睾丸酮丛毛单胞菌JL40中甲酸脱氢酶-O的锑抗性及锑氧化作用
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摘要
利用转座子插入突变技术从睾丸酮丛毛单胞菌(Comamonas testosteroni)JL40中筛选出编码具有锑抗性及锑氧化作用的甲酸脱氢酶-O的fdhO基因。为了进一步验证fdhO基因的功能,在JL40中构建了fdhO基因突变株JL40-fdhO和互补株JL40-fdhO-C,并进行了锑抗性、生长及锑氧化实验。结果表明,fdhO基因突变株的锑抗性及锑氧化速率有所下降,互补株的锑抗性及锑氧化能力得到了一定的恢复。在大肠杆菌中异源表达fdhO基因时发现,与空载对照菌相比,表达fdhO基因的大肠杆菌提高了锑氧化速率。报告基因融合实验表明,fdhO基因的启动子受锑(Ⅲ)的诱导。综上表明,fdhO基因在睾丸酮丛毛单胞菌JL40中具有明显的锑氧化作用,但可能不是唯一的氧化酶;fdhO基因在细菌中的锑氧化功能将会为细菌锑抗性机制的进一步探究提供参考。
We screened gene fdhOcoding formate dehydrogenase-O related to antimony resistance and antimony oxidation by transposon insertion mutagenesis fromComamonas testosteroni JL40.In order to verify the function of gene fdhO,we constructed mutant strain JL40-fdhOand complementary strain JL40-fdhO-C,and experimentally investigated the antimony resistance,growth,and antimony oxidation.The results showed that the antimony resistance and antimony oxidation rate of mutant strain decreased,and those of complementary strain recovered in a certain degree.Heterologous expression assay in Escherichia coli showed that,Escherichia coli expressing gene fdhOcould increase antimony oxidation rate compared with the non-loaded control strain.Moreover,the results of the reporter gene fusion experiment indicated that the promoter of gene fdhO was induced by Sb(Ⅲ).Comprehensive analysis showed that gene fdhOactually had the function of antimony oxidation,but it was not the only oxidase in Comamonas testosteroni JL40.The antimony oxidation function of gene fdhOin bacteria will provide reference for further explore of bacterial antimony resistant mechanism.
We screened gene fdhOcoding formate dehydrogenase-O related to antimony resistance and antimony oxidation by transposon insertion mutagenesis fromComamonas testosteroni JL40.In order to verify the function of gene fdhO,we constructed mutant strain JL40-fdhOand complementary strain JL40-fdhO-C,and experimentally investigated the antimony resistance,growth,and antimony oxidation.The results showed that the antimony resistance and antimony oxidation rate of mutant strain decreased,and those of complementary strain recovered in a certain degree.Heterologous expression assay in Escherichia coli showed that,Escherichia coli expressing gene fdhOcould increase antimony oxidation rate compared with the non-loaded control strain.Moreover,the results of the reporter gene fusion experiment indicated that the promoter of gene fdhO was induced by Sb(Ⅲ).Comprehensive analysis showed that gene fdhOactually had the function of antimony oxidation,but it was not the only oxidase in Comamonas testosteroni JL40.The antimony oxidation function of gene fdhOin bacteria will provide reference for further explore of bacterial antimony resistant mechanism.
引文
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[4]李航彬,杨志辉,袁平夫,等.湘中锑矿区土壤重金属锑的污染特征[J].环境科学与技术,2011,34(1):70-74.
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[9]SAWERS G.The hydrogenases and formate dehydrogenases of Escherichia coli[J].Antonie van Leeuwenhoek,1994,66(1/2/3):57-88.
[10]SOBOH B,PINSKE C,KUHNS M,et al.The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen:benzyl viologen oxidoreductase activity[J].BMC Microbiology,2011,11:173.
[11]JORMAKKA M,TRNROTH S,BYRNE B,et al.Molecular basis of proton motive force generation:structure of formate dehydrogenase-N[J].Science,2002,295(5561):1863-1868.
[12]WEEGER W,LIEVERMONT D,PERRET M,et al.Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment[J].Biometals,1999,12(2):141-149.
[13]LI J,WANG Q,ZHANG S Z,et al.Phylogenetic and genome analyses of antimony-oxidizing bacteria isolated from antimony mined soil[J].International Biodeterioration and Biodegradation,2013,76:76-80.
[14]MILLER J H.Experiments in Molecular Genetics[M].New York:Cold Spring Harbor Laboratory Press,1972:431.
[2]李明顺,李洁,王革娇.微生物对锑的代谢机制研究进展[J].华中农业大学学报,2013,32(5):15-19.
[3]佘玮,揭雨成,邢虎成,等.湖南冷水江锑矿区苎麻对重金属的吸收和富集特性[J].农业环境科学学报,2010,29(1):91-96.
[4]李航彬,杨志辉,袁平夫,等.湘中锑矿区土壤重金属锑的污染特征[J].环境科学与技术,2011,34(1):70-74.
[5]HE M C,YANG J R.Effects of different forms of antimony on rice during the period of germination and growth and antimony concentration in rice tissue[J].Science of the Total Environment,1999,243-244:149-155.
[6]ABIN C A,HOLLIBAUGH J T.Dissimilatory antimonite reduction and production of antimony trioxide microcrystals by a novel microorganism[J].Environental Science and Technology,2013,48(1):681-688.
[7]WANG Q,WARELOW T P,KANG Y S,et al.Arsenite oxidase also functions as an antimonite oxidase[J].Applied and Environmental Microbiology,2015,81(9):3278.
[8]LI J X,WANG Q,LI M S,et al.Proteomics and genetics for identification of a bacterial antimonite oxidase in Agrobacterium tumefaciens[J].Environmental Science and Technology,2015,49(10):5980-5989.
[9]SAWERS G.The hydrogenases and formate dehydrogenases of Escherichia coli[J].Antonie van Leeuwenhoek,1994,66(1/2/3):57-88.
[10]SOBOH B,PINSKE C,KUHNS M,et al.The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen:benzyl viologen oxidoreductase activity[J].BMC Microbiology,2011,11:173.
[11]JORMAKKA M,TRNROTH S,BYRNE B,et al.Molecular basis of proton motive force generation:structure of formate dehydrogenase-N[J].Science,2002,295(5561):1863-1868.
[12]WEEGER W,LIEVERMONT D,PERRET M,et al.Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment[J].Biometals,1999,12(2):141-149.
[13]LI J,WANG Q,ZHANG S Z,et al.Phylogenetic and genome analyses of antimony-oxidizing bacteria isolated from antimony mined soil[J].International Biodeterioration and Biodegradation,2013,76:76-80.
[14]MILLER J H.Experiments in Molecular Genetics[M].New York:Cold Spring Harbor Laboratory Press,1972:431.