nifD Genes Research Articles

Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on a combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, the DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then, the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

Insights into Genetic and Physiological Characteristics of Clover Rhizobia in Afghanistan Soils

Livestock production in Afghanistan highly relies on grazing and clover feed, which is a key component of pastures and forage crops. This study elucidated the genetic diversity of clover-nodulating rhizobia in different ecological regions and their effects on clover growth. A total of 57 rhizobia were isolated and their genetic diversities were studied through 16S rRNA and nifD genes. The isolates were inoculated to clover (Afghan local variety), to investigate the potential of nitrogen fixation and influences of clover growth. The 16S rRNA gene analysis showed two distinct groups of Rhizobium (94.7%) and Ensifer (5.3%) species. The nifD phylogenetic relationship revealed a high similarity to Rhizobium and a novel lineage group close to Rhizobium leguminosarum species. In the plant test, different genotypes significantly (p < 0.01) exhibited an increase in plant biomass production, compared to the un-inoculated plants. Among genotypes, the highest plant biomass was recorded in PC8 (1769.0 mg/plant) and PC9 (1409.2 mg/plant) isolates as compared to un-inoculated plants (144.0 mg/plant). Moreover, these isolates showed maximum nitrogen fixation rates of 8.2 and 6.5 µM/plant, respectively. These isolates were identified as the most promising rhizobial strains for developing biofertilizers in the context of Afghanistan.

Effects of extreme drought on soil microbial functional genes involved in carbon and nitrogen cycling in alpine peatland

Diverse microorganisms drive biogeochemical cycles and consequently influence ecosystem-level processes in alpine peatlands, which are vulnerable to extreme drought induced by climate change. However, there are few reports about the effects of extreme drought on microbial function. Here we identify microbial functional genes associated with carbon and nitrogen metabolisms of extreme drought experiments that occurred at different periods of plant growth, the results show that early extreme drought reduces the abundance of functional genes involved in the decomposition of starch and cellulose; midterm extreme drought increases the abundance of lignin decomposition functional genes; late extreme drought reduces the hemicellulose but increases cellulose decomposition functional genes. In the carbon fixation pathway, extreme drought mainly changes the abundance of functional genes involved in the reductive citrate cycle process, the 3-hydroxy propionate bi-cycle, the dicarboxylate-hydroxybutyrate cycle and the incomplete reductive citrate cycle. Among the nitrogen cycling functional genes, amoA involved in oxidizing ammonia to hydroxylamine significantly increases under early extreme drought; midterm extreme drought reduces nrtC and nifD genes, which participate in nitrate assimilation and nitrogen fixation, respectively; late extreme drought significantly increases hcp genes involved in ammonification. pH and TN had the largest effects on the carbon degradation, fixation and nitrogen cycling functional genes. The composition of microbial community structures involved in carbon fixation differed between treatments in early extreme drought. There is a good linear fit between the diversity of gene abundance and corresponding microbial communities in the reductive citrate cycle, hydroxy propionate-hydroxybutyrate cycle, dicarboxylate-hydroxybutyrate cycle and nitrogen cycling, which suggests that the functional genes and community composition of microorganisms involved in these processes are consistent in response to extreme drought. This study provides new insights into the adaptability and response characteristics of microbial communities and functional genes in plateau peatland ecosystems to extreme drought events.

Impact of waterlogging and heat stress on rice rhizosphere microbiome assembly and potential function in carbon and nitrogen transformation

ABSTRACT The impacts of rhizosphere microorganisms on the turnover of nutrients are affected by many environmental factors. In this study, rice plants were subjected to deep flooding and heat stress for one week at the jointing stage. Rhizospheric soil was sampled for the analysis of rice rhizosphere bacterial assembly and functional groups responsible for carbon and nitrogen transformation. Compared with the control, both deep flooding and heat stress increased bacterial alpha diversity. Moreover, heat stress and the interactions of heat stress and deep flooding improved the bacterial cooperation. Heat stress reduced the relative abundance of Actinobacteria and Firmicutes, which are responsible for organic carbon decomposition. The relative abundance of the gene encoding lignin-degrading catalases under deep flooding and heat stress treatments reduced by 6% and 8%, respectively. The interaction of heat stress and deep flooding increased the relative abundance of the nifD gene by 5%, may slowed down nitrate reduction to nitrite. Our results highlight the effects of heat and flooding stress on the bacteria-mediated mechanisms responsible for regulating organic carbon sequestration and nitrogen cycling in rhizosphere of paddy fields.

Nitrogen and phosphorus significantly alter growth, nitrogen fixation, anatoxin-a content, and the transcriptome of the bloom-forming cyanobacterium, Dolichospermum.

While freshwater cyanobacteria are traditionally thought to be limited by the availability of phosphorus (P), fixed nitrogen (N) supply can promote the growth and/or toxin production of some genera. This study characterizes how growth on N2 (control), nitrate (NO3–), ammonium (NH4+), and urea as well as P limitation altered the growth, toxin production, N2 fixation, and gene expression of an anatoxin-a (ATX-A) – producing strain of Dolichospermum sp. 54. The transcriptomes of fixed N and P-limited cultures differed significantly from those of fixed N-deplete, P-replete (control) cultures, while the transcriptomes of P-replete cultures amended with either NH4+ or NO3– were not significantly different relative to those of the control. Growth rates of Dolichospermum (sp. 54) were significantly higher when grown on fixed N relative to without fixed N; growth on NH4+ was also significantly greater than growth on NO3–. NH4+ and urea significantly lowered N2 fixation and nifD gene transcript abundance relative to the control while cultures amended with NO3– exhibited N2 fixation and nifD gene transcript abundance that was not different from the control. Cultures grown on NH4+ exhibited the lowest ATX-A content per cell and lower transcript abundance of genes associated ATX-A synthesis (ana), while the abundance of transcripts of several ana genes were highest under fixed N and P - limited conditions. The significant negative correlation between growth rate and cellular anatoxin quota as well as the significantly higher number of transcripts of ana genes in cultures deprived of fixed N and P relative to P-replete cultures amended with NH4+ suggests ATX-A was being actively synthesized under P limitation. Collectively, these findings indicate that management strategies that do not regulate fixed N loading will leave eutrophic water bodies vulnerable to more intense and toxic (due to increased biomass) blooms of Dolichospermum.

β-N-Methylamino-L-Alanine (BMAA) Causes Severe Stress in Nostoc sp. PCC 7120 Cells under Diazotrophic Conditions: A Proteomic Study.

Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms’ ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA’s action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected—due to their notable expression differences—for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and β (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes; BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.

Diversity and phylogenetic affinities of Bradyrhizobium isolates from Pampa and Atlantic Forest Biomes

In this work, we investigated Bradyrhizobium strains isolated from soils collected from the rhizosphere of native and exotic legumes species inhabiting two ecoclimatic zones – asubtropical-lowland pasture (Pampa Biome) and a volcanic plateau covered by Araucaria Moist Forests (Atlantic Forest Biome). The rhizobial strains were isolated from the nodules of seven native and one exotic legume species used as rhizobium traps. Single-gene (recA, glnII, dnaK) and combined-gene MLSA analyses (dnaK-glnII-gyrB-recA-rpoB) revealed that nearly 85% of the isolates clustered in B. elkanii supergroup, while the remaining (except for two isolates) in B. japonicum supergroup, albeit, in most cases, separately from the type strains of Bradyrhizobium species. As a symbiotic gene marker, a portion of nifD gene was sequenced for 194 strains. In the nifD-tree, an American branch III.3D (104 isolates), was the most numerous among the isolates. A significant portion of the isolates clustered in American groups; subclade III.4 (40 strains), Clade VII (3 strains), and a new Clade XX (4 strains). Most of the remaining strains belonged to a pantropical III.3C branch (39 isolates). On the other hand, identification of isolates belonging, respectively, to Clade I and Clade II may result of spreading of the Australian (Clade I) and European (Clade II) bradyrhizobia following the introduction of their legume hosts. Our study indicated that the American groups predominated in the symbiotic Bradyrhizobium communities in southern Brazil. However, there is a significant component of exotic lineages, resulting from the dispersal of pantropical Fabaceae taxa and the introduction of exotic legumes.

Anthyllis vulneraria and Lotus corniculatus on calamine heaps form nodules with Bradyrhizobium liaoningense-related strains harboring novel in Europe symbiotic nifD haplotypes

Calamine tailings, a habitat known for high concentrations of zinc and lead, are inhabited by a limited number of plants. The objective of this project was a phylogenetic characterization of Bradyrhizobium strains that form effective symbiosis with Anthyllis vulneraria L. and Lotus corniculatus L. (Fabaceae) spontaneously growing on old calamine tailings in Southern Poland using a multilocus sequence analysis (MLSA) approach based on amplification of selected housekeeping dnaK, glnII, gyrB, recA, and rpoB, and symbiotic nifD genes. The isolates were also tested for their metal tolerance and nitrogenase activity. The analyses have shown enhanced growth of Bradyrhizobium strains in the presence of lead (Pb) or zinc (Zn), unlike uninoculated plants which died within three weeks. In housekeeping gene trees, all heavy metal-resistant bradyrhizobia clustered close to B. liaoningense, a species which until now has not been recorded among Bradyrhizobium symbionts of Loteae tribe. In nifD gene tree, Bradyrhizobium isolates clustered within a group referred to as Clade XVII, encompassing Fabaceae symbionts that originate from different geographical areas but also non-nodulating Bradyrhizobium diazotrophs. Summing up, this study revealed that in these heavy metal-contaminated soils L. corniculatus and A. vulneraria are infected by Bradyrhizobium strains harboring symbiotic nifD gene haplotypes phylogenetically unrelated to Clade II strains that predominate in Europe among bradyrhizobia nodulating Genisteae and Loteae spp.

Excision of the 59-kb fdxN DNA element is required for transcription of the nifD gene in Anabaena PCC 7120 Heterocysts

ABSTRACTIn the heterocyst-forming cyanobacterium Anabaena (also Nostoc) sp. strain PCC 7120, the nitrogen-fixation (nif) genes are expressed specifically in heterocysts during the late stages of development. We used gfp-reporter fusions to examine the regulation of the nifH and nifD genes. Plasmid-borne reporter fusions containing up to 500-bp of the nifH upstream region, which extended into the nifU gene, did not show developmentally regulated GFP fluorescence after removal of a nitrogen source from the growth medium, which indicated that sequences essential for transcriptional regulation are outside of the tested regions. Therefore, a gfp-reporter fusion was engineered into the chromosome at the 5′ end of nifD to produce strain AMC1774. This reporter construct at the native locus showed strong developmentally regulated GFP expression in differentiating heterocysts by 18 h after removal of combined nitrogen. We then screened for UV-induced mutants of AMC1774 that differentiated heterocysts but failed to show GFP-reporter fluorescence. Several dark mutants were obtained and then complemented with an expression library. One mutant was complemented with a clone that contained the xisF and alr1460 genes. The xisF gene is required for excision of a 59,428-bp DNA element present within the fdxN gene in the nifB-fdxN-nifS-nifU gene cluster, which is upstream of the nifHDK gene cluster. Analysis of the original UV-induced mutant showed that it was defective for excision of the fdxN DNA element. Together, our data shows that transcription of the nifHDK genes requires a distant promoter upstream of the fdxN DNA element. We used a gfp transcriptional reporter to demonstrate developmentally regulated promoter activity from the intergenic region upstream of nifB. We also show that the promoter for a second nifH gene, nifH2 (alr0874), lies proximal to nifH2 in its upstream intergenic region.

Molecular and symbiotic characterization of peanut bradyrhizobia from the semi-arid region of Brazil

Peanut (Arachis hypogaea L.) is a legume crop native from South America and grown worldwide. This is well adapted to the unfavorable edaphoclimatic conditions found in Brazilian Semi-Arid region, where is cultivated mainly by smallholders. The selection of new rhizobial isolates is important to increase the rates of biological nitrogen fixation and yield. Furthermore, the diversity evaluation of new bacterial isolates may help us to understand the ecology of these groups in regions where little information are available. In this context, this study aimed to evaluate the phylogenetic relationships of peanut bradyrhizobia from Brazilian Semi-Arid soils by analysis of 16S rRNA, recA, nodC and nifD gene sequences, as well as their symbiotic performance. A trap-host experiment was performed using two soil samples from the municipality of Barbalha, Ceará, Brazil, taken under peanut cropping and one soil sample from the municipality of Juazeiro, Bahia, Brazil, covered by the native Arachis triseminata. Seeds of peanut cv. BR1 were sown and the plants were grown until 52days after emergence. The nodules were inoculated in YMA medium for isolation of slow-growing bacteria. The Box-PCR fingerprinting and the 16S rRNA gene sequences was evaluated for all bacteria and recA, nodC and nifD were sequenced for the six selected isolates. Selected bacteria were evaluated according to their symbiotic characteristics in a greenhouse experiment. Ten slow-growing isolates were obtained and classified within the genus Bradyrhizobium, by the 16S rRNA sequence analysis and showed divergent Box-PCR profiles. All bacteria were clustered within the B. japonicum clade. The bacteria from Juazeiro were closely related to B. yuanmingense, and those from Barbalha showed similarity to B. yuanmingense, B. kavangense and B. guangxiense. The sequences of recA showed partial congruence to the 16S rRNA gene analysis but the phylogeny trees based on the symbiotic genes were not related, indicating horizontal gene transfer. All bacteria were able to efficiently nodulate the peanut and the isolate ESA 83, pointed out regarding the nitrogen fixation ability and were selected for further analysis in non-sterile soils and field conditions.

Nostoc thermotolerans sp. nov., a soil-dwelling species of Nostoc (Cyanobacteria).

A filamentous, soil-dwelling cyanobacterial strain (9C-PST) was isolated from Mandsaur, Madhya Pradesh, India, and is described as a new species of the genus Nostoc. Extensive morphological and molecular characterization along with a thorough assessment of ecology was performed. The style of filament orientation, type and nature of the sheath (e.g. distribution and visibility across the trichome), and vegetative and heterocyte cell dimensions and shape were assessed for over one year using both the laboratory grown culture and the naturally occurring samples. Sequencing of the 16S rRNA gene showed 94 % similarity with Nostocpiscinale CENA21 while analyses of the secondary structures of the 16S-23S ITS region showed unique folding patterns that differentiated this strain from other species of Nostoc. The level of rbcl and rpoC1 gene sequence similarity was 91 and 94 % to Nostocsp. PCC 7524 and Nostocpiscinale CENA21, respectively, while the nifD gene sequence similarity was found to be 99 % with Nostocpiscinale CENA21. The phenotypic, ecological, genetic and phylogenetic observations indicate that the strain 9C-PST represents a novel species of the genus Nostoc with the name proposed being Nostoc thermotolerans sp. nov. according to the International Code of Nomenclature for Algae, Fungi, and Plants.

Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions.

Cyanobacteria is a remarkable group of prokaryotic photosynthetic microorganisms, with several genera capable of fixing atmospheric nitrogen (N2) and presenting a wide range of morphologies. Although the nitrogenase complex is not present in all cyanobacterial taxa, it is spread across several cyanobacterial strains. The nitrogenase complex has also a high theoretical potential for biofuel production, since H2 is a by-product produced during N2 fixation. In this review we discuss the significance of a relatively wide variety of cell morphologies and metabolic strategies that allow spatial and temporal separation of N2 fixation from photosynthesis in cyanobacteria. Phylogenetic reconstructions based on 16S rRNA and nifD gene sequences shed light on the evolutionary history of the two genes. Our results demonstrated that (i) sequences of genes involved in nitrogen fixation (nifD) from several morphologically distinct strains of cyanobacteria are grouped in similarity with their morphology classification and phylogeny, and (ii) nifD genes from heterocytous strains share a common ancestor. By using this data we also discuss the evolutionary importance of processes such as horizontal gene transfer and genetic duplication for nitrogenase evolution and diversification. Finally, we discuss the importance of H2 synthesis in cyanobacteria, as well as strategies and challenges to improve cyanobacterial H2 production.

New species of Nostoc (cyanobacteria) isolated from Pune, India, using morphological, ecological and molecular attributes

Filamentous cyanobacterium (strain NE-PS) isolated from a freshwater body in Pune, India, is being described as new species of the polyphyletic genus Nostoc. Phenotypic and molecular characterizations were performed, and the combined results validated the strain as a new species. Careful observations of the filaments, the presence of a distinct sheath throughout the length of the trichome, prominent differences in the shape and dimensions of the vegetative cells, heterocytes and the akinetes provided reliable morphological signals that the strain differed from rest of the closely related species. Sequencing of the 16S rRNA gene showed 98.66 % sequence similarity with Nostoc linckia, while rbcL and psbA sequencing showed 97 and 94 % similarities with Nostoc sp. PCC 7906 and Nostoc punctiforme PCC 73102, respectively, while the nifD gene sequence similarity was found to be 96 % with N. punctiforme Ind35 and Desmonostoc muscorum. The PC-IGS region was sequenced and concatenated cpcB, IGS and cpcA regions indicated the closest similarity with Nostoc linckia PACC 5085 at 96 %. Subsequent phylogenetic analyses gave a strong pattern of distinct clustering in case of all the molecular markers. The phenotypic, genetic and phylogenetic observations prove conclusively that the strain NE-PS is a new species in the genus Nostoc with the name proposed being Nostoc punensis, sp. nov.

A new species of Scytonema isolated from Bilaspur, Chhattisgarh, India

AbstractFilamentous cyanobacterium (strain 10C‐PS) isolated from a fresh water body of Bilaspur, Chhattisgarh, India is being described as new species of the polyphyletic genus Scytonema. Phenotypic, molecular and phylogenetic characterization was performed and the combined results validated the strain as a new species. Careful observations of the filaments, presence of a distinctly textured sheath throughout the length of the trichome, differences in the shape and dimensions of the vegetative cells, and heterocytes provided reliable morphological signals that the strain differed from rest of the closely related species. Sequencing of the 16S rRNA gene showed 96.89% sequence similarity with Scytonema hofmanni PCC 7110 while rbcl and psbA sequencing showed 95% and 92% similarities with Scytonema hofmanni PCC 7110 and Nostoc sp. PCC 7524 respectively while the nifD gene sequence similarity was found to be 96% with Scytonema hofmanni PCC 7110. The PC‐IGS region was sequenced and concatenated cpcB, IGS and cpcA regions indicated 97% closest similarity with Scytonema sp. PCC 7110 and Scytonema bohnerii Ind24. Subsequent phylogenetic analyses gave a strong pattern of distinct clustering in case of all the molecular markers. The phenotypic, genetic and phylogenetic observations prove conclusively that the strain 10C‐PS is a new species in the genus Scytonema with the name proposed being Scytonema bilaspurensis.

Flooding of the root system in soybean: biochemical and molecular aspects of N metabolism in the nodule during stress and recovery.

Nitrogen fixation of the nodule of soybean is highly sensitive to oxygen deficiency such as provoked by waterlogging of the root system. This study aimed to evaluate the effects of flooding on N metabolism in nodules of soybean. Flooding resulted in a marked decrease of asparagine (the most abundant amino acid) and a concomitant accumulation of γ-aminobutyric acid (GABA). Flooding also resulted in a strong reduction of the incorporation of (15)N2 in amino acids. Nodule amino acids labelled before flooding rapidly lost (15)N during flooding, except for GABA, which initially increased and declined slowly thereafter. Both nitrogenase activity and the expression of nifH and nifD genes were strongly decreased on flooding. Expression of the asparagine synthetase genes SAS1 and SAS2 was reduced, especially the former. Expression of genes encoding the enzyme glutamic acid decarboxylase (GAD1, GAD4, GAD5) was also strongly suppressed except for GAD2 which increased. Almost all changes observed during flooding were reversible after draining. Possible changes in asparagine and GABA metabolism that may explain the marked fluctuations of these amino acids during flooding are discussed. It is suggested that the accumulation of GABA has a storage role during flooding stress.

Cultivable Nitrogen Fixing Bacteria from Extremely Alkaline-Saline Soils

Three soils with different Electrolytic Conductivity (EC) from the former Lake Texcoco (soil with low EC 1.2 dS·m-1, medium with EC 83.1 - 107.8 dS·m-1, and high with EC 137.3 - 152.5 dS•m-1) were used to isolate nitrogen-fixing bacterial strains through enrichment cultures in nitrogen-free media. The medium and high EC in the soil affected negatively the nitrogen-fixing activity, which was generally ten times lower compared to the activity in the soil with low EC. Twenty-one bacterial strains were isolated, identified and characterized for their nitrogen fixation capacity. The diazotrophic genetic potential of all isolates was confirmed by amplification and sequencing of partial nifH and nifD genes and diazotrophic activity quantified by the acetylene reduction assay. Azospirillum brasilense, and several species of Paenibacillus (P. fujiensis, P. durus, P. borealis, P. graminis, P. massiliensis and P. wynnii) were identified. Isolates belonging to the Paenibacillus genus were found in the three soils. Paenibacillus fujiensis and P. durus showed a high nitrogenase activity. The phylograms based on nifH and nifD gene sequences were consistent with 16S rRNA gene phylogeny.

Aquatic nitrogen-fixing cyanobacteria associated with blooms of Didymosphenia geminata: insights from a field study

The nuisance diatom Didymosphenia geminata has recently increased in abundance worldwide and has spread through oligotrophic rivers in the South Island of New Zealand. However, it remains absent from the North Island. Its proliferation in oligotrophic environments has prompted work on phosphorus acquisition, but potential nitrogen acquisition pathways have received little attention. We partially sequenced the nifD gene, encoding a component of nitrogenase, from South Island samples containing D. geminata and compared its diversity with that of North Island sites independently ranked as high, medium and low risk for infestation. Godleyacean cyanobacteria were present in all four South Island provinces investigated, and also in three North Island sites rated medium or high risk. Most high-risk sites were dominated by Nostoc sequences, as were some infested South Island sites. Monte Carlo simulations and the weighted UniFrac metric showed that nifD diversity in low-risk North Island sites differed significantly from sites rated medium and high risk, and from infested South Island sites; however, random resampling showed that the data were insufficient to separate didymo-infested and high-risk sites from medium-risk sites. The relationships we discovered suggest that success of D. geminata in New Zealand could be mediated by indigenous nitrogen-fixing cyanobacteria.

Decoding cyanobacterial phylogeny and molecular evolution using an evonumeric approach.

Forty-one heterocystous cyanobacteria strains representing 12 cyanobacterial genera collected from all across India were assessed in phylogenetic and evolutionary perspectives. The structural gene 16S ribosomal RNA (rRNA) and the functional genes nifD and psbA were selected as molecular chronometers in this study. The phylogenetic analyses demonstrated the monophyly of heterocystous cyanobacteria with significant intermixing, along with establishing the polyphyly of Stigonematales, strongly supporting the need for re-amendments in cyanobacterial taxonomy and systematics. Molecular trends obtained did not clearly reflect the phenotypic affiliations, thus advocating for genetic characterizations using more molecular markers. Large-scale evonumeric extrapolations of gene sequence data of all the three molecular markers was performed to assess the evolutionary pace of heterocystous cyanobacteria on the basis of nucleotide diversity, recombination frequencies, and the DNA divergence between the sampled taxa. The obtained results tilted the evolutionary pace in favor of the less complex Nostocales thus indicating that possibly the simple non-branched forms are more flexible and adaptive for evolutionary diversifications as compared to the more complex and branched ones. This study hence represents a unique blend of molecular phylogeny with evogenomic sequence analyses for understanding the genetic diversity, phylogeny, and evolutionary pace within the heterocystous cyanobacteria.

Mutagenesis at α-423Ile of MoFe protein reduces the catalytic activity of nitrogenase in Klebsiella oxytoca

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia, the central process of biological nitrogen fixation. The mechanism of electron transfer from the P-cluster to the iron-molybdenum cofactor (FeMo-co) during the process of substrate reduction remains unclear. Based on our previous hypothesis, the dual pathway model, three mutants of Klebsiella oxytoca strain M5al (termed Nβ102A, Kα431H and Iα423P) were constructed to map the H+/e− transfer pathway, in which β-102Asn, α-431Lys and α-423Ile of the molybdenum-iron (MoFe) protein of nitrogenases were replaced by alanine, histidine and proline, respectively. All three substitutions affected the diazotrophic growth, especially of the Iα423P strain with very low capability of diazotrophic growth and C2H2 reduction activity. In a 42-L fermenter, the Iα423P strain was induced to the maximal C2H2 reduction activity, which was only ~17 % of that of M5al. However, the transcription level of the nifD gene was approximately four-fold higher than that of the M5al strain, indicating that the low C2H2 reduction activity of Iα423P was not because of downregulation of nifD expression. The maximal C2H2 and H+ reduction activities of the Iα423P MoFe protein were 13 % and 21 % of those of the wild-type MoFe protein, respectively. In conclusion, we propose that the changed properties of the Iα423P MoFe protein are related to the disruption of hydrogen bond formation between α-423Ile and homocitrate, and that α-423Ile serves as a gate to the Mo site in FeMo-co during the H+/e− transfer in nitrogenase.

Impacts of silver nanoparticles on cellular and transcriptional activity of nitrogen‐cycling bacteria

The widespread use of silver nanoparticles (AgNPs) raises the potential for environmental releases that could impact microbial ecosystem services. In the present study, the authors address how the AgNPs and Ag(+) that they release may impact nitrogen-cycling bacteria. The authors studied the cellular and transcriptional response of the denitrifier Pseudomonas stutzeri, the nitrogen fixer Azotobacter vinelandii, and the nitrifier Nitrosomonas europaea exposed to 35 nm (carbon-coated) AgNPs or to Ag(+) (added as AgNO3 ). Based on minimum inhibitory concentrations (MICs), Ag(+) was 20 times to 48 times more toxic to the tested strains than AgNPs (including Ag(+) released during exposure). Exposure to sublethal concentrations of AgNPs or Ag(+) (representing 10% of the respective MIC for AgNO3 ) resulted in no significant effect on the expression of the denitrifying genes narG, napB, nirH, and norB in P. stutzeri or the nitrogen-fixing genes nifD, nifH, vnfD, and anfD in A. vinelandii, whereas nitrifying genes (amoA1 and amoC2) in N. europaea were upregulated (2.1- to 3.3-fold). This stimulatory effect disappeared at higher silver concentrations (60% of the Ag(+) MIC), and toxicity was exerted at concentrations higher than 60% of the Ag(+) MIC. The MIC for N. europaea was 8 times to 24 times lower than for the other strains, indicating higher susceptibility to AgNPs. This was corroborated by the lower half-lethal concentration for N. europaea (87 µg/L) compared with P. stutzeri (124 µg/L) and A. vinelandii (>250 µg/L) when cells were exposed with Ag(+) for 24 h in 1 mM bicarbonate buffer. This suggests that ammonia oxidation would be the most vulnerable nitrogen-cycling process in wastewater treatment plants receiving AgNPs and in agricultural soils amended with biosolids that concentrate them.