We report the changes in the concentrations and "I0 contents of extracellular CO2 and HCO3-in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring iS0 exchange between CO2 and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO2 hydration rate in different samples of cells that were grown on bubbled air (low-CO2 conditions). This activity was only weakly inhibited by ethoxzolamide with an Iso near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with COr starved cells there is considerable generation of CO2 from intracellular stores, a factor that can cause errors in measurement of net CO2 uptake unless accounted for. It was demonstrated that use of`C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO2 readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although (13). In suspensions of cells that contain carbonic anhydrase, depletion of 180 from species of extracellular inorganic carbon reflects the rate of access of the inorganic carbon across the cell membrane to the sites of carbonic anhydrase and the activity of this enzyme in the cells. This method was first used quantitatively by Gerster (4), and subsequently several studies have used the method on red cells and algae (2,14,18).It is known that cyanobacteria similar to Synechococcus have an active uptake mechanism for inorganic carbon (7, 9) which functions in the transport of either CO2 or HCO3 (1) (UTEX 2380) in the absence and presence of light using cells that had been illuminated prior to experiments until all net 02 evolution had ceased (Fig. 1). It is known that this cyanobacterium takes up both CO2 and HCO3-in the light (1), although . Cells were grown with bubbled air (low CO2 conditions) and then suspended (packed cell volume 3.3%) in a solution in the membrane inlet vessel; the solution was sea water with minor elements containing also 15 mM Tricine and 20 mm phosphate buffers at pH 7.9 (except the experiments measuring total inorganic carbon in the top figure which were performed at pH 7.5). The total concentration of all species of CO2 was 2.0 mm with 70% 13C content. Measurements were made at 25°C. The arrows indicate the addition of cells to the inlet vessel and the duration of red light of intensity 400 ME m2 s-'. absence of light, the cells did not take up CO2 to an extent that could be measured by our membrane inlet mass spectrometer. This was true both before and after illumination (Figs. 1 and 2). To determine the rate of uptake of CO2 and to distinguish this process from CO2 generated within the cells, we placed in solution CO2 and HCO3 that were highly enriched in carbon-13 (up to 99% 13C). The natural abundance of 13C iS 1.1% and the cells had not been expo...
A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4' into medium.Mutant strains capable of producing nitrogenase in the presence of NH4' were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, N03 , or low concentrations of NH4' (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4' produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4' when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4' produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment.In free-living, nitrogen-fixing organisms, nitrogenase synthesis and activity are regulated by the presence of NH4' in the medium (3,10,31,45). That NH4+-mediated regulation of nitrogenase synthesis is not exerted by NH4+ itself but is actually a consequence of metabolic products of NH4+ assimilation has been revealed by several lines of evidence.(i) Mutant strains incapable of NH4' assimilation were derepressed for nitrogenase synthesis in the presence of NH4+ (28,31,43,44). (ii) Addition of amino acids to the growth medium repressed nitrogenase synthesis even in nitrogenase-derepressed mutant strains (30). (iii) Inhibition of glutamine synthetase, a primary enzyme responsible for NH4+ assimilation, by a substrate (glutamate) analog, Lmethionine-D,L-sulfoximine (MSX) as well as by other inhibitors led to derepression of nitrogenase synthesis in the presence of NH4+ in all organisms tested (8,13,14,21,31,39). The above described derepression of nitrogenase synthesis is achieved by alteration of cellular physiology leading to a decrease in the rate of glutamate production, since addition of amino acids to the medium reversed the effect (22,29). This set of conditions also derepressed other NH4+-as well as glutamate-producing enzyme systems (NO3 assimilation; histidine and proline utilization) in the cell (16,29).Mutant strains that are blocked at the level of NH4+ assimilation were first described in Klebsiella pneumoniae (32), and such mutant strains not only derepress nitrogenase synthesis but also excrete the NH4+ produce...
G r o w t h o f wheat in a nitrogen-free hydroponic co-culture with a mutant strain o f the cyanobacterium Anabaena variabilis (strain SA-1) was e n h a n c e d over plants g r o w n with the parent strain SA-0. This increase was achieved in the dry weight, grain yield, and total nitrogen content o f the plants. Nitrogenase activity o f the mutant strain SA-1 was increased in a co-culture o f the cyanobacterial mutant with wheat plants c o m p a r e d to the activity o f the wild-type strain in association with wheat.
A marine, unicellular, nitrogen-fixing cyanobacterium was isolated from the blades of a brown alga, Sargassum fluitans. This unicellular cyanobacterium, identified as Synechococcus sp. strain SF1, is capable of photoautotrophic growth with bicarbonate as the sole carbon source and dinitrogen as the sole nitrogen source. Among the organic carbon compounds tested, glucose and sucrose supported growth. Of the nitrogen compounds tested, with bicarbonate serving as the carbon source, both ammonia and nitrate produced the highest growth rates. Most amino acids failed to support growth when present as sole sources of nitrogen. Nitrogenase activity in Synechococcus sp. strain SF1 was induced after depletion of ammonia from the medium. This activity required the photosynthetic utilization of bicarbonate, but pyruvate and hydrogen gas were also effective sources of reductant for nitrogenase activity. Glucose, fructose, and sucrose also supported nitrogenase activity but to a lesser extent. Optimum light intensity for nitrogenase activity was found to be 70 microE/m2 per s, while the optimum oxygen concentration in the gas phase for nitrogenase activity was about 1%. A hydrogenase activity was coinduced with nitrogenase activity. It is proposed that this light- and oxygen-insensitive hydrogenase functions in recycling the hydrogen produced by nitrogenase under microaerobic conditions.
Fructose is specifically taken up by nitrogen-fixing cultures of Anabaena variabilis in the light and lowers the doubling time from 24 to 8 h. The kinetics for both fructose-dependent growth and fructose uptake are exponential. The apparent Km for fructose uptake in N2-fixing cultures is 160 ,IM for cells not previously exposed to fructose and 50,uM in cells adapted to fructose. Picomolar amounts of [14C]fructose are scavenged from the medium and accumulate in filaments. Heterocysts of fructose-adapted filaments accumulate 14C from fructose within 20 min. Short-term experiments with fructose-starved cultures provide evidence that nitrogenase activity, protein, and chlorophyll content change within one generation time upon addition of fructose. In long-term experiments, the amount of fructose initially present in the medium determines heterocyst number and packed-cell volume. Photosynthetic oxygen evolution and amounts of chlorophyll decrease with exogenous fructose concentrations greater than 20 mM.
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