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S10 Mechanisms of microbial mercury methylation and resistance

Tuesday, 26 July, 2011

TS10-P1 — 11:00-12:00 and 17:30-18:30
ASSESSMENT OF BACTERIAL MERCURY RESISTANCE BY THE QUANTIFICATION OF HG(II) REDUCTION
Authors: VASCONCELLOS, Ana C1, BARROCAS, Paulo R1, MOREIRA, Josino C2, JACOB, Silvana C3, DUQUE, Sheila S4, BEZERRA, Adriana L1, WASSERMAN, Julio C5, HACON, Sandra S6
(1) Department of Sanitation and Environmental Health/ ENSP - Oswaldo Cruz Foundation, anavasconcellos@ensp.fiocruz.br; (2) CESTEH - Oswaldo Cruz Foundation; (3) INCQS - Oswaldo Cruz Foundation; (4) IOC - Oswaldo Cruz Foundation; (5) Federal Fluminense University; (6) Oswaldo Cruz Foundation.

Mercury released in aquatic systems causes a selective pressure in the biota, favoring Hg resistant organisms. The most common bacterial mercury resistance mechanism is the enzymatic reduction of Hg(II) to elemental mercury by the mercury reductase, encoded by the merA gene. Our objective was to assess the use of a Hg resistant bacterial strain, Leclercia adenocarboxylata, to remove Hg(II) from aqueous solutions. This strain was isolated from a heavily polluted ecosystem in Brazil. It had a mercury minimum inhibition concentration of 30 µM, while PCR results, using specific primers for the merA gene, suggested the presence of this gene in the isolated DNA strain. Bioassays to quantify Hg(II) reduction were designed using two air samplers impingers in series, connected to an air sampling pump. First impinger had bacterial culture with added Hg, while second impinger had an acidified KMnO4 solution, as a trap for the elemental Hg vapor generated. Samples were collected from both impingers at different times. Total Hg levels were determined after acid digestion, using CVAAS technique. Mass balances, between the Hg levels in the beginning of the experiment and in the end, had recovery rates between 98% and 110%. Results showed a simultaneous decrease of the Hg levels in the first impinger and an increase in the second during the bioassays. The amount of Hg reduced increased as the incubation time went up (from 45% of added Hg after 1 hour until a maximum of 68% after 2 hours of the experiment). Samples taken after 3 and 4 hours of the experiment had the same Hg0 amount as two hour sample. However, the time to reach this plateau and the maximum concentration of elemental mercury generated are influenced by the Hg(II) concentration and the bacterial inoculum added in the beginning of the bioassays. When initial Hg(II) added was doubled (from 5 µM to 10 µM), there was a continuous increase in the Hg0 generated during the experiment (12%, 23% and 34% after 1, 2 and 4 hours, respectively). Alternatively, when we raised the amount of bacterial cells, there was an increase in the Hg(II) reduction efficiency (49%, 64% and 68% using 6,2 x 108, 1,2 x 109 and 1,3 x 109 CFU/mL, respectively). These data suggested that several variables can influence this bioremediation process, which seems to be controlled mostly by the amount of bioavailable Hg in the medium and the number of bacterial cells active.

TS10-P2 — 11:00-12:00 and 17:30-18:30
SOLUBILITY OPTIMIZATION OF THE HG(II)-SPECIFIC TRANSCRIPTIONAL REGULATOR MERR FOR THE STUDY OF MOLECULAR STRUCTURE AND DYNAMICS
Authors: TOMANICEK, Stephen J.1, JOHS, Alexander 1, SUMMERS, Anne O.2, OLLIFF, Lyn2, LIANG, Liyuan1, SMITH, Jeremy C.3
(1)Environmental Sciences Division, Oak Ridge National Laboratory, tomaniceksj@ornl.gov; (2) Department of Microbiology, University of Georgia; (3) University of Tennessee/Oak Ridge National Laboratory Center for Molecular Biophysics.

Mercury (Hg) is a widely found contaminant in the environment and is of particular interest as methyl mercury can enter the food chain and bioaccumulate in higher organisms. Heavy metals such as Hg have no known biological function and are toxic to all living organisms. However, microbes have evolved naturally to deal with heavy metal toxicity resulting in elaborate heavy metal resistance mechanisms. Select bacteria possess specific genes that allow cells to resist poisoning of toxic metals by using multiprotein resistance systems under the control of metal-responsive transcriptional regulators. The transcriptional repressor-activator, MerR, is the archetype of the MerR family of metalloregulators that controls the transcription of a set of genes (the mer operon) providing Hg resistance in many genera of bacteria isolated from Hg-exposed ecosystems. The mer operon encodes specific genes that facilitate transport of Hg species, cleavage of organomercurials and reduction of ionic Hg(II) to volatile, elemental Hg(0). The 144-residue MerR binds to its operator DNA and functions as a homodimer. It represses transcription of the mer operon in the absence of Hg(II) and activates transcription upon Hg(II) binding. Although a wealth of genetic, biochemical, and biophysical data exist for MerR and several structures are known for paralogous members of the MerR family specific for other metals, to date the three-dimensional structure of MerR is unknown. Previous attempts at structure determination of MerR using X-ray crystallography and Small Angle Neutron Scattering (SANS) have been hampered by low protein solubility. Here we present our strategy for overcoming these solubility issues by screening a range of discrete conditions including pH, cations, anions and other additives, to optimize the solubility of MerR. We have successfully obtained a customized formulation that enhances the solubility of Hg(II)-MerR and Hg-free-MerR by factors of 20 and 15, respectively. Dynamic Light Scattering (DLS) showed that samples prepared in the respective optimized buffer conditions are monodisperse. Initial sparse-matrix vapor diffusion crystallization screening of both Hg(II)-MerR and Hg-free-MerR using the optimized buffer formulation revealed multiple crystallization conditions that are currently being optimized to produce diffraction quality crystals for X-ray studies. We anticipate that this solubility optimization strategy will facilitate our current efforts to solve the first X-ray crystallographic structure of MerR. The increased solubility will also enable use of neutron scattering to study MerR dynamics and conformational changes that are essential for understanding its unique mechanism of transcriptional regulation.

TS10-P3 — 11:00-12:00 and 17:30-18:30
EFFECT OF SELENIUM ON THE METHYLATION OF MERCURY BY THE SULFUR-REDUCING BACTERIUM DESULFOVIBRIO DESULFURICANS
Author: BELZILE, Nelson1
(1)Laurentian University, nbelzile@laurentian.ca

The mercury methylation activity of the sulfur-reducing bacterium Desulfovibrio desulfuricans was investigated at environmentally relevant levels of mercury and under controlled laboratory conditions. Particular attention was given to the role of sulfur and selenium on the availability of Hg for the methylation process. A poisoning effect of hydrogen sulfide that evolved during incubation was observed and eventually eliminated with a chemical trap. The formation of methyl mercury was remarkably reduced when selenite was introduced in the culture medium. A proteomic study using polyacryamide 2D gel electrophoresis and mass spectrometry showed repeatedly that, proteins responsible for regulating other enzymes involved in methylation, had disappeared in culture medium containing selenite. Volatile forms of selenium, probably dimethylselenide also evolved during anaerobic culture incubations.

TS10-P4 — 11:00-12:00 and 17:30-18:30
COMPARISON OF METHYLMERCURY PRODUCTION RATES BY DESULFOVIBRIO SPECIES IN SHORT-TERM WASHED CELL ASSAYS
Authors: GRAHAM, Andrew M.1, GILMOUR, Cynthia C.1
(1) Smithsonian Environmental Research Center, grahaman@si.edu

Dissimilatory sulfate-reducing bacteria (DSRB) are thought to be the principal microorganisms responsible for mercury methylation in aquatic ecosystems. Relatively little is known about the prevalence of Hg methylation capability in DSRB at the species or genus level. Here, we report our efforts to determine the distribution of Hg methylation capability within Desulfovibrio, a representative and comparatively well-studied genus of DSRB. Desulfovibrio type strains were obtained from culture collection and grown to mid-log phase on either pyruvate/fumarate (P/F) or sulfate/lactate (S/L) media under strict anoxic conditions. Washed cell suspensions were prepared via repeated centrifugation and cell resuspension with minimal F/P or S/L media containing 500 µM cysteine as a reductant and Hg(II)-complexing agent. Cell suspensions were spiked with ~10 ng/mL of isotopically-enriched 201HgCl2, incubated for 3 h, and excess Me201Hg was quantified at the end of the experiments. Short-term assays in defined minimal media permitted evaluation of MeHg production under conditions of optimal inorganic Hg(II) bioavailability. Utilization of a chemically-defined standardized methylation assay enabled the first direct comparison of protein-normalized MeHg production rates by Desulfovibrio at the species level. This work has implications for our understanding of evolutionary relationships between Desulfovibrio capable and incapable of Hg methylation. Short-term methylation assays also allowed for an assessment of the bioavailability of inorganic Hg(II) species to Desulfovibrio.The impact of dissolved organic mater (DOM) on MeHg production was evaluated for a variety of well-characterized DOM isolates at various Hg/DOM ratios to determine the effect of Hg-DOM complex lability on bacterial MeHg generation.

TS10-P5 — 11:00-12:00 and 17:30-18:30
SMALL THIOLS ENHANCE MERCURY METHYLATION RATES BY SULFATE-REDUCING BACTERIUM DESULFOVIBRIO DESULFURICANS ND132 BY ENHANCING HG SOLUBILITY
Authors: GILMOUR, Cynthia1, GRAHAM, Andrew M.1, ELIAS, Dwayne A2
(1) Smithsonian Environmental Research Center, gilmourc@si.edu; (2) Oak Ridge National Laboratory.

In order to help understand mercury uptake mechanisms in Hg-methylating bacteria, we examined the effect of a variety of sulfur-bearing Hg ligands and amino acids on methylmercury (MeHg) production by Desulfovibrio desulfuricans ND132. Dissimilatory sulfate-reducing bacteria (DSRB) are important mediators of this process in anoxic sediments and soils, and all of the identified Hg-methylators are Deltaproteobacteria.The metabolic pathways and genes involved in Hg uptake MeHg production by DSRB remain poorly understood. The facilitated uptake of Hg bound to amino acids has been proposed as an explanation for the ability to produce MeHg. However, the role of amino acids and small thiols in Hg methylation by DSRB has not been examined.

MeHg production experiments were conducted both in batch culture and with washed cells in minimal medium. Cells were grown up in pyruvate/fumarate medium reduced with TiNTA. In all cases, the Hg partitioning among species (inorganic Hg(II), MeHg and Hg0) and between cells, culture medium, bottle walls was closely monitored. ND132 was used as a model strain because of its high methylation rate. Ligands included small amino acids with and without thiol moieties, and the thiols thioglycolate, DTE and mercaptoethanol. MeHg production serves as a surrogate for Hg uptake, as MeHg production is known to be intracellular.

All of the thiol-bearing ligands tested significantly increased MeHg production by ND132 relative to unamended culture medium, while non-thiol amino acids (his and met) did not. Similarly, all of the thiols enhance Hg solubility, while his and met did not. MeHg production was linearly correlated with the amount of inorganic Hg in solution. In short-term (3h) washed cell assays, ND132 converted almost 100% of the filterable inorganic Hg into MeHg. Differences in MeHg production were not related to effects of the ligands on cell activity. Almost all of the MeHg produced by cells was rapidly excreted.

Small thiols appear to enhance Hg methylation by D. desulfuricans ND132 at least in part by holding inorganic Hg in solution. Since the thiols tested included compounds with a range of additional functional groups, charge and size, it is unlikely that they enhanced Hg uptake by stimulating specific amino acid transport mechanisms.

TS10-P6 — 11:00-12:00 and 17:30-18:30
ANALYTICAL PROBLEMS TO QUANTIFY MERCURY REDUCTASE ACTIVITY AND USE IT TO ASSESS BACTERIA POTENTIAL FOR BIOREMEDIATION OF MERCURY POLLUTION
Authors: BEZERRA, Adriana L1, SILVA FILHO, Moacelio V2, BARROCAS, Paulo R1, VASCONCELLOS, Ana C1, DUQUE, Sheila S3, REBELLO, Raquel C4
(1) Department of Sanitation and Environmental Health, ENSP-Fiocruz, adrianalima@ensp.fiocruz.br; (2) CESTEH, ENSP-Fiocruz; (3) IOC, Fiocruz; (4) Biology Department, ENSP-Fiocruz.

Mercury-resistant bacteria isolated from the environment have been shown to be a very promising tool to bioremediate mercury pollution. The most common microbial resistance mechanism is the transformation of Hg(II) compounds to elemental mercury (Hg0). This biotransformation is catalyzed by the enzyme mercury reductase (MR), product of merA gene. This study reports the analytical problems to quantify MR activity and use it to assess bacteria potential for Hg bioremediation. Two standard Escherichia coli samples (ATCC 35218–Hg resistant strain and ATCC 23724–Hg sensible) and one wild strain (Leclercia adecarboxylata) were used in this research. The main steps in the protocol to quantify MR activity are: 1) Bacteria cells cultivation until mid-log exponential growth, to ensure the same physiological state; 2) Standardization of cell biomass amount that are harvest for the bioassays; 3) Disruption of the bacteria cells, avoiding the presence of viable cells; 4) Isolation of soluble fractions by centrifugation; 5) Measurement of the MR activity. Each of these steps were tested and the main results were: 1) The generation time (38-41min) was determined for all bacteria tested, after a maximal of 5 hours of cell growth, with or without 5 µM of Hg added to the medium; 2) Cell biomass was washed with saline until supernatant had at most 0.1g/L of proteins and diluted to an absorbance of 5.0 at 600nm; 3) The use of glass beads and vortex for cell disruption did not cause a significant cell death and produced turbid homogenates with very variable protein levels; 4) Soluble fractions need to be obtained after a centrifugation of at least 30 minutes at 35,000g; 5) Photometric studies of the MR reaction kinetics showed that Hg(II) reacts with NADPH and with components of bacterial soluble fractions. To avoid interferences, the protocol to measure MR activity needs: a) Checking out NADPH concentration in the reaction medium, by measuring absorption at 340nm, before use; b) Pre-incubating samples with reaction medium for at least 20 minutes; c) Starting the MR reaction by Hg(II) addition, recording A340nm for more 20min. The Absorbance dropped down very fast during the first minutes after Hg(II) addition, as result of Hg(II) reduction. Then, it decreased linearly for the rest of time reaction. For the MR quantification, this linear part of the reaction should be used to avoid mistakes.

TS10-P7 — 11:00-12:00 and 17:30-18:30
SULFATE-REDUCING PROKARYOTES COLONIZING ROOTS OF AQUATIC MACROPHYTES: OCCURRENCE, IMPACT ON MERCURY METHYLATION AND ENVIRONMENTAL RISKS ASSESSMENT.
Authors: GENTÈS, Sophie1, MONPERRUS, Mathilde2, GONI, Maria-Soledad3, LEGEAY, Alexia4, MAURY-BRACHET, Régine5, AMOUROUX, David6, DAVAIL, Stéphane3, ANDRÉ, Jean-Marc3, GUYONEAUD, Rémy3
(1) Environnement et Microbiologie (IPREM UMR 5254),Université de Pau et des Pays de l’Adour, France, sophie.gentes@univ-pau.fr; (2) Chimie Analytique Bio Inorganique et Environnement (IPREM UMR 5254),Université de Pau et des Pays de l’Adour, France; (3) Environnement et Microbiologie (IPREM UMR 5254); (4) Ecotoxicologie Aquatique (EPOC UMR 5805),Université Bordeaux 1, France; (5) Ecotoxicologie Aquatique (EPOC UMR 5805); (6) Chimie Analytique Bio Inorganique et Environnement (IPREM UMR 5254);

Sulfate reducing prokaryotes (SRP) are involved in the mercury biogeochemical cycle through mercury methylation and demethylation. Most of the activity of the SRP takes place in sediments where methylmercury is stored and may be a further source of contamination for food webs. Recent studies showed that SRP colonize aquatic plants roots biofilms in the water column. Since these biofilms accumulate metals through adsorption on inorganic and organic particulate material, the activity of SRP in such matrix can be a contamination source for food webs (suspension feeders or grazers). Indeed, many studies, conducted in tropical environments, showed high mercury methylation and demethylation potentials in such water-roots interfaces. However, plant roots-SRP associations and their impact on mercury bioaccumulation are poorly documented in temperate ecosystems.

In south western France, several shallow lakes and coastal rivers are colonized by invasive aquatic macrophytes (Ludwigia peploides, Lagarosiphon major, Myriophyllum aquaticum), which cause damage to aquatic ecosystems and may interact with mercury biogeochemistry and bioaccumulation. Preliminary results already indicated that mercury concentration in some fishes is close to the European toxicity norm (2.5 mg.kg-1 dw). In the present work, SRP occurrence and diversity as well as mercury methylation and demethylation potentials are investigated in the sediments, the roots biofilms and the water compartments from six different freshwater to brackish ecosystems. SRP occurrence and diversity are first assessed by T-RFLP based on the dsrAB genes. The use of a nested-PCR technique permitted the detection of SRP in all samples, including the water bodies and plant roots. Incubations, realized with stable isotopes of mercury (199Hg2+, CH3201Hg+) demonstrate mercury methylation and demethylation activities in sediments and plant roots whereas they are undetectable in the water column. In order to link mercury speciation and bacterial communities, SRP diversity is assessed by both molecular (cloning-sequencing approaches) and high-through output cultivation methods in the plant-root biofilms in three contrasting sites. SRP representatives are isolated and tested for their mercury transformation potentials (i.e. methylation and demethylation). The comparison between field data and laboratory strain incubations allows a better understanding on the mechanisms of mercury biogeochemical cycle in such aquatic ecosystems. The SRP communities, their influence on mercury speciation and thus the potential contribution of the plant roots ecological niches on methylmercury net production in the water column can be clearly evidenced in this work.

TS10-P8 — 11:00-12:00 and 17:30-18:30
IS THERE A NOVEL MERCURY RESISTANCE MECHANISM AMONG CHEMOSYNTHETIC BACTERIA FROM DEEP SEA HYDROTHERMAL VENTS?
Authors: CRUZ, Kimberly1, CRESPO-MEDINA, Melitza2, BORIN, Sara3, CRUZ, Ramaydalis4, VETRIANI, Costantino5, BARKAY, Tamar6
(1) Joint Graduate Program in Toxicology, Rutgers University & University of Medicine and Dentistry of New Jersey , kimcruz@eden.rutgers.edu; (2) Department of Marine Sciences, University of Georgia; (3) Department of Agriculture Microbiology, Milan University; (4) Graduate Program in Environmental Science, Rutgers University; (5) Institute of Marine and Coastal Sciences, Rutgers University ; (6) Department of Biochemistry and Microbiology, Rutgers University.

Background:
Hydrothermal vents are ecosystems where mercury (Hg) from geothermal sources reach ng/L concentrations suggesting that life in the vent environment must be adapted to Hg toxicity. The Hg tolerance of chemosynthetic microorganisms, those that use inorganic energy and carbon sources are the primary producers in the dark environment of deep-sea vents, is central to life. While heterotrophic bacteria from the vents utilize Hg resistance (mer) operon to detoxify Hg by converting it to the elemental volatile form, little is known about how chemosynthetic microbes tolerate Hg. We therefore, studied the response of chemosynthetic bacteria from diffuse flow vents on the EPR 9°N.

Methods:
Chemosynthetic bacteria were enriched in mineral medium containing 20 mM thiosulfate and 10 µM HgCl2. 16S rRNA genes representing the isolates were used to create a phylogenetic tree. MINEQL+ modeling was used to simulate Hg(II) speciation in the growth media and mer-lux bioreporter assays determined how speciation affected Hg(II) bioavailability. Resistance to Hg(II) was determined by following microbial growth. Mercury reduction by isolates was determined using the x-ray Hg volatilization assays or by following the formation of Hg(0) from incubations of cultures growing with Hg(II).

Results and Conclusion:
Based on 16S rRNA gene sequences, chemosynthetic Hg resistant bacteria were closely related to the Gammaproteobacteria Thiomicrospira crunogena, Thiomicrospira thermophila, Halothiobacillus hydrothermalis, Hydrogenovibrio marinus, and to the Alphaproteobacteria Thioclava pacifica, and Pelagibaca bermudensis. In the growth medium, Hg was present as negatively charged complexes with thiosulfate and this complexation decreased Hg bioavailability by 55%, relative to neutral HgCl2. Strains H hydrothermalis and T.thermophila could tolerate as high as 10 µM Hg(II) and the addition of acetate as a carbon source to the medium decreased the Hg tolerance of H. hydrothermalis and increased it in T.thermophila. Homologs of mer genes could not be detected in the genomes of any of our chemosynthestic deep sea isolates and volatilization assays failed to detect production of Hg(0) when H.hydrothermalis and T.thermophila were exposed to Hg(II). Deep-sea heterotrophic bacteria that were used as controls reduced Hg(II) and had mer gene homologs in their genomes. Thus, chemosynthetic bacteria from deep-sea hydrothermal vents are highly tolerant to Hg by a mechanism that does not involve a mer-mediated reduction to Hg(0); possibly novel mechanism of Hg resistance are employed by these microbes in the unique environment of deep-sea vents.

TS10-P9 — 11:00-12:00 and 17:30-18:30
THE IMPORTANCE OF BIOLOGICAL DEMETHYLATION IN THE MERCURY CYCLING OF A COASTAL POND
Authors: MUNSON, Kathleen M1, LAMBORG, Carl H.2, MINCER, Tracy J.2, BOTHNER, Michael H. 3, HARKNESS, Jennifer S. 4
(1) MIT/WHOI Joint Program in Chemical Oceanography, kmunson@mit.edu; (2) Woods Hole Oceanographic Institution; (3) US Geological Society; (4) Vassar College.

We are combining measurements of potential rates of mercury species transformations with a functional gene expression screen to determine the extent of biologically mediated mercury demethylation and reduction in a Cape Cod pond. Oyster Pond is a seasonally stratified pond that exchanges limited saltwater with Vineyard Sound and has significant freshwater inputs. As a result, a sharp oxycline separates low salinity, oxic upper waters from higher salinity, anoxic bottom waters. Water column concentrations of methylated mercury species are highest in the redox transition zone. We have measured potential mercury methylation, demethylation, and reduction rates from the bacterial communities in and adjacent to the redox transition zone of the pond. Potential transformation rates are measured by determining the incorporation of isotopically labeled mercury into the elemental, inorganic, and methylated mercury pools over time from isotope ratios determined by inductively coupled plasma mass spectrometry. In order to identify possible mechanisms responsible for observed mercury transformations, we collected DNA from corresponding depths in the Oyster Pond water column. We are creating fosmid libraries from this DNA for expression in E. coli hosts. These hosts, which can be grown in a variety of redox conditions, are subjected to varying concentrations of inorganic and methylated mercury species in order to screen for mercury resistance. Genes that convey resistance are sequenced and analyzed for similarity to characterized mercury resistance genes, such as merA and merB. Novel sequences can also be identified using this method. The pairing of the functional gene expression screen with measurements of mercury species transformations provides us with insight into the extent of bacterially driven mercury cycling as well as the identity of mechanisms responsible for mercury demethylation in this coastal environment.

TS10-P10 — 11:00-12:00 and 17:30-18:30
THE DYNAMIC NATURE OF MERCURIC ION REDUCTASE MERA
Authors: JOHS, Alexander1, PARKS, Jerry M.1, HARWOOD, Ian M.2, SMITH, Jeremy C.1, MILLER, Susan M.2, LIANG, Liyuan1
(1)Oak Ridge National Laboratory, johsa@ornl.gov; (2) University of California, San Francisco;

Mercury (Hg) is a ubiquitous contaminant in the environment of particular interest as its conversion to methyl mercury leads to bioaccumulation and toxicity in higher organisms. Some bacteria have naturally evolved elaborate resistance mechanisms to deal with heavy metal toxicity. Bacterial mercury resistance is mediated by the mer operon, a set of genes encoding proteins that specifically facilitate cellular uptake of Hg(II) species, cleavage of organomercurials to hydrocarbons and Hg(II), and the key step in the resistance pathway, reduction of Hg(II) to Hg(0). With its intracellular location, the enzyme catalyzing the reduction, mercuric ion reductase (MerA), not only needs to be an efficient catalyst but also must acquire Hg(II) efficiently from other cellular and pathway proteins. All MerA proteins have a conserved homodimeric catalytic core (~100 kDa), homologous with the NADPH-dependent flavin disulfide oxidoreductases, and many have an N-terminal metallochaperone-like domain, NmerA, which acquires Hg(II) and transfers it using pairs of cysteine residues to an active site in the core homodimer for reduction. Here we have applied small angle neutron and X-ray scattering and molecular dynamics simulations to explore the structure and dynamics of full length MerA and to identify the docking site of NmerA with the core. We have characterized two functionally relevant states of MerA: (1) MerA in the absence of Hg(II) and (2) a multiple Cys to Ala mutant that traps the transient intermediate that would occur during handover of Hg(II) between NmerA and the catalytic core. Our data show first that the two N-terminal domains in dimeric MerA can sample a large number of conformations, consistent with the hypothesis that NmerA serves as a shuttle removing Hg(II) from distant donors in the cytoplasm for delivery to the core. In addition, the results identify the site of interaction between NmerA and the catalytic core during the transient Hg(II) handoff, providing insight into structural features that lead to efficient transfer. The high specificity of NmerA for mercuric mercury facilitates efficient acquisition and directed transport of Hg(II) to the catalytic core of MerA, which actively reduces Hg(II) to less harmful Hg(0).

TS10-P11 — 11:00-12:00 and 17:30-18:30
MOLECULAR DYNAMICS SIMULATIONS OF OPERATOR DNA DISTORTIONS MADE BY TRANSCRIPTIONAL REGULATOR MERR
Authors: GUO, Hao-Bo1, JOHS, Alexander1, PARKS, Jerry M.1, SUMMERS, Anne O.2, LIANG, Liyuan1, SMITH, Jeremy C.1
(1) Oak Ridge National Laboratory, USA , guoh1@ornl.gov; (2) The University of Georgia, Athens ;

Many bacteria possess a mercury resistance (mer) operon that encodes enzymes and transport proteins that effect protonolysis of organomercurials and reduction of Hg(II) to volatile, elemental Hg(0). Transcription of the mer operon is regulated by the Hg(II)-specific regulatory protein MerR. The MerR homodimer binds a palindromic DNA sequence (the MerO operator) that lies within the overlong 19-bp spacer between the -35 and 10 RNA polymerase binding sites of the mer promoter. It is well known that metal-free-MerR bends operator DNA ~40? laterally and, upon binding Hg(II), it also underwinds the DNA axially ~30?, correcting the -10 and -35 sites to the orientation seen in promoters with a typical 17-bp spacer. In vivo and in vitro DNA footprinting have shown that several bases in the central-non-dyadic 4-bp "hyphen" (positions -23 to -26) become distorted upon Hg(II)-activation of MerR. Although there is no 3D structure of MerR yet, we recently used molecular dynamics (MD) to simulate the behavior of a MerR-Hg model based on its small-angle X-ray scattering (SAXS) and crystallographic structures of other MerR family members. In this current work we expanded that model to an activated MerR-DNA complex. Since all 3D MerR family regulator-operator complexes are in the activated form, the initial state of our 23-bp model DNA operator is already bent and underwound and remains so after a 2 ns equilibration. Moreover, two bases in the activated dyad center remain unpaired over a 60 ns trajectory. In contrast, in a 60 ns simulation of the 23-bp operator alone, the activated conformation instantly collapses into the general form of B-DNA with the dyad center bases forming hydrogen bonds. These and other atomic level details arising from the MD simulations of interactions between MerR and its operator DNA were in good agreement with in vivo genetic analysis and protein-DNA footprinting data. Thus, MD will be useful to dissect the energetics and information transduction of the unique activation mode of the widely found MerR family of transcriptional regulators.

TS10-P12 — 11:00-12:00 and 17:30-18:30
ISOLATION AND CHARACTERIZATION OF BACTERIA RESISTANT TO MERCURY COMPOUNDS TO EVALUATE METHYLATION PROCESSES IN CONTAMINATED AREAS – THE PARTICULAR CASE OF TAGUS ESTUARY
Authors: FIGUEIREDO, Neusa1, SERRALHEIRO, Maria Luísa2, LINO, Ana Rosa3, CARVALHO, Cristina4
(1) Faculty of Farmacy, University of Lisbon; (2) Faculty of Sciences, University of Lisbon; (3) Faculty of Sciences; University of Lisbon; (4) Faculty of Farmacy - University of Lisbon.

In Tagus Estuary, one of the largest in Europe, has been showed a high level of contamination by mercury, as a result of a historical industrial discharge. High levels of inorganic mercury (II) (Hg) and monomethylmercury (MeHg) have been reported in surface sediments.

The objective of this study was the characterization of bacteria isolated from the sediments of two hot spot of Tagus Estuary (North Channel and Barreiro) and also Alcochete with lower contamination. To accomplish this aim, the 50 cm core was divided into 16 samples according to the depth of sediments. Anaerobic and aerobic bacteria resistant to MeHg, especially sulfate reducing bacteria (SRB), were isolated and further evaluated for its susceptibility to Hg and MeHg by the determination of minimal inhibitory concentration (MIC).

The results show the distribution of different type of resistant bacteria along the depth, with a particular emphasis to the presence of resistant SRB on the superficial area of the two hot spot areas, where the concentration of SO4-2 tend to be higher. The MIC values of MeHg for aerobic bacteria ranged from 0.02 to 1.23µg/ml whilst for Hg the MIC values were from 0.07 to 13.58µg/ml. Aerobic bacteria isolated from the North Channel were the most resistant (0.06-1.23µg/ml for MeHg; 0.68-13.6µg/ml for Hg).

For anaerobic bacteria the MIC values ranged from 0.04 to 62.80µg/ml for MeHg and 0.42 to 135.8µg/ml for Hg. Anaerobic bacteria from Barreiro presented more resistance (0.78-62.80µg/ml for MeHg; 6.80-135.8µg/ml for Hg).

Biochemical studies are being performed in order to evaluate the involvement of these bacteria in the cycle of mercury.

TS10-P13 — 11:00-12:00 and 17:30-18:30
ARE CERTAIN PERIPHYTON COMMUNITIES MORE RESPONSIBLE OF METHYLMERCURY PRODUCTION THAN OTHERS? AN HYPOTHESIS TESTED IN TWO FLOODPLAIN LAKES IN THE PANTANAL OF MATO GROSSO, BRAZIL
Authors: LÁZARO, Wilkinson L.1, GUIMARÃES, Jean R.D.2, IGNÁCIO, Áurea R.A.3, DA SILVA, Carolina J.4, DÍEZ, Sergi S.5
(1) Pantanal’s Limnology, Biodiversity and Ethnobiology Research Center (CELBE). Mato Grosso State University, Brazil, wilkinsonlopes@hotmail.com; (2) Tracers Laboratory, IBCCF, Rio de Janeiro Federal University (UFRJ), Rio de Janeiro, Brazil; (3) Neurotoxicology laboratory, Mato Grosso State University, Brazil; (4) Pantanal’s Limnology, Biodiversity and Ethnobiology Research Center (CELBE-UNEMAT), Mato Grosso State University, Brazil; (5) Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Barcelona, Spain.

The toxic potential of Hg in aquatic systems is due to the presence and production of methylmercury (MeHg). Recent studies on MeHg production in tropical floodplain environments show that the roots zone of floating macrophytes are able to produce more MeHg than the water column and sediments of the open water zone of lakes and rivers. Indeed, the greater capacity for synthesis of MeHg by the roots of aquatic macrophytes is attributed to the periphyton associated with them. The communities architecture is given from the relationship between the chemical of a primary bacterial carpet and successional stages of a variety of bacterial strains (e.g. sulphate-reducing bacteria), autotrophic microorganisms (algae), and the relationship between the periphytic matrix, the water column and the substrate. We have hypothesized that the algal community structure may explain the potential methylation, given that ecologically distinct communities have different algal and bacterial densities, which can directly affects the formation of MeHg in the macrophytes roots. In this sense, this study aimed to evaluate the production of MeHg in the roots of Eichhornia crassipes (Mart.) Solms in relation to taxonomic structure of the associated periphytic algae assemblage in two Pantanal’s floodplain lakes under different ecological conditions due to their different lateral hydrological connectivity patterns with the Paraguay river. The biological and limnological characteristics showed clear differences between both lakes (i.e. the disconnected lake is more concentrated, showing higher levels of electrical conductivity, higher pH and less dissolved oxygen concentrations in relation to the connected lake). Moreover, the production of MeHg was higher in the disconnected lake and also exhibited a strong positive relationship with the abundance of the class Cyanophyceae and with the total algal biomass, together with a strong negative relationship with the class Zygnemaphyceae in the lake’s community. Apparently, the same ecological characteristics that favor the establishment and development of cyanobacteria (specially some species of the genus Lyngbya) are those that provide higher rates of methylation in aquatic systems. This suggests that cyanobacteria could be active bioindicators of MeHg production in some natural aquatic environments.

TS10-P14 — 11:00-12:00 and 17:30-18:30
DESIGNING A PCR-KIT FOR DETECTION OF POTENTIAL BACTERIA METHYLATING MERCURY
Authors: RANNEKLEV, Sissel Brit1, REFSETH, Unn Hilde1, VEITEBERG BRAATEN, Hans Fredrik1, LARSSEN, Thorjørn1
(1) Norwegian institute for Water Research, SRA@niva.no

The key entry of Hg into the aquatic food web is through MeHg. Anoxic sediments and wetlands are believed to be the parts of the catchment where MeHg is produced, and the main factors controlling Hg methylation is assumed to be: i) sulphate availability, ii) organic matter availability, iii) temperature, iv) bioavailability of mercury, and v) anoxic conditions.

It is generally believed that methylation is principally mediated through sulphate (SRB) and iron reducing bacteria. Today Geobacter sulfurreducens and Desulfovibrio desulfuricans are known bacteria capable of mercury methylation. Unfortunately, the mechanisms and parameters that control the methylation process in aquatic environments are not well understood which may be due to lack of adequate microbial tools. Today most of the studies done on methylation of bacteria are based on conventional growth techniques using plate count and MPN techniques. With the knowledge that about 98% percentages of bacteria in general are non-culturable, it is obvious that the methylation process is not well understood.

In this study a PCR-kit has been designed in order to detect and enumerate species of Geobacter sp., Desulfovibrio sp., and Desulfomicrobium sp. in environmental samples related to Hg methylation. Pairs of primers were designed to amplify particular target regions of the various species. Primer pairs and the particular region to be amplified were selected from 2 to 23 previously known sequences of the various species.

Presences of the potential methylation bacteria were determined in lake sediments from oligotropic and dystrophic lakes. In the dystrophic lake Langtjern, presence of Desulfovibrio sp. was detected. Enumeration of Desulfovibrio sp. under spatial and temporal variation in the sediment will be shown, reflecting the dept, season, and redox potential, and with concurrently analyses of MeHg in the sediments, Hg methylation hotspots can be detected.

Results will be discussed in view of the extensive work that recently has been done on budget estimates of Hg/MeHg in the lake and mercury accumulation in the food chain in the lake and streams in the catchment.

In order to enlarge the numbers of potential methylation bacteria in the PCR-kit, metagenomics focusing on the highly conserved 16S rRNA gene is under progress to be performed on sediments from Lake Langtjern.

TS10-P15 — 11:00-12:00 and 17:30-18:30
BIOGEOCHEMICAL FACTORS INFLUENCING NET MERCURY METHYLATION IN CONTAMINATED FRESHWATER SEDIMENTS FROM THE ST. LAWRENCE RIVER IN CORNWALL, ONTARIO, CANADA
Authors: AVRAMESCU, Mary-Luyza1, YUMIHOZE, Emmanuel1, HINTELMANN, Holger2, RIDAL, Jeff3, FORTIN, Danielle1, LEAN, David1
(1) University of Ottawa; (2) Trent University; (3) St. Lawrence River Institute;

The activity of various anaerobic microbes, including sulfate-reducers (SRB), iron-reducers (FeRP) and methanogens (MPA) has been linked to mercury methylation in aquatic systems, although the relative importance of each microbial group in the overall process is poorly understood in natural sediments. The present study focused on the biogeochemical factors (i.e. the relative importance of various groups of anaerobic microbes (FeRP, SRB, MPA) that affect net monomethylmercury (MMHg) formation in contaminated sediments of the St. Lawrence River (SRL) near Cornwall (Zone 1), Ontario, Canada. Methylation and demethylation potentials were measured separately by using isotope-enriched mercury species (200Hg2+ and MM199Hg+) in sediment microcosms treated with specific microbial inhibitors. Sediments were sampled and incubated in the dark at room temperature in an anaerobic chamber for 96h. The potential methylation rate constants (Km) and demethylation rates (Kd) were found to differ significantly between microcosms. The MPA-inhibited microcosm had the highest potential methylation rate constant (0.016 d-1), whereas the two SRB-inhibited microcosms had comparable potential methylation rate constants (0.003 d-1 and 0.002 d-1, respectively). The inhibition of methanogens stimulated net methylation by inhibiting demethylation and by stimulating methylation along with SRB activity. The inhibition of both methanogens and SRB was found to enhance the iron reduction rates but did not completely stop MMHg production. The strong positive correlation between Km and Sulfate Reduction Rates (SRR) and between Kd and Methane Production Rates (MPR) supports the involvement of SRB in Hg methylation and MPA in MMHg demethylation in the sediments. In contrast, the strong negative correlation between Kd and Iron Reduction Rates (FeRR) shows that the increase in FeRR correspond to a decrease in demethylation, indicating that iron reduction may influence net methylation in the SLR sediments by decreasing demethylation rather than favouring methylation.

TS10-P16 — 11:00-12:00 and 17:30-18:30
METHYLMERCURY AND MERCURY METHYLATION IN PERIPHYTON FROM THE TAPAJOS RIVER (BRAZILIAN AMAZON)
Authors: MIRANDA, Marcio R.1, GUIMARAES, Jean Remy D.2, COELHO-SOUZA, Sergio A.1, POIRIER, Hugo3, LUCOTTE, Marc3, MERGLER, Donna3
(1) Inpetam, topo@biof.ufrj.br ; (2) IBCCF/Universidade Federal do Rio de Janeiro; (3) UQAM;

Periphyton associated to the roots of floating aquatic macrophytes is a major link for mercury methylation in tropical ecosystems. Mercury is methylated mainly by sulfate-reducing, and previous studies found a diverse SRB community associated to the roots of floating macrophytes and high methylation potentials. Methylmercury concentration in the environment is a result of methylmercury production/degradation as well as methylmercury importation/exportation. The aim of this work was to evaluate the concentration of MeHg in periphyton, as well as its relationship with the Me203Hg production. The study area was the lower Tapajós River, a major tributary of the Amazon, where Paspalum repens was the dominant floating macrophyte. Sampling was done at the beginning of the descending waters season. We used radiotracer techniques to measure the net mercury methylation potentials (24 h incubation with 203Hg) and CV-AFS to evaluate methylmercury concentration on macrophyte root-associated periphyton. Net Me203Hg formation in P. repens periphyton, expressed as % of total added 203Hg, was high but varied considerably among sites varying from 2.2 to 44 %MeHg/g dw/24h. Methylmercury concentrations in periphyton of the aquatic macrophyte Paspalum repens sampled in Tapajós river tributaries ranged from 0.9 to 5.22 ng/g dw, and presented a significant correlation (R2= 0.7788; p=0.0199; n=6) with Me203Hg production. Methylmercury produced in the root-associated periphyton may be regarded as highly bioavailable and this complex matrix should receive more attention in the context of Hg biogeochemistry studies.

TS10-P17 — 11:00-12:00 and 17:30-18:30
TWO PATHWAYS FOR THE ENHANCEMENT OF MERCURY METHYLATION BY SYNTROPHIC INTERACTIONS
Authors: YU, Ri-Qing1, HINES, Mark E.2
(1)Rutgers University, rqyu@eden.rutgers.edu; (2) UMass Lowell.

In sulfate limited freshwater ecosystems, syntrophic associations may stimulate growth of SRB and thus microbial Hg methylation. This possibility was supported by the detection of 16S rRNA and dsr genes that were most similar to those of Syntrophobacter spp. in Sphagnum moss enrichments with stimulated Hg methylation. We tested the role of syntrophy in Hg methylation by setting up incubations of propionate-utilizing S. wolinii, S. sulfatireducens, and S. fumaroxidans with Methanospirillum hungatei or with Hg methylating SRB, strains Desulfovibrio desulfuricans ND132 and Desulfovibrio africanus DSM 2603 which cannot grow with propionate. S. wolinii was the only syntroph with a low rate of Hg methylation activity (0.29%/day) when grown with propionate (15.6 mM) and sulfate (19.7 mM). This activity was also demonstrated when S. wolinii grew in a co-culture with M. hungatei (0.20 %/day) in the absence of sulfate. When strains ND132 and DSM 2603 were grown with M. hungatii in a sulfate-free lactate medium, methylation was stimulated by 2 to 4 fold and 7 to 24 fold, respectively, as compared to their activity without the methanogen. Thus, the syntrophy of M. hungatei with a weak Hg methylator (D. africanus DSM 2603) more significantly enhanced methylation as compared to syntrophy with a strong methylator (ND132). Surprisingly, significantly higher potential methylation rates were observed after four days of incubation in co-cultures of S. sulfatireducens with strains ND132 (5.1 %/day) and DSM 2603 (0.4 %/day) in a propionate (15.6 mM) medium with 3.94 mM sulfate in comparison with rates of the Desulfovibrio spp. growing alone (4.3 and 0.2 %/day, respectively). Cell counts of the methylating strains in co-cultures with the syntroph, as determined by flow cytometry cell sorting, increased and sulfate concentrations decreased while biomass and sulfate concentrations of the methylating strains growing alone remained unchanged. The results suggest that S. sulfatireducens by oxidizing propionate provided hydrogen and a carbon source to support growth of methylating Desulfovibrio spp. With increasing sulfate in propionate/sulfate media from 0.39 to 19.71 mM, methyaltion by cocultures of S. sulfatireducens with strain ND132 decreased and with strain DSM 2603 increased, while for both methylation was enhanced in the co-cultures as compared with methylation by the SRB alone. Thus, syntrophy may enhance methylation by SRB growing with methanogens in sulfate-limited environments and with Syntrophobacter spp. in environments where sources of energy and carbon are limiting.

TS10-P18 — 11:00-12:00 and 17:30-18:30
GENETIC APPROACHES IN MERCURY METHYLATION ENZYMOLOGY FOR DESULFOVIBRIO DESULFURICANS ND132
Authors: KUCKEN, Amy M1, SMITH, Steven D1, BROWN, Steven D2, ELIAS, Dwayne A2, WALL, Judy D1
(1) University of Missouri, kuckena@missouri.edu; (2) Oak Ridge National Laboratory;

Methylmercury (MeHg+) is a potent neurotoxin which readily bioaccumulates in the food chain and poses significant risks to top predators in mercury contaminated regions. MeHg+ is primarily produced by anaerobic dissimilatory sulfate-reducing (DSRB) and dissimilatory Fe(III)-reducing bacteria, although interestingly only a subset of species within these groups are capable of mercury-methylation. The DSRB Desulfovibrio desulfuricans ND132 is a known methylator of mercury and is one of only a few strains of mercury-methylating DSRB to have its genome sequenced. The enzymology of mercury methylation remains elusive; hence, we propose to identify the genes and enzymes in ND132 necessary for the methylation activity. We have established genetic manipulation protocols in ND132 to target the deletion of candidate methylation genes and have initiated construction of a random transposon mutant library through conjugation. Thus far ca. 3000 mutants have been isolated and arrayed in a library that will include 10,000 mutants to ensure broad genome coverage. A high-throughput MeHg+ screening assay was developed and optimized to detect mutants altered in methylating capability and over 800 mutants have been screened to date. Additionally, biochemical assays and purification techniques are being applied to identify mercury methylation enzymes from cell extracts of ND132. Determining which genes or pathways are involved in MeHg+ production in ND132 may facilitate the development of new strategies to minimize or eliminate the production of MeHg+ in mercury contaminated environments.

Tuesday, 26 July, 2011