Undervalued Pseudo- nifH Sequences in Public Databases Distort Metagenomic Insights into Biological Nitrogen Fixers

Nitrogen-fixing microbes affect biogeochemical cycling, agricultural productivity, and microbial ecosystems, and their distributions have been investigated intensively using genomic and metagenomic sequencing. Currently, insights into nitrogen fixers in the environment have been acquired by homology searches against nitrogenase genes, particularly the nifH gene, in public databases.

A Taxon-Wise Insight Into Rock Weathering and Nitrogen Fixation Functional Profiles of Proglacial Systems

The Arctic environment is particularly affected by global warming, and a clear trend of the ice retreat is observed worldwide. In proglacial systems, the newly exposed terrain represents different environmental and nutrient conditions compared to later soil stages. Therefore, proglacial systems show several environmental gradients along the soil succession where microorganisms are active protagonists of the soil and carbon pool formation through nitrogen fixation and rock weathering. We studied the microbial succession of three Arctic proglacial systems located in Svalbard (Midtre Lovénbreen), Sweden (Storglaciären), and Greenland (foreland close to Kangerlussuaq). We analyzed 65 whole shotgun metagenomic soil samples for a total of more than 400 Gb of sequencing data. Microbial succession showed common trends typical of proglacial systems with increasing diversity observed along the forefield chronosequence. Microbial trends were explained by the distance from the ice edge in the Midtre Lovénbreen and Storglaciären forefields and by total nitrogen (TN) and total organic carbon (TOC) in the Greenland proglacial system. Furthermore, we focused specifically on genes associated with nitrogen fixation and biotic rock weathering processes, such as nitrogenase genes, obcA genes, and genes involved in cyanide and siderophore synthesis and transport. Whereas we confirmed the presence of these genes in known nitrogen-fixing and/or rock weathering organisms (e.g., Nostoc, Burkholderia), in this study, we also detected organisms that, even if often found in soil and proglacial systems, have never been related to nitrogen-fixing or rock weathering processes before (e.g., Fimbriiglobus, Streptomyces). The different genera showed different gene trends within and among the studied systems, indicating a community constituted by a plurality of organisms involved in nitrogen fixation and biotic rock weathering, and where the latter were driven by different organisms at different soil succession stages.

Comparative genomic insights into culturable symbiotic cyanobacteria from the water fern Azolla

Species of the floating, freshwater fern Azolla form a well-characterized symbiotic association with the non-culturable cyanobacterium Nostoc azollae, which fixes nitrogen for the plant. However, several cyanobacterial strains have over the years been isolated and cultured from Azolla from all over the world. The genomes of 10 of these strains were sequenced and compared with each other, with other symbiotic cyanobacterial strains, and with similar strains that were not isolated from a symbiotic association. The 10 strains fell into three distinct groups: six strains were nearly identical to the non-symbiotic strain, Nostoc ( Anabaena ) variabilis ATCC 29413; three were similar to the symbiotic strain, Nostoc punctiforme , and one, Nostoc sp. 2RC, was most similar to non-symbiotic strains of Nostoc linckia. However, Nostoc sp. 2RC was unusual because it has three sets of nitrogenase genes; it has complete gene clusters for two distinct Mo-nitrogenases and an alternative V-nitrogenase. Genes for Mo-nitrogenase, sugar transport, chemotaxis and pili characterized all the symbiotic strains. Several of the strains infected the liverwort Blasia, including N. variabilis ATCC 29413, which did not originate from Azolla but rather from a sewage pond. However, only Nostoc sp. 2RC, which produced highly motile hormogonia, was capable of high-frequency infection of Blasia. Thus, some of these strains, which grow readily in the laboratory, may be useful in establishing novel symbiotic associations with other plants.

Ecological Divergence Within the Enterobacterial Genus Sodalis: From Insect Symbionts to Inhabitants of Decomposing Deadwood

The bacterial genus Sodalis is represented by insect endosymbionts as well as free-living species. While the former have been studied frequently, the distribution of the latter is not yet clear. Here, we present a description of a free-living strain, Sodalis ligni sp. nov., originating from decomposing deadwood. The favored occurrence of S. ligni in deadwood is confirmed by both 16S rRNA gene distribution and metagenome data. Pangenome analysis of available Sodalis genomes shows at least three groups within the Sodalis genus: deadwood-associated strains, tsetse fly endosymbionts and endosymbionts of other insects. This differentiation is consistent in terms of the gene frequency level, genome similarity and carbohydrate-active enzyme composition of the genomes. Deadwood-associated strains contain genes for active decomposition of biopolymers of plant and fungal origin and can utilize more diverse carbon sources than their symbiotic relatives. Deadwood-associated strains, but not other Sodalis strains, have the genetic potential to fix N2, and the corresponding genes are expressed in deadwood. Nitrogenase genes are located within the genomes of Sodalis, including S. ligni, at multiple loci represented by more gene variants. We show decomposing wood to be a previously undescribed habitat of the genus Sodalis that appears to show striking ecological divergence.

Functional metagenomics of bark microbial communities from avocado trees (Persea americana Mill.) reveals potential for bacterial primary productivity

SummaryBark microbial communities are poorly understood, and information on the metabolic capacities of their inhabitants is lacking. Bark microbial communities share part of their taxonomic composition with soil, but the functional differences and similarities are unknown. By comparing bark microbial communities of avocado trees (Persea americana, Mill.) with rhizospheric soil, functional processes relevant to the bark environment were identified. DNA from bark and soil communities was extracted from the same trees, and shotgun metagenomics sequencing was performed using nextSeq technology. Genes were identified by BLAST methods, and functional annotation was performed with KEGG databases as a reference. Bacterial oxygenic and anoxygenic photosynthesis genes were highly abundant in bark as compared to soil. Furthermore, increased presence of nitrogenase genes suggests a potential for nitrogen fixation. Genes for methanol utilization were abundant in bark, but no evidence of methane utilization potential was observed. Bark microbial communities have the genetic information for potential primary productivity, which might contribute to microbial growth independent of plant-derived carbon substrates.

Diazotrophic Anaeromyxobacter Isolates from Soils

ABSTRACT Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of Anaeromyxobacter (in the class Deltaproteobacteria), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of Anaeromyxobacter in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of Anaeromyxobacter to fix nitrogen. Therefore, we verified the diazotrophy of Anaeromyxobacter based on both genomic and culture-dependent analyses using Anaeromyxobacter sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of nif gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic Geobacter and Pelobacter in the class Deltaproteobacteria contain the minimum set of genes for nitrogenase (nifBHDKEN). These results imply that Anaeromyxobacter species have the ability to fix nitrogen. In fact, Anaeromyxobacter PSR-1 and Red267 exhibited N2-dependent growth and acetylene reduction activity (ARA) in vitro. Transcriptional activity of the nif gene was also detected when both strains were cultured with N2 gas as a sole nitrogen source, indicating that Anaeromyxobacter can fix and assimilate N2 gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that Anaeromyxobacter can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of Anaeromyxobacter, which is a common soil bacterium. IMPORTANCE Anaeromyxobacter is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of Anaeromyxobacter from various soil environments. Although the importance of Anaeromyxobacter as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that Anaeromyxobacter fixes nitrogen. This study demonstrates that Anaeromyxobacter harboring nitrogenase genes exhibits diazotrophic ability; moreover, N2-dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for Anaeromyxobacter to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of Anaeromyxobacter, which is a common bacterium in soils.

Succession of microbial populations and nitrogen-fixation associated with the biodegradation of sediment-oil-agglomerates buried in a Florida sandy beach

The Deepwater Horizon (DWH) oil spill contaminated coastlines from Louisiana to Florida, burying oil up to 70 cm depth in sandy beaches, posing a potential threat to environmental and human health. The dry and nutrient-poor beach sand presents a taxing environment for microbial growth, raising the question how the biodegradation of the buried oil would proceed. Here we report the results of an in-situ experiment that (i) characterized the dominant microbial communities contained in sediment oil agglomerates (SOAs) of DWH oil buried in a North Florida sandy beach, (ii) elucidated the long-term succession of the microbial populations that developed in the SOAs, and (iii) revealed the coupling of SOA degradation to nitrogen fixation. Orders of magnitude higher bacterial abundances in SOAs compared to surrounding sands distinguished SOAs as hotspots of microbial growth. Blooms of bacterial taxa with a demonstrated potential for hydrocarbon degradation (Gammaproteobacteria, Alphaproteobacteria, Actinobacteria) developed in the SOAs, initiating a succession of microbial populations that mirrored the evolution of the petroleum hydrocarbons. Growth of nitrogen-fixing prokaryotes or diazotrophs (Rhizobiales and Frankiales), reflected in increased abundances of nitrogenase genes (nifH), catalyzed biodegradation of the nitrogen-poor petroleum hydrocarbons, emphasizing nitrogen fixation as a central mechanism facilitating the recovery of sandy beaches after oil contamination.

NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501

ABSTRACT Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level. IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

In depth characterization of diazotroph activity across the Western Tropical South Pacific hot spot of N₂ fixation

Here we report quantification of N2 fixation rates over a ~ 4000 km transect in the western and central tropical South Pacific. Water samples were collected along a west to east transect from 160° E to 160° W, covering contrasting trophic regimes, from oligotrophy in the Melanesian archipelagoes (MA) waters to ultra-oligotrophy in the South Pacific Gyre (GY) waters. N2 fixation was detected at all 17 sampled stations with an average rate of 631 ± 286 µmol N m−2 d−1 (range 196–1153 µmol N m−2 d−1) in MA waters and of 85 ± 79 µmol N m−2 d−1 (range 18–172 µmol N m−2 d−1) in GY waters. Exceptionally high rates of N2 fixation in MA waters were favored by availability of both iron and phosphate and the observed warm sea surface temperatures (> 28 °C). Trichodesmium and UCYN-B cyanobacteria dominated the diazotroph community (> 80 %) and gene expression of nitrogenase genes (cDNA > 105 nifH copies L−1) in MA waters, and single-cell isotopic analyses performed by nanoscale secondary ion mass spectrometry at selected stations reveal that Trichodesmium was always the major contributor to N2 fixation in MA waters, accounting for 47.1 to 83.8 % of bulk N2 fixation.

Regulation of fixLJ by Hfq Controls Symbiotically Important Genes in Sinorhizobium meliloti

The RNA-binding chaperone Hfq plays critical roles in the establishment and functionality of the symbiosis between Sinorhizobium meliloti and its legume hosts. A mutation in hfq reduces symbiotic efficiency resulting in a Fix− phenotype, characterized by the inability of the bacterium to fix nitrogen. At least in part, this is due to the ability of Hfq to regulate the fixLJ operon, which encodes a sensor kinase–response regulator pair that controls expression of the nitrogenase genes. The ability of Hfq to bind fixLJ in vitro and in planta was demonstrated with gel shift and coimmunoprecipitation experiments. Two (ARN)2 motifs in the fixLJ message were the likely sites through which Hfq exerted its posttranscriptional control. Consistent with the regulatory effects of Hfq, downstream genes controlled by FixLJ (such as nifK, noeB) were also subject to Hfq regulation in planta.