Genome-Resolved Metagenomics and Detailed Geochemical Speciation Analyses Yield New Insights into Microbial Mercury Cycling in Geothermal Springs

Heat map showing functional proteins from various biogeochemical pathways associated with Hg within genomic bins and unbinned (NGAWHA_1_UNK and NGAWHA_2_UNK) scaffolds from Tiger (NW1) and Cub (NW2) Bath metagenomes. Intensity of color refers to number of genes from each bin that encode the enzyme, operon, or pathway involved in mercury, sulfur, or methane cycling; values are provided in cells for reference. Genomic bins were ordered by the consensus coverage for all scaffolds within the bin (highest to lowest). Analyses and annotations were performed in ggKbase (https://ggkbase.berkeley.edu/).

Maximum likelihood tree showing MerA phylogeny of 31 sequences pulled from assembled Tiger (NW1) and Cub (NW2) Bath metagenomes using ggKbase. Genomic bins or scaffold ID (when MerA was unbinned) are given in bold font. Included in analysis are 113 MerA homologues, including 46 sequences from Yellowstone National Park (YNP) metagenomes (82). Trees were constructed using Le Gascuel amino-acid substitution model with gamma distribution in MEGA6 (77). Data were bootstrapped with 100 replications. The initial neighbor-joining tree was constructed with pairwise distances estimated using a JTT model. Positions with less than 90% site coverage (e.g., alignment gaps, missing data, or ambiguous bases) were excluded. A total of 419 positions were used in the final data set.

Maximum likelihood tree showing HgcA phylogeny of reads obtained from the Cub Bath (NW2) metagenome using HMM search. Included in analysis are 183 sequences, including 56 HgcA homologues from YNP metagenomes, pulled from JGI (see Table S3 in the supplemental material). Amino acid sequences are compared to HgcA homologues from known and predicted methylators. Also included are HgcAB fused proteins from hyperthermophilic bacteria and archaea and carbon monoxide dehydrogenase/acetyl coenzyme A (acetyl-CoA) synthase subunit gamma (HgcA paralogs) from nonmethylators. Trees were inferred from the Le Gascuel amino acid substitution model with gamma distribution in MEGA6 (77). Data were bootstrapped with 100 replications. The initial neighbor-joining tree was constructed with pairwise distances estimated using a JTT model. Positions with less than 93% site coverage (e.g., alignment gaps, missing data, or ambiguous bases) were excluded. A total of 55 positions were used in the final data set. Groups (I to IX) designate distinct subtrees that contain HgcA from hot spring metagenomes. Representative reference sequences are labeled within each subtree, while phylogeny of all branches is indicated by color.

Phylogenetic analysis of DsrAB by maximum likelihood method. The 15 DsrAB homologues in this study were pulled from IDBA-UD assembled Tiger and Cub metagenomes using ggKbase. They were compared to 218 reference DsrAB sequences pulled from the Dome database, including 15 representative DsrAB sequences from thermophilic environments (39). There were six distinct groups of DsrAB homologues found at Ngawha. The tree was constructed using MEGA6 (77) with the Le Gascuel 2008 model with gamma distribution; pairwise distances were estimated using a JTT model. All positions with less than 95% site coverage were eliminated, including alignment gaps, missing data, and ambiguous bases. There was a total of 492 positions in the final data set, with 100 bootstrap replicates.