Liyuan Liang

The Genetic Basis for Bacterial Mercury Methylation

Mercury Methylating Microbes Mercury (Hg) most commonly becomes bioavailable and enters the food web as the organic form methylmercury, where it induces acute toxicity effects that can be magnified up the food chain. But most natural and anthropogenic Hg exists as inorganic Hg2+ and is only transformed into methylmercury by anaerobic microorganisms—typically sulfur-reducing bacteria. Using comparative genomics, Parks et al. (p. 1332, published online 7 February; see the Perspective by Poulain and Barkay) identified two genes that encode a corrinoid and iron-sulfur proteins in six known Hg-methylating bacteria but were absent in nonmethylating bacteria. In two distantly related model Hg-methylating bacteria, deletion of either gene—or both genes simultaneously—reduced the ability for the bacteria to produce methylmercury but did not impair cellular growth. The presence of this two-gene cluster in several other bacterial and lineages for which genome sequences are available suggests the ability to produce methylmercury may be more broadly distributed in the microbial world than previously recognized. A two-gene cluster encodes proteins required for the production of the neurotoxin methylmercury in bacteria. [Also see Perspective by Poulain and Barkay] Methylmercury is a potent neurotoxin produced in natural environments from inorganic mercury by anaerobic bacteria. However, until now the genes and proteins involved have remained unidentified. Here, we report a two-gene cluster, hgcA and hgcB, required for mercury methylation by Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA. In either bacterium, deletion of hgcA, hgcB, or both genes abolishes mercury methylation. The genes encode a putative corrinoid protein, HgcA, and a 2[4Fe-4S] ferredoxin, HgcB, consistent with roles as a methyl carrier and an electron donor required for corrinoid cofactor reduction, respectively. Among bacteria and archaea with sequenced genomes, gene orthologs are present in confirmed methylators but absent in nonmethylators, suggesting a common mercury methylation pathway in all methylating bacteria and archaea sequenced to date.

Mercury reduction and complexation by natural organic matter in anoxic environments

Mercuric Hg(II) species form complexes with natural dissolved organic matter (DOM) such as humic acid (HA), and this binding is known to affect the chemical and biological transformation and cycling of mercury in aquatic environments. Dissolved elemental mercury, Hg(0), is also widely observed in sediments and water. However, reactions between Hg(0) and DOM have rarely been studied in anoxic environments. Here, under anoxic dark conditions we show strong interactions between reduced HA and Hg(0) through thiolate ligand-induced oxidative complexation with an estimated binding capacity of ~3.5 μmol Hg/g HA and a partitioning coefficient >106 mL/g. We further demonstrate that Hg(II) can be effectively reduced to Hg(0) in the presence of as little as 0.2 mg/L reduced HA, whereas production of Hg(0) is inhibited by complexation as HA concentration increases. This dual role played by DOM in the reduction and complexation of mercury is likely widespread in anoxic sediments and water and can be expected to significantly influence the mercury species transformations and biological uptake that leads to the formation of toxic methylmercury.

Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria

The formation of methylmercury (MeHg), which is biomagnified in aquatic food chains and poses a risk to human health, is effected by some iron- and sulfate-reducing bacteria (FeRB and SRB) in anaerobic environments. However, very little is known regarding the mechanism of uptake of inorganic Hg by these organisms, in part because of the inherent difficulty in measuring the intracellular Hg concentration. By using the FeRB Geobacter sulfurreducens and the SRB Desulfovibrio desulfuricans ND132 as model organisms, we demonstrate that Hg(II) uptake occurs by active transport. We also establish that Hg(II) uptake by G. sulfurreducens is highly dependent on the characteristics of the thiols that bind Hg(II) in the external medium, with some thiols promoting uptake and methylation and others inhibiting both. The Hg(II) uptake system of D. desulfuricans has a higher affinity than that of G. sulfurreducens and promotes Hg methylation in the presence of stronger complexing thiols. We observed a tight coupling between Hg methylation and MeHg export from the cell, suggesting that these two processes may serve to avoid the build up and toxicity of cellular Hg. Our results bring up the question of whether cellular Hg uptake is specific for Hg(II) or accidental, occurring via some essential metal importer. Our data also point at Hg(II) complexation by thiols as an important factor controlling Hg methylation in anaerobic environments.

Mercury Reduction and Oxidation by Reduced Natural Organic Matter in Anoxic Environments

Natural organic matter (NOM)-mediated redox cycling of elemental mercury Hg(0) and mercuric Hg(II) is critically important in affecting inorganic mercury transformation and bioavailability. However, these processes are not well understood, particularly in anoxic water and sediments where NOM can be reduced and toxic methylmercury is formed. We show that under dark anoxic conditions reduced organic matter (NOM(re)) simultaneously reduces and oxidizes Hg via different reaction mechanisms. Reduction of Hg(II) is primarily caused by reduced quinones. However, Hg(0) oxidation is controlled by thiol functional groups via oxidative complexation, which is demonstrated by the oxidation of Hg(0) by low-molecular-weight thiol compounds, glutathione, and mercaptoacetic acid, under reducing conditions. Depending on the NOM source, oxidation state, and NOM:Hg ratio, NOM reduces Hg(II) at initial rates ranging from 0.4 to 5.5 h(-1), which are about 2 to 6 times higher than those observed for photochemical reduction of Hg(II) in open surface waters. However, rapid reduction of Hg(II) by NOM(re) can be offset by oxidation of Hg(0) with an estimated initial rate as high as 5.4 h(-1). This dual role of NOM(re) is expected to strongly influence the availability of reactive Hg and thus to have important implications for microbial uptake and methylation in anoxic environments.

The X-ray crystal structure of Shewanella oneidensis OmcA reveals new insight at the microbe–mineral interface

The X‐ray crystal structure of Shewanella oneidensis OmcA, an extracellular decaheme cytochrome involved in mineral reduction, was solved to a resolution of 2.7 Å. The four OmcA molecules in the asymmetric unit are arranged so the minimum distance between heme 5 on adjacent OmcA monomers is 9 Å, indicative of a transient OmcA dimer capable of intermolecular electron transfer. A previously identified hematite binding motif was identified near heme 10, forming a hydroxylated surface that would bring a heme 10 electron egress site to ∼10 Å of a mineral surface.

Mechanism of Hg-C protonolysis in the organomercurial lyase MerB.

Demethylation is a key reaction in global mercury cycling. The bacterial organomercurial lyase, MerB, catalyzes the demethylation of a wide range of organomercurials via Hg-C protonolysis. Two strictly conserved cysteine residues in the active site are required for catalysis, but the source of the catalytic proton and the detailed reaction mechanism have not been determined. Here, the two major proposed reaction mechanisms of MerB are investigated and compared using hybrid density functional theory calculations. A model of the active site was constructed from an X-ray crystal structure of the Hg(II)-bound MerB product complex. Stationary point structures and energies characterized for the Hg-C protonolysis of methylmercury rule out the direct protonation mechanism in which a cysteine residue delivers the catalytic proton directly to the organic leaving group. Instead, the calculations support a two-step mechanism in which Cys96 or Cys159 first donates a proton to Asp99, enabling coordination of two thiolates with R-Hg(II). At the rate-limiting transition state, Asp99 protonates the nascent carbanion in a trigonal planar, bis thiol-ligated R-Hg(II) species to cleave the Hg-C bond and release the hydrocarbon product. Reactions with two other substrates, vinylmercury and cis-2-butenyl-2-mercury, were also modeled, and the computed activation barriers for all three organomercurial substrates reproduce the trend in the experimentally observed enzymatic reaction rates. Analysis of atomic charges in the rate-limiting transition state structure using Natural Population Analysis shows that MerB lowers the activation free energy in the Hg-C protonolysis reaction by redistributing electron density into the leaving group and away from the catalytic proton.

Characterization of the Decaheme c-type Cytochrome OmcA in Solution and on Hematite Surfaces by Small Angle X-ray Scattering and Neutron Reflectometry

The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (alpha-Fe(2)O(3)). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 x 90 x 65 A(3). To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by approximately 7 A (the maximum dimension was 96 A for oxidized OmcA, and 89 A for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcAs role in mediating redox processes.

Characterization of Archaeal Community in Contaminated and Uncontaminated Surface Stream Sediments

Archaeal communities from mercury and uranium-contaminated freshwater stream sediments were characterized and compared to archaeal communities present in an uncontaminated stream located in the vicinity of Oak Ridge, TN, USA. The distribution of the Archaea was determined by pyrosequencing analysis of the V4 region of 16S rRNA amplified from 12 streambed surface sediments. Crenarchaeota comprised 76% of the 1,670 archaeal sequences and the remaining 24% were from Euryarchaeota. Phylogenetic analysis further classified the Crenarchaeota as a Freshwater Group, Miscellaneous Crenarchaeota group, Group I3, Rice Cluster VI and IV, Marine Group I and Marine Benthic Group B; and the Euryarchaeota into Methanomicrobiales, Methanosarcinales, Methanobacteriales, Rice Cluster III, Marine Benthic Group D, Deep Sea Hydrothermal Vent Euryarchaeota 1 and Eury 5. All groups were previously described. Both hydrogen- and acetate-dependent methanogens were found in all samples. Most of the groups (with 60% of the sequences) described in this study were not similar to any cultivated isolates, making it difficult to discern their function in the freshwater microbial community. A significant decrease in the number of sequences, as well as in the diversity of archaeal communities was found in the contaminated sites. The Marine Group I, including the ammonia oxidizer Nitrosopumilus maritimus, was the dominant group in both mercury and uranium/nitrate-contaminated sites. The uranium-contaminated site also contained a high concentration of nitrate, thus Marine Group I may play a role in nitrogen cycle.

Stoichiometry and temperature sensitivity of methanogenesis and CO2 production from saturated polygonal tundra in Barrow, Alaska

Arctic permafrost ecosystems store ~50% of global belowground carbon (C) that is vulnerable to increased microbial degradation with warmer active layer temperatures and thawing of the near surface permafrost. We used anoxic laboratory incubations to estimate anaerobic CO2 production and methanogenesis in active layer (organic and mineral soil horizons) and permafrost samples from center, ridge and trough positions of water-saturated low-centered polygon in Barrow Environmental Observatory, Barrow AK, USA. Methane (CH4 ) and CO2 production rates and concentrations were determined at -2, +4, or +8 °C for 60 day incubation period. Temporal dynamics of CO2 production and methanogenesis at -2 °C showed evidence of fundamentally different mechanisms of substrate limitation and inhibited microbial growth at soil water freezing points compared to warmer temperatures. Nonlinear regression better modeled the initial rates and estimates of Q10 values for CO2 that showed higher sensitivity in the organic-rich soils of polygon center and trough than the relatively drier ridge soils. Methanogenesis generally exhibited a lag phase in the mineral soils that was significantly longer at -2 °C in all horizons. Such discontinuity in CH4 production between -2 °C and the elevated temperatures (+4 and +8 °C) indicated the insufficient representation of methanogenesis on the basis of Q10 values estimated from both linear and nonlinear models. Production rates for both CH4 and CO2 were substantially higher in organic horizons (20% to 40% wt. C) at all temperatures relative to mineral horizons (<20% wt. C). Permafrost horizon (~12% wt. C) produced ~5-fold less CO2 than the active layer and negligible CH4 . High concentrations of initial exchangeable Fe(II) and increasing accumulation rates signified the role of iron as terminal electron acceptors for anaerobic C degradation in the mineral horizons.

Oxidation of Dissolved Elemental Mercury by Thiol Compounds under Anoxic Conditions

Mercuric ion, Hg(2+), forms strong complexes with thiolate compounds that commonly dominate Hg(II) speciation in natural freshwater. However, reactions between dissolved aqueous elemental mercury (Hg(0)aq) and organic ligands in general, and thiol compounds in particular, are not well studied although these reactions likely affect Hg speciation and cycling in the environment. In this study, we compared the reaction rates between Hg(0)aq and a number of selected organic ligands with varying molecular structures and sulfur (S) oxidation states in dark, anoxic conditions to assess the role of these ligands in Hg(0)aq oxidation. Significant Hg(0)aq oxidation was observed with all thiols but not with ligands containing no S. Compounds with oxidized S (e.g., disulfide) exhibited little or no reactivity toward Hg(0)aq either at pH 7. The rate and extent of Hg(0)aq oxidation varied greatly depending on the chemical and structural properties of thiols, thiol/Hg ratios, and the presence or absence of electron acceptors. Smaller aliphatic thiols and higher thiol/Hg ratios resulted in higher Hg(0)aq oxidation rates than larger aromatic thiols at lower thiol/Hg ratios. The addition of electron acceptors (e.g., humic acid) also led to substantially increased Hg(0)aq oxidation. Our results suggest that thiol-induced oxidation of Hg(0)aq is important under anoxic conditions and can affect Hg redox transformation and bioavailability for microbial methylation.