Organoarsenical Biotransformations by Shewanella putrefaciens

Abstract

Microbes play a critical role in the global arsenic biogeocycle. Most studies have focused on redox cycling of inorganic arsenic in bacteria and archaea. The parallel cycles of organoarsenical biotransformations are less well characterized. Here we describe organoarsenical biotransformations in the environmental microbe Shewanella putrefaciens. Under aerobic growth conditions, S. putrefaciens reduced the herbicide MSMA (methylarsenate or MAs(V)) to methylarsenite (MAs(III)). Even though it does not contain an arsI gene, which encodes the ArsI C-As lyase, S. putrefaciens demethylated MAs(III) to As(III). It cleaved the C-As bond in aromatic arsenicals such as the trivalent forms of the antimicrobial agents roxarsone (Rox(III)), nitarsone (Nit(III)) and phenylarsenite (PhAs(III)), which have been used as growth promoters for poultry and swine. S. putrefaciens thiolated methylated arsenicals, converting MAs(V) into the more toxic metabolite monomethyl monothioarsenate (MMMTAs(V)), and transformed dimethylarsenate (DMAs(V)) into dimethylmonothioarsenate (DMMTAs(V)). It also reduced the nitro groups of Nit(V), forming p-aminophenyl arsenate (p-arsanilic acid or p-AsA(V)), and Rox(III), forming 3-amino-4-hydroxybenzylarsonate (3A4HBzAs(V)). Elucidation of organoarsenical biotransformations by S. putrefaciens provides a holistic appreciation of how these environmental pollutants are degraded.

Figure 1

Organoarsenic accumulation in LB-grown cells of S. putrefaciens incubated in M9 medium was assayed as described in Materials and Methods. Organoarsenicals were each added at 10 µM, final concentration: (o), MAs(III); (□), MAs(V); (▽), DMAs(V); (◊), Nit(III); (Δ), Rox(III) and (▲), Rox(V). Data are the mean ± SE (n = 3).

Figure 2

Cultures of S. putrefaciens were cultured to the density of A₆₀₀ = 0.6 at 30 °C with aeration in LB medium, washed and suspended at the same density in 5x-ST 10⁻¹ medium supplemented with 0.2% glucose as carbon source, then induced with MAs(III) or As(III) at 2 µM, final concentration, for 14 h 30 °C. Arsenic uptake of (A) MAs(III) or (B) As(III) at 10 µM, final concentration, was assayed as described in Materials and Methods. (o), no induction; (▽), induced with As(III); (□), induced with MAs(III). Data are the mean ± SE (n = 3).

Figure 3

S. putrefaciens is a highly versatile soil and marine microbe that has multiple pathways of arsenic biotransformations. As(III), As(V) and Rox(III) are transported into cells of S. putrefaciens by unidentified carriers. As(V) is reduced either by the respiratory arsenate reductase for energy generation or by the ArsC resistance arsenate reductase. MAs(V) is reduced to MAs(III) by an unknown process, and both MAs(III) and MAs(V) are thiolated to MMMTAs-(V). The As(III) and nitro groups of Rox(III) are reduced, producing 3A4HBzAs(V). The C–As bond in Rox(III) and MAs(III) are cleaved to release inorganic As(III), which is extruded from cells by the ArsB permease.