Glutathione Is Involved in the Reduction of Methylarsenate to Generate Antibiotic Methylarsenite in Enterobacter sp. Strain CZ-1

Abstract

Methylarsenate (MAs(V)) is a product of microbial arsenic (As) biomethylation and has also been widely used as an herbicide. Some microbes are able to reduce nontoxic MAs(V) to highly toxic methylarsenite (MAs(III)) possibly as an antibiotic. The mechanism of MAs(V) reduction in microbes has not been elucidated. Here, we found that the bacterium Enterobacter sp. CZ-1 isolated from an As-contaminated paddy soil has a strong ability to reduce MAs(V) to MAs(III). Using a MAs(III)-responsive biosensor to detect MAs(V) reduction in E. coli Trans5α transformants of a genomic library of Enterobacter sp. CZ-1, we identified gshA, encoding a glutamate-cysteine ligase, as a key gene involved in MAs(V) reduction. Heterologous expression of gshA increased the biosynthesis of glutathione (GSH) and MAs(V) reduction in E. coli Trans5α. Deletion of gshA in Enterobacter sp. CZ-1 abolished its ability to synthesize GSH and decreased its MAs(V) reduction ability markedly, which could be restored by supplementation of exogenous GSH. In the presence of MAs(V), Enterobacter sp. CZ-1 was able to inhibit the growth of Bacillus subtilis 168; this ability was lost in the gshA-deleted mutant. In addition, deletion of gshA greatly decreased the reduction of arsenate to arsenite. These results indicate that GSH plays an important role in MAs(V) reduction to generate MAs(III) as an antibiotic. IMPORTANCE Arsenic is a ubiquitous environmental toxin. Some microbes detoxify inorganic arsenic through biomethylation, generating relatively nontoxic pentavalent methylated arsenicals, such as methylarsenate. Methylarsenate has also been widely used as an herbicide. Surprisingly, some microbes reduce methylarsenate to highly toxic methylarsenite possibly to use the latter as an antibiotic. How microbes reduce methylarsenate to methylarsenite is unknown. Here, we show that gshA encoding a glutamate-cysteine ligase in the glutathione biosynthesis pathway is involved in methylarsenate reduction in Enterobacter sp. CZ-1. Our study provides new insights into the crucial role of glutathione in the transformation of a common arsenic compound to a natural antibiotic.

Keywords: Enterobacter sp. CZ-1; antibiotic; arsenic; glutathione; gshA; methylarsenate; methylarsenite.

FIG 1

Reduction of MAs(V) by Enterobacter sp. CZ-1. Cultures were incubated at 30°C for 24 h in ST10⁻¹ medium containing 10 μM MAs(V). Arsenic species were determined using HPLC-ICP-MS.

FIG 2

The glutamate-cysteine ligase gene gshA from Enterobacter sp. CZ-1 is involved in MAs(V) reduction. (A) The transformant E. coli Trans5α (pUC118-MR) showed increased fluorescence response when cultured in ST10⁻¹ medium supplemented with 2 μM MAs(V). Uninoculated ST10⁻¹ medium containing 2 μM MAs(V) and E. coli Trans5α (pUC118) were used as controls. Data are means ± SD (n = 4). (B) HPLC-ICP-MS chromatograms showing MAs(V) reduction by E. coli Trans5α (pUC118), E. coli Trans5α (pUC118-MR), E. coli Trans5α (pUC118-yqaA), and E. coli Trans5α (pUC118-gshA) after these strains were incubated in ST10⁻¹ medium with 2 μM MAs(V). (C) The DNA fragment MR contains a putative inner membrane protein gene and a putative glutamate-cysteine ligase gene. (D) Intracellular concentrations of GSH and γ-EC of E. coli Trans5α (pUC118) and E. coli Trans5α (pUC118-gshA). ND, not detectable. Data are means ± SD (n = 3).

FIG 3

HPLC analysis of cellular thiol compounds in wild type Enterobacter sp. CZ-1 and gshA-deleted mutant. Cell extracts from Enterobacter sp. CZ-1 (A), gshA-deleted mutant (B), and standards consisting of GSH and γ-EC (C).

FIG 4

Glutathione is involved in MAs(V) reduction in Enterobacter sp. CZ-1. (A–B) HPLC-ICP-MS chromatograms (A) and quantitative analysis (B) of arsenic species in the cultures of wild-type Enterobacter sp. CZ-1, gshA-deleted mutant, and gshA-complemented strain incubated in ST10⁻¹ medium with 10 μM MAs(V). Data are means ± SD (n = 3). (C–D) Effect of exogenous GSH on MAs(V) reduction (C) and the percentage of MAs(V) reduced (D) by the gshA-deleted mutant of Enterobacter sp. CZ-1 cultured in ST10⁻¹ medium supplemented with the indicated concentrations of GSH. Uninoculated ST10⁻¹ medium containing 10 μM MAs(V) and 250 μM GSH was used as a negative control. Data are means ± SD (n = 3).

FIG 5

Assay of the antibacterial activity by wild-type Enterobacter sp. CZ-1 (A) and the gshA-deleted mutant (B) against Bacillus subtilis 168 in the presence or absence of 10 μM MAs(V).

FIG 6

Glutathione is required for the reduction and detoxification of As(V) by Enterobacter sp. CZ-1. (A) Reduction of As(V) by wild type Enterobacter sp. CZ-1 and gshA-deleted mutant incubated with 600 μM As(V) at 30°C for 12 h. (B) Growth of Enterobacter sp. CZ-1 and gshA-deleted mutant in ST10⁻¹ medium supplemented with different concentrations of As(V). (C) Effect of exogenous GSH on the growth of Enterobacter sp. CZ-1 and gshA-deleted mutant incubated in ST10⁻¹ medium with or without 50 μM GSH supplementation. Data are means ± SD (n = 3).