Diazotrophic Cyanobacterium Nostoc Research Articles

Enhanced cyanophycin accumulation in diazotrophic cyanobacterium through random mutagenesis and tailored selection under varying phosphorus availability.

This study explored a sustainable alternative to the Haber-Bosch process by enhancing the production of the nitrogen-rich polymer cyanophycin (CGP) in the diazotrophic cyanobacterium Nostoc sp. PCC 7120. Applying UV-mutagenesis followed by canavanine selection, we isolate an initial mutant with enhanced CGP accumulation. Subsequently, a secondary selection under phosphorus-limited conditions was employed to decrease cellular ploidy, yielding stable mutants. Among these, strain 44 exhibited an improved CGP accumulation, achieving up to 34% of cellular dry weight in batch cultures. Under continuous phosphorus-limited cultivation, this mutant demonstrated a CGP productivity of 63mg L-1 day-1, approximately a fourfold improvement over the wild type. Genomic analysis of the mutants revealed mutations unrelated to known CGP biosynthetic pathways, suggesting that the observed enhancement in CGP may arise from complex, synergistic effects of multiple genetic changes. This integrative approach-combining mutagenesis, screening, and cultivation techniques-successfully increased CGP accumulation from atmospheric nitrogen over threefold compared to the wild-type.

Unravelling HetC as a peptidase-based ABC exporter driving functional cell differentiation in the cyanobacterium Nostoc PCC 7120.

The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.

Toxicity assessment of arsenate and arsenite on growth, chlorophyll a fluorescence and antioxidant machinery in Nostoc muscorum

The present study deals with impact of varied doses of arsenite (AsIII; 50, 100 and 150 µM) and arsenate (AsV; 50, 100 and 150 mM) on growth, photosynthetic pigments, photochemistry of photosystem II, oxidative biomarkers, (O2•¯, H2O2 and MDA equivalents contents) and activity of antioxidant enzymes in diazotrophic cyanobacterium Nostoc muscorum after 48 and 96 h of the treatments. The reduction in growth, pigment contents (Chl a, Phy and Car) and PS II photochemistry was found to increase with enhanced accumulation of test metal in cells, and the damaging effect on photosynthetic pigments showed the order (Phy > chl a> Car). The negative effect on PS II photochemistry was due to significant decrease in the value of JIP kinetics ϕP0, FV/F0, ϕE0,Ψ0 and PIABS except F0/FV and significant rise in values of energy flux parameters such as ABS/RC, TR0/RC, ET0/RC and DI0/RC. Both the species of arsenic caused significant rise in oxidative biomarkers as evident by in vitro and in vivo analysis of (O2•¯, H2O2 and MDA equivalents contents) despite of appreciable rise in the activity antioxidative enzymes such as SOD, POD, CAT and GST. The study concludes that in among both forms of arsenic, arsenite effect was more dominant on growth, photosynthetic pigments; oxidative stress biomarkers as evident by weak induction of anti-oxidative defense system to overcome the stress as compared to arsenate.

Cloning and Overexpression Studies on the Glutathione Peroxidase-Like Protein from the Cyanobacterium Nostoc punctiforme ATCC 29133

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes possessing a wide range of enzymatic antioxidants to tackle oxidative stress if and when it arises in cells. Glutathione peroxidase is an important enzyme in the antioxidative machinery of cells and may confer protection to various stress conditions. In the diazotrophic cyanobacterium Nostoc punctiforme ATCC 29133 (hereafter N.punctiforme), an open reading frame (ORF) encoding for a putative glutathione peroxidase was identified as NpunR_4660 by bioinformatics approach. This 486 bp ORF was amplified from the genomic DNA of N.punctiforme by polymerase chain reaction. The forward and reverse primers used for cloning the ORF contained NdeI and XhoI at their 5’ and 3’ end, respectively. The PCR amplified product was sub-cloned into pJET cloning vector. Further the ORF was introduced into pET-20B vector for overexpression of the corresponding protein in Escherichia coli BL21 DE3 strain. After induction of E.coli cultures by Isopropylthiogalactoside (IPTG) and, Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of cell-free extracts, an approximately 18 kDa protein was detected in the cytoplasm, which matched with the theoretical molecular weight of the protein. This protein was not present in control cultures transformed with pET-20B without the cyanobacterial ORF. These results suggest that foreign enzymes such as glutathione peroxidase can be produced in large amounts for biotechnological purposes successfully even in cells of a different origin like E.coli.

Diazotrophic specific cytochrome c oxidase required to overcome light stress in the cyanobacterium Nostoc muscorum.

Diazotrophic, filamentous and heterocystous cyanobacterium Nostoc muscorum perform photosynthesis in vegetative whereas nitrogen fixation occurs in heterocyst only. However, despite their metabolic plasticity, respiration takes place both in vegetative cells and heterocysts. The role of the respiratory electron transport system and terminal oxidases under light stress is not evident so far. As compared to the diazotrophically grown cultures, the non-diazotrophically grown cultures of the N. muscorum show a slight decrease in their growth, chlorophyll a contents and photosynthetic O2 evolution under light stress. Whereas respiratory O2 uptake under identical stress condition increases several fold. Likewise, nitrogen fixing enzyme i.e. nitrogenase over-expresses itself under light stress condition. The terminal enzyme of respiratory electron transport chain i.e. cytochrome c oxidase shows more activity under light stress, whilst light stress has no impact on Ca(++)-dependent ATPase activity. This leads to the conclusion that under light stress, cytochrome c oxidase plays a vital role in mitigating given light stress.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production by the diazotrophic cyanobacterium Nostoc muscorum Agardh: Process optimization and polymer characterization

Polyhydroxyalkanoate (PHA) homo- and copolymers are accumulated as cytoplasmic inclusions in a wide variety of bacteria. Cyanobacteria are emerging as a novel source for production of PHA polymers. In this report, response surface methodology (RSM) was used to evaluate the relationships between the critical variables that enhanced the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer accumulation in a N2-fixing cyanobacterium, Nostoc muscorum Agardh. Using multifactor optimization strategy, a yield of 69% of dry cell weight (dcw) (61 mgL−1day−1) was achieved at reduced level of nutrients for an incubation period of 7days. The polymer productivity was increased up to 98.3 mg L-1day-1 (71% dcw) and 109.7mgL−1day−1 (78% dcw), respectively under P- and N-deficiencies. Moreover, the poly-β-hydroxybutyrate (PHB) homopolymer and P(3HB-co-3HV) copolymer films produced by N. muscorum, were analyzed for their material (thermal and mechanical) properties, and were compared with the PHAs obtained from other cyanobacterial and bacterial sources. Structural details were investigated by wide angle X-ray diffraction (WAXRD), which showed semicrystalline nature for PHB as well as P(3HB-co-3HV) co-polymers. Thermal and mechanical properties of the polymer films produced by the test cyanobacterium are comparable with the polymers obtained from other cyanobacterial (Aulosira fertilissima CCC 444), bacterial (Cupriavidus necator) and the commercial polypropylene, and thus could be ensued for large-scale production.

The slow S to M fluorescence rise in cyanobacteria is due to a state 2 to state 1 transition

In dark-adapted plants and algae, chlorophyll a fluorescence induction peaks within 1s after irradiation due to well documented photochemical and non-photochemical processes. Here we show that the much slower fluorescence rise in cyanobacteria (the so-called “S to M rise” in tens of seconds) is due to state 2 to state 1 transition. This has been demonstrated in particular for Synechocystis PCC6803, using its RpaC− mutant (locked in state 1) and its wild-type cells kept in hyperosmotic suspension (locked in state 2). In both cases, the inhibition of state changes correlates with the disappearance of the S to M fluorescence rise, confirming its assignment to the state 2 to state 1 transition. The general physiological relevance of the SM rise is supported by its occurrence in several cyanobacterial strains: Synechococcus (PCC 7942, WH 5701) and diazotrophic single cell cyanobacterium (Cyanothece sp. ATCC 51142). We also show here that the SM fluorescence rise, and also the state transition changes are less prominent in filamentous diazotrophic cyanobacterium Nostoc sp. (PCC 7120) and absent in phycobilisome-less cyanobacterium Prochlorococcus marinus PCC 9511. Surprisingly, it is also absent in the phycobiliprotein rod containing Acaryochloris marina (MBIC 11017). All these results show that the S to M fluorescence rise reflects state 2 to state 1 transition in cyanobacteria with phycobilisomes formed by rods and core parts. We show that the pronounced SM fluorescence rise may reflect a protective mechanism for excess energy dissipation in those cyanobacteria (e.g. in Synechococcus PCC 7942) that are less efficient in other protective mechanisms, such as blue light induced non-photochemical quenching. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Response of diazotrophic cyanobacterium Nostoc carneum under pesticide and UV-B stress

A study was under taken, under controlled laboratory conditions, to investigate the influence of non-ionizing radiation (UV-B) and an organochlorine pesticide on the growth, photosynthetic pigments, protein content and DCPIP photoreduction of a cyanobacterium Nostoc carneum. Test algae was isolated from rice field soils of Sambalpur, Western Orissa, India and grown in nitrogen free BG 11 culture medium. Culture of algae from log phase of growth was treated with 5ppm of the insecticide, Endodhan and UV-B (20mWm−2) for 2h daily, separately and in combination of insecticide and UV-B radiation. Algal samples treated with UV-B and pesticide separately showed distinct inhibitory effects on growth, pigments, protein content and DCPIP reduction of the test samples. However, when pesticide treated samples were subjected to UV-B exposure, the effect showed additive as well as synergetic effect. Experiment conducted to check the ability of the organism to recover from the stress, exposed for various time periods, suggest their ability to partially recover from the stress.

Assessment of Salinity-Induced Antioxidative Defense System of Diazotrophic Cyanobacterium Nostoc muscorum

The present study examines the salinity-induced oxidative damage and differential response of enzymatic and non-enzymatic antioxidants of Nostoc muscorum. As compared to carotenoid content which showed induction the chlorophyll and phycocyanin contents were inhibited after salt stress. Acceleration of lipid peroxidation and peroxide production suggested onset of oxidative damage. The activities of all studied enzymatic antioxidants were significantly increased by salt stress with maximum induction of superoxide dismutase (154.8% at 200 mM NaCl treatment). Interestingly under severe stress condition (250 mM NaCl) ascorbate peroxidase seems to be more crucial than catalase for peroxide scavenging. Among the studied non-enzymatic antioxidants alpha-tocopherol was induced maximally (56.0%), however, ascorbate and reduced glutathione were increased by only 8.9% after 250 mM NaCl treatment as compared to control cells. Therefore, salinity was found to induce antioxidative defense system of N. muscorum.

Energy-dependent Ca(2+) efflux from the cells of nostoc calcicola Breb: role of modifying factors

Energy-dependent Ca(2+) efflux and its regulation from the diazotrophic cyanobacterium Nostoc calcicola Breb has been investigated. Like Ca(2+) uptake, Ca(2+) efflux pattern also reflected a rapid phase for the first 10 min followed by a slower one lasting up to 1 h with a total of 80 nmol Ca(2+) mg(-1) protein (31% of the Ca(2+) concentration taken in by such cells at 1 h). Ca(2+) efflux kinetics remained hyperbolic with a K(m) of 1.9 mM and V(max) 5.5 nmol mg(-1) protein min(-1). Ca(2+) efflux to a major extent depended on photosynthetic energy generation as the cells facing dark incubation and addition of 3-(3, 4-dichlorophenyl)-1-dimethyl urea (DCMU) to light-grown cells showed significant reduction in Ca(2+) extrusion. The strong inhibition in Ca(2+) efflux by addition of metabolic inhibitors like carbonyl cyanide-p-nitrofluoromethoxylphenyl hydrazone (FCCP) and N,N, -dicyclohexylcarbo-diimide (DCCD) suggested the vital role of membrane potential and ATP hydrolysis in driving this process. Verapamil (Ca(2+) antagonist) had insignificant effect on Ca(2+) efflux, whereas the addition of Calmodulin antagonists like trifluoroperazine, W-7 and compound 48/80 resulted in the enhancement in Ca(2+) efflux over control sets, thus suggesting that this increase may be owing to the additional extrusion of intracellular free calcium that was unable to bind with calmodulin in the presence of these antagonists.http://link.springer-ny. com/link/service/journals/00284/bibs/39n5p254.html</HEA

Partial purification and some properties of urease from the alkaliphilic cyanobacteriumNostoc calcicola

Urease extracted from an alkaliphilic diazotrophic cyanobacteriumNostoc calcicola was partially purified and some of its properties were studied. Urease purified 39-fold from the crude enzyme extract showed its optimum activity at pH 7.5 and at 40°C with aK m value of 120 μmol/L. The enzyme was found to be sensitive to metal cations, particularly Hg2+, Ag+ and Cu2+. 4-Hydroxymercuribenzoate (a mercapto-group inhibitor) and acetohydroxamic acid (a chelating agent of nickel) inhibited, the enzyme activity completely. These results suggest the involvement of an SH-group and Ni2+ in the activity of urease fromN. calcicola.

Toxicity potential of a textile dye toward the diazotrophic cyanobacteriumNostoc muscorum ISU

The impact of graded concentrations (5, 10, 15, and 20 mg L−1) of Omega Chrome Red ME, a common dye used in woollen, carpet, and textile industries, on protein production, pigment content, and photosynthetic oxygen evolution in a diazotrophic cyanobacterium Nostoc muscorum ISU, was studied. All concentrations were found to be inhibitory, with lower concentrations (5 mg L−1) producing a negligible effect, while higher concentrations produced a dramatic decrease in the parameters observed. The highest concentration of dye tested (20 mg L−1) resulted in a decrease in protein, chlorophyll a, phycocyanin, and carotenoid content of 82.20, 82.69, 46.20 and 41% of control, respectively. Photosynthetic oxygen evolution was decreased by 83.50% of control at the highest dye concentration. © 1994 by John Wiley & Sons, Inc..

Assessment of the effect of the toxicity of a textile dye on Nostoc muscorum ISU, a diazotrophic cyanobacterium

The impact of graded concentrations (5, 10, 15 and 20 mg dm −3 of acid mordant Metomega Chrome Orange GL, a common dye used in woollen, carpet and textile industries, was studied on protein and pigment content and photosynthetic oxygen evolution in a diazotrophic cyanobacterium Nostoc muscorum. The lower concentration of 5 mg dm −3 showed a negligible effect on protein and pigment content and photosynthetic oxygen evolution, whereas at higher concentrations a drastic decrease in the above parameters was observed. The highest concentration of 20 mg dm −3 resulted in a decrease of protein, chlorophyll a, phycocyanin and carotenoid content by 72, 76, 54 and 17% of the control, respectively. Photosynthetic oxygen evolution also decreased by 92% of the control at this concentration of the dye.

Protective effects of Ca 2+, Mg 2+, Cu 2+, and Ni 2+ on mercury and methylmercury toxicity to a cyanobacterium

Toxicological investigations of the impact of inorganic mercury (Hg 2+) and methylmercury (CH 3Hg +) in terms of growth, NH 4 + uptake, in vivo glutamine synthetase (transferase) activity, and regulation of toxicity by Ca 2+, Mg 2+, Cu 2+, and Ni 2+ in the diazotrophic cyanobacterium Nostoc calcicola Bréb. have been completed. Photoautotrophic growth of the cyanobacterium was extremely sensitive to both mercury compounds, CH 3Hg + being 2.5 times more toxic than Hg 2+. Although NH 4 + uptake was 6 times more sensitive than in vivo GS activity against the two mercurials, both processes had a greater susceptibility toward CH 3Hg +. On the basis of K m and V max, it is suggested that both mercury species inhibit such metabolic events noncompetitively. Ca 2+, Mg 2+, Cu 2+, and Ni 2+ did not change the nature of inhibition and effectively antagonized the Hg 2+ and CH 3Hg + toxicities in the sequence Ca 2+ > Mg 2+ ⪡ Cu 2+ > Ni 2+.

Cu uptake in a cyanobacterium: Fate of selected photochemical reactions

Cu uptake in the diazotrophic cyanobacteriumNostoc calcicola Breb. was accompanied by inhibitions in the in vivo activities of photosystem (PS) II, PS I,14CO2-fixation, and decline in the ATP pool. Cyanobacterial cells, while saturated for Cu uptake within 1 h at 40 μM Cu, showed more than 50% inhibition of PS II and 95.4% of14CO2 fixation compared with only 15.5% decrease in the PS I activity. The total extractable ATP content also declined by 32.2% within 1 h. In a subsequent follow-up study lasting 72 h, PS II activity and14CO2 fixation showed complete inhibition, in contrast to 34.4% of PS I activity and 4.2% of ATP still remaining unaffected. The results have been discussed in the light of multiple effects of Cu during and subsequent to its uptake by the cyanobacterium.

Ammonium Transport in the Alkalophilic Diazotrophic Cyanobacterium Nostoc calcicola: Influence of Phosphate Limitation and Metabolic Inhibitors

Ammonium transport in the diazotrophic cyanobacterium Nostoc calcicola was investigated in phosphate-sufficient and deficient conditions. Cells grown under phosphate-deficient conditions were characterized by a decrease in specific growth rate, phycocyanin content (50 %), oxygen evolution (50 %), reduced ATP pool and derepressed alkaline phosphatase activity. Under these conditions ammonium transport exhibited no change in K m (10.0 µM) in comparison to phosphate-sufficient cells, but a change in V max (41.0 nmol mg -1 protein min -1 ) was observed. The reduced rate of ammonium transport was attributed to unavailability of sufficient energy. Conditions that lowered or abolished the ATP pool (dark incubation or treatment with metabolic inhibitors DCMU, DCCD or CCCP) also reduced ammonium transport via two transport systems. The results indicated that unavailability of phosphate in the immediate environment may limit ammonium transport by a diazotrophic cyanobacterium.

Nutritional control of copper uptake in the cyanobacteriumNostoc calcicola Br�b

The kinetics of Cu uptake in nutritionally starved cells of the diazotrophic cyanobacteriumNostoc calcicola Breb. have been compared with those in cells recovering from starvation. Unstarved cyanobacterial cells assimilated 97.0 nmol Cu mg−1 protein within 1 h when incubated in medium containing 40 μM Cu. Uptake was markedly inhibited in carbon-starved cells and, to a lesser extent, in cells starved of nitrogen or sulphur. The intracellular concentrations of protein and photopigments were markedly lower in cells starved of carbon, nitrogen, sulphur or phosphorus, whilst that of carbohydrate was lower in cells starved of carbon, sulphur or phosphorus, but almost doubled in cells starved of nitrogen. The ability to assimilate Cu was partially restored in cells after 72 h of recovery from phosphorus or sulphur deprivation, but showed little improvement during recovery from carbon or nitrogen starvation. A possible role of phosphorus in regulating Cu transport and accumulation is discussed.

Factors regulating copper uptake in a cyanobacterium

Copper uptake in the diazotrophic cyanobacteriumNostoc calcicola was found to be typically biphasic, comprising rapid binding of the cations to the cell wall (during the first 10 min) followed by the subsequent metabolism-dependent intracellular uptake for at least 1 h, with a curvilinear kinetics saturating at 40 µM (Km 25.0 µM, Vmax 3.0 nmol Cu mg−1 protein min−1). The cellular Cu uptake was light- and ATP-dependent, and the addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or exogenous ATP proved that the energy to drive Cu transport was derived mainly through PS II reactions. The application of metabolic inhibitors and uncouplers like carbonylcyanidep-nitrofluoromethoxylphenyl hydrazone, N,N′-dicyclohexycarbodiimide, azide, and p-chloromercuribenzoate revealed that -SH group(s), proton gradient across the cell membrane, and ATP hydrolysis were involved in the transmembrane movement of Cu inN. calcicola. While monothiol (2-mercaptoethanol) caused a twofold reduction in Cu uptake rate, dithiol (dithiothreitol) contributed towards a further drop in the cation uptake rate.

Effect of cyanophage N-1 development on nitrogen metabolism of cyanobacterium Nostoc muscorum

The impact of cyanophage N-1 development on nitrogenase, glutamine synthetase (GS) and aminotransferases activities in the diazotrophic cyanobacterium Nostoc muscorum was investigated during its latent period. The nitrogenase activity was inhibited after 2 h of infection, suggesting that phage development does not require the product of nitrogenase activity. GS activity was not inhibited until 4 h of infection; however, a decline in activity was subsequently observed. Glutamate oxaloacetate transaminase was inhibited after 1 h of infection and no activity was detectable during the entire latent period. In contrast, glutamate pyruvate transaminase activity increased 2-fold by 4 h of infection and remained higher than the background level until the end of the latent period. The results suggested that under nitrogen fixing conditions, N-1 multiplication proceeds in the absence of nitrogen fixation and that the metabolism of amino acids is altered in favour of phage multiplication.