New insights into microbial oxidation of antimony and arsenic

Abstract

Sb(III) oxidation was documented in an Agrobacterium tumefaciens isolate that can also oxidize As(III). Equivalent Sb(III) oxidation rates were observed in the parental wild-type organism and in two well-characterized mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting that Sb(III) and As(III) may be biochemical analogs, Sb(III) oxidation is catalyzed by a pathway different than that used for As(III). Sb(III) and As(III) oxidation was also observed for an eukaryotic acidothermophilic alga belonging to the order Cyanidiales, implying that the ability to oxidize metalloids may be phylogenetically widespread.

FIG. 1.

A. tumefaciens oxidation of Sb(III). (A) Sb(V) formation in the wild-type strain 5A (□), the aoxR mutant (•), the mrpB mutant (○), and uninoculated controls (▪). Data are averages from duplicate cultures, and error bars (where visible) represent the range of the two values. (B) RT-PCR amplicons documenting expression of aoxAB in strain 5A but not in the aoxR mutant strain MSUAt1. Protocols for RNA extraction from mid- to late-log-phase cells, RT-PCR conditions and primers, and absence of DNA contamination were all as previously described (12). Lanes: 1, molecular mass standards in kb; 2, wild-type strain 5A total RNA amplified with aoxAB-spanning primers (arrow denotes aoxAB RT-PCR amplicon); 3, aoxR mutant MSUAt1 total RNA amplified with aoxAB-spanning primers; 4, wild-type strain 5A total RNA amplified with 16S rRNA gene primers; 5, aoxR mutant total RNA amplified with 16S rRNA gene primers. All strains were cultured in a minimal mannitol medium as previously described (12). Cultures were amended with 50 μM Sb(III) (as potassium antimonyl tartrate). All experiments were repeated at least twice, and in each case experimental treatments (inoculated and uninoculated) were at least in duplicate.

FIG. 2.

Comparison of the toxicities of As(III) and Sb(III) to the wild-type A. tumefaciens strain 5A. Toxicity was estimated by growth response. Cells were first cultured in minimal mannitol ammonium broth in the presence (preexposed) or absence (naïve) of 50 μM As(III) or Sb(III) and then inoculated into the same medium amended with or without 50 μM As(III) or Sb(III). □, As-naïve strain 5A grown in the absence of 50 μM As(III); ▪, As-naïve strain 5A cultured with 50 μM As(III); ▵, As(III)-preexposed strain 5A cultured with 50 μM As(III); ⋄, Sb-naïve strain 5A cultured with 50 μM Sb(III); ⧫, Sb(III)-preexposed strain 5A cultured with 50 μM Sb(III). Symbols represent the means of at least duplicate cultures, and error bars (where visible) represent the range of the two values.

FIG. 3.

Growth and As(V) formation by the Cyanidiales alga isolate CCMEE 5508. •, growth in the presence of 20 μM As(III); ○, growth in the absence of As(III); □, As(V) accumulated as a result of As(III) oxidation. No As(III) oxidation occurred in uninoculated controls or in killed controls (results not shown). Data represent the mean of duplicate cultures, and error bars (where visible) represent the range of the two values. The thermoacidophilic alga used in this work was grown at 42°C under constant illumination from cool-white fluorescent lamps (80 μE·m⁻²·s⁻²) in Allen medium (1) amended to contain 250 μM phosphate and 20 μM As(III) in closed serum bottles with headspace containing filter (0.2 μm)-sterilized CO₂ (100% initial concentration). As controls, serum bottles containing uninoculated medium or autoclave-killed cells were included, with the headspace of each connected to that of the inoculated serum bottle via sterile Tygon tubing. This precaution was taken to verify that the Sb(III) oxidation observed in these experiments was not abiotic auto-oxidation due to the photosynthetically derived O₂ produced by the algae.

FIG. 4.

Growth response to Sb(III) and Sb(V) formation by the Cyanidiales alga isolate CCMEE 5508. (A) Growth of Sb(III)-naïve (○) and Sb(III)-preexposed (•) algal cultures. (B) Sb(V) formation in CCMEE 5508 cultures that were Sb(III) naïve (○) or Sb(III) preexposed (•). No Sb(III) oxidation occurred in uninoculated controls (□) or in killed controls (▪). Cultures were grown as described in the legend to Fig. 3 except that 20 μM Sb(III) was substituted for 20 μM As(III). Data in both panels represent the mean of four replicate cultures. Error bars (where visible) represent 1 standard deviation of the mean.