Permafrost contains an estimated 1672 Pg carbon (C),metagenomes converged to be more similar to each other than while they were frozen. We found that multiple genes involved in cycling of C and nitrogen shifted rapidly during thaw. We also constructed the first draft genome from a complex soil metagenome, which corresponded to a novel methanogen. Methane previously accumulated in permafrost was released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost.We collected three intact frozen permafrost soil cores with their overlying seasonally thawed active layers from Hess Creek (HC), Alaska. This is a black spruce forest site containing many meters of frozen peat and the C was dated to 1200 ybp 10 .Other soil properties and microbial respiration rates were previously characterized 10 .Frozen active layer and permafrost layer samples from each core were thawed and incubated for 7 days at 5°C under a He headspace. During the incubations, CH 4 ( Fig. 1a) and CO 2 ( Supplementary Fig 1) concentrations were monitored in the headspace and DNA was extracted for 16S ribosomal RNA (rRNA) and metagenome sequencing.There was a burst of CH 4 from the permafrost within 48 h of thaw, followed by a significant (P = 0.05) decrease in concentration from day 2 to day 7 (Fig. 1a). To determine (a) if methane release was due to post-thaw production or from trapped gas, (b)whether the methane was consumed by methanotrophs or anaerobic methane oxidizers, and (c) the CH 4 oxidation potential over time, we treated additional samples with 1500 3 ppm CH 4 and 2-bromoethane sulphonic acid (BES) and measured CH 4 levels daily. BES is an inhibitor of archaeal methanogenesis and methyl-coenzyme M reductase (MCR)-dependent anaerobic methane oxidation. Rapid release of CH 4 from samples treated with BES suggested that the CH 4 primarily originated from gas present in the permafrost prior to thaw ( Supplementary Fig. 2), as previously reported 11 . Subsequent CH 4 consumption in both BES and non-BES-treated samples was indicative of CH 4 oxidation by methanotrophic bacteria (Fig. 1b). The oxygen utilized for methane oxidation presumably originated from permafrost water or aerobic microsites in the samples 12 . Together these data indicate CH 4 levels are dynamic in thawing permafrost.To determine the phylogenetic and functional gene repertoire before and after thaw, we performed deep metagenome sequencing of samples from two of the three replicate cores (cores 1 and 2). DNA was extracted from frozen active layer and permafrost samples and from samples thawed at 5°C for 2 and 7 days. This resulted in 12 samples for metagenome sequencing.Due to low DNA yield we used emulsion PCR (emPCR) to generate random shotgun short insert libraries with minimum amplification bias 13 . Sequencing yielded a total of 176 million reads and 39.8 Gb of raw sequence. The individual metagenome reads were annotated by comparison to protein-coding...

Abstract:In a 26-year soil warming experiment in a mid-latitude hardwood forest, we 14 documented changes in soil carbon cycling to investigate the potential consequences for the 15 climate system. We found that soil warming results in a four-phase pattern of soil organic matter

In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 Å resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

Soil microbes are major drivers of soil carbon cycling, yet we lack an understanding of how climate warming will affect microbial communities. Three ongoing field studies at the Harvard Forest Long-term Ecological Research (LTER) site (Petersham, MA) have warmed soils 5°C above ambient temperatures for 5, 8, and 20 years. We used this chronosequence to test the hypothesis that soil microbial communities have changed in response to chronic warming. Bacterial community composition was studied using Illumina sequencing of the 16S ribosomal RNA gene, and bacterial and fungal abundance were assessed using quantitative PCR. Only the 20-year warmed site exhibited significant change in bacterial community structure in the organic soil horizon, with no significant changes in the mineral soil. The dominant taxa, abundant at 0.1% or greater, represented 0.3% of the richness but nearly 50% of the observations (sequences). Individual members of the Actinobacteria, Alphaproteobacteria and Acidobacteria showed strong warming responses, with one Actinomycete decreasing from 4.5 to 1% relative abundance with warming. Ribosomal RNA copy number can obfuscate community profiles, but is also correlated with maximum growth rate or trophic strategy among bacteria. Ribosomal RNA copy number correction did not affect community profiles, but rRNA copy number was significantly decreased in warming plots compared to controls. Increased bacterial evenness, shifting beta diversity, decreased fungal abundance and increased abundance of bacteria with low rRNA operon copy number, including Alphaproteobacteria and Acidobacteria, together suggest that more or alternative niche space is being created over the course of long-term warming.

Roots moving through soil induce physical and chemical changes that differentiate rhizosphere from bulk soil, and the effects of these changes on soil microorganisms have long been a topic of interest. The use of a high-density 16S rRNA microarray (PhyloChip) for bacterial and archaeal community analysis has allowed definition of the populations that respond to the root within the complex grassland soil community; this research accompanies compositional changes reported earlier, including increases in chitinase- and protease-specific activity, cell numbers and quorum sensing signal. PhyloChip results showed a significant change compared with bulk soil in relative abundance for 7% of the total rhizosphere microbial community (147 of 1917 taxa); the 7% response value was confirmed by16S rRNA terminal restriction fragment length polymorphism analysis. This PhyloChip-defined dynamic subset was comprised of taxa in 17 of the 44 phyla detected in all soil samples. Expected rhizosphere-competent phyla, such as Proteobacteria and Firmicutes, were well represented, as were less-well-documented rhizosphere colonizers including Actinobacteria, Verrucomicrobia and Nitrospira. Richness of Bacteroidetes and Actinobacteria decreased in soil near the root tip compared with bulk soil, but then increased in older root zones. Quantitative PCR revealed rhizosphere abundance of β-Proteobacteria and Actinobacteria at about 108 copies of 16S rRNA genes per g soil, with Nitrospira having about 105 copies per g soil. This report demonstrates that changes in a relatively small subset of the soil microbial community are sufficient to produce substantial changes in functions observed earlier in progressively more mature rhizosphere zones.

A recent paper by Martiny argues that “high proportions” of bacteria in diverse Earth environments have been cultured. Here we reanalyze a portion of the data in that paper, and argue that the conclusion is based on several technical errors, most notably a calculation of sequence similarity that does not account for sequence gaps, and the reliance on 16S rRNA gene amplicons that are known to be biased towards cultured organisms. We further argue that the paper is also based on a conceptual error: namely, that sequence similarity cannot be used to infer “culturability” because one cannot infer physiology from 16S rRNA gene sequences. Combined with other recent, more reliable studies, the evidence supports the conclusion that most bacterial and archaeal taxa remain uncultured.

Rapidly fluctuating environmental conditions can significantly stress organisms, particularly when fluctuations cross thresholds of normal physiological tolerance. Redox potential fluctuations are common in humid tropical soils, and microbial community acclimation or avoidance strategies for survival will in turn shape microbial community diversity and biogeochemistry. To assess the extent to which indigenous bacterial and archaeal communities are adapted to changing in redox potential, soils were incubated under static anoxic, static oxic or fluctuating redox potential conditions, and the standing (DNA-based) and active (RNA-based) communities and biogeochemistry were determined. Fluctuating redox potential conditions permitted simultaneous CO₂ respiration, methanogenesis, N₂O production and iron reduction. Exposure to static anaerobic conditions significantly changed community composition, while 4-day redox potential fluctuations did not. Using RNA:DNA ratios as a measure of activity, 285 taxa were more active under fluctuating than static conditions, compared with three taxa that were more active under static compared with fluctuating conditions. These data suggest an indigenous microbial community adapted to fluctuating redox potential.

Low-molecular-weight organic compounds in root exudates play a key role in plant-microorganism interactions by influencing the structure and function of soil microbial communities. Model exudate solutions, based on organic acids (OAs) (quinic, lactic, maleic acids) and sugars (glucose, sucrose, fructose), previously identified in the rhizosphere of Pinus radiata, were applied to soil microcosms. Root exudate compound solutions stimulated soil dehydrogenase activity and the addition of OAs increased soil pH. The structure of active bacterial communities, based on reverse-transcribed 16S rRNA gene PCR, was assessed by denaturing gradient gel electrophoresis and PhyloChip microarrays. Bacterial taxon richness was greater in all treatments than that in control soil, with a wide range of taxa (88-1043) responding positively to exudate solutions and fewer (<24) responding negatively. OAs caused significantly greater increases than sugars in the detectable richness of the soil bacterial community and larger shifts of dominant taxa. The greater response of bacteria to OAs may be due to the higher amounts of added carbon, solubilization of soil organic matter or shifts in soil pH. Our results indicate that OAs play a significant role in shaping soil bacterial communities and this may therefore have a significant impact on plant growth.