Single-Cell-Genomics-Facilitated Read Binning of Candidate Phylum EM19 Genomes from Geothermal Spring Metagenomes

Abstract

The vast majority of microbial life remains uncatalogued due to the inability to cultivate these organisms in the laboratory. This "microbial dark matter" represents a substantial portion of the tree of life and of the populations that contribute to chemical cycling in many ecosystems. In this work, we leveraged an existing single-cell genomic data set representing the candidate bacterial phylum "Calescamantes" (EM19) to calibrate machine learning algorithms and define metagenomic bins directly from pyrosequencing reads derived from Great Boiling Spring in the U.S. Great Basin. Compared to other assembly-based methods, taxonomic binning with a read-based machine learning approach yielded final assemblies with the highest predicted genome completeness of any method tested. Read-first binning subsequently was used to extract Calescamantes bins from all metagenomes with abundant Calescamantes populations, including metagenomes from Octopus Spring and Bison Pool in Yellowstone National Park and Gongxiaoshe Spring in Yunnan Province, China. Metabolic reconstruction suggests that Calescamantes are heterotrophic, facultative anaerobes, which can utilize oxidized nitrogen sources as terminal electron acceptors for respiration in the absence of oxygen and use proteins as their primary carbon source. Despite their phylogenetic divergence, the geographically separate Calescamantes populations were highly similar in their predicted metabolic capabilities and core gene content, respiring O 2 , or oxidized nitrogen species for energy conservation in distant but chemically similar hot springs.T he vast majority of the diversity of microbial life on Earth remains undiscovered; the core metabolisms of yet-uncultivated species, interguild interactions within natural and managed ecosystems, and the contributions of microbial populations to the geochemistry of the environment remain poorly understood (1, 2). There are currently over 60 bacterial and archaeal phylumlevel groups that have been observed through the use of 16S rRNA gene sequencing and phylogenetics, with over half containing no cultivated representatives (3). This so-called microbial dark matter comprises a substantial proportion of the tree of life and of microbial communities that likely play significant roles in biogeochemical cycles in a variety of environments (4-9).Metagenomic analyses of low-diversity microbial communities have yielded robust, near-complete genomic assemblies representative of the abundant populations, expanding the knowledge of the metabolic potential of predominant organisms in these ecosystems (10-14). Nucleotide word frequencies calculated from metagenomic contiguous assembled sequences (contigs) have been used to separate population-specific clusters, or "bins," from the community DNA pool (15-17), which has greatly advanced our understanding of the genomic diversity in natural environments and how populations differ between chemically distinct environments and along environmental gradients (18,19). Even using modern sequencing techniques, ...

Life in High-Temperature Environments

“…Although each of these techniques has its own strengths and weaknesses, there is much potential for synergistic application of single-cell genomics and metagenomics (171). These techniques have yielded genomic data from dozens of major lineages of microorganisms (172), including several that contain (hyper)thermophiles (173)(174)(175)(176)(177)(178)(179). Several of these candidate phyla ("Acetothermia," "Aigarchaeota," "Calesmantes," "Fervidibacteria," and "Korarchaeota") appear to be restricted to thermal environments and thus likely add to the list of lineages composed primarily or exclusively of thermophilic specialists.…”

Solagigasbacteria: Lone genomic giants among the uncultured bacterial phyla

“…An increasing body of evidence shows that this assumption is not valid in many microorganismal lineages and environments, with genomic rearrangements and horizontal gene transfer being more prevalent than previously thought (Ochman et al, 2000; Feldgarden et al, 2003; Shapiro, 2010; Kashtan et al, 2014; Labonte et al, 2015a). By recovering data from the most fundamental units of biological organization, single-cell genomics does not rely on the assumption of clonality, offers an opportunity to improve our understanding of microbial microevolutionary processes (Garrity and Lyons, 2003; Engel et al, 2014; Kashtan et al, 2014), and helps calibrating the performance and interpretation of metagenomics tools when working with complex, natural microbial assemblages (Becraft et al, 2015).…”

“…Technological innovations in single-cell genomics and metagenomics have led to a rapid improvement in our understanding of the genomic features, evolutionary histories and metabolic capabilities of tens of phylum-level branches of Archaea, Bacteria and Eukarya that lack cultured representatives (Yoon et al, 2011; Rinke et al, 2013; Becraft et al, 2015; Brown et al, 2015; Castelle et al, 2015). In these efforts, the subsurface has emerged as a bountiful reservoir of undiscovered, deeply branching microbial lineages that may hold clues to the emergence and evolution of life on our planet (Kallmeyer et al, 2012; Colwell and D’Hondt, 2013).…”

“…In these efforts, the subsurface has emerged as a bountiful reservoir of undiscovered, deeply branching microbial lineages that may hold clues to the emergence and evolution of life on our planet (Kallmeyer et al, 2012; Colwell and D’Hondt, 2013). Many of the recently discovered candidate phyla are composed of microorganisms with streamlined genomes lacking some core metabolic pathways, which may be a factor contributing to the inability to obtain pure cultures of these organisms (Rinke et al, 2013; Becraft et al, 2015; Brown et al, 2015; Castelle et al, 2015). This apparent genomic reduction has given rise to hypotheses of genome-streamlining, parasitism, symbiotic lifestyles, and large-scale community metabolic inter-dependence (Giovannoni et al, 2014; Castelle et al, 2015; Anantharaman et al, 2016).…”

Genomes from uncultivated prokaryotes: a comparison of metagenome-assembled and single-amplified genomes

“…Studies have successfully combined the SAG and MAG approaches to reach conclusions about organisms and ecosystems [38, 39]. The approaches have also been combined to methodologically improve either the quality of the single-cell assemblies [40] or the metagenome binning performance [41]. However, with the exception of a study that focused on a single phylum and that did not use abundance patterns over multiple samples for the MAG construction [38] the performance of the two approaches have to our knowledge not been thoroughly compared.…”