An alternate pathway of arsenate resistance in E. coli mediated by the glutathione S-transferase GstB

Abstract

Microbial arsenate resistance is known to be conferred by specialized oxidoreductase enzymes termed arsenate reductases. We carried out a genetic selection on media supplemented with sodium arsenate for multicopy genes that can confer growth to E. coli mutant cells lacking the gene for arsenate reductase (E. coli ΔarsC). We found that overexpression of glutathione S-transferase B (GstB) complemented the ΔarsC allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type E. coli arsenate reductase, arsenate resistance via GstB was not dependent on reducing equivalents provided by glutaredoxins or a catalytic cysteine residue. Instead, two arginine residues, which presumably coordinate the arsenate substrate within the electrophilic binding site of GstB, were found to be critical for transferase activity. We provide biochemical evidence that GstB acts to directly reduce arsenate to arsenite with reduced glutathione (GSH) as the electron donor. Our results reveal a pathway for the detoxification of arsenate in bacteria that hinges on a previously undescribed function of a bacterial glutathione S-transferase.

Figure 1

GstB overexpression confers arsenate resistance to E. coli ΔarsC cells. (a) Arsenate resistance of DHB4 ΔarsC cells expressing gstB, cpsB, dksA, and fruR selected from the genetic screen. Strains are plated on MOPS agar plates containing 1 mM sodium arsenate. (b) Growth of varied selected mutant strains expressing either the gstB construct (pGstB) or the ASKA arsC construct (pArsC_ASKA). Mutants were grown for 24 h on MOPS plates containing between 0 and 5 mM sodium arsenate. DHB4 ΔarsCΔgrxABC represents mutants lacking the arsC gene and the glutaredoxin genes grxA, grxB, and grxC; ΔarsRBC represents mutants lacking the arsRBC operon.

Figure 2

Mutational analysis of GstB residues involved in arsenate resistance. (a) Crystal structure of the E. coli GstB homologue, YliJ, from Salmonella enterica (PDB 4KH7). Three arginine residues (blue stick models), R7, R111, and R119, form the putative electrophilic binding site near glutathione (GSH). (b) Growth curves of E. coli EQ301 (DHB4 ΔarsCΔgstB) expressing selected GstB variants from the multicopy pCA24N construct in liquid minimal MOPS media containing either 0 mM sodium arsenate (top panel) or 1 mM sodium arsenate (bottom panel). Growth curves are plotted as the average absorbance values from three independent growth studies. Error bars represent the standard deviation in absorbance at each time point.

Figure 3

GstB overexpression results in the accumulation of arsenite in culture supernatant. Levels of arsenite accumulated in the supernatant of bacteria cultured in MOPS minimal media containing 1 mM sodium arsenate were measured using a two-step colorimetric assay. Exported arsenite was isolated from arsenate in the media using anion exchange. Separated arsenite was subsequently detected using a colorimetric assay with mercuric bromide. Panels 1–6 illustrate the levels of arsenite produced by selected E. coli mutant strains containing either no plasmid (−), the ASKA arsC construct (pArsC_ASKA), the gstB construct (pGstB), or the ASKA gstA construct (pGst_ASKA). Panel 7 represents the detection of arsenic in uninoculated growth media containing only 1 mM sodium arsenate.

Figure 4

Reduction of arsenate to arsenite by GstB in vitro. GstB directed reduction of arsenate via GSH was measured using two separate in vitro assays. (a) Levels of arsenite were measured using the two-step colorimetric assay after a 30 or 60 min reaction. Reactions were performed using 50 mM sodium arsenate and 1 mM GSH. When indicated, active GstB or inactive GstB_R111Q/R119Q was added to the reaction at a final concentration of 0.12 mM (Panels 3 and 4, respectively). (b) NADPH redox-coupled oxidation assays monitor the oxidation of NADPH at an absorbance of 340 nm. The plot shows the rate of NADPH oxidation in the presence of GOR, 50 mM sodium arsenate and 1 mM GSH. When indicated, active GstB and inactive GstB_R111Q/R119Q were added to the reaction at a concentration of 0.12 mM. Error bars represent the standard deviation in absorbance across three replicate samples.

Scheme 1. Proposed Mechanisms of Arsenate Reduction by GstB

Route A. GstB directly enhances the rate of GS-arsenate conjugation. Once formed, the intermediate undergoes a spontaneous reaction with a second equivalent of GSH, which results in reduction to arsenite and oxidized GSH (GSSG). Route B. Conjugation of one reduced GSH molecule to arsenate occurs spontaneously in solution. The presence of GstB then catalyzes the conjugation of a second GSH molecule to arsenate and subsequent reduction of (GS)₂–arsenate intermediate into oxidized GSSG and arsenite.