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S10 (I) Mechanisms of microbial mercury methylation

Wednesday, 27 July, 2011

WS10-O1 — 8:30-9:00
MICROBIAL MERCURY METHYLATION: HISTORICAL PERSPECTIVES, CURRENT UNDERSTANDINGS, AND FUTURE DIRECTIONS
Author: BARKAY, Tamar1
(1) Rutgers University, barkay@aesop.rutgers.edu

Mercury methylation by anaerobic microorganisms was discovered in the late 1960’s shortly after the severity of methylmercury toxicosis was realized following the Minamata Bay incidence. Initial research efforts were focused on the role of methanogens in methylation due to the importance of transmethylation of negatively charged methyl groups in their metabolism; these studies have defined a role for methyl cobalamine (vitamin B12) as the methyl donor in methylation. It was not until intact environmental samples were examined that the surprising role of sulfate reducing bacteria (SRB) in methylation was discovered and confirmed with pure cultures and field investigations. A detailed mechanistic study demonstrated that in Desulfovibrio desulfuricans methylation occurred when a methyl group was enzymatically transferred to Hg2+ rather than to CoA-SH in the acetylcoenzyme (CoA) pathway. An elegant series of studies that combined chemical speciation modeling with pure cultures and field observations identified neutral and aqueous soluble complexes of Hg with sulfides as the form that is available to methylating SRB by passive diffusion through their cell envelope.

However, findings reported in the last decade showed that (i) iron reducing bacteria that are affiliated with the Deltaproteobacteria may methylate Hg, (ii) among some SRB, methylation may not involve methyl B12 or the CoA pathway, (iii) there is little relationship between the phylogeny of SRB and their ability to methylate Hg, and (iv) active transport of thiolated Hg(II) is involved in methylation. These observations have resulted in an appreciation of how complex Hg methylation is; however, a clear picture on the mechanism of methylation is yet to emerge. Future studies based on these recent findings will be made even more exciting by the availability of sequenced genomes of several Hg methylating bacteria and the promise these hold for directed genetic and biochemical studies on methylation. In addition, the diversity of methylating microbes may be expanding with the discovery of water column (aerobic?) methylation in the marine environment and demonstrations of methylation by methanogens and by syntrophic associations. Thus, new directions are opening that will enhance the depth and breadth of our understanding of microbial Hg methylation.

WS10-O3 — 9:00-9:15
MICROBIAL METHYLMERCURY PRODUCTION IN ECOSYSTEMS: NEW INSIGHTS ON ECOPHYSIOLOGICAL, CELLULAR AND GENETIC ASPECTS
Author: GUYONEAUD, Rémy1
(1)Environmental Microbiology (EEM), UMR IPREM 5254, University Pau, BP1155, 64013 Pau, France , remy.guyoneaud@univ-pau.fr

Microbial activities play a major role in mercury biogeochemical cycle. Hg methylation, demethylation and reduction control the methylmercury concentrations in the aquatic environments and microorganisms are implicated in all of these processes. The pathways, genes and regulation processes are well described for some of these activities, i.e., mercury reductive demethylation and mercury reduction. It is not the case for mercury methylation even if many environmental data are available. These data, together with physiological characterizations led to exemplify the role of sulfate-reducers, and iron-reducers, in methylmercury production. Due to the ecological niches of these microorganisms, these data tended to restrict biological mercury methylation to anoxic environments (sediments, hypoliminions), considered as main methylmercury sources. This paradigm suffers some gaps. Recent results demonstrated that sulfate-reducers could inhabit (and be active) in oxic water bodies where they can produce methylmercury. Besides, some unexpected organisms, such as some Citrobacter species were identified are potential mercury methylators. In addition, it has been demonstrated that many organisms that methylate mercury may also participate in mercury demethylation without involving the “classic” reductive demethylation pathway. To follow such simultaneous and reversible reactions, the use of multiple stable isotopic tracers is very useful. It allows not only to determine mercury methylation and demethylation potentials in environmental samples, co-cultures and pure strains but also to localize mercury species in sub-cellular fractions. All these data modify our view on the global mercury biogeochemical cycle and thus on the potential methylmercury sources.

Despite our knowledge on the organisms responsible for methylmercury production, it remains extremely difficult to linked microbial communities composition at the species/genus level, or microbial activities in situ with net methylmercury production. We have only a vague idea of the overall environmental parameters and the underlying mechanisms (uptake, transformation rates, regulation) that control the net methylmercury production. Efforts must be placed in determining the environmental factors (biotic and abiotic) enhancing methylmercury production Microbial communities are complex physiological networks in which one organisms may shift from one metabolism to another depending on the environmental conditions. Efforts must also be placed in identifying and characterizing the genetic/enzymatic systems, controlling mercury methylation. This concerns the enzymes (and their corresponding genes) responsible for mercury methylation but also the genetic regulation behind, in order to gain a better understanding on methylmercury production in environmental samples.

WS10-O4 — 9:15-9:30
INVESTIGATION OF MERCURY METHYLATION PATHWAYS IN BIOFILM VERSUS PLANKTONIC CULTURES OF DESULFOVIBRIO DESULFURICANS
Authors: LIN, Chu-Ching1, KAMPALATH, Rita 2, ZHANG, Ming3, JAY, Jennifer A.2
(1) National Central University Taiwan, chuching@gmail.com; (2) UCLA; (3) East China Normal University;

Almost all pure culture mercury methylation work to date has been conducted with pure cultures; however, the majority of organisms in subsurface environments are living in biofilms. Our previous study showed that methylation rates in biofilm cultures were up to an order of magnitude greater than those in planktonic cultures of the same strain of sulfate-reducing bacteria. The objective of this study is to probe whether the differential methylation rates resulted from metabolic differences between these two cultures. Mercury methylation assays following molybdate or chloroform inhibition (a specific inhibitor of the acetyl-CoA pathway) were conducted on biofiom and planktonic cultures of Desulfovibrio desulfuricans strains M8 and ND132. Molybdate was as effective in inhibiting Hg methylation in both planktonic and biofilm cultures as in inhibiting growth. Unlike planktonic cultures, addition of chloroform only impacted mercury methylation in biofilm cultures. We screened for the presence of cooS, a gene used in the acetyl-CoA pathway, in several strains of sulfate reducing bacteria. RT-qPCR was used to quantify amplication of this gene. Although D. desulfuricans did not methylate mercury via the acetyl-CoA pathway in planktonic growth, this pathway appeared to be employed in mercury methylation in biofilm cultures.

WS10-O5 — 9:30-9:45
STUDY OF THE TRANSFORMATION RATES AND LOCALIZATION OF HG SPECIES AT SUB-CELLULAR LEVEL IN ANAEROBIC BACTERIA
Authors: PEDRERO ZAYAS, Zoyne 1, BRIDOU, Romain2, PERROT, Vincent1, MOUNICOU, Sandra1, GUYONEAUD, Remy2, MONPERRUS, Mathilde1, AMOUROUX, David1
(1) LCABIE CNRS Pau, zoyne.pedrerozayas@univ-pau.fr; (2) Equipe Environnement et Microbiologie, CNRS UMR 5254 IPREM, Université de Pau et des Pays de l’Adour, Pau, France;

Bacterial methylation is one of the major processes which transform inorganic mercury into toxic monomethylmercury. The methylation potential of some bacteria, as well as their MeHg assimilation potential could be directive to evaluate their impact on the ecosystem. Despite the understanding of the microbial of Hg methylation in aquatic ecosystems and the characterization of the involved bacteria, its mechanism at cellular scale is still poorly understood. The advantages of the use of stable isotopically labeled Hg species are exploited in this work to evaluate the extent and the localization of such processes.

Two pure bacteria strains, Desulfovibrio desulfuricans (G200) and Desulfovibrio sp. BerOc1 (EU137840), were incubated in presence of isotopically labelled mercury species (199IHg and 201MeHg) under controlled anoxic conditions. The first one is used as a non methylating biotic control, while BerOc1 is well recognised for its methylating potential. A kinetic experiment was also carried out to follow the distribution of added and formed species during 24h of incubation.

Hg speciation analyses in each sub-cellular fraction (extracellular, membranes, cytoplasm) were carried out by GC-ICP-MS, and quantification was performed by reverse species-specific isotope dilution analysis. The use of isotopically enriched Hg species 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 is observed. In general terms, MeHg added and formed show a similar pattern, being mainly present in the extracellular fraction. In contrast, IHg added to the culture is associated to the cells but when resulting from demethylation processes it is found in the extracellular fraction. The analysis of the cytosol and the extracellular fraction by size exclusion chromatography-ICP-MS revealed divergences on the profile of biomolecules binding mercury corresponding to the control and the methylating strain. The results confirm the use of isotopic tracers as a unique tool to investigate the relative rate of the cell uptake and further metabolic transformation, contributing to the elucidation of such metabolic mechanisms.

WS10-O6 — 9:45-10:00
THE INFLUENCE OF KINETICALLY-LIMITED MERCURY SULFIDE PRECIPITATION ON MERCURY BIOAVAILABILITY FOR METHYLATION BY PURE CULTURES OF SULFATE REDUCING BACTERIA
Authors: ZHANG, Tong1, HSU-KIM, Heileen1, DESHUSSES, Marc A.1
(1) DUKE UNIVERSITY, tong.zhang@duke.edu

In natural aquatic systems, the predominant mechanism of monomethylmercury (MeHg) production is microbial methylation of inorganic mercury (Hg) by sulfate-reducing bacteria (SRB). Integration of field and lab observations suggest that MeHg production rates are largely controlled by the availability of Hg species for uptake into SRB, in addition to microbial activity. The objective of our research is to identify the chemical forms of Hg that are bioavailable to methylating bacteria and susceptible to methylation. In sediments where MeHg production occurs, sulfide controls Hg speciation due to its high abundance and strong affinity for Hg. Previous studies have demonstrated that natural organic matter (NOM) interferes with the precipitation of HgS(s) by preventing aggregation of HgS nanoparticles that are formed during the initial stages of precipitation. As a result of this kinetically-limited reaction, HgS nanoparticles are expected to persist and exhibit higher methylation potential than larger, more crystalline metacinnabar particles due to the large specific surface areas of nanoparticles that enable greater contact with methylating bacteria. Additionally, recent field studies showed that ‘newly’ deposited Hg appeared to be more readily transformed to MeHg than the ‘older’ Hg pool that existed in sediments, which implies a previously unrecognized role of the ‘aging’ effects in Hg methylation. Therefore, we hypothesize that bioavailable Hg concentration is related to the kinetics of HgS precipitation, and not necessarily to equilibrium speciation of Hg in sulfidic porewater. We conducted methylation bioassays using pure cultures of two methylating SRB, Desulfobulbus propionicus (1pr3) and Desulfovibrio desulfuricans (ND132). The bacterial cultures were exposed to three different forms of Hg-sulfides, including dissolved Hg(NO3)2 and Na2S, humic-coated HgS nanoparticles (<30 nm), and metacinnabar particles (>1000 nm), which were formulated to represent three different ‘aging’ states of mercury in sulfidic sediments. Our results revealed that methylation rates were greatest with the dissolved Hg-sulfide treatment. In the treatments with HgS nanoparticles, Hg methylation was observed at a rate that was significantly faster than the micro-scale metacinnabar treatment, possibly due to higher solubility and more surface contact with the bacterial cells. During methylation, Hg appeared to partition onto bacterial cells or large particles; however, ‘intracelluar Hg’ (dissolved Hg released from cell pellets after freeze-thaw cycles and sonication) was minimal. Investigations on how nanoparticles deliver mercury to bacterial cells and become more available than bulk metacinnabar will also be presented and discussed.

WS10-O7 — 10:00-10:15
INVESTIGATIONS ON THE ROLE OF MAJOR CATIONS ON THE UPTAKE OF INORGANIC HG SPECIES (HG2+ AND HG0) BY A GRAM-NEGATIVE BACTERIUM
Authors: DAGUENÉ, Valérie1, YUMVIHOZE, Emmanuel1, AMYOT, Marc2, POULAIN, Alexandre1
(1) University of Ottawa, valerie.daguene@gmail.com; (2) Université de Montréal;

The pathways leading to the intracellular accumulation of mercury remain poorly understood. Detailed knowledge of these pathways is important for two reasons: i) to gain insights into the availability of Hg to bacteria responsible for methylation (gram-negative sulphate reducers), a process thought to occur in the cytoplasm and ii) to better predict the relative availability of inorganic vs. organic mercury species to improve models of bioaccumulation and bioamplification trends within foodwebs.

Our work focuses on the availability of inorganic mercury species to bacteria. Both passive diffusion and facilitated transport have been suggested as ways for inorganic Hg complexes to cross bacterial membranes. In this context, it is critical to consider the chemistry of inorganic mercury in solution. It is also critical, although often overlooked, to take into consideration how biological membranes are affected by their chemical environment and therefore ultimately control Hg uptake.

By carefully manipulating the chemistry in solution and the speciation of mercury, we investigated the role of major cations (i.e. Ca2+, Mg2+, Sr2+, Na+, and K+) on Hg uptake. Our model bacterium was E. coli transformed with a plasmid-born mer-lux fusion construct. This bacterial bioreporter, emits light proportionally to intracellular Hg levels. All species were provided at environmentally relevant levels (i.e. nM to mM for major ions and pM for Hg).

Preliminary results suggest that increasing concentrations of divalent cations significantly alter mercury uptake. We did not observe a similar effect in the presence of similar levels of monovalent cations. The use of various counter anions (e.g., Cl-, SO42-), the use of specific inhibitors of Ca2+ transporters and the use of Hg0, for its lipophilic properties, instead of Hg2+, suggest that the observed effect is not associated with the presence of a specific mercury species but rather with a discriminating effect of the cell membrane. Our results suggest i) the need to take into consideration variables that affect cell membranes to better model Hg accumulation by biota and ii) point out to the role of major divalent cations as potentially controlling Hg uptake. Therefore, processes that control the mobility of such cations in the environment may also indirectly control mercury methylation.

WS10-O8 — 10:15-10:30
A MULTIPRONGED APPROACH RANGING FROM PURE CULTURES TO MICROBIAL COMMUNITIES TO DETERMINE THE MECHANISM OF MERCURY METHYLATION.
Authors: ELIAS, Dwayne1, MOSHER, Jennifer1, VISHNIVETSKAYA, Tatiana1, BROWN, Steven1, BRANDT, Craig1, BROOKS, Scott1, PODAR, Mircea1, GILMOUR, Cynthia2, KUCKEN, Amy3, WALL, Judy3, PALUMBO, Anthony1
(1) Oak Ridge National Laboratory, eliasda@ornl.gov; (2) Smithsonian Environmental Research Center; (3) University of Missouri;

In microbial transformation and genetic studies, we have undertaken ecological and pure culture investigations to discern the organisms and genes responsible for methylmercury (MeHg) generation. Physiological characterizations are being coordinated with Hg methylation/demethylation in Desulfovibrio africanus and Desulfobulbus propionicus. Using D. desulfuricans ND132, we sequenced the genome and are constructing a transposon library to identify the methylating gene(s). In ecological studies, MeHg generating microbial communities were examined using a functional gene array (FGA) and phylogenetically via 454 amplification and sequencing of the V4 region of 16S genes. Analysis of 59 samples revealed pronounced phylogenetic and functional differences that appear to be related to seasonal trends. Geochemical principal component analysis showed that one area was substantially different due to the presence of U(VI) and nitrate, and this was reflected in the microbial community. Virtually all of the microbial communities in the other five sites trended towards dissolved Hg. Further, a correlation of the 454 data with geochemistry at the phylum and genus level showed that some Hg methylating bacteria such as Geobacter spp. do not correlate with Hg or MeHg. However, the Delta- and Epsilon- Proteobacteria as well as Verrucomicrobia all trended towards dissolved Hg, and Desulfobulbus spp. strongly trended towards MeHg. This is significant in that Desulfobulbus proprionicus is a known Hg methylator. Current ecological efforts include enrichment and isolation of methylating bacteria from these sites as well as six metagenomic, metatranscriptomic, and metaproteomic analyses from three background, Hg(II) contaminated, and MeHg generating sites so as to more comprehensively ascertain the genes and gene products that are differentially abundant and expressed in active MeHg generating ecosystems.

Wednesday, 27 July, 2011