The subcellular distribution of nickel in Ni-sensitive and Ni-resistant strains of Saccharomyces cerevisiae.

Examination of the subcellular distribution of nickel in a Ni-resistant strain N08 of Saccharomyces cerevisiae showed that 70% of the nickel is distributed in the vascular fraction, which contains large amounts of histidine. The nickel taken up by cells grown in medium containing a high concentration of histidine was preferentially distributed to the vacuole. Arginine and lysine did not affect the intracellular distribution of Ni. In a Ni-sensitive strain 0605-S6, the distribution of nickel into the vacuole was lower than that observed in strain N08. Strain 0605-S6 exhibited no increase in the histidine content of the vacuolar fraction when grown in a Ni-supplemented medium. The Ni-resistant mechanism appears to involve the sequestration of nickel to the vacuole, and histidine could play an important role in the reduction of free nickel in the vacuole by the formation of histidine-nickel complexes.     

Effect of four heavy metals on the biology of Nostoc muscorum

Summary This study presents the effects of Cr, Pb, Ni and Ag on growth, pigments, protein, DNA, RNA, heterocyst frequency, uptake of NH₄ ⁺ and N0₃ ^–, loss of electrolytes (Na⁺ and K⁺), nitrate reductase and glutamine synthetase activities ofNostoc muscorum. The statistical tests revealed a direct positive correlation between the metal concentration and inhibition of different processes. Ni was found to be more toxic against growth, pigments and heterocyst differentiation compared to the other metals. Inhibition of pigment showed the following trend: chlorophyll > phycocyanin > carotenoid. No generalized trend for inhibition of macromolecules was observed. The loss of K⁺ and Na⁺ as affected by Cr, Ni and Pb was similar but more pronounced for K⁺ than Na⁺. The inhibition of physiological variables depicted the following trend: Na⁺ loss > K⁺ loss > glutamine synthetase > NH₄ uptake > growth > N0₃ ^– uptake > nitrate reductase > heterocyst frequency. This study therefore suggests that loss of electrolytes can be used as a first signal of metal toxicity in cyanobacteria. However, further study is needed to confirm whether the abnormality induced by nickel (branch formation) is a physiological or genetic phenomenon.     

Genetic transformation of glutamine auxotrophy to prototrophy in the cyanobacterium Nostoc muscorum

Glutamine auxotrophic (Gln ^-) and l-methionine d,l-sulfoximine (MSX) resistant (MSX ^r) mutants of N. muscorum were isolated and characterized for nitrogen nutrition, nitrogenase activity, glutamine synthetase (GS) activity and glutamine amide, -keto-glutarate amido transferase (GOGAT) activity. The glutamine auxotroph was found to the GOGAT-containing GS-defective, incapable of growth with N₂ or NH ₄ ⁺ but capable of growth with glutamine as nitrogen source, thus, suggesting GS to be the primary enzyme of both ammonia assimilation and glutamine formation in the cyanobacterium. The results of transformation and reversion studies suggests that glutamine auxotrophy is the result of a mutation in the gln A gene and that gln A gene can be transferred from one strain to another by transformation.     

Reconstitution of electron transport by cytochrome c-553 in a cell-free system of Nostoc muscorum

Treatment of spheroplasts of Nostoc museorum with hypotonic buffer results in membranes depleted of cytochrome c-553, but still active in photosynthetic and respiratory electron transport. These membranes retain full photosystem II activity (H₂ODAD_ox). Complete linear electron transport (H₂ONADP⁺), however, is decreased as compared with untreated spheroplasts. Addition of basic Nostoc cytochrome c-553 to depleted membranes reconstitutes NADP⁺ reduction and redox reactions of the photosystem I region as well.Using NADPH as electron donor, respiration of depleted membranes is also stimulated by adding cytochrome c-553, indicative of its function in respiratory electron transport.Cytochrome c-553 from Bumilleriopsis filiformis, Spirulina platensis (acidic types), Phormidium foveolarum (basic type), and mitochondrial horse-heart cytochrome c-550 are not effective in reconstituting both photosynthetic and respiratory electron transport, which points to a specific role of Nostoc cytochrome c-553.Abbreviations BSA bovine serum albumin - DAD 3,6-diaminodurene - DAD_ox 3,6-diaminodurene oxidized by potassium ferricyanide - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Fd ferredoxin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(-N-morpholino)-ethanesulfonic acid - MV methylviologen (1,1-dimethyl-4,4-bipyridylium dichloride) - PS I photosystem I - PS II photosystem II - Tris tris-(hydroxymethyl)-aminomethane     

Metabolic changes associated with cyanophage N-1 infection of the cyanobacterium Nostoc muscorum

The development of cyanophage N-1 in the N₂-fixing cyanobacterium Nostoc muscorum is dependent on light. The redox state of thioredoxin m was altered in phage infected cells, with the proportion of reduced thioredoxin increasing during the eclipse period. In one step growth experiments, the specific activity of glucose-6-phosphate dehydrogenase increased transiently during the eclipse period, whereas that of glutamine synthetase increased towards the end of the eclipse period (2–4h after infection) then remained high until the end of the latent period (about 7 h after infection). The rate of respiratory O₂ uptake was maintained until the end of the latent period. In contrast, the specific activity of phosphoribulokinase and the rate of photosynthetic O₂ evolution began to decrease towards the end of the eclipse period and later than the level of extractable protein began to decrease. Nitrogenase activity remained high throughout the eclipse period then decreased rapidly after 5 h. The level of glutamine synthetase protein decreased in parallel with the decrease in total extractable protein, whereas the level of thioredoxin m protein decreased more slowly.     

Response of two strains of Nostoc muscorum to metal stress and salinity

Two strains of a cyanobacterium Nostoc muscorum, wild-type N. muscorum (Cd^s) and an isolate having resistance to the heavy metal cadmium (Cd^r), were selected for characterisation of their growth potential and physiological assays in certain defined stress environments. The chosen determinants were copper (heavy metal) and NaCl (salt stress). The observations on growth, heterocyst frequency, chlorophyll and nucleic acid contents, photosynthetic O₂ evolution, ¹⁴C incorporation and acetylene reduction suggested that the strain Cd^r was also resistant to copper. This strain, however, was found to be more sensitive to NaCl in comparison to its wild-type counterpart. NaCl was found to enhance sugar accumulation in Cd^s and was more inhibitory to acetylene reduction rates than to the photosynthetic activities. The interaction between Cu and NaCl appeared to be antagonistic as the depression of growth and physiological activities by a mixture of the two was lesser than that caused by either of these. These observations form the first report on the response of a metal resistant strain of cyanobacterium to salinity.     

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.     

Common nitrogen control of caesium uptake, caesium toxicity and ammonium (methylammonium) uptake in the cyanobacterium Nostoc muscorum

Abstract Studies were carried out to examine the role of ammonium transport activity in the control of caesium uptake and toxicity in Nostoc muscorum . The results showed a definite specific role of the ammonium-repressible/derepressible ammonium transport system of the cyanobacterium in caesium uptake, accumulation and toxicity. Furthermore, the results showed that N. muscorum can acquire resistance against diazotrophically-associated caesium toxicity when supplied with ammonium as a nitrogen source. In addition, alternatively, a mutant strain was Cs-resistant in the absence of any effect on NH₄⁺-transport, suggesting that Cs⁺ resistance may be determined at more than one cellular site.     

Biotransformation of hydrocortisone by a natural isolate of Nostoc muscorum

Hydrocortisone was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some androstane and pregnane derivatives. The microorganism was, isolated during a screening program from soil samples collected from paddy fields of north of Iran. The bioproducts obtained were purified using chromatographic methods and identified as 11beta-hydroxytestosterone, 11beta-hydroxyandrost-4-en-3,17-dione and 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one on the basis of their spectroscopic features.     

Structural aspects of the adaptation of Nostoc muscorum to salt

The physiological and biochemical changes during the adaptation of Nostoc muscorum to salt are accompanied by specific structural changes. Cells of Nostoc muscorum exposed to saline medium vary in size and envelope organization. There are also drastic changes in the intracellular organization of the thylakoidal assembly. The heterocysts exhibit a preferential tolerance to NaCl rather than mannitol. These findings suggest that Nostoc muscorum is equipped with a specific physiological capacity for NaCl tolerance.     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

Genetic control of heterocyst formation in the blue-Green algae Nostoc muscorum and nostoc linckia

Non-heterocystous, non-nitrogenfixing (het ^- nif^-), heterocystous, non-nitrogenfixing (het ⁺ nif^-) and multiple heterocystous, nitrogen-fixing (M-het ⁺ nif⁺) mutants of heterocystous, nitrogen-fixing (het ⁺ nif⁺) wild-type Nostoc muscorum and Nostoc linckia were isolated and characterized with respect to (a) nitrogenfixing activity, (b) reversion frequency, (c) ammonium repressibility of heterocyst formation, (d) heterocyst spacing pattern, and (e) action of L-methionine-DL-sulphoximine (MSO), an inhibitor of glutamine synthetase (GS), on heterocyst regulation. The mutant and revertant results suggest: (i) either involvement of a common genetic determinant in the formation of heterocyst and nitrogenase or the organization of het genes and nif genes in a single operon prone to complete inactivation by a single polar mutation, (ii) non-participation of active nitrogenase in regulation of heterocyst spacing; (iii) involvement of genetic factor(s) in the control of heterocyst spacing pattern in N. linckia, and (iv) apparently different nature of the mechanism of heterocyst inhibition by proheterocyst from that of heterocyst inhibition by NO ₃ ^- or NH ₄ ⁺ . L-Methionine-DL-sulphoximine inhibits growth and causes heterocyst formation in chains in N. linckia growing in nitrogen-free, NO ₃ ^- , NO ₂ ^- or NH ₄ ⁺ medium, thus indicating a close physiological linkage between heterocyst and inorganic nitrogen metabolism regulation.     

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     

Formation of Giant and Ultramicroscopic Forms of Nostoc muscorum CALU 304 during Cocultivation with Rauwolfia Tissues

The study of heteromorphic Nostoc muscorum CALU 304 cells, whose formation was induced by 6- to 7-week cocultivation with the Rauwolfia callus tissues under unfavorable conditions, revealed the occurrence of giant cell forms (GCFs) with a volume which was 35–210 times greater than that of standard cyanobacterial cells. Some GCFs had an impaired structure of the murein layer of the cell wall, which resulted in a degree of impairment of the cell wall ranging from the mere loss of its rigidity to its profound degeneration with the retention of only small peptidoglycan fragments. An analysis of thin sections showed that all GCFs had enlarged nucleoids. The photosynthetic membranes of spheroplast-like GCFs formed vesicles with contents analogous to that of nucleoids (DNA strands and ribosomes). About 60% of the vesicles had a size exceeding 300 nm. With the degradation of GCFs, the vesicles appeared in the intercellular slimy matrix. It is suggested that the vesicles are analogous to elementary bodies, which are the minimal and likely primary reproductive elements of L-forms. The data obtained in this study indicate that such L-forms may be produced in the populations of the cyanobionts of natural and model syncyanoses. Along with the other known cyanobacterial forms induced by macrosymbionts, L-forms may represent specific adaptive cell forms generated in response to the action of plant symbionts.     

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.     

Metabolic interconversion of ferredoxin-nitrate reductase and NADP reductase of Nostoc muscorum.

Ammonia is a physiological uncoupler of photophosphorylation which drastically inhibits the photosynthetic reduction of nitrate by Nostoc muscorum particles by promoting the conversion of ferredoxin-nitrate reductase into its reduced inactive form. Ammonia promotes also, after an initial stimulation, the total inhibition of the photosynthetic reduction of NADP⁺. Like its nitrate counterpart, ferredoxin-NADP reductase is reversibly inactivated by reduction and reactivated by oxidation.     

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.     

Cytochemical changes in the developmental process of Nostoc sphaeroides (cyanobacterium)

There are several apparent developmental stages in the life cycle of Nostoc sphaeroides Kützing, an edible cyanobacterium found mainly in paddy fields in central China. The cytochemical changes in developmental stages such as hormogonia, aseriate stage, filamentous stage and colony in N. sphaeroides were examined using fluorescent staining and colorimetric methods. The staining of acidic and sulfated polysaccharides increased with development when hormogonia were used as the starting point. Acidic polysaccharides (AP) were most abundant at the aseriate stage and then decreased, while the sulfated polysaccharides (SP) were highest at the colony stage. Quantitatively, along the developmental process from hormogonia to colony, total carbohydrates first increased, then became stable, and then reached their highest level at the colony stage, while reducing sugars were highest at the hormogonia stage and then decreased sharply once development began. SP were not detectable in the hot water soluble polysaccharides (HWSP), and hormogonia had the lowest content of AP, while old colonies had the highest. The AP content of the aseriate stage, filamentous stage and young colony stage were very similar. The evolutionary relationships reflected in the developmental stages of N. sphaeroides are discussed.