Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations

Abstract

Methylmercury (MeHg) is a bioaccumulative toxic contaminant in many ecosystems, but factors governing its production are poorly understood. Recent work has shown that the anaerobic microbial conversion of mercury (Hg) to MeHg requires the Hg-methylation genes hgcAB and that these genes can be used as biomarkers in PCR-based estimators of Hg-methylator abundance. In an effort to determine reliable methods for assessing hgcA abundance and diversity and linking them to MeHg concentrations, multiple approaches were compared including metagenomic shotgun sequencing, 16S rRNA gene pyrosequencing and cloning/sequencing hgcAB gene products. Hg-methylator abundance was also determined by quantitative hgcA qPCR amplification and metaproteomics for comparison to the above measurements. Samples from eight sites were examined covering a range of total Hg (HgT; 0.03–14 mg kg–1 dry wt. soil) and MeHg (0.05–27 μg kg–1 dry wt. soil) concentrations. In the metagenome and amplicon sequencing of hgcAB diversity, the Deltaproteobacteria were the dominant Hg-methylators while Firmicutes and methanogenic Archaea were typically ∼50% less abundant. This was consistent with metaproteomics estimates where the Deltaproteobacteria were steadily higher. The 16S rRNA gene pyrosequencing did not have sufficient resolution to identify hgcAB + species. Metagenomic and hgcAB results were similar for Hg-methylator diversity and clade-specific qPCR-based approaches for hgcA are only appropriate when comparing the abundance of a particular clade across various samples. Weak correlations between Hg-methylating bacteria and soil Hg concentrations were observed for similar environmental samples, but overall total Hg and MeHg concentrations poorly correlated with Hg-cycling genes.

An improvedhgcABprimer set and direct high-throughput sequencing expand Hg-methylator diversity in nature

“…We still know relatively little about the distribution of Hg-methylating organisms in the environment, although the discovery of the hgcAB gene pair conferring Hg-methylation capability has greatly improved their identification (Parks et al, 2013;Bravo and Cosio, 2019). Mercury methylation is a species (and possibly strain)-specific trait that is not well predicted by 16S rRNA taxonomic classification (Ranchou-Peyruse et al, 2009;Gilmour et al, 2013;Yu et al, 2013;Gilmour et al, 2018;Christensen et al, 2019). The presence or abundance of groups of microbes that include Hg methylators is not predictive of the abundance of hgcAB + microbes.…”

“…In addition to the Hg-methylators confirmed in culture, sulfate-and iron-reducing Deltaproteobacteria, acetogenic and syntrophic Firmicutes, and methanogenic Archaea (Compeau and Bartha, 1985;Fleming et al, 2006;Kerin et al, 2006;Ranchou-Peyruse et al, 2009;Gilmour et al, 2013;Yu et al, 2013;Gilmour et al, 2018;Goñi-Urriza et al, 2019)genomic analyses have identified hgcAB (and thus inferred the ability to produce MeHg) in many other lineages including Chloroflexi, Chrysiogenetes, Aminicenantes (Candidate Phylum OP8), Atribacteria (Candidate Phylum OP9), Nitrospira, Nitrospina, Elusimicrobia, Thermotogae and Spirochaetes (Podar et al, 2015a;Gionfriddo et al, 2016;Liu et al, 2018a;Bowman et al, 2019;Christensen et al, 2019;Jones et al, 2019). The occurrence of hgcAB in Bacterial and Archaeal genomes is rare but widely dispersed across phyla (Parks et al, 2013;Podar et al, 2015a;Jones et al, 2019).…”

“…The occurrence of hgcAB in Bacterial and Archaeal genomes is rare but widely dispersed across phyla (Parks et al, 2013;Podar et al, 2015a;Jones et al, 2019). Hg-methylating microorganisms often constitute less than 5% of the bulk microbial community (Christensen et al, 2019), yet in these same environments MeHg can comprise up to 10% of the total Hg within a system (Christensen et al, 2019). Since MeHg readily biomagnifies up aquatic food webs, it poses a significant risk to ecosystem and public health even at picomolar concentrations in the water column and sediments (Chen et al, 2014;Lee and Fisher, 2017).…”

“…PCR primers and reference libraries for hgcAB have improved with the increasing number of hgcAB sequences identified. Early studies relied on sequences from a small number of cultured methylators (Bae et al, 2014;Liu et al, 2014;Schaefer et al, 2014) while more recent work has captured a wider diversity (Christensen et al, 2016;Bravo and Cosio, 2019;Christensen et al, 2019;Jones et al, 2019). Improved techniques for capturing hgcAB presence and diversity are necessary for identifying both the major players in environmental MeHg production as well as the evolutionary history of the gene pair.…”

“…The new primer set is a modification of the Christensen broad-range hgcAB primer set, which was designed using a database of 84 known and predicted Hg-methylator genomes (Christensen et al, 2016). While the highly divergent primers amplified hgcAB from many types of pure cultures, amplification efficiencies from natural environments were often low (Christensen et al, 2019). PCR primer degeneracy is an important tool for capturing genetic diversity while minimizing biases (von Wintzingerode et al, 1997;Sipos et al, 2007), but high levels of degeneracy can reduce amplification of individual primer matches.…”