Research ArticleENVIRONMENTAL STUDIES

Methylmercury uptake and degradation by methanotrophs

See allHide authors and affiliations

Science Advances  31 May 2017:
Vol. 3, no. 5, e1700041
DOI: 10.1126/sciadv.1700041

Abstract

Methylmercury (CH3Hg+) is a potent neurotoxin produced by certain anaerobic microorganisms in natural environments. Although numerous studies have characterized the basis of mercury (Hg) methylation, no studies have examined CH3Hg+ degradation by methanotrophs, despite their ubiquitous presence in the environment. We report that some methanotrophs, such as Methylosinus trichosporium OB3b, can take up and degrade CH3Hg+ rapidly, whereas others, such as Methylococcus capsulatus Bath, can take up but not degrade CH3Hg+. Demethylation by M. trichosporium OB3b increases with increasing CH3Hg+ concentrations but was abolished in mutants deficient in the synthesis of methanobactin, a metal-binding compound used by some methanotrophs, such as M. trichosporium OB3b. Furthermore, addition of methanol (>5 mM) as a competing one-carbon (C1) substrate inhibits demethylation, suggesting that CH3Hg+ degradation by methanotrophs may involve an initial bonding of CH3Hg+ by methanobactin followed by cleavage of the C–Hg bond in CH3Hg+ by the methanol dehydrogenase. This new demethylation pathway by methanotrophs indicates possible broader involvement of C1-metabolizing aerobes in the degradation and cycling of toxic CH3Hg+ in the environment.

Keywords
  • Methylmercury production
  • uptake
  • demethylation
  • bioaccumulation
  • organomercurial lyase
  • aquatic environments

INTRODUCTION

Methylmercury (CH3Hg+) toxin is predominantly produced by certain anaerobic microorganisms (for example, Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA) having two key genes (hgcA and hgcB) necessary for converting inorganic mercury (Hg) to CH3Hg+ (13). It can accumulate and biomagnify at high levels in fish as well as in rice grains, and human consumption can cause neurological damage (47). Our understanding of the mechanisms responsible for Hg methylation has greatly improved recently through the identification of the genetic basis (13) and factors affecting Hg methylation (812). However, net CH3Hg+ levels in the environment depend on two competing biological processes—CH3Hg+ production and demethylation (1317), although demethylation can also take place photochemically in surface waters (18).

To date, much attention has focused on Hg methylation, but fewer studies have examined microbial demethylation, except the process mediated by the mer operon (17, 19), in which demethylation is carried out by an organomercurial lyase (MerB). MerB cleaves off the methyl group to form methane (CH4) and Hg(II), whereas a mercuric reductase (MerA) reduces the released Hg(II) to volatile elemental Hg(0) (7, 13, 20). However, only certain aerobic prokaryotes have this CH3Hg+ degradation pathway. mer-mediated pathway is operative only at extremely high Hg concentrations (that is, micromolar) (7, 17, 21), conditions that are largely irrelevant to most natural waters and sediments, where Hg or CH3Hg+ concentrations are usually at picomolar to low nanomolar ranges (7, 17). In addition, in vitro experiments with the isolated MerB enzyme showed that demethylation by MerB occurs generally at above neutral pH conditions, with an optimal pH of ~10 (21).

However, degradation of CH3Hg+ has been observed in anoxic sediments and in a limited number of pure cultures at relatively low Hg concentrations (for example, nanomolar) (1417, 22). Anaerobic sulfate reducers and methanogens are thought to be primarily responsible for this oxidative demethylation because Hg(II), CH4, and carbon dioxide (CO2) have been identified as major products (1416). Despite the finding of CH4 and CO2 evolution, methanotrophs are not considered as important players in the oxidative demethylation of CH3Hg+. The possible involvement of methanotrophs has never been directly tested, and the bacteria involved and pathways leading to oxidative demethylation remain unexplored.

Methanotrophs can thrive under a wide range of redox conditions, particularly at the oxic-anoxic interface where CH4 and CH3Hg+ are commonly observed (2325). They are widespread and found in diverse locations, such as freshwater and marine sediments, bogs, forest and agricultural soils, and volcanic soils (26, 27). Many methanotrophs also produce an extracellular metal-binding peptide called methanobactin that has been shown to bind CH3Hg+ (28, 29). In addition, most methanotrophs can use methanol as a one-carbon (C1) growth substrate, and some can also grow on methylamine (24, 30), and we therefore hypothesized that at least some methanotrophs can take up and possibly degrade CH3Hg+.

RESULTS AND DISCUSSION

CH3Hg+ uptake and degradation were first examined in representative strains of α-(Methylosinus trichosporium OB3b) and γ-proteobacterial (Methylococcus capsulatus Bath) methanotrophs. Both methanotrophs were found to sorb substantial amounts of CH3Hg+, with M. trichosporium OB3b showing slightly higher sorption affinity and kinetics than M. capsulatus Bath (Fig. 1A). Within 1 hour, ~95% of the CH3Hg+ was sorbed or associated with M. trichosporium OB3b, whereas only ~65% was associated with M. capsulatus Bath cells, although the sorption increased to ~85% on M. capsulatus Bath cells in 4 hours. Analyses of Hg species distributions indicated that a large percentage of the CH3Hg+ was internalized or taken up by both M. trichosporium OB3b and M. capsulatus Bath cells in 4 hours, leaving only a small percentage of the CH3Hg+ in solution (Fig. 1B). These results are in contrast to the rapid export and little sorption of CH3Hg+ observed with known mercury methylators, such as D. desulfuricans ND132 (10, 31, 32), suggesting that both M. trichosporium OB3b and M. capsulatus Bath have a high affinity to sorb or take up CH3Hg+.

Fig. 1 Methylmercury (CH3Hg+) sorption, degradation, and species distribution.

(A) CH3Hg+ sorption kinetics and (B) Hg species distributions (at 4 and 120 hours) by methanotrophs M. trichosporium OB3b and M. capsulatus (MC) Bath in 5 mM MOPS buffer. The total added CH3Hg+ concentration (HgT) was 5 nM at t = 0, and the cell concentration was 108 cells ml−1. CH3Hg+sol, soluble CH3Hg+; CH3Hg+ad, cell surface–adsorbed CH3Hg+; CH3Hg+up, cell uptake of or internalized CH3Hg+. IHg results from degradation of CH3Hg+. Error bars represent 1 SD from triplicate samples.

We found that, with increasing incubation time (120 hours), a substantial amount of CH3Hg+ (~43%) was degraded and converted to inorganic Hg (IHg) by M. trichosporium OB3b, but not by M. capsulatus Bath cell (Fig. 1B). This observation was confirmed by additional detailed time- and concentration-dependent studies of CH3Hg+ degradation by both M. trichosporium OB3b (Fig. 2, A and B) and M. capsulatus Bath (Fig. 2, C and D). We found no demethylation at all with M. capsulatus Bath cultures, regardless of the reaction time (up to 120 hours) and CH3Hg+ concentration (from 5 to 125 nM). However, CH3Hg+ was degraded progressively by M. trichosporium OB3b with time and CH3Hg+ concentrations up to 75 nM (Fig. 2, A and B). The pseudo–first-order rate constants at the initial CH3Hg+ concentrations of 5, 25, and 75 nM were 0.017 (±0.001), 0.032 (±0.008), and 0.037 (±0.003) hour−1, respectively, and approximately 55, 62, and 73% of the added CH3Hg+ were degraded after 5 days. Again, CH3Hg+ was converted to IHg (fig. S1A), but no gaseous Hg(0) was observed (fig. S1B). The amount of the cell-associated CH3Hg+, particularly the adsorbed CH3Hg+ad, decreased with time, whereas the proportion of IHg increased with time. The produced IHg mostly remained inside the cell, with less than 6% of the IHg either left in solution or sorbed on the cell surface because Hg(II) is known to strongly sorb or interact with thiol functional groups of proteins and cellular materials (33). Note that, at the highest added CH3Hg+ concentration (125 nM), the reaction rate decreased to 0.011 (±0.001) hour−1 (Fig. 2A), and demethylation was inhibited in the first 8 to 24 hours. However, with a longer incubation time (120 hours), the cells were able to recover and degrade a substantial amount of CH3Hg+ (71%). This initially inhibited CH3Hg+ degradation may be interpreted as a result of potential toxic effects of CH3Hg+ on M. trichosporium OB3b, similar to that observed with Geobacter bemidjiensis Bem (17).

Fig. 2 Time- and concentration-dependent degradation of methylmercury (CH3Hg+) by methanotrophs.

(A and B) M. trichosporium OB3b at 30°C and (C and D) M. capsulatus Bath at 45°C in 5 mM MOPS buffer. The added cell concentration was 108 cells ml−1 (washed), and the CH3Hg+ concentration was varied from 0 to 125 nM. Data points at 5 nM CH3Hg+ in (A) represent an average of replicate samples (10 to 15) from five independent batch experiments, and all other data points represent an average of triplicate samples. Error bars represent 1 SD from all replicate samples.

Because demethylation was observed neither in M. trichosporium OB3b spent medium (fig. S1B) nor in M. capsulatus Bath cultures (Fig. 2, C and D), the results signify that demethylation was biologically mediated and methanotroph strain specific. However, neither M. trichosporium OB3b nor M. capsulatus Bath contains a homolog of merB (encoding for the organomercurial lyase) in their genome, suggesting that CH3Hg+ degradation by M. trichosporium OB3b relies on an as yet unknown mechanism and that this mechanism does not exist in M. capsulatus Bath.

To elucidate this mechanism, we first considered the fact that both M. trichosporium OB3b and M. capsulatus Bath are sensitive to the availability of copper. That is, the copper-to-biomass ratio is a key factor in regulating the expression of the following: (i) genes encoding for the soluble and particulate methane monooxygenases (MMOs), with soluble MMO only expressed in the absence of copper (25, 34); and (ii) genes encoding for the chalkophore methanobactin with expression greatest in the absence of copper (24, 25). Genes encoding for the chalkophore are found in M. trichosporium OB3b, but not in M. capsulatus Bath, which contains a different class of chalkophores (35). Furthermore, methanobactin from M. trichosporium OB3b has been found to bind Hg(II) and CH3Hg+ (29) and may thus be involved in CH3Hg+ degradation.

We subsequently investigated CH3Hg+ degradation by M. trichosporium OB3b in the presence of a known MMO inhibitor, acetylene, but no apparent inhibitory effects were observed (fig. S2). We next considered CH3Hg+ degradation by cells grown either in the absence (0 μM) or in the presence (1 μM) of copper. Although CH3Hg+ degradation was observed under both conditions (Fig. 3 and table S1), greater degradation of CH3Hg+ was evident in the absence than in the presence of copper. We then examined several mutant strains of M. trichosporium OB3b defective in methanobactin production (mbnA::Gmr and ΔmbnAN) (25) to determine whether methanobactin is directly involved in CH3Hg+ degradation. We also examined two additional methanotrophs—one (Methylocystis strain SB2) makes methanobactin and the other (Methylocystis parvus OBBP) does not (36). Results show that Methylocystis strain SB2 degraded CH3Hg+, whereas all methanobactin mutants did not regardless of the culture conditions in the presence or absence of Cu2+ ions (Fig. 3 and table S1). In addition, no demethylation was observed with M. parvus OBBP. These findings strongly suggest that methanobactin plays a critical role in degrading CH3Hg+.

Fig. 3 Methylmercury (CH3Hg+) degradation by different methanotrophs and mutants.

Comparisons of the time-dependent degradation of CH3Hg+ by washed cells of M. trichosporium OB3b and its mutant strains (mbnA::Gmr and ΔmbnAN), M. capsulatus Bath, and M. parvus OBBP in 5 mM MOPS buffer. The added cell concentration was 108 cells ml−1, and the CH3Hg+concentration was ~5 nM. Data points represent an average of all replicate samples (3 to 15), and error bars represent 1 SD.

Although methanobactin is clearly needed for CH3Hg+ degradation by M. trichosporium OB3b, subsequent studies indicate that it is not sufficient. That is, when CH3Hg+ was incubated with the purified methanobactin, no appreciable CH3Hg+ degradation was observed in the same MOPS buffer used in whole-cell studies (fig. S3). This result suggests that methanobactin likely served as a carrier or as a binding agent for CH3Hg+ in the cell where it is degraded by some as yet unknown enzyme(s). We subsequently considered possible involvement of methanol dehydrogenase (MeDH), which is responsible for oxidation of methanol to formaldehyde in the central pathway of CH4 oxidation, because M. trichosporium OB3b may take up CH3Hg+ and use its methyl (–CH3) group as a supplementary C1 source and energy. We found that addition of methanol (5 mM or higher) completely inhibited CH3Hg+ degradation (fig. S4). This inhibition cannot be attributed to decreased metabolism of M. trichosporium OB3b because no inhibitory effects were observed in this organism even at methanol concentrations as high as 990 mM (37). The result suggests that MeDH, in conjunction with methanobactin, degraded CH3Hg+, similar to the degradation of methanol where the methyl group is cleaved and possibly oxidized via this periplasmic enzyme. Hence, the methyl group of CH3Hg+ may serve as an auxiliary C1 source for methanotrophs, as speculated by others (19).

In conclusion, we present evidence that strongly suggests the presence of a novel biological pathway of CH3Hg+ demethylation by methanotrophs, which warrants further investigation. This pathway is remarkably different from the canonical organomercurial lyase found in some aerobic microorganisms (13, 20). Unlike the organomercurial lyase in Hg-resistant bacteria, methanotrophs take up and degrade CH3Hg+ at environmentally relevant Hg concentrations (that is, picomolar to nanomolar). Methanotrophic-mediated CH3Hg+ degradation was also evident at circumneutral pH, unlike organomercurial lyase that has an optimal pH of ~10 (21). These findings suggest that methanotrophs may play an important role in controlling Hg transformation or net CH3Hg+ production and toxicity in situ, thereby providing new insights into as yet unknown but potentially widespread biological mechanisms of CH3Hg+ uptake and demethylation in the environment.

MATERIALS AND METHODS

The methanotrophs M. trichosporium OB3b and M. capsulatus Bath were grown in nitrate minimal salts medium at 30° and 45°C, respectively, either without added copper or with 1 μM copper (as CuCl2) (29, 38). Cells were harvested at the late exponential phase, washed once, and then resuspended in 5 mM MOPS buffer solution at pH 7.3. Methanobactin was isolated from M. trichosporium OB3b, as previously described (39).

Methylmercury (CH3Hg+) sorption, uptake, and demethylation assays were conducted in 4-ml amber glass vials (National Scientific) by mixing washed cells with CH3Hg+ in 5 mM MOPS buffer under ambient conditions. To determine whether MMOs were involved in CH3Hg+ demethylation, we added 100 μl of acetylene to the headspace (through a septum) and allowed it to equilibrate with the cells first for 30 min in one subset of assays because acetylene is a strong and selective inhibitor of MMO activity (25, 40). CH3Hg+ working solution (10 nM) was prepared by diluting 5 μM stock solution (CH3HgOH in 0.5% acetic acid and 0.2% HCl from Brooks Rand Labs) in MOPS. The reaction was initiated by mixing 0.5 ml of CH3Hg+ working solution with 0.5 ml of washed cells to give a final concentration of CH3Hg+ at 5 nM and of cells at 1 × 108 cells ml−1 (17), or otherwise specified. Samples were then placed on a rotary shaker, kept at 30°C for M. trichosporium OB3b and its mutants, and at 45°C for M. capsulatus Bath. Replicate sample vials were taken at selected time points and analyzed as follows. For CH3Hg+ sorption (or uptake) analysis, triplicate samples were filtered through 0.2-μm syringe filters (to remove cells) and analyzed for CH3Hg+sol (17, 32). The unfiltered samples were used to determine the total Hg and total CH3Hg+ (CH3Hg+Total) so that the cell-associated or total sorbed Hg can be calculated by their difference. For Hg species distribution analyses, six replicate samples (in separate vials) were taken, and three of them were filtered as above and analyzed for total soluble Hg (Hgsol) and CH3Hg+sol (17, 33). The remaining three samples were used to determine cellular uptake of CH3Hg+ (CH3Hg+up) and cell surface–adsorbed CH3Hg+ (CH3Hg+ad). This was accomplished by adding 2,3-dimercapto-1-propanesulfonic acid (DMPS), a Hg-chelating agent, at 150 μM to wash off the sorbed CH3Hg+ad at each time point and then analyzing CH3Hg+ in filtered samples (17, 33), so that CH3Hg+up can be calculated by subtracting CH3Hg+ad and CH3Hg+sol from CH3Hg+Total. The inorganic IHg species, resulting from degradation of CH3Hg+, were analyzed in the same manner, in which the adsorbed IHgad and cellular uptake of IHgup were determined following DMPS washing, and soluble IHg (IHgsol) was calculated by subtracting CH3Hg+sol from Hgsol (17, 33). Selected samples (before filtration) were determined for purgeable elemental Hg(0), but none was detected. Additional experiments were performed with cell spent medium and MOPS buffer as controls. Demethylation experiments were repeated at least once to ensure data quality, and error bars in all figures represent 1 SD of all replicate samples. Demethylation rate constants (kdemeth) were calculated on the basis of the pseudo–first-order rate law: d[CH3Hg+]/dt = −kdemeth[CH3Hg+], where kdemeth was determined by the slope of the linear regression between natural logarithm of the CH3Hg+ concentration and time (12, 41).

A modified EPA Method 1630 was used for CH3Hg+ analysis, in which isotope dilution with enriched CH3200Hg+ was used as an internal standard, and an inductively coupled plasma mass spectrometer (ELAN DRC-e, PerkinElmer Inc.) was used to separate the various Hg isotopes to determine CH3Hg+ concentrations (17, 32, 33). The recovery of spiked CH3Hg+ standards was 100 ± 10%, and the detection limit was about 3 × 10−5 nM CH3Hg+. Gaseous Hg(0) was directly determined by inserting needles through the septa of the 4-ml glass vials and then purging with ultrapure N2 for 2 min into a gaseous Hg(0) analyzer (Lumex 915+, Ohio Lumex). Total Hg and Hgsol were analyzed via SnCl2 reduction and detection by the Lumex analyzer after samples were oxidized in BrCl (5%, v/v) overnight at 4°C (11, 12, 42). The detection limit was ~2.5 × 10−4 nM.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1700041/DC1

table S1. Methylmercury degradation by washed cells of methanotrophs, including M. trichosporium OB3b and its two methanobactin (mb) defective mutants (mbnA::Gmr and ΔmbnAN), Methylocystis strain SB2, M. capsulatus Bath, and M. parvus OBBP in 5 mM MOPS at pH 7.3.

fig. S1. Methylmercury (CH3Hg+) and inorganic mercury (IHg) species distribution during CH3Hg+ degradation assays with M. trichosporium OB3b.

fig. S2. Effects of acetylene addition (as an inhibitor of MMOs) on methylmercury (CH3Hg+) degradation by washed cells of M. trichosporium OB3b (108 cells ml−1) in 5 mM MOPS buffer at 30°C.

fig. S3. Reactions between methylmercury (CH3Hg+, 5 nM) and purified methanobactin (1 μM) from M. trichosporium OB3b in 5 mM MOPS buffer (pH 7.3) at 30°C.

fig. S4. Effects of methanol addition on methylmercury (CH3Hg+) degradation by washed cells of M. trichosporium OB3b (108 cells ml−1) in 5 mM MOPS buffer at 30°C.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

REFERENCES AND NOTES

Acknowledgments: We thank X. Yin for Hg and MeHg analyses, Y. Liu and Z. Yang for technical assistance and discussion, and A. Johs for critical review of the manuscript. Funding: This research was sponsored by the U.S. Department of Energy (DOE) Office of Science, Office of Biological and Environmental Research, as part of the Mercury Science Focus Area at the Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC under contract no. DE-AC05-00OR22725 with DOE and grant no. DE-SC0006630 to J.D.S. and A.A.D. Author contributions: X.L., B.G., J.D.S., and A.A.D. designed the research. X.L., W.G., L.Z., and M.F.U.H. performed the experiments and analyses. B.G., X.L., J.D.S., and A.A.D. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
View Abstract

Navigate This Article