An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers.

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H2 Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic ß-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

Hypolithic microbial communities: between a rock and a hard place.

Drylands are the largest terrestrial biome on Earth and a ubiquitous feature is desert pavement terrain, comprising rocks embedded in the mineral soil surface. Quartz and other translucent rocks are common and microbial communities termed hypoliths develop as biofilms on their ventral surfaces. In extreme deserts these represent major concentrations of biomass, and are emerging as key to geobiological processes and soil stabilization. These highly specialized communities are dominated by cyanobacteria that support diverse heterotrophic assemblages. Here we identify global-scale trends in the ecology of hypoliths that are strongly related to climate, particularly with regard to shifts in cyanobacterial assemblages. A synthesis of available data revealed a linear trend for colonization with regard to climate, and we suggest potential application for hypoliths as 'biomarkers' of aridity on a landscape scale. The potential to exploit the soil-stabilizing properties of hypolithic colonization in environmental engineering on dryland soils is also discussed.

Highly specialized microbial diversity in hyper-arid polar desert.

The McMurdo Dry Valleys in Antarctica are a cold hyperarid polar desert that present extreme challenges to life. Here, we report a culture-independent survey of multidomain microbial biodiversity in McKelvey Valley, a pristine example of the coldest desert on Earth. We demonstrate that life has adapted to form highly-specialized communities in distinct lithic niches occurring concomitantly within this terrain. Endoliths and chasmoliths in sandstone displayed greatest diversity, whereas soil was relatively depauperate and lacked a significant photoautotrophic component, apart from isolated islands of hypolithic cyanobacterial colonization on quartz rocks in soil contact. Communities supported previously unreported polar bacteria and fungi, but archaea were absent from all niches. Lithic community structure did not vary significantly on a landscape scale and stochastic moisture input due to snowmelt resulted in increases in colonization frequency without significantly affecting diversity. The findings show that biodiversity near the cold-arid limit for life is more complex than previously appreciated, but communities lack variability probably due to the high selective pressures of this extreme environment.

Hypolithic microbial community of quartz pavement in the high-altitude tundra of central Tibet.

The hypolithic microbial community associated with quartz pavement at a high-altitude tundra location in central Tibet is described. A small-scale ecological survey indicated that 36% of quartz rocks were colonized. Community profiling using terminal restriction fragment length polymorphism revealed no significant difference in community structure among a number of colonized rocks. Real-time quantitative PCR and phylogenetic analysis of environmental phylotypes obtained from clone libraries were used to elucidate community structure across all domains. The hypolithon was dominated by cyanobacterial phylotypes (73%) with relatively low frequencies of other bacterial phylotypes, largely represented by the chloroflexi, actinobacteria, and bacteriodetes. Unidentified crenarchaeal phylotypes accounted for 4% of recoverable phylotypes, while algae, fungi, and mosses were indicated by a small fraction of recoverable phylotypes.

Endolithic microbial colonization of limestone in a high-altitude arid environment.

The morphology of endolithic colonization in a limestone escarpment and surrounding rocky debris (termed float) at a high-altitude arid site in central Tibet was documented using scanning electron microscopy. Putative lichenized structures and extensive coccoid bacterial colonization were observed. Absolute and relative abundance of rRNA gene signatures using real-time quantitative polymerase chain reaction and phylogenetic analysis of environmental phylotypes were used to characterize community structure across all domains. Escarpment endoliths were dominated by eukaryotic phylotypes suggestive of lichenised associations (a Trebouxia lichen phycobiont and Leptodontidium lichen mycobiont), whereas float endoliths were dominated by bacterial phylotypes, including the cyanobacterium Chroococcidiopsis plus several unidentified beta proteobacteria and crenarchaea. Among a range of abiotic variables tested, ultraviolet (UV) transmittance by rock substrates was the factor best able to explain differences in community structure, with eukaryotic lichen phylotypes more abundant under conditions of greater UV-exposure compared to prokaryotes. Variously pigmented float rocks did not support significantly different communities. Estimates of in situ carbon fixation based upon (14)C radio-labelled bicarbonate uptake indicated endolithic productivity of approximately 2.01 g C/m(2)/year(-1), intermediate between estimates for Antarctic and temperate communities.

Genome-centric resolution of novel microbial lineages in an excavated Centrosaurus dinosaur fossil bone from the Late Cretaceous of North America.

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.

Thaumarchaea Genome Sequences from a High Arctic Active Layer.

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.

Vertical partitioning and expression of primary metabolic genes in a thermophilic microbial mat.

A thermophilic microbial mat with a relatively simple morphological composition was used to study the expression of key metabolic genes between mat layers. Mats comprised Roseiflexus castenholzii, Synechococcus sp., a Sphingomonas-like proteobacterial taxon and an unidentified member of the Thermotogae as determined by 16S rRNA phylotypes. The diversity of expressed loci for key genes involved in oxygenic photosynthesis (cbbL), anoxygenic photosynthesis (pufM) and nitrogen fixation (nifH) was assessed. The cyanobacterial surface layer supported two cbbL transcripts, with closest phylogenetic affinity to those from the cyanobacterium Synechococcus sp. and a proteobacterium Nitrobacter sp. This indicates that both photoautotrophic and chemolithoautotrophic carbon dioxide fixation may occur in this mat layer. Lower layers did not support cbbL transcripts. Anoxygenic photosynthesis was indicated by a single pufM transcript with closest affinity to that of R. castenholzii. Expression occurred in all layers beneath the cyanobacterial surface layer. Expression of a single nifH transcript with closest affinity to a proteobacterial nitrogenase occurred in samples throughout all mat layers.

Bacterial community composition in thermophilic microbial mats from five hot springs in central Tibet.

Despite detailed study of selected thermophilic taxa, overall community diversity of bacteria in thermophilic mats remains relatively poorly understood. A sequence-based survey of bacterial communities from several hot spring locations in central Tibet was undertaken. Diversity and frequency of occurrence for 140 unique 16S rRNA gene phylotypes were identified in clone libraries constructed from environmental samples. A lineage-per-time plot revealed that individual locations have evolved to support relatively large numbers of phylogenetically closely related phylotypes. Application of the F ( ST ) statistic and P test to community data was used to demonstrate that phylogenetic divergence between locations was significant, thus emphasizing the status of hot springs as isolated habitats. Among phylotypes, only the Chlorobi were ubiquitous to all mats, other phototrophs (Cyanobacteria and Chloroflexi) occurred in most but not all samples and generally accounted for a large number of recovered phylotypes. Phylogenetic analyses of phototrophic phylotypes revealed support for location-specific lineages. The alpha, beta and gamma proteobacteria were also frequently recovered phyla, suggesting they may be abundant phylotypes in mats, a hitherto unappreciated aspect of thermophilic mat biodiversity. Samples from one location indicated that where phototrophic bacteria were rare or absent due to niche disturbance, the relative frequency of proteobacterial phylotypes increased.

Aspartic acid racemization constrains long-term viability and longevity of endospores.

Certain microorganisms survive long periods of time as endospores to cope with adverse conditions. Since endospores are metabolically inactive, the extent of aspartic acid (Asp) racemization will increase over time and might kill the spores by preventing their germination. Therefore, understanding the relationship between endospore survivability and Asp racemization is important for constraining the long-term survivability and global dispersion of spore-forming bacteria in nature. Geobacillus stearothermophilus was selected as a model organism to investigate racemization kinetics and survivability of its endospores at 65°C, 75°C and 98°C. This study found that the Asp racemization rates of spores and autoclaved spores were similar at all temperatures. The Asp racemization rate of spores was not significantly different from that of vegetative cells at 65°C. The Asp racemization rate of G. stearothermophilus spores was not significantly different from that of Bacillus subtilis spores at 98°C. The viability of spores and vegetative cells decreased dramatically over time, and the mortality of spores correlated exponentially with the degree of racemization (R2 = 0.9). This latter correlation predicts spore half-lives on the order of hundreds of years for temperatures typical of shallow marine sediments, a result consistent with studies about the survivability of thermophilic spores found in these environments.

Uncultured Nitrospina-like species are major nitrite oxidizing bacteria in oxygen minimum zones.

Oxygen minimum zones (OMZs) are marine regions where O2 is undetectable at intermediate depths. Within OMZs, the oxygen-depleted zone (ODZ) induces anaerobic microbial processes that lead to fixed nitrogen loss via denitrification and anammox. Surprisingly, nitrite oxidation is also detected in ODZs, although all known marine nitrite oxidizers (mainly Nitrospina) are aerobes. We used metagenomic binning to construct metagenome-assembled genomes (MAGs) of nitrite oxidizers from OMZs. These MAGs represent two novel Nitrospina-like species, both of which differed from all known Nitrospina species, including cultured species and published MAGs. Relative abundances of different Nitrospina genotypes in OMZ and non-OMZ seawaters were estimated by mapping metagenomic reads to newly constructed MAGs and published high-quality genomes of members from the Nitrospinae phylum. The two novel species were present in all major OMZs and were more abundant inside ODZs, which is consistent with the detection of higher nitrite oxidation rates in ODZs than in oxic seawaters and suggests novel adaptations to anoxic environments. The detection of a large number of unclassified nitrite oxidoreductase genes in the dataset implies that the phylogenetic diversity of nitrite oxidizers is greater than previously thought.

The Discovery of Twenty-Eight New Encapsulin Sequences, Including Three in Anammox Bacteria.

Many prokaryotes encode protein-based encapsulin nanocompartments, including anaerobic ammonium oxidizing (anammox) bacteria. This study expands the list of known anammox encapsulin systems from freshwater species to include the marine genus Scalindua. Two novel systems, identified in "Candidatus Scalindua rubra" and "Candidatus Scalindua sp. SCAELEC01 167" possess different architectures than previously studied freshwater anammox encapsulins. Characterization of the S. rubra encapsulin confirms that it can self-assemble to form compartments when heterologously expressed in Escherichia coli. BLASTp and HMMER searches of additional genomes and metagenomes spanning a range of environments returned 26 additional novel encapsulins, including a freshwater anammox encapsulin identified in "Candidatus Brocadia caroliniensis". Phylogenetic analysis comparing these 28 new encapsulin sequences and cargo to that of their closest known relatives shows that encapsulins cluster by cargo protein type and therefore likely evolved together. Lastly, prokaryotic encapsulins may be more common and diverse than previously thought. Through searching a small sample size of all public metagenomes and genomes, many new encapsulin systems were unearthed by this study. This suggests that many additional encapsulins likely remain to be discovered.

Metagenome-Assembled Genome of USCα AHI, a Potential High-Affinity Methanotroph from Axel Heiberg Island, Canadian High Arctic.

Metagenomic sequencing of active-layer cryosols from the Canadian High Arctic has yielded a nearly complete genome for an atmospheric CH4-oxidizing bacterium belonging to upland soil cluster α (USCα). This genome contains genes involved in CH4 metabolism, H2 metabolism, and multiple carbon assimilation pathways.

The genome of a subterrestrial nematode reveals adaptations to heat.

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 basis of heat tolerance in a complete subterrestrial eukaryotic genome.

Taxonomic and Functional Compositions Impacted by the Quality of Metatranscriptomic Assemblies.

Metatranscriptomics has recently been applied to investigate the active biogeochemical processes and elemental cycles, and in situ responses of microbiomes to environmental stimuli and stress factors. De novo assembly of RNA-Sequencing (RNA-Seq) data can reveal a more detailed description of the metabolic interactions amongst the active microbial communities. However, the quality of the assemblies and the depiction of the metabolic network provided by various de novo assemblers have not yet been thoroughly assessed. In this study, we compared 15 de novo metatranscriptomic assemblies for a fracture fluid sample collected from a borehole located at 1.34 km below land surface in a South African gold mine. These assemblies were constructed from total, non-coding, and coding reads using five de novo transcriptomic assemblers (Trans-ABySS, Trinity, Oases, IDBA-tran, and Rockhopper). They were evaluated based on the number of transcripts, transcript length, range of transcript coverage, continuity, percentage of transcripts with confident annotation assignments, as well as taxonomic and functional diversity patterns. The results showed that these parameters varied considerably among the assemblies, with Trans-ABySS and Trinity generating the best assemblies for non-coding and coding RNA reads, respectively, because the high number of transcripts assembled covered a wide expression range, and captured extensively the taxonomic and metabolic gene diversity, respectively. We concluded that the choice of de novo transcriptomic assemblers impacts substantially the taxonomic and functional compositions. Care should be taken to obtain high-quality assemblies for informing the in situ metabolic landscape.

Draft Genome Sequence of "Candidatus Bathyarchaeota" Archaeon BE326-BA-RLH, an Uncultured Denitrifier and Putative Anaerobic Methanotroph from South Africa's Deep Continental Biosphere.

Metagenomic sequencing of fracture fluid from South Africa recovered a nearly complete "Candidatus Bathyarchaeota" archaeon genome. The metagenome-assembled genome of BE326-BA-RLH contains genes involved in methane metabolism and dissimilatory nitrate reduction. This study presents the first genomic evidence for potential anaerobic methane oxidation in the phylum "Ca. Bathyarchaeota."

Stochastic and Deterministic Effects of a Moisture Gradient on Soil Microbial Communities in the McMurdo Dry Valleys of Antarctica.

Antarctic soil supports surface microbial communities that are dependent on ephemeral moisture. Understanding the response to availability of this resource is essential to predicting how the system will respond to climate change. The McMurdo Dry Valleys are the largest ice-free soil region in Antarctica. They are a hyper-arid polar desert with extremely limited moisture availability. Microbial colonization dominates this ecosystem but surprisingly little is known about how communities respond to changing moisture regimes. We utilized the natural model system provided by transiently wetted soil at lake margins in the Dry Valleys to interrogate microbial responses along a well-defined contiguous moisture gradient and disentangle responses between and within phyla. We identified a striking non-linear response among bacteria where at low moisture levels small changes resulted in a large impact on diversity. At higher moister levels community responses were less pronounced, resulting in diversity asymptotes. We postulate that whilst the main drivers of observed community diversity were deterministic, a switch in the major influence occurred from abiotic factors at low moisture levels to biotic interactions at higher moisture. Response between and within phyla was markedly different, highlighting the importance of taxonomic resolution in community analysis. Furthermore, we resolved apparent stochasticity at high taxonomic ranks as the result of deterministic interactions taking place at finer taxonomic and spatial scales. Overall the findings provide new insight on the response to moisture and this will be useful in advancing understanding of potential ecosystem responses in the threatened McMurdo Dry Valleys system.

Draft Genome Sequence of Uncultured Upland Soil Cluster Gammaproteobacteria Gives Molecular Insights into High-Affinity Methanotrophy.

Aerated soils form the second largest sink for atmospheric CH4 A near-complete genome of uncultured upland soil cluster Gammaproteobacteria that oxidize CH4 at <2.5 ppmv was obtained from incubated Antarctic mineral cryosols. This first genome of high-affinity methanotrophs can help resolve the mysteries about their phylogenetic affiliation and metabolic potential.

Global Diversity of Desert Hypolithic Cyanobacteria.

Global patterns in diversity were estimated for cyanobacteria-dominated hypolithic communities that colonize ventral surfaces of quartz stones and are common in desert environments. A total of 64 hypolithic communities were recovered from deserts on every continent plus a tropical moisture sufficient location. Community diversity was estimated using a combined t-RFLP fingerprinting and high throughput sequencing approach. The t-RFLP analysis revealed desert communities were different from the single non-desert location. A striking pattern also emerged where Antarctic desert communities were clearly distinct from all other deserts. Some overlap in community similarity occurred for hot, cold and tundra deserts. A further observation was that the producer-consumer ratio displayed a significant negative correlation with growing season, such that shorter growing seasons supported communities with greater abundance of producers, and this pattern was independent of macroclimate. High-throughput sequencing of 16S rRNA and nifH genes from four representative samples validated the t-RFLP study and revealed patterns of taxonomic and putative diazotrophic diversity for desert communities from the Taklimakan Desert, Tibetan Plateau, Canadian Arctic and Antarctic. All communities were dominated by cyanobacteria and among these 21 taxa were potentially endemic to any given desert location. Some others occurred in all but the most extreme hot and polar deserts suggesting they were relatively less well adapted to environmental stress. The t-RFLP and sequencing data revealed the two most abundant cyanobacterial taxa were Phormidium in Antarctic and Tibetan deserts and Chroococcidiopsis in hot and cold deserts. The Arctic tundra displayed a more heterogenous cyanobacterial assemblage and this was attributed to the maritime-influenced sampling location. The most abundant heterotrophic taxa were ubiquitous among samples and belonged to the Acidobacteria, Actinobacteria, Bacteroidetes, and Proteobacteria. Sequencing using nitrogenase gene-specific primers revealed all putative diazotrophs were Proteobacteria of the orders Burkholderiales, Rhizobiales, and Rhodospirillales. We envisage cyanobacterial carbon input to the system is accompanied by nitrogen fixation largely from non-cyanobacterial taxa. Overall the results indicate desert hypoliths worldwide are dominated by cyanobacteria and that growing season is a useful predictor of their abundance. Differences in cyanobacterial taxa encountered may reflect their adaptation to different moisture availability regimes in polar and non-polar deserts.

A metagenomic window into carbon metabolism at 3 km depth in Precambrian continental crust.

Subsurface microbial communities comprise a significant fraction of the global prokaryotic biomass; however, the carbon metabolisms that support the deep biosphere have been relatively unexplored. In order to determine the predominant carbon metabolisms within a 3-km deep fracture fluid system accessed via the Tau Tona gold mine (Witwatersrand Basin, South Africa), metagenomic and thermodynamic analyses were combined. Within our system of study, the energy-conserving reductive acetyl-CoA (Wood-Ljungdahl) pathway was found to be the most abundant carbon fixation pathway identified in the metagenome. Carbon monoxide dehydrogenase genes that have the potential to participate in (1) both autotrophic and heterotrophic metabolisms through the reversible oxidization of CO and subsequent transfer of electrons for sulfate reduction, (2) direct utilization of H2 and (3) methanogenesis were identified. The most abundant members of the metagenome belonged to Euryarchaeota (22%) and Firmicutes (57%)-by far, the highest relative abundance of Euryarchaeota yet reported from deep fracture fluids in South Africa and one of only five Firmicutes-dominated deep fracture fluids identified in the region. Importantly, by combining the metagenomics data and thermodynamic modeling of this study with previously published isotopic and community composition data from the South African subsurface, we are able to demonstrate that Firmicutes-dominated communities are associated with a particular hydrogeologic environment, specifically the older, more saline and more reducing waters.