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Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N₂‐fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P. flexilis–AAB association is consistent across years, we re‐sampled P. flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N₂, we incubated twigs with ¹³N₂‐enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using ¹³N. We used the acetylene reduction assay to test for nitrogenase activity within P. flexilis twigs four times from June to September. We found evidence for N₂ fixation in P. flexilis foliage. N₂ diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8–13.6 μg N m⁻² d⁻¹ to P. flexilis stands. AAB dominated the P. flexilis needle endophyte community. We propose that foliar endophytes represent a low‐cost, evolutionarily stable N₂‐fixing strategy for long‐lived conifers. This novel source of biological N₂ fixation has fundamental implications for understanding forest N budgets.

Biological nitrogen fixation, performed by the enzyme nitrogenase, supplies nearly 50% of the bioavailable nitrogen pool on Earth, yet the structural nature of the enzyme intermediates involved in this cycle remains ambiguous. Here we present four high resolution cryoEM structures of the nitrogenase MoFe-protein, sampled along a time course of alkaline reaction mixtures under an acetylene atmosphere. This series of structures reveals a sequence of salient changes including perturbations to the inorganic framework of the FeMo-cofactor; depletion of the homocitrate moiety; diminished density around the S2B belt sulfur of the FeMo-cofactor; rearrangements of cluster-adjacent side chains; and the asymmetric displacement of the FeMo-cofactor. We further demonstrate that the nitrogenase associated factor T protein can recognize and bind an alkaline inactivated MoFe-protein in vitro. These time-resolved structures provide experimental support for the displacement of S2B and distortions of the FeMo-cofactor at the E 0 -E 3 intermediates of the substrate reduction mechanism, prior to nitrogen binding, highlighting cluster rearrangements potentially relevant to nitrogen fixation by biological and synthetic clusters. The mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, remains enigmatic. Time-resolved cryoEM structures under alkaline turnover conditions highlight distortions in the active site of relevance to substrate reduction.

Biological nitrogen fixation (BNF) performed by moss-associated cyanobacteria is one of the main sources of new nitrogen (N) input in pristine, high-latitude ecosystems. Yet, the nutrients that limit BNF remain elusive. Here, we tested whether this important ecosystem function is limited by the availability of molybdenum (Mo), phosphorus (P), or both. BNF in dominant mosses was measured with the acetylene reduction assay (ARA) at different time intervals following Mo and P additions, in both laboratory microcosms with mosses from a boreal spruce forest and field plots in subarctic tundra. We further used a 15N2 tracer technique to assess the ARA to N2 fixation conversion ratios at our subarctic site. BNF was up to four-fold higher shortly after the addition of Mo, in both the laboratory and field experiments. A similar positive response to Mo was found in moss colonizing cyanobacterial biomass. As the growing season progressed, nitrogenase activity became progressively more P limited. The ARA: 15N2 ratios increased with increasing Mo additions. These findings show that N2 fixation activity as well as cyanobacterial biomass in dominant feather mosses from boreal forests and subarctic tundra are limited by Mo availability.

Acetylene (IUPAC name: ethyne) is a colorless, gaseous hydrocarbon, composed of two triple bonded carbon atoms attached to hydrogens (C2H2). When microbiologists and biogeochemists think of acetylene, they immediately think of its use as an inhibitory compound of certain microbial processes and a tracer for nitrogen fixation. However, what is less widely known is that anaerobic and aerobic microorganisms can degrade acetylene, using it as a sole carbon and energy source and providing the basis of a microbial food web. Here, we review what is known about acetylene degrading organisms and introduce the term 'acetylenotrophs' to refer to the microorganisms that carry out this metabolic pathway. In addition, we review the known environmental sources of acetylene and postulate the presence of an hidden acetylene cycle. The abundance of bacteria capable of using acetylene and other alkynes as an energy and carbon source suggests that there are energy cycles present in the environment that are driven by acetylene and alkyne production and consumption that are isolated from atmospheric exchange. Acetylenotrophs may have developed to leverage the relatively high concentrations of acetylene in the pre-Cambrian atmosphere, evolving later to survive in specialized niches where acetylene and other alkynes were produced.

Nitrification is a central component of the global nitrogen cycle. Ammonia oxidation, the first step of nitrification, is performed in terrestrial ecosystems by both ammonia‐oxidizing bacteria (AOB) and ammonia‐oxidizing archaea (AOA). Published studies indicate that soil pH may be a critical factor controlling the relative abundances of AOA and AOB communities. In order to determine the relative contributions of AOA and AOB to ammonia oxidation in two agricultural acidic Scottish soils (pH 4.5 and 6), the influence of acetylene (a nitrification inhibitor) was investigated during incubation of soil microcosms at 20 °C for 1 month. High rates of nitrification were observed in both soils in the absence of acetylene. Quantification of respective amoA genes (a key functional gene for ammonia oxidizers) demonstrated significant growth of AOA, but not AOB. A significant positive relationship was found between nitrification rate and AOA, but not AOB growth. AOA growth was inhibited in the acetylene‐containing microcosms. Moreover, AOA transcriptional activity decreased significantly in the acetylene‐containing microcosms compared with the control, whereas no difference was observed for the AOB transcriptional activity. Consequently, growth and activity of only archaeal but not bacterial ammonia oxidizer communities strongly suggest that AOA, but not AOB, control nitrification in these two acidic soils.

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 °C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.

It has been hypothesized that nitrogen fixation occurs in the human gut. However, whether the gut microbiota truly has this potential remains unclear. We investigated the nitrogen-fixing activity and diversity of the nitrogenase reductase (NifH) genes in the faecal microbiota of humans, focusing on Papua New Guinean and Japanese individuals with low to high habitual nitrogen intake. A 15 N 2 incorporation assay showed significant enrichment of 15 N in all faecal samples, irrespective of the host nitrogen intake, which was also supported by an acetylene reduction assay. The fixed nitrogen corresponded to 0.01% of the standard nitrogen requirement for humans, although our data implied that the contribution in the gut in vivo might be higher than this value. The nifH genes recovered in cloning and metagenomic analyses were classified in two clusters: one comprising sequences almost identical to Klebsiella sequences and the other related to sequences of Clostridiales members. These results are consistent with an analysis of databases of faecal metagenomes from other human populations. Collectively, the human gut microbiota has a potential for nitrogen fixation, which may be attributable to Klebsiella and Clostridiales strains, although no evidence was found that the nitrogen-fixing activity substantially contributes to the host nitrogen balance.

Bacteria-mediated acquisition of atmospheric N₂ serves as a critical source of nitrogen in terrestrial ecosystems. Here we reveal that symbiotic nitrogen fixation facilitates the cultivation of specialized fungal crops by leaf-cutter ants. By using acetylene reduction and stable isotope experiments, we demonstrated that N₂ fixation occurred in the fungus gardens of eight leaf-cutter ant species and, further, that this fixed nitrogen was incorporated into ant biomass. Symbiotic N₂-fixing bacteria were consistently isolated from the fungus gardens of 80 leaf-cutter ant colonies collected in Argentina, Costa Rica, and Panama. The discovery of N₂ fixation within the leaf-cutter ant-microbe symbiosis reveals a previously unrecognized nitrogen source in neotropical ecosystems.

Cyanobacteria from Subsection V (Stigonematales) are important components of microbial mats in non-acidic terrestrial hot springs. Despite their diazotrophic nature (N 2 fixers), their impact on the nitrogen cycle in such extreme ecosystems remains unknown. Here, we surveyed the identity and activity of diazotrophic cyanobacteria in the neutral hot spring of Porcelana (Northern Patagonia, Chile) during 2009 and 2011–2013. We used 16S rRNA and the nif H gene to analyze the distribution and diversity of diazotrophic cyanobacteria. Our results demonstrate the dominance of the heterocystous genus Mastigocladus (Stigonematales) along the entire temperature gradient of the hot spring (69–38 °C). In situ nitrogenase activity (acetylene reduction), nitrogen fixation rates (cellular uptake of 15 N 2 ) and nif H transcription levels in the microbial mats showed that nitrogen fixation and nif H mRNA expression were light-dependent. Nitrogen fixation activities were detected at temperatures ranging from 58 °C to 46 °C, with maximum daily rates of 600 nmol C 2 H 4 cm −2 per day and 94.1 nmol N cm −2 per day. These activity patterns strongly suggest a heterocystous cyanobacterial origin and reveal a correlation between nitrogenase activity and nif H gene expression during diurnal cycles in thermal microbial mats. N and C fixation in the mats contributed ~3 g N m −2 per year and 27 g C m −2 per year, suggesting that these vital demands are fully met by the diazotrophic and photoautotrophic capacities of the cyanobacteria in the Porcelana hot spring.

We observed that the maize pathogenic fungus Ustilago maydis grew in nitrogen (N)-free media at a rate similar to that observed in media containing ammonium nitrate, suggesting that it was able to fix atmospheric N2. Because only prokaryotic organisms have the capacity to reduce N2, we entertained the possibility that U. maydis was associated with an intracellular bacterium. The presence of nitrogenase in the fungus was analyzed by acetylene reduction, and capacity to fix N2 by use of 15N2. Presence of an intracellular N2-fixing bacterium was analyzed by PCR amplification of bacterial 16S rRNA and nifH genes, and by microscopic observations. Nitrogenase activity and 15N incorporation into the cells proved that U. maydis fixed N2. Light and electron microscopy, and fluorescence in situ hybridization (FISH) experiments revealed the presence of intracellular bacteria related to Bacillus pumilus, as evidenced by sequencing of the PCR-amplified fragments. These observations reveal for the first time the existence of an endosymbiotic N2-fixing association involving a fungus and a bacterium.