Genome-resolved metagenomics identifies genetic mobility, metabolic interactions, and unexpected diversity in perchlorate-reducing communities

Abstract

Dissimilatory perchlorate reduction is an anaerobic respiratory pathway that in communities might be influenced by metabolic interactions. Because the genes for perchlorate reduction are horizontally transferred, previous studies have been unable to identify uncultivated perchlorate-reducing populations. Here we recovered metagenome-assembled genomes from perchlorate-reducing sediment enrichments and employed a manual scaffolding approach to reconstruct gene clusters for perchlorate reduction found within mobile genetic elements. De novo assembly and binning of four enriched communities yielded 48 total draft genomes. In addition to canonical perchlorate reduction gene clusters and taxa, a new type of gene cluster with an alternative perchlorate reductase was identified. Phylogenetic analysis indicated past exchange between these gene clusters, and the presence of plasmids with either gene cluster shows that the potential for gene transfer via plasmid persisted throughout enrichment. However, a majority of genomes in each community lacked perchlorate reduction genes. Putative chlorate-reducing or sulfur-reducing populations were dominant in most communities, supporting the hypothesis that metabolic interactions might result from perchlorate reduction intermediates and byproducts. Other populations included a novel phylum-level lineage (Ca. Muirbacteria) and epibiotic prokaryotes with no known role in perchlorate reduction. These results reveal unexpected genetic diversity, suggest that perchlorate-reducing communities involve substantial metabolic interactions, and encourage expanded strategies to further understand the evolution and ecology of this metabolism.

Fig. 1

Summary of energy metabolism genes, abundance, and activity of metagenome-assembled genomes from perchlorate-reducing enrichments (1, 3, 5, and 7% salinity) and sediment inoculum. Genomes predicted to be dissimilatory perchlorate-reducing bacteria are shown in red. a Gene content based on presence (filled squares) or absence (empty squares) of key genes involved different metabolisms. Genes in the same vertical division perform the same function. Gene abbreviations: pcr perchlorate reductase, pnar periplasmic Nar nitrate reductase, cld chlorite dismutase, yedYZ periplasmic methionine sulfoxide reductase, clr chlorate reductase, nap periplasmic nitrate reductase, cbb₃ cbb₃-type cytochrome c oxidase, aa₃ aa₃-type cytochrome c oxidase or cytochrome bo₍₃₎ ubiquinol oxidase, bd cytochrome bd quinol oxidase, psr polysulfide reductase, pMHC periplasmic multi-heme cytochrome c, ack-pta acetate kinase and phosphotransacetylase, acs acetyl-CoA synthase, nqo proton-translocating NADH:quinol oxidoreductase, nqr sodium-translocating NADH:quinol oxidoreductase. b Relative abundance of metagenome-assembled genomes within samples (coverage/coverage of most abundant community member). c Relative activity across samples as measured by index of replication (iRep). Slash indicates the iRep value could not be determined

Fig. 2

Maximum-likelihood tree of perchlorate and chlorate reduction enzymes obtained from metagenome-assembled genomes (bold) and the NCBI and IMG databases. Trees were constructed using MUSCLE and PhyML(100 bootstraps) and visualized with FigTree. Branch length and local support values provided by bootstrapping are indicated. Blue text indicates taxa isolated from marine environments. a Perchlorate reductase alpha subunit. b Chlorite dismutase; colored squares indicate the closest perchlorate or chlorate reductase gene to the chlorite dismutase gene. c Chlorate reductase alpha subunit

Fig. 3

Characterization of periplasmic nitrate reductase (pNar) genes with possible perchlorate reductase activity. a Gene clusters containing both pnarGHJI and cld. b Alignment of selected Nar-type enzymes constructed using MUSCLE and edited using AliView. Perchlorate reductase residue positions α, β, and γ are structurally conserved and functionally significant [59]. Substrate utilization:+, reduced substrate in vivo or in vitro; −, did not reduce substrate; (+), predicted to reduce substrate. Tree: position in the phylogenetic tree at right. c Maximum-likelihood phylogeny of Nar-type molybdoenzymes constructed using MUSCLE and PhyML with 100 bootstraps. Four lineages are found in gene clusters with chlorite dismutase (blue background): group 1 PcrA, group 1 ClrA (SerA-like), group 2 ClrA (DdhA-like), and group 2 PcrA (pNarG-like). Genome bins encoding proteins from each group are indicated

Fig. 4

Use of contig graphs to manually assemble perchlorate reduction genes within mobile genetic elements. a Each sample was assembled individually and inspected for perchlorate reduction genes in contig graphs. Contig graphs depict possible connections between contigs produced by de Bruijn graph assembly. The real connections between contigs may be determined using information such as coverage (line weight) and gene content (line colors). PRI sequences including group 1 pcrA (red), group 2 pcrA (“pnarG”, green), and cld (blue) that were clearly connected to contigs from genome bins (arrows) were added to those bins. Mean coverage for particular plasmid sequences and bins is indicated in parenthesis. For simplicity, only contigs within a few connections of PRI sequences are shown, and some contigs are not shown in their entirety. b Reconstructed PRI from contig graphs. Shaded genes indicate sequences that were highly similar and assembled as a consensus sequence from multiple strains. Each genome was annotated independently using Prokka

Fig. 5

Metabolic potential of perchlorate-reducing communities. (a) The relative abundance, among high-quality bins, of genomes with each predicted metabolism: dissimilatory perchlorate-reducing bacteria (red), dissimilatory chlorate-reducing bacteria (orange), dissimilatory elemental sulfur-reducing bacteria (gray), free-living fermentative bacteria (dark blue), and epibiotic fermentative bacteria (light blue). Communities differ in salinity (% NaCl w/v), sampling time (days), and perchlorate removed (%). (b). Possible metabolic interactions and their commonality among the four enriched communities