Diverse Methylmercury (MeHg) Producers and Degraders Inhabit Acid Mine Drainage Sediments, but Few Taxa Correlate with MeHg Accumulation

Abstract

Methylmercury (MeHg) is a notorious neurotoxin, and its production and degradation in the environment are mainly driven by microorganisms. A variety of microbial MeHg producers carrying the gene pair hgcAB and degraders carrying the merB gene have been separately reported in recent studies. However, surprisingly little attention has been paid to the simultaneous investigation of the diversities of microbial MeHg producers and degraders in a given habitat, and no studies have been performed to explore to what extent these two contrasting microbial groups correlate with MeHg accumulation in the habitat of interest. Here, we collected 86 acid mine drainage (AMD) sediments from an area spanning approximately 500,000 km² in southern China and profiled the sediment-borne putative MeHg producers and degraders using genome-resolved metagenomics. 46 metagenome-assembled genomes (MAGs) containing hgcAB and 93 MAGs containing merB were obtained, including those from various taxa without previously known MeHg-metabolizing microorganisms. These diverse MeHg-metabolizing MAGs were formed largely via multiple independent horizontal gene transfer (HGT) events. The putative MeHg producers from Deltaproteobacteria and Firmicutes as well as MeHg degraders from Acidithiobacillia were closely correlated with MeHg accumulation in the sediments. Furthermore, these three taxa, in combination with two abiotic factors, explained over 60% of the variance in MeHg accumulation. Most of the members of these taxa were characterized by their metabolic potential for nitrogen fixation and copper tolerance. Overall, these findings improve our understanding of the ecology of MeHg-metabolizing microorganisms and likely have implications for the development of management strategies for the reduction of MeHg accumulation in the AMD sediments. IMPORTANCE Microorganisms are the main drivers of MeHg production and degradation in the environment. However, little attention has been paid to the simultaneous investigation of the diversities of microbial MeHg producers and degraders in a given habitat. We used genome-resolved metagenomics to reveal the vast phylogenetic and metabolic diversities of putative MeHg producers and degraders in AMD sediments. Our results show that the diversity of MeHg-metabolizing microorganisms (particularly MeHg degraders) in AMD sediments is much higher than was previously recognized. Via multiple linear regression analysis, we identified both microbial and abiotic factors affecting MeHg accumulation in AMD sediments. Despite their great diversity, only a few taxa of MeHg-metabolizing microorganisms were closely correlated with MeHg accumulation. This work underscores the importance of using genome-resolved metagenomics to survey MeHg-metabolizing microorganisms and provides a framework for the illumination of the microbial basis of MeHg accumulation via the characterization of physicochemical properties, MeHg-metabolizing microorganisms, and the correlations between them.

Keywords: acid mine drainage (AMD); demethylator; metagenome; methylator; methylmercury; sediment.

FIG 1

Geographic location, total mercury (THg), and methymercury (MeHg) of the study sites. (A) Geographic locations of the 20 studied mine sites where we collected a total of 86 acid mine drainage (AMD) sediments. Abbreviations of the study sites are shown, and detailed information on these sites is provided in Table S1. (B) Concentrations (mean ± standard deviation) of THg in the studied AMD sediments. (C) Concentrations of MeHg in the studied AMD sediments. The log₁₀-transformed concentration data are shown.

FIG 2

Analysis of the 46 good-quality or high-quality metagenome-assembled genomes (MAGs) of putative MeHg producers. (A) Phylogenic analysis of the 46 good-quality (completeness > 75% and contamination < 10%) or high-quality (completeness > 90% and contamination < 5%) hgcAB-carrying MAGs obtained in this study. The bootstrap values were based on 100 replicates, and those greater than 50% are marked with black circles. The taxonomy of individual MAGs is generally shown at the phylum level, with those affiliated with Proteobacteria being shown at the class level. Each of the eight taxonomic lineages is indicated by a separate color, as shown in panel C. A taxonomic lineage without previously known MeHg producers is marked in red font. The number of MAGs belonging to each of the eight taxonomic lineages is also given at the bracket following the name of that lineage. MAGs containing dsrAB and merB are indicated with red stars and blue circles, respectively. The frequencies of occurrence of the individual MAGs in all samples as well as their average relative abundances are indicated on the right of the phylogenetic tree with pink and green bars, respectively. (B) The relative abundance of the MAGs affiliated with each of the eight taxonomic lineages in each study site. The color coding for the taxonomic lineages is the same as that in panel C. (C) Distribution of merA, merB, and sulfate reduction-associated genes in the MAGs affiliated with each of the eight taxonomic lineages.

FIG 3

Analysis of the 93 good-quality or high-quality MAGs of putative MeHg degraders. (A) Phylogenic analysis of the merB-carrying good-quality MAGs obtained in this study. Bootstrap values were based on 100 replicates, and those greater than 50% are marked with black circles. The taxonomy of individual MAGs is generally shown at the phylum level, with those affiliated with Proteobacteria being shown at the class level. Each of the 12 taxonomic lineages is indicated by a separate color, as shown in panel C. Three phyla without previously known MeHg degraders are marked in red font. The number of MAGs belonging to each of the 12 taxonomic lineages is also given at the bracket following the name of that lineage. MAGs containing dsrAB and hgcAB are indicated with red stars and blue circles, respectively. The frequencies of occurrence of individual MAGs in all samples as well as their relative abundances are indicated on the right of the phylogenetic tree with pink and green bars, respectively. (B) Relative abundance of the MAGs affiliated with each of the 12 taxonomic lineages in each study site. The color coding for the taxonomic lineages is the same as that in panel C. (C) Distribution of merA, hgcAB, and sulfate reduction-associated genes in the MAGs affiliated with each of the 12 taxonomic lineages.

FIG 4

Comparison of the genome-based phylogenetic tree and concatenated HgcAB protein tree for putative MeHg producers. (A) Phylogenic analysis of hgcAB-carrying MAGs and the reference genome. (B) Phylogenic analysis of the concatenated HgcAB proteins and the reference HgcAB proteins. Sequences are generally grouped at the phylum level, with those of Proteobacteria being grouped at the class level. Each phylum or class is indicated by a separate color to identify horizontal gene transfer (HGT), based on inconsistent branching patterns. The numbers in panel A represent the total numbers of independent HGT events associated with putative hgcAB genes in specific phyla or classes in AMD sediments. The right triangles in panel B represent the HGT events of putative HgcAB in specific phyla or classes in AMD sediments. The bootstrap values were based on 100 replicates, and those greater than 50% are shown with black circles. Asterisks indicate the branches obtained in this study that contained hgcAB genes. The HgcAB protein tree was rerooted with fusion HgcAB of Streptomyces sp. CNQ-509.

FIG 5

Comparison of the genome-based phylogenetic tree and the MerB protein tree for putative MeHg degraders. (A) Phylogenic analysis of merB-containing MAGs and the reference genome. (B) Phylogenic analysis of putative MerB proteins and the reference MerB proteins. Sequences are generally grouped at the phylum level, with those of Proteobacteria being grouped at the class level. Each phylum or class is indicated by a separate color to identify HGT, based on inconsistent branching patterns. The numbers in panel A represent the total numbers of independent HGT events associated with putative merB genes in specific phyla or classes in AMD sediments. The right triangles in panel B represent the HGT events of putative MerB in specific phyla or classes in AMD sediments. The bootstrap values were based on 100 replicates, and those greater than 50% are shown with black circles. Asterisks indicate the branches obtained in this study that contained merB genes. The MerB protein tree was rerooted with MerA of Bacillus sp. RC607.

FIG 6

Key microbial and environmental factors correlating with MeHg accumulation in AMD sediments. (A–C) Pearson correlations between the relative abundances of putative MeHg producers affiliated with Deltaproteobacteria, Nitrospirae, or Firmicutes and the MeHg concentrations. (D) Pearson correlations between the relative abundance of putative MeHg degraders affiliated with Acidithiobacillia and the MeHg concentrations. (E and F) Pearson correlations between the total carbon (TC) content or the Fe²⁺/Fe³⁺ ratio and the MeHg concentrations. (G) The relative importance of individual factors in explaining MeHg accumulation, as assessed by a multiple linear regression model. (H) The relative importance of the key microbial and environmental factors contributing to MeHg accumulation, as assessed by a variance decomposition analysis. The mean values of the MeHg concentrations and the microbial and environmental factors of the individual study sites were used in the analysis.

FIG 7

Distribution of the genes responsible for nitrogen metabolism and metal tolerance in putative MeHg producers and degraders. The taxonomy of individual MAGs is generally shown at the phylum level, with those affiliated with Proteobacteria being shown at the class level. Four taxonomic lineages that were identified as important determinants of MeHg accumulation in AMD sediments are marked in red font. The color of each cell refers to the percentage of MAGs affiliated with each taxonomic lineage containing the gene(s) involved in the individual pathways of nitrogen metabolism or metal tolerance. As, arsenic; Cd, cadmium; Co, cobalt; Cu, copper; Zn, zinc.