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The Genetic Basis for Bacterial Mercury Methylation

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Science  15 Mar 2013:
Vol. 339, Issue 6125, pp. 1332-1335
DOI: 10.1126/science.1230667

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.

Abstract

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 (Hg) is a pervasive global pollutant; in the form of methylmercury (CH3Hg+), it bioaccumulates in the food web and is highly toxic to humans and other organisms (1). Unlike inorganic forms of Hg, which originate from atmospheric deposition and point discharges, methylmercury is generated in the environment predominantly by anaerobic microorganisms (2). Sulfate-reducing bacteria are the main producers of CH3Hg+ (3, 4), although iron-reducing bacteria (57) and methanogens (8, 9) can also be involved.

Production of CH3Hg+ by the model methylating bacteria Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA involves cellular uptake of Hg(II) by active transport, methylation of Hg(II) in the cytosol, and export of CH3Hg+ from the cell (10). Hg methylation is an enzyme-catalyzed process proposed to be associated with the reductive acetyl–coenzyme A (CoA) pathway [also called the Wood-Ljungdahl pathway (11)] and potentially linked to corrinoid proteins involved in this pathway (12). However, no direct evidence firmly connects the acetyl-CoA pathway and the ability of bacteria to methylate Hg (13). Furthermore, phylogenetic analyses have not revealed any distinctive trends or clustering of methylating versus nonmethylating microorganisms (1416).

To understand the genetic and biochemical basis of microbial Hg methylation, we analyzed the genomes of methylating and nonmethylating bacteria in the context of biochemical pathways involved in single-carbon metabolism. The well-characterized corrinoid iron-sulfur protein (CFeSP) is known to transfer methyl groups to a NiFeS cluster in acetyl-CoA synthase (17). Therefore, recognizing that a corrinoid protein associated with the acetyl-CoA pathway could be required for Hg methylation, we reasoned that a protein similar to CFeSP might transfer a methyl group to a Hg substrate to yield CH3Hg+, and that genes encoding such a protein should be recognizable in the genome sequences of Hg-methylating bacteria. Complete genome sequences are available for six methylating and eight closely related nonmethylating bacterial species (tables S1 and S2). Furthermore, molecular structures and functions have been determined for various enzymes of the reductive acetyl-CoA pathway, including CFeSP from Moorella thermoacetica (18, 19) and Carboxydothermus hydrogenoformans (20, 21). Accordingly, we performed a BLASTP search with the sequence of the large subunit of CFeSP (CfsA, locus tag CHY_1223) from C. hydrogenoformans Z-2901 against the translated genome sequence of D. desulfuricans ND132 (22). Sequence similarity was found between the C-terminal corrinoid-binding domain of CfsA and the N terminus of DND132_1056 (fig. S1), although DND132_1056 lacks both the TIM barrel domain and the C-terminal [4Fe-4S] binding motif of CfsA. The C-terminal region showed no detectable similarity to any proteins of known structure, but exhibited features characteristic of a transmembrane domain (fig. S2).

We also performed comparative genomic analyses of known Hg methylators and nonmethylators on the basis of Pfam classifications (23), with an emphasis on enzyme families known to be involved in methyl transfer reactions. The distribution of Pfam domains in the genomes is heterogeneous and, for the most part, does not coincide with the mercury methylation phenotype (table S3). However, the distribution of proteins of the CdhD family (PF03599, annotated as CO dehydrogenase/acetyl-CoA synthase delta subunit) encoded in the genomes correlates with the ability or inability of those organisms to methylate mercury. DND132_1056 is annotated as encoding a CdhD member, as are its close relatives in all five other confirmed methylators.

Analysis of the genomic context in the confirmed Hg methylators revealed genes similar to both the putative corrinoid protein–encoding gene and an additional, ferredoxin-like gene located downstream, which suggests that these two genes might be coexpressed and functionally related (Fig. 1). In D. desulfuricans ND132, the annotated coding sequences of the two genes (DND132_1056 and DND132_1057) are on the same strand and are separated by only 14 base pairs. Similar gene pairs were found in the genomes of 52 organisms with sequence translations available in public databases (table S4). The two genes are present in all sequenced, confirmed methylators and absent in the sequenced, confirmed nonmethylators. The other 46 organisms in which the genes are present have not been tested for Hg methylation (table S4).

Fig. 1

Putative mercury methylation gene cluster and genomic context for six confirmed mercury methylators with sequenced genomes.

We hypothesized that these two genes are key components of the bacterial Hg methylation pathway, with the putative corrinoid protein facilitating methyl transfer and the ferredoxin carrying out corrinoid reduction. Therefore, we deleted these genes individually, and also together, from D. desulfuricans ND132 (supplementary text). Additionally, we deleted the orthologs GSU1440 and GSU1441 together, and GSU1440 individually, from G. sulfurreducens PCA. In both of these organisms, CH3Hg+ production decreased in the deletion mutants by >99% relative to the parental strains (Fig. 2). Complementation of the two-gene deletions by reincorporation of the genes into the chromosomes restored 26% and 87% of the wild-type methylation activity in D. desulfuricans ND132 and G. sulfurreducens PCA, respectively, as measured by inductively coupled plasma mass spectrometry (ICP-MS) (Fig. 2). Deletion of DND132_1057 alone yielded <0.2% of wild-type methylation activity, and subsequent complementation showed 97% methylation activity. Complementation of either gene alone into the double deletion mutant did not restore detectable methylation activity (Fig. 2). Restoration of ΔDND132_1056 was not performed. Although the relative location of the two genes is consistent with cotranscription, reverse transcription polymerase chain reaction confirmed the transcription of DND132_1056 in the ΔDND132_1057 strain and DND132_1057 in the ΔDND132_1056 mutant (fig. S3).

Fig. 2

Production of methylmercury by D. desulfuricans ND132 and G. sulfurreducens PCA, by deletions of hgcA, hgcB, and hgcAB, and by complements in both bacteria. CH3Hg+ concentrations (ng/liter) were determined after overnight incubations and measured by ICP-MS. The prefix Δ indicates a gene deletion; the symbol :: indicates complementation by chromosomal insertion. Values plotted are the average CH3Hg+ concentrations detected per strain from triplicate assays; error bars denote SD. Note that ΔPCA_hgcAB complemented with PCA_hgcA+ or PCA_hgcB+ is still deleted for PCA_hgcB or PCA_hgcA, respectively.

The above findings are consistent with both genes being required for Hg methylation activity, although other unidentified genes are also likely to be involved. Hereafter, we refer to the DND132_1056 gene and its inferred orthologs as hgcA, encoding putative corrinoid proteins required for CH3Hg+ production, and DND132_1057 and its inferred orthologs as hgcB, encoding putative corrinoid protein–associated 2[4Fe-4S] ferredoxins. To determine whether gene loss impairs metabolism on a more general scale, we obtained comparative growth curves. The deletion mutants showed no impairment in rate or extent of growth (fig. S4). Thus, under the conditions tested, the construction of the deletions did not cause major growth aberrations that might interfere with the detection of methylation activity. The native functions of hgcA and hgcB remain unknown, but these genes are not essential for cell survival or proliferation.

The above findings merit some mechanistic considerations. The requirement for HgcA and HgcB in methylation is largely consistent with a previously proposed Hg methylation pathway by Bartha and co-workers (11), which we revise (fig. S5 and supplementary text). The methyl group in CH3Hg+ originates from CH3-H4-folate in D. desulfuricans LS (11) and is likely first transferred (as CH3+) to cob(I)alamin-HgcA to form CH3-cob(III)alamin-HgcA. This step may be catalyzed by a folate-binding methyltransferase similar to the CH3-H4-folate:CFeSP methyltransferase (MeTr) from the reductive acetyl-CoA pathway (24, 25), or by an unknown enzyme (fig. S5 and supplementary text). The high affinity of thiolate ligands for Hg2+ [formation constants for Hg(SR)2, log K = 40 to 43] (26) suggests that a possible substrate for HgcA could be a Hg(II) bis-thiolate complex involving either free cellular thiols or cysteine residues from a protein. Methyl transfer from CH3-cob(III)alamin-HgcA to a Hg substrate likely involves either CH3· or CH3. Although transfer of a carbanion from methylcobalamin to Hg(II) nonenzymatically is known to occur (27, 28), enzymatic transfer of CH3 by a corrinoid protein has never been observed.

Further sequence analysis of the 52 HgcA orthologs (Fig. 3) revealed a highly conserved motif, Asn-(Val/Ile)-Trp-Cys-Ala-(Ala/Gly)-Gly-Lys, in the region of highest similarity to the CfsA subunit of CFeSP. This region corresponds to the cap helix of CFeSP, which is located near the lower axial face of the corrin ring (20). In all HgcA sequences, a strictly conserved cysteine (Cys93 in D. desulfuricans ND132) occupies the position corresponding to Thr374 in CFeSP from C. hydrogenoformans Z-2901 (Fig. 3 and fig. S1). Although Thr374 is not considered a ligand for Co (19, 20), a cysteine might coordinate to Co, depending on its location relative to the cofactor and the Co oxidation state. Homology modeling (fig. S6) and ultraviolet-visible spectra (fig. S7) of the cobalamin-binding domain of HgcA suggest that Co-S coordination may be present in HgcA. Lower axial coordination of alkyl-cob(III)alamin by a biological thiolate has been proposed previously (29) but has never been observed for a corrinoid protein. The likely role of the ferredoxin-like protein HgcB is to accomplish the thermodynamically difficult reduction of Co(II) to Co(I) required for turnover, consistent with a previously suggested need for a ferredoxin as a reductant in Hg methylation (11). The mechanistic details of methyl transfer (supplementary text), the integration of these two gene products into carbon and energy metabolism, and their functioning with other potential, as yet unidentified, proteins remain to be determined.

Fig. 3

Multiple sequence alignments of 52 HgcA and HgcB orthologs from bacteria and archaea, including six confirmed methylating bacteria (arrowheads). Red boxes indicate highly conserved regions including the putative cap helix [consensus sequence motif, N(V/I)WCA(A/G)GK] in HgcA, two strictly conserved CX2CX2CX3C motifs characteristic of [4Fe-4S] clusters, and a conserved vicinal pair of cysteines located at the C terminus of HgcB. Abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr; X, any amino acid.

In the absence of genome sequences for all Hg-methylating organisms, the generality of the present findings cannot yet be ascertained. However, our interpretation is in agreement with all currently available sequence information for methylating bacteria and archaea. The presence of the hgcAB cluster in the genomes of several sequenced, but so far untested, microorganisms (table S4) leads us to hypothesize that these organisms are also capable of methylating mercury. The gene cluster appears to be quite sporadically distributed across two phyla of bacteria (Proteobacteria and Firmicutes) and one phylum of archaea (Euryarchaeota). Organisms possessing the two-gene cluster include 24 strains of Deltaproteobacteria, 16 Clostridia, 1 Negativicutes, and 11 Methanomicrobia. Interestingly, we also found these genes in a psychrophile (30), in a thermophile (31), and in a human commensal methanogen (32) (Fig. 3). The sparse phylogenetic gene distribution of the hgcAB system may be due to gene loss or lateral gene transfer (or both) across distant taxa and may be linked to environmental and community-structure factors. The sporadic distribution of these genes and the lack of an obvious selective advantage related to mercury toxicity (15) raise important questions regarding their physiological roles. Identification of these genes is a critical step linking specific microorganisms and environmental factors that influence microbial Hg methylation in aquatic ecosystems.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1230667/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S9

Tables S1 to S8

References (3369)

References and Notes

  1. U.S. EPA, Method 1630 (2001).
  2. U.S. EPA, Method 1631 (2002).
  3. Acknowledgments: We thank S. Miller, C. Gilmour, and T. Barkay for helpful discussions, and K. Rush, X. Yin, G. Christensen, and Q. Gui for experimental assistance. Supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, through the Mercury Scientific Focus Area Program at Oak Ridge National Laboratory (ORNL). ORNL is managed by UT Battelle, LLC, for DOE under contract DE-AC05-00OR22725. All other data are available online in the supplementary materials. Author contributions: J.M.P., A.J., R.B., J.C.S., A.V.P., D.A.E., S.D.B., M.P., J.D.W., and L.L. designed the research. M.P., S.D.B., and C.C.B. performed the comparative genomic analyses. A.J. and J.M.P. performed the bioinformatics and biochemical interpretations. R.B., R.A.H., S.D.S., S.J.T., A.J., and Y.Q. performed the experiments. J.M.P., A.J., R.B., J.C.S., D.A.E., J.D.W., and L.L. wrote the paper.
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