An improvedhgcABprimer 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 broadrange 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 Hgmethylator 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. 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. Contribution to the Field StatementThe gene pair hgcAB is 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 capturing hgcAB diversity are necessary for identifying the major environmental p...

Expanded Phylogenetic Diversity and Metabolic Flexibility of Mercury-Methylating Microorganisms

“…Gene gain/loss mediated through several horizontal gene transfer (HGT) events was previously suggested to underpin the sparse and divergent phylogenetic distribution of hgcAB ( 17 , 19 – 21 , 29 ). Metabolic functions can be horizontally transferred across diverse phyla through numerous mechanisms, such as phage transduction, transposon-mediated insertion of genomic islands or plasmid exchanges, direct conjugation, and/or gene gain/loss events.…”

“…Discordance between the HgcAB and ribosomal protein phylogenies suggests that nonspecific amplicon approaches and broad-range qPCR assays will not yield easily interpretable results. Although new broad-range hgcAB primers have been developed based on an updated reference database of hgcAB sequences ( 21 ), the apparent HGT dynamics of this gene pair makes assigning the taxonomy of resulting hits challenging. Instead, finer-scale primers of specific methylating groups could be constructed based on hgcAB sequences from assembled population genomes from specific environments.…”

“…This discovery has allowed for high-throughput identification of microorganisms with methylation potential in cultures or diverse environmental data sets based upon hgcAB sequence presence (17,20). Clone and amplicon sequencing has been used as a low-cost method to retrieve hgcAB sequences with sufficient sequencing depth to assess the abundance and diversity of the hgcAB gene pair in the environment (18,(21)(22)(23)(24)(25)(26). Broad-range and clade-specific quantitative PCR (qPCR) primers have been developed to screen for and quantify specific methylating groups in a given site (18,21,22).…”

Deltaproteobacteria and Spirochaetes-Like Bacteria Are Abundant Putative Mercury Methylators in Oxygen-Deficient Water and Marine Particles in the Baltic Sea

“…Only a fraction of these 3,215 genes correspond to hgcA and hgcB genes and we therefore performed a manual check of their amino acid sequences using the knowledge from the seminal paper of Parks et al (2013) that described unique motifs in hgcA (NVWCA(A/G/S)GK) and hgcB amino acid sequences (C(M/I)EC(G/S)(A/G)C). To ensure the reliability of the outputs obtained using the HMM profiles of hgcAB amino acid sequences from Podar et al (2015) to capture all hgcAB -like genes from the metagenomic dataset, we applied the same procedure using the HMM profiles provided by the ORNL hgcAB library ( Gionfriddo et al, 2020 ). The two procedures resulted in the recovery of the same hgcA genes and hgcAB gene clusters from the BARM dataset.…”

“…Recent advances in metagenomics have yielded new insights into the microbial taxonomic and functional diversity in various aquatic ecosystems (e.g., Mehrshad et al, 2016 ; Haro-Moreno et al, 2018 ; Nowinski et al, 2019 ). The approach has for example been applied to broadly assess the presence and diversity of genes central to biological Hg cycling in marine systems ( Podar et al, 2015 ; Gionfriddo et al, 2016 , 2020 ; Bowman et al, 2019 ; Lin et al, 2020 ; Tada et al, 2020 ; Villar et al, 2020 ). Podar et al (2015) only detected hgcAB genes in a few metagenomes from marine pelagic waters (seven out of 138 metagenomes) but highlighted that limited sequencing depths of these metagenomes could have hampered detection.…”