Mercury methylation by metabolically versatile and cosmopolitan marine bacteria

Abstract

Microbes transform aqueous mercury (Hg) into methylmercury (MeHg), a potent neurotoxin that accumulates in terrestrial and marine food webs, with potential impacts on human health. This process requires the gene pair hgcAB, which encodes for proteins that actuate Hg methylation, and has been well described for anoxic environments. However, recent studies report potential MeHg formation in suboxic seawater, although the microorganisms involved remain poorly understood. In this study, we conducted large-scale multi-omic analyses to search for putative microbial Hg methylators along defined redox gradients in Saanich Inlet, British Columbia, a model natural ecosystem with previously measured Hg and MeHg concentration profiles. Analysis of gene expression profiles along the redoxcline identified several putative Hg methylating microbial groups, including Calditrichaeota, SAR324 and Marinimicrobia, with the last the most active based on hgc transcription levels. Marinimicrobia hgc genes were identified from multiple publicly available marine metagenomes, consistent with a potential key role in marine Hg methylation. Computational homology modelling predicts that Marinimicrobia HgcAB proteins contain the highly conserved amino acid sites and folding structures required for functional Hg methylation. Furthermore, a number of terminal oxidases from aerobic respiratory chains were associated with several putative novel Hg methylators. Our findings thus reveal potential novel marine Hg-methylating microorganisms with a greater oxygen tolerance and broader habitat range than previously recognized.

Fig. 1. Map of Saanich Inlet and mercury profiles of station “S3”.

A Map of Saanich Inlet showing locations of the station “S2”, “S3” and “S4” by triangles. B Concentration of total dissolved Hg at station “S3”. C Concentration of dissolved MeHg at station “S3”. D MeHg as a percentage of total dissolved Hg at station “S3”.

Fig. 2. Maximum-likelihood phylogenetic tree of HgcA amino acid sequences (1000 ultrafast bootstrap replicates; values >90% are shown by black dots at the nodes).

HgcA sequences recovered in this study are highlighted in blue. HgcA sequences retrieved from public databases are shown in black. Experimentally confirmed HgcA from previous studies are shown in brown. HgcA paralogues from non-methylating bacteria were used as outgroups and are shown in gray. The 15 representative HgcA sequences used in this study are indicated by stars. Taxonomic classifications of the hgcA-carrying genomes are labeled in the outer circle by different colors. Scale bar indicates substitutions per site.

Fig. 3. Relative abundance of representative hgcA genes and transcripts in different samples from Saanich Inlet.

Gene abundance is normalized by gene length and genome equivalent for each sample, and transcript abundances are represented as RPKM values. A Relative abundance of the sum of representative hgcA genes for different depths. B Relative abundance of different representative hgcA genes from different metagenomic datasets. Larger circles indicate a higher percentage of the whole microbial community. Different shades of blue indicate different depths from which the samples were taken. C RPKM values of the sum of representative hgcA transcripts in different depths. D RPKM values of different representative hgcA transcripts in different metatranscriptomic samples. HgcA sequences from metaproteomic samples are indicated with stars.

Fig. 4. DNA and RNA abundance of merB in Saanich Inlet datasets changing with depth.

A merB gene abundance in Saanich Inlet metagenomic datasets. Percentage abundance was normalized by gene length and genome equivalent for each sample. Line plot depicts the average abundance of merB with depth; shaded area depicts 95% confidence interval. B merB transcript abundance in Saanich Inlet metatranscriptomic datasets, as represented by RPKM values. Line plot depicts mean RPKM value of merB with depth; shaded area depicts 95% confidence interval. C Comparison between RPKM values for hgcA (orange) and merB (green) in Saanich Inlet metatranscriptomic datasets.

Fig. 5. Phylogeny and global distribution of phylum Marinimicrobia.

A Maximum-likelihood phylogenetic tree of phylum Marinimicrobia based on concatenated housekeeping genes (1000 ultrafast bootstrap replicates; values >90% are shown at the nodes). Scale bar indicates substitutions per site. A total of 424 Marinimicrobia genomes were used for the tree; Marinimicrobia lineages without hgcA genes were collapsed to simplify presentation (see Fig. S6 for a more detailed tree). B Distribution of hgcA-carrying Marinimicrobia in various environments globally, shown as different colors; total numbers of BioProjects from each environment are shown in brackets.

Fig. 6. Three-dimensional homology models of Marinimicrobia-HgcA and -HgcB proteins.

A Model of full-length Marinimicrobia-HgcA, shown relative to the cell membrane. An enlarged view of the functional domain bound to cobalamin is shown, with the ionic interaction between cysteine and cobalt represented by a dotted line. B Ramachandran plot of Marinimicrobia-HgcA model showing that 93.2%, 5.9%, and 0.8% of residues lie within favoured, allowed, and outlier regions, respectively. The backbone Phi and Psi (φ and ψ) dihedral angles are shown, along with the energetically favoured regions. C Model of full-length Marinimicrobia-HgcB in complex with two [4Fe4S] clusters. Interactions between the protein and iron are shown by dotted lines. D Ramachandran plot of Marinimicrobia-HgcB model is shown, revealing 94.1%, 5.9%, and 0% of residues lay within favoured, allowed, and outlier regions, respectively.

Fig. 7. KEGG pathways of the hgcA-carrying MAGs.

Taxonomic classifications of MAGs are represented at bottom of heatmap by different colors. Categories of pathways are represented at left side of heatmap by different colors. Color of each cell refers to completeness of enzymes involved in each pathway. Corresponding genes involved in each pathway are shown in parentheses.