An ImprovedhgcABPrimer Set and Direct High-Throughput Sequencing Expand Hg-Methylator Diversity in Nature

The 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.Combined with high-throughput amplicon specific sequencing, the new primer set also indentified novelhgcABsequences similar toLentisphaerae,Bacteroidetes,Atribacteria, and candidate phyla WOR-3 and KSB1 bacteria. 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.