The initial organic products of fixation of 13N-labeled nitrogen gas by the blue-green alga Anabaena cylindrica

Thomas, Joseph; Wölk, C. Peter; Shaffer, Paul W.; Austin, Sam M.; Galonsky, A.

doi:10.1016/0006-291x(75)90840-2

Cited by 49 publications

(27 citation statements)

References 16 publications

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“…This apparent conundrum, that is the juxtaposition of oxygen-sensitive functions with oxygen-producing cells, has long been a focus of cyanobacterial biology, and a variety of experimental approaches have been used to decipher the functional and physiological differences between heterocysts and vegetative cells (Thomas et al, 1975Wolk et al, 1976;Meeks et al, 1977;Kumar et al, 1983;Flores et al, 2006). Pulse-chase experiments and autoradiography using the short-lived radioisotope 13 N have demonstrated that heterocysts are the site of N 2 -fixation and showed both incorporation of N 2 by heterocyst and export of recently fixed organic N to vegetative cells (Wolk et al, 1974).…”

Section: Introductionmentioning

confidence: 99%

Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides

et al. 2007

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Filamentous nitrogen fixing cyanobacteria are key players in global nutrient cycling, but the relationship between CO 2 -and N 2 -fixation and intercellular exchange of these elements remains poorly understood in many genera. Using high-resolution nanometer-scale secondary ion mass spectrometry (NanoSIMS) in conjunction with enriched H 13 CO 3 À and 15 N 2 incubations of Anabaena oscillarioides, we imaged the cellular distributions of C, N and P and 13 C and 15 N enrichments at multiple time points during a diurnal cycle as proxies for C and N assimilation. The temporal and spatial distributions of the newly fixed C and N were highly heterogeneous at both the intra-and inter-cellular scale, and indicative of regions performing active assimilation and biosynthesis. Subcellular components such as the neck region of heterocycts, cell division septae and putative cyanophycin granules were clearly identifiable by their elemental composition. Newly fixed nitrogen was rapidly exported from heterocysts and was evenly allocated among vegetative cells, with the exception of the most remote vegetative cells between heterocysts, which were N limited based on lower 15 N enrichment. Preexisting functional heterocysts had the lowest levels of 13 C and 15 N enrichment, while heterocysts that were inferred to have differentiated during the experiment had higher levels of enrichment. This innovative approach, combining stable isotope labeling and NanoSIMS elemental and isotopic imaging, allows characterization of cellular development (division, heterocyst differentiation), changes in individual cell composition and cellular roles in metabolite exchange.

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Section: Introductionmentioning

confidence: 99%

Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides

et al. 2007

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“…These carriers have been suggested to be 2-oxo-glutarate, glutamate, glutamine, aspartate, and arginine. The product of nitrogen fixation, ammonia, is added to glutamate to make glutamine; that much has been established by studies of the incorporation of radioactive N 2 (5,6). How the heterocyst acquires enough glutamate is still a mystery (2).…”

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The Sakaguchi Reaction Product Quenches Phycobilisome Fluorescence, Allowing Determination of the Arginine Concentration in Cells of Anabaena Strain PCC 7120

Haselkorn

2013

J Bacteriol

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The filamentous cyanobacterium Anabaena fixes nitrogen in specialized cells called heterocysts. The immediate product of fixation, ammonia, is known to be assimilated by addition to glutamate to make glutamine. How fixed nitrogen is transported along the filament to the 10 to 20 vegetative cells that separate heterocysts is unknown. N-fixing heterocysts accumulate an insoluble polymer containing aspartate and arginine at the cell poles. Lockau's group has proposed that the polymer is degraded at the poles to provide a mobile carrier, arginine, to the vegetative cells (R. Richter, M. Hejazi, R. Kraft, K. Ziegler, and W. Lockau, Eur. J. Biochem. 263:163-169, 1999). We wished to use the Sakaguchi reaction for arginine to determine the relative cellular concentration of arginine along the filament. At present, the methods for measuring absorption of the Sakaguchi reaction product at 520 nm are insufficiently sensitive for that purpose. However, that product quenches the fluorescence of phycobiliproteins, which we have adapted to a determination of arginine. Our results are consistent with the proposal that arginine is a principal nitrogen carrier from heterocysts to vegetative cells in Anabaena.

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“…These results suggest that ammoniumperse is an important in vivo regulator ofnitrogen fixation and its function can be mimicked by methylamine. Previous studies employing methionine sulfoximine may have to be re-evaluated in light of the inhibitory effects of methionine sulfoximine on the ammonium transporter.In the absence ofcombined nitrogen, heterocystous blue-green algae fix atmospheric N2 and produce NH3 which is subsequently assimilated by the GS2/GOGAT pathway (2,8,20,21). Exogenous NH3 inhibits nitrogenase activity and heterocyst differentiation in nitrogen fixing algae (3).…”

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In Vivo Nitrogenase Regulation by Ammonium and Methylamine and the Effect of MSX on Ammonium Transport in Anabaena flos-aquae

1984

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Ammonium suppresses nitrogenase activity in Anabaena flos-aquae (Lyng) Breb. at all pH values tested. L-Methionine-DL-sulfoximine at 1 millimolar totally inhibited glutamine synthetase, and 10 micromolar partially inhibited. Both concentrations protected nitrogenase activity from ammonium-induced suppression at pH 7.1 and 8.1. At pH 93 and 10.2, methionine sulfoximine did not alleviate the suppression of nitrogenase by ammonium. This pH-dependent protection of nitrogenase activity is a result of the noncompetitive inhibition of the ammonium transporter by methionine sulfoximine. At pH 7.1 and 8.2, ammonium is protonated and methionine sulfoximine inhibits its entry into the cell. At pH 93 and 10.2, unprotonated ammonia is abundant and may enter the cell independent of the transport system. The effects of ammonium are closely mimicked by the ammonium analog methylamine. These results suggest that ammoniumperse is an important in vivo regulator ofnitrogen fixation and its function can be mimicked by methylamine. Previous studies employing methionine sulfoximine may have to be re-evaluated in light of the inhibitory effects of methionine sulfoximine on the ammonium transporter.In the absence ofcombined nitrogen, heterocystous blue-green algae fix atmospheric N2 and produce NH3 which is subsequently assimilated by the GS2/GOGAT pathway (2,8,20,21). Exogenous NH3 inhibits nitrogenase activity and heterocyst differentiation in nitrogen fixing algae (3). Stewart and Rowell (20) have shown that this effect can be alleviated by the presence ofthe GS inhibitor L-methionine-DL-sulfoximine (MSX). This result prompted Stewart and Rowell (20) to suggest that the inhibition of nitrogen fixation was not caused by ammonium directly but rather by GS, GOGAT, or a product ofammonium assimilation. Subsequent work (14) suggested that the size of the glutamine pool was an important factor in nitrogenase regulation. Recent Cultures were maintained at 30°C in 400-ml culture bottles bubbled with filter sterilized laboratory air which had been passed through an activated charcoal filter in series with two gas scrubbers containing 1 N H2SO4 and distilled H20, respectively. This treatment removed particulate matter and gaseous ammonia while at the same time saturating the air with water vapor. Continuous illumination was provided by two Gro-lux fluorescent tubes. The quantum flux density was attenuated to 40 gE m-2 s-' with neutral density screening. Experiments were carried out using 20-ml portions of algal cultures in mid-logarithmic phase of growth (1-2 ,ug Chl a ml-'). These portions were incubated in 40-ml air-bubbled test tubes. Culture pH was adjusted to pH 7.1, 8.1, and 10.2 with phosphate (20 mM), bicine (20 mM), and CHES (20 mM), respectively.Nitrogenase Activity. Nitrogenase activity was determined by acetylene reduction. Acetylene reduction was measured using 2-ml aliquots placed in 1 5-ml test tubes sealed with rubber septa. The tubes were preincubated for 10 min in a shaking water bath and illuminated with a quantum...

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The initial organic products of fixation of 13N-labeled nitrogen gas by the blue-green alga Anabaena cylindrica

Cited by 49 publications

References 16 publications

Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides

Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides

The Sakaguchi Reaction Product Quenches Phycobilisome Fluorescence, Allowing Determination of the Arginine Concentration in Cells of Anabaena Strain PCC 7120

In Vivo Nitrogenase Regulation by Ammonium and Methylamine and the Effect of MSX on Ammonium Transport in Anabaena flos-aquae

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