MvaT调控铜绿假单胞菌吩嗪的合成机制
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摘要
【背景】属于H-NS家族的MvaT转录因子参与了铜绿假单胞菌的许多重要代谢过程,如吩嗪合成代谢,但其调控方式仍不十分明确。【目的】确定转录调控因子MvaT是否直接调控铜绿假单胞菌的吩嗪合成过程,即该蛋白是否可以直接结合2个吩嗪-1-羧酸合成基因簇(phzA1G1和phzA2G2)与3个分支转化基因(phzH、phzS和phzM)的上游启动子区域。【方法】以铜绿假单胞菌SJTD-1和其mvaT基因敲除突变株SJTD-1(ΔmvaT)为研究对象,检测其在不同培养基条件下吩嗪化合物的合成量差异。通过体外异源表达与亲和纯化,获得重组蛋白MvaT。利用凝胶阻滞实验,确定MvaT重组蛋白对5个吩嗪代谢基因簇/基因上游启动子的结合情况。【结果】mvaT基因敲除突变株SJTD-1(ΔmvaT)的吩嗪产量较野生型显著提升。MvaT重组蛋白被有效表达与纯化,体外凝胶阻滞实验结果显示,该重组蛋白可与phzA1G1、phzA2G2、phzM、phzS和phzH的上游启动子区域均发生特异性结合。其中,重组蛋白MvaT与phzA1G1和phzA2G2的结合区域位于其上游启动子的200 bp以内,而该蛋白与phzM、phzS和phzH的结合区域则位于其上游启动子的100 bp以内。【结论】MvaT蛋白通过直接结合吩嗪合成代谢基因的上游启动子区域来直接调控假单胞菌的吩嗪类化合物合成。
[Background] MvaT protein belonging to the H-NS transcription factor family is involved in many important metabolic processes of Pseudomonas aeruginosa, such as phenazine synthesis pathway.However, up to now its regulation mode is still unclear. [Objective] The goal of this work was to determine if MvaT could directly regulate the two phenazine-1-carboxylic acid synthesis gene clusters(phzA1 G1 and phzA2 G2) and three transforming genes(phzH, phzS and phzM) of phenazine compounds,by detecting the binding capability of MvaT to the promoter regions of these genes. [Methods]Pseudomonas aeruginosa SJTD-1 was used as target and the mvaT-knockout strain SJTD-1(ΔmvaT) was constructed by homologous recombination. The yield of phenazine compounds of the two strains in different media was detected. Further the recombinant MvaT protein was obtained by the heterologous expression and affinity purification. The electrophoretic mobility shift assay(EMSA) was performed to determine if the recombinant MvaT protein could bind to the promoter regions of the five phenazine synthetic gene clusters/genes. [Results] The yield of phenazine compounds of strain SJTD-1(ΔmvaT) was significantly higher than that of the wild type SJTD-1 strain. The recombinant MvaT protein could be expressed and purified efficiently. In vitro EMSA results indicated that the recombinant MvaT protein could directly bind to the promoter regions of phenazine synthetic gene clusters/genes. The binding sites of MvaT to the promoter of phzA1 G1 and phzA2 G2 gene clusters were within the upstream 200 bp region,and that to the promoter of phzM, phzS and phzH genes were within its upstream 100 bp region.[Conclusion] MvaT protein can directly bind the upstream promoter regions of the five phenazine synthetic gene clusters/genes and regulate the synthesis of phenazine compounds.
[Background] MvaT protein belonging to the H-NS transcription factor family is involved in many important metabolic processes of Pseudomonas aeruginosa, such as phenazine synthesis pathway.However, up to now its regulation mode is still unclear. [Objective] The goal of this work was to determine if MvaT could directly regulate the two phenazine-1-carboxylic acid synthesis gene clusters(phzA1 G1 and phzA2 G2) and three transforming genes(phzH, phzS and phzM) of phenazine compounds,by detecting the binding capability of MvaT to the promoter regions of these genes. [Methods]Pseudomonas aeruginosa SJTD-1 was used as target and the mvaT-knockout strain SJTD-1(ΔmvaT) was constructed by homologous recombination. The yield of phenazine compounds of the two strains in different media was detected. Further the recombinant MvaT protein was obtained by the heterologous expression and affinity purification. The electrophoretic mobility shift assay(EMSA) was performed to determine if the recombinant MvaT protein could bind to the promoter regions of the five phenazine synthetic gene clusters/genes. [Results] The yield of phenazine compounds of strain SJTD-1(ΔmvaT) was significantly higher than that of the wild type SJTD-1 strain. The recombinant MvaT protein could be expressed and purified efficiently. In vitro EMSA results indicated that the recombinant MvaT protein could directly bind to the promoter regions of phenazine synthetic gene clusters/genes. The binding sites of MvaT to the promoter of phzA1 G1 and phzA2 G2 gene clusters were within the upstream 200 bp region,and that to the promoter of phzM, phzS and phzH genes were within its upstream 100 bp region.[Conclusion] MvaT protein can directly bind the upstream promoter regions of the five phenazine synthetic gene clusters/genes and regulate the synthesis of phenazine compounds.
引文
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[11]Diggle SP,Stacey RE,Dodd C,et al.The galactophilic lectin,LecA,contributes to biofilm development in Pseudomonas aeruginosa[J].Environmental Microbiology,2006,8(6):1095-1104
[12]Wu XG,Wei YR,Liu JC,et al.MvaT and MvaVtranscriptionally regulate PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24[J].Acta Microbiologica Sinica,2012,52(6):710-717(in Chinese)吴小刚,魏亚蕊,刘九成,等.生防假单胞菌2P24中mvaT和mvaV基因对PcoI/PcoR群体感应系统的调控作用[J].微生物学报,2012,52(6):710-717
[13]Li CR,Wally H,Miller SJ,et al.The multifaceted proteins MvaT and MvaU,members of the H-NS family,control arginine metabolism,pyocyanin synthesis,and prophage activation in Pseudomonas aeruginosa PAO1[J].Journal of Bacteriology,2009,191(20):6211-6218
[14]Liu H,Liang RB,Tao F,et al.Genome sequence of Pseudomonas aeruginosa strain SJTD-1,a bacterium capable of degrading long-chain alkanes and crude oil[J].Journal of Bacteriology,2012,194(17):4783-4784
[15]Liu H,Sun WB,Liang RB,et al.iTRAQ-based quantitative proteomic analysis of Pseudomonas aeruginosa SJTD-1:Aglobal response to n-octadecane induced stress[J].Journal of Proteomics,2015,123:14-28
[16]You T,Liu JH,Liang RB,et al.Survival elongation of Pseudomonas aeruginosa improves power output of microbial fuel cells[J].Chinese Journal of Biotechnology,2017,33(4):601-608(in Chinese)游婷,刘季华,梁如冰,等.铜绿假单胞菌存活时间延长可提高生物燃料电池的产电量[J].生物工程学报,2017,33(4):601-608
[17]Liang HH,Li LL,Dong ZL,et al.The YebC family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production[J].Journal of Bacteriology,2008,190(18):6217-6227
[18]Lu Z,Liang R,Liu X,et al.RNase HIII from Chlamydophila pneumoniae can efficiently cleave double-stranded DNAcarrying a chimeric ribonucleotide in the presence of manganese[J].Molecular Microbiology,2012,83(5):1080-1093
[19]Withers H,Swift S,Williams P.Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria[J].Current Opinion in Microbiology,2001,4(2):186-193
[20]Passador L,Cook JM,Gambello MJ,et al.Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication[J].Science,1993,260(5111):1127-1130
[21]Bertani I,?evo M,Kojic M,et al.Role of GacA,LasI,RhlI,Ppk,PsrA,Vfr and ClpXP in the regulation of the stationary-phase sigma factor rpoS/RpoS in Pseudomonas[J].Archives of Microbiology,2003,180(4):264-271
[22]Baehler E,Maurhofer M,Keel C.Henolic signal metabolites from bacteria and plants affect production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0 in Abstr[A]//The Proceedings of the 9th International Sympoium Microbiology Ecology[C].Amsterdam,2001:266
[23]Sarniguet A,Kraus J,Henkels MD,et al.The sigma factorσSaffects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5[J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(26):12255-12259
[24]Zhou JF,Ge YH,Liu T,et al.Effect of rpoS mutation on two gene cluster of phenazine in Pseudomonas aeruginosa PAO1[J].Acta Microbiologica Sinica,2010,50(3):411-417(in Chinese)周金凤,葛宜和,刘婷,等.rpoS基因插入突变对铜绿假单胞菌两个吩嗪合成基因簇的调控[J].微生物学报,2010,50(3):411-417
[25]Miao J,Chi XY,Wang YH,et al.Regulation of pyocyanin biosynthesis by transcriptional factor sigma38 in Pseudomonas aeruginosa PAO1[J].Acta Microbiologica Sinica,2017,57(2):229-239(in Chinese)缪静,迟晓艳,王艳华,等.转录调节因子σ38介导铜绿假单胞菌绿脓菌素合成代谢调控[J].微生物学报,2017,57(2):229-239
[26]Diggle SP,Winzer K,Chhabra SR,et al.The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy,regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR[J].Molecular Microbiology,2003,50(1):29-43
[27]McKnight SL,Iglewski BH,Pesci EC.The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa[J].Journal of Bacteriology,2000,182(10):2702-2708
[28]Rabaey K,Boon N,Siciliano SD,et al.Biofuel cells select for microbial consortia that self-mediate electron transfer[J].Applied and Environmental Microbiology,2004,70(9):5373-5382
[29]Pham TH,Boon N,Aelterman P,et al.Metabolites produced by Pseudomonas sp.enable a Gram-positive bacterium to achieve extracellular electron transfer[J].Applied Microbiology and Biotechnology,2008,77(5):1119-1129
[30]Yong XY,Shi DY,Chen YL,et al.Enhancement of bioelectricity generation by manipulation of the electron shuttles synthesis pathway in microbial fuel cells[J].Bioresource Technology,2014,152:220-224
[31]Nie HY.Studies on characteristics of the secretion of extracellular small active compounds by P.aeruginosa NY3and their promotion mechanisms to the efficiency of biodegradation of hydrocarbon[D].Xi’an:Doctoral Dissertation of Xi’an University of Architecture and Technology,2017(in Chinese)聂红云.铜绿假单胞菌NY3胞外小分子活性物及其促进烃类降解的作用机制研究[D].西安:西安建筑科技大学博士学位论文,2017
[2]Mavrodi DV,Blankenfeldt W,Thomashow LS.Phenazine compounds in fluorescent Pseudomonas spp.biosynthesis and regulation[J].Annual Review of Phytopathology,2006,44:417-445
[3]Haas D,Défago G.Biological control of soil-borne pathogens by Fluorescent pseudomonads[J].Nature Reviews Microbiology,2005,3(4):307-319
[4]Pierson LS,Pierson EA.Metabolism and function of phenazines in bacteria:impacts on the behavior of bacteria in the environment and biotechnological processes[J].Applied Microbiology and Biotechnology,2010,86(6):1659-1670
[5]Mavrodi DV,Bonsall RF,Delaney SM,et al.Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1[J].Journal of Bacteriology,2001,183(21):6454-6465
[6]Hunter RC,Klepac-Ceraj V,Lorenzi MM,et al.Phenazine content in the cystic fibrosis respiratory tract negatively correlates with lung function and microbial complexity[J].American Journal of Respiratory Cell and Molecular Biology,2012,47(6):738-745
[7]Britigan EB,Railsback MA,Cox CD.The Pseudomonas aeruginosa secretory product pyocyanin inactivatesα1 protease inhibitor:implications for the pathogenesis of cystic fibrosis lung disease[J].Infection and Immunity,1999,67(3):1207-1212
[8]Tendeng C,Soutourina OA,Danchin A,et al.MvaT proteins in Pseudomonas spp.:a novel class of H-NS-like proteins[J].Microbiology,2003,149(Pt11):3047-3050
[9]Diggle SP,Winzer K,Lazdunski A,et al.Advancing the quorum in Pseudomonas aeruginosa:MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression[J].Journal of Bacteriology,2002,184(10):2576-2586
[10]Vallet I,Diggle SP,Stacey RE,et al.Biofilm formation in Pseudomonas aeruginosa:fimbrial cup gene clusters are controlled by the transcriptional regulator MvaT[J].Journal of Bacteriology,2004,186(9):2880-2890
[11]Diggle SP,Stacey RE,Dodd C,et al.The galactophilic lectin,LecA,contributes to biofilm development in Pseudomonas aeruginosa[J].Environmental Microbiology,2006,8(6):1095-1104
[12]Wu XG,Wei YR,Liu JC,et al.MvaT and MvaVtranscriptionally regulate PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24[J].Acta Microbiologica Sinica,2012,52(6):710-717(in Chinese)吴小刚,魏亚蕊,刘九成,等.生防假单胞菌2P24中mvaT和mvaV基因对PcoI/PcoR群体感应系统的调控作用[J].微生物学报,2012,52(6):710-717
[13]Li CR,Wally H,Miller SJ,et al.The multifaceted proteins MvaT and MvaU,members of the H-NS family,control arginine metabolism,pyocyanin synthesis,and prophage activation in Pseudomonas aeruginosa PAO1[J].Journal of Bacteriology,2009,191(20):6211-6218
[14]Liu H,Liang RB,Tao F,et al.Genome sequence of Pseudomonas aeruginosa strain SJTD-1,a bacterium capable of degrading long-chain alkanes and crude oil[J].Journal of Bacteriology,2012,194(17):4783-4784
[15]Liu H,Sun WB,Liang RB,et al.iTRAQ-based quantitative proteomic analysis of Pseudomonas aeruginosa SJTD-1:Aglobal response to n-octadecane induced stress[J].Journal of Proteomics,2015,123:14-28
[16]You T,Liu JH,Liang RB,et al.Survival elongation of Pseudomonas aeruginosa improves power output of microbial fuel cells[J].Chinese Journal of Biotechnology,2017,33(4):601-608(in Chinese)游婷,刘季华,梁如冰,等.铜绿假单胞菌存活时间延长可提高生物燃料电池的产电量[J].生物工程学报,2017,33(4):601-608
[17]Liang HH,Li LL,Dong ZL,et al.The YebC family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production[J].Journal of Bacteriology,2008,190(18):6217-6227
[18]Lu Z,Liang R,Liu X,et al.RNase HIII from Chlamydophila pneumoniae can efficiently cleave double-stranded DNAcarrying a chimeric ribonucleotide in the presence of manganese[J].Molecular Microbiology,2012,83(5):1080-1093
[19]Withers H,Swift S,Williams P.Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria[J].Current Opinion in Microbiology,2001,4(2):186-193
[20]Passador L,Cook JM,Gambello MJ,et al.Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication[J].Science,1993,260(5111):1127-1130
[21]Bertani I,?evo M,Kojic M,et al.Role of GacA,LasI,RhlI,Ppk,PsrA,Vfr and ClpXP in the regulation of the stationary-phase sigma factor rpoS/RpoS in Pseudomonas[J].Archives of Microbiology,2003,180(4):264-271
[22]Baehler E,Maurhofer M,Keel C.Henolic signal metabolites from bacteria and plants affect production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0 in Abstr[A]//The Proceedings of the 9th International Sympoium Microbiology Ecology[C].Amsterdam,2001:266
[23]Sarniguet A,Kraus J,Henkels MD,et al.The sigma factorσSaffects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5[J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(26):12255-12259
[24]Zhou JF,Ge YH,Liu T,et al.Effect of rpoS mutation on two gene cluster of phenazine in Pseudomonas aeruginosa PAO1[J].Acta Microbiologica Sinica,2010,50(3):411-417(in Chinese)周金凤,葛宜和,刘婷,等.rpoS基因插入突变对铜绿假单胞菌两个吩嗪合成基因簇的调控[J].微生物学报,2010,50(3):411-417
[25]Miao J,Chi XY,Wang YH,et al.Regulation of pyocyanin biosynthesis by transcriptional factor sigma38 in Pseudomonas aeruginosa PAO1[J].Acta Microbiologica Sinica,2017,57(2):229-239(in Chinese)缪静,迟晓艳,王艳华,等.转录调节因子σ38介导铜绿假单胞菌绿脓菌素合成代谢调控[J].微生物学报,2017,57(2):229-239
[26]Diggle SP,Winzer K,Chhabra SR,et al.The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy,regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR[J].Molecular Microbiology,2003,50(1):29-43
[27]McKnight SL,Iglewski BH,Pesci EC.The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa[J].Journal of Bacteriology,2000,182(10):2702-2708
[28]Rabaey K,Boon N,Siciliano SD,et al.Biofuel cells select for microbial consortia that self-mediate electron transfer[J].Applied and Environmental Microbiology,2004,70(9):5373-5382
[29]Pham TH,Boon N,Aelterman P,et al.Metabolites produced by Pseudomonas sp.enable a Gram-positive bacterium to achieve extracellular electron transfer[J].Applied Microbiology and Biotechnology,2008,77(5):1119-1129
[30]Yong XY,Shi DY,Chen YL,et al.Enhancement of bioelectricity generation by manipulation of the electron shuttles synthesis pathway in microbial fuel cells[J].Bioresource Technology,2014,152:220-224
[31]Nie HY.Studies on characteristics of the secretion of extracellular small active compounds by P.aeruginosa NY3and their promotion mechanisms to the efficiency of biodegradation of hydrocarbon[D].Xi’an:Doctoral Dissertation of Xi’an University of Architecture and Technology,2017(in Chinese)聂红云.铜绿假单胞菌NY3胞外小分子活性物及其促进烃类降解的作用机制研究[D].西安:西安建筑科技大学博士学位论文,2017