skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: An Improved hgcAB Primer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature

Abstract

The gene pair hgcAB is essential for microbial mercury methylation. Our understanding of its abundance and diversity in nature is rapidly evolving. In this study we developed a new broad-range primer set for hgcAB, plus an expanded hgcAB reference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy to hgcA sequences from clone, amplicon, and metagenomic datasets. We evaluated potential biases introduced in primer design, sequence length, and classification, and suggest best practices for studying Hg-methylator diversity. Our study confirms the emerging picture of an expanded diversity of HgcAB-encoding microbes in many types of ecosystems, with abundant putative mercury methylators Nitrospirae and Chloroflexi in several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB included Phycisphaerae, Aminicenantes, Spirochaetes, and Elusimicrobia. Combined with high-throughput amplicon specific sequencing, the new primer set also indentified novel hgcAB sequences similar to Lentisphaerae, Bacteroidetes, Atribacteria, and candidate phyla WOR-3 and KSB1 bacteria. Gene abundance data also corroborate the important role of two “classic” groups of methylators (Deltaproteobacteria and Methanomicrobia) in many environments, but generally show a scarcity of hgcAB+ Firmicutes. The new primer set was developed to specifically target hgcABmore » sequences found in nature, reducing degeneracy and providing increased sensitivity while maintaining broad diversity capture. We evaluated mock communities to confirm primer improvements, including culture spikes to environmental samples with variable DNA extraction and PCR amplification efficiencies. For select sites, this new workflow was combined with direct high-throughput hgcAB sequencing. The hgcAB diversity generated by direct amplicon sequencing confirmed the potential for novel Hg-methylators previously identified using metagenomic screens. A new phylogenetic analysis using sequences from freshwater, saline, and terrestrial environments showed Deltaproteobacteria HgcA sequences generally clustered among themselves, while metagenome-resolved HgcA sequences in other phyla tended to cluster by environment, suggesting horizontal gene transfer into many clades. HgcA from marine metagenomes often formed distinct subtrees from those sequenced from freshwater ecosystems. Overall the majority of HgcA sequences branch from a cluster of HgcAB fused proteins related to Thermococci, Atribacteria (candidate division OP9), Aminicenantes (OP8), and Chloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity of hgcAB+ microbes in nature.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1670477
Alternate Identifier(s):
OSTI ID: 1671431
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Published Article
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Name: Frontiers in Microbiology Journal Volume: 11; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Research Foundation
Country of Publication:
Switzerland
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; amplicon sequencing; hgcAB; primer development; mercury methylation; environmental microbiology; microbial diversity

Citation Formats

Gionfriddo, Caitlin M., Wymore, Ann M., Jones, Daniel S., Wilpiszeski, Regina L., Lynes, Mackenzie M., Christensen, Geoff A., Soren, Ally, Gilmour, Cynthia C., Podar, Mircea, and Elias, Dwayne A. An Improved hgcAB Primer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature. Switzerland: N. p., 2020. Web. https://doi.org/10.3389/fmicb.2020.541554.
Gionfriddo, Caitlin M., Wymore, Ann M., Jones, Daniel S., Wilpiszeski, Regina L., Lynes, Mackenzie M., Christensen, Geoff A., Soren, Ally, Gilmour, Cynthia C., Podar, Mircea, & Elias, Dwayne A. An Improved hgcAB Primer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature. Switzerland. https://doi.org/10.3389/fmicb.2020.541554
Gionfriddo, Caitlin M., Wymore, Ann M., Jones, Daniel S., Wilpiszeski, Regina L., Lynes, Mackenzie M., Christensen, Geoff A., Soren, Ally, Gilmour, Cynthia C., Podar, Mircea, and Elias, Dwayne A. Tue . "An Improved hgcAB Primer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature". Switzerland. https://doi.org/10.3389/fmicb.2020.541554.
@article{osti_1670477,
title = {An Improved hgcAB Primer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature},
author = {Gionfriddo, Caitlin M. and Wymore, Ann M. and Jones, Daniel S. and Wilpiszeski, Regina L. and Lynes, Mackenzie M. and Christensen, Geoff A. and Soren, Ally and Gilmour, Cynthia C. and Podar, Mircea and Elias, Dwayne A.},
abstractNote = {The gene pair hgcAB is essential for microbial mercury methylation. Our understanding of its abundance and diversity in nature is rapidly evolving. In this study we developed a new broad-range primer set for hgcAB, plus an expanded hgcAB reference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy to hgcA sequences from clone, amplicon, and metagenomic datasets. We evaluated potential biases introduced in primer design, sequence length, and classification, and suggest best practices for studying Hg-methylator diversity. Our study confirms the emerging picture of an expanded diversity of HgcAB-encoding microbes in many types of ecosystems, with abundant putative mercury methylators Nitrospirae and Chloroflexi in several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB included Phycisphaerae, Aminicenantes, Spirochaetes, and Elusimicrobia. Combined with high-throughput amplicon specific sequencing, the new primer set also indentified novel hgcAB sequences similar to Lentisphaerae, Bacteroidetes, Atribacteria, and candidate phyla WOR-3 and KSB1 bacteria. Gene abundance data also corroborate the important role of two “classic” groups of methylators (Deltaproteobacteria and Methanomicrobia) in many environments, but generally show a scarcity of hgcAB+ Firmicutes. The new primer set was developed to specifically target hgcAB sequences found in nature, reducing degeneracy and providing increased sensitivity while maintaining broad diversity capture. We evaluated mock communities to confirm primer improvements, including culture spikes to environmental samples with variable DNA extraction and PCR amplification efficiencies. For select sites, this new workflow was combined with direct high-throughput hgcAB sequencing. The hgcAB diversity generated by direct amplicon sequencing confirmed the potential for novel Hg-methylators previously identified using metagenomic screens. A new phylogenetic analysis using sequences from freshwater, saline, and terrestrial environments showed Deltaproteobacteria HgcA sequences generally clustered among themselves, while metagenome-resolved HgcA sequences in other phyla tended to cluster by environment, suggesting horizontal gene transfer into many clades. HgcA from marine metagenomes often formed distinct subtrees from those sequenced from freshwater ecosystems. Overall the majority of HgcA sequences branch from a cluster of HgcAB fused proteins related to Thermococci, Atribacteria (candidate division OP9), Aminicenantes (OP8), and Chloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity of hgcAB+ microbes in nature.},
doi = {10.3389/fmicb.2020.541554},
journal = {Frontiers in Microbiology},
number = ,
volume = 11,
place = {Switzerland},
year = {2020},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.3389/fmicb.2020.541554

Save / Share:

Works referenced in this record:

Mercury-methylating genes dsrB and hgcA in soils/sediments of the Three Gorges Reservoir
journal, December 2016

  • Du, Hongxia; Ma, Ming; Sun, Tao
  • Environmental Science and Pollution Research, Vol. 24, Issue 5
  • DOI: 10.1007/s11356-016-8213-9

Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations
journal, July 2019

  • Christensen, Geoff A.; Gionfriddo, Caitlin M.; King, Andrew J.
  • Environmental Science & Technology, Vol. 53, Issue 15
  • DOI: 10.1021/acs.est.8b06389

Challenges and opportunities for managing aquatic mercury pollution in altered landscapes
journal, January 2018


Overview of Mercury Methylation Capacities among Anaerobic Bacteria Including Representatives of the Sulphate-Reducers: Implications for Environmental Studies
journal, January 2009

  • Ranchou-Peyruse, M.; Monperrus, M.; Bridou, R.
  • Geomicrobiology Journal, Vol. 26, Issue 1, p. 1-8
  • DOI: 10.1080/01490450802599227

Microbial community structure with trends in methylation gene diversity and abundance in mercury-contaminated rice paddy soils in Guizhou, China
journal, January 2018

  • Vishnivetskaya, Tatiana A.; Hu, Haiyan; Van Nostrand, Joy D.
  • Environmental Science: Processes & Impacts, Vol. 20, Issue 4
  • DOI: 10.1039/c7em00558j

The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay
journal, January 2010

  • Stadhouders, Ralph; Pas, Suzan D.; Anber, Jeer
  • The Journal of Molecular Diagnostics, Vol. 12, Issue 1
  • DOI: 10.2353/jmoldx.2010.090035

Quantification of Mercury Bioavailability for Methylation Using Diffusive Gradient in Thin-Film Samplers
journal, June 2018

  • Ndu, Udonna; Christensen, Geoff A.; Rivera, Nelson A.
  • Environmental Science & Technology, Vol. 52, Issue 15
  • DOI: 10.1021/acs.est.8b00647

Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies
journal, July 2016

  • Gohl, Daryl M.; Vangay, Pajau; Garbe, John
  • Nature Biotechnology, Vol. 34, Issue 9
  • DOI: 10.1038/nbt.3601

Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis
journal, July 1999


Cognitive deficits at age 22 years associated with prenatal exposure to methylmercury
journal, January 2016


The assessment and remediation of mercury contaminated sites: A review of current approaches
journal, March 2020


Syntrophs Dominate Sequences Associated with the Mercury Methylation-Related Gene hgcA in the Water Conservation Areas of the Florida Everglades
journal, August 2014

  • Bae, Hee-Sung; Dierberg, Forrest E.; Ogram, Andrew
  • Applied and Environmental Microbiology, Vol. 80, Issue 20
  • DOI: 10.1128/aem.01666-14

MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data
journal, June 2016


Mercury Methylation by Novel Microorganisms from New Environments
journal, September 2013

  • Gilmour, Cynthia C.; Podar, Mircea; Bullock, Allyson L.
  • Environmental Science & Technology, Vol. 47, Issue 20
  • DOI: 10.1021/es403075t

Genomic reconstruction of a novel, deeply branched sediment archaeal phylum with pathways for acetogenesis and sulfur reduction
journal, January 2016

  • Seitz, Kiley W.; Lazar, Cassandre S.; Hinrichs, Kai-Uwe
  • The ISME Journal, Vol. 10, Issue 7
  • DOI: 10.1038/ismej.2015.233

Genomic resolution of linkages in carbon, nitrogen, and sulfur cycling among widespread estuary sediment bacteria
journal, April 2015


Trimmomatic: a flexible trimmer for Illumina sequence data
journal, April 2014


Robust Mercury Methylation across Diverse Methanogenic Archaea
journal, April 2018

  • Gilmour, Cynthia C.; Bullock, Allyson L.; McBurney, Alyssa
  • mBio, Vol. 9, Issue 2
  • DOI: 10.1128/mBio.02403-17

Hidden Markov model speed heuristic and iterative HMM search procedure
journal, August 2010


Ironing out the wrinkles in the rare biosphere through improved OTU clustering: Ironing out the wrinkles in the rare biosphere
journal, March 2010


Novel Microbial Assemblages Dominate Weathered Sulfide-Bearing Rock from Copper-Nickel Deposits in the Duluth Complex, Minnesota, USA
journal, June 2017

  • Jones, Daniel S.; Lapakko, Kim A.; Wenz, Zachary J.
  • Applied and Environmental Microbiology, Vol. 83, Issue 16
  • DOI: 10.1128/AEM.00909-17

Detection of a key Hg methylation gene, hgcA , in wetland soils : Detection of the Hg methylation gene, hgcA, in soils
journal, January 2014

  • Schaefer, J. K.; Kronberg, R. -M.; Morel, F. M. M.
  • Environmental Microbiology Reports, Vol. 6, Issue 5
  • DOI: 10.1111/1758-2229.12136

Database resources of the National Center for Biotechnology Information
journal, January 2009

  • Sayers, E. W.; Barrett, T.; Benson, D. A.
  • Nucleic Acids Research, Vol. 37, Issue Database
  • DOI: 10.1093/nar/gkn741

Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment
journal, July 2016

  • Christensen, Geoff A.; Wymore, Ann M.; King, Andrew J.
  • Applied and Environmental Microbiology, Vol. 82, Issue 19
  • DOI: 10.1128/aem.01271-16

Differential depth distribution of microbial function and putative symbionts through sediment-hosted aquifers in the deep terrestrial subsurface
journal, January 2018

  • Probst, Alexander J.; Ladd, Bethany; Jarett, Jessica K.
  • Nature Microbiology, Vol. 3, Issue 3
  • DOI: 10.1038/s41564-017-0098-y

The Impact of DNA Polymerase and Number of Rounds of Amplification in PCR on 16S rRNA Gene Sequence Data
journal, May 2019


Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.
journal, January 1994


Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sediment †
journal, January 1985


Marine mercury-methylating microbial communities from coastal to Capbreton Canyon sediments (North Atlantic Ocean)
journal, July 2020


The Genetic Basis for Bacterial Mercury Methylation
journal, February 2013


Global prevalence and distribution of genes and microorganisms involved in mercury methylation
journal, October 2015

  • Podar, Mircea; Gilmour, Cynthia C.; Brandt, Craig C.
  • Science Advances, Vol. 1, Issue 9
  • DOI: 10.1126/sciadv.1500675

Biotic formation of methylmercury: A bio–physico–chemical conundrum
journal, November 2019

  • Bravo, Andrea G.; Cosio, Claudia
  • Limnology and Oceanography, Vol. 65, Issue 5
  • DOI: 10.1002/lno.11366

Search and clustering orders of magnitude faster than BLAST
journal, August 2010


phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data
journal, April 2013


History of mercury use and environmental contamination at the Oak Ridge Y-12 Plant
journal, January 2011


RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies
journal, January 2014


Characterization of the Deltaproteobacteria in contaminated and uncontaminated stream sediments and identification of potential mercury methylators
journal, July 2012

  • Mosher, Jj; Vishnivetskaya, Ta; Elias, Da
  • Aquatic Microbial Ecology, Vol. 66, Issue 3
  • DOI: 10.3354/ame01563

The NCBI Taxonomy database
journal, December 2011


Mercury Methylation from Unexpected Sources: Molybdate-Inhibited Freshwater Sediments and an Iron-Reducing Bacterium
journal, January 2006


VSEARCH: a versatile open source tool for metagenomics
journal, January 2016


Periphyton Biofilms Influence Net Methylmercury Production in an Industrially Contaminated System
journal, September 2016

  • Olsen, Todd A.; Brandt, Craig C.; Brooks, Scott C.
  • Environmental Science & Technology, Vol. 50, Issue 20
  • DOI: 10.1021/acs.est.6b01538

Mercury Methylation by the Methanogen Methanospirillum hungatei
journal, August 2013

  • Yu, Ri-Qing; Reinfelder, John R.; Hines, Mark E.
  • Applied and Environmental Microbiology, Vol. 79, Issue 20
  • DOI: 10.1128/AEM.01556-13

PCR inhibitors - occurrence, properties and removal
journal, July 2012


Unraveling Microbial Communities Associated with Methylmercury Production in Paddy Soils
journal, October 2018

  • Liu, Yu-Rong; Johs, Alexander; Bi, Li
  • Environmental Science & Technology, Vol. 52, Issue 22
  • DOI: 10.1021/acs.est.8b03052

Methylmercury production in a Chesapeake Bay salt marsh: METHYLMERCURY PRODUCTION IN A SALT MARSH
journal, June 2008

  • Mitchell, Carl P. J.; Gilmour, Cynthia C.
  • Journal of Geophysical Research: Biogeosciences, Vol. 113, Issue G2
  • DOI: 10.1029/2008JG000765

Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities
journal, October 2009

  • Schloss, P. D.; Westcott, S. L.; Ryabin, T.
  • Applied and Environmental Microbiology, Vol. 75, Issue 23, p. 7537-7541
  • DOI: 10.1128/AEM.01541-09

Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis
journal, November 1997


Microbial mercury methylation in Antarctic sea ice
journal, August 2016


Modulators of mercury risk to wildlife and humans in the context of rapid global change
journal, January 2018


Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics
journal, February 2018

  • Ardui, Simon; Ameur, Adam; Vermeesch, Joris R.
  • Nucleic Acids Research, Vol. 46, Issue 5
  • DOI: 10.1093/nar/gky066

Genome insights of mercury methylation among Desulfovibrio and Pseudodesulfovibrio strains
journal, January 2020

  • Goñi-Urriza, Marisol; Klopp, Christophe; Ranchou-Peyruse, Magali
  • Research in Microbiology, Vol. 171, Issue 1
  • DOI: 10.1016/j.resmic.2019.10.003

The EMBL-EBI bioinformatics web and programmatic tools framework
journal, April 2015

  • Li, Weizhong; Cowley, Andrew; Uludag, Mahmut
  • Nucleic Acids Research, Vol. 43, Issue W1
  • DOI: 10.1093/nar/gkv279

pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree
journal, October 2010

  • Matsen, Frederick A.; Kodner, Robin B.; Armbrust, E. Virginia
  • BMC Bioinformatics, Vol. 11, Issue 1
  • DOI: 10.1186/1471-2105-11-538

MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004

  • Edgar, R. C.
  • Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
  • DOI: 10.1093/nar/gkh340

Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system
journal, October 2016

  • Anantharaman, Karthik; Brown, Christopher T.; Hug, Laura A.
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms13219

Analysis of the Microbial Community Structure by Monitoring an Hg Methylation Gene (hgcA) in Paddy Soils along an Hg Gradient
journal, February 2014

  • Liu, Y. -R.; Yu, R. -Q.; Zheng, Y. -M.
  • Applied and Environmental Microbiology, Vol. 80, Issue 9
  • DOI: 10.1128/aem.04225-13

Insights into the phylogeny and coding potential of microbial dark matter
journal, July 2013

  • Rinke, Christian; Schwientek, Patrick; Sczyrba, Alexander
  • Nature, Vol. 499, Issue 7459
  • DOI: 10.1038/nature12352

CD-HIT Suite: a web server for clustering and comparing biological sequences
journal, January 2010


Mechanisms Regulating Mercury Bioavailability for Methylating Microorganisms in the Aquatic Environment: A Critical Review
journal, February 2013

  • Hsu-Kim, Heileen; Kucharzyk, Katarzyna H.; Zhang, Tong
  • Environmental Science & Technology, Vol. 47, Issue 6
  • DOI: 10.1021/es304370g

Geobacteraceae are important members of mercury-methylating microbial communities of sediments impacted by waste water releases
journal, January 2018


Genome-wide selective sweeps and gene-specific sweeps in natural bacterial populations
journal, January 2016

  • Bendall, Matthew L.; Stevens, Sarah LR; Chan, Leong-Keat
  • The ISME Journal, Vol. 10, Issue 7
  • DOI: 10.1038/ismej.2015.241

Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons
journal, January 2011


Molecular evidence for novel mercury methylating microorganisms in sulfate-impacted lakes
journal, February 2019


A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform
journal, July 2017


Under-detection of endospore-forming Firmicutes in metagenomic data
journal, January 2015

  • Filippidou, Sevasti; Junier, Thomas; Wunderlin, Tina
  • Computational and Structural Biotechnology Journal, Vol. 13
  • DOI: 10.1016/j.csbj.2015.04.002