Fractionation of phycobilisomes from the blue-green alga Nostoc muscorum

Phycobilisomes (PBS) isolated from Nostoc muscorum contain eight spectral forms of phycobiliproteins. PBS partially dissociated by osmotic or temperature shock, were separated into 42S and 34S particles by centrifugation in sucrose density gradient. The 42S particles are enriched with phycocyanin, the 34S ones--with allophycocyanin. The 42S particles dissociate to free pigments by repeated osmotic or temperature shock and the 34S ones dissociate to subparticles I and II having the constant pigments composition. The integrity of PBS and isolated substructures is controlled by the yield of energy transfer between pigment molecules. PBS from Nostoc muscorum was found to contain the rods of three different types. The model PBS containing different spectral forms of biliproteins is proposed.     

Isolation of a sulphate reductase-less mutant of the blue-green alga Nostoc muscorum.

The wild type Nostoc muscorum (UW strain) has yielded various physiological mutants altered in utilization of sulphate, following mutagenic treatments with N-methyl, N'-nitro N-nitrosoguanidine (NTG). One of the mutant strains designated as Sat-20 failed to grow in a medium containing sulphate (MgSO4.7 H2O). However, the mutant strain could grow when supplemented with thiosulphate (Na2S2O3.5 H2O), while methionine could fulfil the sulphur requirement only partially. On comparative reasons, the wild type as well as the mutant showed preference for thiosulphate over other sulphur sources employed.     

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     

Phosphomonoesterase activity of the cyanobacterium (blue-green alga) Calothrix parietina

Cellular and extracellular phosphomonoesterase activities were compared in Calothrix parietina D550, a strain whose original environment has been studied in detail. Activity in both fractions became detectable at about the same stage in batch culture. Differences in the influence of environmental factors between the two were slight, suggesting a common origin. The optimum temperatures for cellular and extracellular activities were 40 degrees C and 30 degrees C, respectively, and the upper limits for detectable activity were 80 degrees C and 65 degrees C. The pH optimum for both cellular and extracellular activity was 10.0-10.2. When P-limited cultures were tested with p-nitrophenyl phosphate (pNPP) as substrate, Km values for cellular and extracellular activities were 43 and 33 microM pNPP, respectively. Eleven ions were tested for their influence on activity. In most cases the effect was low or negligible at concentrations likely to be present in nature or freshwater laboratory media. Where obvious effects occurred, these were usually apparent at lower concentrations with extracellular than cellular activity. One mM Ca led to a 40% increase in extracellular activity in comparison with 0.1 mM Ca, but had no effect on cellular activity. However, inorganic phosphate, which had a marked inhibitory effect at concentrations above 10 microM, brought about a similar response with cellular and extracellular activities (approximately 60% decrease with 100 microM).     

Effect of nutrients on the toxicity of pesticides carbofuran and hexachlorocyclohexane to blue-green alga Nostoc muscorum.

The effects of various levels of nutrients like potassium phosphate (dibasic), calcium nitrate, and calcium chloride individually and in combinations were studied on the toxicity of the commonly used pesticides carbofuran and hexachlorocyclohexane (HCH) in growth medium to the N2-fixing blue-green alga Nostoc muscorum. It was observed that all these chemicals had some effects on toxicity. The toxicity of both carbofuran and HCH was reduced to some extent in the presence of higher concentrations of the nutrients when compared to normal levels of the chemicals in the medium. The higher doses of nutrients in combinations antagonized the effect of individual treatment and enhanced the toxicity of pesticides.     

Role of calcium in the inhibition of nitrogenase activity by Methylparathion and Benthiocarb in the cyanobacterium Nostoc muscorum

Methylparathion and Benthiocarb inhibition of N₂ fixation in the cyanobacterium Nostoc muscorum was reversed by Ca²⁺ at 1 mm but not at 0.1 mm. The concentration of intracellular Ca²⁺ was relatively high in the presence of these pesticides when 1 mm Ca²⁺ was also present, indicating that intracellular Ca²⁺ may participate in protecting nitrogenase activity against Methylparathion and Benthiocarb.The authors are with the Department of Biochemistry, University College of Science, 35 Ballygunge Circular Road, Calcutta 700 019, India.     

Effect of lead on nitrogenase and enzymes of nitrogen assimilation in a cyanobacterium Nostoc muscorum.

Lead decreased the growth rates, total cell mass, heterocyst frequency, total cell protein, nitrogenase activity, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities in N:muscorum. However, lead at 0.01 and 10 micrograms ml-1 conc. enhanced nitrogenase as well as GS activity of the cells. On transfer to excess lead (100 micrograms ml-1), nitrogenase and GS activities ceased almost after 24 hr in the cyanobacterium. It is deduced that lead has a two step effect on stimulation and inhibition of metabolic activity at 0.01 and 10 micrograms ml-1 concentration and 0.1 and 100 micrograms ml-1 concentration respectively indicating a close interaction between nitrogen fixation and GS activity. However, GOGAT activity is an exception to this two step stimulation and inhibition process.     

The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cyclindrica

Exogenous pyruvate added to cultures of the bluegreen alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO₂ (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O₂, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O₂. Malate, citrate, α-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of ¹⁴C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.     

Powerful mutagenicity of a bipyridylium herbicide in a nitrogen-fixing blue-green alga Nostoc muscorum

Malondialdehyde (MDA), an in vivo metabolite of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in Salmonella typhimurium. It is a reactive electrophile that can form interstrand cross-links in DNA. To explore the possibility that MDA-induced interstrand cross-links are the pre-mutagenic lesion, we have quantitated the ability of highly purified preparations of MDA to form interstrand cross-links when reacted with linear plasmid DNA. At physiological temperature and pH, MDA did not form DNA cross-links as determined by DNA denaturation followed by agarose gel electrophoresis. DNA cross-links were formed, however, when incubations with MDA were carried out at either pH 4.2 or temperatures exceeding 60 degrees. alpha-Methylmalondialdehyde (CH3MDA) was found to cross-link DNA more efficiently than MDA, but was not mutagenic in any tester strain of Salmonella. MDA polymers, formed by acid incubation of MDA, also were capable of inducing cross-links. However, an inverse relationship was observed between mutagenicity and extent of polymerization. The pattern of mutagenic response for MDA in different strains of Salmonella was compared with mitomycin C, an established mutagenic cross-linking agent. Error-prone repair and a UvrB+ phenotype, which are needed for the induction of mutations by mitomycin C, were not required for MDA mutagenesis. These findings, taken together, dissociate the mutagenicity of MDA from its ability to form interstrand cross-links with DNA.     

Histidine sensitive variant of the blue-green alga Nostoc muscorum: response to corepressors of histidine biosynthesis.

Summary A model has been proposed to account for growth inhibition by L-histidine in a variant strain of Nostoc muscorum. This strain has been characterized for its response to 3-amino-1,2,4-triazole and 1,2,4-triazole-3-alanine known to act as false corepressors of the histidine biosynthesis genes. The histidine sensitive strain retained its sensitivity to triazole alanine while the inhibitory effects of aminotriazole were much reduced indicating a change in regulation of his genes. The probable interactions between nif and his genes in cyanobacteria (blue-green algae) have been discussed.     

Components and activity of the photosynthetic electron transport system of intact heterocysts isolated from the blue-green alga Nostoc muscorum

Heterocysts of the blue-green alga Nostoc muscorum have been isolated by prolonged treatment with lysozyme. Quantitative data are presented which show the occurrence of cytochromes c-553, f-557 and b-563 in heterocysts in amounts comparable to vegetative cells. Particularly the content of the water-soluble cytochrome c-553 can be used to evaluate the intactness of a heterocyst preparation. Cytochrome f-557 has been partially purified and found to be a c-type cytochrome corresponding to cytochrome f of higher plants and other algae. Cytochrome b-559 is present in vegetative cells but not in heterocysts. The content of plastoquinone in heterocysts is reduced to 42% of the amount present in vegetative cells. These data suggest a degradation of Photosystem II during heterocyst differentiation. Measurements of photosynthetic electron transport in heterocysts proved the inability of the photosynthetic apparatus to carry out electron transport with electrons donated by water or diphenylcarbazide. In Tris-washed thylakoids from vegetative cells, however, diphenylcarbazide can act as an electron donor to Photosystem II.     

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)