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.

Microbial Antimony Biogeochemistry: Enzymes, Regulation, and Related Metabolic Pathways

“…The disruption of anoA reduced resistance to Sb(III) and also decreased the Sb(III) oxidation rate by ϳ27% compared with that of the wild-type strain, while the overexpression of anoA increased the Sb(III) oxidation rate by ϳ34%. In addition, heterologous expression of AnoA significantly increased the Sb(III) oxidation rate in E. coli (117). Acting as a novel Sb(III) oxidase, AnoA could also oxidize As(III) in vitro, with a K m of 103.2 Ϯ 17.7 M and a maximum rate of metabolism (V max ) of 88.23 Ϯ 6 nmol min Ϫ1 mg Ϫ1 (Fig.…”

“…This indicates that AnoA in Comamonas has a distant phylogenetic relationship from that in Agrobacterium, Rhizobium, and Sinorhizobium strains. Based on the literature published to date, the deletion of either aioBA or anoA only reduces the Sb(III) oxidation rate but does not completely eliminate Sb(III) oxidation, implying (but not proving) the possible existence of another mechanism(s) of Sb(III) oxidation (117,123).…”

“…Due to an increased focus on bacterial Sb(III) oxidation, Ͼ60 Sb(III)-oxidizing strains were isolated from mining soil (112)(113)(114) and contaminated sediments (115)(116)(117). Chemoautotrophy, as defined by Sb(III)-dependent growth and inorganic carbon fixation, is more difficult to achieve experimentally.…”

“…Based on genome analysis, the antimonite oxidase gene anoA (117) exists in all of the arsenite-oxidizing Agrobacterium and Comamonas strains tested thus far, and the gene exists in other asyet-untested bacterial strains. To understand the phylogenetic relationship among AnoA in different bacteria, we performed phylogenetic analysis based on amino acid sequences of the putative AnoA from 10 Agrobacterium strains, together with five Rhizobium strains, three Sinorhizobium strains, and two Comamonas strains (see Fig.…”

“…(135)(136)(137)(138), Miscanthus sinensis (139), and Agrobacterium tumefaciens (117). The proteomics analysis in Leishmania spp.…”