土壤中群体感应淬灭细菌的原位分离与筛选
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摘要
【背景】许多革兰氏阴性细菌通常以N-酰基高丝氨酸内酯(N-acylhomoserine lactones,AHLs)作为群体感应主要的信号分子。【目的】从土壤中筛选和鉴定新型群体感应淬灭细菌。【方法】通过"垫圈法"从土壤中原位培养分离细菌,采用琼脂条法、报告菌平板法及β-半乳糖苷酶活性测定筛选群体感应淬灭细菌,根据16S rRNA基因序列同源性分析确定菌株系统发育地位。【结果】从不同地区土样中原位培养共分离获得细菌502株。以根癌土壤杆菌Agrobacterium tumefaciens NTL4 (pZLR4)作为报告菌,最终得到11株具有较强降解AHLs能力的细菌,包括假单胞菌5株、不动杆菌4株、变形杆菌和莱茵海默氏菌各1株。大部分细菌可完全降解N-3-羰基十二酰基高丝氨酸内酯(3OC12-HSL),部分细菌对N-(3-氧代己酰)高丝氨酸内酯(3OC6-HSL)和N-3-氧代辛酰高丝氨酸内酯(3OC8-HSL)具有一定降解活性。【结论】Proteus和Rheinheimera可降解AHLs,为今后防治依赖群体感应的植物细菌病害提供新型生防资源。
[Background] N-acylhomoserine lactones(AHLs) are used as the main quorum-sensing signaling molecules in many Gram-negative bacteria. [Objective] To screen and identify new quorum quenching(QQ) bacteria from soil organisms. [Methods] We used "washer method" to grow organisms by cultivation in situ. QQ bacteria were screened through "agar slice", "reporter plate" and the assay ofβ-galactosidase activity. The phylogenetic status was determined based on 16 S rRNA gene homology analysis. [Results] Here, 502 organisms were obtained from the soil samples in situ and screened for AHL-degrading bacteria using A. tumefaciens NTL4(pZLR4) as a reporter strain. Eleven isolates degraded AHLs within 24 h. Based on their 16 S rRNA gene sequences similarities, five isolates(3-49,3-58, 61, 66, 120) and four isolates(3-59, 151, 3-41, 2-34) were assigned to the genera Pseudomonas and Acinetobacter. Strain 2-63 and 129 were identified as Proteus sp. and Rheinheimera sp., respectively. Most of these strains degraded strongly 3 OC12-HSL and were active against 3 OC6-HSL and 3 OC8-HSL.[Conclusion] This study showed that Proteus sp. and Rheinheimera sp. may degrade AHLs, which might provide new biocontrol resources to control of the quorum sensing-dependent bacterial diseases.
[Background] N-acylhomoserine lactones(AHLs) are used as the main quorum-sensing signaling molecules in many Gram-negative bacteria. [Objective] To screen and identify new quorum quenching(QQ) bacteria from soil organisms. [Methods] We used "washer method" to grow organisms by cultivation in situ. QQ bacteria were screened through "agar slice", "reporter plate" and the assay ofβ-galactosidase activity. The phylogenetic status was determined based on 16 S rRNA gene homology analysis. [Results] Here, 502 organisms were obtained from the soil samples in situ and screened for AHL-degrading bacteria using A. tumefaciens NTL4(pZLR4) as a reporter strain. Eleven isolates degraded AHLs within 24 h. Based on their 16 S rRNA gene sequences similarities, five isolates(3-49,3-58, 61, 66, 120) and four isolates(3-59, 151, 3-41, 2-34) were assigned to the genera Pseudomonas and Acinetobacter. Strain 2-63 and 129 were identified as Proteus sp. and Rheinheimera sp., respectively. Most of these strains degraded strongly 3 OC12-HSL and were active against 3 OC6-HSL and 3 OC8-HSL.[Conclusion] This study showed that Proteus sp. and Rheinheimera sp. may degrade AHLs, which might provide new biocontrol resources to control of the quorum sensing-dependent bacterial diseases.
引文
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[20]Chan KG,Atkinson S,Mathee K,et al.Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale(ginger)rhizosphere:co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia[J].BMC Microbiology,2011,11:51
[21]Sun SW,Dai XY,Sun J,et al.A diketopiperazine factor from Rheinheimera aquimaris QS102 exhibits anti-quorum sensing activity[J].Scientific Reports,2016,6:39637
[22]Zhang QX,Zhang Y,Ji YY,et al.Isolation and characterization of quorum-quenching genes from bacterium Pseudomonas sp.WX14[J].Chinese Journal of Biological Control,2018,341(1):109-116(in Chinese)张清霞,张迎,吉艳艳,等.假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究.中国生物防治学报,2018,341(1):109-116
[23]Tannières M,Beury-Cirou A,Vigouroux A,et al.A metagenomics study highlights phylogenetic proximity of quorum-quenching and xenobiotic-degrading amidases of the AS-family[J].PLoSOne,2013,8(6):e65473
[2]Zhang LQ,Tian T,Mei GY.Quorum quenching,a new strategy for controlling plant bacterial diseases[J].Chinese Journal of Biological Control,2010,26(3):241-247(in Chinese)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
[3]Chen F,Gao YX,Chen XY,et al.Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection[J].International Journal of Molecular Sciences,2013,14(9):17477-17500
[4]Fetzner S.Quorum quenching enzymes[J].Journal of Biotechnology,2015,201:2-14
[5]Stevenson BS,Eichorst SA,Wertz JT,et al.New strategies for cultivation and detection of previously uncultured microbes[J].Applied and Environmental Microbiology,2004,70(8):4748-4755
[6]Kaeberlein T,Lewis K,Epstein SS.Isolating“uncultivable”microorganisms in pure culture in a simulated natural environment[J].Science,2002,296(5570):1127-1129
[7]Nichols D,Cahoon N,Trakhtenberg EM,et al.Use of ichip for high-throughput in situ cultivation of“uncultivable”microbial species[J].Applied and Environmental Microbiology,2010,76(8):2445-2450
[8]Wei HL,Zhang LQ.Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24[J].Antonie Van Leeuwenhoek,2006,89(2):267-280
[9]Cha C,Gao P,Chen YC,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
[10]Mei GY,Yan XX,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
[11]Chilton MD,Currier TC,Farrand SK,et al.Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors[J].Proceedings of the National Academy of Sciences of the United States of America,1974,71(9):3672-3676
[12]Gavrish E,Bollmann A,Epstein S,et al.A trap for in situ cultivation of filamentous actinobacteria[J].Journal of Microbiological Methods,2008,72(3):257-262
[13]Marchesi JR,Sato T,Weightman AJ,et al.Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16s r RNA[J].Applied and Environmental Microbiology,1998,64(2):759-799
[14]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
[15]Gao A,Mei G,Liu S,et al.High-resolution structures of AidHcomplexes provide insights into a novel catalytic mechanism for N-acyl homoserine lactonase[J].Acta Crystallographica Section D,Biological Crystallography,2013,69(1):82-91
[16]Lewis K.Platforms for antibiotic discovery[J].Nature Reviews Drug Discovery,2013,12:371-387
[17]Wahjudi M,Papaioannou E,Hendrawati O,et al.PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily[J].Microbiology,2011,157:2042-2055
[18]Shepherd RW,Lindow SE.Two dissimilar N-acyl-homoserine lactone acylases of Pseudomonas syringae influence colony and biofilm morphology[J].Applied and Environmental Microbiology,2009,75(1):45-53
[19]Ochiai S,Yasumoto S,Morohoshi T,et al.AmiE,a novel N-acylhomoserine lactone 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
[20]Chan KG,Atkinson S,Mathee K,et al.Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale(ginger)rhizosphere:co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia[J].BMC Microbiology,2011,11:51
[21]Sun SW,Dai XY,Sun J,et al.A diketopiperazine factor from Rheinheimera aquimaris QS102 exhibits anti-quorum sensing activity[J].Scientific Reports,2016,6:39637
[22]Zhang QX,Zhang Y,Ji YY,et al.Isolation and characterization of quorum-quenching genes from bacterium Pseudomonas sp.WX14[J].Chinese Journal of Biological Control,2018,341(1):109-116(in Chinese)张清霞,张迎,吉艳艳,等.假单胞菌WX14群体感应淬灭酶基因的克隆及其功能研究.中国生物防治学报,2018,341(1):109-116
[23]Tannières M,Beury-Cirou A,Vigouroux A,et al.A metagenomics study highlights phylogenetic proximity of quorum-quenching and xenobiotic-degrading amidases of the AS-family[J].PLoSOne,2013,8(6):e65473