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Nitrogen Starvation Acclimation in Synechococcus elongatus: Redox-Control and the Role of Nitrate Reduction as an Electron Sink.

Klotz A, Reinhold E, Doello S, Forchhammer K - Life (Basel) (2015)

Bottom Line: This study established a method that allows uncoupling metabolic and redox-signals involved in nitrogen-starvation acclimation.This study demonstrates that nitrogen starvation is perceived not only through metabolic signals, but requires a redox signal indicating over-reduction of PSI-reduced electron acceptors.It further establishes a cryptic role of nitrate/nitrite reductases as electron sinks to balance conditions of over-reduction.

View Article: PubMed Central - PubMed

Affiliation: Interfakultäres Institut für Mikrobiologie und Infektionsmedizin der Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany. alexander.klotz@uni-tuebingen.de.

ABSTRACT
Nitrogen starvation acclimation in non-diazotrophic cyanobacteria is characterized by a process termed chlorosis, where the light harvesting pigments are degraded and the cells gradually tune down photosynthetic and metabolic activities. The chlorosis response is governed by a complex and poorly understood regulatory network, which converges at the expression of the nblA gene, the triggering factor for phycobiliprotein degradation. This study established a method that allows uncoupling metabolic and redox-signals involved in nitrogen-starvation acclimation. Inhibition of glutamine synthetase (GS) by a precise dosage of l-methionine-sulfoximine (MSX) mimics the metabolic situation of nitrogen starvation. Addition of nitrate to such MSX-inhibited cells eliminates the associated redox-stress by enabling electron flow towards nitrate/nitrite reduction and thereby, prevents the induction of nblA expression and the associated chlorosis response. This study demonstrates that nitrogen starvation is perceived not only through metabolic signals, but requires a redox signal indicating over-reduction of PSI-reduced electron acceptors. It further establishes a cryptic role of nitrate/nitrite reductases as electron sinks to balance conditions of over-reduction.

No MeSH data available.


Related in: MedlinePlus

(A) Measurement of variable fluorescence from PSII by PAM-fluorometry after 4, 20, and 24 hours of MSX-treatment. (B) Determination of the colony forming units (CFU) after 24 h treatment of S. elongatus cells with different concentrations of MSX.
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life-05-00888-f004: (A) Measurement of variable fluorescence from PSII by PAM-fluorometry after 4, 20, and 24 hours of MSX-treatment. (B) Determination of the colony forming units (CFU) after 24 h treatment of S. elongatus cells with different concentrations of MSX.

Mentions: To determine, how MSX affects the viability of the cells, cultures were treated with different concentrations of MSX and at different time points; variable fluorescence from PSII was measured by PAM fluorometry as an indication of functional PSII repair. Loss of viability leads to cessation of PSII repair, and in consequence to a loss of variable PSII fluorescence [39]. As shown in Figure 4A, there is a gradual loss of variable fluorescence with increasing MSX concentrations at prolonged incubation times, whereas after 4 h of incubation, MSX, even at higher concentrations, did not severely affect PSII fluorescence. Cells, which were incubated for 24 h with 5 µM MSX or more, were not able to maintain variable fluorescence, indicating a severe loss of viability. To directly assess viability, viable counts were determined by measuring the colony forming units after culturing the cells for 24 h in presence of different concentrations of MSX. As shown in Figure 4B, 5 µM causes a severe loss of CFUs, in agreement with the vanishing variable fluorescence. By comparison, at 2 µM MSX, more than two orders of magnitude more viable cells were detected. Therefore, we decided to use MSX at 2 µM in the following experiments, a concentration, which maximally induces nblA expression and completely inhibits GS activity. Furthermore, we focused on the first hours of MSX treatment in order to minimize artifactual results due to toxic side effects. A further control experiment confirmed that cells treated for 5 h with 2 µM MSX are able to fully recover from this treatment (Supplementary Figure S1).


Nitrogen Starvation Acclimation in Synechococcus elongatus: Redox-Control and the Role of Nitrate Reduction as an Electron Sink.

Klotz A, Reinhold E, Doello S, Forchhammer K - Life (Basel) (2015)

(A) Measurement of variable fluorescence from PSII by PAM-fluorometry after 4, 20, and 24 hours of MSX-treatment. (B) Determination of the colony forming units (CFU) after 24 h treatment of S. elongatus cells with different concentrations of MSX.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390884&req=5

life-05-00888-f004: (A) Measurement of variable fluorescence from PSII by PAM-fluorometry after 4, 20, and 24 hours of MSX-treatment. (B) Determination of the colony forming units (CFU) after 24 h treatment of S. elongatus cells with different concentrations of MSX.
Mentions: To determine, how MSX affects the viability of the cells, cultures were treated with different concentrations of MSX and at different time points; variable fluorescence from PSII was measured by PAM fluorometry as an indication of functional PSII repair. Loss of viability leads to cessation of PSII repair, and in consequence to a loss of variable PSII fluorescence [39]. As shown in Figure 4A, there is a gradual loss of variable fluorescence with increasing MSX concentrations at prolonged incubation times, whereas after 4 h of incubation, MSX, even at higher concentrations, did not severely affect PSII fluorescence. Cells, which were incubated for 24 h with 5 µM MSX or more, were not able to maintain variable fluorescence, indicating a severe loss of viability. To directly assess viability, viable counts were determined by measuring the colony forming units after culturing the cells for 24 h in presence of different concentrations of MSX. As shown in Figure 4B, 5 µM causes a severe loss of CFUs, in agreement with the vanishing variable fluorescence. By comparison, at 2 µM MSX, more than two orders of magnitude more viable cells were detected. Therefore, we decided to use MSX at 2 µM in the following experiments, a concentration, which maximally induces nblA expression and completely inhibits GS activity. Furthermore, we focused on the first hours of MSX treatment in order to minimize artifactual results due to toxic side effects. A further control experiment confirmed that cells treated for 5 h with 2 µM MSX are able to fully recover from this treatment (Supplementary Figure S1).

Bottom Line: This study established a method that allows uncoupling metabolic and redox-signals involved in nitrogen-starvation acclimation.This study demonstrates that nitrogen starvation is perceived not only through metabolic signals, but requires a redox signal indicating over-reduction of PSI-reduced electron acceptors.It further establishes a cryptic role of nitrate/nitrite reductases as electron sinks to balance conditions of over-reduction.

View Article: PubMed Central - PubMed

Affiliation: Interfakultäres Institut für Mikrobiologie und Infektionsmedizin der Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany. alexander.klotz@uni-tuebingen.de.

ABSTRACT
Nitrogen starvation acclimation in non-diazotrophic cyanobacteria is characterized by a process termed chlorosis, where the light harvesting pigments are degraded and the cells gradually tune down photosynthetic and metabolic activities. The chlorosis response is governed by a complex and poorly understood regulatory network, which converges at the expression of the nblA gene, the triggering factor for phycobiliprotein degradation. This study established a method that allows uncoupling metabolic and redox-signals involved in nitrogen-starvation acclimation. Inhibition of glutamine synthetase (GS) by a precise dosage of l-methionine-sulfoximine (MSX) mimics the metabolic situation of nitrogen starvation. Addition of nitrate to such MSX-inhibited cells eliminates the associated redox-stress by enabling electron flow towards nitrate/nitrite reduction and thereby, prevents the induction of nblA expression and the associated chlorosis response. This study demonstrates that nitrogen starvation is perceived not only through metabolic signals, but requires a redox signal indicating over-reduction of PSI-reduced electron acceptors. It further establishes a cryptic role of nitrate/nitrite reductases as electron sinks to balance conditions of over-reduction.

No MeSH data available.


Related in: MedlinePlus