The deep subsurface is an enormous repository of microbial life. However, the metabolic capabilities of these microorganisms and the degree to which they are dependent on surface processes are largely unknown. Due to the logistical difficulty of sampling and inherent heterogeneity, the microbial populations of the terrestrial subsurface are poorly characterized. In an effort to better understand the biogeochemistry of deep terrestrial habitats, we evaluate the energetic yield of chemolithotrophic metabolisms and microbial diversity in the Sanford Underground Research Facility (SURF) in the former Homestake Gold Mine, SD, USA. Geochemical data, energetic modeling, and DNA sequencing were combined with principle component analysis to describe this deep (down to 8100 ft below surface), terrestrial environment. SURF provides access into an iron-rich Paleoproterozoic metasedimentary deposit that contains deeply circulating groundwater. Geochemical analyses of subsurface fluids reveal enormous geochemical diversity ranging widely in salinity, oxidation state (ORP 330 to −328 mV), and concentrations of redox sensitive species (e.g., Fe2+ from near 0 to 6.2 mg/L and Σ S2- from 7 to 2778μg/L). As a direct result of this compositional buffet, Gibbs energy calculations reveal an abundance of energy for microorganisms from the oxidation of sulfur, iron, nitrogen, methane, and manganese. Pyrotag DNA sequencing reveals diverse communities of chemolithoautotrophs, thermophiles, aerobic and anaerobic heterotrophs, and numerous uncultivated clades. Extrapolated across the mine footprint, these data suggest a complex spatial mosaic of subsurface primary productivity that is in good agreement with predicted energy yields. Notably, we report Gibbs energy normalized both per mole of reaction and per kg fluid (energy density) and find the later to be more consistent with observed physiologies and environmental conditions. Further application of this approach will significantly expand our understanding of the deep terrestrial biosphere.

The terrestrial deep subsurface is a huge repository of microbial biomass, but in relation to its size and physical heterogeneity, few sites have been investigated in detail. Here, we applied a culture-independent metagenomic approach to characterize the microbial community composition in deep (1500 meters below surface) terrestrial fluids. Samples were collected from a former gold mine in Lead, South Dakota, USA, now Sanford Underground Research Facility (SURF). We reconstructed 74 genomes from metagenomes (MAGs), enabling the identification of common metabolic pathways. Sulfate and nitrate/nitrite reduction were the most common putative energy metabolisms. Complete pathways for autotrophic carbon fixation were found in more than half of the MAGs, with the reductive acetyl-CoA pathway by far the most common. Nearly 40% (29 of 74) of the recovered MAGs belong to bacterial phyla without any cultivated members-microbial dark matter. Three of our MAGs constitute two novel phyla previously only identified in 16 S rRNA gene surveys. The uniqueness of this data set-its physical depth in the terrestrial subsurface, the relative abundance and completeness of microbial dark matter genomes and the overall diversity of this physically deep, dark, community-make it an invaluable addition to our knowledge of deep subsurface microbial ecology.

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO 2 and N 2 , have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)-the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules-led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.

The record of the coevolution of oxygenic phototrophs and the environment is preserved in three forms: genomes of modern organisms, diverse geochemical signals of surface oxidation and diagnostic Proterozoic microfossils. When calibrated by fossils, genomic data form the basis of molecular clock analyses. However, different interpretations of the geochemical record, fossil calibrations and evolutionary models produce a wide range of age estimates that are often conflicting. Here, we show that multiple interpretations of the cyanobacterial fossil record are consistent with an Archean origin of crown-group Cyanobacteria. We further show that incorporating relative dating information from horizontal gene transfers greatly improves the precision of these age estimates, by both providing a novel empirical criterion for selecting evolutionary models, and increasing the stringency of sampling of posterior age estimates. Independent of any geochemical evidence or hypotheses, these results support oxygenic photosynthesis evolving at least several hundred million years before the Great Oxygenation Event (GOE), a rapid diversification of major cyanobacterial lineages around the time of the GOE, and a post-Cryogenian origin of extant marine picocyanobacterial diversity.

The phylum Cyanobacteria includes free-living bacteria and plastids, the descendants of cyanobacteria that were engulfed by the ancestral lineage of the major photosynthetic eukaryotic group Archaeplastida. Endosymbiotic events that followed this primary endosymbiosis spread plastids across diverse eukaryotic groups. The remnants of the ancestral cyanobacterial genome present in all modern plastids, enable the placement of plastids within Cyanobacteria using sequence-based phylogenetic analyses. To date, such phylogenetic studies have produced conflicting results and two competing hypotheses: (1) plastids diverge relatively recently in cyanobacterial evolution and are most closely related to nitrogen-fixing cyanobacteria, or (2) plastids diverge early in the evolutionary history of cyanobacteria, before the divergence of most cyanobacterial lineages. Here, we use phylogenetic analysis of ribosomal proteins from an expanded data set of cyanobacterial and representative plastid genomes to infer a deep placement for the divergence of the plastid ancestor lineage. We recover plastids as sister to Gloeomargarita and show that the group diverges from other cyanobacterial groups before Pseudanabaena , a previously unreported placement. The tree topologies and phylogenetic distances in our study have implications for future molecular clock studies that aim to model accurate divergence times, especially with respect to groups containing fossil calibrations. The newly sequenced cyanobacterial groups included here will also enable the use of novel cyanobacterial microfossil calibrations.

Earth's deep subsurface biosphere (DSB) is home to a vast number and wide variety of microorganisms. Although difficult to access and sample, deep subsurface environments have been probed through drilling programs, exploration of mines and sampling of deeply sourced vents and springs. In an effort to understand the ecology of deep terrestrial habitats, we examined bacterial diversity in the Sanford Underground Research Facility (SURF), the former Homestake gold mine, in South Dakota, USA. Whole genomic DNA was extracted from deeply circulating groundwater and corresponding host rock (at a depth of 1.45 km below ground surface). Pyrotag DNA sequencing of the 16S rRNA gene revealed diverse communities of putative chemolithoautotrophs, aerobic and anaerobic heterotrophs, numerous candidate phyla and unique rock-associated microbial assemblage. There was a clear and near-total separation of communities between SURF deeply circulating fracture fluids and SURF host-rocks. Sequencing data from SURF compared against five similarly sequenced terrestrial subsurface sites in Europe and North America revealed classes Clostridia and Betaproteobacteria were dominant in terrestrial fluids. This study presents a unique analysis showing differences in terrestrial subsurface microbial communities between fracture fluids and host rock through which those fluids permeate.

Deep subsurface biofilms are estimated to host the majority of prokaryotic life onEarth, yet fundamental aspects of their ecology remain unknown. An inherent difficulty in studying subsurface biofilms is that of sample acquisition. While samples from marine and terrestrial deep subsurface fluids have revealed abundant and diverse microbial life, limited work has described the corresponding biofilms on rock fracture and pore space surfaces. The recently established Deep Mine Microbial Observatory (DeMMO) is a long-term monitoring network at which we can explore the ecological role of biofilms in fluid-filled fractures to depths of 1.5 km. We carried out in situ cultivation experiments with single minerals representative of DeMMO host rock to explore the ecological drivers of biodiversity and biomass in biofilm communities in the continental subsurface. Coupling cell densities to thermodynamic models of putative metabolic reactions with minerals suggests a metabolic relationship between biofilms and the minerals they colonize. Our findings indicate that minerals can significantly enhance biofilm cell densities and promote selective colonization by taxa putatively capable of extracellular electron transfer. In turn, minerals can drive significant differences in biodiversity between fluid and biofilm communities. Given our findings at DeMMO, we suggest that host rock mineralogy is an important ecological driver in deep continental biospheres.

The cyanobacterial genus Trichodesmium is biogeochemically significant because of its dual role in nitrogen and carbon fixation in the oligotrophic ocean. Trichodesmium species form colonies that can be easily enriched from the water column and used for shipboard rate measurements to estimate their contribution to oceanic carbon and nitrogen budgets. During a July 2010 cruise near the Hawaiian Islands in the oligotrophic North Pacific Subtropical Gyre, a specific morphology of Trichodesmium puff-form colonies were examined under epifluorescent microscopy and found to harbor a colonial endobiont, morphologically identified as the heterocystous diazotrophic cyanobacterium Calothrix. Using unialgal enrichments obtained from this cruise, we show that these Calothrix-like heterocystous cyanobionts (hetDA for 'Trichodesmium-associated heterocystous diazotroph') fix nitrogen on a diurnal cycle (maximally in the middle of the light cycle with a detectable minimum in the dark). Gene sequencing of nifH from the enrichments revealed that this genus was likely not quantified using currently described quantitative PCR (qPCR) primers. Guided by the sequence from the isolate, new hetDA-specific primers were designed and subsequent qPCR of environmental samples detected this diazotroph from surface water to a depth of 150 m, reaching densities up to B9 Â 10 3 l À 1 . Based on phylogenetic relatedness of nifH and 16S rRNA gene sequences, it is predicted that the distribution of this cyanobiont is not limited to subtropical North Pacific but likely reaches to the South Pacific and Atlantic Oceans. Therefore, this previously unrecognized cohabitation, if it reaches beyond the oligotrophic North Pacific, could potentially influence Trichodesmium-derived nitrogen fixation budgets in the world ocean.