一株高拮抗活性菌株的鉴定及其抗菌活性缺失突变体的获得
|
摘要
本研究以黄瓜枯萎病菌(Fusarium oxysporum)和青枯劳尔氏菌(Ralstonia solanacearum)为指示菌,从大豆植物根际土壤中筛选到一株拮抗活性较高的菌株(D-7),经Rec A基因序列分析,鉴定其为新洋葱伯克霍尔德氏菌(Burkholderia cenocepacia)。经PCR检测,该菌株不存在毒性基因BCESM(esmR)和cbl A。致病性分析发现该菌株对苜蓿幼苗和洋葱没有致病性。为了解该菌株的抗菌物质合成相关基因,利用Tn5转座子突变法构建菌株D-7的突变体库,并筛选获得10株对黄瓜枯萎病菌抗菌活性减弱的突变体。对各突变株插入位点分析发现,其中5个突变株的Tn5转座子插入到同一基因,该基因编码葡萄糖抑制的分裂蛋白A(Glucose-inhibited division protein A,Gid A),其余5株突变体的插入位点分别位于不同的基因上,编码的蛋白包括糖基转移酶(Glycosyl transferase)、Ⅱ型分泌系统蛋白(typeⅡsecretion system protein)等,表明菌株D-7的抗真菌能力有多个基因参与。
A bacteria strain D-7 was isolated from soybean rhizosphere soil and showed high anti-microbial activity to plant pathogenic fungi and bacteria, such as Fusarium oxysporum and Ralstonia solanacearum. The strain was identified to be Burkholderia cenocepacia based on Rec A gene sequence and phylogenetic analysis. Specific PCR was constructed to amplify virulence genes,such as BCESM(esmR) and cbl A,and the results showed the strain D-7 did not have the two genes. It also had no pathogenicity to alfalfa and onion. In order to understand the genes related to antibacterial substances of the strain D-7,a random mutant library was constructed using Tn5 transposon mutation method,and 10 mutants with reduced antimicrobial activity to Fusarium oxysporum were obtained. By the mutant insertion site analysis,we found that the Tn5 transposon in 5 mutants were inserted into the same gene,which encoded glucose-inhibited division protein A(Gid A),while the insertion sites of the remaining 5 mutants were located on different genes,and the encoded proteins included glycosyl transferase,typeⅡ secretion system protein and so on. It indicated that there were multiple genes involved in the antifungal activity of strain D-7.
A bacteria strain D-7 was isolated from soybean rhizosphere soil and showed high anti-microbial activity to plant pathogenic fungi and bacteria, such as Fusarium oxysporum and Ralstonia solanacearum. The strain was identified to be Burkholderia cenocepacia based on Rec A gene sequence and phylogenetic analysis. Specific PCR was constructed to amplify virulence genes,such as BCESM(esmR) and cbl A,and the results showed the strain D-7 did not have the two genes. It also had no pathogenicity to alfalfa and onion. In order to understand the genes related to antibacterial substances of the strain D-7,a random mutant library was constructed using Tn5 transposon mutation method,and 10 mutants with reduced antimicrobial activity to Fusarium oxysporum were obtained. By the mutant insertion site analysis,we found that the Tn5 transposon in 5 mutants were inserted into the same gene,which encoded glucose-inhibited division protein A(Gid A),while the insertion sites of the remaining 5 mutants were located on different genes,and the encoded proteins included glycosyl transferase,typeⅡ secretion system protein and so on. It indicated that there were multiple genes involved in the antifungal activity of strain D-7.
引文
[1]Chiarini L,Bevivino A,Dalmastri C,et al.Burkholderia cepacia complex species:health hazards and biotechnological potential[J].Trends Microbiol.,2006,14(6):277-286.
[2]Parke J L,Gurian-Sherman D.Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains[J].Annu.Rev.Phytopathol.,2001,39(1):225-258.
[3]Caballero-Mellado J,Martínez-Aguilar L,Paredes-Valdez G,et al.Burkholderia unamae sp.nov.,an N2-fixing rhizospheric and endophytic species[J].Int.J.Syst.Evol.Microbiol.,2004,54(4):1165-1172.
[4]Leahy J G,Byrne A M,Olsen R H.Comparison of factors influencing trichloroethylene degradation by toluene-oxidizing bacteria[J].Appl.Environ.Microb.,1996,62(3):825-833.
[5]Zú1iga A,Poupin M J,Donoso R,et al.Quorum sensing and indole-3-acetic acid degradation play a role in colonization and plant growth promotion of Arabidopsis thaliana by Burkholderia phytofirmans Ps JN[J].Mol.Plant.Microbe.Interact.,2013,26(5):546-553.
[6]Kim S,Lowman S,Hou G,et al.Growth promotion and colonization of switchgrass(Panicum virgatum)cv.Alamo by bacterial endophyte Burkholderia phytofirmans strain Ps JN[J].Biotechnol.Biofuels,2012,5:37.
[7]任嘉红,吴小芹,刘辉,等.吡咯伯克霍尔德氏菌JKSH007抗菌蛋白的分离纯化[J].微生物学通报,2010,37(6):872-880.
[8]Estrada-De los Santos P,Vinuesa P,Martínez-Aguilar L,et al.Phylogenetic analysis of Burkholderia species by multilocus sequence analysis[J].Curr.Microbiol.,2013,67(1):51-60.
[9]Hauser A R,Jain M,Bar-Meir M,et al.Clinical significance of microbial infection and adaptation in cystic fibrosis[J].Clin.Microbiol.Rev.,2011,24(1):29-70.
[10]Mahenthiralingam E,Bischof J,Byrne S K,et al.DNAbased diagnostic approaches for identification of Burkholderia cepacia complex,Burkholderia vietnamiensis,Burkholderia multivorans,Burkholderia stabilis,and Burkholderia cepacia genornovars I and III[J].J.Clin.Microbiol.,2000,38(9):3165-3173.
[11]Mahenthiralingam E,Simpson D A,Speert D P.Identification and characterization of a novel DNA maker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis[J].J.Clin.Microbiol.,1997,35(4):808-816.
[12]Richardson J,Stead D E,Coutts R H.Incidence of the cbl A major subunit pilin gene amongst Burkholderia species[J].Fems microbiol.Lett.,2001,196(1):61-66.
[13]Mahenthiralingam E,Urban T A,Goldberg J B.The multifarious,multireplicon Burkholderia cepacia complex[J].Nat.Rev.Microbiol.,2005,3(2):144-156.
[14]Sajjan U S,Sun L,Goldstein R,et al.Cable(cbl)type II pili of cystic fibrosis-associated Burkholderia(Pseudomonas)cepacia:nucleotide sequence of the cbl A major subunit pilin gene and novel morphology of the assembled appendage fibers[J].J.Bacteriol.,1995,177(4):1030-1038.
[15]Sun L,Jiang R Z,Steinbach S,et al.The emergence of a highly transmissible lineage of cbl+Pseudomonas(Burkholderia)cepacia causing CF centre epidemics in North America and Britain[J].Nat.Med.,1995,1:661-666.
[16]韩超,武贵元,刘爱新,等.生姜茎基腐病病原拮抗细菌的筛选与鉴定[J].山东农业科学,2012,44(11):84-89.
[17]Bergmann S,Wild D,Diekmann O,et al.Identification of a novel plasmin(ogen)-binding motif in surface displayedα-enolase of Streptococcus pneumoniae[J].Mol.Microbiol.,2003,49(2):411-423.
[18]张岚岚,徐春燕,徐昌杰.大肠杆菌感受态细胞转化能力的影响因素[J].细胞生物学杂志,2004,26(4):429-432.
[19]Guentas L,Gensous S,Cavaloc Y,et al.Burkholderia novacaledonica sp.nov.and B.ultramafica sp.nov.isolated from roots of Costularia spp.pioneer plants of ultramafic soils in New Caledonia[J].Syst.and Appl.Microbiol.,2016,39(3):151-159.
[20]Wang X Q,Liu A X,Guerrero A,et al.Occidiofungin is an important component responsible for the antifungal activity of Burkholederia pyrrocinia strain Lyc2[J].J.Appl.Microbiol.,2016,120(3):607-618.
[2]Parke J L,Gurian-Sherman D.Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains[J].Annu.Rev.Phytopathol.,2001,39(1):225-258.
[3]Caballero-Mellado J,Martínez-Aguilar L,Paredes-Valdez G,et al.Burkholderia unamae sp.nov.,an N2-fixing rhizospheric and endophytic species[J].Int.J.Syst.Evol.Microbiol.,2004,54(4):1165-1172.
[4]Leahy J G,Byrne A M,Olsen R H.Comparison of factors influencing trichloroethylene degradation by toluene-oxidizing bacteria[J].Appl.Environ.Microb.,1996,62(3):825-833.
[5]Zú1iga A,Poupin M J,Donoso R,et al.Quorum sensing and indole-3-acetic acid degradation play a role in colonization and plant growth promotion of Arabidopsis thaliana by Burkholderia phytofirmans Ps JN[J].Mol.Plant.Microbe.Interact.,2013,26(5):546-553.
[6]Kim S,Lowman S,Hou G,et al.Growth promotion and colonization of switchgrass(Panicum virgatum)cv.Alamo by bacterial endophyte Burkholderia phytofirmans strain Ps JN[J].Biotechnol.Biofuels,2012,5:37.
[7]任嘉红,吴小芹,刘辉,等.吡咯伯克霍尔德氏菌JKSH007抗菌蛋白的分离纯化[J].微生物学通报,2010,37(6):872-880.
[8]Estrada-De los Santos P,Vinuesa P,Martínez-Aguilar L,et al.Phylogenetic analysis of Burkholderia species by multilocus sequence analysis[J].Curr.Microbiol.,2013,67(1):51-60.
[9]Hauser A R,Jain M,Bar-Meir M,et al.Clinical significance of microbial infection and adaptation in cystic fibrosis[J].Clin.Microbiol.Rev.,2011,24(1):29-70.
[10]Mahenthiralingam E,Bischof J,Byrne S K,et al.DNAbased diagnostic approaches for identification of Burkholderia cepacia complex,Burkholderia vietnamiensis,Burkholderia multivorans,Burkholderia stabilis,and Burkholderia cepacia genornovars I and III[J].J.Clin.Microbiol.,2000,38(9):3165-3173.
[11]Mahenthiralingam E,Simpson D A,Speert D P.Identification and characterization of a novel DNA maker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis[J].J.Clin.Microbiol.,1997,35(4):808-816.
[12]Richardson J,Stead D E,Coutts R H.Incidence of the cbl A major subunit pilin gene amongst Burkholderia species[J].Fems microbiol.Lett.,2001,196(1):61-66.
[13]Mahenthiralingam E,Urban T A,Goldberg J B.The multifarious,multireplicon Burkholderia cepacia complex[J].Nat.Rev.Microbiol.,2005,3(2):144-156.
[14]Sajjan U S,Sun L,Goldstein R,et al.Cable(cbl)type II pili of cystic fibrosis-associated Burkholderia(Pseudomonas)cepacia:nucleotide sequence of the cbl A major subunit pilin gene and novel morphology of the assembled appendage fibers[J].J.Bacteriol.,1995,177(4):1030-1038.
[15]Sun L,Jiang R Z,Steinbach S,et al.The emergence of a highly transmissible lineage of cbl+Pseudomonas(Burkholderia)cepacia causing CF centre epidemics in North America and Britain[J].Nat.Med.,1995,1:661-666.
[16]韩超,武贵元,刘爱新,等.生姜茎基腐病病原拮抗细菌的筛选与鉴定[J].山东农业科学,2012,44(11):84-89.
[17]Bergmann S,Wild D,Diekmann O,et al.Identification of a novel plasmin(ogen)-binding motif in surface displayedα-enolase of Streptococcus pneumoniae[J].Mol.Microbiol.,2003,49(2):411-423.
[18]张岚岚,徐春燕,徐昌杰.大肠杆菌感受态细胞转化能力的影响因素[J].细胞生物学杂志,2004,26(4):429-432.
[19]Guentas L,Gensous S,Cavaloc Y,et al.Burkholderia novacaledonica sp.nov.and B.ultramafica sp.nov.isolated from roots of Costularia spp.pioneer plants of ultramafic soils in New Caledonia[J].Syst.and Appl.Microbiol.,2016,39(3):151-159.
[20]Wang X Q,Liu A X,Guerrero A,et al.Occidiofungin is an important component responsible for the antifungal activity of Burkholederia pyrrocinia strain Lyc2[J].J.Appl.Microbiol.,2016,120(3):607-618.