Nitrogen and ammonia assimilation in the cyanobacteria

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1978

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Abstract

A marine filamentous cyanobacterium capable of rapid growth under nitrogen-fixing conditions has been isolated in axenic culture from the Texas Gulf Coast. This organism appears to be an Anabaena species and has been given the strain designation CA. Cultures grown on mineral salts media with 1% CO₂-enriched air at 42°C show a growth rate of 5.6 ± 0.05 generations per day with molecular nitrogen as the sole nitrogen source. This growth rate is higher than that reported in the literature to date for heterocystous cyanobacteria growing on dinitrogen. Nitrogen fixation rates, as measured by acetylene reduction, show maximum activity values in the range of 50-100 nmoles/min/mg protein. These values compare favorably with those obtained from heterotrophic bacteria and are much higher than values reported for other cyanobacteria. Because of its high growth rate on dinitrogen, this newly isolated organism appears ideal for studying nitrogen metabolism and heterocyst development among the cyanobacteria. Anabaena sp. CA has been used to study the control of heterocyst development, an attractive model for examining cellular differentiation. Heterocyst synthesis is repressed by the presence of exogenous combined nitrogen. The tryptophan analogue D,L-7-azatryptophan (Aza-T) is capable of relieving the repressive effect of exogenous NH₄NO₃ on heterocyst and nitrogenase synthesis. The glutamate analogue, L-methionine-D, L-sulfoximine (MSX), has also been shown to cause derepression in the synthesis of heterocysts and nitrogenase. However, unlike MSX, Aza-T does not exert its effects by inhibiting primary ammonia assimilation. Moreover, MSX and Aza-T produce an additive effect when added together to cultures of Anabaena sp. CA. The evidence indicates that Aza-T may be exerting its effect at some novel control site of heterocyst development. Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA. The purification requires only three steps and results in a preparation that is electrophoretically homogeneous. The enzyme appears to have a molecular weight of 590,000 consisting of twelve identical subunits of 50,000 daltons. No adenylylation system controlling glutamine synthetase activity was found. Repression of enzyme synthesis by exogenous ammonia is slight and unlikely to be of physiological importance. In order of decreasing effectiveness, Cd²⁺ , Ca²⁺ , Zn²⁺ , and Mn²⁺ were found to inhibit activity, while Co²⁺ stimulated activity over five-fold. The enzyme is inhibited by the feedback modifiers L-alanine, glycine, L-serine, L-aspartate, and 5'-AMP. Inhibition by L-serine and L-aspartate is linear, non-competitive with respect to L-glutamate with K[subscript i] values of 3 mM and 13 mM, respectively. Cumulative inhibition is seen with mixtures of L-serine, L-aspartate, and 5'-AMP. The results indicate that; in vivo, the effect of divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and, hence, ammonia assimilation in nitrogen-fixing cyanobacteria

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