Universal qualitative and clade specific quantitative (Deltaproteobacteria, Firmicutes, or methanogenic Archaea) DNA and mRNA primers were developed to determine the species involved in Hg-methylation and to quantify the genes for each clade, respectively, since each has shown a different level of methylation in the lab. All primers were validated on pure bacterial strains that methylate as positive controls and those that do not methylate as negative controls. Environmental samples were further used to validate the primers and to determine corrective calculations for DNA extraction and polymerase chain reaction (PCR) amplification efficiencies.
These primers are a substantial improvement over earlier reports in that both qualitative and quantitative primers were developed by the same team, and the quantitative primers take into consideration the different degrees of methylation potential for each clade which ranges from ~10% in the Archaea to ~90% in some Deltaproteobacterial species. These findings, combined with the determination of the corrective factors for DNA extraction and PCR amplification will now allow for a more realistic picture to be generated regarding the possible levels of methylmercury generation that may occur in a given environment. These data can now be used for more accurate risk management assessments and can be more confidently used in the generation of hydrobiogeochemical models of methylmercury generation.
Two genes, hgcA and hgcB, are essential for microbial mercury (Hg)-methylation. Detection and estimation of their abundance in conjunction with quantification of Hg species and other geochemical factors is 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 broad-range primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea. A distinct PCR product at the expected ~950 base pair size was confirmed for all hgcAB+ strains tested and validated via Sanger sequencing. Additionally, clade-specific, degenerate quantitative primers (qPCR) targeted hgcA in the three dominant Hg-methylating clades (Deltaproteobacteria, Firmicutes and Archaea) using the same cultures and resulted in amplification of hgcA from 64%, 88%, and 86% of the tested species, respectively. Amplification was specific for the designed clade using the tested conditions. Sensitivity was lower in the methanogens due to a lack of sequence conservation. The two matrices were amended with representative strains from the three clades in equimolar ratios to demonstrate the qPCR primer specificity and sensitivity in complex environments.
Christensen, G.A., A. M. Wymore, A. J. King, M. Podar, R. A. Hurt Jr., E. U. Santillan, A. Soren, C. C. Brandt, S. D. Brown, A. V. Palumbo, J. D. Wall, C. C. Gilmour, and D. A. Elias. Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment. Applied Environmental Microbiology. Accepted manuscript posted online 15 July 2016. DOI:10.1128/ AEM.01271-16