Effects of nitrogen fertilization on diazotrophic activity of microorganisms associated with Sphagnum magellanicum

In pristine ombrotrophic Sphagnum-dominated peatland ecosystems nitrogen (N) is often a limiting nutrient, which is replenished by biological N-2 fixation and atmospheric N deposition. It is, however, unclear which impact long-term N deposition has on microbial N-2 fixing activity and diazotrophic diversity, and whether phosphorus (P) modulates the response. Therefore, we studied the impact of increased N deposition and N depletion on microbial N-2 fixation and diazotrophic diversity associated with the peat moss Sphagnum magellanicum, and their interaction with P availability.Nitrogenase activities of S. magellanicum-associated microorganisms were determined by acetylene reduction assays (ARA) and N-15(2) tracer methods on mosses from two geographically distinct locations with different N deposition histories, high or low N deposition, and in samples depleted in N (grown 3 years in the greenhouse) versus recent field samples. The short-term response to increased N deposition was tested for mosses differing in N and P fertilization histories. In addition, diversity of diazotrophic microorganisms was assessed by nifH gene amplicon sequencing of N-depleted mosses.We showed distinct and persistent differences in diazotrophic communities and their activities associated with S. magellanicum from sites with high versus low N deposition. Initially, diazotrophic activity was six times higher for the low N site. During incubation and repeated ARA, however, this activity strongly decreased, while it remained stable for the high N site. Activity for the high N site could not be increased by long-term experimental N deprivation. Short-term, experimental N application had an inhibitory effect on N-2 fixation for both sites, which was not observed in mosses with high indirect P availability.We conclude that although N deposition negatively affects N-2 fixation as also shown in previous studies, long-term effects of N deprivation on the diazotrophic activity and community are more complex. Furthermore, our results indicated that P availability might be an important factor in modulating the response of Sphagnum-associated diazotrophs to N deposition.</p

Methylacidimicrobium thermophilum AP8, a Novel Methane- and Hydrogen-Oxidizing Bacterium Isolated From Volcanic Soil on Pantelleria Island, Italy

The Favara Grande is a geothermal area located on Pantelleria Island, Italy. The area is characterized high temperatures in the top layer of the soil (60\ub0C), low pH (3\u20135) and hydrothermal gas emissions mainly composed of carbon dioxide (CO2), methane (CH4), and hydrogen (H2). These geothermal features may provide a suitable niche for the growth of chemolithotrophic thermoacidophiles, including the lanthanide-dependent methanotrophs of the phylum Verrucomicrobia. In this study, we started enrichment cultures inoculated with soil of the Favara Grande at 50 and 60\ub0C with CH4 as energy source and medium containing sufficient lanthanides at pH 3 and 5. From these cultures, a verrucomicrobial methanotroph could be isolated via serial dilution and floating filters techniques. The genome of strain AP8 was sequenced and based on phylogenetic analysis we propose to name this new species Methylacidimicrobium thermophilum AP8. The transcriptome data at \u3bcmax (0.051 \ub1 0.001 h 121, doubling time ~14 h) of the new strain showed a high expression of the pmoCAB2 operon encoding the membrane-bound methane monooxygenase and of the gene xoxF1, encoding the lanthanide-dependent methanol dehydrogenase. A second pmoCAB operon and xoxF2 gene were not expressed. The physiology of strain AP8 was further investigated and revealed an optimal growth in a pH range of 3\u20135 at 50\ub0C, representing the first thermophilic strain of the genus Methylacidimicrobium. Moreover, strain AP8 had a KS(app) for methane of 8 \ub1 1 \u3bcM. Beside methane, a type 1b [NiFe] hydrogenase enabled hydrogen oxidation at oxygen concentrations up to 1%. Taken together, our results expand the knowledge on the characteristics and adaptations of verrucomicrobial methanotrophs in hydrothermal environments and add a new thermophilic strain to the genus Methylacidimicrobium

FACIL: Fast and Accurate Genetic Code Inference and Logo

Motivation: The intensification of DNA sequencing will increasingly unveil uncharacterized species with potential alternative genetic codes. A total of 0.65% of the DNA sequences currently in Genbank encode their proteins with a variant genetic code, and these exceptions occur in many unrelated taxa. Results: We introduce FACIL (Fast and Accurate genetic Code Inference and Logo), a fast and reliable tool to evaluate nucleic acid sequences for their genetic code that detects alternative codes even in species distantly related to known organisms. To illustrate this, we apply FACIL to a set of mitochondrial genomic contigs of Globobulimina pseudospinescens. This foraminifer does not have any sequenced close relative in the databases, yet we infer its alternative genetic code with high confidence values. Results are intuitively visualized in a Genetic Code Logo

A novel mesocosm set-up reveals strong methane emission reduction in submerged peat moss Sphagnum cuspidatum by tightly associated methanotrophs

Wetlands present the largest natural sources of methane (CH_4) and their potential CH_4 emissions greatly vary due to the activity of CH_4-oxidizing bacteria associated with wetland plant species. In this study, the association of CH_4-oxidizing bacteria with submerged Sphagnum peat mosses was studied, followed by the development of a novel mesocosm set-up. This set-up enabled the precise control of CH_4 input and allowed for monitoring the dissolved CH_4in a Sphagnum moss layer while mimicking natural conditions. Two mesocosm set-ups were used in parallel: one containing a Sphagnum moss layer in peat water, and a control only containing peat water. Moss-associated CH_4 oxidizers in the field could reduce net CH_4 emission up to 93%, and in the mesocosm set-up up to 31%. Furthermore, CH_4 oxidation was only associated with Sphagnum, and did not occur in peat water. Especially methanotrophs containing a soluble methane monooxygenase enzyme were significantly enriched during the 32 day mesocosm incubations. Together these findings showed the new mesocosm setup is very suited to study CH_4 cycling in submerged Sphagnum moss community under controlled conditions. Furthermore, the tight associated between Sphagnum peat mosses and methanotrophs can significantly reduce CH_4 emissions in submerged peatlands

Complete Genome Sequence of the Aerobic Facultative Methanotroph Methylocella tundrae Strain T4

Methylocella tundrae T4T is a facultative aerobic methanotroph which was isolated from an acidic tundra wetland and possesses only a soluble methane monooxygenase. The complete genome, which includes two megaplasmids, was sequenced using a combination of Illumina and Nanopore technologies. One of the megaplasmids carries a propane monooxygenase gene cluster

A novel mesocosm set-up reveals strong methane emission reduction in submerged peat moss Sphagnum cuspidatum by tightly associated methanotrophs

Wetlands present the largest natural sources of methane (CH_4) and their potential CH_4 emissions greatly vary due to the activity of CH_4-oxidizing bacteria associated with wetland plant species. In this study, the association of CH_4-oxidizing bacteria with submerged Sphagnum peat mosses was studied, followed by the development of a novel mesocosm set-up. This set-up enabled the precise control of CH_4 input and allowed for monitoring the dissolved CH_4in a Sphagnum moss layer while mimicking natural conditions. Two mesocosm set-ups were used in parallel: one containing a Sphagnum moss layer in peat water, and a control only containing peat water. Moss-associated CH_4 oxidizers in the field could reduce net CH_4 emission up to 93%, and in the mesocosm set-up up to 31%. Furthermore, CH_4 oxidation was only associated with Sphagnum, and did not occur in peat water. Especially methanotrophs containing a soluble methane monooxygenase enzyme were significantly enriched during the 32 day mesocosm incubations. Together these findings showed the new mesocosm setup is very suited to study CH_4 cycling in submerged Sphagnum moss community under controlled conditions. Furthermore, the tight associated between Sphagnum peat mosses and methanotrophs can significantly reduce CH_4 emissions in submerged peatlands

A single evolutionary innovation drives the deep evolution of symbiotic N₂-fixation in angiosperms

Symbiotic associations occur in every habitat on earth, but we know very little about their evolutionary histories. Current models of trait evolution cannot adequately reconstruct the deep history of symbiotic innovation, because they assume homogenous evolutionary processes across millions of years. Here we use a recently developed, heterogeneous and quantitative phylogenetic framework to study the origin of the symbiosis between angiosperms and nitrogen-fixing (N2) bacterial symbionts housed in nodules. We compile the largest database of global nodulating plant species and reconstruct the symbiosis’ evolution. We identify a single, cryptic evolutionary innovation driving symbiotic N2-fixation evolution, followed by multiple gains and losses of the symbiosis, and the subsequent emergence of ‘stable fixers’ (clades extremely unlikely to lose the symbiosis). Originating over 100 MYA, this innovation suggests deep homology in symbiotic N2-fixation. Identifying cryptic innovations on the tree of life is key to understanding the evolution of complex traits, including symbiotic partnerships

Spontaneous symbiotic reprogramming of plant roots triggered by receptor-like kinases

Symbiosis Receptor-like Kinase (SYMRK) is indispensable for the development of phosphate-acquiring arbuscular mycorrhiza (AM) as well as nitrogen-fixing root nodule symbiosis, but the mechanisms that discriminate between the two distinct symbiotic developmental fates have been enigmatic. In this study, we show that upon ectopic expression, the receptor-like kinase genes Nod Factor Receptor 1 (NFR1), NFR5, and SYMRK initiate spontaneous nodule organogenesis and nodulation-related gene expression in the absence of rhizobia. Furthermore, overexpressed NFR1 or NFR5 associated with endogenous SYMRK in roots of the legume Lotus japonicus. Epistasis tests revealed that the dominant active SYMRK allele initiates signalling independently of either the NFR1 or NFR5 gene and upstream of a set of genes required for the generation or decoding of calcium-spiking in both symbioses. Only SYMRK but not NFR overexpression triggered the expression of AM-related genes, indicating that the receptors play a key role in the decision between AM- or root nodule symbiosis-development

Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth—Application to the Eif3 Complex

BACKGROUND: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis. METHODOLOGY/PRINCIPAL FINDINGS: Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. CONCLUSION/SIGNIFICANCE: eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis

Supplemental Information 3: Ribosomal protein S3 protein alignment

Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO2. However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10–20 cm and 30–40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. We also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation. Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. The new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics