathology, 2002, 40: 443-465.

pmid: 12147767
[9]
JONES J B, JACKSON L E, BALOGH B, OBRADOVIC A, IRIARTE F B, MOMOL M T. Bacteriophages for plant disease control. Annual Review of Phytopathology, 2007, 45: 245-262.

pmid: 17386003
[10]
GARCÍA P, MARTÍNEZ B, OBESO J M, RODRÍGUEZ A. Bacteriophages and their application in food safety. Letters in Applied Microbiology, 2008, 47(6): 479-485.

doi: 10.1111/j.1472-765X.2008.02458.x pmid: 19120914
[11]
李洪涛, 张静文, 盛强, 唐章虎, 张祥林, 张春竹, 罗明. 我国20个梨品种(种质)对国外梨火疫病菌的抗病性评价. 果树学报, 2019, 36(5): 629-637.
LI H T, ZHANG J W, SHENG Q, TANG Z H, ZHANG X L, ZHANG C Z, LUO M. Resistance evaluation of 20 pear varieties (germplasms) in China to foreign strains of Erwinia amylovora. Journal of Fruit Science, 2019, 36(5): 629-637. (in Chinese)
[12]
YOUNG R. Bacteriophage lysis: Mechanism and regulation. Microbiological Reviews, 1992, 56(3): 430-481.

doi: 10.1128/mr.56.3.430-481.1992 pmid: 1406491
[13]
YOUNG R. Bacteriophage holins: Deadly diversity. Journal of Molecular Microbiology and Biotechnology, 2002, 4(1): 21-36.

pmid: 11763969
[14]
WANG I N, SMITH D L, YOUNG R. Holins: The protein clocks of bacteriophage infections. Annual Review of Microbiology, 2000, 54: 799-825.

pmid: 11018145
[15]
BERNHARDT T G, ROOF W D, YOUNG R. Genetic evidence that the bacteriophage phi X174 lysis protein inhibits cell wall synthesis. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(8): 4297-4302.
[16]
CATALAO M J, GIL F, MONIZ-PEREIRA J, PIMENTEL M. The mycobacteriophage Ms6 encodes a chaperone-like protein involved in the endolysin delivery to the peptidoglycan. Molecular Microbiology, 2010, 77(3): 672-686.

doi: 10.1111/j.1365-2958.2010.07239.x pmid: 20545844
[17]
CHEN S, ZHOU Y, CHEN Y, GU J. Fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34(17): i884-i890.

doi: 10.1093/bioinformatics/bty560
[18]
ANTIPOV D, RAIKO M, LAPIDUS A, PEVZNER P A. Metaviral SPAdes: Assembly of viruses from metagenomic data. Bioinformatics, 2020, 36(14): 4126-4129.

doi: 10.1093/bioinformatics/btaa490 pmid: 32413137
[19]
AZIZ R K, BARTELS D, BEST A A, DEJONGH M, DISZ T, EDWARDS R A, FORMSMA K, GERDES S, GLASS E M, KUBAL M, et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genomics, 2008, 9: 75.

doi: 10.1186/1471-2164-9-75 pmid: 18261238
[20]
SULLIVAN M J, PETTY N K, BEATSON S A. Easyfig: A genome comparison visualizer. Bioinformatics, 2011, 27(7): 1009-1010.

doi: 10.1093/bioinformatics/btr039 pmid: 21278367
[21]
NCBI Resource Coordinators. Database resources of the National Center for Biotechnology Information. Nucleic Acids Research, 2016, 44(D1): D7-D19.

doi: 10.1093/nar/gkv1290
[22]
KUMAR S, STECHER G, LI M, KNYAZ C, TAMURA K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 2018, 35(6): 1547-1549.

doi: 10.1093/molbev/msy096 pmid: 29722887
[23]
FINN R D, ATTWOOD T K, BABBITT P C, BATEMAN A, BORK P, BRIDGE A J, CHANG H Y, DOSZTANYI Z, EL-GEBALI S, FRASER M, et al. InterPro in 2017-beyond protein family and domain annotations. Nucleic Acids Research, 2017, 45(D1): D190-D199.

doi: 10.1093/nar/gkw1107
[24]
KROGH A, LARSSON B, VON HEIJNE G, SONNHAMMER E L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. Journal of Molecular Biology, 2001, 305(3): 567-580.

doi: 10.1006/jmbi.2000.4315 pmid: 11152613
[25]
NIELSEN H. Predicting secretory proteins with SignalP. Methods in Molecular Biology, 2017, 1611: 59-73.

doi: 10.1007/978-1-4939-7015-5_6 pmid: 28451972
[26]
LETUNIC I, KHEDKAR S, BORK P. SMART: Recent updates, new developments and status in 2020. Nucleic Acids Research, 2021, 49(D1): D458-D460.

doi: 10.1093/nar/gkaa937 pmid: 33104802
[27]
OLIVER D B, CABELLI R J, DOLAN K M, JAROSIK G P. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proceedings of the National Academy of Sciences of the United States of America, 1990, 87(21): 8227-8231.
[28]
徐琳赟, 古丽孜热·曼合木提, 韩剑, 蒋萍, 黄伟, 罗明. 香梨内生拮抗细菌的筛选及对梨火疫病的生防潜力. 西北植物学报, 2021, 41(1): 132-141.
XU L Y, GULIZZIER·MANHEMUTI, HAN J, JIANG P, HUANG W, LUO M. Screening of endophytic antagonistic bacteria from Kuerlexiangli pear and their biocontrol potential against fire blight disease. Acta Botanica Boreali-Occidentalia Sinica, 2021, 41(1): 132-141. (in Chinese)
[29]
鲁晏宏, 郝金辉, 罗明, 黄伟, 盛强, 王宁, 詹发强, 龙宣杞, 包慧芳. 梨火疫病拮抗菌筛选及温室防效测定. 微生物学通报, 2021, 48(10): 3690-3699.
LU Y H, HAO J H, LUO M, HUANG W, SHENG Q, WANG N, ZHAN F Q, LONG X Q, BAO H F. Screening of antagonistic bacteria against Erwinia amylovora and its control effect in greenhouse. Microbiology China, 2021, 48(10): 3690-3699. (in Chinese)
[30]
CUI Z, HU L, ZENG L, MENG W, GUO D, SUN L. Isolation and characterization of Priestia megaterium KD7 for the biological control of pear fire blight. Frontiers in Microbiology, 2023, 14: 1099664.

doi: 10.3389/fmicb.2023.1099664
[31]
WITTMANN J, TURNER D, MILLARD A D, MAHADEVAN P, KROPINSKI A M, ADRIAENSSENS E M. From orphan phage to a proposed new Family - The diversity of N4-like viruses. Antibiotics, 2020, 9(10): 663.

doi: 10.3390/antibiotics9100663
[32]
BRIERS Y, PEETERS L M, VOLCKAERT G, LAVIGNE R. The lysis cassette of bacteriophage ΦKMV encodes a signal-arrest-release endolysin and a pinholin. Bacteriophage, 2011, 1(1): 25-30.

doi: 10.4161/bact.1.1.14868
[33]
BRIERS Y, VOLCKAERT G, CORNELISSEN A, LAGAERT S, MICHIELS C W, HERTVELDT K, LAVIGNE R. Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages ΦKZ and EL. Molecular Microbiology, 2007, 65(5): 1334-1344.

doi: 10.1111/mmi.2007.65.issue-5
[34]
XU M, STRUCK D K, DEATON J, WANG I N, YOUNG R. A signal-arrest-release sequence mediates export and control of the phage P1 endolysin. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(17): 6415-6420.
[35]
OLIVEIRA H, MELO L D, SANTOS S B, NOBREGA F L, FERREIRA E C, CERCA N, AZEREDO J, KLUSKENS L D. Molecular aspects and comparative genomics of bacteriophage endolysins. Journal of Virology, 2013, 87(8): 4558-4570.

doi: 10.1128/JVI.03277-12 pmid: 23408602
[36]
SAVVA C G, DEWEY J S, MOUSSA S H, TO K H, HOLZENBURG A, YOUNG R. Stable micron-scale holes are a general feature of canonical holins. Molecular Microbiology, 2014, 91(1): 57-65.

doi: 10.1111/mmi.12439 pmid: 24164554
[37]
SUMMER E J, BERRY J, TRAN T A, NIU L, STRUCK D K, YOUNG R. Rz/Rz1 lysis gene equivalents in phages of Gram- negative hosts. Journal of Molecular Biology, 2007, 373(5): 1098-1112.

doi: 10.1016/j.jmb.2007.08.045
[38]
DY R L, RIGANO L A, FINERAN P C. Phage-based biocontrol strategies and their application in agriculture and aquaculture. Biochemical Society Transactions, 2018, 46(6): 1605-1613.

doi: 10.1042/BST20180178 pmid: 30514766
[39]
JAMAL M, BUKHARI S, ANDLEEB S, ALI M, RAZA S, NAWAZ M A, HUSSAIN T, RAHMAN S U, SHAH S S A. Bacteriophages: An overview of the control strategies against multiple bacterial infections in different fields. Journal of Basic Microbiology, 2019, 59(2): 123-133.

doi: 10.1002/jobm.201800412 pmid: 30485461
[40]
POLASKA M, SOKOLOWSKA B. Bacteriophages - a new hope or a huge problem in the food industry. AIMS Microbiology, 2019, 5(4): 324-346.

doi: 10.3934/microbiol.2019.4.324
[41]
CARSTENS A B, DJURHUUS A M, KOT W, HANSEN L H. A novel six-phage cocktail reduces Pectobacterium atrosepticum soft rot infection in potato tubers under simulated storage conditions. FEMS Microbiology Letters, 2019, 366(9): fnz101.

doi: 10.1093/femsle/fnz101
[42]
LEE C, KIM J, SON B, RYU S. Development of advanced chimeric endolysin to control multidrug-resistant Staphylococcus aureus through domain shuffling. ACS Infectious Diseases, 2021, 7(8): 2081-2092.

doi: 10.1021/acsinfecdis.0c00812
[43]
ANDERSSON H, VON HEIJNE G., Membrane protein topology: Effects of delta mu H+ on the translocation of charged residues explain the ‘positive inside’ rule. The EMBO Journal, 1994, 13(10): 2267-2272.

doi: 10.1002/embj.1994.13.issue-10
[44]
CAHILL J, YOUNG R. Phage lysis: Multiple genes for multiple barriers. Advances in Virus Research, 2019, 103: 33-70.

doi: S0065-3527(18)30056-3 pmid: 30635077
[45]
TO K H, DEWEY J, WEAVER J, PARK T, YOUNG R. Functional analysis of a class I holin, P2 Y. Journal of Bacteriology, 2013, 195(6): 1346-1355.

doi: 10.1128/JB.01986-12 pmid: 23335412
[1] LU Li-ming. Identification and Phylogenetic Analysis of Potassium Transporters in Major Crops#br# [J]. Scientia Agricultura Sinica, 2009, 42(7): 2271-2279 .
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd