Mutagenesis of the blue-green alga,Anabaena doliolum Bharadwaja

Mutagenesis in the blue-green alga, Anabaena doliolum Bharadwaja has been investigated with particular reference to N₂ fixation. Several types of mutant have been isolated after induction with UV, NG, acridine orange and acriflavine. From a comparative characterization it is concluded that the heterocyst is not the sole site of N₂ fixation. There does not appear to be a linkage between N₂ fixation and heterocyst or spore differentiation: they seem to be independent processes probably regulated either by different genes or by a single regulatory gene with independent operons. A common genetic determinant has also been suggested for nitrogenase and nitrate and nitrite reductases.     

Pigment variations in ultraviolet-treated strains of the blue-green alga Anabaena doliolum

Summary Two kinds of cultures were raised from clones of Anabaena doliolum surviving on selective medium following exposure of spores to ultraviolet radiation. The pigments of these cultures have been characterized with respect to those of controls.     

Genetic control of sporulation in the blue-green alga Anabaena doliolum Bharadwaja

Summary Two genetically distinct, non-sporulating mutant strains of Anabaena doliolum were isolated following exposure of the alga to ultraviolet radiation. Among the progeny of a cross between the two non-sporulating strains, some filaments were able to sporulate. The spores of such filaments were of two types. Type 1 upon germination produced the parental phenotype, type 2 gave rise to wild phenotype. This suggests the involvement of heterozygosity in genetic recombination in A. doliolum. The results further indicate that the formation of spores in this alga is under the control of more than one genetic determinant, and that nuclear segregation occurs during sporulation.     

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.     

Effect of certain herbicides on survival, growth, and nitrogen fixation of blue-green alga Anabaena doliolum Bharadwaja

Growth and fixation of nitrogen were retarded in Anabaena doliolum when grown on elemental nitrogen in presence of four different herbicides. The alga was found to be intolerant to these chemicals under nitrogen fixing conditions even in low concentrations. An inhibition of photosynthesis has been regarded as the main basis of herbicidal activity besides other interactions.     

The utilization of molecular hydrogen by the blue-green alga Anabaena cylindrica

The blue-green alga Anabaena cylindrica is found to consume molecular hydrogen in a hydrogenase dependent reaction. This hydrogen uptake proceeds in the dark and is strictly dependent on oxygen, thus representing a Knallgas reactions. Its rate is almost as high as that of the endogenous respiration in Anabaena. Studies with inhibitors reveal that hydrogen is utilized via the complete respiratory chain providing additional energy for the alga. CO plus C₂H₂ completely block the Knallgas reaction which explains the previously reported considerable increase in the total H₂ formation representing the difference between the nitrogenase-dependent H₂-evolution and the reutilization of the gas catalysed by the hydrogenase in intact Anabaena.H₂ is able to support the C₂H₂-reduction in the dark in a reaction again strictly dependent on oxygen. Moreover, H₂ is also consumed in experiments carried out under far red light and in the presence of dichlorophenyl-dimenthyl-urea (DCMU) where the energy for nitrogen fixation is no longer provided by respiration but by cyclic photophosphorylation. Under these conditions, H₂ is found to supply electrons for the formation of C₂H₄ from C₂H₂ in a reaction no longer dependent on the presence of oxygen. Moreover, in these experiments, the presence of H₂ stabilizes the C₂H₂-reduction activity against the deleterious effect of oxygen.Thus, this communication provides evidence for a triplicate function of the H₂-uptake catalysed by hydrogenase in intact Anabaena which is (a) to provide energy by the Knallgas reaction, (b) to supply reducing equivalents for nitrogenase, (c) to protect nitrogenase from damage by oxygen.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-dimethylurea - DNP 2-4-dinitrophenol - FCCP carbonylcyanid-p-trifluormethoxyphenyl-hydrazone(=p-CF₃-CCP) - Chl chlorophyll     

Ribosomes from the blue-green alga Anabaena variabilis

Summary Extracts from the blue-green alga Anabaena variabilis were prepared by ultrasonic disintegration or by extrusion through a French pressure cell; examination by sucrose density gradient centrifugation and analytical ultracentrifugation indicated the existence of a procaryotic (70S) ribosome. However both the ribosomes and their sub-units were found to be relatively unstable after isolation and examination in tris buffer pH 7.4 but not in 10^-3 m-sodium phosphate buffer pH 7.0 containing 10^-1 m-potassium chloride. Experiments with ³²P and ³⁵S isotopes indicated that this instability was associated with the loss of ³⁵S labelled material, presumably therefore protein rather than nucleic acid. A comparison of behaviour with that of bacterial ribosomes is presented and the existence of certain similarities to ribosome preparations from chloroplasts of several plant species discussed.     

Effect of nitrate on nitrogen-fixation by the blue-green alga Anabaena cylindrica

The effect of nitrate on nitrogen-fixation in the blue-greenalga Anabaena cylindrica Lemm (Fogg strain) was investigated.At concentrations up to 2x10^–2 M, nitrate neither inhibitedthe activity of nitrogenase nor repressed its formation. Atthe late logarithmic phase, more than 50% of cell nitrogen wasprovided by nitrogen-fixation when the cells were grown in thepresence of nitrate. Ammonia at a concentration of 1x10^–3M completely repressed the formation of nitrogenase, but hadno effect on its activity. Nitrogen-fixing activity in the algavaried to a considerable extent during growth on N₂ and themaximum activity was attained at the middle logarithmic phase.However, atmospheric nitrogen did not directly affect the inductionof nitrogenase. The formation of nitrogenase in A. cylindricaappears to be controlled by the intracellular level of a certainnitrogenous metabolite. ¹ This work was supported by grant No. 38814 from the Ministryof Education. (Received January 26, 1972; )     

Effect of ammonia on nitrogen fixation by the blue-green alga Anabaena cylindrica

Ammonia at a concentration of 1 ? 10^–3M completely inhibitednitrogenase activity, as measured by acetylene reduction, inthe blue-green alga Anabaena cylindrica. Free ammonia was undetectablein cells grown either on N₂ or ammonia within the limits ofprecision of the method used. Glutamic acid formed a major aminoacid pool in N₂-grown cells, and basic amino acids, i.e. lysine,histidine and arginine were abundant in ammonia-grown cells.A 10-fold increase in the amounts of labile amino compound(s)was observed when N₂-grown cells were exposed to ammonia. When cells were incubated under anaerobic conditions, the acetylene-reducingactivity increased 2-fold or more; ammonia had no effect. Oxygenwas required for ammonia to inhibit acetylene reduction. Modes of inhibition by ammonia on acetylene reduction were comparedwith those by chloramphenicol, puromycin, cycloheximide, DCMUand CCCP. On the basis of these comparisons we concluded thatammonia not only acts as a suppressor of nitrogenase synthesisbut also inhibits acetylene-reducing activity by lowering thesupply of reductant and/or of energy for the nitrogenase system. ¹This work was supported by grant No. 38814 from the Ministryof Education. (Received July 30, 1973; )     

Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

Urease fromAnabaena doliolum andAnacystic nidulans showed maximum activity at pH 7.0–7.4 at 40°C when measured in cell-free, phosphate-buffered extracts. It is a soluble enzyme located in cytoplasm. The apparent K_m forA. doliolum urease was 120 M. Anacystis nidulans urease exhibited biphasic kinetics (K_m=250 M and 1.66 mM). Enzyme, fully expressed in cells grown with urea, nitrate, or N₂, was repressed in ammonia-grown cells, but ammonia did not inhibit the activity in vitro. Incubation of algal cells in N₂ medium with chloramphenicol for 12 h caused degradation of urease. Cu²⁺ at 1 M inhibited the enzyme activity by 50%, whereas Co²⁺ and Ni²⁺ up to 20 M had no effect.p-Hydroxymercuribenzoate appeared to be a more powerful inhibitor of urease than acetohydroxamic acid.Address reprint requests to: c/o Prof. Robert Tabita, Department of Microbiology, Experimental Science Building #319, The University of Texas at Austin, Austin, TX 78712, USA.     

The effect of chloramphenicol on the production of cyanophycin granule polypeptide in the blue-green alga Anabaena cylindrica

Summary The cyanophycin or structured granule of the blue-green algae is composed of polypeptides which are copolymers of aspartic acid and arginine. The addition of chloramphenicol to an exponentially growing culture of the blue-green alga Anabaena cylindrica at concentrations which completely inhibit protein synthesis results both in the inhibition of growth and in the accumulation of the cyanophycin granule polypeptide (CGP). The chloramphenicol induced increase in CGP content is energy dependent. Removal of the chloramphenicol results in resumption of growth and the hydrolysis of the stored CGP. The data presented indicate that CGP is synthesized via a non-ribosomal system and are consistent with the idea that CGP serves as a cellular nitrogen reserve.     

Nitrogen fixation and heterocysts in the blue-green alga Anabaena cylindrica

The site of nitrogen fixation in the blue-green alga Anabaenacylindrica Lemra (Fogg strain) was investigated. Less than 4%of the total nitrogen fixed during a relatively short period(5-15 min) was recovered in heterocysts. When estimated on thecellular nitrogen basis, vegetative cells can fix molecularnitrogen at the same rate as do heterocysts. There was no positivecorrelation between nitrogen fixation and heterocyst formation.Results do not support the hypothesis that the heterocyst isthe main site for nitrogen fixation in blue-green algae. ¹ This work was supported by grant (No. 38814) from the Ministryof Education. (Received July 23, 1971; )