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 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 ...

Fecal Methylmercury Correlates With Gut Microbiota Taxa in Pacific Walruses (Odobenus rosmarus divergens)

“…While hgcAB has been reported in Bacteria, it has also been found in multiple Archaea (including the gut commensal, M. luminyensis ) ( Parks et al, 2013 ), and thus some potentially important microbes, relevant to Hg metabolism, were not likely represented in the sequence dataset. There was an insufficient mass of remaining fecal sample for analyses of the hgcAB gene cluster ( Gionfriddo et al, 2020 ) or MeHg demethylation ( Lu et al, 2016 ); however, these assays will be included in future studies.…”

Microbial Diversity and Mercury Methylation Activity in Periphytic Biofilms at a Run-of-River Hydroelectric Dam and Constructed Wetlands

“…More recently, a combination of methylation assays using metabolic inhibitors and molecular genetic surveys of microbial assemblages broadened the diversity of putative Hg methylators to include iron-reducing bacteria (FeRB) (15) and methanogens (16). The discovery of the genetic basis for Hg methylation (hgcA and hgcB genes that encode a corrinoid iron-sulfur protein and a ferredoxin protein, respectively) (17) has facilitated studies that have expanded the diversity of putative Hg methylators and explored their distributions in the environment (18)(19)(20)(21)(22). Numerous additional bacterial phyla are now known to be implicated in MMHg production, including Actinobacteria, Bacteroidetes, Firmicutes, and Chloroflexi (17,18,(23)(24)(25).…”

Nutrient Exposure Alters Microbial Composition, Structure, and Mercury Methylating Activity in Periphyton in a Contaminated Watershed

“…The bacterial phylum Proteobacteria has previously been found to dominate periphyton ( Bae et al, 2019 ) but not Atribacteria. Sediment samples collected from the same study site (EFPC) also found an abundance of Proteobacteria, but rather than Candidatus Atribacteria, the sediment community had an abundance of Nitrospirae and Chloroflexi that dominated the Hg-methylating community ( Gionfriddo et al, 2020 ) Candidatus Atribacteria are a ubiquitous and difficult to cultivate phylum ( Katayama et al, 2020 ). Given the vast interaction networks the periphyton microbiome demonstrated, networks of the periphyton may aid in future isolation endeavors of these taxa as well as other difficult-to-cultivate microorganisms.…”

“…After extraction, a two-step PCR approach was used with barcode tagged templates and primers targeting the V4 region of the 16S rRNA gene for archaea and bacteria and the ITS2 region of the ribosomal operon for fungi using pooled primer sets ( Supplementary Table S1 ) to increase coverage of archaeal, bacterial, and fungal marker genes ( Cregger et al, 2018 ). Hg methylation genes ( hgcAB ) were amplified with a two-step PCR following methods and primers ( Supplementary Table S1 ) previously described ( Gionfriddo et al, 2020 ). After PCRs, all experimental units were pooled equally and purified with Agencourt AMPure XP beads (0.7:1 bead-to-DNA ratio; Beckman Coulter Inc., Pasadena, CA, United States).…”

“…hgcAB sequences were filtered and trimmed to 201-nt base pairs using Trimmomatic ( Bolger et al, 2014 ) and dereplicated, and singletons and chimeras were removed with VSEARCH ( Rognes et al, 2016 ). SVs were generated with dada2 and taxonomy was assigned using the reference package ORNL_HgcA_201.refpkg ( Gionfriddo et al, 2020 ).…”