Article pubs.acs.org/est
Transformation, Localization, and Biomolecular Binding of Hg Species at Subcellular Level in Methylating and Nonmethylating Sulfate-Reducing Bacteria Zoyne Pedrero,*,† Romain Bridou,‡ Sandra Mounicou,† Remy Guyoneaud,‡ Mathilde Monperrus,*,† and David Amouroux† †
Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, Institut Pluridisciplinaire de Recherche sur l'Environnement et les Matériaux UMR 5254, CNRS Université de Pau et des Pays de l’Adour, 2 Avenue Pierre Angot, 64053 Pau, France ‡ Equipe Environnement et Microbiologie, Institut Pluridisciplinaire de Recherche sur l'Environnement et les Matériaux, UMR 5254,CNRS Université de Pau et des Pays de l’Adour, Bâtiment IBEAS, BP1155, 64013 Pau Cedex, France S Supporting Information *
ABSTRACT: Microbial activity is recognized to play an important role on Hg methylation in aquatic ecosystems. However, the mechanism at the cellular level is still poorly understood. In this work subcellular partitioning and transformation of Hg species in two strains: Desulfovibrio sp. BerOc1 and Desulfovibrio desulf uricans G200 (which exhibit different Hg methylation potential) are studied as an approach to the elucidation of Hg methylation/demethylation processes. The incubation with isotopically labeled Hg species (199Hgi and Me201 Hg) not only allows the determination of methylation and demethylation rates simultaneously, but also the comparison of the localization of the originally added and resulting species of such metabolic processes. A dissimilar Hg species distribution was observed. In general terms, monomethylmercury (MeHg) is preferentially localized in the extracellular fraction; meanwhile inorganic mercury (Hgi) is associated to the cells. The investigation of Hg binding biomolecules on the cytoplasmatic and extracellular fractions (size exclusion chromatography coupled to ICP-MS) revealed noticeable differences in the pattern corresponding to the Hg methylating and nonmethylating strains.
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INTRODUCTION Mercury methylation in aquatic ecosystems is a critical step in the accumulation of this hazardous species in fish, leading to human exposure. It is well established that microbial methylation is one of the major biotic processes that transform inorganic mercury (Hgi) into toxic MeHg. Sulfate-reducing bacteria (SRB) are recognized as the principal Hg methylators in anoxic waters and sediments,1,2 which hold up to 98% of mercury in aquatic ecosystems.3 Despite being predominantly anaerobic, recent studies have revealed methylation activity in oxygen rich environments,4 such as the peryphiton of Amazonian macrophytes.5 Due to the direct impact on the ecosystem, several studies focused on the investigation of Hg methylation by SRB, most of them concentrated on the influence of environmental parameters6−8 and biogeochemical factors.9 However, Hg methylation mechanisms remain unknown and it has been suggested that it could be a protective mechanism against Hgi toxicity developed during the early evolution of some microorganisms.10 It should be also considered that some SRB exhibit both Hgi methylation and MeHg demethylation potentials.11 To estimate the net methylation, the demethylation process should be also © 2012 American Chemical Society
studied. It has been proposed that MeHg demethylation could be produced by reductive pathways under oxic conditions, mediated by the mer-operon system and leading to the formation of Hg0 and CH4.12,13 In contrast, oxidative demethylation dominates under anoxic conditions, producing Hgi and CO2.12,14,15 One of the main hypothesized mechanisms for SRB suggests that Hgi can be methylated through the acetyl-coenzyme A (CoA) pathway,16 which implies the participation of carbon monoxide dehydrogenase (CODH) to produce the methyl moiety. However, Ekstrom et al.17 demonstrated later that it cannot be extrapolated to incomplete oxidizing (converting carbon substrates to acetate rather than to CO2) SRB strains, where Hg methylation seems to be independent of acetyl-CoA. Apparently, Hg methylation is related to the acetyl-CoA in complete oxidizers, meanwhile in incomplete oxidizers further efforts are required to determine the biochemical pathways. Received: Revised: Accepted: Published: 11744
June 15, 2012 September 27, 2012 October 10, 2012 October 10, 2012 dx.doi.org/10.1021/es302412q | Environ. Sci. Technol. 2012, 46, 11744−11751
Environmental Science & Technology
Article
its capacity to methylate Hg,18 was grown using abovedescribed medium and NaCl was added to a final concentration of 10 g L−1. This halophilic strain is closely related to the Hg methylating Desulfovibrio desulf uricans strain ND132, recently proposed as a model microorganism to study Hg methylation.27,28 Logarithmic phase growing cells were incubated 96 h at 30 °C to an optical density (A600 nm) of 0.3. The resulting culture was diluted 1:10 in 200 mL of anoxic fresh medium and incubated overnight at 30 °C to an optical density of 0.1 in four different 250-mL flasks. The overnight cultures were then carefully transferred in 250-mL PTFE centrifuge flasks under anoxic conditions and centrifuged at 8000g, 25 °C for 15 min. Cells pellets were pooled, washed into mineral base medium three times sequentially. Washed cells were gently homogenized in 45 mL of anoxic fresh medium without carbon and energy source (e.g., pyruvate), transferred into a 45-mL PTFE flask and spiked with 100 ng of 199HgCl2 per g of culture media and 10 ng of Me201HgCl per g of culture media. The starting resting cell density was determined by cell numeration with a Malassez cell counting chamber. Samples were collected after 5 h incubation (37 °C) to determine the concentrations and isotopic composition of Hg species in the different incubating fractions as described later. (2) Strain G200. D. desulf uricans, which is closely related to strain G20, was isolated from an oil well souring site.29 It is a Desulfovibrio model microorganism that was intensely involved in the development of genetic studies for this genus over the last 20 years.30−33 In this study, it was used as a negative Hg methylating control and was grown in the same medium and conditions as described for strain BerOc1 except that the NaCl concentration was kept at 3 g L−1. Incubation with Hg species was performed under the same conditions previously described for the methylating strain BerOc1. Experiments were reproducible as similar results concerning Hg distribution in cell and media have been observed in two other repetitions of these experiments, with a variation of approximately 10%. Cell Fractionation. After 5 h of incubation, the cell cultures were centrifuged at 10 000g at 4 °C for 15 min for the separation of cells (pellets) and extracellular fraction (supernatant). Pellets were resuspended in 20 mL of 200 mM ammonium acetate buffer, pH 7.5. Cell disruption was performed by action of French Press (French Press, Thermo spectronic) at an equivalent pressure of 20 Kpsi applied in a 35-mL standard cell unit FA-032 (40K Standard). The bacterial cell lysate was collected in a 30-mL Teflon PTFE tube maintained in ice to prevent proteolysis and enzymatic subcellular degradation. It was immediately distributed in 1.5-mL Eppendorf tubes (Eppendorf LoBind Protein) and centrifuged at 25 000g at 4 °C (Eppendorf, 5417R) for 2 hours (adapted from 16) in order to separate the supernatant (later referred as cytoplasm) and cell debris fractions (Supporting Information). Separation of the cell-associated and extracellular fractions, cell integrity after centrifugation of the culture, and the cell disruption efficiency of French press were followed by microscopic observations (×1000, Olympus BX60), using each previous sequential step as reference control. Reagents and Standards. All solutions were prepared using ultrapure water (18 MΩ cm, Millipore). For the preparation of samples, standards, and blanks, trace metal grade acids (HNO3 and HCl) were purchased from Fisher Scientific (Illkirch, France).
In addition, the capability of Hg methylation by SRB is strain dependent, rather than dependent on genus or species,11,18 which reflects the necessity of performing pure cells culture studies. Unfortunately, the interpretation and comparison of most of the published data is difficult, due to the differences in cell culture conditions (Hg concentration, incubation time, etc.) and results expression units. The incubation with multiple stable isotopic tracers represents a powerful tool to deal with the simultaneous microbial Hg methylation and demethylation determination.11,19,20 It has been on real environmental samples such as water,21 sediments,9 and periphyton successfully applied to the study of Hg methylation/demethylation in complex.5 This experimental approach, characterized by its high precision, is confirmed as a valuable tool for tracing environmental and metabolic processes.22 The transmembrane uptake of Hgi has been proposed as a decisive step for MeHg production.23 Schaefer and Morel have demonstrated that Hg methylation in Geobacter sulf ureducens is greatly promoted by Hg speciation.23 However, there are divergences with respect to the Hgi uptake, meanwhile some authors consider it as a passive process.24 Among the different Hg ligands investigated, organic ligands enhance uptake potential, where the highest one corresponds to cysteine (Cys) through formation of Hg−thiol complex.23 These results suggest the influence of Hg binding biomolecules and its specific role on its uptake. The identification of molecular targets of Hg contributes to the understanding of metabolic pathways, since it determines their toxicity, mobility, and transformation, etc. As a first step, the study of Hg species distribution among the protein fractions of the bacterial cell will provide useful and original information as, so far, most of the speciation studies are limited to the quantification of Hgi and MeHg. In this work, the advantage of using multiple isotopic labeled species is exploited to investigate not only the methylation and demethylation potential of two pure bacterial strains: Desulfovibrio sp. BerOc1 and Desulfovibrio desulf uricans G200 (Hg methylating control), but also the localization and origin of the different Hg species at subcellular levels. Cytoplasmic and extracellular fractions were screened for Hg species binding biomolecules by HPLC-ICP-MS in combination with GC-ICPMS.
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EXPERIMENTAL SECTION Culture Conditions. All experiments were performed in autoclaved glass or Teflon flasks that were cleaned by ultrasonication in successive baths of 10% HNO3 and HCl of 2-hour cycles followed by rinsing three times in 18 MΩ deionized water. Sulfate-reducing bacterial strains were grown in the dark at 30 °C and pH 7.0−7.1 under fumarate respiration with 10 mM pyruvate and fumarate in order to avoid complexation of Hg species with sulfides produced under sulfate-reducing conditions. Defined mineral base optimal growth media contained ( in g L−1 unless otherwise indicated): NaCl, 3; MgCl2·6 H2O, 0.40; CaCl2·2 H2O, 0.10; NH4Cl, 0.25; KH2PO4, 0.20; KCl, 0.50; SL12 B, 1 mL L−1; selenite tungstate solution, 1 mL L−1; NaHCO3, 30; vitamins solution (V725), 0.25 mL L−1. In addition, precise growth and experimental procedure were followed as described below. (1) Strain BerOc1, Desulfovibrio sp. an incomplete oxidizer isolated from brackish lagoon sediments,26 previously tested for 11745
dx.doi.org/10.1021/es302412q | Environ. Sci. Technol. 2012, 46, 11744−11751
Environmental Science & Technology
Article
Table 1. Hg Species Concentrations (ng g−1) in the Bulk Culture at the Initial (t = 0 h) and Final (t = 5 h) Incubation Timesa Me201Hg bulk 0 h bulk 5 h a
Me199Hg
201
199
Hgi
Hgi
BerOcl
G 200
BerOcl
G 200
BerOcl
G 200
BerOcl
G 200
10.2 ± 0.5 9.4 ± 0.5
9.5 ± 0.5 6.8 ± 0.7
