假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究

doi:10.16409/j.cnki.2095-039x.2018.01.013

中国生物防治学报 ›› 2018, Vol. 34 ›› Issue (1): 109-116.DOI: 10.16409/j.cnki.2095-039x.2018.01.013

假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究

张清霞, 张迎, 吉艳艳, 童蕴慧, 纪兆林

扬州大学园艺与植物保护学院, 扬州 225009

收稿日期:2017-08-10 出版日期:2018-02-08 发布日期:2018-02-06
通讯作者: 张清霞
作者简介:张清霞,博士,副教授;E-mail:zqx817@126.com
基金资助:
国家自然科学基金（31772210）；江苏省重点研发计划（现代农业）重点项目（BE2017344，BE2015354）

Isolation and Characterization of Quorum-quenching Genes from Bacterium Pseudomonas sp. WX14

ZHANG Qingxia, ZHANG Ying, JI Yanyan, TONG Yunhui, JI Zhaolin

College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China

Received:2017-08-10 Online:2018-02-08 Published:2018-02-06

摘要/Abstract

摘要： 本研究采用报告菌平板法及β-半乳糖苷酶活性测定并筛选到10株具有较强降解酰基高丝氨酸内酯（AHLs）能力的细菌，其中菌株WX14降解AHLs能力较强，且菌体和菌体外泌物均具有降解活性。利用PCR法从菌株WX14中克隆到hacA和hacB两个群体感应淬灭酶基因，基因全长分别为2286和2370 bp，氨基酸序列比对分析结果表明，HacA属于阿库来菌素A酰化酶蛋白家族，HacB属于青霉素G酰化酶蛋白家族。这两个基因编码产物均为N末端亲核（N-terminal nucleophile，Ntn）水解酶家族成员。将hacA和hacB导入到软腐果胶杆菌Pectobacterium carotovorum后，可减弱该细菌在马铃薯块和白菜上的致病性，因此这2种AHLs降解酶对依赖于群体感应的软腐病有一定防病作用。经16S rDNA序列同源性分析鉴定，3株为假节杆菌Pseudarthrobacter spp.、1株节杆菌Paenarthrobacter sp.、4株假单胞菌Pseudomonas和2株芽胞杆菌Bacillus spp.。

关键词: 群体感应淬灭, 酰基高丝氨酸内酯, 酰基转移酶, N末端亲核水解酶

Abstract: In this study, ten strains with strong AHLs-degrading activity were obtained through "reporter plate" and the assay of β-galactosidase activity. Strain WX14 displayed the highest AHLs-degrading activity and the AHLs-degrading capacity was observed in bacterial cells and extracellular secretions. Quorum quenching genes hacA and hacB were identified and cloned from strain WX14, and full lengths of these two genes are 2286 bp and 2370 bp, respectively. Analysis of amino sequence similarities showed that HacA belongs to aculeacin A acylase family, and HacB belongs to penicillin G acylase family. Both HacA and HacB are members of the N-terminal nucleophile hydrolase superfamily. It was observed that Pectobacterium carotovorum Z3-3 after introduced hacA or hacB attenuated its pathogenicity in potato tubers and Chinese cabbages, indicating these two AHLs degrading enzymes might have potential to control of the QS-dependent soft rot disease. BLASTn analysis of bacterial 16S rDNA sequences showed that ten isolates were identified as Pseudarthrobacter spp. (3 isolates), Paenarthrobacter sp. (1 isolate), Pseudomonas spp. (4 isolates) and Bacillus spp. (2 isolates), respectively.

Key words: quorum quenching, acyl-homoserine lactones, acylase, N-terminal nucleophile hydrolytic enzymes

中图分类号:

S476

张清霞, 张迎, 吉艳艳, 童蕴慧, 纪兆林. 假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究[J]. 中国生物防治学报, 2018, 34(1): 109-116.

ZHANG Qingxia, ZHANG Ying, JI Yanyan, TONG Yunhui, JI Zhaolin. Isolation and Characterization of Quorum-quenching Genes from Bacterium Pseudomonas sp. WX14[J]. journal1, 2018, 34(1): 109-116.

参考文献

[1] Tang K, Zhang Y, Yu M, et al. Evaluation of a new high-throughputmethod for identifying quorum quenching bacteria[J]. Scientific Report, 2013, 3(10):2935.
[2] Crépin A, Beury-Cirou A, Barbey C, et al. N-acyl homoserine lactones indiverse Pectobacterium and Dickeya plant pathogens:diversity, abundance, and involvement in virulence[J]. Sensors, 2012, 12(3):3484-3497.
[3] Cui X, Harling R. N-acyl-homoserine lactone-mediated quorum sensing blockage, a novel strategy for attenuating pathogenicity of gram-negative bacterial plant pathogens[J]. European Journal of Plant Pathology, 2005, 111(4):327-339.
[4] Schell M A. To be or not to be:how Pseudomonas solanacearum decides whether or not to express virulence genes[J]. European Journal of Plant Pathology, 1996, 102:459-469.
[5] 任争光, 林敏, 姜文君, 等. 群体感应系统对甜瓜果斑病菌MH21致病力的影响[J]. 植物病理学报, 2012, 42(6):608-619.
[6] LaSarre B, Federle M J. Exploiting quorum sensing to confuse bacterial pathogens[J]. Microbiology and Molecular Biology Reviews, 2013, 77(1):73-111.
[7] 张力群, 田涛, 梅桂英. 群体感应淬灭——防治植物细菌病害的新策略[J]. 中国生物防治, 2010, 26(3):241-247.
[8] Kalia V C, Raju S C, Purohit H J. Genomic analysis reveals versatile organisms for quorum quenching enzymes:acyl-homoserine lactone-acylase and -lactonase[J]. Open Microbiology Journal, 2011, 5(1):1-13.
[9] Dong Y H, Xu J L, Li X Z, et al. AiiA, an enzyme that inactivates the acylhomoserine lactonequorum-sensing signal and attenuates the virulence of Erwinia carotovora[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(7):3526-3531.
[10] Dong Y H, Wang L H, Xu J L, et al. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase[J]. Nature, 2001, 411:813-817.
[11] Fan J, Qian G L, Chen T, et al. Biocontrol of bacterial soft rot of callalily by elicitor Harpin_xoo and N-acyl homoserine lactonase (AttM)[J]. World Journal of Microbiology and Biotechnology, 2010, 27(2):401-410.
[12] Christiaen S E A, Brackman G, Nelis H J, et al. Isolation and identification of quorum quenching bacteria from environmental samples[J]. Journal of Microbiological Methods, 2011, 87:213-219.
[13] Wei H L, Zhang L Q. Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24[J]. Antonie van Leeuwenhoek, 2006, 89(2):267-280.
[14] Mei G Y, Yan X X, Turak A, et al. AidH, an alpha/beta-hydrolase fold family member from an Ochrobactrum sp. strain, is a novel N-acylhomoserine lactonase[J]. Applied and Environmental Microbiology, 2010, 76(15):4933-4942.
[15] Heeb S, Blumer C, Haas D. Regulatory RNAas mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0[J]. Journal of Bacteriology, 2002, 184(4):1046-1056.
[16] Marchesi J R, Sato T, Weightman A J, et al. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA[J]. Applied and Environmental Microbiology, 1998, 64(2):795-799.
[17] Tamura K, Peterson D, Peterson N, et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology Evolution, 2011, 28(10):2731-2739.
[18] Ochiai S, Yasumoto S, Morohoshi T, et al. AmiE, a novel N-acylhomoserinelactone acylase belonging to the amidase family, from the activated-sludge isolate Acinetobacter sp. strain Ooi24[J]. Applied and Environmental Microbiology, 2014, 80(22):6919-6925.
[19] Fetzner S. Quorum quenching enzymes[J]. Journal of Biotechnology, 2015, 201:2-14.
[20] Lin Y H, Xu J L, Hu J Y, et al. Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a noveland potent class of quorum-quenching enzymes[J]. Molecular Microbiology, 2003, 47(3):849-860.
[21] Huang J J, Petersen A, Whiteley M, et al. Identification of QuiP, the product of gene PA1032, asthe second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1[J]. Applied and Environmental Microbiology, 2006, 72(2):1190-1197.
[22] Wahjudi M, Papaioannou E, Hendrawati O, et al. PA0305 of Pseudomonas aeruginosaisa quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily[J]. Microbiology, 2011, 157(7):2042-2055.
[23] Park S Y, Kang H O, Jang H S, et al. Identifcation of extracellular N-acylhomoserine lactoneacylase from a Streptomyces sp. and its application to quorum quenching[J]. Applied and Environmental Microbiology, 2005, 71(5):2632-2641.
[24] Romero M, Diggle S P, Heeb S, et al. Quorum quenching activity in Anabaena sp. PCC7120:identification of AiiC, a novel AHL-acylase[J]. FEMS Microbiology Letters, 2008, 280(1):73-80.
[25] Shepherd R W, Lindow S E. Two dissimilar N-acyl-homoserine lactone acylases ofPseudomonas syringae influence colony and biofilm morphology[J]. Applied and Environmental Microbiology, 2009, 75(1):45-53.
[26] Dong Y H, Wang L Y, Zhang L H. Quorum-quenching microbial infections:mechanisms and implications[J]. Philosophical Transactions of the Royal Society of London[J]. 2007, 362(1483):1201-1211.
[27] Molina L, Constantinescu F, Michel L, et al. Degradation of pathogen quorum-sensing molecules by soil bacteria:a preventive and curative biological control mechanism[J]. FEMS Microbiology Ecology, 2003, 45(1):71-81.
[28] Uroz S, Heinonsalo J. Degradation of N-acyl homoserine lactone quorum sensing signal molecules by forest root-associated fungi[J]. FEMS Microbiology Ecology, 2008, 65(2):271-278.
[29] Cha C, Gao P, Chen Y C, et al. Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria[J]. Molecular Plant-Microbe Interactions, 1998, 11(11):1119-1129.

假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究

Isolation and Characterization of Quorum-quenching Genes from Bacterium Pseudomonas sp. WX14

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics

本文评价

联系我们

[1]	杨欣, 张鹏飞, 白变霞, 叶建仁, 任嘉红. 群体感应信号物质对吡咯伯克霍尔德氏菌JK-SH007在杨树内定殖的影响[J]. 中国生物防治学报, 2019, 35(6): 949-957.
[2]	张力群1 ;田涛2 ;梅桂英1 . 群体感应淬灭——防治植物细菌病害的新策略[J]. , 2010, 26(3): 241-247.