The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and non-targeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaA and, to a lesser extent, in the ΔkaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.
The rotation of the Earth results in predictable environmental changes that pose challenges for organisms and force them to adapt. To address this daily rhythm, organisms from all kingdoms of life have evolved diverse timing mechanisms. In the cyanobacterium Synechococcus elongatus PCC 7942, the three proteins KaiA, KaiB, and KaiC constitute the central timing mechanism that drives circadian oscillations. In addition to the standard oscillator, named KaiAB1C1, Synechocystis sp. PCC 6803 harbors several, diverged clock homologs. The nonstandard KaiB3-KaiC3 system was suggested to impact the metabolic switch in response to darkness. Here, we demonstrate the direct interaction of KaiC3 with Sll0485, which is a potential new chimeric KaiA homolog that we named KaiA3. The existence of a functional link between these proteins is further supported by the co occurrence of genes encoding KaiA3 with the KaiB3-KaiC3-like gene products in 10 cyanobacterial and five other bacterial species. KaiA3 is annotated as a NarL-type response regulator due to its similarity to the response regulator receiver domains. However, its similarity to canonical NarL drastically decreases in the C-terminal domain, which resembles the circadian clock protein KaiA. In line with this, we detected the stimulation of KaiC3 phosphorylation by KaiA3 in vitro. Furthermore, we showed that deletion of the kaiA3 gene led to growth defects during mixotrophic growth conditions and, like a kaiC3-deficient mutant, viability was impaired during chemoheterotrophic growth in complete darkness. In summary, we suggest KaiA3 as a novel, nonstandard KaiA homolog within the cyanobacterial phylum, extending the KaiB3-KaiC3 system in Cyanobacteria and other prokaryotes.
The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and nontargeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaA and, to a lesser extent, in the ΔkaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.
hängig von der Kohlenstoff-und Stickstoffverfügbarkeit, einen Komplex entweder mit dem zentralen Regulatorprotein PII oder der Phosphoglyceratmutase, die fixiertes CO 2 in Form von Glycerat-3-Phosphat in die Glykolyse einspeist. Unter Stickstoff-Mangel wird der PII-PirC-Komplex aufgelöst; PirC kann nun die Phosphoglyceratmutase binden und hemmen. Kohlenstoff, der unter diesen Bedingungen normalerweise zum Aufbau des Speicherstoffs Glykogen dient, wird so in der Zelle in Gen in den SchlagzeilenDie Rolle von KAT7 bei der zellulären Alterung
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