Arsenic detoxification and evolution of trimethylarsine gas by a microbial arsenite S-adenosylmethionine methyltransferase

Abstract

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.

Fig. 1.

The ars genes of R. palustris CGA009 Shown are the operons of R. palustris that appear to be regulated by an ArsR-type repressor. The first operon (RPA2256, 2257, 2258, 2259) resembles ars operons found in Pseudomonas or Bacillus of the type arsRCBH (ArsR-repressor, ArsC-As(V) reductase, ArsB-As(III) efflux pump, and ArsH-unknown arsenic resistance protein). The arsRM-operon (RPA3561, 3562) encodes the As(III)-methyl-transferase (ArsM) described in this report regulated by an ArsR-type repressor. The third operon (RPA3553–RPA3559) of R. palustris contains two putative arsR genes, two putative As(V) reductases, a putative As(III) permease (acr3), and two genes where the function of the proteins is unknown. The two putative As(III) permeases ArsB and Acr3belong to two unrelated protein families.

Fig. 2.

ArsM confers resistance to As(III) in E. coli.Resistance to As(III) was assayed in cells of E. coli AW3110(DE3) (ΔarsRBC), bearing vector plasmid pET28a(+) (circle), pET28arsM (encoding wild-type ArsM) (square), or pET28arsMC2 (encoding the ArsMC2 variant) (triangle) were grown with no (white), 50μM (gray), or 70 μM (black) sodium As(III) at 37°C, with shaking. Absorbance at 600 nm was monitored by using a SPECTRA max 340PC microplate reader (Molecular Devices) with a path length of 024 cm. Shown is the average of three independent assays with standard deviation.

Fig. 3.

In vivo formation of methylated arsenicals. (A) Volatilization of arsenic. Cells of E. coli strain AW3110(DE3) with vector plasmid pET28a(+) (circle) or plasmid pET28arsMC2 (triangle) were grown in 4 ml of LB medium in the presence of 25μM sodium As(III), and growth was monitored as optical density at 600nm (gray). Total arsenic in the culture medium (white) and volatilized arsenic (black) were determined as described above. (B) Speciation of arsenic in the culture medium. Cultures were grown for 18 h after the determination of the arsenic species in the medium by HPLC-ICP-MS using known standards of As(III), As(V), DMA(V), and TMAO. The unknown peak did not correspond to any of the standards. Black bars indicate the arsenic species at time point 0, white bars, after 18 h of cultivation.

Fig. 4.

Purified ArsM catalyzes As(III) methylation. Arsenic speciation of the reaction solution was performed by anion exchange HPLC-ICP-MS, with the relative amounts of arsenic expressed as cps. As indicated, each assay contained 5μM As(III) or DMA(III), 0.5 mM AdoMet, 8 mM GSH, and 08 μM wild-type ArsM in 50 mM phosphate buffer, pH 7.4. Shown are the arsenic species produced with As(III) as substrate without GSH (A), As(III) as substrate with GSH (B), and DMA(III) as substrate with GSH (C).

Fig. 5.

Formation of volatile TMA(III) by purified ArsM. The activity of wild-type ArsM and mutant ArsMC2 were assayed as described in the legend to Fig. 4, except that the concentration of ArsM was increased to 10μM. Wild-type ArsM was used in A and C, and mutant ArsMC2was used in B and D. Arsenic speciation of the reaction solution (A and B) and volatilized gas (C and D) was performed by anion exchange HPLC-ICP-MS. The reaction products were analyzed after 3h and 17h in the presence of ArsM or buffer only.