Deletion of the Synechocystis sp. PCC 6803 kaiAB1C1 gene cluster causes impaired cell growth under light–dark conditions

Abstract

In contrast to Synechococcus elongatus PCC 7942, few data exist on the timing mechanism of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiAB1C1 operon present in this organism was shown to encode a functional KaiC protein that interacted with KaiA, similar to the S. elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect when grown under light–dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown under constant light. Microarray experiments performed with cells grown for 1 day under a light–dark cycle revealed many differentially regulated genes with known functions in the ΔkaiABC mutant in comparison with the WT. We identified the genes encoding the cyanobacterial phytochrome Cph1 and the light-repressed protein LrtA as well as several hypothetical ORFs with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in ΔkaiABC cells in comparison with the WT. In general, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark. Strikingly, deletion of the ΔkaiABC operon led to a much stronger phenotype under light–dark cycles in Synechocystis sp. PCC 6803 than in Synechococcus sp. PCC 7942.

The role of the Synechocystis sp. PCC 6803 homolog of the circadian clock output regulator RpaA in day–night transitions

“…The discrepancies between the ∆ rpaA and ∆ sasA strain can be explained by a different experimental design, as our samples were taken in the middle of the light and dark periods, whereas Singh and Sherman () have analyzed gene expression after 1 h in the dark (for microarray experiments) and up to 3 h darkness (in Northern blot experiments). Remarkably, the mutants Δ sasA and Δ kaiAB1C1 show strongly affected transcript accumulation of cph1 and its response regulator rcp1 (Singh and Sherman, ; Dörrich et al ., ), similar to the Δ rpaA deletion mutant in our work. However, a ∆ cph1/rcp1 strain did not show any phenotype in a light‐dark cycle or under mixotrophic growth conditions (Singh and Sherman, ).…”

“…Eventually, reduced transcript accumulation of genes encoding enzymes of the OPPP or the glycolytic pathway can lead to shortages in carbon metabolism, favoring growth defects. In accordance with an earlier microarray analysis of the ∆ kaiAB1C1 mutant (Dörrich et al ., ), the gene of the sigma factor SigE (Sll1689), a positive regulator of sugar catabolic genes of the OPPP and glycogen catabolism (Osanai et al ., ), showed a 1.8 log 2 ‐fold reduction in expression during the light phase (Table S4). From previous reports it is known, that sigE mutants show reduced viability under heterotrophic growth (Osanai et al ., ).…”

“…However, these studies did not analyze the behavior of the mutant in a light‐dark cycle. Similar to the kaiAB1C1 and sasA mutants, the ∆ rpaA strain in our study showed a pronounced growth deficiency in light‐dark cycles, especially under mixotrophic conditions (Singh and Sherman, ; Dörrich et al ., ). Singh and Sherman () have analyzed the expression of several genes encoding enzymes involved in glycolysis, OPPP and glycogen metabolism in their ∆ sasA mutant under photoautotrophic and mixotrophic growth.…”

The overproduction ofSynechocystissp. PCC 6803 heat-shock protein (Sll0170) protectsEscherichia coliagainst high-temperature stress

“…[7,23,24] It has been suggested that dnaK2 is unique among the three dnaK genes exhibiting a stress response, although they are expressed in the same cellular compartment. [25] This suggestion strongly supports the functional assignment of these DnaK proteins, and the highly conserved sequence of DnaK2 implies that it has the most essential Hsp70 functions. [25] In this study, E. coli was used as a model system to test whether the recombinant DnaK2 can protect the microorganism cells against high-temperature stress.…”

“…[25] This suggestion strongly supports the functional assignment of these DnaK proteins, and the highly conserved sequence of DnaK2 implies that it has the most essential Hsp70 functions. [25] In this study, E. coli was used as a model system to test whether the recombinant DnaK2 can protect the microorganism cells against high-temperature stress. Such stress was produced in vitro by transferring the cell cultures from 37 C to 45 C (heat-shock stress) for 15 min and then back to 37 C. The cells were also exposed to high-temperature stress (52 C) for different lengths of time.…”