Effect of pH on intracellular accumulation of trace concentrations of Hg(II) in Escherichia coli under anaerobic conditions, as measured using a mer-lux bioreporter

Abstract

The effects of pH on the uptake and accumulation of Hg(II) by Escherichia coli were determined at trace, environmentally relevant, concentrations of Hg and under anaerobic conditions. Hg(II) accumulation was measured using inducible light production from E. coli HMS174 harboring a mer-lux bioreporter plasmid (pRB28). The effect of pH on the toxicity of higher concentrations of Hg(II) was measured using a constitutive lux plasmid (pRB27) in the same bacterial host. In this study, intracellular accumulation and toxicity of Hg(II) under anaerobic conditions were both significantly enhanced with decreasing pH over the pH range of 8 to 5. The pH effect on Hg(II) accumulation was most pronounced at pHs of <6, which substantially enhanced the Hg(II)-dependent light response. This enhanced response did not appear to be due to pH stress, as similar results were obtained whether cells were grown at the same pH as the assay or at a different pH. The enhanced accumulation of Hg(II) was also not related to differences in the chemical speciation of Hg(II) in the external medium resulting from the changes in pH. Experiments with Cd(II), also detectable by the mer-lux bioreporter system, showed that Cd(II) accumulation responded differently to pH changes than the net accumulation of Hg(II). Potential implications of these findings for our understanding of bacterial accumulation of Hg(II) under anaerobic conditions and for bacteria-mediated cycling of Hg(II) in aquatic ecosystems are discussed. Arguments are provided suggesting that this differential accumulation is due to changes in uptake of mercury.

FIG. 1.

Effect of varying pH on the Hg(II)-induced light response of 1 ng liter⁻¹ in E. coli HMS174(pRB28) (left) and on constitutive light production from E. coli HMS174(pRB27) (right). Light production was measured following 120 min of incubation. Error bars represent the standard deviations of two independent experiments (n = 4).

FIG. 2.

Effect of varying pH on the Hg(II)-induced light response from E. coli HMS174(pRB28) preconditioned in pH 6 to 8 growth medium (left) and on constitutive light production from E. coli HMS174(pRB27) (right). Hg(II) was added to a final concentration of 1 ng liter⁻¹ in the bioreporter assays, and light production was measured following 120 min of incubation. Error bars for the mer-lux bioreporter assays represent the standard deviations of duplicate samples. Error bars for the constitutive lux assays represent the standard errors of three independent assays (n= 6).

FIG. 3.

Effect of pH on Cd(II)- and Hg(II)-induced light production in E. coli HMS174(pRB28). The assay medium contained a final concentration of 6.7 mM phosphate. Cd(II) and Hg(II) were added to final concentrations of 112 μg liter⁻¹ and 1 ng liter⁻¹, respectively. Error bars represent the standard deviations of two independent assays (n = 4).

FIG. 4.

Effect of pH on Hg(II) toxicity. Hg(II) was added to a final concentration of 2,000 ng liter⁻¹. The amount of light measured in the Hg(II)-spiked medium was compared to the amount of light in the nonamended medium, at the same pH, and is expressed as a percentage of light remaining. Error bars represent the standard deviations of three independent assays (n = 5).

FIG. 5.

Effect of pH on Hg(II) speciation in the assay medium.

FIG. 6.

Effects of varying the pH on Hg(II) uptake in E. coli MG1655(pRB28) and E. coli RKP2922(pRB28). Samples were incubated 120 min prior to measurement. Error bars represent the standard deviations of two independent assays (n = 4).

FIG. 7.

Effects of varying the concentration of HgHPO₄ independently of pH on Hg(II) uptake (pRB28) (A) and the effect of varying the concentration of phosphate and (NH₄)₂SO₄ on constitutive light production (pRB27) (B) in E. coli HMS174 and E. coli RKP2922. Assays were performed in pH 5.9 assay medium containing 10 ng liter⁻¹ Hg(II). The concentrations of phosphate and (NH₄)₂SO₄ in the media were as follows: data set 1, 67 mM and 9 mM; 2, 33.5 mM and 9 mM; 3, 17.5 mM and 9 mM; 4, 8.75 mM and 9 mM; 5, 67 mM and 4.5 mM; 6, 67 mM and 2.25 mM. Samples were incubated for 120 min prior to measurement. Error bars represent the standard deviations of two independent assays of triplicate samples (n = 6) (A) and two independent assays of duplicate samples (n = 4) (B).