Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment

Abstract

Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB ؉ strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB. IMPORTANCEThe neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair, hgcA and hgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivationindependent strategy. T he trace metal mercury (Hg) is a widespread global pollutant with a Ͼ4-fold increase in atmospheric emissions since antiquity (1). While all forms of Hg are toxic, the neurotoxin monomethyl Hg (MeHg) poses the most health risks due to its toxicity and biomagnification in food webs (2). MeHg production in nature is predominantly microbial (3) and is limited to strictly anaerobic microorganisms (4, 5). Methylation has commonly been tied to microbial sulfate reduction (6-8), but only a subset of sulfate-reducing microbes are capable of Hg methylation (4, 9). Hg methylation has also...

Distribution of mercury‐cycling genes in the Arctic and equatorial Pacific Oceans and their relationship to mercury speciation

“…For PCR, Platinum Taq DNA polymerase (Invitrogen) was used in a 20 μ L reaction with 1 μ mol L −1 of forward primer ORNL‐HgcAB‐uni‐F, 1 μ mol L −1 reverse primer ORNL‐HgcAB‐uni‐R, 1.5 mmol L −1 MgCl 2 , 0.2 mmol L −1 dNTPs, 10 ng gDNA template, and 10 6 copies of a positive control. Reaction conditions from Christensen et al () were followed, with one exception: the initial denaturation temperature was lowered to 95°C. Amplification of 10 5 copies of hgcAB from G. sulfurreducens in gDNA template was achieved, indicating no matrix interference ( n = 3).…”

Recent advances in the study of mercury methylation in aquatic systems

“…However, they were unable to relate gene expression to potential methylation rates. Although in its infancy, with the advent of new molecular techniques, including the recent development of clade-specific quantitative molecular probes (Christensen et al 2016), future studies should Until recently, research has focused on one or two environmental factors as a means of understanding the controls of MeHg production. Recent developments of the use of stable isotope tracers and geonomic work have begun to examine the synergies of multiple factors.…”

Mercury geochemistry and microbial diversity in meromictic Glacier Lake, Jamesville, NY

“…There were not enough hgcA sequences to capture the entire diversity of microbes containing hgc A due to a combination of known primer biases (the primers used primarily capture Proteobacteria (Christensen et al . ; Schaefer et al . ) and low cloning success.…”