An improvedhgcABprimer set and direct high-throughput sequencing expand Hg-methylator diversity in nature
Authors: 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. 
Published: March 2020

AbstractThe gene pairhgcABis 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 forhgcAB, plus an expandedhgcABreference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy tohgcAsequences 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 methylatorsNitrospiraeandChloroflexiin several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB includedPhycisphaerae, Aminicenantes, Spirochaetes, andElusimicrobia. Gene abundance data also corroborate the important role of two “classic” groups of methylators (DeltaproteobacteriaandMethanomicrobia) in many environments, but generally show a scarcity ofhgcAB+Firmicutes. The new primer set was developed to specifically targethgcABsequences 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-throughputhgcABsequencing. ThehgcABdiversity 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 showedDeltaproteobacteriaHgcA 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 toThermococci, Atribacteria(candidate division OP9),Aminicenantes(OP8), andChloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity ofhgcAB+ microbes in nature.Contribution to the Field StatementThe gene pairhgcABis essential for microbial production of the neurotoxin methylmercury. In recent years these genes have been used as biomarkers to determine the potential of a microbiome to generate methylmercury via PCR amplification using degenerate primers from several research groups. However, improved techniques for capturinghgcABdiversity are necessary for identifying the major environmental producers of the neurotoxin as well as the expanding diversity of novel putative methylators, and the genes’ evolutionary history. The work described herein advanceshgcABdetection in environmental samples through an updated primer set coupled with a direct high-throughput sequencing method that enables broader diversity capture. We provide an expandedhgcABsequence reference library that allows for more sensitive and robust estimations of Hg-methylator diversity and potential for MeHg generation in the environment. ThehgcABdiversity generated by high-throughput sequencing confirms the potential for novel Hg-methylators previously only identified using metagenomic screens. This study provides a significantly improved assessment of the abundance and diversity ofhgcAB+ microbes in nature. By expanding our understanding of the microbial metabolic clades associated with mercury methylation, this work improves our ability to predict environmental conditions that drive production and accumulation of the neurotoxin in aquatic ecosystems.