Recent studies have shown that a high proportion of the organisms in the subsurface have yet to be cultured, known only by culture-independent genetic evidence such as metagenomic shotgun sequencing of whole genomic DNA Wrighton et al. In addition, newly identified microbial lineages are now commonly implicated in major geochemical cycles Rasigraf et al.
It follows that there is substantial metabolic diversity yet to be discovered within the marine and terrestrial subsurface ecosystems. To better describe and model the microbial networks that catalyze carbon, nitrogen, and sulfur cycling in the subsurface, we need to incorporate newly identified microbial lineages along with their metabolic capabilities. Molecular environmental surveys have provided a wealth of data elucidating microbial phylogenetic diversity, especially in previously poorly sampled habitats.
Advances in cultivation-independent methods for examining uncultured microbes, including single-cell genomics and deep sequencing of environmental samples metagenomics , have begun yielding complete or near-complete genomes from many novel lineages Iverson et al.
These candidate lineages, previously recognized only through SSU rRNA sequencing and for which we have no cultured representatives, are providing a more complete view of the tree of life and a better understanding of global microbial ecology Rinke et al. A few of the recently discovered and thoroughly described candidate phyla include the Korarchaeota Elkins et al.
However, in many cases, an in-depth analysis of the metabolic potential and environmental interaction of these newly discovered lineages is lacking Parks et al. To that end, we present the phylogenetic and metabolic analysis of the first two genomes of a novel bacterial lineage that was recently identified in the deep terrestrial subsurface Momper et al. We follow proposed standards Konstantinidis et al. Candidatus applies both to the organism as well as the potential new lineage. This allows us to combine environmentally derived genome sequence taxonomic classification with currently accepted nomenclature standards, as proposed by Hedlund et al.
In this study, we use full genome sequencing and metabolic reconstruction to elucidate the probable ecological importance of this newly identified candidate lineage. We show that members of this lineage are globally distributed in terrestrial and marine subsurface environments and possess putative functional adaptations that enable them to thrive in these dark, often anoxic environments. Geochemical measurements were made either on site, or upon return to the laboratory Osburn et al.
A comprehensive description of the fluid sampling methods can be found in Osburn et al. In brief, the biomass in borehole fluids was collected on 47 mm, 0. Whole genomic DNA was extracted using a modified phenol-chloroform extraction with ethanol precipitation as previously described in Osburn et al.
DNA concentration was checked on a Qubit 2. Reads were quality trimmed and filtered using Trimmomatic version 0. Sequences from both fluid samples were co-assembled in order to implement differential coverage binning methods for genome bin analysis. Minimum contig length for the co-assembly was set at 10, bp. Coverage information was then attained by individually mapping the paired-end reads of each of the two samples to this co-assembly using Bowtie2 Langmead and Salzberg, Coverage information was extracted using SAMtools 0.
After refinement, genome bin completeness and contamination were re-calculated using five widely accepted marker gene suites compiled from Creevey et al. Closest neighbors generated from these searches were used to generate a 16S phylogenetic species tree Supplementary Figure 1.
A phylogenetic tree was constructed using a concatenation of 16 syntenic and co-located highly conserved ribosomal proteins according to Hug et al. A full list of all proteins used to build the phylogenetic tree can be found in Supplementary Data File 1. The culled tree was then rebuilt using the RAxML maximum likelihood method with the GTR model of nucleotide substitution under the gamma- and invariable- models of rate heterogeneity Stamatakis, According to recent published standards Bowers et al.
This difference in phylogenetic placement between the single 16S rRNA gene and a concatenation of ribosomal proteins illustrates the limited reliability of reconstructed 16S sequences alone.
Indeed, previous studies have noted the discrepancy between the percent identity of partial or reconstructed 16S rRNA sequences versus phylogeny based on full length 16S or multiple concatenated ribosomal sequences Schloss, ; Parks et al. TABLE 1. General characteristics of metagenome assembled genomes analyzed in this study. Bacterial phylogeny estimated from the maximum likelihood phylogeny of 16 concatenated ribosomal proteins is shown. Scale bar indicates 0.
The list of proteins used to build the tree can be found in Supplementary Data File 1. Pathways for both assimilatory and dissimilatory sulfur metabolisms were identified. An enlarged version of the flagellar assembly, chemotaxis cassette and complete gene annotations can be found in Supplementary Figure 2 and Supplementary Data File 3.
Completeness of a given pathway or metabolism is calculated by identifying the requisite genes for that pathway that are not involved in other cellular processes, and calculating the percentage of those genes that are present in each of the five genomes.
Darker red boxes indicate a larger percentage of requisite genes are present, with decreasing percentages represented by lighter shades. A complete list of 16S gene results, including accession numbers, query coverage, identity and collection source can be found in Supplementary Data File 4. These cells were collected from an anaerobic terephthalate-degrading sludge bioreactor Rinke et al. It also requires the lowest energy input of all the six known carbon fixation pathways Berg et al.
Furthermore, the reductive acetyl-CoA pathway requires anoxic conditions, as some of its enzymes, especially the crucial acetyl-CoA synthase, are highly oxygen sensitive Berg et al. Because of energetic efficiency and the necessity for anoxia, the reductive acetyl-CoA pathway is the ideal mode of inorganic carbon fixation in highly reducing, aphotic and energy-deplete deep subsurface fluids, including those encountered at SURF, where the oxidation-reduction potential was assessed at to mV Osburn et al.
Additionally, both genomes contain both pdh- and por- encoded pyruvate dehydrogenase and ferredoxin oxidoreductase genes. These couple the reductive acetyl-CoA pathway to the Embden-Meyerhof glycolysis pathway, and subsequently to the TCA cycle, by catalyzing the oxidation of pyruvate to acetyl-coA or acetate Ragsdale and Pierce, Figure 2. Typically, the por gene suite is used in acetogens and other anaerobes because these enzymes use low potential electron transfer proteins like ferredoxin and flavodoxin, which likely makes the pyruvate synthase reaction feasible as the first step for converting acetyl-CoA into cell material.
Given the presence of por genes and a complete reductive acetyl-CoA pathway which is often used by acetogens in both SURF genomes, acetogenesis is a possible energy metabolism.
Autotrophic growth using the reductive acetyl-CoA pathway does not produce ATP by substrate-level phosphorylation: it is an energy-requiring process and must be coupled to an exergonic anaerobic respiratory process Ragsdale and Pierce, Note that the reductive acetyl-CoA pathway can operate in reverse, with heterotrophs using carbon monoxide dehydrogenase and acetyl-CoA synthase to oxidize acetyl-CoA Rabus et al.
We searched for energy-yielding pathways and found evidence for a complex oxidative phosphorylation pathway in both MAGs, which suggests aerobic respiration and the transfer of electrons to molecular oxygen Figures 3 , 4 and Supplementary Data File 3.
We searched for further evidence of oxygen respirations, such as cytochrome c oxidases, but could not identify any in either MAG. In denitrification, energy is conserved as nitrate NO 3 - , nitrite NO 2 - , nitric oxide NO and nitrous oxide N 2 O are sequentially reduced to dinitrogen gas N 2 , each step catalyzed by one or more metalloenzymes Mahne and Tiedje, ; Zumft, The NO 2 - produced by this reaction is also a highly reactive possible electron acceptor. We hypothesize that the other nrf genes have been lost in SURF genomes, and instead of DNRA they are likely performing other nitrogen, sulfur or carbon based metabolisms that are more exergonic in the subsurface fluids from which they were collected.
However, because NO is a highly toxic molecule, interfering with cellular processes, we cannot rule out the possibility that the function of the nitric oxide reductase, qNOR, is for detoxification rather than an energy yielding process. Although this metabolic strategy is not widespread, anaerobic methylotrophy coupled to denitrification is exhibited in several marine and wastewater-associated species, including Methylophaga nitratireducenticrescens , Hyphomicrobium denitrificans , and Methyloversatilis spp.
Urakami et al. This pathway can also be run in reverse, catalyzing the stepwise reduction of sulfate to sulfite and then sulfide. However, a genome-wide search revealed only the presence of assimilatory sulfite reductases. Neither the dissimilatory sulfite reductase genes dsrAB , nor the requisite companion gene, dsrD , was identified. However, given that sulfate reduction with H 2 is only moderately exergonic in SURF fluids, and that sulfate reduction fell near anaerobic heterotrophy in a principal component analysis of thermodynamic favorability using in situ fluid geochemistry Osburn et al.
Given the presence of genes that encode enzymes for hydrogenases and methylotrophy, electron donor utilization appears to be versatile in SURF genomes, is most likely varied depending upon the electron acceptor concentration, and on subsurface geochemical conditions in situ.
The retention of genes encoding multiple putative methods of carbon, nitrogen and sulfur metabolisms may be a way for Ca. Abyssubacteria to cope with variable geochemistry and intermittent energetically unfavorable conditions. In contrast, the two genomes analyzed here were collected from deep continental subsurface fluids, in a relatively natural and undisturbed environment, compared to anaerobic sludge bioreactors.
Their closest relatives, according to the vast repository of 16S rRNA clone sequences, reside in similar continental and marine subsurface environments Figure 4 and Supplementary Data File 4.
This discrepancy in habitat is reflected in the putative metabolisms encoded by available genomes Figure 3. In contrast, the genomes from SURF contain all genes necessary for autotrophic carbon fixation via the reductive acetyl-CoA pathway Figures 2 , 3.
Although genome reconstruction was not possible, Nobu et al. None of these appear to be the most likely metabolisms for the genomes collected from SURF. Cells are motile, and have versatile carbon and energy-yielding chemotrophic metabolic potential, enabling them to survive in dark, energy deplete subsurface environments under varying levels of oxygen, nitrogen, carbon, and sulfur. LM analyzed the metagenomic data, performed the phylogenetic analyses, and reconstructed the metabolism of genomes analyzed in the study and wrote the manuscript.
HA analyzed the global distribution, performed the phylogenomic analyses, POCP calculations and contributed to results and discussion. JA funded sample retrieval and provided guidance during analysis and writing of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Many thanks to Dr.
Laura Zinke for insightful discussion of microbial metabolisms. We also thank Elaina Graham and Dr. Benjamin Tully at the University of Southern California for their invaluable assistance with bioinformatic analyses.
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