Diazotrophic Cyanobacterium Anabaena Research Articles

The LysR-type transcriptional factor PacR controls heterocyst differentiation and C/N metabolism in the cyanobacterium Anabaena PCC 7120

PacR (All3953) has previously been identified as a global transcriptional regulator of carbon assimilation in cyanobacteria. In the facultative diazotrophic and filamentous cyanobacterium Anabaena PCC 7120 (Anabaena), inactivation of pacR has been shown to affect cell growth under various conditions. Nitrogen fixation in Anabaena occurs in heterocysts, cells differentiated semiregularly along the filaments following deprivation of combined nitrogen such as nitrate or ammonium. Here, we created a markerless deletion mutant of pacR. In addition to its growth defects observed under different light and nitrogen conditions, the mutant could form a high frequency of heterocysts, including heterocyst doublets, even in the presence of nitrate. Inactivation of pacR led to the upregulation of ntcA, a global regulator of nitrogen metabolism and heterocyst formation, as well as downregulation of genes involved in nitrate uptake and assimilation. These changes led to N-limited cells in the presence of nitrate. PacR also regulates most of the genes encoding bicarbonate transport systems. The promoter regions of ntcA, and several other genes involved in nitrogen or carbon uptake and assimilation, as well as patS and hetN involved in heterocyst patterning can be directly recognized by PacR in vitro. These findings, along with previously reported ChIP-seq data, establish PacR as a crucial transcriptional regulator for balancing carbon and nitrogen metabolism in cyanobacteria.

Pretilachlor-induced physiological, biochemical and morphological changes in Indian paddy field agroecosystem inhabited Anabaena doliolum

Pretilachlor is a systemic, pre-emergence herbicide applied in the paddy fields to kill narrow and broadleaf weeds. The present study evaluates the toxicity of pretilachlor on the non-target diazotrophic free-living cyanobacterium Anabaena doliolum, commonly found in the paddy fields of eastern Uttar Pradesh (India) and used as a biofertilizer. A.doliolum was subjected to several doses (0, 2, 5, 7, 10, 20 and 40 μg/ml) of pretilachlor and its effects were examined in terms of alterations in cellular morphology, ultrastructure, physiology, and biochemical attributes. The treatment of pretilachlor decreased the growth, total pigment content and photosynthetic efficiency of the test organism in a dose-dependent manner. The decline in growth was observed on 20th day at 2, 5, 7, 10, 20 and 40 μg/ml of pretilachlor concentration by 4, 9, 26, 47, 71 and 92%, respectively. Furthermore, Chlorophyll a and phycocyanin levels were noticeably declined. As a result, the photosynthetic performance also registered a similar decline as measured by chlorophyll fluorescence. However, carotenoid content increased by 13%, 41% and 53% at 5, 10 and 20 μg/ml on 5th day reflecting its protective property. A marked increase in fluorescence intensity and malondialdehyde content by 2.65 and 2.45 folds at 10 and 20 μg/ml on 7th day was registered. The enzymatic antioxidants (SOD and CAT) and a concurrent increase in glutathione reductase activity were registered (1.75 and 2.11-fold at 20 and 40 μg/ml on 5th day), indicating pretilachlor mediated ROS generation. Moreover, ultrastructural studies done by SEM and TEM revealed plasma membrane and thylakoid membrane damage and fragmentation. These findings have contributed to the broader comprehension of the stress responses triggered by pretilachlor in cyanobacteria. Moreover, they can aid in the evaluation of the detrimental impact of pretilachlor on A. doliolum, given their crucial function as a nitrogen contributor in paddy fields.

A tRNA t6A modification system contributes to the sensitivity towards the toxin β-N-methylamino-L-alanine (BMAA) in the cyanobacterium Anabaena sp. PCC 7120.

Cyanobacteria are oxygen-evolving photosynthetic autotrophs essential for nutrient cycling in the environment. They possess multiple control mechanisms for their cellular activities in order to adapt to the environment. While protein translation is essential for cell survival and adaptation, the regulation and the flexibility of this process are poorly understood in cyanobacteria. β-N-methylamino-L-alanine (BMAA), an amino acid analog proposed as an environmental neurotoxin, is highly toxic to the filamentous diazotrophic cyanobacterium Anabaena PCC 7120. In this study, through genetic analysis of BMAA-resistant mutants, we demonstrate that the system responsible for modification of ANN-decoding tRNAs with N(6)-threonylcarbamoyl adenosine (t6A) is involved in BMAA sensitivity through the control of translation. Both BMAA and inactivation of the t6A biosynthesis pathway affect translational fidelity and ribosome assembly. However, the two factors display either additive effects on translational elongation, or attenuate each other over translational fidelity or the resistance/sensitivity to antibiotics that inhibit different steps of the translational process. BMAA has a broad effect on translation and transcription, and once BMAA enters the cells, the presence of the t6A biosynthesis pathway increases the sensitivity of the cells towards this toxin. BMAA-resistant mutants screening is an effective method for getting insight into the toxic mechanisms of BMAA. In addition, BMAA is a useful tool for probing translational flexibility of cyanobacteria, and the characterization of the corresponding resistant mutants should help us to reveal translational mechanism allowing cyanobacteria to adapt to changing environments.

Physicochemical behavioral changes in consort with nitrogen metabolism of cyanobacterium Anabaena PCC 7120 under arsenite regimes.

The present study was undertaken to investigate the arsenite (As III)-induced changes in the diazotrophic cyanobacterium Anabaena PCC 7120. It was observed that the growth of cyanobacterial decreased with increase in As (III) concentration. The cells exposed to As (III) showed morphological variation (deformity) due to the formation of deeper constrictions in vegetative cells. Strain showed increased heterocyst differentiation (1.6-fold higher) whereas decreased nitrogenase activity at the concentration of 40ppm As (III). The activities of NR, NiR, urease and GS decreased with increase in As (III) concentrations and attained their minimum levels at 40ppm of As (III). The Ca2+-dependent ATPase activity increased with increase in As (III) concentration and attained its about 2.72-fold higher level at 40ppm of As (III). In contrast, sharp decline in Mg2+-dependent ATPase activity (28%) was recorded at 1ppm of As (III) over untreated control. The rates of photosynthetic O2 evolution and respiration decreased with increase in As (III) concentration and attained its minimal level at 40ppm of As (III). Therefore, this study highlighted arsenite regimes efficiently correlated with behavioral changes in consort with strain.

Reconstruction of a regulated two-cell metabolic model to study biohydrogen production in a diazotrophic cyanobacterium Anabaena variabilis ATCC 29413.

Anabaena variabilis is a diazotrophic filamentous cyanobacterium that differentiates to heterocysts and produces hydrogen as a byproduct. Study on metabolic interactions of the two differentiated cells provides a better understanding of its metabolism especially for improving hydrogen production. To this end, a genome-scale metabolic model for Anabaena variabilis ATCC 29413, iAM957, was reconstructed and evaluated in this research. Then, the model and transcriptomic data of the vegetative and heterocyst cells were applied to construct a regulated two-cell metabolic model. The regulated model improved prediction for biomass in high radiation levels. The regulated model predicts that heterocysts provide an oxygen-free environment and then, this model was used to find strategies for improving hydrogen production in heterocysts. The predictions indicate that the removal of uptake hydrogenase improves hydrogen production which is consistent with previous empirical research. Furthermore, the regulated model proposed activation of some reactions to provide redox cofactors which are required for improving hydrogen production up to 60% by bidirectional hydrogenase.

Continuous Cultivation as a Method to Assess the Maximum Specific Growth Rate of Photosynthetic Organisms.

Modeling the growth of photosynthetic organisms is challenging, due to the complex role of light, which can be limiting because of self-shading, or photoinhibiting in the case of high intensities. A case of particular interest is represented by nitrogen-fixing cyanobacteria, whose growth is controlled not only by the light intensity, but also by the availability of atmospheric nitrogen in the liquid medium. The determination of the maximum specific growth rate is often affected by many variables that, in batch growth systems, may change significantly. On the other hand, in a continuous system, once the steady state is reached the values of all the process variables remain constant, including the biomass concentration and the specific light supply rate. In this work, the diazotrophic cyanobacterium Anabaena PCC 7122 was cultivated in continuous photobioreactors, to investigate the role of nitrogen, light and residence time on growth kinetics, and to retrieve the value of the maximum specific growth rate of this organism. In addition, the kinetic parameters for temperature and the half saturation constant for nitrogen (3 mg L−1) were measured by respirometric tests. Based on the results of continuous experiments, the specific maintenance rate was found to depend on the light intensity supplied to the reactor, ranging between 0.5 and 0.8 d−1. All these parameters were used to develop a kinetic model able to describe the biomass growth in autotrophic conditions. The maximum specific growth rate could hence be determined by applying the kinetic model in the material balances of the continuous photobioreactor, and resulted equal to 8.22 ± 0.69 d−1.

Isolation and characterization of high temperature tolerant mutant from the cyanobacterium Anabaena doliolum.

The mutant strain of the diazotrophic cyanobacterium Anabaena doliolum able to tolerate high temperature was isolated by induced mutation techniques using ethyl methane sulphonate. This mutant strain exhibited higher temperature tolerance than the wild type. The wild type was able tolerate temperature up to 40 °C whereas the mutant was able to grow at an elevated temperature of 48 °C. This mutant exhibited higher growth rate, heterocyst frequency, and nitrogen fixation. Mutant strains exhibited comparable levels of chlorophyll, phycocyanin, PS II activity, and O2 evolution as compared to unexposed control. Results also showed that the mutant accumulated low levels of peroxides and lipid peroxidation products with enhanced activity of antioxidant enzymes. The FAME analysis revealed quantitative and qualitative changes in the profile of fatty acids in the mutant strain. Maximum number of saturated fatty acids was observed in the mutant strain followed by control whereas the wild type exposed to elevated temperature showed least diversity of fatty acids. Enhanced level of antioxidant enzymes coupled with efficient modulation of fatty acid profile could therefore enhance the mutant to resist the high temperature stress. The results could be exploited further to decipher molecular mechanisms underlying the temperature tolerance and enhancing the utility of A. doliolum as efficient biofertilizer for rice paddy keeping in view of the future climatic change scenario.

Anti-Oxidative Response of Cyanobacterium Anabaena sp. strain PCC 7120 to Arsenite (As(III))

Arsenite (As(III)) induced changes were investigated in the diazotrophic cyanobacterium Anabaena PCC 7120. The cells of Anabaena sp. strain PCC 7120 exposed to As(III) produced H2O2 and its production increased with increase in As(III) concentration. As a part of resistant mechanism and also to counteract the deleterious effect of H2O2, the cells of Anabaena sp. strain PCC 7120 produced more ascorbate peroxidase (APx) whose level also increased in response to As(III) concentrations. The increase in APx activity was directly proportional to the increase in H2O2 production and maximum APx activity was recorded at 40 ppm of As(III). In contrast, glutathione reductase (GR) activity decreases with increase in As(III) concentrations and attained its minimum level at 40 ppm of As(III). Lipid peroxidation increased with increase in As(III) concentration and maximum peroxidation (which was about two fold higher than that of the untreated control cells) was recorded at 40 ppm of As(III). Exposure of the Anabaena sp. strain PCC 7120cells to As(III) has also resulted in a significant increase in ascorbate and dehydro-ascorbate contents which was about 6.44 and 1.97 fold higher than that of the untreated control cells, respectively at 40 ppm of As(III).

Decoding the role of hypothetical protein All3255 of Anabaena PCC7120 in heavy metal stress management in Escherichia coli.

Cadmium is a non-essential toxic heavy metal for organisms, including plants and cyanobacteria. Cadmium resistance transporters involved in resistance of cells against various toxicants such as drugs and effluxes cytotoxic compounds from cells. However, cadmium resistance-associated protein (CadD) has never been reported from a diazotrophic cyanobacterium Anabaena sp. To test whether the hypothetical protein All3255 of Anabaena sp. PCC7120 a homolog of cadmium resistance-associated protein (CadD) involved in cadmium or heavy metal resistance or not, cloning and heterologous expression analysis of all3255 performed in Escherichia coli BL21 (DE3). Our results revealed that the strain transformed with pGEX-5X-2 + all3255 showed resistant towards not only to cadmium but also other heavy metals such as nickel, copper, zinc, lead and cobalt in addition to arsenic than those of transformed with empty vector (pGEX-5X-2). Furthermore, the results of metal accumulation analysis of these cells unveil a lower accumulation of tested heavy metals in all3255-overexpressing E. coli cells than those transformed with empty vector. This study strongly supports the role of All3255 of Anabaena sp. PCC7120 as a CadD efflux pump of heavy metals in E.coli.

Unusual Versatility of the Filamentous, Diazotrophic Cyanobacterium Anabaena torulosa Revealed for Its Survival during Prolonged Uranium Exposure.

Reports on interactions between cyanobacteria and uranyl carbonate are rare. Here, we present an interesting succession of the metabolic responses employed by a marine, filamentous, diazotrophic cyanobacterium, Anabaena torulosa for its survival following prolonged exposure to uranyl carbonate extending up to 384 h at pH 7.8 under phosphate-limited conditions. The cells sequestered uranium (U) within polyphosphates on initial exposure to 100 μM uranyl carbonate for 24 to 28 h. Further incubation until 120 h resulted in (i) significant degradation of cellular polyphosphates causing extensive chlorosis and cell lysis, (ii) akinete differentiation followed by (iii) extracellular uranyl precipitation. X-ray diffraction (XRD) analysis, fluorescence spectroscopy, X-ray absorption near edge structure (XANES), and extended X-ray absorption fine structure (EXAFS) spectroscopy established the identity of the bioprecipitated uranium as a U(VI) autunite-type mineral, which settled at the bottom of the vessel. Surprisingly, A. torulosa cells resurfaced as small green flakes typical of actively growing colonies on top of the test solutions within 192 to 240 h of U exposure. A consolidated investigation using kinetics, microscopy, and physiological and biochemical analyses suggested a role of inducible alkaline phosphatase activity of cell aggregates/akinetes in facilitating the germination of akinetes leading to substantial regeneration of A. torulosa by 384 h of uranyl incubation. The biomineralized uranium appeared to be stable following cell regeneration. Altogether, our results reveal novel insights into the survival mechanism adopted by A. torulosa to resist sustained uranium toxicity under phosphate-limited oxic conditions.IMPORTANCE Long-term effects of uranyl exposure in cyanobacteria under oxic phosphate-limited conditions have been inadequately explored. We conducted a comprehensive examination of the metabolic responses displayed by a marine cyanobacterium, Anabaena torulosa, to cope with prolonged exposure to uranyl carbonate at pH 7.8 under phosphate limitation. Our results highlight distinct adaptive mechanisms harbored by this cyanobacterium that enabled its natural regeneration following extensive cell lysis and uranium biomineralization under sustained uranium exposure. Such complex interactions between environmental microbes such as Anabaena torulosa and uranium over a broader time range advance our understanding on the impact of microbial processes on uranium biogeochemistry.

In silico characterization and transcriptomic analysis of nif family genes from Anabaena sp. PCC7120.

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.

Exploring the membrane proteome of the diazotropic cyanobacterium Anabaena PCC7120 through gel-based proteomics and in silico approaches

This paper focuses on the gel-based membrane proteomics from diazotrophic cyanobacterium Anabaena PCC7120 by modifying the protocol of Hall et al. [1]. The bioinformatic analysis revealed that 59 (29 integral, 30 peripheral) of the 67 proteins identified were membrane proteins. Of the 29 integral proteins, except Alr0834, the remaining 28 contained 1–12 transmembrane helices. Sixteen integral proteins harboring signal peptides (Sec/TAT/LipoP) suggest that protein targeting in Anabaena involves both sec-dependent and sec-independent pathways. While majority of photosynthesis and respiration proteins (21 of 24) were confined to broad pH gradient the hypothetical and unknown (12 of 13), and cell envelope proteins (3 of 3) preferred the narrow pH range. Of the 5 transporters and binding proteins, Na+/H+-exchanging protein and Alr2372 were present in broad, pstS1 and cmpD in narrow and cmpA was common to both pH ranges. The distribution of proteins across pH gradient, thus clearly indicates the functional and structural diversity in membrane proteome of Anabaena. It requires mention that protochlorophyllide oxido-reductase, Na+/H+-exchanging protein, All1355, Alr2055, Alr3514, Alr2903 and Alr2751 were new entries to the 2DE membrane protein profile of Anabaena. This study demonstrates suitability of the modified protocol for the study of membrane protein from filamentous cyanobacteria. SignificanceAnabaena sp. PCC7120 is used as a model organism due to its agriculture significance as biofertilizer, close resemblance with higher plant chloroplast and availability of full genome sequence. Although cytosolic proteome has been explored a lot membrane proteins are still understudied as they are notoriously difficult to display using 2-D technology. Identification and characterization of these proteins is therefore required to elucidate and understand cellular mechanisms. The purpose of this study was to develop a protocol suitable for membrane protein extraction from Anabaena. Additionally, by homology comparison or domain assignment a possible function could be ascribed to novel uncharacterized proteins which will serve as a useful reference for further detailed studies of membrane system in filamentous cyanobacteria. Resolution of membrane proteins ranging from least (single transmembrane helix) to highly hydrophobic (several transmembrane helices) one on 2D gels recommends the gel based approach for identification of membrane proteomics from filamentous cyanobacteria.This article is part of a Special Issue entitled: Proteomics in India.

UV-B stress induced metabolic rearrangements explored with comparative proteomics in three Anabaena species

Comparative proteomics together with physiological variables revealed different responses among three species of diazotrophic cyanobacterium Anabaena exposed to UV-B stress at the same time points. Perceptible decline in PSII activity, ATP pool, nitrogenase activity and respiration rate was observed for all the three species; this being maximum in Anabaena doliolum, followed by Anabaena sp. PCC 7120 and minimum in Anabaena L31. Statistical analysis of the protein abundance divided majority of them as early accumulated in A. L31, late accumulated in A. sp. PCC 7120 and downregulated in A. doliolum. Tolerance of A. L31 may be ascribed to post-translational modification reflected through the highest number of protein isoforms in its proteome followed by A. PCC 7120 and A. doliolum. Furthermore, increase in abundance of cyanophycinase, glutamine synthetase and succinate semialdehyde dehydrogenase in A. L31 suggests operation of an alternate pathway for assimilation of nitrogen and carbon under UV-B stress. An early accumulation of four proteins viz., glutamate ammonia ligase (Alr2328), transketolase (Alr3344), inorganic pyrophosphatase (All3570), and trigger protein (Alr3681) involved respectively in amino acid metabolism, energy metabolism, biosynthesis of cofactor and trigger protein and chaperone like activity across three species, suggests them to be marker of UV-B stress in Anabaena spp.This article is part of a Special Issue entitled: Proteomics in India.

Involvement of thioredoxin on the scaffold activity of NifU in heterocyst cells of the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120.

The diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 (A.7120) differentiates into specialized heterocyst cells that fix nitrogen under nitrogen starvation conditions. Although reducing equivalents are essential for nitrogen fixation, little is known about redox systems in heterocyst cells. In this study, we investigated thioredoxin (Trx) networks in Anabaena using TrxM, and identified 16 and 38 candidate target proteins in heterocysts and vegetative cells, respectively, by Trx affinity chromatography (Motohashi et al. (Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA, 2001; 98: , 11224-11229)). Among these, the Fe-S cluster scaffold protein NifU that facilitates functional expression of nitrogenase in heterocysts was found to be a potential TrxM target. Subsequently, we observed that the scaffold activity of N-terminal catalytic domain of NifU is enhanced in the presence of Trx-system, suggesting that TrxM is involved in the Fe-S cluster biogenesis.

Arsenic and cadmium are inhibitors of cyanobacterial dinitrogenase reductase (nifH1) gene.

The enzyme nitrogenase complex is a key component conferring nitrogen fixation in all known diazotrophs. This study for the first time examines the impact of As, Na, Cd, Cu and butachlor on component II (dinitrogenase reductase, nifH1) of nitrogenase from diazotrophic cyanobacterium Anabaena sp. PCC7120 using in silico and wet lab approaches. The nifH1 of Anabaena is a glycine-rich stable protein having DNA-binding properties and shows close similarity with free living compared with symbiotic diazotrophs. Phylogenetic tree revealed an adverse effect of the selected stresses on close homologs across the diazotroph community. The protein interaction network demonstrated the presence of nirA, glnA, glnB, alr4255 and alr2485 proteins besides nif proteins, suggesting their involvement in nitrogen fixation along with nifH1. Homology modelling and docking under As, Na, Cd, Cu and butachlor revealed an interaction between stressors and nifH1 protein which was further validated by a transcript of the gene through quantitative real-time PCR (qRT-PCR). Presence of binding sites for As, Na, Cd and Cu on oxyR promoter attested their adverse affects on nifH1. Maximum down-regulation of nifH1 in Cd and As followed by salt, copper and butachlor revealed that arsenic and cadmium were most potential inhibitors of nitrogenase of diazotrophic community, which might negatively affect crop yield.

Regulation of iron-siderophore uptake in a diazotrophic cyanobacterium Anabaena anabaena 7120

The regulation of 55Fe-siderophore uptake of was studied in a diazotrophic cyanobacterium Anabaena 7120. The uptake up to 20 min was found to be biphasic, a rapid first and steady second phase. A single kinetic system mediated the uptake of iron-siderophore with an apparent Km of 200 µM and a Vmax of 20 nmol iron taken up mg-1 protein min-1. 3-(3,4dichlorophenyl)-1, 1-dimethyl urea (DCMU), 2-n-heptyl-4-hydroxy quinoline (HOQNO), dicyclohexyl carbodiimide (DCCD), carbonyl cyanide p-fluoromethoxy phenyl hydrazone (FCCP) and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) inhibited the iron-siderophore uptake significantly, whereas KCN inhibited marginally. No iron-siderophore uptake was observed under dark-aerobic and dark-anaerobic conditions. The siderophore schizokinen supported the maximum iron-siderophore uptake. However, the siderophore aerobactin and desferal could support only 53 and 3.4% iron-siderophore uptake, respectively as compared to its schizokinen counterpart.

Regulation of photosynthesis during heterocyst differentiation in Anabaena sp. strain PCC 7120 investigated in vivo at single-cell level by chlorophyll fluorescence kinetic microscopy

Changes of photosynthetic activity in vivo of individual heterocysts and vegetative cells in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 during the course of diazotrophic acclimation were determined using fluorescence kinetic microscopy (FKM). Distinct phases of stress and acclimation following nitrogen step-down were observed. The first was a period of perception, in which the cells used their internally stored nitrogen without detectable loss of PSII activity or pigments. In the second, the stress phase of nitrogen limitation, the cell differentiation occurred and an abrupt decline of fluorescence yield was observed. This decline in fluorescence was not paralleled by a corresponding decline in photosynthetic pigment content and PSII activity. Both maximal quantum yield and sustained electron flow were not altered in vegetative cells, only in the forming heterocysts. The third, acclimation phase started first in the differentiating heterocysts with a recovery of PSII photochemical yields [Formula: see text] Afterwards, the onset of nitrogenase activity was observed, followed by the restoration of antenna pigments in the vegetative cells, but not in the heterocysts. Surprisingly, mature heterocysts were found to have an intact PSII as judged by photochemical yields, but a strongly reduced PSII-associated antenna as judged by decreased F 0. The possible importance of the functional PSII in heterocysts is discussed. Also, the FKM approach allowed to follow in vivo and evaluate the heterogeneity in photosynthetic performance among individual vegetative cells as well as heterocysts in the course of diazotrophic acclimation. Some cells along the filament (so-called "superbright cells") were observed to display transiently increased fluorescence yield, which apparently proceeded by apoptosis.

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min⁻¹ mg protein⁻¹) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min⁻¹ mg protein⁻¹) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (∆arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.

The freshwater cyanobacterium Anabaena doliolum transformed with ApGSMT-DMT exhibited enhanced salt tolerance and protection to nitrogenase activity, but became halophilic

Glycine betaine (GB) is an important osmolyte synthesized in response to different abiotic stresses, including salinity. The two known pathways of GB synthesis involve: 1) two step oxidation of choline (choline → betaine aldehyde → GB), generally found in plants, microbes and animals; and 2) three step methylation of glycine (glycine → sarcosine → dimethylglycine → GB), mainly found in halophilic archaea, sulphur bacteria and the cyanobacterium Aphanothece (Ap.) halophytica. Here, we transformed a salt-sensitive freshwater diazotrophic filamentous cyanobacterium Anabaena (An.) doliolum with N-methyltransferase genes (ApGSMT-DMT) from Ap. halophytica using the triparental conjugation method. The transformed An. doliolum synthesized and accumulated GB in cells, and showed increased salt tolerance and protection to nitrogenase activity. The salt responsiveness of the transformant was also apparent as GB synthesis increased with increasing concentrations of NaCl in the nutrient solution, and maximal [12.92 µmol (g dry weight)(-1)] in cells growing at 0.5 M NaCl. Therefore, the transformed cyanobacterium has changed its behaviour from preferring freshwater to halophily. This study may have important biotechnological implications for the development of stress tolerant nitrogen-fixing cyanobacteria as biofertilizers for sustainable agriculture.

Facultative diazotrophy increases Cylindrospermopsis raciborskii competitiveness under fluctuating nitrogen availability

Relative fitness of three bloom-forming and potentially toxic cyanobacteria from the subtropical St. John's River, Florida was investigated under a range of nutrient conditions, during a bloom dominated by Cylindrospermopsis raciborskii. Nitrogen (N) was the primary nutrient limiting phytoplankton primary productivity and biomass. Phytoplankton biomass was also enhanced by phosphorus (P) added either alone or jointly with N, suggesting different components of the phytoplankton experienced distinct nutrient limitations. Based on quantitative PCR, the diazotrophic cyanobacteria Anabaena sp. and C.raciborskii were responsible for the primary production response to P additions, while the nondiazotrophic Microcystis aeruginosa appeared to benefit from N released from the diazotrophs. Cylindrospermopsis raciborskii maintained high net growth rates under diazotrophic and nondiazotrophic conditions, while Anabaena sp. growth was significantly reduced under DIN enrichment. C.raciborskii appears to be a generalist with regard to N source, a lifestyle traditionally not considered a viable ecological strategy among diazotrophs. Using facultative diazotrophy, C.raciborskii gains a growth advantage under fluctuating DIN conditions, such as systems that are under the influence of anthropogenic N loading events. The described niche differentiation may be a key factor explaining the recent global expansion of C.raciborskii.