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

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.

Figure 1

Treatment of ammonium-grown WT-C 103 cells with different concentrations of MSX (0–500 µM) (A,B) and measurement of luciferease reporter expression at time point 0, and after 1, 2, 3, 4, and 5 (A) as well as 24 h (B) of MSX treatment at concentrations indicated by color coding. (C) Chlorosis of WT-C 103 cells caused by treatment with MSX at the indicated concentrations for one day.

Figure 2

GS activity of Synechococcus WT-C 103 cells with different concentrations of MSX (0‑50 µM) after 4 h of incubation.

Figure 3

Expression of the glnB::luxAB reporter gene within 5 h after MSX addition at concentrations indicated using the FAM2 reporter strain and measuring luciferease activity.

Figure 4

(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.

Figure 5

Effect of nitrate and nitrite compared to ammonia on MSX-induced nblA::luxAB or glnB::luxAB expression. (A) nblA::luxAB expression of WT-C 103 cells growing in media with different nitrogen sources within 5 h after MSX treatment (2 µM): 17.7 mM nitrate, 5 mM nitrite and 5 mM ammonia. (B) Highlighting of the results of nblA::luxAB expression of WT-C 103 in nitrate (17.7 mM) or nitrite (5 mM) containing media. (C) glnB::luxAB expression of FAM2 cells in media with nitrate (17.7 mM) and ammonia (5 mM) within 5 h after MSX treatment (2 µM).

Figure 6

Absorbance spectra between 550 and 750 nm of S. elongatus PCC 7942 grown for 3 days in nitrate (A) or ammonia (B) containing medium after treatment with 2 µM MSX. To visualize the specific absorption of the pigments, the spectra were first normalized to an OD₇₅₀ of 1, then subtracted by 1 (to set the OD₇₅₀ to zero). Subsequently, a baseline correction was performed between OD₇₅₀ and OD₅₅₀ to eliminate light scattering effects.

Figure 7

Expression of the nblA:luxAB reporter in strain WT-C 103 incubated in BG₀ medium for 5 h after addition of 2 µM MSX together with different amounts of nitrate as indicated.

Figure 8

Effect of reduced PPDF on MSX-induced nblA and glnB expression. WT-C 103 and FAM2 cells were treated with 2 µM MSX prior to measurement and further propagated for 5 h under different light intensities (10 and 35 µmol photons m⁻² s⁻¹).

Figure 9

Bioluminescence measurement of nblA expression within 5 h after 2 µM MSX treatment and different amounts of ammonia (5 mM and 25 mM).

Figure 10

Determination of intracellular glycogen content in WT-C 103 cells incubated in BG₀ with nitrate or ammonia with or without 2 µM MSX within 24 h.