In this article, a mechanism of arsenite [As(III)] resistance through methylation and subsequent volatization is described. Heterologous expression of arsM from Rhodopseudomonas palustris was shown to confer As(III) resistance to an arsenic-sensitive strain of Escherichia coli. ArsM catalyzes the formation of a number of methylated intermediates from As(III), with trimethylarsine as the end product. The net result is loss of arsenic, from both the medium and the cells. Because ArsM homologues are widespread in nature, this microbial-mediated transformation is proposed to have an important impact on the global arsenic cycle.As(III) ͉ ArsM ͉ methylation A s genomes are sequenced, it is becoming clear that nearly all bacteria and archaea have arsenic-resistance (ars) operons that confer resistance to arsenite [As(III)] and arsenate [As(V)] (1). The widespread occurrence of ars genes reflects the fact that arsenic is a ubiquitous environmental toxic metal. In most cases, these operons encode transport proteins that extrude As(III) from cells. In eukaryotes, As(III) detoxification involves glutathionylation coupled to removal of the As(GS) 3 complex from the cytosol by ABC transporters, such as the Saccharomyces cerevisiae Ycf1p vacuolar pump (2) or mammalian biliary extrusion pump MRP2 (3). In many mammals, including humans, an alternate metabolic fate of As(III) is methylation in the liver, followed by urinary excretion of the methylated species (4). In the past, this process was considered a detoxification mechanism (5), but more recent data suggest that the methylation actually increases toxicity by producing the more toxic monomethylarsenite [MMA(III)] and dimethylarsenite [DMA(III)], calling into question whether the process is, in fact, a detoxification process (6). An enzyme (termed Cyt19 or As3MT) that catalyzes As(III)-S-adenosylmethyltransferase activity has been identified recently in rats and humans (7-9). The enzyme has been characterized in vitro, but its physiological role is unknown.Bacteria and fungi are known to produce volatile and toxic arsines (10) but the physiological roles of arsenic methylation in microorganisms are likewise unclear, and the biochemical basis is unknown. While examining microbial genomes, we identified large number of genes for bacterial and archaeal homologues of Cyt19. We have termed a subset of these genes arsM and their protein product ArsM (As(III) S-adenosylmethyltransferase). What sets these arsM genes apart from genes for other homologues is that they are each downstream of an arsR gene, encoding the archetypal arsenic-responsive transcriptional repressor that controls expression of ars operons (11), suggesting that these ArsMs evolved to confer arsenic resistance.The gene for the 283-residue ArsM (29,656 Da) (accession no. NP948900.1) was cloned from Rhodopseudomonas palustris and expressed in an arsenic-hypersensitive strain of Escherichia coli. As(III)-resistance cells in E. coli expressing recombinant arsM correlated with conversion of medium arsenic to the methy...

BackgroundArsenic is known as a toxic metalloid, which primarily exists in inorganic form [As(III) and As(V)] and can be transformed by microbial redox processes in the natural environment. As(III) is much more toxic and mobile than As(V), hence microbial arsenic redox transformation has a major impact on arsenic toxicity and mobility which can greatly influence the human health. Our main purpose was to investigate the distribution and diversity of microbial arsenite-resistant species in three different arsenic-contaminated soils, and further study the As(III) resistance levels and related functional genes of these species.ResultsA total of 58 arsenite-resistant bacteria were identified from soils with three different arsenic-contaminated levels. Highly arsenite-resistant bacteria (MIC > 20 mM) were only isolated from the highly arsenic-contaminated site and belonged to Acinetobacter, Agrobacterium, Arthrobacter, Comamonas, Rhodococcus, Stenotrophomonas and Pseudomonas. Five arsenite-oxidizing bacteria that belonged to Achromobacter, Agrobacterium and Pseudomonas were identified and displayed a higher average arsenite resistance level than the non-arsenite oxidizers. 5 aoxB genes encoding arsenite oxidase and 51 arsenite transporter genes [18 arsB, 12 ACR3(1) and 21 ACR3(2)] were successfully amplified from these strains using PCR with degenerate primers. The aoxB genes were specific for the arsenite-oxidizing bacteria. Strains containing both an arsenite oxidase gene (aoxB) and an arsenite transporter gene (ACR3 or arsB) displayed a higher average arsenite resistance level than those possessing an arsenite transporter gene only. Horizontal transfer of ACR3(2) and arsB appeared to have occurred in strains that were primarily isolated from the highly arsenic-contaminated soil.ConclusionSoils with long-term arsenic contamination may result in the evolution of highly diverse arsenite-resistant bacteria and such diversity was probably caused in part by horizontal gene transfer events. Bacteria capable of both arsenite oxidation and arsenite efflux mechanisms had an elevated arsenite resistance level.

The genome sequence of Halobacterium sp. strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic. These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC and arsR2M) and on the chromosome (arsB). We studied the role of these ars genes in arsenic resistance genetically by construction of gene knockouts. Deletion of the arsADRC gene cluster in a Halobacterium NRC-1 ⌬ura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate. In contrast, knockout of the chromosomal arsB gene did not show significantly increased sensitivity to arsenite or arsenate. We also found that knockout of the arsM gene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase. These results indicate that Halobacterium sp. strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts. Deletion analysis was facilitated by an improved method for gene knockouts/replacements in Halobacterium that relies on both selection and counterselection of ura3 using a uracil dropout medium and 5-fluoroorotic acid. The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal. Northern hybridization and reverse transcription-PCR analyses showed that arsA, arsD, arsR, arsM, arsC, and arsB, but not arsR2, are inducible by arsenite and antimonite. We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome.The halophilic archaeon (haloarchaeon) Halobacterium sp. strain NRC-1 is an excellent model for postgenomic analysis of heavy metal resistance. Its genome is completely sequenced, and a large number of genetic tools are available for characterization of this extreme halophile (3, 9, 10). It is easily grown in the laboratory in hypersaline medium containing about a 10-fold concentration of seawater (2), and its natural environment is usually rich in heavy metals, many of which are toxic to cells. The genome sequence of Halobacterium sp. strain NRC-1 revealed multiple putative metal ion transporter genes, including arsenic, cadmium, copper, cobalt, zinc, and iron (9), indicating an excellent ability to handle metal ions in its environment. However, none of these hypothetical genes has been shown to be functional, and very few studies have been directed at the understanding of heavy metal resistance in haloarchaea.The Halobacterium sp. strain NRC-1 genome contains a 2-Mb chromosome and two megaplasmids/minichromosomes, pNRC100 and pNRC200 (191 and 365 kb, respectively) (6-9).

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e Antimony (Sb) is a toxic metalloid that occurs widely at trace concentrations in soil, aquatic systems, and the atmosphere. Nowadays, with the development of its new industrial applications and the corresponding expansion of antimony mining activities, the phenomenon of antimony pollution has become an increasingly serious concern. In recent years, research interest in Sb has been growing and reflects a fundamental scientific concern regarding Sb in the environment. In this review, we summarize the recent research on bacterial antimony transformations, especially those regarding antimony uptake, efflux, antimonite oxidation, and antimonate reduction. We conclude that our current understanding of antimony biochemistry and biogeochemistry is roughly equivalent to where that of arsenic was some 20 years ago. This portends the possibility of future discoveries with regard to the ability of microorganisms to conserve energy for their growth from antimony redox reactions and the isolation of new species of "antimonotrophs."