Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Altenburger, Rolf; Abarzua, Sibylle; Callies, R.; Grimme, L. Horst; Mayer, A.; Leibfritz, Dieter

doi:10.1007/bf00245394

Cited by 15 publications

(4 citation statements)

References 19 publications

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“…The rationale used earlier for 13 C-NMR of carbon fluxes was applied similarly to nitrogen fluxes: living organisms fed with natural abundance nutrients (99.6% 14 N, 0.4% 15 N) are suddenly exposed to 15 N-labeled precursors; 15 N-NMR spectroscopy then reports the integration of 15 N atoms in the first metabolic products, the natural abundance background signal being close to zero. Using this 15 N-NMR approach, nitrogen metabolism was characterized in live microorganisms such as the fungus Neurospora crassa [873,874], C. glutamicum, a bacterium used for industrial production of amino acids [875,876], Pseudomonas [758], or cyanobacteria [877], and even once in mammalian cells [878]. From the late 1980's, most 15 N-NMR studies on living organisms were concerned with nitrogen assimilation by plants, focusing on symbiotic bacteria [879] or fungi [880][881][882], algae [883], spruce buds [884] or cell cultures [885], maize root [886][887][888], duckweed [889], invasive weed [890,891] and even carrot cell suspensions [892,893].…”

Section: Nitrogen Flux Studied Bymentioning

confidence: 99%

In-cell NMR: Why and how?

Theillet

Luchinat

2022

Progress in Nuclear Magnetic Resonance Spectroscopy

105

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Section: Nitrogen Flux Studied Bymentioning

confidence: 99%

In-cell NMR: Why and how?

Theillet

Luchinat

2022

Progress in Nuclear Magnetic Resonance Spectroscopy

105

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“…Paramagnetic ions exert a significant influence on the 15 N NOE by their contributing to the relaxation of the 15 N nucleus. This can lead to a variable behavior of 15 N signal intensities depending on environmental conditions such as medium mineral composition and cell density (1,15) as observed in this study (cf. Fig.…”

Section: Discussionmentioning

confidence: 62%

In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by ¹⁵ N Nuclear Magnetic Resonance

Tesch

Graaf

Sahm

1999

Appl Environ Microbiol

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Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicumstrains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4 + was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.

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“…This process runs over a period of several hours, and is caused by a changing magnitude of the NOE (nuclear Overhauser effect) when the protons bound to the ' 'N in the ' "^NHI molecules are decoupled (Altenburger et al 1991). In order to understand this phenomenon, Altenburger et al (1991) used cells of Chlorella fusca to check if intracellular ' "^NHJ contributes to the resonance at -353.9 ppm. They concluded that the inverted '^NHI signal comes from extracellular ''^NH4 associated with the cells.…”

Section: Discussionmentioning

confidence: 99%

“…The spectra were plotted with positive peak orientation, even though NOEs for ' 'N are usually negative in small molecules. Further conditions were as described by Altenburger et al (1991). During the measurement, between acquisition times, the cells were aerated with 100% N2 or 95% N. + 5% CO2.…”

Section: '^N-nmr Spectroscopymentioning

confidence: 99%

Ammonium rhythm in cultures of the cyanobacterium Microcystis firma

Abarzua¹,

Altenburger²,

Callies³

et al. 1993

Physiol Plant

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Schiewer, U. 1993. Ammonium rhythm in cultures of the cyanobacterium Microcystis firma. -Physiol. Plant. 89: 659-663. Over a period of several days, rhythmic changes in extracellular NHl concentration take place in cultures of the cyanobacterium Microcystis firma (Breb et Lenorm.) Schmidle, strain Gromov/St. Petersb. 398, under conditions of restricted CO. supply and light/dark alternation. The changes are enhanced by nitrate supply. Among the various processes generating intracellular NHt (NH:J uptake, NOj reduction, protein and amino acid degradation, photorespiration), NO3 reduction appears as the one most important. This can be concluded from experiments with and without nitrate and/or ammonium in the medium. In the presence of saturating CO2, continuous light, or continuous darkness, rhythmic NH^ oscillations are not induced. Studies of the incorporation of NHJ nitrogen by in vivo '"^N-NMR show that if CO2 is supplied, '^N is accumulated in several components with the following time course: in the first hour in Gin (5), in the second hour in the a-amino groups of most nonbranched amino acids, in the third hour in y-aminobutyric acid (GABA), Om (d) and Lys (e), and in the sixth hour in Ala. Carbon limitation, however, results in accumulation of label in the amide nitrogen of glutamine only.

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Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cited by 15 publications

References 19 publications

In-cell NMR: Why and how?

In-cell NMR: Why and how?

In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by ¹⁵ N Nuclear Magnetic Resonance

Ammonium rhythm in cultures of the cyanobacterium Microcystis firma

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Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cited by 15 publications

References 19 publications

In-cell NMR: Why and how?

In-cell NMR: Why and how?

In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by 15 N Nuclear Magnetic Resonance

Ammonium rhythm in cultures of the cyanobacterium Microcystis firma

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In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by ¹⁵ N Nuclear Magnetic Resonance