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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 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 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 size was confirmed for all hgcAB+ strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative primers (qPCR) 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 inmore » this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. Here, 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.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [2];  [2];  [1];  [1];  [1];  [3];  [2]; ORCiD logo [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Smithsonian Environmental Research Center, Edgewater, MD (United States)
  3. Univ. of Missouri, Columbia, MO (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1326544
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 82; Journal Issue: 19; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Hg SFA; Hg; methylmercury; hgcAB; primers; qPCR; metagenomics

Citation Formats

Christensen, Geoff A., Wymore, Ann M., King, Andrew J., Podar, Mircea, Hurt, Jr., Richard A., Santillan, Eugenio U., Soren, Ally, Brandt, Craig C., Brown, Steven D., Palumbo, Anthony V., Wall, Judy D., Gilmour, Cynthia C., and Elias, Dwayne A. Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment. United States: N. p., 2016. Web. https://doi.org/10.1128/AEM.01271-16.
Christensen, Geoff A., Wymore, Ann M., King, Andrew J., Podar, Mircea, Hurt, Jr., Richard A., Santillan, Eugenio U., Soren, Ally, Brandt, Craig C., Brown, Steven D., Palumbo, Anthony V., Wall, Judy D., Gilmour, Cynthia C., & Elias, Dwayne A. Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment. United States. https://doi.org/10.1128/AEM.01271-16
Christensen, Geoff A., Wymore, Ann M., King, Andrew J., Podar, Mircea, Hurt, Jr., Richard A., Santillan, Eugenio U., Soren, Ally, Brandt, Craig C., Brown, Steven D., Palumbo, Anthony V., Wall, Judy D., Gilmour, Cynthia C., and Elias, Dwayne A. Fri . "Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment". United States. https://doi.org/10.1128/AEM.01271-16. https://www.osti.gov/servlets/purl/1326544.
@article{osti_1326544,
title = {Development and validation of broad-range qualitative and clade-specific quantitative molecular probes for assessing mercury methylation in the environment},
author = {Christensen, Geoff A. and Wymore, Ann M. and King, Andrew J. and Podar, Mircea and Hurt, Jr., Richard A. and Santillan, Eugenio U. and Soren, Ally and Brandt, Craig C. and Brown, Steven D. and Palumbo, Anthony V. and Wall, Judy D. and Gilmour, Cynthia C. and Elias, Dwayne A.},
abstractNote = {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 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 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 size was confirmed for all hgcAB+ strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative primers (qPCR) 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. Here, 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.},
doi = {10.1128/AEM.01271-16},
journal = {Applied and Environmental Microbiology},
number = 19,
volume = 82,
place = {United States},
year = {2016},
month = {7}
}

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