Letter pubs.acs.org/journal/estlcu
Unexpected Effects of Gene Deletion on Interactions of Mercury with the Methylation-Deficient Mutant ΔhgcAB Hui Lin,*,† Richard A. Hurt, Jr.,‡ Alexander Johs,† Jerry M. Parks,‡ Jennifer L. Morrell-Falvey,‡ Liyuan Liang,† Dwayne A. Elias,‡ and Baohua Gu*,† †
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
‡
S Supporting Information *
ABSTRACT: The hgcA and hgcB gene pair is essential for mercury (Hg) methylation by certain anaerobic bacteria, but little is known about how deletion of hgcAB affects the cell surface interactions and intracellular uptake of Hg. Here, we compare ΔhgcAB mutants with the wild-type (WT) strains of both Geobacter sulf urreducens PCA and Desulfovibrio desulf uricans ND132 and observe differences in Hg redox transformations, adsorption, and uptake in laboratory incubation studies. In both strains, deletion of hgcAB increased the rate of reduction of Hg(II) but decreased the rate of oxidation of Hg(0) under anaerobic conditions. The measured cellular thiol content in ΔhgcAB mutants was lower than that in the WT, accounting for the decreased rates of adsorption and uptake of Hg. Despite the lack of methylation activity, uptake of Hg by the ΔhgcAB mutant continued, albeit at a rate slower than that of the WT. These findings demonstrate that deletion of the hgcAB gene pair not only eliminates Hg methylation but also alters cell physiology, resulting in changes to Hg redox reactions, sorption, and uptake by cells.
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INTRODUCTION Methylmercury (MeHg), recognized as being bioaccumulative and neurotoxic to humans, is primarily produced by Gramnegative microorganisms such as iron- or sulfate-reducing bacteria1−4 and methanogens5 in anaerobic environments. The mechanism of mercury (Hg) methylation has been intensively studied for decades,2,3,6−8 leading to the recent identification of a gene pair, hgcA and hgcB, that is necessary for Hg methylation.1,9 It has been shown that deletion of either or both genes in Desulfovibrio desulf uricans ND132 and Geobacter sulfurreducens PCA abolishes mercury methylation. In addition to the genetic requirement, Hg−cell surface interactions and uptake by cells have been shown to influence Hg methylation.7,8,10 Intracellular uptake of Hg has recently been proposed to involve an active transport process and is sensitive to Hg speciation7,8 compared to passive diffusion.11,12 Interactions of mercury with cell surfaces (e.g., adsorption, reduction, and oxidation) can change Hg speciation, thus affecting the uptake and methylation of Hg.10,13−15 For example, Hg methylation by G. sulf urreducens PCA is enhanced in the presence of cysteine,7,8 whereas oxidation of Hg(0) by D. desulf uricans ND132 increases the rate of MeHg production.10,13 Adsorption of Hg onto cell surface thiols, however, inhibits Hg(II) reduction14,16 and promotes Hg(0) oxidation.10 Sequence analysis suggests that HgcA and HgcB are associated with the cytoplasmic membrane, with HgcA including both a cytosolic cobalamin binding domain and a © 2014 American Chemical Society
transmembrane domain, whereas HgcB may assume the role of a cytosolic electron shuttle between an unknown electron donor and HgcA.1 We do not know, however, if the cell surface physiology might be affected by the absence of HgcA and HgcB or its impact on Hg speciation. It is unclear whether deletion of these genes could also cause changes in how Hg interacts with cells, which may subsequently affect Hg uptake. Here, we report on the Hg−cell interactions in the methylation-deficient mutant strains (ΔhgcAB) 1 of D. desulf uricans ND132 and G. sulf urreducens PCA and compare them to those of their respective wild-type (WT) strains.
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MATERIALS AND METHODS Bacterial Strains and Hg−Cell Interaction Assays. Procedures for the deletion of genes hgcA and hgcB in D. desulf uricans ND132 and G. sulf urreducens PCA and growth conditions for both the WT and the mutant strains of ND132 and PCA have been described previously,1,14,17 and additional details are provided in the Supporting Information. All washed cell assays were conducted in 4 mL amber glass vials under anaerobic conditions.10,14 The final cell density in each vial was 108 cells/mL in all Hg(II) or Hg(0) assays except, in the Hg(0) assay with PCA (both WT and ΔhgcAB), a higher cell density Received: February 13, 2014 Accepted: April 11, 2014 Published: April 11, 2014 271
dx.doi.org/10.1021/ez500107r | Environ. Sci. Technol. Lett. 2014, 1, 271−276
Environmental Science & Technology Letters
Letter
(10 9 cells/mL) was used. Unless otherwise specified, phosphate-buffered saline (PBS) at pH 7.4 (Supporting Information)10,14 was used for all assays and supplemented with 1 mM electron donor and acceptor (acetate and fumarate for PCA and pyruvate and fumarate for ND132, respectively).13,14 Hg stock solutions of either Hg(II) (as HgCl2) or dissolved Hg(0) were used in the Hg(II) or Hg(0) assays, respectively, to yield a final concentration of ∼25 nM. The final reaction volume in each vial was 1 mL. All vials were sealed immediately, placed on an orbital shaker, and kept in the dark. Identical Hg(II) assays with spent medium, heat-killed (3 h at 55 °C)7,8 and live PCA ΔhgcAB cells without the electron donor or acceptor, were conducted as controls. The spent medium was obtained by filtration (0.2 μm) of cultures after cell growth under anaerobic conditions. At selected time points, samples were immediately analyzed for purgeable gaseous Hg(0) without opening the vials.10,14 An aliquot of the purged samples was then analyzed for the total nonpurgeable Hg (HgNP) (with cells), and another aliquot was filtered through a 0.2 μm, 13 mm Supor syringe filter (to remove cells) and analyzed for nonpurgeable, soluble Hg (Hgsol) (including MeHg, if any). The cell-associated Hg (Hgcell) was calculated as the difference between HgNP and Hgsol. The loss of Hg via sorption onto vials and filters was small (2−7%) as determined by mass balance analysis. For MeHg analysis, samples in separate vials were preserved with trace metal grade H2SO4 [0.2% (v/v)] and held at −20 °C until analysis.1,8,10 Hg uptake studies by the PCA mutant and the WT were investigated in parallel in separate sets of sacrificial sample vials taken at preselected time intervals. After Hg(0) had been purged out, the cell-adsorbed Hgad was determined using established methods of sequential washing with an EDTA (50 mM) and sodium oxalate (100 mM) solution followed by a solution of glutathione (1 mM) and sodium ascorbate (1 mM) in PBS.7,8 Cells were suspended for 15 min in each solution before being filtered with 0.2 μm syringe filters. The filtrate solution containing both the washed-off Hg (i.e., Hgad) and Hgsol was analyzed, and the adsorbed Hgad was calculated by subtracting Hgsol from that in the washing solution. Intracellular Hg uptake at each time point was then calculated as the difference between Hgad and Hgcell (i.e., intracellular Hg = Hgcell − Hgad). Analytical Methods. The purgeable Hg(0) in each vial was determined by direct purging with ultrapure N2 for 2 min using a Hg(0) analyzer (Lumex915+, Ohio Lumex).14,18−20 The 2 min purging time was sufficient to purge >99% of the Hg(0) out of a freshly prepared Hg(0) stock (25 nM) but
