AmoA、AmoE和AmoF蛋白在嗜水气单胞菌铁载体合成中的作用研究
|
摘要
铁载体被认为是嗜水气单胞菌的毒力因子之一,其由amoCEBFAGH七个基因编码, AmoCGH在前人的研究中已证实参与铁载体的合成。RT-PCR实验表明amoAEF基因的表达受到铁的调控。为进一步探究amoAEF基因的功能,利用融合PCR和基因同源重组原理,以自杀性质粒PRE112为载体构建基因缺失株ΔamoA、ΔamoE和ΔamoF。通过CAS平板检测实验以及arnow实验来检测野生株WT与各基因缺失突变株铁载体的合成情况,并比较野生株与各缺失株在低铁培养基中的生长差异。结果显示,成功构建了基因缺失株ΔamoA、ΔamoE和ΔamoF;在富铁条件下,基因缺失株ΔamoA、ΔamoE和ΔamoF的生长与野生株无显著性差异,但在低铁条件下,基因缺失株ΔamoA、ΔamoE和ΔamoF的生长能力、铁载体合成能力显著低于野生株。可见, amoA、amoE和amoF基因是嗜水气单胞菌铁载体合成的关键基因,其缺失会导致细菌在低铁环境中的生长受到抑制。
Siderophore is considered as one of the virulence factors of Aeromonas hydrophila and is encoded by seven genes of amoCEBFAGH. Amo CGH have been confirmed to be involved in the synthesis of siderophore in previous studies. RT-PCR assay indicated that the expression of amoAEF genes were regulated by iron. In order to further explore the function of genes amoAEF, in this study, we used the suicide plasmid PRE112 to construct gene deletion mutant strains ΔamoA, ΔamoE and ΔamoF based on the infusion PCR and gene homologous recombination principle.The detection of the siderophore synthesis in the wild strains and mutant strains were performed using CAS plate test and arnow test. The results showed that Aeromonas hydrophila ΔamoA, ΔamoE and ΔamoF were successfully constructed. The growth of wild strain, ΔamoA, ΔamoE and ΔamoF had no significant difference in iron-riched medium,while in iron-depleted medium, the growth and siderophore synthesis capacity of ΔamoA, ΔamoE and ΔamoF were significantly lower than that of the wild strain. The results suggested that amoA, amoE and amoF are the critical synthesis genes of siderophore, and the growth of the deletion mutants were inhibited under low iron conditions.
Siderophore is considered as one of the virulence factors of Aeromonas hydrophila and is encoded by seven genes of amoCEBFAGH. Amo CGH have been confirmed to be involved in the synthesis of siderophore in previous studies. RT-PCR assay indicated that the expression of amoAEF genes were regulated by iron. In order to further explore the function of genes amoAEF, in this study, we used the suicide plasmid PRE112 to construct gene deletion mutant strains ΔamoA, ΔamoE and ΔamoF based on the infusion PCR and gene homologous recombination principle.The detection of the siderophore synthesis in the wild strains and mutant strains were performed using CAS plate test and arnow test. The results showed that Aeromonas hydrophila ΔamoA, ΔamoE and ΔamoF were successfully constructed. The growth of wild strain, ΔamoA, ΔamoE and ΔamoF had no significant difference in iron-riched medium,while in iron-depleted medium, the growth and siderophore synthesis capacity of ΔamoA, ΔamoE and ΔamoF were significantly lower than that of the wild strain. The results suggested that amoA, amoE and amoF are the critical synthesis genes of siderophore, and the growth of the deletion mutants were inhibited under low iron conditions.
引文
[1]Thune R L,Stanley L A,Cooper R K.Pathogenesis of gram-negative bacterial infections in warm water fish[J].Annual Reviews of Fish Diseases,1993,3:145-185
[2]Zhang L L,Zhang Q Q,Chen H,et al.Construction and biological characteristics analysis of cpxRA mutant in Aeromonas hydrophila[J].Acta Hydrobiologica Sinica,2018,42(1):1-10[张兰梨,张倩倩,陈辉,等.嗜水气单胞菌cpxR-A缺失株构建及生物学特性研究.水生生物学报,2018,42(1):1-10]
[3]Funahashi T,Tanabe T,Maki J,et al.Identification and characterization of Aeromonas hydrophila genes encoding the outer membrane receptor of ferrioxamine B and an AraC-type transcriptional regulator[J].Bioscience Biotechnology and Biochemistry,2014,78(10):1777-1787
[4]Jiang Q,Chen W,Qin Y,et al.AcuC,a histone deacetylase,contributes to the pathogenicity of Aeromonas hydrophila[J].Microbiologyopen,2017,6(4):1-12
[5]Pang M,Jiang J,Xie X,et al.Novel insights into the pathogenicity of epidemic Aeromonas hydrophila ST251clones from comparative genomics[J].Scientific Reports,2015,5:9833-9847
[6]Krewulak K D,Vogel H J.Structural biology of bacterial iron uptake[J].Biochimica et Biophysica Acta,2008,1778(9):1781-1804
[7]Funahashi T,Tanabe T,Miyamoto K,et al.Characterization of a gene encoding the outer membrane receptor for ferric enterobactin in Aeromonas hydrophila ATCC7966(T)[J].Bioscience Biotechnology and Biochemistry,2013,77(2):353-360
[8]Najimi M,Lemos M L,Osorio C R.Identification of iron regulated genes in the fish pathogen Aeromonas salmonicida subsp.salmonicida:genetic diversity and evidence of conserved iron uptake systems[J].Veterinary Microbiology,2009,133(4):377-382
[9]Balado M,Souto A,Vences A,et al.Two catechol siderophores,acinetobactin and amonabactin,are simultaneously produced by Aeromonas salmonicida subsp.salmonicida sharing part of the biosynthetic pathway[J].ACS Chemical Biology,2015,10(12):2850-2860
[10]Visca P,Imperi F,Lamont I L.Pyoverdine siderophores:from biogenesis to biosignificance[J].Trends in Microbiology,2007,15(1):22-30
[11]George M,Arceneaux J E L,Byers B R.Acquisition of iron from host sources by mesophilic Aeromonas species[J].Microbiology,1991,137(2):237-241
[12]Telford J R,Leary J A,Tunstad L M G,et al.Amonabactin:characterization of a series of siderophores from Aeromonas hydrophila[J].Journal of the American Chemical Society,1994,116:4499-4500
[13]Seshadri R,Joseph S W,Chopra A K,et al.Genome sequence of Aeromonas hydrophila ATCC 7966T:jack of all trades[J].Journal of Bacteriology,2006,188(23):8272-8282
[14]Barghouthi S,Payne S M,Arceneaux J E L,et al.Cloning,mutagenesis,and nucleotide sequence of a siderophore biosynthetic gene(amoA)from Aeromonas hydrophila[J].Journal of Bacteriology,1991,173:5121-5128
[15]Esmaeel Q,Chevalier M,Chataigne G,et al.Nonribosomal peptide synthetase with a unique iterative-alternative-optional mechanism catalyzes amonabactin synthesis in Aeromonas[J].Applied Microbiology and Biotechnology,2016,100(19):8453-8463
[16]Zhang X J,Yang W M,Li T T,et al.The genetic diversity and virulence characteristics of Aeromonas hydrophila isolated from fishponds with disease outbreaks in Hubei province[J].Acta Hydrobiologica Sinica,2013,37(3):458-466[张旭杰,杨五名,李彤彤,等.湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究.水生生物学报,2013,37(3):458-466]
[17]Liu L Y,Nie P,Yu H B,et al.Regulation of typeⅢsecretion of translocon and effector proteins by the EsaB/EsaL/EsaM complex in Edwardsiella tarda[J].Infection and Immunity,2017,85(9):1-15
[18]Hoa S N,Hunt H D,Horton R M,et al.Site-directed mutagenesis by overlap extension using the polymerase chain reaction[J].Gene,1989,77(1):51-59
[19]Arnow L E.Colorimetric determination of the components of 3,4-dihydroxyphenylalaninetyrosine mixtures[J].Journal of Biological Chemistr,1937,118(2):531-537
[20]Schwyn B,Neilands J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56
[21]Barghouthi S,Young R,Arceneaux J E.Physiological control of amonabactin biosynthesis in Aeromonas hydrophila[J].Biometals,1989,2(3):155-160
[22]Keating T A,Marshall C G,Walsh C T.Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB,VibE,VibF,and VibH[J].Biochemistry,2000,39(50):15522-15530
[23]Wyckoff E E,Stoebner J A,Reed K E,et al.Cloning of a Vibrio cholerae vibriobactin gene cluster:identification of genes required for early steps in siderophore biosynthesis[J].Journal of Bacteriology,1997,179:7055-7062
[24]Zhang C G.Cristal structure and function of VibB and its interaction with VibD[D].Master’s thesis,Shandong University.2010[张从刚.铁载体合成蛋白VibB的结构和功能以及VibB_VibD的相互作用分析.硕士学位论文,山东大学,山东,2010]
[25]Janda J M,Abbott S L.The genus Aeromonas:taxonomy,pathogenicity,and infection[J].Clinical Microbiology Reviews,2010,23(1):35-73
[26]Scott W Bearden,Fetherston J D,Perry R D.Genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis[J].Infection and Immunity,1997,65(5):1659-1668
[27]Heesemann J,Hantke K,Vocke T,et al.Virulence of Yersinia enterocolitica is closely associated with siderophore production,expression of an iron-repressible outer membrane polypeptide of 65000 Da and pesticin sensitivity[J].Molecular Microbiology,1993,8(2):397-408
[28]Abdelhamed H,Lawrence M L,Karsi A.The Role of TonB Gene in Edwardsiella ictaluri Virulence[J].Frontiers in Physiology,2017,8:1066-1073
[29]Oh M H,Lee S M,Lee D H,et al.Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence[J].Infection and Immunity,2009,77(3):1208-1215
[30]Takase H,Nitanai H,Hoshino K,et al.Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice[J].Infection and Immunity,2000,68(4):1834-1839
[31]Zywno S R,Arceneaux J E,Altwegg M,et al.Siderophore production and DNA hybridization groups of Aeromonas spp.[J].Journal of Clinical Microbiology,1992,30(3):619-622
[2]Zhang L L,Zhang Q Q,Chen H,et al.Construction and biological characteristics analysis of cpxRA mutant in Aeromonas hydrophila[J].Acta Hydrobiologica Sinica,2018,42(1):1-10[张兰梨,张倩倩,陈辉,等.嗜水气单胞菌cpxR-A缺失株构建及生物学特性研究.水生生物学报,2018,42(1):1-10]
[3]Funahashi T,Tanabe T,Maki J,et al.Identification and characterization of Aeromonas hydrophila genes encoding the outer membrane receptor of ferrioxamine B and an AraC-type transcriptional regulator[J].Bioscience Biotechnology and Biochemistry,2014,78(10):1777-1787
[4]Jiang Q,Chen W,Qin Y,et al.AcuC,a histone deacetylase,contributes to the pathogenicity of Aeromonas hydrophila[J].Microbiologyopen,2017,6(4):1-12
[5]Pang M,Jiang J,Xie X,et al.Novel insights into the pathogenicity of epidemic Aeromonas hydrophila ST251clones from comparative genomics[J].Scientific Reports,2015,5:9833-9847
[6]Krewulak K D,Vogel H J.Structural biology of bacterial iron uptake[J].Biochimica et Biophysica Acta,2008,1778(9):1781-1804
[7]Funahashi T,Tanabe T,Miyamoto K,et al.Characterization of a gene encoding the outer membrane receptor for ferric enterobactin in Aeromonas hydrophila ATCC7966(T)[J].Bioscience Biotechnology and Biochemistry,2013,77(2):353-360
[8]Najimi M,Lemos M L,Osorio C R.Identification of iron regulated genes in the fish pathogen Aeromonas salmonicida subsp.salmonicida:genetic diversity and evidence of conserved iron uptake systems[J].Veterinary Microbiology,2009,133(4):377-382
[9]Balado M,Souto A,Vences A,et al.Two catechol siderophores,acinetobactin and amonabactin,are simultaneously produced by Aeromonas salmonicida subsp.salmonicida sharing part of the biosynthetic pathway[J].ACS Chemical Biology,2015,10(12):2850-2860
[10]Visca P,Imperi F,Lamont I L.Pyoverdine siderophores:from biogenesis to biosignificance[J].Trends in Microbiology,2007,15(1):22-30
[11]George M,Arceneaux J E L,Byers B R.Acquisition of iron from host sources by mesophilic Aeromonas species[J].Microbiology,1991,137(2):237-241
[12]Telford J R,Leary J A,Tunstad L M G,et al.Amonabactin:characterization of a series of siderophores from Aeromonas hydrophila[J].Journal of the American Chemical Society,1994,116:4499-4500
[13]Seshadri R,Joseph S W,Chopra A K,et al.Genome sequence of Aeromonas hydrophila ATCC 7966T:jack of all trades[J].Journal of Bacteriology,2006,188(23):8272-8282
[14]Barghouthi S,Payne S M,Arceneaux J E L,et al.Cloning,mutagenesis,and nucleotide sequence of a siderophore biosynthetic gene(amoA)from Aeromonas hydrophila[J].Journal of Bacteriology,1991,173:5121-5128
[15]Esmaeel Q,Chevalier M,Chataigne G,et al.Nonribosomal peptide synthetase with a unique iterative-alternative-optional mechanism catalyzes amonabactin synthesis in Aeromonas[J].Applied Microbiology and Biotechnology,2016,100(19):8453-8463
[16]Zhang X J,Yang W M,Li T T,et al.The genetic diversity and virulence characteristics of Aeromonas hydrophila isolated from fishponds with disease outbreaks in Hubei province[J].Acta Hydrobiologica Sinica,2013,37(3):458-466[张旭杰,杨五名,李彤彤,等.湖北地区暴发病池塘中嗜水气单胞菌的遗传多样性和毒力特征研究.水生生物学报,2013,37(3):458-466]
[17]Liu L Y,Nie P,Yu H B,et al.Regulation of typeⅢsecretion of translocon and effector proteins by the EsaB/EsaL/EsaM complex in Edwardsiella tarda[J].Infection and Immunity,2017,85(9):1-15
[18]Hoa S N,Hunt H D,Horton R M,et al.Site-directed mutagenesis by overlap extension using the polymerase chain reaction[J].Gene,1989,77(1):51-59
[19]Arnow L E.Colorimetric determination of the components of 3,4-dihydroxyphenylalaninetyrosine mixtures[J].Journal of Biological Chemistr,1937,118(2):531-537
[20]Schwyn B,Neilands J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160(1):47-56
[21]Barghouthi S,Young R,Arceneaux J E.Physiological control of amonabactin biosynthesis in Aeromonas hydrophila[J].Biometals,1989,2(3):155-160
[22]Keating T A,Marshall C G,Walsh C T.Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB,VibE,VibF,and VibH[J].Biochemistry,2000,39(50):15522-15530
[23]Wyckoff E E,Stoebner J A,Reed K E,et al.Cloning of a Vibrio cholerae vibriobactin gene cluster:identification of genes required for early steps in siderophore biosynthesis[J].Journal of Bacteriology,1997,179:7055-7062
[24]Zhang C G.Cristal structure and function of VibB and its interaction with VibD[D].Master’s thesis,Shandong University.2010[张从刚.铁载体合成蛋白VibB的结构和功能以及VibB_VibD的相互作用分析.硕士学位论文,山东大学,山东,2010]
[25]Janda J M,Abbott S L.The genus Aeromonas:taxonomy,pathogenicity,and infection[J].Clinical Microbiology Reviews,2010,23(1):35-73
[26]Scott W Bearden,Fetherston J D,Perry R D.Genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis[J].Infection and Immunity,1997,65(5):1659-1668
[27]Heesemann J,Hantke K,Vocke T,et al.Virulence of Yersinia enterocolitica is closely associated with siderophore production,expression of an iron-repressible outer membrane polypeptide of 65000 Da and pesticin sensitivity[J].Molecular Microbiology,1993,8(2):397-408
[28]Abdelhamed H,Lawrence M L,Karsi A.The Role of TonB Gene in Edwardsiella ictaluri Virulence[J].Frontiers in Physiology,2017,8:1066-1073
[29]Oh M H,Lee S M,Lee D H,et al.Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence[J].Infection and Immunity,2009,77(3):1208-1215
[30]Takase H,Nitanai H,Hoshino K,et al.Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice[J].Infection and Immunity,2000,68(4):1834-1839
[31]Zywno S R,Arceneaux J E,Altwegg M,et al.Siderophore production and DNA hybridization groups of Aeromonas spp.[J].Journal of Clinical Microbiology,1992,30(3):619-622