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Nanomolar Copper Enhances Mercury Methylation by Desulfovibrio desulfuricans ND132 Xia Lu, Alexander Johs, Linduo Zhao, Lihong Wang, Eric M. Pierce, and Baohua Gu Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00232 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Environmental Science & Technology Letters

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Nanomolar Copper Enhances Mercury Methylation by

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Desulfovibrio desulfuricans ND132

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Xia Lu,1,2 Alexander Johs,1 Linduo Zhao,1 Lihong Wang,1 Eric M. Pierce,1 Baohua Gu1,3*

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1

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States

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Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, China 730000

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Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN 37996, United States

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To whom correspondence should be addressed: Email: [email protected]; Phone: (865)-574-7286

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ABSTRACT Methylmercury (MeHg) is produced by certain anaerobic microorganisms, such as the

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sulfate-reducing bacterium Desulfovibrio desulfuricans ND132, but environmental factors

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affecting inorganic mercury [Hg(II)] uptake and methylation remain unclear. We report that the

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presence of a small amount of copper ions [Cu(II), 0.05), regardless of the reaction time (2 or 24 h) (Figure 1b), since both Mg(II) and Mn(II) are

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nutrients for microbes.

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The enhanced Hg(II) methylation by nanomolar concentrations of Cu(II), but not by

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Zn(II) or Ni(II), is of particular interest and indicates that the enhancement is highly metal ion

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specific. Therefore, we further investigated Hg(II) methylation at varying concentrations of

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Cu(II), Zn(II), and Ni(II) (Figure 2; SI Figure S1). MeHg production increased significantly with

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increasing Cu(II) concentrations up to 75–100 nM (p < 0.01), but decreased with further

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increasing Cu(II) concentrations (>100 nM) (Figure 2a). At 50–100 nM Cu(II) (or Cu:Hg molar

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ratios of 2 to 4), MeHg production nearly doubled, but then decreased consistently at Cu(II)

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concentrations >200 nM. Methylation was completely inhibited at a Cu(II) concentration of 1

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µM. Similarly, Zn(II) and Ni(II) at concentrations >200 nM inhibited Hg(II) methylation (SI

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Figure S1) but, unlike Cu(II), lower Zn(II) or Ni(II) concentrations cannot enhance Hg(II)

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methylation.



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The effect of Cu(II) on Hg(II) methylation was also demonstrated in time-dependent

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experiments; MeHg production again doubled within 24 h at a Cu(II) concentration of 75 nM

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(Figure 2b). However, significant inhibition occurred at a Cu(II) concentration of 500 nM at all

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time points (p < 0.05). Careful examination of the initial methylation kinetics (within 0.5 h) also

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revealed an inhibition of Hg(II) methylation even at low Cu(II) concentrations (25–75 nM)

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(Figure 2b inset). MeHg production at 0.5 h decreased from 1.3 (±0.04) to 1.2 (±0.04) and

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1.1(±0.03) nM with the addition of 0, 25, and 75 nM Cu(II), respectively, but then increased and

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reached the same level as the Cu(II)-free control after 2 h, and ultimately exceeded that of the

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control after 8 h. However, at a high Cu(II) concentration (500 nM), cells never completely

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recovered, and methylation was inhibited even after 48 h. Therefore, the Cu(II)-enhanced or

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inhibited methylation depends on both the Cu(II) concentration and exposure time, which can be

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attributed again to both beneficial and toxic effects of Cu(II) on microbial metabolism. 24,29 The

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initial, rapid uptake of Cu(II) and Hg(II) (described below) likely led to a metal stress response

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by the cells, which is reflected in decreased MeHg production within the first 0.5 h. Over time,

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the cells slowly recovered and detoxified, leading to enhanced methylation. These observations

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suggest that microbial methylation activity was more sensitive to increasing Cu(II)

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concentrations or Cu(II) toxicity than its overall metabolic activity, as evidenced by a substantial

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decrease in Hg(II) methylation at 500 nM Cu(II) (Figure 2) but similar cell growth and

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consumption rates of pyruvate and fumarate (or production rates of acetate and succinate) (SI

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Figure S2). While exact mechanisms of Cu-enhanced Hg(II) methylation are unclear, it is known

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that bacteria are extremely sensitive to Cu(II) toxicity and tightly control Cu resistance and

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homeostasis.24, 31-33 Excess amounts of Cu(II) can trigger the expression of the genes for Cu

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resistance such as Cu-efflux pumps to maintain tolerable copper levels in cells. 31,34,35 Gram-


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negative bacteria may utilize several mechanisms to mitigate copper toxicity, such as tripartite

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efflux pumps in the resistance-nodulation-cell division (RND) family (e.g., CusCBA), 36,37

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periplasmic multicopper oxidases (e.g., CueO, PcoA),38,39 and metallochaperones (e.g., CopZ,

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CueP, etc.).32 The genome of D. desulfuricans ND132 encodes several homologs related to

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copper efflux pumps, metallochaperones, and a putative Cu-responsive transcriptional regulator

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(SI Table S1).

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Since no apparent changes in cell growth or metabolic activity were observed over 72 h

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either in the absence or presence of 75 nM Cu(II) (SI Figure S2), the results suggest that,

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although it is not required, a small amount of Cu(II) may facilitate Hg(II) uptake and thus

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methylation by a yet uncharacterized mechanism. This argument is supported by the following

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lines of evidence; firstly, we found that addition of Cu(II) (75 and 200 nM) significantly

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increased the initial rates of Hg(II) uptake by ND132 cells (p < 0.05) (Figure 3). When the cells

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were added to a mixed solution of Cu(II) (75 nM) and Hg(II) (25 nM), cells took up ~65% of the

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Hg(II) within a minute, as compared to ~30% of the Hg(II) uptake in Cu-free controls (Figure 3).

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Hg(II) sorption and uptake by ND132 cells are fairly rapid (SI Figure S3), even in the absence of

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Cu(II), as previously reported.23,26,27 Therefore, nearly 100% of the added Hg(II) was taken up

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after 24 h, regardless of the presence or absence of Cu(II). At a high level of Cu(II) (200 nM),

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Hg(II) uptake also increased from 8.6 nM [no Cu(II)] to 17.6 nM in 1 min, and from 15.9 nM to

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19.7 nM in 30 min (Figure 3). Secondly, as described earlier, the Cu(II)-enhanced Hg(II) uptake

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and methylation are metal ion specific since no enhancement was observed with the addition of

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Zn(II) or Ni(II) at the same concentrations (Figure 1a; SI Figures S1 and S4). Only inhibitory

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effects of Zn(II) and Ni(II) were observed at concentrations above 200 nM. The enhanced Hg(II)

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methylation was observed neither with the addition of Mg(II) or Mn(II) (Figure 1b) at both low



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and high concentrations. We also determined the effects of competition between Cu(II) (50 nM)

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and Mg(II) (50 µM) on Hg(II) methylation and found that addition of Cu(II) (50 nM) together

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with a high concentration of Mg(II) (50 µM) did not show any inhibitory effects on Hg(II)

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methylation either (SI Figure S5), suggesting that there was no competition between Cu(II) and

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Mg(II) and thus no effects on MeHg production.

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Taken together our results indicate: (1) The presence of Cu(II) facilitates an initial rapid

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uptake of Hg(II), but this effect diminishes over time, regardless of the presence or absence of

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Cu(II) (Figure 3), as cells may have other pathways for Hg(II) uptake; (2) The initial, rapid

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uptake of Hg(II) results in decreased Hg(II) methylation within a short time period (0.5 h)

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(Figure 2b, inset). However, over time (> 2 h), methylation increases in the presence of low

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Cu(II) concentrations ( 500 nM), cell

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methylation activity was unable to recover, leading to a consistent decrease or cease of Hg(II)

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methylation; (4) The Cu(II)-enhanced Hg(II) uptake and methylation are highly specific and not

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observed with divalent metal ions such as Zn(II), Ni(II), Mg(II), and Mn(II) under the same

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experimental conditions. Whether the initial increase in Hg(II) uptake involves copper

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transporters or metallochaperones is unknown at present. Future studies may target specific

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genes implicated with copper transport and homeostasis to confirm the potential roles of copper-

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transporters and chaperones in Hg(II) uptake and methylation in ND132.

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI:

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XXX. Additional details on cell culture medium and conditions; Tables showing proteins

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implicated with Cu transport and homeostasis; Figures showing metal ion effects on Hg(II)

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methylation, cell growth and metabolic activity, and Hg(II) sorption and uptake.

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Notes

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The authors declare no competing financial interest. The U.S. Department of Energy (DOE) will

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provide public access to these results of federally sponsored research in accordance with the

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DOE Public Access Plan (http://energy.gov/downloads/doepublic-access-plan).

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ACKNOWLEDGMENTS

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We thank Xiangping Yin for technical assistance in Hg(II) and MeHg analyses. This research

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was sponsored by US Department of Energy (DOE) Office of Science, Office of Biological and

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Environmental Research, as part of the Mercury Science Focus Area at the Oak Ridge National

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Laboratory (ORNL), which is managed by UT-Battelle, LLC under Contract No. DE-AC05-

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00OR22725 with DOE.

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FIGURE CAPTIONS:

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Figure 1. Methylmercury (MeHg) production by washed cells of D. desulfuricans ND132 at 2 h and 24 h in the presence of various divalent metal ions: (a) 50 nM and (b) 50 µM each. The initial added Hg(II) (as HgCl2) concentration was 25 nM, which was mixed first with the metal ion solution in PBS. Cells were added last at 5×108 cells mL-1. Error bars represent one standard deviation of replicate samples (3–5) from two independent batch experiments. An asterisk denotes a significant difference from the control (PBS only), p < 0.01, using pairwise comparison (Bonferroni test) in the one-way ANOVA.

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Figure 2. (a) Copper concentration-dependent Hg(II) methylation with washed cells of D. desulfuricans ND132 (5×108 cells/mL) in 24 h, and (b) MeHg production kinetics in the presence of Cu(II) at 0, 25, 75, and 500 nM under the same experimental conditions described in Figure 1. The Inset shows MeHg production in the first 8 h. Error bars in (a) represent one standard deviation of 6–8 replicate samples from three independent batches and (b) from 2–3 replicate samples. The statistical differences were tested at the 0.05 significance level by the one-way ANOVA (a) or two-way repeated measures ANOVA (b).

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Figure 3. Effects of Cu(II) ions on time-dependent Hg(II) uptake by washed cells of D. desulfuricans ND132 in PBS. Hg(II) uptake (or internalized) was determined by the difference between the total Hg(II) and the wash-off inorganic Hg(II) using a strong Hgchelator, 2,3-dimercapto-1-propanesulfonic acid (DMPS) at various time points. Other experimental conditions are the same as described in Figure 1. Error bars represent one standard deviation of 2–3 replicate samples. The statistical differences were tested at the 0.05 significance level by the two-way repeated measures ANOVA.

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