The Combined Effect of Hg(II) Speciation, Thiol Metabolism, and Cell Physiology on Methylmercury Formation by Geobacter sulfurreducens

The chemical and biological factors controlling microbial formation of methylmercury (MeHg) are widely studied separately, but the combined effects of these factors are largely unknown. We examined how the chemical speciation of divalent, inorganic mercury (Hg(II)), as controlled by low-molecular-mass thiols, and cell physiology govern MeHg formation by Geobacter sulfurreducens. We compared MeHg formation with and without addition of exogenous cysteine (Cys) to experimental assays with varying nutrient and bacterial metabolite concentrations. Cysteine additions initially (0-2 h) enhanced MeHg formation by two mechanisms: (i) altering the Hg(II) partitioning from the cellular to the dissolved phase and/or (ii) shifting the chemical speciation of dissolved Hg(II) in favor of the Hg(Cys)2 complex. Nutrient additions increased MeHg formation by enhancing cell metabolism. These two effects were, however, not additive since cysteine was largely metabolized to penicillamine (PEN) over time at a rate that increased with nutrient addition. These processes shifted the speciation of dissolved Hg(II) from complexes with relatively high availability, Hg(Cys)2, to complexes with lower availability, Hg(PEN)2, for methylation. This thiol conversion by the cells thereby contributed to stalled MeHg formation after 2-6 h Hg(II) exposure. Overall, our results showed a complex influence of thiol metabolism on microbial MeHg formation and suggest that the conversion of cysteine to penicillamine may partly suppress MeHg formation in cysteine-rich environments like natural biofilms

The Role of Oxygen in Stimulating Methane Production in Wetlands

Methane (CH4), a potent greenhouse gas, is the second most important greenhouse gas contributor to climate change after carbon dioxide (CO2). The biological emissions of CH4 from wetlands are a major uncertainty in CH4 budgets. Microbial methanogenesis by Archaea is an anaerobic process accounting for most biological CH4 production in nature, yet recent observations indicate that large emissions can originate from oxygenated or frequently oxygenated wetland soil layers. To determine how oxygen (O2) can stimulate CH4 emissions, we used incubations of Sphagnum peat to demonstrate that the temporary exposure of peat to O2 can increase CH4 yields up to 2000-fold during subsequent anoxic conditions relative to peat without O2 exposure. Geochemical (including ion cyclotron resonance mass spectrometry, X-ray absorbance spectroscopy) and microbiome (16S rDNA amplicons, metagenomics) analyses of peat showed that higher CH4 yields of redox-oscillated peat were due to functional shifts in the peat microbiome arising during redox oscillation that enhanced peat carbon (C) degradation. Novosphingobium species with O2-dependent aromatic oxygenase genes increased greatly in relative abundance during the oxygenation period in redox-oscillated peat compared to anoxic controls. Acidobacteria species were particularly important for anaerobic processing of peat C, including in the production of methanogenic substrates H2 and CO2. Higher CO2 production during the anoxic phase of redox-oscillated peat stimulated hydrogenotrophic CH4 production by Methanobacterium species. The persistence of reduced iron (Fe(II)) during prolonged oxygenation in redox-oscillated peat may further enhance C degradation through abiotic mechanisms (e.g., Fenton reactions). The results indicate that specific functional shifts in the peat microbiome underlie O2 enhancement of CH4 production in acidic, Sphagnum-rich wetland soils. They also imply that understanding microbial dynamics spanning temporal and spatial redox transitions in peatlands is critical for constraining CH4 budgets; predicting feedbacks between climate change, hydrologic variability, and wetland CH4 emissions; and guiding wetland C management strategies

Effect of Thiols, Zinc, and Redox Conditions on Hg Uptake in Shewanella oneidensis

Mercury uptake in bacteria represents a key first step in the production and accumulation of methylmercury in biota. Previous experiments with mercury methylating bacteria have shown that Hg uptake is enhanced by some thiols, in particular cysteine, and that it is an energy-dependent process through heavy metal transporters [Schaefer et al. Environ. Sci. Technol.2014, 48, 3007]. In this study, we examine Hg uptake in the nonmethylating facultative aerobe, Shewanella oneidensis, under both anaerobic and aerobic conditions. Our results demonstrate similar characteristics of the Hg uptake system to those of the Hg methylating strains: (1) uptake is enhanced in the presence of some thiols but not others; (2) uptake is energy dependent as evidenced by inhibition by a protonophore, and (3) uptake is inhibited by high Zn(II) concentrations. Initial cellular uptake rates in S. oneidensis were remarkably similar under aerobic and fumarate-reducing conditions. These data support a similar Hg(II) uptake mechanism within the proteobacteria of accidental Hg(II) transport through heavy metal transporters with similar rates of uptake but differences in the ability to take up Hg bound to different thiols

The combined effect of Hg(II) speciation, thiol metabolism, and cell physiology on methylmercury formation by Geobacter sulfurreducens

The chemical and biological factors controlling microbial formation of methylmercury (MeHg) are widely studied separately, but the combined effects of these factors are largely unknown. We examined how the chemical speciation of divalent, inorganic mercury (Hg(II)), as controlled by low-molecular-mass thiols, and cell physiology govern MeHg formation by Geobacter sulfurreducens. We compared MeHg formation with and without addition of exogenous cysteine (Cys) to experimental assays with varying nutrient and bacterial metabolite concentrations. Cysteine additions initially (0–2 h) enhanced MeHg formation by two mechanisms: (i) altering the Hg(II) partitioning from the cellular to the dissolved phase and/or (ii) shifting the chemical speciation of dissolved Hg(II) in favor of the Hg(Cys)2 complex. Nutrient additions increased MeHg formation by enhancing cell metabolism. These two effects were, however, not additive since cysteine was largely metabolized to penicillamine (PEN) over time at a rate that increased with nutrient addition. These processes shifted the speciation of dissolved Hg(II) from complexes with relatively high availability, Hg(Cys)2, to complexes with lower availability, Hg(PEN)2, for methylation. This thiol conversion by the cells thereby contributed to stalled MeHg formation after 2–6 h Hg(II) exposure. Overall, our results showed a complex influence of thiol metabolism on microbial MeHg formation and suggest that the conversion of cysteine to penicillamine may partly suppress MeHg formation in cysteine-rich environments like natural biofilms

Effect of Divalent Metals on Hg(II) Uptake and Methylation by Bacteria

The production of methylmercury by some bacteria is a key first step in the accumulation and biomagnification of this toxic substance in aquatic food webs, a major human health concern. By direct measurement of cellular Hg­(II) uptake in model iron and sulfate reducing bacteria, we have observed that specific trace metals, such as Zn­(II) and Cd­(II), inhibit uptake and methylation in these organisms, whereas other metals, such as Ni­(II), Co­(II), or Fe­(II), do not. The inhibition of Hg­(II) methylation by Zn­(II) was competitive in nature and related to the concentration of inorganically complexed Zn­(II) (Zn′). The inhibition of Hg­(II) methylation was alleviated by decreasing the free Zn′ concentration through complexation with nitrilotriacetic acid without altering the speciation of Hg­(II). The inhibitory effect by Zn­(II) was observed when either Hg-cysteine complexes or neutral HgCl₂ dominated the speciation of Hg­(II), demonstrating that both charged and neutral species are transported into the cytosol by an active rather than passive process. We propose that Hg­(II) uptake is the result of its accidental uptake by metal transporter(s), possibly one effecting the transport of Zn­(II)

Metabolic turnover of cysteine-related thiol compounds at environmentally relevant concentrations by Geobacter sulfurreducens

Low-molecular-mass (LMM) thiol compounds are known to be important for many biological processes in various organisms but LMM thiols are understudied in anaerobic bacteria. In this work, we examined the production and turnover of nanomolar concentrations of LMM thiols with a chemical structure related to cysteine by the model iron-reducing bacterium Geobacter sulfurreducens. Our results show that G. sulfurreducens tightly controls the production, excretion and intracellular concentration of thiols depending on cellular growth state and external conditions. The production and cellular export of endogenous cysteine was coupled to the extracellular supply of Fe(II), suggesting that cysteine excretion may play a role in cellular trafficking to iron proteins. Addition of excess exogenous cysteine resulted in a rapid and extensive conversion of cysteine to penicillamine by the cells. Experiments with added isotopically labeled cysteine confirmed that penicillamine was formed by a dimethylation of the C-3 atom of cysteine and not via indirect metabolic responses to cysteine exposure. This is the first report of de novo metabolic synthesis of this compound. Penicillamine formation increased with external exposure to cysteine but the compound did not accumulate intracellularly, which may suggest that it is part of G. sulfurreducens’ metabolic strategy to maintain cysteine homeostasis. Our findings highlight and expand on processes mediating homeostasis of cysteine-like LMM thiols in strict anaerobic bacteria. The formation of penicillamine is particularly noteworthy and this compound warrants more attention in microbial metabolism studies

Effect of Thiols, Zinc, and Redox Conditions on Hg Uptake in <i>Shewanella oneidensis</i>

Mercury uptake in bacteria represents a key first step in the production and accumulation of methylmercury in biota. Previous experiments with mercury methylating bacteria have shown that Hg uptake is enhanced by some thiols, in particular cysteine, and that it is an energy-dependent process through heavy metal transporters [Schaefer et al. <i>Environ. Sci. Technol.</i> <b>2014</b>, <i>48</i>, 3007]. In this study, we examine Hg uptake in the nonmethylating facultative aerobe, <i>Shewanella oneidensis</i>, under both anaerobic and aerobic conditions. Our results demonstrate similar characteristics of the Hg uptake system to those of the Hg methylating strains: (1) uptake is enhanced in the presence of some thiols but not others; (2) uptake is energy dependent as evidenced by inhibition by a protonophore, and (3) uptake is inhibited by high Zn­(II) concentrations. Initial cellular uptake rates in <i>S. oneidensis</i> were remarkably similar under aerobic and fumarate-reducing conditions. These data support a similar Hg­(II) uptake mechanism within the proteobacteria of accidental Hg­(II) transport through heavy metal transporters with similar rates of uptake but differences in the ability to take up Hg bound to different thiols

Mechanisms of Methyl Mercury Net Degradation in Alder Swamps: The Role of Methanogens and Abiotic Processes

Wetlands are common net producers of the neurotoxin monomethylmercury (MeHg) and are largely responsible for MeHg bioaccumulation in aquatic food-webs. However, not all wetlands net produce MeHg; notable exceptions are black alder (Alnus glutinosa) swamps, which net degrade MeHg. Here we report the mechanisms of MeHg demethylation in one such swamp (EHT), shown to be a sink for MeHg during four consecutive years. The potential demethylation rate constant (<i>k</i>_d) in soil incubations was ∼3 times higher in the downstream (EHT-D: <i>k</i>_d ∼ 0.14 d^–1) as compared to the upstream part of the swamp (EHT-U: <i>k</i>_d ∼ 0.05 d^–1). This difference concurred with increased stream and soil pH, and a change in plant community composition. Electron acceptor and inhibitor addition experiments revealed that abiotic demethylation dominated at EHT-U while an additional and equally large contribution from biotic degradation was observed at EHT-D, explaining the increase in MeHg degradation. Biotic demethylation (EHT-D) was primarily due to methanogens, inferred by a decrease in <i>k</i>_d to autoclaved levels following selective inhibition of methanogens. Though methanogen-specific transcripts (<i>mcrA</i>) were found throughout the wetland, transcripts clustering with <i>Methanosaetaceae</i> were exclusive to EHT-D, suggesting a possible role for these acetoclastic methanogens in the degradation of MeHg