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Mercury (Hg) methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-cycling microorganisms in aquatic and terrestrial ecosystems (Hg-MATE) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce marky-coco, a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on coassembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a coassembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

Multiomics approaches need to be applied in the central Arctic Ocean to benchmark biodiversity change and to identify novel species and their genes. As part of MOSAiC, EcoOmics will therefore be essential for conservation and sustainable bioprospecting in one of the least explored ecosystems on Earth.

To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single-cell genomes and a 5-year metagenomic time series. The single-cell genomes grouped into 15 monophyletic clusters (termed "tribes") that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI Actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.

Although microbes mediate much of the biogeochemical cycling in freshwater, the categories of carbon and nutrients currently used in models of freshwater biogeochemical cycling are too broad to be relevant on a microbial scale. One way to improve these models is to incorporate microbial data. Here, we analyze both genes and genomes from three metagenomic time series and propose specific roles for microbial taxa in freshwater biogeochemical cycles. Our metagenomic time series span multiple years and originate from a eutrophic lake (Lake Mendota) and a humic lake (Trout Bog Lake) with contrasting water chemistry. Our analysis highlights the role of polyamines in the nitrogen cycle, the diversity of diazotrophs between lake types, the balance of assimilatory vs. dissimilatory sulfate reduction in freshwater, the various associations between types of phototrophy and carbon fixation, and the density and diversity of glycoside hydrolases in freshwater microbes. We also investigated aspects of central metabolism such as hydrogen metabolism, oxidative phosphorylation, methylotrophy, and sugar degradation. Finally, by analyzing the dynamics over time in nitrogen fixation genes and Cyanobacteria genomes, we show that the potential for nitrogen fixation is linked to specific populations in Lake Mendota. This work represents an important step towards incorporating microbial data into ecosystem models and provides a better understanding of how microbes may participate in freshwater biogeochemical cycling.

Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, and yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions and greatly expand knowledge of the genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly)saccharide degraders in freshwater, uncovered interesting genomic features for this lifestyle, and suggested their adaptation to nutrient availabilities in their environments. Verrucomicrobia populations differ significantly between the two lakes in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially derived allochthonous carbon sources of the two ecosystems, respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humus-rich environment. Notably, most epilimnion genomes have large numbers of so-called "Planctomycete-specific" cytochrome c-encoding genes, which exhibited distribution patterns nearly opposite to those seen with glycoside hydrolase genes, probably associated with the different levels of environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes represent a major step toward understanding the role, ecophysiology, and distribution of Verrucomicrobia in freshwater. IMPORTANCE Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called "Planctomycete-specific" cytochrome c-encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.

An explosion in the number of available genome sequences obtained through metagenomics and single-cell genomics has enabled a new view of the diversity of microbial life, yet we know surprisingly little about how microbes interact with each other or their environment. In fact, the majority of microbial species remain uncultivated, while our perception of their ecological niches is based on reconstruction of their metabolic potential. In this work, we demonstrate how the "seed set framework," which computes the set of compounds that an organism must acquire from its environment (E. Borenstein, M. Kupiec, M. W. Feldman, and E. Ruppin, Proc Natl Acad Sci U S A 105:14482-14487, 2008, https://doi.org/10.1073/pnas.0806162105), enables computational analysis of metabolic reconstructions while providing new insights into a microbe's metabolic capabilities, such as nutrient use and auxotrophies. We apply this framework to members of the ubiquitous freshwater actinobacterial lineage acI, confirming and extending previous experimental and genomic observations implying that acI bacteria are heterotrophs reliant on peptides and saccharides. We also present the first metatranscriptomic study of the acI lineage, revealing high expression of transport proteins and the light-harvesting protein actinorhodopsin. Putative transport proteins complement predictions of nutrients and essential metabolites while providing additional support of the hypothesis that members of the acI are photoheterotrophs. IMPORTANCE The metabolic activity of uncultivated microorganisms contributes to numerous ecosystem processes, ranging from nutrient cycling in the environment to influencing human health and disease. Advances in sequencing technology have enabled the assembly of genomes for these microorganisms, but our ability to generate reference genomes far outstrips our ability to analyze them. Common approaches to analyzing microbial metabolism require reconstructing the entirety of an organism's metabolic pathways or performing targeted searches for genes involved in a specific process. This paper presents a third approach, in which draft metabolic reconstructions are used to identify compounds through which an organism may interact with its environment. These compounds can then guide more-intensive metabolic reconstruction efforts and can also provide new hypotheses about the specific contributions that microbes make to ecosystem-scale metabolic processes.

To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single cell genomes and a 5-year metagenomic time series. The single cell genomes grouped into 15 monophyletic clusters (termed “tribes”) that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.

Most free-living planktonic cells are streamlined and in spite of their limitations in functional flexibility, their vast populations have radiated into a wide range of aquatic habitats. Here we compared the metabolic potential of subgroups in the Alphaproteobacteria lineage SAR11 adapted to marine and freshwater habitats. Our results suggest that the successful leap from marine to freshwaters in SAR11 was accompanied by a loss of several carbon degradation pathways and a rewiring of the central metabolism. Examples for these are C1 and methylated compounds degradation pathways, the Entner-Doudouroff pathway, the glyoxylate shunt and anapleuretic carbon fixation being absent from the freshwater genomes. Evolutionary reconstructions further suggest that the metabolic modules making up these important freshwater metabolic traits were already present in the gene pool of ancestral marine SAR11 populations. The loss of the glyoxylate shunt had already occurred in the common ancestor of the freshwater subgroup and its closest marine relatives, suggesting that the adaptation to freshwater was a gradual process. Furthermore, our results indicate rapid evolution of TRAP transporters in the freshwater clade involved in the uptake of low molecular weight carboxylic acids. We propose that such gradual tuning of metabolic pathways and transporters toward locally available organic substrates is linked to the formation of subgroups within the SAR11 clade and that this process was critical for the freshwater clade to find and fix an adaptive phenotype.

Multiple models describe the formation and evolution of distinct microbial phylogenetic groups. These evolutionary models make different predictions regarding how adaptive alleles spread through populations and how genetic diversity is maintained. Processes predicted by competing evolutionary models, for example, genome-wide selective sweeps vs gene-specific sweeps, could be captured in natural populations using time-series metagenomics if the approach were applied over a sufficiently long time frame. Direct observations of either process would help resolve how distinct microbial groups evolve. Here, from a 9-year metagenomic study of a freshwater lake (2005-2013), we explore changes in single-nucleotide polymorphism (SNP) frequencies and patterns of gene gain and loss in 30 bacterial populations. SNP analyses revealed substantial genetic heterogeneity within these populations, although the degree of heterogeneity varied by >1000-fold among populations. SNP allele frequencies also changed dramatically over time within some populations. Interestingly, nearly all SNP variants were slowly purged over several years from one population of green sulfur bacteria, while at the same time multiple genes either swept through or were lost from this population. These patterns were consistent with a genome-wide selective sweep in progress, a process predicted by the 'ecotype model' of speciation but not previously observed in nature. In contrast, other populations contained large, SNP-free genomic regions that appear to have swept independently through the populations prior to the study without purging diversity elsewhere in the genome. Evidence for both genome-wide and gene-specific sweeps suggests that different models of bacterial speciation may apply to different populations coexisting in the same environment.

To understand the forces driving differentiation and diversification in wild bacterial populations, we must be able to delineate and track ecologically relevant units through space and time. Mapping metagenomic sequences to reference genomes derived from the same environment can reveal genetic heterogeneity within populations, and in some cases, be used to identify boundaries between genetically similar, but ecologically distinct, populations. Here we examine population-level heterogeneity within abundant and ubiquitous freshwater bacterial groups such as the acI Actinobacteria and LD12 Alphaproteobacteria (the freshwater sister clade to the marine SAR11) using 33 single cell genomes and a 5-year metagenomic time series. The single cell genomes grouped into 15 monophyletic clusters (termed “tribes”) that share at least 97.9% 16S rRNA identity. Distinct populations were identified within most tribes based on the patterns of metagenomic read recruitments to single-cell genomes representing these tribes. Genetically distinct populations within tribes of the acI actinobacterial lineage living in the same lake had different seasonal abundance patterns, suggesting these populations were also ecologically distinct. In contrast, sympatric LD12 populations were less genetically differentiated. This suggests that within one lake, some freshwater lineages harbor genetically discrete (but still closely related) and ecologically distinct populations, while other lineages are composed of less differentiated populations with overlapping niches. Our results point at an interplay of evolutionary and ecological forces acting on these communities that can be observed in real time.