title: 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 IMPORTANCE: The 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 cultivation-independent strategy.
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Geoff A
authors.initials: GA
authors.lastname: Christensen
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Ann M
authors.initials: AM
authors.lastname: Wymore
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: King
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Mircea
authors.initials: M
authors.lastname: Podar
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Hurt
authors.affiliation: Smithsonian Environmental Research Center, Edgewater, Maryland, USA.
authors.forename: Eugenio U
authors.initials: EU
authors.lastname: Santillan
authors.affiliation: Smithsonian Environmental Research Center, Edgewater, Maryland, USA.
authors.forename: Ally
authors.initials: A
authors.lastname: Soren
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Craig C
authors.initials: CC
authors.lastname: Brandt
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Brown
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
authors.forename: Anthony V
authors.initials: AV
authors.lastname: Palumbo
authors.affiliation: University of Missouri, Columbia, Missouri, USA.
authors.forename: Judy D
authors.initials: JD
authors.lastname: Wall
authors.affiliation: Smithsonian Environmental Research Center, Edgewater, Maryland, USA.
authors.forename: Cynthia C
authors.initials: CC
authors.lastname: Gilmour
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA eliasda@ornl.gov.
authors.forename: Dwayne A
authors.identifier: 0000-0002-4469-6391
authors.initials: DA
authors.lastname: Elias
mesh_terms: D001105:Archaea / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D020564:Deltaproteobacteria / Q000235:genetics / Q000378:metabolism; D004784:Environmental Monitoring / Q000379:methods*; D000068536:Bacillota / Q000235:genetics / Q000378:metabolism; D019015:Geologic Sediments / Q000382:microbiology; D008628:Mercury / Q000378:metabolism*; D008745:Methylation; D008767:Methylmercury Compounds / Q000378:metabolism*; D015336:Molecular Probe Techniques / Q000592:standards*; D015335:Molecular Probes / Q000235:genetics / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D008767:Methylmercury Compounds; D015335:Molecular Probes; D008628:Mercury