Nitrogen Fixation Genes Research Articles

Depth-dependent variability of biological nitrogen fixation and diazotrophic communities in mangrove sediments

BackgroundNitrogen-fixing prokaryotes (diazotrophs) contribute substantially to nitrogen input in mangrove sediments, and their structure and nitrogen fixation rate (NFR) are significantly controlled by environmental conditions. Despite the well-known studies on diazotrophs in surficial sediments, the diversity, structure, and ecological functions of diazotrophic communities along environmental gradients of mangrove sediment across different depths are largely unknown. Here, we investigated how biological nitrogen fixation varied with the depth of mangrove sediments from the perspectives of both NFR and diazotrophic communities.ResultsThrough acetylene reduction assay, nifH gene amplicon and metagenomic sequencing, we found that the NFR increased but the diversity of diazotrophic communities decreased with the depth of mangrove sediments. The structure of diazotrophic communities at different depths was largely driven by salinity and exhibited a clear divergence at the partitioning depth of 50 cm. Among diazotrophic genera correlated with NFR, Agrobacterium and Azotobacter were specifically enriched at 50–100 cm sediments, while Anaeromyxobacter, Rubrivivax, Methylocystis, Dickeya, and Methylomonas were more abundant at 0–50 cm. Consistent with the higher NFR, metagenomic analysis demonstrated the elevated abundance of nitrogen fixation genes (nifH/D/K) in deep sediments, where nitrification genes (amoA/B/C) and denitrification genes (nirK and norB) became less abundant. Three metagenome-assembled genomes (MAGs) of diazotrophs from deep mangrove sediments indicated their facultatively anaerobic and mixotrophic lifestyles as they contained genes for low-oxygen-dependent metabolism, hydrogenotrophic respiration, carbon fixation, and pyruvate fermentation.ConclusionsThis study demonstrates the depth-dependent variability of biological nitrogen fixation in terms of NFR and diazotrophic communities, which to a certain extent relieves the degree of nitrogen limitation in deep mangrove sediments.BxKuS27-gQH4xs4TJqgBb7Video

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus

Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter by 13,8% and 88,6% respectively, as well as root dry matter by 61,4%, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems.

Nitrogeniibacter aestuarii sp. nov., a Novel Nitrogen-Fixing Bacterium Affiliated to the Family Zoogloeaceae and Phylogeny of the Family Zoogloeaceae Revisited.

Members of the family Zoogloeaceae within the order Rhodocyclales are found to play vital roles in terrestrial and aquatic ecosystems by participating in biofloc formation in activated sludge, polycyclic aromatic hydrocarbon degradation, and nitrogen metabolism, such as denitrification and nitrogen fixation. Here, two bacterial strains designated H1-1-2AT and ZN11-R3-1 affiliated to the family Zoogloeaceae were isolated from coastal wetland habitats. The 16S rRNA gene sequences of the two strains were 100% identical and had maximum similarity with Nitrogeniibacter mangrovi M9-3-2T of 98.4% and ≤94.5% with other species. Phylogenetic analysis suggested that the two strains belonged to a single species and formed a novel monophyletic branch affiliated to the genus Nitrogeniibacter. The average nucleotide identity (ANI) value and digital DNA-DNA hybridization (dDDH) estimate between the two strains and N. mangrovi M9-3-2T were 78.5–78.7% and 21.4–21.6%, respectively, indicating that the two strains represent a novel species. The genomes of strain H1-1-2AT (complete genome) and ZN11-R3-1 (draft genome) were 4.7Mbp in length encoding ~4,360 functional genes. The DNA G+C content was 62.7%. Nitrogen fixation genes were found in the two strains, which were responsible for the growth on nitrogen-free medium, whereas denitrification genes found in N. mangrovi M9-3-2T were absent in the two strains. The respiratory quinone was ubiquinone-8. The major polar lipids consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and aminophospholipid. The major fatty acids were summed feature 3 (C16:1ω7c and C16:1ω6c), C16:0, C12:0, and C10:0 3-OH. Based on genomic, phenotypic, and chemotaxonomic characterizations, strains H1-1-2AT and ZN11-R3-1 represent a novel species of the genus Nitrogeniibacter, for which the name Nitrogeniibacter aestuarii sp. nov. is proposed. The type strain is H1-1-2AT (=MCCC 1K04284T=KCTC 82672T), and additional strain is ZN11-R3-1 (=MCCC 1A17971=KCTC 82671). Additionally, phylogenomic analysis of the members of the family Zoogloeaceae including type strains and uncultivated bacteria was performed, using the Genome Taxonomic Database toolkit (GTDB-Tk). Combined with the 16S rRNA gene phylogeny, four novel genera, Parazoarcus gen. nov., Pseudazoarcus gen. nov., Pseudothauera gen. nov., and Cognatazoarcus gen. nov., were proposed. This study provided new insights to the taxonomy of the family Zoogloeaceae.

(Keynote) Engineering Cyanobacteria for Nitrogen Reduction

Ammonia is currently the second most widely produced chemical in the world (second to sulfuric acid). Most ammonia (over 66%) is now produced through the Haber−Bosch process, consuming approximately 5% of the global natural gas production and contributes to over 3% of global carbon dioxide emissions. In an effort to develop an electrochemical alternative, we have engineered cyanobacterium to perform nitrogen fixation under ambient conditions without consuming natural gas, in stark contrast to the Haber-Bosch process that requires extreme temperatures and pressures to the same end. To realize the heterologous expression of nitrogenase, we transformed the cyanobacterium S. elongatus PCC 7942 with the minimal nitrogen fixation (nif) gene cluster. Nitrogenase can reduce N2 to NH3 at mild conditions (< 40 oC, atmospheric pressure). There are three known types of nitrogenase, in which the central composition of their active-site metalloclusters are molybdenum(Mo), vanadium (V), and iron (Fe), respectively. Most biological nitrogen fixation is catalyzed by the Mo-based nitrogenase enzyme. Biosynthesis of Mo-nitrogenase generally requires a large number of nif genes. However, the transformation and transcription of such a larger set of gene cluster into the cyanobacterium is challenging. In this work, a minimal nif cluster with reduced genetic complexity was chosen to accomplish the transformation of the cyanobacterium S. elongatus PCC 7942. The cyanobacterium S. elongatus PCC 7942 was transformed with nif genes and it was shown to be able to express active nitrogenase. The engineered S. elongatus PCC 7942 was immobilized on an electrode to accelerate the microbial conversion of nitrogen into ammonium using electrochemical driving forces. Methyl viologen (MV) was added as an electron mediator to shuttle electrons from the electrode to the enzyme active center, since MV can go through the cell membrane and deliver continuous electrons to nitrogenase.

Selective influence of cyflumetofen in degradation and ecological risk assessment

AbstractPesticide pollution has gradually caused land degradation. In order to avoid this problem, it is recommended to use enantiomeric pesticides that have less impact on the soil. Cyflumetofen's (CYF) microecological environmental assessment at the enantiomer level is unclear. In our study, the degradation of CYF enantiomers and the effect on soil functions are closely related to microorganisms. (+)‐CYF enantiomer is degradable preferred and further discovered that related microorganism that degrades enantiomers. CYF enantiomers alter the bacteria structure and decreased the bacteria abundance. The combination of high‐throughput and quantitative PCR results showed that the diversity of the (+)‐CYF treatment was significantly lower than that of the (−)‐CYF (−30.41 to 44.60) treatment, and the (+)‐CYF treatment (−27.80% to 56.70%) was more capable of causing the decrease in the number of soil microorganisms. In addition, (+)‐CYF severely interferes with nitrogen cycling–related functions. Furthermore, the soil microbial structure was changed to its original level by enantiomers posed. In the study of nitrogen cycle functions, we found that both enantiomers can restrain the abundance of nitrogen cycle–related genes, especially the (+)‐CYF treatment decreased more. Canonical correspondence analysis (CCA) showed that g‐Massilia and g‐Arthrobacter are closely related to nitrogen fixation genes and nitrification genes, and degradation of the two enantiomers of CYF by g‐Arthrobacter is closely related. The biological effects of CYF enantiomers remain unclear. Bioassay results show that enantiomers have similar virulence to Tetranychus cinnabarinus. Therefore, while achieving the prevention and control effect, the use of a single isomer (+)‐CYF has a higher potential risk to the soil ecosystem.

Whole genome analysis of Gluconacetobacter azotocaptans DS1 and its beneficial effects on plant growth.

The online version contains supplementary material available at 10.1007/s13205-021-02996-1.

Phylogenetic study of endophytic bacterial strains using gyrB, rpoD, FEN and nifH genes

Tomato is one of the most important commercial crops in the world especially Iran. The use of entophytic bacteria is one of the new ways to manage this disease. In this study, eight tomato endophytic bacteria namely Pseudomonas fluorescens NZ105, Bacillus pumilus strains NZ103 and 106, Bacillus subtilis NZ104, Bacillus safensis NZ107, Enterobacter ludwigii NZ108, Serratia marcescens NZ102 and Pseudomonas baetica NZ101were identified based on 16S rRNA gene sequence analysis. These were further examined for conserved genes of gyrB and rpoD, two functional antibiotic-producing FEN and nitrogen-stabilizing nifH genes. Among these, only P. fluorescens NZ105 have gyrB and rpoD genes and B. safensis and S. marcescens have antibiotic-producing FEN gene. None of the eight isolates harbouring nitrogen-fixing nifH gene compared to the positive control Azotobacter chroococum. The phylogeny of eight endophytic bacteria formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. safensis and B. albus.

Bio-conversion of kitchen waste into bacterial cellulose using a new multiple carbon utilizing Komagataeibacter rhaeticus: Fermentation profiles and genome-wide analysis

A cellulose-producing bacterium Komagataeibacter rhaeticus K15 was isolated from kombucha tea, and its metabolic pathways and cellulose synthesis operon were analyzed by genome sequencing. Different from the reported K. rhaeticus, the K15 produced little gluconic acid (2.26 g/L) when glucose was the sole carbon source and has the capacity for high cellulose production (4.76 g/L) with other carbon sources. Furthermore, six nitrogen-fixing genes were found to be responsible for the survival of K15 on a nitrogen-free medium. Based on its fermentation characteristics, K15 was cultured in a kitchen waste medium as a strategy for green and sustainable bacterial cellulose production. The SEM, XRD, and FTIR results indicated that synthesized cellulose has a mean diameter of 40–50 nm nanofiber, good crystallinity, and the same chemical structure. The K15 strain provides a highly viable alternative strategy to reduce the costs of bacterial cellulose production using agro-industrial residues as nutrient sources.

Polystyrene nanoplastics change the functional traits of biofilm communities in freshwater environment revealed by GeoChip 5.0

There is an increasing concern regarding the potential effects of nanoplastics (NPs) on freshwater ecosystems. Considering the functional values of biofilms in freshwater, knowledge on whether and to what extent NPs can influence the ecosystem processes of biofilms were still limited. Herein, the freshwater biofilms cultured in lab were exposed to 100 nm polystyrene NPs (PS-NPs) of different dosages (1 and 10 mg/L) for 14 days. Confocal laser scanning microscope observation indicated that biofilms were dominated by filamentous, and spiral algae species and the intensity of extracellular polymeric substances increased under PS-NPs exposure. GeoChip 5.0 analysis revealed that PS-NPs exposure triggered a significant increase in functional genes α diversity (p < 0.05) and altered biofilms’ functional structure. Furthermore, the abundance of genes involved in the total carbon and nitrogen cycling were increased under PS-NPs exposure. The abundance of nitrogen fixation genes experienced the most pronounced increase (24.4%) under 1 mg/L PS-NPs treatment, consistent with the increase of ammonium in overlying water. Whereas antibiotic resistance genes and those related to photosynthetic pigments production were suppressed. These results provided direct evidence for PS-NPs’ effects on the biofilm functions in terms of biogeochemical cycling, improving our understanding of the potentials of NPs on freshwater ecosystems.

Final Destination? Pinpointing Hyella disjuncta sp. nov. PCC 6712 (Cyanobacteria) Based on Taxonomic Aspects, Multicellularity, Nitrogen Fixation and Biosynthetic Gene Clusters.

Unicellular cyanobacteria inhabit a wide range of ecosytems and can be found throughout the phylum offering space for taxonomic confusion. One example is strain PCC 6712 that was described as Chlorogloea sp. (Nostocales) and later assigned to the genus Chroococcidiopsis (Chroococcidiopsidales). We now show that this strain belongs to the order Pleurocapsales and term it Hyella disjuncta based on morphology, genome analyses and 16S-23S ITS rRNA phylogeny. Genomic analysis indicated that H. disjuncta PCC 6712 shared about 44.7% orthologue genes with its closest relative H. patelloides. Furthermore, 12 cryptic biosynthetic gene clusters (BGCs) with potential bioactivity, such as a mycosporine-like amino acid BGC, were detected. Interestingly, the full set of nitrogen fixation genes was found in H. disjuncta PCC 6712 despite its inability to grow on nitrogen-free medium. A comparison of genes responsible for multicellularity was performed, indicating that most of these genes were present and related to those found in other cyanobacterial orders. This is in contrast to the formation of pseudofilaments—a main feature of the genus Hyella—which is weakly expressed in H. disjuncta PCC 6712 but prominent in Hyella patelloides LEGE 07179. Thus, our study pinpoints crucial but hidden aspects of polyphasic cyanobacterial taxonomy.

Resource Allocation During the Transition to Diazotrophy in Klebsiella oxytoca

Free-living nitrogen-fixing bacteria can improve growth yields of some non-leguminous plants and, if enhanced through bioengineering approaches, have the potential to address major nutrient imbalances in global crop production by supplementing inorganic nitrogen fertilisers. However, nitrogen fixation is a highly resource-costly adaptation and is de-repressed only in environments in which sources of reduced nitrogen are scarce. Here we investigate nitrogen fixation (nif) gene expression and nitrogen starvation response signaling in the model diazotroph Klebsiella oxytoca (Ko) M5a1 during ammonium depletion and the transition to growth on atmospheric N2. Exploratory RNA-sequencing revealed that over 50% of genes were differentially expressed under diazotrophic conditions, among which the nif genes are among the most highly expressed and highly upregulated. Isotopically labelled QconCAT standards were designed for multiplexed, absolute quantification of Nif and nitrogen-stress proteins via multiple reaction monitoring mass spectrometry (MRM-MS). Time-resolved Nif protein concentrations were indicative of bifurcation in the accumulation rates of nitrogenase subunits (NifHDK) and accessory proteins. We estimate that the nitrogenase may account for more than 40% of cell protein during diazotrophic growth and occupy approximately half the active ribosome complement. The concentrations of free amino acids in nitrogen-starved cells were insufficient to support the observed rates of Nif protein expression. Total Nif protein accumulation was reduced 10-fold when the NifK protein was truncated and nitrogenase catalysis lost (nifK1–1203), implying that reinvestment of de novo fixed nitrogen is essential for further nif expression and a complete diazotrophy transition. Several amino acids accumulated in non-fixing ΔnifLA and nifK1–1203 mutants, while the rest remained highly stable despite prolonged N starvation. Monitoring post-translational uridylylation of the PII-type signaling proteins GlnB and GlnK revealed distinct nitrogen regulatory roles in Ko M5a1. GlnK uridylylation was persistent throughout the diazotrophy transition while a ΔglnK mutant exhibited significantly reduced Nif expression and nitrogen fixation activity. Altogether, these findings highlight quantitatively the scale of resource allocation required to enable the nitrogen fixation adaptation to take place once underlying signaling processes are fulfilled. Our work also provides an omics-level framework with which to model nitrogen fixation in free-living diazotrophs and inform rational engineering strategies.

Analysis of Nitrogenase Fe Protein Activity in Transplastomic Tobacco

Integration of prokaryotic nitrogen fixation (nif) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting of Nif proteins within plant and yeast cells. Here we use tobacco plants with the Azotobacter vinelandii nifH and nifM genes integrated into the plastid genome. NifH and its maturase NifM were constitutively produced in leaves, but not roots, during light and dark periods. Nif protein expression in transplastomic plants was stable throughout development. Chloroplast NifH was soluble, but it only showed in vitro activity when isolated from leaves collected at the end of the dark period. Exposing the plant extracts to elevated temperatures precipitated NifM and apo-NifH protein devoid of [Fe4S4] clusters, dramatically increasing the specific activity of remaining NifH protein. Our data indicate that the chloroplast endogenous [Fe-S] cluster biosynthesis was insufficient for complete NifH maturation, albeit a negative effect on NifH maturation due to excess NifM in the chloroplast cannot be excluded. NifH and NifM constitutive expression in transplastomic plants did not affect any of the following traits: seed size, germination time, germination ratio, seedling growth, emergence of the cotyledon and first leaves, chlorophyll content and plant height throughout development.

Molecular Insights into Beneficial Effects of Tea-Plant Growth and Selenium Enrichment by Herbaspirillum camelliae

Herbaspirillum camelliae WT00C, as a tea-plant endophytic bacterium, not only colonizes specifically in tea plants but also promotes tea-plant growth and selenium enrichment. Different from diazotrophic endophytes H. seropedicae, H. frisingense and H. rubrisubalbicans, H. camelliae WT00C does not display nitrogen-fixing activity. To understand the molecular mechanisms of promoting the growth of tea plant and Se-enrichment, we sequenced and annotated the genome of H. camelliae WT00C. The results showed that the genome was composed of 6,079,821 base pairs with a total of 5,537 genes. The genomic survey also revealed that H. camelliae WT00C was a multifunctional bacterium metabolizing a variety of carbon and nitrogen sources and defending against biotic and abiotic stress. Although this bacterium did not have intact nitrogen-fixing genes, its genome held the genes responsible for indole-3-acetic acid (IAA) biosynthesis, 1-aminocyclopropane-1-carboxylate (ACC) deamination, siderophore synthesis, ammonia formation, urea metabolism, glutathione and selenocompound metabolisms. Biosynthesis of IAA, siderophore, ammonia, urea and ACC deaminase could explain why two bacterial strains promote tea-plant growth and development. Selenocompound metabolism in this bacterium might also benefit tea-plant growth and Se-enrichment. In addition, the genome of H. camelliae also contained a multitude of protein secretion systems T1SS, T3SS, T4SS and T6SS, in which T4SS did not exhibit in other members of the genus Herbaspirillum.

Molecular Components of Nitrogen Fixation Gene Cluster and Associated Enzymatic Activities of Non-Heterocystous Thermophilic Cyanobacterium Thermoleptolyngbya sp.

Thermoleptolyngbya is a genus of non-heterocystous cyanobacteria that are typical inhabitants of hot spring microbial mats. These filamentous cyanobacteria are capable of nitrogen fixation. In this study, we examined the genome sequences of five publicly available Thermoleptolyngbya strains to explore their nitrogen fixation gene cluster. Analysis of the nitrogen-fixation clusters in these extremophilic strains revealed that the cluster is located in a single locus in Thermoleptolyngbyace. The average nucleotide and amino acid identities of the nitrogen-fixation cluster combined with phylogenetic reconstructions support that nitrogen fixation genes in Thermoleptolyngbyaceae are closely related to one another but also heterogeneous within the genus. The strains from Asia, and China more specifically, generate a separate clade within the genus. Among these strains Thermoleptolyngbya sp. PKUAC-SCTB121 has been selected for experimental validation of clade’s nitrogen fixation capacity. The acetylene reduction experiments of that strain shown that the strain can reduce acetylene to ethylene, indicating a fully functional nitrogenase. The activity of nitrogenase has been tested using different gas compositions across 72 h and exhibited a two-phase trend, high nitrogenase activity at the beginning of the assay that slowed down in the second phase of the analysis.

Regulatory Switching by Concerted Motions on the Microsecond Time Scale of the Oxygen Sensor Protein FixL.

Signal transduction proteins perceive external stimuli in their sensor module and regulate the biological activities of the effector module, allowing cellular adaptation in response to environmental changes. FixL is a dimeric heme protein kinase that senses the oxygen level in plant root nodules to regulate the transcription of nitrogen fixation genes via the phosphorylation of its cognate transcriptional activator. Dissociation of oxygen from the heme induces conformational changes in the protein, converting it from the inactive form for phosphorylation to the active form. However, how FixL undergoes conformational change to regulate kinase activity upon oxygen dissociation remains poorly understood. Here we report time-resolved ultraviolet resonance Raman spectra showing conformational changes for FixL from Sinorhizobium meliloti. We observed spectral changes with a time constant of about 3 μs, which were oxygen-specific. Furthermore, we found that the conformational changes in the sensor and kinase domains are coupled, enabling allosteric control of kinase activity. Our results demonstrate that concerted structural changes on the microsecond time scale serve as the regulatory switch in FixL.

Morphological, Biochemical and Molecular Characterization of Rhizobia of Faba Bean Plants Grown in North Nile Delta Egypt.

&lt;b&gt;Background and Objective:&lt;/b&gt; Rhizobia are bacteria including genes codes for enzymes involved in the fixing of the atmospheric nitrogen. A set of twenty rhizobial isolates were studied to determine their morphological, biochemical, molecular characteristics using the 16S rRNA gene in addition to assess their growth and symbiotic performance. &lt;b&gt;Materials and Methods:&lt;/b&gt; Rhizobial isolates were isolated from root nodules of &lt;i&gt;Vicia faba &lt;/i&gt;L. plants. The isolates were morphologically characterized by determining cell shapes, size, Gram stain reaction, motility, sporulation, bacterial growth performance was determined by IAA production and biomass density. Symbiotic performance was measured by evaluation of nodulation status and shoot/root dry weight. Sequencing of 16S rRNA and phylogenetic analysis were done for the five promising isolates. Statistical analysis was performed using a one-sample Student t-test. &lt;b&gt;Results:&lt;/b&gt; Only five rhizobial isolates (Rh 32, Rh 6-A, Rh 3-4, Rh RL3 and Rh 8-A) were selected according to their growth and symbiotic performance and subjected to further molecular characterizations. All isolates were found to have remarkable nodulation status, IAA production, nitrogenase activity and increasing the root and shoot dry weight. The five selected rhizobial isolates were identified by partial sequencing of 16S rRNA genes and registered in the GenBank database. The alignment and phylogenetic analyses of 16S rRNA sequences closely related in the GenBank revealed that all isolates belonging to &lt;i&gt;Rhizobium leguminosarum&lt;/i&gt; bv. viciae. &lt;b&gt;Conclusion:&lt;/b&gt; The results confirmed that the five Rhizobial strains will be promising as a source of genes for nitrogen fixation and plant growth promotion.

Disrupting hierarchical control of nitrogen fixation enables carbon-dependent regulation of ammonia excretion in soil diazotrophs.

The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation (nif) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii. The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A. vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family.

Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1T, 162S2 and 66S1MBT have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1T (=LMG 29930T=HAMBI 3676T) and 66S1MBT (=LMG 31547T=HAMBI 3720T) as type strains, respectively.

Draft Genome Sequences of Switchgrass Diazotrophs.

ABSTRACTWe report the draft genome sequences of five native nitrogen-fixing bacteria associated with roots of switchgrass isolated from the tallgrass prairies of Oklahoma. Nitrogen-fixing genes, including the nif cluster, are conserved across the Klebsiella and Kosakonia strains.

Effects of long-term discharge of acid mine drainage from abandoned coal mines on soil microorganisms: microbial community structure, interaction patterns, and metabolic functions

More than twenty abandoned coal mines in the Yudong River Basin of Guizhou Province have discharged acid mine drainage (AMD) for a long time. The revelation of microbial community composition, interaction patterns, and metabolic functions can contribute to abetter understanding ofsuch ecosystems, which in its turn can be helpfulin the development of strategies aiming atthe ecological remediation of AMD pollution. In this study, reference and contaminated soil samples were collected along the AMD flow path for high-throughput sequencing. Results showed that the long-term AMD pollution promoted the evolution of γ-Proteobacteria, and the acidophilic iron-oxidizing bacteria Ferrovum (relative abundance of 15.50%) and iron-reducing bacteria Metallibacterium (9.87%) belonging to this class became the dominant genera. Co-occurrence analysis revealed that the proportion of positive correlations among bacteria increased from 51.02 (reference soil) to 75.16% (contaminated soil), suggesting that acidic pollution promotes the formation of mutualistic interaction networks of microorganisms. Metabolic function prediction (Tax4Fun) revealed that AMD contamination enhanced microbial functions such as translation, repair, and biosynthesis of peptidoglycan and lipopolysaccharide, etc., which may be an adaptive mechanism for microbial survival in extremely acidic environment. In addition, acidic pollution promoted the high expression of nitrogen-fixing genes in soil, and the discovery of autotrophic nitrogen-fixing bacteria such as Ferrovum highlights the possibility of using this taxon for bioremediation of AMD pollution.