Homologs of Circadian Clock Proteins Impact the Metabolic Switch Between Light and Dark Growth in the Cyanobacterium Synechocystis sp. PCC 6803

Abstract

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 role of the 5’ sensing function of ribonuclease E in cyanobacteria

“…Comparison of the growth under different conditions of mutant strains to the WT, revealed that the application of glucose in light-dark cycles had the strongest effect on the growth of rne (5p). A similar phenotype has been described for Synechocystis mutants lacking genes encoding components of the circadian clock [ 96 ], the sRNA Ncr0700/PmgR1 [ 75 ]; or alternative sigma factors [ 97 ], regulatory factors which all target a large number of genes. However, due to the large variety of transcript types and segments affected by RNase E, we are not able to relate growth defects with any specific targets.…”

Diurnal rhythm causes metabolic crises in the cyanobacterial mutants of c-di-AMP signalling cascade

“…3 ). Furthermore, elevated ROS and glutathione levels is another aspect of the metabolic crisis, which the mutants undergo in diurnal growth and which, besides others, ultimately leads to cell death, similar to what observed in the mutant of the circadian clock regulator, RpaA (Diamond et al 2017, Scheurer et al 2021). Notably, our proteomics dataset revealed a slight reduction of RpaA ( slr0115 ) levels in Δ dacA ( Supplemental Table S3 ).…”

Positional13C Enrichment Analysis of Aspartate by GC-MS to Determine PEPC ActivityIn Vivo

“…To find these limits of E 13 C determination, all positional aspartic acid standards were combined equally and then isotopically diluted by natural aspartic acid in different proportions (Table S1). Injections of 1.25 ng up to 250 ng of each mixture were analyzed to cover the abundance-range of our analyses in agreement with the expected biological variation 28, 39 . Most fragment ions showed small absolute value deviations from expected E 13 C when 12.5 ng or more were injected ( Figure 2 A).…”