Pleiotropic effect of a histidine kinase on carbohydrate metabolism in Synechocystis sp. strain PCC 6803 and its requirement for heterotrophic growth

Abstract

The deletion of a gene coding for a histidine kinase (sll0750, Hik8) in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 resulted in a conditional lethal phenotype with a pleiotropic effect on the expression of genes involved in glucose metabolism. This mutant had comparable doubling times to wild type (WT) in continuous-light-grown photoautotrophic and mixotrophic cultures, whereas it grew poorly under mixotrophic conditions with different light and dark cycles. Growth was completely stopped, and cells eventually died, when the light duration was less than 6 h on a 24-h regimen. Northern blot analysis demonstrated that steady-state transcript levels of genes encoding key enzymes of glycolysis, gluconeogenesis, the oxidative pentose phosphate pathway, and glycogen metabolism were significantly altered in a strain with mutant hik8 (Deltahik8) grown with or without glucose. In some cases, differential expression was dependent on growth conditions (photoautotrophic versus mixotrophic). The enzyme activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphofructokinase were significantly reduced in Deltahik8 compared to WT. Glycogen determination indicated that Deltahik8 accumulated glycogen under mixotrophic conditions but was unable to utilize these reserves for heterotrophic growth. The results suggest that the loss of gap1 transcription in the absence of Hik8 was the key factor that rendered cells unable to catabolize glucose and grow heterotrophically. Additionally, the transcript levels of the phytochrome gene (cph1) and its cotranscribed response regulator gene (rcp1) were significantly reduced and its dark inducibility was lost in Deltahik8. The results demonstrated that Hik8 plays an important role in glucose metabolism and is necessary for heterotrophic growth.

FIG. 1.

Simplified scheme depicting the glycolytic, OPP, and gluconeogenic pathways in Synechocystis sp. strain PCC 6803. The key enzymes that were examined in the present study are shown with possible redundant genes, based on the Kazusa annotation. The dotted, solid single, and double arrows indicate the direction of glucose (Glc) metabolism under either HT or PA growth conditions or both, respectively.

FIG. 2.

Northern blots of Synechocystis sp. strain PCC 6803 genes encoding proteins involved in (i) glycolysis, (ii) gluconeogenesis and glycogen metabolism, (iii) OPP, and (iv) other functions. Strains monitored were WT, Δhik8, and Δcph1rcp1 grown under PA (A) or MT (B) conditions in LL (lane 1) or after transfer to dark for 0.25 (lane 2), 1 (lane 3), and 3 h (lane 4) and subsequently reillumination for 1 (lane 5) or 3 h (lane 6). Five micrograms of total RNA isolated from these samples was electrophoresed on a 1.2% agarose gel, transferred to a nylon membrane, and hybridized with the respective gene probes. The data are representative of three separate biological replicates. The autoradiograms of these genes were exposed for different time periods for the sake of clarity.

FIG. 3.

Glycogen content in WT (dotted bars), Δcph1rcp1 (line bars), and Δhik8 (cross line bars). Exponentially grown cells under PA (A) or MT (B) conditions were transferred to the dark (1, 3, and 5 h) and then reilluminated with light (1, 3, and 5 h) followed by 24 h of growth in the dark. The glycogen content was measured as described in Materials and Methods. The values for PA and MT are means ± standard errors for five and three separate experiments, respectively. Chl, chlorophyll.

FIG. 4.

Electron micrographs of Synechocystis sp. strain PCC 6803 PA-grown WT (A) and Δhik8 (B) cells. Cells were grown to mid-logarithmic phase under PA conditions, transferred to the dark for 4 h, and prepared for microscopy as described in Materials and Methods. Arrows indicate glycogen granules.

FIG. 5.

Diagrammatic model for Hik8 function. Hik8 is required for the transcription of cph1-rcp1 and various genes involved in glucose metabolism. The activation may be direct or indirect and possibly involves an unidentified response regulator. A possible interaction between Hik8 and KaiABC is shown based on the report of Iwasaki et al. (9). The dotted line indicates the potential, but nonproven, involvement of a Hik8 and Kai complex in the regulation of glycolytic genes. The question mark indicates the unknown genes regulated by Cph1-Rcp1.