Syntrophs dominate sequences associated with the mercury methylation-related gene hgcA in the water conservation areas of the Florida Everglades

Abstract

The mechanisms and rates of mercury methylation in the Florida Everglades are of great concern because of potential adverse impacts on human and wildlife health through mercury accumulation in aquatic food webs. We developed a new PCR primer set targeting hgcA, a gene encoding a corrinoid protein essential for Hg methylation across broad phylogenetic boundaries, and used this primer set to study the distribution of hgcA sequences in soils collected from three sites along a gradient in sulfate and nutrient concentrations in the northern Everglades. The sequences obtained were distributed in diverse phyla, including Proteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia; however, hgcA clone libraries from all sites were dominated by sequences clustering within the order Syntrophobacterales of the Deltaproteobacteria (49 to 65% of total sequences). dsrB mRNA sequences, representing active sulfate-reducing prokaryotes at the time of sampling, obtained from these sites were also dominated by Syntrophobacterales (75 to 89%). Laboratory incubations with soils taken from the site low in sulfate concentrations also suggested that Hg methylation activities were primarily mediated by members of the order Syntrophobacterales, with some contribution by methanogens, Chloroflexi, iron-reducing Geobacter, and non-sulfate-reducing Firmicutes inhabiting the sites. This suggests that prokaryotes distributed within clades defined by syntrophs are the predominant group controlling methylation of Hg in low-sulfate areas of the Everglades. Any strategy for managing mercury methylation in the Everglades should consider that net mercury methylation is not limited to the action of sulfate reduction.

FIG 1

Maximum likelihood phylogenetic tree representing phylogenetic distribution of hgcA-based OTUs from soil samples at three Everglades sites. Clade colors black, gray, and white represent clades composed of only reference sequences from GenBank, reference sequences and our sequences, and our sequences only, respectively. Taxon names inferred from reference sequences were presented for individual clades, with the number of OTUs included in the clades. Reference sequences and representative OTUs in each clade are provided in Table S3 in the supplemental material. Bootstrap values higher than 50% of 1,000 reassemblages are placed at branch points. Bar represents 0.1 substitution per deduced amino acid sequences of hgcA. Pie charts show relative proportions of major clades within clone libraries. ChFlx, Chloroflexi; FIRM, Firmicutes; EuArch, Euryarchaeota.

FIG 2

Phylogenetic distribution of dsrB mRNA sequences from soil samples at three Everglades sites. Maximum likelihood phylogenetic trees were generated using deduced amino acid sequences from the dsrB transcripts. Bar represents 0.2 substitution per deduced amino acid sequences of dsrB. The taxon name in each clade was inferred from the reference sequences inside the clade (detailed reference sequences are provided in Table S4 in the supplemental material). The clades containing only our sequences or together with known reference sequences are indicated by white or gray, respectively; clades containing none of our sequences are shown in black. Pie charts show relative proportions of major clades within clone libraries.

FIG 3

Methylmercury (MeHg) production, sulfate (SO₄²⁻) consumption and the transcript concentration of mcrA and dsrB during the laboratory incubations of WCA-3A soils with and without specific inhibitors. All controls and treatments received 139 ng liter⁻¹ of Hg²⁺ as HgCl₂ at the beginning of the incubation. D0, D7, and D14 represent the initial, day 7, and day 14 concentrations, respectively, of MeHg and sulfate in the water column. The transcript concentrations are from the soil after 14 days of incubation. CT, control (no inhibitors added); Mo, 20 mM molybdate (MoO₄²⁻); BES, 50 mM bromoethanesulfonate; ND, not determined; ns, no sample. Error bars represent the standard errors from triplicate incubation vessels.