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Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared >99.2% average nucleotide identity, low frequency of SNP’s, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders.

Ten distinct isolates from the genus Pseudomonas were isolated in culture. The genomes of these isolates were sequenced using the Illumina MiSeq platform and assembled in order to provide insight into the metabolic and carbon-degrading potential of bacteria residing in soils at high latitudes.

Methane emissions from organic-rich soils in the Arctic have been extensively studied due to their potential to increase the atmospheric methane burden as permafrost thaws1–3. However, this methane source might have been overestimated without considering high-affinity methanotrophs (HAMs; methane-oxidizing bacteria) recently identified in Arctic mineral soils4–7. Herein we find that integrating the dynamics of HAMs and methanogens into a biogeochemistry model8–10 that includes permafrost soil organic carbon dynamics3 leads to the upland methane sink doubling (~5.5 Tg CH4 yr−1) north of 50 °N in simulations from 2000–2016. The increase is equivalent to at least half of the difference in net methane emissions estimated between process-based models and observation-based inversions11,12, and the revised estimates better match site-level and regional observations5,7,13–15. The new model projects doubled wetland methane emissions between 2017–2100 due to more accessible permafrost carbon16–18. However, most of the increase in wetland emissions is offset by a concordant increase in the upland sink, leading to only an 18% increase in net methane emission (from 29 to 35 Tg CH4 yr−1). The projected net methane emissions may decrease further due to different physiological responses between HAMs and methanogens in response to increasing temperature19,20. Models overestimate Arctic methane emissions compared to observations. Incorporating microbial dynamics into biogeochemistry models helps reconcile this discrepancy; high-affinity methanotrophs are an important part of the Arctic methane budget and double previous estimates of methane sinks.

Background Exceptional preservation of endogenous organics such as collagens and blood vessels has been frequently reported in Mesozoic dinosaur fossils. The persistence of these soft tissues in Mesozoic fossil bones has been challenged because of the susceptibility of proteins to degradation and because bone porosity allows microorganisms to colonize the inner microenvironments through geological time. Although protein lability has been studied extensively, the genomic diversity of microbiomes in dinosaur fossil bones and their potential roles in bone taphonomy remain underexplored. Genome-resolved metagenomics was performed, therefore, on the microbiomes recovered from a Late Cretaceous Centrosaurus bone and its encompassing mudstone in order to provide insight into the genomic potential for microbial alteration of fossil bone. Results Co-assembly and binning of metagenomic reads resulted in a total of 46 high-quality metagenome-assembled genomes (MAGs) affiliated to six bacterial phyla (Actinobacteria, Proteobacteria, Nitrospira, Acidobacteria, Gemmatimonadetes and Chloroflexi) and 1 archaeal phylum (Thaumarchaeota). The majority of the MAGs represented uncultivated, novel microbial lineages from class to species levels based on phylogenetics, phylogenomics and average amino acid identity. Several MAGs from the classes Nitriliruptoria, Deltaproteobacteria and Betaproteobacteria were highly enriched in the bone relative to the adjacent mudstone. Annotation of the MAGs revealed that the distinct putative metabolic functions of different taxonomic groups were linked to carbon, nitrogen, sulfur and iron metabolism. Metaproteomics revealed gene expression from many of the MAGs, but no endogenous collagen peptides were identified in the bone that could have been derived from the dinosaur. Estimated in situ replication rates among the bacterial MAGs suggested that most of the microbial populations in the bone might have been actively growing but at a slow rate. Conclusions Our results indicate that excavated dinosaur bones are habitats for microorganisms including novel microbial lineages. The distinctive microhabitats and geochemistry of fossil bone interiors compared to that of the external sediment enrich a microbial biomass comprised of various novel taxa that harbor multiple gene sets related to interconnected biogeochemical processes. Therefore, the presence of these microbiomes in Mesozoic dinosaur fossils urges extra caution to be taken in the science of paleontology when hunting for endogenous biomolecules preserved from deep time.

Approximately 87% of the Arctic consists of low-organic carbon mineral soil, but knowledge of microbial activity in low-carbon permafrost (PF) and active layer soils remains limited. This study investigated the taxonomic composition and genetic potential of microbial communities at contrasting depths of the active layer (5, 35, and 65 cm below surface, bls) and PF (80 cm bls). We showed microbial communities in PF to be taxonomically and functionally different from those in the active layer. 16S rRNA gene sequence analysis revealed higher biodiversity in the active layer than in PF, and biodiversity decreased significantly with depth. The reconstructed 91 metagenome-assembled genomes showed that PF was dominated by heterotrophic, fermenting Bacteroidota using nitrite as their main electron acceptor. Prevalent microbes identified in the active layer belonged to bacterial taxa, gaining energy via aerobic respiration. Gene abundance in metagenomes revealed enrichment of genes encoding the plant-derived polysaccharide degradation and metabolism of nitrate and sulfate in PF, whereas genes encoding methane/ammonia oxidation, cold-shock protein, and two-component systems were generally more abundant in the active layer, particularly at 5 cm bls. The results of this study deepen our understanding of the low-carbon Arctic soil microbiome and improve prediction of the impacts of thawing PF.

Permafrost microbes may be metabolically active in microscopic layers of liquid brines, even in ancient soil. Metagenomics can help discern whether permafrost microbes show adaptations to this environment. Thirty-three metagenome-assembled genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly cored permafrost from the Siberian Kolyma-Indigirka Lowland region. These soils have been continuously frozen for ∼20,000 to 1,000,000 years. Eight of these MAGs were ≥80% complete with

We review the abundance and diversity of terrestrial rock hosted life, the environments it inhabits, the evolution of its metabolisms, and its fossil biomarkers to provide guidance in the search for the biomarkers of rock hosted life on Mars. Key finds are metabolic pathways for chemolithotrophic microorganisms evolved much earlier in Earth history than those of surface dwelling phototrophic microorganisms,the emergence of the former occurred at a time when Mars was habitable, whereas that of the latter occurred at a time when the martian surface would have been uninhabitable, subsurface biomass do not correlate with organic carbon and tends to be highest at interfaces where chemical redox gradients are most pronounced, deep subsurface metabolic activity does not rely upon the respiration of organic photosynthate but upon the flux of inorganic energy and the abiotic and biotic recycling of metabolic waste products, and the rock record reveals examples of subsurface life back to 3.45 Ga with several examples of good preservation potential in rock types that are quite different from those preserving the photospheric supported biosphere.These findings suggest that rock hosted life would have likely to emerge and be preserved in a martian context. We thus propose a Mars exploration strategy that scales spatially, focusing initially on identifying rocks with evidence for groundwater flow and low temperature mineralization, then identifying redox and permeability interfaces preserved within rock outcrops, and finally focusing on finding minerals associated with redox reactions and traces of carbon and diagnostic biosignatures. The lessons from Earth show that ancient rock hosted life is preserved in the fossil record and confirmable via a suite of morphologic, organic, mineralogical and isotopic fingerprints and microscopic textures., 55 pages, 5 figures, 2 tables

The surface of Mars has been well mapped and characterized, yet the subsurface — the most likely place to find signs of extant or extinct life and a repository of useful resources for human exploration — remains unexplored. In the near future this is set to change.

Observations of trace methane (CH4) in the Martian atmosphere are significant to the astrobiology community given the overwhelming contribution of biological methanogenesis to atmospheric CH4 on Earth. Previous studies have shown that methanogenic Archaea can generate CH4 when incubated with perchlorates, highly oxidizing chaotropic salts which have been found across the Martian surface. However, the regulatory mechanisms behind this remain completely unexplored. In this study we performed comparative transcriptomics on the methanogen Methanosarcina barkeri, which was incubated at 30˚C and 0˚C with 10–20 mM calcium-, magnesium-, or sodium perchlorate. Consistent with prior studies, we observed decreased CH4 production and apparent perchlorate reduction, with the latter process proceeding by heretofore essentially unknown mechanisms. Transcriptomic responses of M. barkeri to perchlorates include up-regulation of osmoprotectant transporters and selection against redox-sensitive amino acids. Increased expression of methylamine methanogenesis genes suggest competition for H2 with perchlorate reduction, which we propose is catalyzed by up-regulated molybdenum-containing enzymes and maintained by siphoning diffused H2 from energy-conserving hydrogenases. Methanogenesis regulatory patterns suggest Mars’ freezing temperatures alone pose greater constraints to CH4 production than perchlorates. These findings increase our understanding of methanogen survival in extreme environments and confers continued consideration of a potential biological contribution to Martian CH4.

Background Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost. Results Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26–120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost. Conclusions Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars.

In Earth's deep continental subsurface, where groundwaters are often isolated for >106 to 109 years, energy released by radionuclides within rock produces oxidants and reductants that drive metabolisms of non-photosynthetic microorganisms. Similar processes could support past and present life in the martian subsurface. Sulfate-reducing microorganisms are common in Earth's deep subsurface, often using hydrogen derived directly from radiolysis of pore water and sulfate derived from oxidation of rock-matrix-hosted sulfides by radiolytically derived oxidants. Radiolysis thus produces redox energy to support a deep biosphere in groundwaters isolated from surface substrate input for millions to billions of years on Earth. Here, we demonstrate that radiolysis by itself could produce sufficient redox energy to sustain a habitable environment in the subsurface of present-day Mars, one in which Earth-like microorganisms could survive wherever groundwater exists. We show that the source localities for many martian meteorites are capable of producing sufficient redox nutrients to sustain up to millions of sulfate-reducing microbial cells per kilogram rock via radiolysis alone, comparable to cell densities observed in many regions of Earth's deep subsurface. Additionally, we calculate variability in supportable sulfate-reducing cell densities between the martian meteorite source regions. Our results demonstrate that martian subsurface groundwaters, where present, would largely be habitable for sulfate-reducing bacteria from a redox energy perspective via radiolysis alone. We present evidence for crustal regions that could support especially high cell densities, including zones with high sulfide concentrations, which could be targeted by future subsurface exploration missions.

Radiokrypton dating using the long-lived natural isotope 81Kr has been developed for determining the age of ancient groundwater. 81Kr is attractive for this purpose because it is generally thought to be produced solely in the upper atmosphere. Its 229,000-year half-life, a spatially homogeneous distribution in the atmosphere, and the absence of anthropogenic sources makes it an ideal tracer to determine ages up to ∼1.3 million years. As a noble gas, it is inert and thus not subject to interfering geochemical reactions that may alter the age information. We sought to date groundwater samples collected from deep (0.6–1.9 km) rock fractures in the Kaapvaal Craton, South Africa. Previous studies using other dating methods have estimated groundwater ages at these sites in the 1–100 million year range. Surprisingly, three of the four samples collected from flowing boreholes in gold and diamond mines showed 81Kr isotopic abundances at 2–5 times the atmospheric value. This is the first time that underground production of 81Kr has been detected, indicating that 81Kr can be generated underground in measurable quantities in contrast to the long-held paradigm that such production is insignificant in natural rocks. A radionuclide production and release model is proposed in order to quantify the importance of different factors that affect the concentrations of 81Kr in the groundwater. It is not only the high effective uranium content of the rock but also a higher 81Kr fission yield than previously anticipated that are likely causing the elevated 81Kr values in this case. In less extreme environments with average crustal composition (i.e. moderate U concentration), however, we anticipate that the underground production minimally affects groundwater 81Kr dating as demonstrated.

Additional file 2: Table S1. Statistical summary for MAGs recovered from permafrost sediment samples at 3.4, 5.8 and 14.8 m. Note: CDS refers to protein coding sequence.

Charles D. Edwards (Jet Propulsion Laboratory, California Institute of Technology). Co-Authors: 1. Vlada Stamenkovic Jet Propulsion Laboratory, California Institute of Technology; 2. Penelope Boston NASA Ames; 3. Kennda Lynch LPI/USRA … et al.

Additional file 6: Table S5. Summary of identified proteins from ancient permafrost at 3.4, 5.8 and 14.8m.

Bulletin of the AAS, 53 (4)

Additional file 9. The scripts used for the analyses in this study.

Additional file of Genomic reconstruction of fossil and living microorganisms in ancient Siberian permafrost

Additional file 1: Figure S1. Geographic location of the sampling site at Cape Chukochii near the East Siberian Sea coast. Figure S2. Image of the drilling site and the schematic of the sediment core (~22 m) from borehole Ch1-17. The red stars indicate the depth of the sediment samples (3.4, 5.8 and 14.8 m, meters below land surface) that were selected for metagenomic sequencing of iDNA and eDNA with and without DNA repair. Figure S3 Temperature and geochemistry profiles of the permafrost sediment collected at various depths from borehole Ch1-17. Figure S4. Concentration of low-molecular-weight organic acids in the permafrost sediment collected at various depths from borehole Ch1-17. Figure S5 Yield of iDNA and eDNA fractions from ancient permafrost sediment at 3.4, 5.8 and 14.8 m. The green bars represent the estimated cell numbers from the intracellular DNA fraction by assuming 2×10-15 g DNA/cell. Figure S6. Size distribution of DNA fragments in iDNA (3.4i, 5.8i and 14.8i) and eDNA (3.4e, 5.8e and 14.8e) fractions from ancient permafrost sediment at 3.4, 5.8 and 14.8 m. The top peaks at 10380 bp for 3.4iDNA and 3.4eDNA sampels were cropped out due to the much higher concentration. Figure S7. Live/Dead cell staining of separated cells from ancient permafrost sediments at 3.4 (top), 5.8 (middle) and 14.8 m (bottom). The green stained live cells by Syto9 are shown in the left panel whereas the red stained dead cells are depicted in the right panel. Figure S8. Relative abundance of MAGs from each metagenome of iDNA and eDNA at 3.4 m with and without PreCR DNA repair. The scale bar indicates the relative abundance of each MAG normalized to the individual sample size as genome copies per million reads. Figure S9. Relative abundance of MAGs from each metagenome of iDNA and eDNA at 5.8 m with and without PreCR DNA repair. The scale bar indicates the relative abundance of each MAG normalized to the individual sample size as genome copies per million reads. Figure S10. Relative abundance of MAGs from each metagenome of iDNA and eDNA at 14.8 m with and without PreCR DNA repair. The scale bar indicates the relative abundance of each MAG normalized to the individual sample size as genome copies per million reads. Figure S11. Percentage of unique reads in each metagenome generated from iDNA and eDNA fractions of the 3.4, 5.8 and 14.8 m samples with and without PreCR DNA repair. Figure S12. Heatmap of the global similarity of each iDNA and eDNA derived metagenomes from the 3.4, 5.8 and 14.8 m samples with and without PreCR DNA repair. The similarity matrix was calculated from the similarity of reads in each metagenome. The scale bar represents the normalized percentage of similarity between two metagenomes with respect to the total number of reads in each metagenome. Figure S13. Principal coordinate analyses (PCoA) of weighted UniFrac distances derived from the microbial community based on the 16S rRNA genes retrieved from each metagenomic dataset of iDNA and eDNA fraction with and without PreCR DNA repair. Figure S14. Number of quality-filtered reads in each metagenome generated from iDNA and eDNA fractions extracted from ancient permafrost sediment at 3.4, 5.8 and 14.8 m with and without PreCR DNA repair. Figure S15. Plot of GC content and contig abundance in each MAG recovered from individual metagenomes of the iDNA and eDNA extracted from the 14.8 m with and without PreCR DNA repair. Figure S16. Groups of carbohydrates active enzymes identified in MAGs recovered from three depths at 3.4, 5.8 and 14.8 m, respectively. The abbreviations for the enzymes classes are as follow: The glycoside hydrolases (GHs), glycosyl transferases (GTs), carbohydrate esterases (CEs) and auxiliary activities (AAs). The relative abundance represents the number of carbohydrates active enzymes identified in each specific subgroup. Figure S17. Relative abundance of proteins identified in the metaproteome from each MAG recovered from the 5.8 m sample. These proteins were involved in carbon and energy metabolism and coping with cold, osmotic and oxidative stresses. The scale bar indicates the balanced spectral counts of proteins. Figure S18. Number of identified proteins in the metaproteomic dataset at 14.8 m when all genes from the metagenome were used as database for search. Figure S19. Number of identified proteins from each MAG recovered from 14.8m. Figure S20 GRiD measurement of bacterial MAGs from the iDNA metagenomic datasets from ancient permafrost sediment at 3.4, 5.8 and 14.8m. The criterion of valid GRiD values (dnaA/ori and ter/dif ratios > 0.8) for each MAGs was selected according to the output results from the GRiD tool.

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells’ growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB’s ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist’s toolkit, with potentially widespread application in the field of environmental microbiology.

The role of archaeal ammonia oxidizers often exceeds that of bacterial ammonia oxidizers in marine and terrestrial environments but has been understudied in permafrost, where thawing has the potential to release ammonia. Here, three thaumarchaea genomes were assembled and annotated from metagenomic data sets from carbon-poor Canadian High Arctic active-layer cryosols.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of minimal cellular energy transduction mechanisms that potentially precede the evolution of respiration, thus relying solely on fermentation for energy conservation.

The nematode Halicephalobus mephisto was originally discovered inhabiting a deep terrestrial aquifer 1.3 km underground. H. mephisto can thrive under conditions of abiotic stress including heat and minimal oxygen, where it feeds on a community of both chemolithotrophic and heterotrophic prokaryotes in an unusual ecosystem isolated from the surface biosphere. Here we report the comprehensive genome and transcriptome of this organism, identifying a signature of adaptation: an expanded repertoire of 70 kilodalton heat-shock proteins (Hsp70) and avrRpt2 induced gene 1 (AIG1) proteins. The expanded Hsp70 genes are transcriptionally induced upon growth under heat stress, and we find that positive selection is detectable in several members of this family. We further show that AIG1 may have been acquired by horizontal gene transfer (HGT) from a rhizobial fungus. Over one-third of the genes of H. mephisto are novel, highlighting the divergence of this nematode from other sequenced organisms. This work sheds light on the genomic strategies of adaptation to heat in the first complete subterrestrial eukaryotic genome.

Additional file 6: Table S5 Taxonomic identification of MAGs based on homology of 16S rRNA gene.

Additional file 2: Table 1. Geochemical characteristics in water extract from the bone and mudstone.

Additional file 3: Table S2. Statistical summary for MAGs recovered from microbiome in dinosaur fossil bone.

Dissolved sulfate is a crucial electron acceptor for the subsurface biosphere, particularly for the living microbial ecosystems in the long-isolated (on the order of millions to billions of years) deep subsurface fracture waters in Precambrian cratons, e.g., in the Witwatersrand Basin of the Kaapvaal Craton, South Africa. Aiming to understand the role of sulfate in the sustainability of the subsurface habitat and the spatial extent of the terrestrial subsurface biosphere, we carried out a basin-scale examination on the sources and producing mechanisms of dissolved sulfur (sulfate and sulfide) in the subsurface fracture waters in the Witwatersrand Basin using multiple sulfur isotopes (32,33,34,36S) and oxygen isotopes (16,18O). Dissolved sulfates in 14 fracture water samples collected from 900 to 3,413 meters below the surface at 6 sites show isotopic ranges from −5.3‰ to +19.4‰ for δ34S, −0.40‰ to +0.50‰ for Δ33S, and −1.1‰ to +10.9‰ for δ18O. These isotopic signatures are distinct to those of the sulfate minerals (e.g., the carbonate-associated sulfate in local Transvaal Supergroup dolomite sequences: δ34S = +31.4‰ to +39.2‰; Δ33S = −0.01‰ to +0.16‰), but identical to those of the sulfide minerals in the host rocks. This indicates that the dissolved sulfate in the fracture waters were dominantly generated by in situ oxidation of sulfide minerals at basin scale, although mixing of a small amount of surface sulfate at some sites cannot be completely ruled out. The dissolved sulfates in the less deep and less saline fracture waters are in oxygen isotope disequilibrium with their host waters, a surprising result given that the water residence times are orders of magnitude longer than the time required for oxygen isotope exchange to reach equilibrium. This implies that vigorous in situ sulfate production has occurred after the fracture waters were isolated. In contrast, the dissolved sulfate in the deeper, more saline waters are in apparent oxygen isotope equilibration or close to equilibration with their host waters. This might be attributed to a combined effect of faster oxygen isotope exchange between sulfate and water at higher temperatures, larger extent of sulfate reduction, and/or less efficient sulfate production. The dissolved sulfide in the fracture waters has similar Δ33S values but is 3.0‰ to 26.4‰ lower in δ34S than coexisting sulfate, suggesting that the dissolved sulfide is mostly generated from bacterial sulfate reduction, which is consistent with the widespread existence of sulfate-reducing bacteria down to >3.4 km below surface in the Witwatersrand Basin. Overall, these novel isotopic results demonstrate that geological processes can provide a steady long-term sulfate source in deep fracture fluids by in situ oxidation of sulfide minerals in their host rocks, and thereby a mechanism to sustain the terrestrial subsurface biosphere, even in deep, high-temperature, long-isolated water systems. Thus, sulfate as a terminal electron acceptor is not the limiting factor for the spatial expansion of terrestrial subsurface biosphere.

The composition of dissolved organic matter (DOM) in extremely deep groundwaters has rarely been investigated. Focusing on deep fracture waters in billion year old rocks in South Africa, we hypothesized the DOM would be predominantly derived from in situ microbial sources, consistent with H2-driven lithoautotrophic ecosystems previously documented in these fluids. We collected groundwater from 0.6 to 3.4 km depths via boreholes in mines, and characterized the DOM by a variety of analytical methods. Dissolved organic carbon concentrations ranged from 0.25 to 4.9 mg C/L. Low molecular weight organic acids (formate, acetate, lactate, propanoate) and amino acids (after hydrolysis) accounted for varying fractions of the total dissolved organic carbon. UV spectrophotometry, scanning fluorimetry, and nuclear magnetic resonance (NMR) spectra indicated lower concentrations of aromatic compounds than are commonly found in surface waters and shallow groundwaters. Molecular weights of aromatic compounds measured by size-exclusion chromatography were dominantly 500–1000 Da. Scanning fluorimetry revealed humic substances in two shallow (578 and 1300 m) samples; deeper samples showed peaks indicating aromatic amino acids. Intensive study of one sample from a previously sampled borehole in Beatrix mine resulted in large NMR peaks for aliphatics and carboxyls with lesser aromatics peaks; negative electrospray ion Fourier transform ion cyclotron resonance-mass spectrometry detected protein-, lipid-, and lignin-like compounds. These results are consistent with DOM in the form of microbial metabolites, and modified cell components (membrane lipids and proteins). These findings support previous geomicrobiological reports of chemolithoautotrophic microbial ecosystems functioning in isolation from photosynthetic primary production.

Few traces of Earth’s geologic record are preserved from the time of life’s emergence, over 3,800 million years ago. Consequently, what little we understand about abiogenesis — the origin of life on Earth — is based primarily on laboratory experiments and theory. The best geological lens for understanding early Earth might actually come from Mars, a planet with a crust that’s overall far more ancient than our own. On Earth, surface sedimentary environments are thought to best preserve evidence of ancient life, but this is mostly because our planet has been dominated by high photosynthetic biomass production at the surface for the last ~2,500 million years or more. By the time oxygenic photosynthesis evolved on Earth, Mars had been a hyperarid, frozen desert with a surface bombarded by high-energy solar and cosmic radiation for more than a billion years, and as a result, photosynthetic surface life may never have occurred on Mars. Therefore, one must question whether searching for evidence of life in Martian surface sediments is the best strategy. This Perspective explores the possibility that the abundant hydrothermal environments on Mars might provide more valuable insights into life’s origins. Ancient hydrothermal deposits formed in the Martian subsurface may be the best targets for finding evidence for ancient life on Mars, and clues about the origin of life on Earth.