The Mechanisms Of Enzymatic And H₂O₂-mediated Non-enzymatic Antimonite Oxidation In Agrobacterium Tumefaciens GW4

Antimony?Sb?is a toxic metalloid and shares similar biochemical characters with arsenic?As?.Nowadays,with the development of its wide industrial applications and the corresponding expansion of antimony mining activities,antimony pollution has become an increasingly serious concern to human's heath.Antimonite [Sb???]-oxidizing bacteria can transform the toxic Sb???into the less toxic antimonite [Sb?V?],which is a detoxification process for microorganisms and environments.Thus,studying bacterial antimony transformations,especially Sb???oxidation is very significant for understanding Sb biogeochemical cycle and bioremediation of Sb contaminated environments.In recent years,the research interest in microbial Sb transformation has been growing in the world,but before I started my Master study?year 2012?,the knowledge of mechanism for microbial Sb???oxidation was still unknown.Our group has found many heterotrophic Sb???-oxidizing strains in rencet years.The work of this thesis is to determine mian mechanism of Sb???oxidation and energy metabolism in the heterotrophic As???/Sb???-oxidizing strain Agrobacterium tumefaciens GW4.The main results are as follows:1)The first discovery of a NADP+ dependent Sb???oxidase AnoA.Comparative proteomics showed that addition of Sb???up-regulated AnoA expression.Quantitative reverse transcriptase-PCR and lacZ reporter gene analyses indicated that the transctription of anoA was induced by different concentrations of Sb???.Deletion of the anoA gene reduced Sb???resistance and decreased Sb???oxidation by ?27%,whereas the anoA complemented strain was similar to the wild type GW4 and a GW4 anoA overexpressing strain increased Sb???oxidation by ?34%.A His-tag purified AnoA was found to require NADP+ as cofactor,and exhibited a Km for Sb???of 64 ± 10 ?M and a Vmax of 150 ± 7 nmol min-1 mg-1.Moreover,the addition of Sb???yielded Km and Vmax values of 103.2 ± 17.7 ?M and 88.23 ± 6 nmol min-1 mg-1,suggesting that AnoA is prone to oxidize Sb???.Heterologous expression of anoA in Escherichia coli demonstrated direct transferability of this activity.These results indicated that AnoA is a novel Sb???oxidase in strain GW4,but other mechanism?s?for bacterial Sb???oxidation may also exits.2)The cellular H₂O₂ acts as a non-enzymatic factor for bacterial Sb???oxidation.Sb???induces bacterial oxidative stress and results in a accumulation of cellular H₂O₂.The catalase gene katA,which responsible for H₂O₂ degradation,was induced by different concentrations of Sb???and H₂O₂,and was essential for H₂O₂ resistance.Deletion of katA significantly increased both Sb???oxidation rate and the cellular H₂O₂ content.Furthermore,linear correlations were observed between cellular H₂O₂ and Sb?V?content in vivo and chemical H₂O₂ and Sb?V?content in vitro?R2= 0.93 and 0.94,respectively,and P<0.05?.These results indicated that bacterial oxidative stress induced by Sb???is associated with Sb???oxidation and resistance.The cellular H₂O₂ mediated bacterial Sb???oxidation is an smart detoxifcation process of Sb???-oxidizing bacteria through the“using poison against poison” strategy,which could transform the toxic Sb???to the much less toxic Sb?V?and consume the toxic cellular H₂O₂ simultaneously3)Identification of the role of the arsenite oxidase AioAB on Sb???oxidation in strain GW4.AioAB is responsible for oxidizing As???to As?V?in periplasm.Previous study showed that AioAB could oxidize Sb???in A.tumefaciens 5A,and deletion of aioA decreased Sb???oxidation rate in strain 5A.In this study,in contrast,deletion of aioA increased Sb???oxidation rate in A.tumefaciens GW4 and the transcription of anoA and katA was increased.In addition,the cellular H₂O₂ content was increased in mutant strain GW4-?aioA compared with that of wild-type strain,and the phynotype of complemented strain was recovered,revealing that deletion of aioA could increase the AnoA catalyzed-and H₂O₂ mediated-Sb???oxidation.These results indicated that the fuction of AioAB on Sb???oxidation in strain GW4 is not obvious,but it is involved in Sb???oxidation by influencing the expression of AnoA and Kat A.4)Deternimation of the effects upon metabolic pathways and energy production by Sb???and AioAB in strain GW4.Proteomics and transcriptional analyses showed that proteins/genes involved in Sb???oxidation and resistance,stress responses,carbon metabolism,cell mobility,phosphonate and phosphinate metabolism,and amino acid and nucleotide metabolism were induced by Sb???and were more significantly induced in the aioA mutant.The results suggested that Sb???oxidation may produce energy.In addition,without periplasmic AioAB,more Sb???would enter bacterial cells,however,the cytoplasmic AnoA and the oxidative stress response proteins were significantly up-regulated,which may contribute to the increased Sb???oxidation efficiency.Moreover,the carbon metabolism was also activated to generate more energy against Sb???stress.The generated energy may be used in Sb transportation,DNA repair,amino acid synthesis,and cell mobility.In summary,based on these systematic studies in heterotrophic Sb???-oxidizing strain A.tumefaciens GW4,the work of this thesis discovered and identified a novel Sb???oxidase for the first time,and proposed that microbial Sb???oxidation is a co-metabolism process catalyzed by AioAB,AnoA?enzymatic?and mediated by H₂O₂?non-enzymatic?.In addition,we also determined the effects upon metabolic pathways and energy production by Sb???and AioAB in strain GW4.The results of this thesis have been published in a series of international journals,which greatly enriched the understanding of the mechanism of microbial Sb???oxidation.Based on these results and research progress in the world,we published a review on microbial Sb transformation for the first time.It represents an important step toward understanding of how microorganisms participate in antimony biogeochemical cycling in nature,and provides theoretical basis for bioremediation of antimony contaminated environments.