西瓜噬酸菌血红素氧化酶基因hemO功能研究
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摘要
在生物体中,血红素氧化酶(Heme oxygenase,HemO)在血红素代谢、铁循环和氧化应激等过程中发挥重要作用。在一些病原菌中,血红素氧化酶可参与调控其致病能力。在西瓜噬酸菌(Acidovorax citrulli)中,存在一个编码血红素氧化酶的基因hemO,然而其功能尚未可知。以西瓜噬酸菌Aac5菌株为研究对象,通过基因敲除构建hemO缺失突变体菌株,分析致病力及生物学表型,并检测致病相关基因表达量,初步探究HemO在西瓜噬酸菌中的功能。结果表明,hemO基因缺失会促进菌株生物膜的形成,并导致菌株运动性、生长能力、致病力降低,显著影响致病相关基因的表达,但不影响病原菌诱导烟草过敏性反应的能力。这表明hemO基因参与西瓜噬酸菌的致病力调控过程。
Heme oxygenase(HemO) plays an important role in the process of heme metabolism, iron cycle and oxidative stress in living organisms. Heme oxygenase can be involved in the regulation of pathogenicity in some pathogenic bacteria.The hemO gene was identified in Acidovorax citrulli genome. However, the biological function of the gene remains unclear. In this study, the hemO mutant strain was constructed by gene knockout, the pathogenicity and biological phenotype were analyzed, and the expression levels of related pathogenetic genes was detected to explore the function of HemO based on the wild type strain Aac5. The results showed that the deletion of hemO gene promoted biofilm formation,caused the decline in motility, growth ability and pathogenicity, and significantly influenced the expression of related pathogenic genes, but did not affect the ability to induce hypersensitive reaction in non-host Nicotiana tabacum. These results indicated hemO is involved in the pathogenic regulation process of A. citrulli.
Heme oxygenase(HemO) plays an important role in the process of heme metabolism, iron cycle and oxidative stress in living organisms. Heme oxygenase can be involved in the regulation of pathogenicity in some pathogenic bacteria.The hemO gene was identified in Acidovorax citrulli genome. However, the biological function of the gene remains unclear. In this study, the hemO mutant strain was constructed by gene knockout, the pathogenicity and biological phenotype were analyzed, and the expression levels of related pathogenetic genes was detected to explore the function of HemO based on the wild type strain Aac5. The results showed that the deletion of hemO gene promoted biofilm formation,caused the decline in motility, growth ability and pathogenicity, and significantly influenced the expression of related pathogenic genes, but did not affect the ability to induce hypersensitive reaction in non-host Nicotiana tabacum. These results indicated hemO is involved in the pathogenic regulation process of A. citrulli.
引文
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[29]OCHSNER U A,VASIL M L.Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa:cycle selection of iron-regulated genes[J].Proceedings of the National Academy of Sciences,1996,93(9):4409-4414.
[30]MAZMANIAN S K,SKAAR E P,GASPAR A H,et al.Passage of heme-iron across the envelope of Staphylococcus aureus[J].Science,2003,299(5608):906-909.
[31]SKAAR E P.The battle for iron between bacterial pathogens and their vertebrate hosts[J].Plos Pathogens,2010,6(8):e1000949.
[32]WANDERSMAN C,DELEPELAIRE P.Bacterial iron sources:from siderophores to hemophores[J].Annual Review of Microbiology,2004,58:611-647.
[33]LIU H,DONG C,ZHAO T,et al.Functional analysis of the ferric uptake regulator gene fur in Xanthomonas vesicatoria[J].Plos One,2017,11(2):e0149280.
[34]CHA J Y,LEE J S,OH J I,et al.Functional analysis of the role of Fur in the virulence of Pseudomonas syringae pv.tabaci11528:Fur controls expression of genes involved in quorum-sensing[J].Biochemical and Biophysical Research Communications,2008,366(2):281-287.
[2]赵廷昌,孙福在,王兵万,等.哈密瓜细菌性果斑病病原菌鉴定[J].植物病理学报,2001,31(4):357-364.
[3]阎莎莎.瓜类细菌性果斑病菌种内遗传多样性的研究[D].北京:中国农业科学院,2011.
[4]DUTTA B,GITAITIS R,SMITH S,et al.Interactions of seedborne bacterial pathogens with host and non-host plants in relation to seed infestation and seedling transmission[J].PloSOne,2014,9(6):e99215.
[5]JIANG C H,WU F,YU Z Y,et al.Study on screening and antagonistic mechanisms of Bacillus amyloliquefaciens 54 against bacterial fruit blotch(BFB)caused by Acidovorax avenae subsp.citrulli[J].Microbiological Research,2015,170:95-104.
[6]FRANKENBERG-DINKEL N.Bacterial heme oxygenases[J].Antioxidants&Redox Signaling,2004,6(5):825-834.
[7]KIKUCHI G,YOSHIDA T,NOGUCHI M,et al.Heme oxygenase and heme degradation[J].Biochemical and Biophysical Research Communications,2005,338(1):558-567.
[8]RYTER S W,ALAM J,CHOI A M.Heme oxygenase-1/carbon monoxide:from basic science to therapeutic applications[J].Physiological Reviews,2006,86(2):583-650.
[9]WILKS A J A,SIGNALING R.Heme oxygenase:evolution,structure,and mechanism[J].Antioxidants&Redox Signaling,2002,4(4):603-614.
[10]税芸,刘马峰,程安春.细菌血红素降解蛋白结构特点及作用机制[J].中国生物化学与分子生物学报,2015,31(8):806-814.
[11]YAN S,YANG Y,WANG T,et al.Genetic diversity analysis of Acidovorax citrulli in China[J].European Journal of Plant Pathology,2013,136(1):171-181.
[12]ZHANG X,ZHAO M,YAN J,et al.Involvement of hrpXand hrpG in the virulence of Acidovorax citrulli strain Aac5,causal agent of bacterial fruit blotch in cucurbits[J].Frontiers in Microbiology,2018,9:507.
[13]SCHAFER A,TAUCH A,JAGER W,et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum[J].Gene,1994,145(1):69-73.
[14]KOVACH M E,ELZER P H,HILL D S,et al.Four new derivatives of the broad-host-range cloning vector pBBR1MCS,carrying different antibiotic-resistance cassettes[J].Gene,1995,166(1):175-176.
[15]ZHANG Y,WEI C,JIANG W,et al.The HD-GYP domain protein RpfG of Xanthomonas oryzae pv.oryzicola regulates synthesis of extracellular polysaccharides that contribute to biofilm formation and virulence on rice[J].PloS one,2013,8(3):e59428.
[16]KVITKO B H,COLLMER A.Construction of Pseudomonas syringae pv.tomato DC3000 mutant and polymutant strains[M].Methods in Molecular Biology,2011,712:109-128.
[17]WALCOTT R R,GITAITIS R D.Detection of Acidovorax avenae subsp.citrulli in watermelon seed using immunomagnetic separation and the polymerase chain reaction[J].Plant Disease,2000,84(4):470-474.
[18]JOHNSON K L,MINSAVAGE G V,LE T,et al.Efficacy of a nonpathogenic Acidovorax citrulli strain as a biocontrol seed treatment for bacterial fruit blotch of cucurbits[J].Plant Disease,2011,95(6):697-704.
[19]王健超,袁云云,代园凤,等.西瓜噬酸菌吡哆醛磷酸(VB6)营养缺陷型与致病性相关[J].农业生物技术学报,2013,21(9):1093-1102.
[20]STEPANOVIC S,VUKOVIC D,HOLA V,et al.Quantification of biofilm in microtiter plates:overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci[J].APMIS,2007,115(8):891-899.
[21]BAHAR O,GOFFER T,BURDMAN S.Type IV pili are required for virulence,twitching motility,and biofilm formation of Acidovorax avenae subsp.citrulli[J].Molecular Plant-microbe Interactions,2009,22(8):909-920.
[22]WANG T,GUAN W,HUANG Q,et al.Quorum-sensing contributes to virulence,twitching motility,seed attachment and biofilm formation in the wild type strain Aac-5 of Acidovorax citrulli[J].Microbial Pathogenesis,2016,100:133-140.
[23]张爱萍,张晓晓,吴林娜,等.西瓜噬酸菌趋化性及鞭毛素基因ΔcheAΔfliC双突变体构建及功能[J].农业生物技术学报,2017,25(11):1838-1850.
[24]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[25]MCGRANE R,BEATTIE G A.Pseudomonas syringae pv.syringae B728a regulates multiple stages of plant colonization via the bacteriophytochrome BphP1[J].MBio,2017,8(5):e01178.
[26]WU L,MCGRANE R S,BEATTIE G A.Light regulation of swarming motility in Pseudomonas syringae integrates signaling pathways mediated by a bacteriophytochrome and a LOV protein[J].MBio,2013,4(3):e00334-13.
[27]BONOMI H R,TOUM L,SYCZ G,et al.Xanthomonas campestris attenuates virulence by sensing light through a bacteriophytochrome photoreceptor[J].EMBO Rep,2016,17(11):1565-1577.
[28]程兴军,刘马峰,程安春.革兰氏阴性菌血红素转运系统结构及功能特点[J].生物化学与分子生物学报,2014,30(9):848-855.
[29]OCHSNER U A,VASIL M L.Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa:cycle selection of iron-regulated genes[J].Proceedings of the National Academy of Sciences,1996,93(9):4409-4414.
[30]MAZMANIAN S K,SKAAR E P,GASPAR A H,et al.Passage of heme-iron across the envelope of Staphylococcus aureus[J].Science,2003,299(5608):906-909.
[31]SKAAR E P.The battle for iron between bacterial pathogens and their vertebrate hosts[J].Plos Pathogens,2010,6(8):e1000949.
[32]WANDERSMAN C,DELEPELAIRE P.Bacterial iron sources:from siderophores to hemophores[J].Annual Review of Microbiology,2004,58:611-647.
[33]LIU H,DONG C,ZHAO T,et al.Functional analysis of the ferric uptake regulator gene fur in Xanthomonas vesicatoria[J].Plos One,2017,11(2):e0149280.
[34]CHA J Y,LEE J S,OH J I,et al.Functional analysis of the role of Fur in the virulence of Pseudomonas syringae pv.tabaci11528:Fur controls expression of genes involved in quorum-sensing[J].Biochemical and Biophysical Research Communications,2008,366(2):281-287.