title: Proteomics and Genetics for Identification of a Bacterial Antimonite Oxidase in Agrobacterium tumefaciens.
abstract: Antimony (Sb) and its compounds are listed by the United States Environmental Protection Agency (USEPA, 1979) and the European Union (CEC, 1976) as a priority pollutant. Microbial redox transformations are presumed to be an important part of antimony cycling in nature; however, regulation of these processes and the enzymology involved are unknown. In this study, comparative proteomics and reverse transcriptase-PCR analysis of Sb(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 revealed an oxidoreductase (anoA) is widely distributed in microorganisms, including at least some documented to be able to oxidize Sb(III). Deletion of the anoA gene reduced Sb(III) resistance and decreased Sb(III) oxidation by ∼27%, whereas the anoA complemented strain was similar to the wild type GW4 and a GW4 anoA overexpressing strain increased Sb(III) oxidation by ∼34%. Addition of Sb(III) up-regulated anoA expression and cloning anoA to Escherichia coli demonstrated direct transferability of this activity. A His-tag purified AnoA was found to require NADP(+) as cofactor, and exhibited a K(m) for Sb(III) of 64 ± 10 μM and a V(max) of 150 ± 7 nmol min(-1) mg(-1). This study contributes important initial steps toward a mechanistic understanding of microbe-antimony interactions and enhances our understanding of how microorganisms participate in antimony biogeochemical cycling in nature.
authors.affiliation: †State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Jingxin
authors.initials: J
authors.lastname: Li
authors.affiliation: †State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: †State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Mingshun
authors.initials: M
authors.lastname: Li
authors.affiliation: †State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Birong
authors.initials: B
authors.lastname: Yang
authors.affiliation: †State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Manman
authors.initials: M
authors.lastname: Shi
authors.affiliation: †State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
authors.affiliation: ‡Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana 59717, United States.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: McDermott
authors.affiliation: §Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, DK-1871, Denmark.
authors.forename: Christopher
authors.initials: C
authors.lastname: Rensing
authors.affiliation: †State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China.
authors.forename: Gejiao
authors.initials: G
authors.lastname: Wang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D000965:Antimony / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005838:Genotype; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010802:Phylogeny; D040901:Proteomics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; C061374:antimonite; D000965:Antimony; D010088:Oxidoreductases