Biochemical Characterization of Molybdenum-Cofactor in a Cyanobacterium, Nostoc muscorum

Cell-free extracts of nitrate-grown Nostoc muscorum containnitrate reductase and molybdenum-cofactor activities. Whilenitrate reductase activity is associated with the paniculatefraction, cofactor activity is found predominantly in the solublefraction. This activity was distributed between two pools. Inone pool, the molybdenumcofactor is associated with a carrier(protein) of approximately 30,000 Da with an S_{20, w} between2.3 and 2.5. The carrier-bound cofactor is non-dialyzable andis found along with the major proteins during filtration inSephadex G-25 and G-100. The second pool contains free or unboundcofactor. It is separated from soluble proteins by dialysiswith a membrane with a pore-size of 10 to 15 kDa. However, itis retained with a membrane with a pore size of 1 kDa. It isin the included volume during chromatography through SephadexG-25. Its molecular mass is estimated to be between 1,000 and5,000 Da. The molybdenum content was proportional to cofactoractivity in both pools. Reducing agents increased cofactor activity.However, activity in both pools was sensitive to heat, acid,and oxidative treatments. The carrier protein appears to givesome protection. ¹Fulbright Scholar from Department of Biological Sciences, R.D. University, Jabalpur-482001, India. To whom reprint requestsshould be addressed. (Received June 22, 1987; Accepted August 21, 1987)     

Potential Use of Rice Field Cyanobacterium Nostoc muscorum in the Evaluation of Butachlor Induced Toxicity and their Degradation

In the present study, butachlor (5, 10, 20, 40 and 80 ppm) induced toxicity in Nostoc muscorum and their degradation was evaluated. The dose of butachlor dependent decreased in the cell survival and growth of N. muscorum was noticed. Scanning electron microscopy revealed the adverse impact on the cell size and shapes. Low concentrations of butachlor (10 and 20 ppm) induced the over expression of a polypeptides of 31.0 K Da and 42.7 K Da, respectively which could be responsible for developing resistance in the organism up to certain level. Further, the degradation product of butachlor as a result of metabolic activities of N. muscorum, identified by GC-MS analysis includes phenols and benzene dicarboxylic acid indicating the utilization of herbicide during active growth.     

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.     

Increase of Nitrogenase Activity in the Blue-Green Alga Nostoc muscorum (Cyanobacterium)

Preincubation of the blue-green alga (cyanobacterium) Nostoc muscorum under hydrogen or argon (nongrowing conditions, neither CO₂ nor N₂ or bound nitrogen present) in the light resulted in a two- to fourfold increase of light-induced hydrogen evolution and a 30% increase of acetylene reduction. Preincubation under the same gases in the dark led to a decrease of both activities. Cultivation of algae under a hydrogen-containing atmosphere (N₂, H₂, CO₂) increased neither hydrogen nor ethylene evolution by the cells. Formation of both ethylene and hydrogen is due to nitrogenase activity, which apparently was induced by the absence of N₂ or bound nitrogen and not by the presence of hydrogen. Inhibitors of protein biosynthesis prevented the increase of nitrogenase activity. Hydrogen uptake by the cells was almost unaffected under all of these conditions. With either ammonia or chloramphenicol present, nitrogenase activity decreased under growing conditions (i.e., an atmosphere of N₂ and CO₂). The kinetics of decrease were the same with ammonia or chloramphenicol, which was interpreted as being due to rapid protein breakdown with a half-life of approximately 4 h. The decay of nitrogenase activity caused by chloramphenicol could be counteracted by nitrogenase-inducing conditions, i.e., by the absence of N₂ or bound nitrogen. A cell-free system from preconditioned algae with an adenosine 5′-triphosphate-generating system exhibited the same increase or decrease of nitrogenase activity as the intact cell filaments, indicating that this effect resided in the nitrogenase complex only. We tentatively assume that not the whole nitrogenase complex, but merely a subunit or a special protein with regulatory function, is susceptible to fast turnover.     

Adaptation of the Cyanobacterium Microcystis aeruginosa to Light Intensity

Light intensity adaptation (20 to 565 microeinsteins per square meter per second) of Microcystis aeruginosa (UV-027) was examined in turbidostat culture. Chlorophyll a and phycocyanin concentrations decreased with increasing light intensity while carotenoid, cellular carbon, and nitrogen contents did not vary. Variation in the number but not the size of photosynthetic units per cell, based on chlorophyll a/P₇₀₀ ratios, occurred on light intensity adaptation. Changes in the numbers of photosynthetic units partially dampened the effects of changes in light intensity on growth rates.     

Toxicity of carbofuran to soil isolates of Chlorella vulgaris,Nostoc linckia and N. muscorum

A microalga, Chlorella vulgaris, and two diazotrophic cyanobacteria, Nostoc linckia and N. muscorum, all isolated from a rice soil, were compared for their response in terms of growth and metabolic activities to the application of carbofuran. The toxicity criteria included cell constituents (chlorophyll a, total protein, carbohydrate), ¹⁴CO₂ uptake and nitrate reductase, besides nitrogenase activity (acetylene reduction) in the cyanobacteria. C. vulgaris and N. muscorum were more sensitive to carbofuran than was N. linckia. The significant toxicity of the insecticide, observed with higher concentrations of 20 and 50 g ml^–1, to nitrogenase activity in N. linckia was reversed by the addition of ATP at 10 M. Transmission electron microscopy of the cultures, exposed to 50 g carbofuran ml^–1 showed certain cellular abnormalities, indicating interference of the insecticide with membrane properties. Correspondence to: K. Venkateswarlu     

Osmoregulation and cell composition in salt-adaptation of Nostoc muscorum

Adaptation to salt in the cyanobacterium Nostocmuscorum, is composed of a few mechanisms which together lead to the generation of a salt-tolerant cell. The initial mechanism combines a stimulation of photosynthetic activity with the accumulation of sucrose as an osmoregulator. The secondary mechanism involves the adaptation of N₂ fixation activity and protein biosynthesis. The adaptation is most efficient in response to NaCl-induced stress and functions only partially under stress induced by either KCl or a nonionic osmoticum such as mannitol.     

Nitrogenase activity in Nostoc muscorum; recovery from desiccation

Summary Nitrogenase activity in Nostoc crusts desiccated to below 10 per cent saturation for 24 hours and 7 days recovered on rewetting. Recovery over a 3 day period was greater in crusts returned to higher saturation levels. Possible reasons for, and limitations to, recovery are considered. re]19760420     

Pathways of hydrogen uptake in the cyanobacterium Nostoc muscorum

Two pathways of hydrogen uptake in Nostoc muscorum are apparent using either oxygen or nitrogen as electron acceptor. Hydrogen uptake (under argon with some oxygen as electron acceptor assayed in the dark; oxyhydrogen reaction) is found to be more active in dense, light-limited cultures than in thin cultures when light is not limiting. Addition of bicarbonate inhibits this hydrogen uptake, because photosynthesis is stimulated. In a cell-free hydrogenase assay, a 10-fold increase of the activity can be measured, after the cells having been kept under lightlimiting conditions. After incubation under light-saturating conditions, no hydrogen uptake is found, when filaments are assayed under argon plus some oxygen. Assaying these cells under a nitrogen atmosphere, a strong hydrogen uptake occurs. The corresponding cell-free hydrogenase assay exhibits low hydrogenase activity. Furthermore, the hydrogen uptake by intact filaments under nitrogen in the light apparently is correlated with nitrogenase activity. These studies give evidence that, under certain physiological conditions, hydrogen uptake of heterocysts proceeds directly via nitrogenase, with no hydrogenase involved.Abbreviations Chl chlorophyll - DCMU (diuron) 3-3,4-dichlorophenyl)-1,1-dimethylurea - pev packed cell volume     

PHB accumulation in Nostoc muscorum under different carbon stress situations

Use of algae for intracellular poly-β-hydroxybutyrate (PHB) accumulation for bioplastic production offers an opportunity in economic efficiency by reduced costs. The cyanobacterium Nostoc muscorum is a PHB accumulator which presents a great potential as raw material supplier because of short generation cycles. Here, we examined a range of experimental conditions including different growth conditions of phosphate-starved cells with the addition of external carbon sources. The highest, absolute PHB accumulation was measured in a phosphate-starved medium with 1% (w/w) glucose and 1% (w/w) acetate. PHB accumulated inside algae cells. After 23 days of growth in phosphate-starved medium, 1 L of culture contained up to 145.1 mg PHB. The highest PHB accumulation based on the cell dry weight was in an experiment with aeration and CO₂ addition. The intracellular level of PHB was up to 21.5% cell dry weight after 8 days.     

Studies on Diuron Uptake in a Blue-Green Alga Nostoc muscorum

A mutant of Nostoc muscorum that is resistant to 3-(3.4-dichlorophenyl)-I.1-dimethylurea (diuron) has been selected. This mutant maylack the step in photosynthesis that is affected by diuron (DCMU).but it can also use DCMU as a source of carbon and nitrogen.Another mutant of this organism resistant to L-methionine-DL-sulphoximine(MSO), that was isolated previously, also shows some cross-resistanceto DCMU. Key words: Nostoc muscorum, Diuron resistant mutants, MSO resistant mutants     

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum: effects of metabolic inhibitors

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.     

Physiological Significance of Vanadium Uptake during N2 and NC3- Metabolism in Various Strains of a N2-Fixing Cyanobacterium Nostoc muscorum

The nitrogenase and nitrate reductase activities of the molybdenum(Mo)-requiring parent Nostoc muscorum and its tungsten (W)-and chromium (Cr)-requiring mutant strains, growing with Mo,W and Cr, respectively, were significantly enhanced by the additionof 0.063 µM vanadium (V). Such interactions were not observedwith NO₂^- as a nitrogen source. (Received October 17, 1983; Accepted May 2, 1984)     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum and Spirulina platensis under phosphate limitation

Nostoc muscorum and Spirulina platensis were grown under phosphate deficiency in order to investigate the role of internal phosphate pool and activity of alkaline phosphatase on poly-β-hydroxybutyrate (PHB) accumulation. PHB accumulation in N. muscorum increased to 22.7% of dry weight (dw) after 4 day of phosphate deficiency, while the internal phosphate pool reduced to the level of 0.02 μM mg dw⁻¹ at a maximum APase activity of 2.57 nM PNP mg dw⁻¹ h⁻¹. In contrary, S. platensis depicted maxima of 1.39 nM PNP mg dw⁻¹ h⁻¹ on day 30 of incubation, which was about 2 fold lower than the observed value of N. muscorum. PHB content in S. platensis remained low even after prolonged phosphate starvation, and a rise only up to 3.5% of dw was recorded on day 60 of phosphate deficiency. Supplementation of NADPH exogenously to S. platensis cultures grown under phosphate deficiency favoured PHB accumulation in 10, 20 and 30 days old cultures, but not in the cultures grown under phosphate deficiency for 60 days. The possible role of phosphate limitation on PHB accumulation is discussed.