Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H 2 . 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 CH 4 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 β-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 H 2 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.active subsurface environment | metabolic interactions | sulfur-driven autotrophic denitrifiers | syntrophy | inverted biomass pyramid M icroorganisms living in deep-subsurface ecosystems acquire energy through chemosynthesis and carbon from organic or inorganic sources. Whereas heterotrophs use dissolved organic carbon (DOC) transported from the surface and/or produced in situ, detrital organic deposits buried along with the sediments, and hydrocarbons migrating into petroleum reservoirs, chemolithoautotrophs fix dissolved inorganic carbon (DIC). In oligotrophic systems, subsurface lithoautotrophic microbial ecosystems (SLiMEs) (1) that are fueled by H 2 support the occurrence of autotrophic methanogens, acetogens, and sulfate reducers (2-5). These environments can host highly diverse communities, consisting mostly of prokaryotes, but also multicellular microeukaryotes and viral particles (6-13). Due to the limitation of available nutrients and energy substrates in the oligotrophic subsurface, it is reasonable to hypothesize that subsurface inhabitants with diverse functional traits cooperate syntrophically to maximize energy yield SignificanceMicroorganisms are known to live in the deep ...

Fibrillarin is a nucleolar protein known to be involved in the processing of ribosomal RNA precursors. We isolated AtFbr1, a cDNA encoding a homolog of fibrillarin in Arabidopsis. The cDNA is 1.2 kb in size and encodes a polypeptide of 310 amino acid residues with a molecular mass of 33 kD. AtFbr1 is expressed at high levels in the flower and root tissue and at a slightly lower level in leaf tissue, whereas it was nearly undetectable in siliques. Expression of AtFbr1 was compared with that of the FLP (fibrillarin-like protein) gene identified by the Arabidopsis genome project. Abscisic acid treatment resulted in the down-regulation of the expression of both AtFbr1 and FLP genes in seedlings, although the degree of suppression was higher for FLP than for AtFbr1. In addition, the expression level of FLP decreased with the age of the seedlings, whereas AtFbr1 did not exhibit any detectable change. The subcellular localization of AtFbrl was studied with an in vivo targeting approach using a fusion protein, and was found to be correctly targeted to the nucleolus in protoplasts when expressed as a green fluorescent fusion protein (GFP). Deletion experiments showed that the N-terminal glycine-and arginine-rich region is necessary and sufficient to target AtFbr1 to the nucleolus.

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.