Nitrogen-fixing Cyanobacteria Research Articles

A fluorescence method to detect cyanophycin in the symbiotic cyanobacterium, Anabaena azollae

ABSTRACT The cyanophycin content of the heterocystous nitrogen-fixing symbiotic cyanobacterium, Anabaena azollae, which inhabits an ovoid cavity in the dorsal leaf lobes of the fern, Azolla filiculoides, is seldom analyzed. To study the cyanophycin content in vegetative cells and heterocysts of A. azollae, we used three fluorochromes: aluminum trichloride, lead citrate and Wilson citroboric solution and Coomassie brilliant blue. Blue and yellow fluorescence were emitted from the polar nodes and cytoplasm cyanophycin granules of the heterocysts when stained with the three fluorochromes. The cyanophycin observed without staining or with Coomassie brilliant blue staining did not alter the results obtained using the fluorochromes. We found that aluminum trichloride, lead acetate and Wilson citroboric solution could be used to detect cyanophycin.

An integrated approach for the phycoremediation of Pb(II) and the production of biofertilizer using nitrogen-fixing cyanobacteria.

In recent years, growing attention has been directed toward the phycoremediation of heavy metals from bodies of water; however, many challenges remain. The nitrogen requirements for algal growth in nutrient-poor waters can lead to substantial costs. Moreover, proper management of the metal-loaded biomass is a concern. This study assessed the performance of two nitrogen-fixing cyanobacteria, Anabaena sp. and Nostoc muscorum, in treating Pb(II)-contaminated water without nitrogen under batch and fed-batch modes, as well as the subsequent utilization of the produced biomass as a biofertilizer. After 12 days of the batch mode with initial Pb(II) concentrations of 10, 20, 35, and 60mg/L, Pb(II) removal efficiencies were 98.90%, 98.95%, 97.20%, and 84.98% by Anabaena sp. and 88.00%, 73.10%, 54.54%, and 26.83% by N. muscorum, respectively. Anabaena sp. sustained growth and Pb(II) removal under the fed-batch mode by adjusting hydraulic retention time based on the influent Pb(II) concentration. Decontamination of the metal-loaded Anabaena sp. biomass was performed and resulted in a Pb(II) desorption of 93%. The desorbed Anabaena sp. extract provided the nutrient requirements for Chlorella vulgaris. The proposed strategy provides simultaneous Pb(II) bioremediation and biofertilizer production in a system driven by light energy, atmospheric N2, and CO2.

Investigating the Unique Ability of Trichodesmium To Fix Carbon and Nitrogen Simultaneously Using MiMoSA.

The open ocean is an extremely competitive environment, partially due to the dearth of nutrients. Trichodesmium erythraeum, a marine diazotrophic cyanobacterium, is a keystone species in the ocean due to its ability to fix nitrogen and leak 30 to 50% into the surrounding environment, providing a valuable source of a necessary macronutrient to other species. While there are other diazotrophic cyanobacteria that play an important role in the marine nitrogen cycle, Trichodesmium is unique in its ability to fix both carbon and nitrogen simultaneously during the day without the use of specialized cells called heterocysts to protect nitrogenase from oxygen. Here, we use the advanced modeling framework called multiscale multiobjective systems analysis (MiMoSA) to investigate how Trichodesmium erythraeum can reduce dimolecular nitrogen to ammonium in the presence of oxygen. Our simulations indicate that nitrogenase inhibition is best modeled as Michealis-Menten competitive inhibition and that cells along the filament maintain microaerobia using high flux through Mehler reactions in order to protect nitrogenase from oxygen. We also examined the effect of location on metabolic flux and found that cells at the end of filaments operate in distinctly different metabolic modes than internal cells despite both operating in a photoautotrophic mode. These results give us important insight into how this species is able to operate photosynthesis and nitrogen fixation simultaneously, giving it a distinct advantage over other diazotrophic cyanobacteria because they can harvest light directly to fuel the energy demand of nitrogen fixation. IMPORTANCE Trichodesmium erythraeum is a marine cyanobacterium responsible for approximately half of all biologically fixed nitrogen, making it an integral part of the global nitrogen cycle. Interestingly, unlike other nitrogen-fixing cyanobacteria, Trichodesmium does not use temporal or spatial separation to protect nitrogenase from oxygen poisoning; instead, it operates photosynthesis and nitrogen fixation reactions simultaneously during the day. Unfortunately, the exact mechanism the cells utilize to operate carbon and nitrogen fixation simultaneously is unknown. Here, we use an advanced metabolic modeling framework to investigate and identify the most likely mechanisms Trichodesmium uses to protect nitrogenase from oxygen. The model predicts that cells operate in a microaerobic mode, using both respiratory and Mehler reactions to dramatically reduce intracellular oxygen concentrations.

Primer design for the amplification of the ammonium transporter genes from the uncultured haptophyte algal species symbiotic with the marine nitrogen-fixing cyanobacterium UCYN-A1.

The multiple symbiotic partnerships between closely related species of the haptophyte algae Braarudosphaera bigelowii and the nitrogen-fixing cyanobacteria Candidatus Atelocyanobacterium thalassa (UCYN-A) contribute importantly to the nitrogen and carbon cycles in vast areas of the ocean. The diversity of the eukaryotic 18S rDNA phylogenetic gene marker has helped to identify some of these symbiotic haptophyte species, yet we still lack a genetic marker to assess its diversity at a finer scale. One of such genes is the ammonium transporter (amt) gene, which encodes the protein that might be involved in the uptake of ammonium from UCYN-A in these symbiotic haptophytes. Here, we designed three specific PCR primer sets targeting the amt gene of the haptophyte species (A1-Host) symbiotic with the open ocean UCYN-A1 sublineage, and tested them in samples collected from open ocean and near-shore environments. Regardless of the primer pair used at Station ALOHA, which is where UCYN-A1 is the pre-dominant UCYN-A sublineage, the most abundant amt amplicon sequence variant (ASV) was taxonomically classified as A1-Host. In addition, two out of the three PCR primer sets revealed the existence of closely-related divergent haptophyte amt ASVs (>95% nucleotide identity). These divergent amt ASVs had higher relative abundances than the haptophyte typically associated with UCYN-A1 in the Bering Sea, or co-occurred with the previously identified A1-Host in the Coral Sea, suggesting the presence of new diversity of closely-related A1-Hosts in polar and temperate waters. Therefore, our study reveals an overlooked diversity of haptophytes species with distinct biogeographic distributions partnering with UCYN-A, and provides new primers that will help to gain new knowledge of the UCYN-A/haptophyte symbiosis.

Emergence of hydrocarbon-degrading bacteria in crude oil-contaminated soil in a hyperarid ecosystem: Effect of phosphate addition and augmentation with nitrogen-fixing cyanobacteria on oil bioremediation

Hyperarid hot deserts impose limiting conditions for microbial activity, thus delaying crude oil biodegradation resulting from accidental spills. This study examined the emergence of oil-degrading bacterial species in the soil of a hyperarid area in the southern Negev desert, Israel, which was exposed to a major oil spill. Amplicon sequencing of the 16S rRNA gene showed dominance of the phyla Proteobacteria and Actinobacteria in both contaminated and control soils. Significant differences were seen in the abundance of the phylum Firmicutes. Classes Actinobacteria, Gammaproteobacteria and Bacilli dominated the oil-contaminated soil. Bacterial species capable of degrading aromatic and aliphatic oil constituents were isolated from the contaminated soil. Contaminated soil microcosms were amended separately with nitrogen, phosphorus, or nitrogen-fixing cyanobacteria (NFC) biomass; the highest reduction in total petroleum hydrocarbons was observed in the phosphorus-supplemented soil. The abundance of alkB gene, encoding alkane monooxygenase, increased significantly in the control, NFC and phosphate treatments, but not in the nitrogen treatment. However, nahA abundance (coding for naphthalene dioxygenase) was only considerably increased with the phosphorus amendment. While nitrogen amendment via NFC bioaugmentation is a sustainable and novel biostimulation method, phosphorus amendment significantly accelerates the biodegradation of crude oil under extremely arid conditions.

The balance between photosynthesis and respiration explains the niche differentiation between Crocosphaera and Cyanothece

Crocosphaera and Cyanothece are both unicellular, nitrogen-fixing cyanobacteria that prefer different environments. Whereas Crocosphaera mainly lives in nutrient-deplete, open oceans, Cyanothece is more common in coastal, nutrient-rich regions. Despite their physiological similarities, the factors separating their niches remain elusive. Here we performed physiological experiments on clone cultures and expand upon a simple ecological model to show that their different niches can be sufficiently explained by the observed differences in their photosynthetic capacities and rates of carbon (C) consumption. Our experiments revealed that Cyanothece has overall higher photosynthesis and respiration rates than Crocosphaera. A simple growth model of these microorganisms suggests that C storage and consumption are previously under-appreciated factors when evaluating the occupation of niches by different marine nitrogen fixers.

Cell-specific measurements show nitrogen fixation by particle-attached putative non-cyanobacterial diazotrophs in the North Pacific Subtropical Gyre

Biological nitrogen fixation is a major important source of nitrogen for low-nutrient surface oceanic waters. Nitrogen-fixing (diazotrophic) cyanobacteria are believed to be the primary contributors to this process, but the contribution of non-cyanobacterial diazotrophic organisms in oxygenated surface water, while hypothesized to be important, has yet to be demonstrated. In this study, we used simultaneous 15N-dinitrogen and 13C-bicarbonate incubations combined with nanoscale secondary ion mass spectrometry analysis to screen tens of thousands of mostly particle-associated, cell-like regions of interest collected from the North Pacific Subtropical Gyre. These dual isotope incubations allow us to distinguish between non-cyanobacterial and cyanobacterial nitrogen-fixing microorganisms and to measure putative cell-specific nitrogen fixation rates. With this approach, we detect nitrogen fixation by putative non-cyanobacterial diazotrophs in the oxygenated surface ocean, which are associated with organic-rich particles (<210 µm size fraction) at two out of seven locations sampled. When present, up to 4.1% of the analyzed particles contain at least one active putative non-cyanobacterial diazotroph. The putative non-cyanobacterial diazotroph nitrogen fixation rates (0.76 ± 1.60 fmol N cell−1 d−1) suggest that these organisms are capable of fixing dinitrogen in oxygenated surface water, at least when attached to particles, and may contribute to oceanic nitrogen fixation.

Phosphate limitation intensifies negative effects of ocean acidification on globally important nitrogen fixing cyanobacterium

Growth of the prominent nitrogen-fixing cyanobacterium Trichodesmium is often limited by phosphorus availability in the ocean. How nitrogen fixation by phosphorus-limited Trichodesmium may respond to ocean acidification remains poorly understood. Here, we use phosphate-limited chemostat experiments to show that acidification enhanced phosphorus demands and decreased phosphorus-specific nitrogen fixation rates in Trichodesmium. The increased phosphorus requirements were attributed primarily to elevated cellular polyphosphate contents, likely for maintaining cytosolic pH homeostasis in response to acidification. Alongside the accumulation of polyphosphate, decreased NADP(H):NAD(H) ratios and impaired chlorophyll synthesis and energy production were observed under acidified conditions. Consequently, the negative effects of acidification were amplified compared to those demonstrated previously under phosphorus sufficiency. Estimating the potential implications of this finding, using outputs from the Community Earth System Model, predicts that acidification and dissolved inorganic and organic phosphorus stress could synergistically cause an appreciable decrease in global Trichodesmium nitrogen fixation by 2100.

Comparative analysis of the endophytic bacteria inhabiting the phyllosphere of aquatic fern Azolla species by high-throughput sequencing

BackgroundAzolla is a small floating fern living in symbiosis with nitrogen-fixing cyanobacteria and provides a variety of important ecosystem benefits. Previous studies have presented that Azolla harbors diverse bacteria that may play a key role in host fitness and productivity. However, the characteristics of endophytic bacteria inhabiting the phyllosphere of different species of Azolla have not yet been fully understood.ResultsIn this study, the 16S ribosomal DNA (rDNA) V5-V7 region of bacteria was determined by Illumina high-throughput sequencing platform to study the diversity and richness of endophytic bacterial communities in the phyllosphere of five Azolla species collected from different countries. A total of 1150 operational taxonomic units (OTUs) were detected for the endophytic bacteria community. According to the α diversity indices, the diversity of bacteria was ordered as Azolla imbricata > A. pinnata > A. filiculoides > A. mexicana > A. caroliniana. The PCoA results displayed that the bacterial communities of A. mexicana and A. caroliniana shared the highest similarity, followed by the similarity between A. pinnata and A. imbricata, and they were significantly distinct from the community of A. filiculoides. The dominant bacteria of Azolla mainly belonged to the phylum of Proteobacteria, followed by Actinobacteria, Chlorobillobacteria, and Firmicutes. In detail, the relative abundance of Proteobacteria in A. imbricata was 52.23%, whereas it was more than 80.00% in the other four species of Azolla. Notably, Herbaspirillum (45.91%, 44.08%) and Methylophilus (29.97%, 37.96%) were the main genera inhabiting A. mexicana and A. caroliniana respectively. Ferrovibrio (18.54%) and Rhizobium (16.68%) were the dominant genera inhabiting A. filiculoides. The group of unidentified genera (41.63%, 44.92%) consisted most of the bacteria in A. imbricata and A. pinnata respectively. Further analysis suggested that the significant different bacteria identified in LDA Effect Size analysis existed Azolla species-specific patterns.ConclusionsIn summary, all results suggested that the diversity and composition of the endophytic bacterial communities were different in Azolla species.

Identification and toxicity towards aquatic primary producers of the smallest fractions released from hydrolytic degradation of polycaprolactone microplastics.

Bioplastics are thought as a safe substitute of non-biodegradable polymers. However, once released in the environment, biodegradation may be very slow, and they also suffer abiotic fragmentation processes, which may give rise to different fractions of polymer sizes. We present novel data on abiotic hydrolytic degradation of polycaprolactone (PCL), tracking the presence of by-products during 132 days by combining different physicochemical techniques. During the study a considerable amount of two small size plastic fractions were found (up to∼6mg of PCL by-product/g of PCL beads after 132 days of degradation); and classified as submicron-plastics (sMPs) from 1μm to 100nm and nanoplastics (NPs, <100nm) as well as oligomers. The potential toxicity of the smallest fractions, PCL by-products < 100nm (PCL-NPs+PCL oligomers) and the PCL oligomers single fraction, was tested on two ecologically relevant aquatic primary producers: the heterocystous filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, and the unicellular cyanobacterium Synechococcus sp. PCC 7942. Upon exposure to both, single and combined fractions, Reactive Oxygen Species (ROS) overproduction, intracellular pH and metabolic activity alterations were observed in both organisms, whilst membrane potential and morphological damages were only observed upon PCL-NPs+PCL oligomers exposure. Notably both PCL by-products fractions inhibited nitrogen fixation in Anabaena, which may be clearly detrimental for the aquatic trophic chain. As conclusion, fragmentation of bioplastics may render a continuous production of secondary nanoplastics as well as oligomers that might be toxic to the surrounding biota; both PCL-NPs and PCL oligomers, but largely the nanoparticulate fraction, were harmful for the two aquatic primary producers. Efforts should be made to thoroughly understand the fragmentation of bioplastics and the toxicity of the smallest fractions resulting from that degradation.

The occurrence of the cis/trans geometric isomerism of myxoxanthophyll and 4-ketomyxoxanthophyll in the cyanobacterium Anabaena sp. PCC7120.

The cyanobacteria of the genus Anabaena have recently been recognized as a potential source of secondary metabolites of pharmacological and biotechnological importance. In particular, myxoxanthophylls - specific carotenoid glycosides that accumulate in cyanobacterial cells, are attracting increasing interest. Anabaena (Nostoc) sp. PCC7120, a filamentous, mesophilic, nitrogen-fixing cyanobacterium, is a model organism used in biochemical and genetic studies. The carotenoid pool of Anabaena sp. PCC7120 consists of five main species of pigments, namely β-carotene, echinenone, canthaxanthin and two derivatives of myxoxanthophyll: myxoxanthophyll ((3R,2'S)-myxol 2'-fucoside) and 4-ketomyxoxanthophyll ((3S,2'S)-4-ketomyxol 2'-fucoside). Recent findings show that the carotenoid biosynthesis pathway functions in Anabaena sp. PCC7120 cells are affected by environmental factors. Specifically, the balance between β-carotene and ketocarotenoids alters according to the temperature conditions. In this study, a new method, based on single-step liquid adsorption chromatography was developed and applied to separate a fraction containing myxoxanthophyll and 4-ketomyxoxanthophyll from Anabaena sp. PCC7120 cells. It was found that this method allowed a high purity fraction of carotenoid glycosides to be obtained from pigment pools as extracted from cyanobacterial cells. The subsequent analysis using the methods HPLC and LC/MS demonstrated that this fraction consists of a mixture of compounds with different retention times. On the basis of their fragmentation spectra and optical properties, these compounds were identified as geometrical isomers of myxoxanthophyll and 4-ketomyxoxanthophyll, including the dominant all-trans forms and less abundant cis forms. Proposals regarding the structures of myxoxanthophyll isomers are made.

Molecular mechanisms underlying iron and phosphorus co-limitation responses in the nitrogen-fixing cyanobacterium Crocosphaera.

In the nitrogen-limited subtropical gyres, diazotrophic cyanobacteria, including Crocosphaera, provide an essential ecosystem service by converting dinitrogen (N2) gas into ammonia to support primary production in these oligotrophic regimes. Natural gradients of phosphorus (P) and iron (Fe) availability in the low-latitude oceans constrain the biogeography and activity of diazotrophs with important implications for marine biogeochemical cycling. Much remains unknown regarding Crocosphaera's physiological and molecular responses to multiple nutrient limitations. We cultured C. watsonii under Fe, P, and Fe/P (co)-limiting scenarios to link cellular physiology with diel gene expression and observed unique physiological and transcriptional profiles for each treatment. Counterintuitively, reduced growth and N2 fixation resource use efficiencies (RUEs) for Fe or P under P limitation were alleviated under Fe/P co-limitation. Differential gene expression analyses show that Fe/P co-limited cells employ the same responses as single-nutrient limited cells that reduce cellular nutrient requirements and increase responsiveness to environmental change including smaller cell size, protein turnover (Fe-limited), and upregulation of environmental sense-and-respond systems (P-limited). Combined, these mechanisms enhance growth and RUEs in Fe/P co-limited cells. These findings are important to our understanding of nutrient controls on N2 fixation and the implications for primary productivity and microbial dynamics in a changing ocean.

Yield and Yield Attributes of Chickpea as Influenced by Phosphorus and Bio-fertilizer Application under Rainfed Conditions

Background: Biofertilizers contain an effective tool in rural environments as a beneficial, sustainable and ecofriendly source of plant supplements. These are arranged on premise of the particular supplement accessibility concern. A few microorganisms are normally utilized as biofertilizers including nitrogen-fixing soil microbes (Azotobacter, Rhizobium), nitrogen-fixing cyanobacteria (Anabaena), phosphate-solubilizing microscopic organisms (Pseudomonas sp.) and AM fungi. Biofertilizers are minimal expense, effective, eco-friendly and sustainable source of plant supplements to enhance fertilizer. Methods: A field experiment was conducted on rainfed chickpea at crop research center of career point University, Kota during 2016-17 and 2017-18 with four levels of phosphorus and four bio-fertilizer levels. Result: The results revealed that crop fertilized with 40 kg P2O5/ha and combined application of Rhizobium + PSB proved significantly better than other treatment regarding yield attributes and yield (Grain and straw). The economics in the form of net return and B: C ratio was also computed which was confirmed the results, recorded as yield attributes and yield.

The influence of trophic status and seasonal environmental variability on morpho-functional traits in tropical man-made shallow lakes

In the tropics, seasons are delimitated by the extent of rainfall resulting in seasonal differences in water parameters shaping phytoplankton community dynamics. Dry periods can intensify eutrophication and often result in seasonal or even perennial cyanobacterial dominance. This study was developed to evaluate phytoplankton response to trophic state and seasonal differences of environmental filters (dry and rainy periods) using the morphology-based functional groups (MBFG) approach. We also aimed at identifying environmental thresholds of each MBFG dominance in six man-made lakes located in the tropical semiarid region of Brazil. Our results showed clear MBFG association with lakes’ trophic states. The dominant groups in mesotrophic conditions were members of MBFGs V (unicellular flagellates) and VI (non-flagellated with a siliceous exoskeleton), and in meso-eutrophic MBFG IV (medium size without specialized traits) dominated. Conversely, MBFG VII (with mucilage and aerotopes) and VIII (nitrogen-fixing cyanobacteria) dominated mostly under eutrophic conditions, though linked to shallower euphotic zones. Light and phosphorous were the most important environmental thresholds associated with MBFG’s dominance. Overall, most of the lakes displayed seasonal differences in environmental filters. In contrast to what was expected, the rainy season was associated with higher nutrients, suspended solids, and reduced euphotic depth compared to the dry season. Our results, overall, show that the effects of seasonality varied across lakes and highlight eutrophication as the main environmental factor for MBFG selection suggesting reduced seasonality effects during dry years in the tropics.

Arginine inhibition of the argininosuccinate lyases is conserved among three orders in cyanobacteria.

This study revealed different catalytic efficiencies of cyanobacterial argininosuccinate lyases in non-nitrogen-fixing and nitrogen-fixing cyanobacteria, demonstrating that L-arginine inhibition of L-argininosuccinate lyase is conserved among enzymes of three cyanobacterial orders. Arginine is a nitrogen-rich amino acid that uses a nitrogen reservoir, and its biosynthesis is strictly controlled by feedback inhibition. Argininosuccinate lyase (EC 4.3.2.1) is the final enzyme in arginine biosynthesis that catalyzes the conversion of argininosuccinate to L-arginine and fumarate. Cyanobacteria synthesize intracellular cyanophycin, which is a nitrogen reservoir composed of aspartate and arginine. Arginine is an important source of nitrogen for cyanobacteria. We expressed and purified argininosuccinate lyases, ArgHs, from Synechocystis sp. PCC 6803, Nostoc sp. PCC 7120, and Arthrospira platensis NIES-39. The catalytic efficiency of the Nostoc sp. PCC 7120 ArgH was 2.8-fold higher than those of Synechocystis sp. PCC 6803 and Arthrospira platensis NIES-39. All three ArgHs were inhibited in the presence of arginine, and their inhibitory effects were lowered at pH 7.0, compared to those at pH 8.0. These results indicate that arginine inhibition of ArgH is widely conserved among the three cyanobacterial orders. The current results demonstrate the conserved regulation of enzymes in the cyanobacterial aspartase/fumarase superfamily.

Fate of nitrogen fixed by nitrogen-fixing cyanobacteria in rice and soil during the vegetative growth period of rice

Fate of nitrogen fixed by nitrogen-fixing cyanobacteria in rice and soil during the vegetative growth period of rice

Cascading effects of predators on algal size structure

The presence of edible and inedible prey species in a food web can influence the strength that nutrients (bottom-up) or herbivores (top-down) have on primary production. In boreal peatlands, wetter more nutrient-rich conditions associated with ongoing climate change are expanding consumer access to aquatic habitat and promoting sources of primary production (i.e., algae) that are susceptible to trophic regulation. Here, we used an in situ mesocosm experiment to evaluate the consequences of enhanced nutrient availability and food-web manipulation (herbivore and predator exclusion) on algal assemblage structure in an Alaskan fen. Owing to the potential for herbivores to selectively consume edible algae (small cells) in favor of more resistant forms, we predicted that the proportion of less-edible algae (large cells) would determine the strength of top-down or bottom-up effects. Consistent with these expectations, we observed an increase in algal-cell size in the presence of herbivores (2-tiered food web) that was absent in the presence of a trophic cascade (3-tiered food web), suggesting that predators indirectly prevented morphological changes in the algal assemblage by limiting herbivory. Increases in algal-cell size with herbivory were driven by a greater proportion of filamentous green algae and nitrogen-fixing cyanobacteria, whose size and morphological characteristics mechanically minimize consumption. While consumer-driven shifts in algal assemblage structure were significant, they did not prevent top-down regulation of biofilm development by herbivores. Our findings show that increasing wet periods in northern peatlands will provide new avenues for trophic regulation of algal production, including directly through consumption and indirectly via a trophic cascade.

AzuR From the SmtB/ArsR Family of Transcriptional Repressors Regulates Metallothionein in Anabaena sp. Strain PCC 7120.

Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.

Co-cultivation Approach to Decipher the Influence of Nitrogen-Fixing Cyanobacterium on Growth and N Uptake in Rice Crop.

The present study was performed to evaluate the efficacy of selected potential nitrogen-fixing cyanobacterial strain (Anabaenasp.), isolated from rhizospheric soil of rice plants on growth, pigments, N uptake, root architecture, and image-based phenotypic traits of rice crop using co-cultivation approach under controlled sand culture conditions. We studied the beneficial interaction of cyanobacterium to rice using sensor image-based Phenomics approach as well as conventional methods. Co-cultivation experiment revealed that inoculation with Anabaena sp. significantly improved plant growth, chlorophyll, leaf area, % nitrogen, and protein of rice by ~ 70%, ~ 22%, ~ 60%, and ~ 25% under 100% nitrogen input in comparison with un-inoculated control. Further, comparative evaluation revealed superior performance of Anabaena sp. at 100% and 75% N followed by 50% N input improving below-ground parameters as well as phenotypic traits as compared to control treatment. Hence, inoculation performed better with inorganic nitrogen input for overall growth of rice crop. Therefore, cyanobacterial strain can be used as an efficient bio-inoculant for sustainable rice production under integrated nutrient management.

Nitrogen fixing blue-green algae as pioneer plants on Surtsey 1968-1973

Nitrogen fixing blue-green algae as pioneer plants on Surtsey 1968-1973