Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses

Abstract

Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP(+) showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium.

Importance: Cyanobacteria are photosynthetic microbes that use energy from sunlight and CO2 as feedstock. Certain cyanobacterial strains are amenable to facile genetic manipulation, thus enabling synthetic biology and metabolic engineering applications. Such strains are being developed as a chassis for the sustainable production of food, feed, and fuel. To this end, a holistic knowledge of cyanobacterial physiology and its correlation with gene expression patterns under the diurnal cycle is warranted. In this report, a genomewide transcriptional analysis of Synechocystis PCC 6803, the most widely studied model cyanobacterium, sheds light on the global coordination of cellular processes during diurnal periods. Furthermore, we found that, in addition to light, the redox level of NADP(H) is an important endogenous regulator of diurnal entrainment of Synechocystis PCC 6803.

FIG 1

Cycling of gene expression in Synechocystis PCC 6803. Almost two-third of the cyclic genes peaked at the end of the dark period (i.e., D11) or the early light period (i.e., L1 and L3). The time point of the peak expression of any gene was calculated by averaging its expression levels over two consecutive diurnal cycles. Percentages are of all cycling genes.

FIG 2

Expression profiles of genes with cyclic patterns involved in PSI (A), PSII (B), and membrane transport (C). L/D cycles are indicated as gray and white bars below the x axis, respectively. The log₂ ratios of transcript abundance to the pooled sample control are plotted on the y axis.

FIG 3

Relative changes in the expression levels (A) and metabolic fluxes (B) of major metabolic genes over the L/D cycle. Here, both gene expression and metabolic flux were scaled between 0 and 1.

FIG 4

Glycogen and ATP levels (A) and NADPH/NADP⁺ ratios (B) in cells during diurnal cycles. The transcriptional oscillation of genes involved in glycogen metabolism and ATP synthesis is shown in the insets in panel A. Cells were autotrophically cultured in BG11 medium under alternating 12-h L/D cycle conditions. Data points are the mean ± the standard deviation of three biological replicates.

FIG 5

Schematic representations of the differences in Synechocystis PCC 6803 during light and dark periods. Four phases of the diurnal cycles are highlighted: D1 through D9, D11, L1/L3, and L5 to L11. The thicknesses of the arrows in the metabolic map are proportional to the activities of specific metabolic or transport reactions under given conditions. Gray and black regulatory functions represent inactive and active regulatory genes, respectively. G6P, glucose 6-phosphate; Ru5p, ribulose 5-phosphate; Rubp, ribulose-1,5-bisphosphate; 6pgl, 6-phosphogluconolactone; 6pgn, 6-phosphogluconate; F6p, fructose 6-phosphate; X5p, xylulose 5-phosphate; R5p, ribose 5-phosphate; Fdp, fructose 1,6-bisphosphate; G3p, glyceraldehyde 3-phosphate; S7p, sedoheptulose 7-phosphate; Gp, glycerone phosphate; E4p, erythrose 4-phosphate; 13pdg, 1,3-bisphospho-d-glycerate; 3pg, 3-phosphoglyceric acid; Sbp, sedoheptulose-1,7-bisphosphate; Pep, phosphoenolpyruvate; Pyr, pyruvate; Accoa, acetyl CoA-carboxylase; 2kg, 2-ketoglutarate; Cbp, carbamoyl phosphate; Cynphy, cyanophycin; TM, transmembrane; ETC, electron transport chain; PC, plastocyanin; PQ, plastoquinone; Cyt b6f, cytochrome b6f complex.