Many organisms survive stressful conditions via entry into a dormant state that can be rapidly exited when the stressor disappears; this ability provides a strong selective advantage. In the cyanobacterium sp. PCC 6803, the exit from nitrogen chlorosis takes less than 48 h and is enabled by the impressive metabolic flexibility of these cyanobacteria, which pass through heterotrophic and mixotrophic phases before reentering photoautotrophic growth. Switching between these states requires delicate coordination of carbohydrate oxidation, CO fixation, and photosynthesis. Here, we investigated the contribution of the different carbon catabolic routes by assessing mutants of these pathways during nitrogen chlorosis and resuscitation. The addition of nitrate to nitrogen-starved cells rapidly starts the awakening program. Metabolism switches from maintenance metabolism, characterized by residual photosynthesis and low cellular ATP levels, to an initial heterotrophic phase, characterized by respiration and an immediate increase in ATP levels. This respiration relies on glycogen breakdown catalyzed by the glycogen phosphorylase GlgP2. In the following transient mixotrophic phase, photosynthesis and CO fixation restart and glycogen is consumed. During the mixotrophic phase, parallel operation of the oxidative pentose phosphate cycle and the Entner-Doudoroff pathway is required for resuscitation to proceed; the glycolytic route via the Embden-Meyerhof-Parnas pathway has minor importance. Our data suggest that, during resuscitation, only the Entner-Doudoroff and oxidative pentose phosphate pathways supply the metabolic intermediates necessary for the anabolic reactions required to reconstitute a vegetative cell. Intriguingly, the key enzymes for glycogen catabolism are already expressed during the preceding chlorotic phase, in apparent preparation for rapid resuscitation.

Polyhydroxybutyrate (PHB) is a polymer of great interest as a substitute for conventional plastics, which are becoming an enormous environmental problem. PHB can be produced directly from CO2 in photoautotrophic cyanobacteria. The model cyanobacterium Synechocystis sp. PCC 6803 produces PHB under conditions of nitrogen starvation. However, it is so far unclear which metabolic pathways provide the precursor molecules for PHB synthesis during nitrogen starvation. In this study, we investigated if PHB could be derived from the main intracellular carbon pool, glycogen. A mutant of the major glycogen phosphorylase, GlgP2 (slr1367 product), was almost completely impaired in PHB synthesis. Conversely, in the absence of glycogen synthase GlgA1 (sll0945 product), cells not only produced less PHB, but were also impaired in acclimation to nitrogen depletion. To analyze the role of the various carbon catabolic pathways (EMP, ED and OPP pathways) for PHB production, mutants of key enzymes of these pathways were analyzed, showing different impact on PHB synthesis. Together, this study clearly indicates that PHB in glycogen-producing Synechocystis sp. PCC 6803 cells is produced from this carbon-pool during nitrogen starvation periods. This knowledge can be used for metabolic engineering to get closer to the overall goal of a sustainable, carbon-neutral bioplastic production.

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.

Highlights d Synechocystis engages sodium bioenergetics during nitrogen starvation d Sodium-dependent ATP synthesis is only essential during metabolic dormancy d ATP levels are finely tuned according to the metabolic requirements