As global temperatures rise, large amounts of carbon sequestered in permafrost are becoming available for microbial degradation. Accurate prediction of carbon gas emissions from thawing permafrost is limited by our understanding of these microbial communities. Here we use metagenomic sequencing of 214 samples from a permafrost thaw gradient to recover 1,529 metagenome-assembled genomes, including many from phyla with poor genomic representation. These genomes reflect the diversity of this complex ecosystem, with genus-level representatives for more than sixty per cent of the community. Meta-omic analysis revealed key populations involved in the degradation of organic matter, including bacteria whose genomes encode a previously undescribed fungal pathway for xylose degradation. Microbial and geochemical data highlight lineages that correlate with the production of greenhouse gases and indicate novel syntrophic relationships. Our findings link changing biogeochemistry to specific microbial lineages involved in carbon processing, and provide key information for predicting the effects of climate change on permafrost systems.

The northwestern Sargasso Sea undergoes annual cycles of productivity with increased production in spring corresponding to periods of upwelling, and oligotrophy in summer and autumn, when the water column becomes highly stratified. The biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorus and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary liquid chromatography (LC)-tandem mass spectrometry to detect microbial proteins in surface samples collected in September 2005. A total of 2215 peptides that mapped to 236 SAR11 proteins, 1911 peptides that mapped to 402 Prochlorococcus proteins and 2407 peptides that mapped to 404 Synechococcus proteins were detected. Mass spectra from SAR11 periplasmic substrate-binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars and spermidine. Proteins implicated in the prevention of oxidative damage and protein refolding were also abundant. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme, but nutrient flux is sufficient to sustain microbial community activity.

SUMMARY In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

Summary In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases, Pol IV and Pol V and the putative RNA-dependent RNA polymerase, RDR2. Here, we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to alpha-amanitin and differ in their ability to displace the non-template DNA strand during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs), which guide cytosine methylation to corresponding sequences, requires both Pol IV and RDR2, which physically associate in vivo. Whereas Pol IV does not require RDR2 for activity, RDR2 is non-functional in the absence of associated Pol IV. These results suggest that the physical and mechanistic coupling of Pol IV and RDR2 results in the channeled synthesis of double-stranded precursors for 24 nt siRNA biogenesis.

SignificanceChlamydomonas reinhardtii is the premier reference organism for understanding unicellular green algae. Chlamydomonas is an important model for photosynthesis as well as fermentation and other anaerobic pathways under dark anoxic conditions. We have produced a diurnal transcriptome, validated by subproteomic analyses, and matched with measurements of pigments, select metabolites, and physiological parameters. We report that the majority of the algal genome is differentially expressed over the course of the day and the timing of specific genes is dictated by their biological function. We also discovered that fermentation rather than respiration is the preferred metabolic fate of starch-derived glycolytic pyruvate. We offer our rich dataset to the algal and plant communities.

Fermentation-based metabolism is an important ecosystem function often associated with environments rich in organic carbon, such as wetlands, sewage sludge and the mammalian gut. The diversity of microorganisms and pathways involved in carbon and hydrogen cycling in sediments and aquifers and the impacts of these processes on other biogeochemical cycles remain poorly understood. Here we used metagenomics and proteomics to characterize microbial communities sampled from an aquifer adjacent to the Colorado River at Rifle, CO, USA, and document interlinked microbial roles in geochemical cycling. The organic carbon content in the aquifer was elevated via acetate amendment of the groundwater occurring over 2 successive years. Samples were collected at three time points, with the objective of extensive genome recovery to enable metabolic reconstruction of the community. Fermentative community members include organisms from a new phylum, Melainabacteria, most closely related to Cyanobacteria, phylogenetically novel members of the Chloroflexi and Bacteroidales, as well as candidate phyla genomes (OD1, BD1-5, SR1, WWE3, ACD58, TM6, PER and OP11). These organisms have the capacity to produce hydrogen, acetate, formate, ethanol, butyrate and lactate, activities supported by proteomic data. The diversity and expression of hydrogenases suggests the importance of hydrogen metabolism in the subsurface. Our proteogenomic data further indicate the consumption of fermentation intermediates by Proteobacteria can be coupled to nitrate, sulfate and iron reduction. Thus, fermentation carried out by previously unknown members of sediment microbial communities may be an important driver of nitrogen, hydrogen, sulfur, carbon and iron cycling.

Marine phytoplankton produce ~109 tons of dimethylsulfoniopropionate (DMSP) per year1,2, an estimated 10% of which is catabolized by bacteria through the DMSP cleavage pathway to the climatically active gas dimethyl sulfide (DMS)3,4. SAR11 Alphaproteobacteria (order Pelagibacterales), the most abundant chemoorganotrophic bacteria in the oceans, have been shown to assimilate DMSP into biomass, thereby supplying this cell’s unusual requirement for reduced sulfur5,6. Here we report that Pelagibacter HTCC1062 produces the gas methanethiol (MeSH) and that simultaneously a second DMSP catabolic pathway, mediated by a cupin-like DMSP lyase, DddK, shunts as much as 59% of DMSP uptake to DMS production. We propose a model in which the allocation of DMSP between these pathways is kinetically controlled to release increasing amounts of DMS as the supply of DMSP exceeds cellular sulfur demands for biosynthesis

In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample.