假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰（英文）
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摘要
【目的】DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。【方法】首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spf BCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了Spf B蛋白与DNA结合序列。【结果】spf B基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spf B基因的回补能够恢复该表型,证明spf B基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在?spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spf BCDE的启动子区域5′-TGTTTGT-3′相结合。【结论】SpfB作为转录调控因子负调控磷硫酰化修饰基因spf BCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。
[Objective] DNA phosphorothioate(PT) modification, in which the sulfur replaces a nonbridging oxygen, occurs naturally in diverse bacteria, archaea and human pathogens as a new kind of epigenetic modification. However, the regulatory mechanism of PT modification has not been fully characterized. In this study, the regulatory mechanism of spf B on DNA phosphorothioate(PT) modification in Pseudomonas fluorescens Pf0-1 was demonstrated. [Methods] Firstly, the spf B genetic interruption and complementary strains were constructed by homologous recombination and then tested the modification frequency in these strains by iodine cleavage. The operons within spf gene cluster were grouped by RT-PCR and the transcriptional level was analyzed in the Δspf B mutant by quantitative real-time PCR. Finally, the possible regulatory region of Spf B on the spf operon was characterized by EMSA and DNase I footprinting assay. [Results] The inactivation of spf B led to more dispersed small fragments in genomic DNA of Δspf B mutant and its complementation obviously restored the phenotype of wild type strain. Genes in spf gene cluster were assigned into one co-transcription unit, and the disruption of spf B directly up-regulated the transcription of the operon. In vitro Spf B directly protected two separate sequences within the spf promoter region from DNase I cleavage, and each protected sequence contained a direct repeat(5′-TGTTTGT-3′). [Conclusion] Spf B in Pseudomonas fluorescens Pf0-1 was a negative regulator in DNA phosphorothioate modification.
[Objective] DNA phosphorothioate(PT) modification, in which the sulfur replaces a nonbridging oxygen, occurs naturally in diverse bacteria, archaea and human pathogens as a new kind of epigenetic modification. However, the regulatory mechanism of PT modification has not been fully characterized. In this study, the regulatory mechanism of spf B on DNA phosphorothioate(PT) modification in Pseudomonas fluorescens Pf0-1 was demonstrated. [Methods] Firstly, the spf B genetic interruption and complementary strains were constructed by homologous recombination and then tested the modification frequency in these strains by iodine cleavage. The operons within spf gene cluster were grouped by RT-PCR and the transcriptional level was analyzed in the Δspf B mutant by quantitative real-time PCR. Finally, the possible regulatory region of Spf B on the spf operon was characterized by EMSA and DNase I footprinting assay. [Results] The inactivation of spf B led to more dispersed small fragments in genomic DNA of Δspf B mutant and its complementation obviously restored the phenotype of wild type strain. Genes in spf gene cluster were assigned into one co-transcription unit, and the disruption of spf B directly up-regulated the transcription of the operon. In vitro Spf B directly protected two separate sequences within the spf promoter region from DNase I cleavage, and each protected sequence contained a direct repeat(5′-TGTTTGT-3′). [Conclusion] Spf B in Pseudomonas fluorescens Pf0-1 was a negative regulator in DNA phosphorothioate modification.
引文
[1]Zhou XF,He XY,Liang JD,Li AY,Xu TG,Kieser T,Helmann JD,Deng ZX.A novel DNA modification by sulphur.Molecular Microbiology,2005,57(5):1428-1438.
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[8]Hu W,Wang CK,Liang JD,Zhang TL,Hu ZP,Wang ZJ,Lan WX,Li F,Wu HM,Ding JP,Wu G,Deng ZX,Cao CY.Structural insights into Dnd E from Escherichia coli B7Ainvolved in DNA phosphorothioation modification.Cell Research,2012,22(7):1203-1206.
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[10]Cao B,Zheng XQ,Cheng QX,Yao F,Zheng T,Ramesh Babu I,Zhou HC,Dedon P,You DL.In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.Scientific Reports,2015,5:12513.
[11]Xu TG,Yao F,Zhou XF,Deng ZX,You DL.A novel host-specific restriction system associated with DNAbackbone S-modification in Salmonella.Nucleic Acids Research,2010,38(20):7133-7141.
[12]Cao B,Cheng QX,Gu C,Yao F,De Mott MS,Zheng XQ,Deng ZX,Dedon PC,You DL.Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella.Molecular Microbiology,2014,93(4):776-785.
[13]Kellner S,De Mott MS,Cheng CP,Russell BS,Cao B,You DL,Dedon PC.Oxidation of phosphorothioate DNAmodifications leads to lethal genomic instability.Nature Chemical Biology,2017,13(8):888-894.
[14]Cheng QX,Cao B,Yao F,Li JL,Deng ZX,You DL.Regulation of DNA phosphorothioate modifications by the transcriptional regulator Dpt B in Salmonella.Molecular Microbiology,2015,97(6):1186-1194.
[15]Haas D,Blumer C,Keel C.Biocontrol ability of fluorescent pseudomonads genetically dissected:importance of positive feedback regulation.Current Opinion in Biotechnology,2000,11(3):290-297.
[16]Schnider U,Keel C,Voisard C,Défago G,Haas D.Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0:mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.Applied and Environmental Microbiology,1995,61(11):3856-3864.
[17]Zhang WK,Wang L,Kong LX,Wang T,Chu YW,Deng ZX,You DL.Unveiling the post-PKS redox tailoring steps in biosynthesis of the Type II polyketide antitumor antibiotic xantholipin.Chemistry&Biology,2012,19(3):422-432.
[18]Thompson JD,Higgins DG,Gibson TJ.CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice.Nucleic Acids Research,1994,22(22):4673-4680.
[19]Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,2011,28(10):2731-2739.
[20]Xu TG,Liang JD,Chen S,Wang LR,He XY,You DL,Wang ZJ,Li AY,Xu ZL,Zhou XF,Deng ZX.DNAphosphorothioation in Streptomyces lividans:mutational analysis of the dnd locus.BMC Microbiology,2009,9:41.
[21]Cao B,Chen C,De Mott,MS,Cheng QX,Clark TA,Xiong XL,Zheng XQ,Butty V,Levine SS,Yuan G,Boitano M,Luong K,Song Y,Zhou XF,Deng ZX,Turner SW,Korlach J,You DL,Wang LR,Chen S,Dedon PC.Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences.Nature Communications,2014,5:3951.
[22]Rojo F.Mechanisms of transcriptional repression.Current Opinion in Microbiology,2001,4(2):145-151.
[23]Studier FW,Rosenberg AH,Dunn JJ,Dubendorff JW.Use of T7 RNA polymerase to direct expression of cloned genes.Methods in Enzymology,1990,185:60-89.
[24]Milton DL,Norqvist A,Wolf-Watz H.Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.Journal of Bacteriology,1992,174(22):7235-7244.
[25]Malakhov MP,Mattern MR,Malakhova OA,Drinker M,Weeks SD,Butt TR.SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins.Journal of Structural and Functional Genomics,2004,5(1/2):75-86.
[26]Sch?fer A,Tauch A,J?ger W,Kalinowski J,Thierbach G,Pühler A.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids p K18 and p K19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,1994,145(1):69-73.
[27]Kovach ME,Elzer PH,Hill DS,Robertson GT,Farris MA,Roop II RM,Peterson KM.Four new derivatives of the broad-host-range cloning vector p BBR1MCS,carrying different antibiotic-resistance cassettes.Gene,1995,166(1):175-176.
[2]He XY,Ou HY,Yu Q,Zhou XF,Wu J,Liang JD,Zhang W,Rajakumar K,Deng ZX.Analysis of a genomic island housing genes for DNA S-modification system in Streptomyces lividans 66 and its counterparts in other distantly related bacteria.Molecular Microbiology,2007,65(4):1034-1048.
[3]Wang LR,Chen S,Xu TG,Taghizadeh K,Wishnok JS,Zhou XF,You DL,Deng ZX,Dedon PC.Phosphorothioation of DNA in bacteria by dnd genes.Nature Chemical Biology,2007,3(11):709-710.
[4]Wang LR,Chen S,Vergin KL,Giovannoni SJ,Chan SW,De Mott MS,Taghizadeh K,Cordero OX,Cutler M,Timberlake S,Alm EJ,Polz,MF,Pinhassi J,Deng ZX,Dedon PC.DNA phosphorothioation is widespread and quantized in bacterial genomes.Proceedings of the National Academy of Sciences of the United States of America,2011,108(7):2963-2968.
[5]R?mling U,Tümmler B.Achieving 100%typeability of Pseudomonas aeruginosa by pulsed-field gel electrophoresis.Journal of Clinical Microbiology,2000,38(1):464-465.
[6]You DL,Wang LR,Yao F,Zhou XF,Deng ZX.A novel DNAmodification by sulfur:?Dnd A is a Nif S-like cysteine desulfurase capable of assembling Dnd C as an iron-sulfur cluster protein in Streptomyces lividans.Biochemistry,2007,46(20):6126-6133.
[7]Yao F,Xu TG,Zhou XF,Deng ZX,You DL.Functional analysis of spf D gene involved in DNA phosphorothioation in Pseudomonas fluorescens Pf0-1.FEBS Letters,2009,583(4):729-733.
[8]Hu W,Wang CK,Liang JD,Zhang TL,Hu ZP,Wang ZJ,Lan WX,Li F,Wu HM,Ding JP,Wu G,Deng ZX,Cao CY.Structural insights into Dnd E from Escherichia coli B7Ainvolved in DNA phosphorothioation modification.Cell Research,2012,22(7):1203-1206.
[9]Zheng T,Jiang P,Cao B,Cheng QX,Kong LX,Zheng XQ,Hu QH,You DL.Dnd Ei exhibits helicase activity essential for DNA phosphorothioate modification and ATPase activity strongly stimulated by DNA substrate with a GAAC/GTTCmotif.The Journal of Biological Chemistry,2016,291(3):1492-1500.
[10]Cao B,Zheng XQ,Cheng QX,Yao F,Zheng T,Ramesh Babu I,Zhou HC,Dedon P,You DL.In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.Scientific Reports,2015,5:12513.
[11]Xu TG,Yao F,Zhou XF,Deng ZX,You DL.A novel host-specific restriction system associated with DNAbackbone S-modification in Salmonella.Nucleic Acids Research,2010,38(20):7133-7141.
[12]Cao B,Cheng QX,Gu C,Yao F,De Mott MS,Zheng XQ,Deng ZX,Dedon PC,You DL.Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella.Molecular Microbiology,2014,93(4):776-785.
[13]Kellner S,De Mott MS,Cheng CP,Russell BS,Cao B,You DL,Dedon PC.Oxidation of phosphorothioate DNAmodifications leads to lethal genomic instability.Nature Chemical Biology,2017,13(8):888-894.
[14]Cheng QX,Cao B,Yao F,Li JL,Deng ZX,You DL.Regulation of DNA phosphorothioate modifications by the transcriptional regulator Dpt B in Salmonella.Molecular Microbiology,2015,97(6):1186-1194.
[15]Haas D,Blumer C,Keel C.Biocontrol ability of fluorescent pseudomonads genetically dissected:importance of positive feedback regulation.Current Opinion in Biotechnology,2000,11(3):290-297.
[16]Schnider U,Keel C,Voisard C,Défago G,Haas D.Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0:mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.Applied and Environmental Microbiology,1995,61(11):3856-3864.
[17]Zhang WK,Wang L,Kong LX,Wang T,Chu YW,Deng ZX,You DL.Unveiling the post-PKS redox tailoring steps in biosynthesis of the Type II polyketide antitumor antibiotic xantholipin.Chemistry&Biology,2012,19(3):422-432.
[18]Thompson JD,Higgins DG,Gibson TJ.CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice.Nucleic Acids Research,1994,22(22):4673-4680.
[19]Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,2011,28(10):2731-2739.
[20]Xu TG,Liang JD,Chen S,Wang LR,He XY,You DL,Wang ZJ,Li AY,Xu ZL,Zhou XF,Deng ZX.DNAphosphorothioation in Streptomyces lividans:mutational analysis of the dnd locus.BMC Microbiology,2009,9:41.
[21]Cao B,Chen C,De Mott,MS,Cheng QX,Clark TA,Xiong XL,Zheng XQ,Butty V,Levine SS,Yuan G,Boitano M,Luong K,Song Y,Zhou XF,Deng ZX,Turner SW,Korlach J,You DL,Wang LR,Chen S,Dedon PC.Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences.Nature Communications,2014,5:3951.
[22]Rojo F.Mechanisms of transcriptional repression.Current Opinion in Microbiology,2001,4(2):145-151.
[23]Studier FW,Rosenberg AH,Dunn JJ,Dubendorff JW.Use of T7 RNA polymerase to direct expression of cloned genes.Methods in Enzymology,1990,185:60-89.
[24]Milton DL,Norqvist A,Wolf-Watz H.Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.Journal of Bacteriology,1992,174(22):7235-7244.
[25]Malakhov MP,Mattern MR,Malakhova OA,Drinker M,Weeks SD,Butt TR.SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins.Journal of Structural and Functional Genomics,2004,5(1/2):75-86.
[26]Sch?fer A,Tauch A,J?ger W,Kalinowski J,Thierbach G,Pühler A.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids p K18 and p K19:selection of defined deletions in the chromosome of Corynebacterium glutamicum.Gene,1994,145(1):69-73.
[27]Kovach ME,Elzer PH,Hill DS,Robertson GT,Farris MA,Roop II RM,Peterson KM.Four new derivatives of the broad-host-range cloning vector p BBR1MCS,carrying different antibiotic-resistance cassettes.Gene,1995,166(1):175-176.
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