Role of sigma factors in controlling global gene expression in light/dark transitions in the cyanobacterium Synechocystis sp. strain PCC 6803

Abstract

We report on differential gene expression in the cyanobacterium Synechocystis sp. strain PCC 6803 after light-dark transitions in wild-type, DeltasigB, and DeltasigD strains. We also studied the effect of day length in the presence of glucose on a DeltasigB DeltasigE mutant. Our results indicated that the absence of SigB or SigD predominately altered gene expression in the dark or in the light, respectively. In the light, approximately 350 genes displayed transcript levels in the DeltasigD strain that were different from those of the wild type, with over 200 of these up-regulated in the mutant. In the dark, removal of SigB altered more than 150 genes, and the levels of 136 of these were increased in the mutant compared to those in the wild type. The removal of both SigB and SigE had a major impact on gene expression under mixotrophic growth conditions and resulted in the inability of cells to grow in the presence of glucose with 8-h light and 16-h dark cycles. Our results indicated the importance of group II sigma factors in the global regulation of transcription in this organism and are best explained by using the sigma cycle paradigm with the stochastic release model described previously (R. A. Mooney, S. A. Darst, and R. Landick, Mol. Cell 20:335-345, 2005). We combined our results with the total protein levels of the sigma factors in the light and dark as calculated previously (S. Imamura, S. Yoshihara, S. Nakano, N. Shiozaki, A. Yamada, K. Tanaka, H. Takahashi, M. Asayama, and M. Shirai, J. Mol. Biol. 325:857-872, 2003; S. Imamura, M. Asayama, H. Takahashi, K. Tanaka, H. Takahashi, and M. Shirai, FEBS Lett. 554:357-362, 2003). Thus, we concluded that the control of global transcription is based on the amount of the various sigma factors present and able to bind RNA polymerase.

FIG. 1.

(A) Photoautotrophic growth of cultures from the ΔsigB and ΔsigD experiments. The bar (top) represents the light-dark regimen used in these experiments. Cells were grown for 72 h in continuous light before beginning the 12-h light/12-h dark cycles. The growth curves for the wild-type (square), ΔsigB (triangle), and ΔsigD (diamond) strains, as measured by the optical density at 750 nm (OD₇₅₀), are shown to scale beneath the bar. The data are the averages ± standard errors of three independent measurements. The arrows represent the time points L1 and D1, at which cells were harvested and RNA was extracted and used for microarray analysis. (B and C) Histogram of genes differentially expressed 1.5-fold or more (FDR = 0.05) at L1 and D1. The numbers of differentially expressed genes for different functional categories are plotted on the x axis, with genes up-regulated in the mutant shown above the zero line on the y axis and genes down-regulated shown beneath zero line. (B) Comparison of the ΔsigB strain to the wild type. (C) Comparison of the ΔsigD strain to the wild type. Genes are presented as Cyanobase functional categories, as defined in the key beneath the graphs. (D) The light-dark regimen for mixotrophic growth of wild-type and ΔsigB ΔsigE strains (top). The cells were grown for 72 h in the light before transfer to a 16-h dark/8-h light cycle. The addition of glucose (Glu) to a final concentration of 5 mM is indicated. The arrows on the top bar represent time points L7, D1, D8, and D16, the points at which wild-type cells were harvested and RNA was extracted and used for microarray analysis. The arrows on the second bar represent time points D1 and L1, the points at which wild-type and ΔsigB ΔsigE cells were harvested and RNA was extracted and used for microarray analysis. The growth curves for the wild type (square) and ΔsigB ΔsigE (diamond), as measured by the OD₇₅₀, are shown to scale beneath the bar. The data are the averages ± standard errors of three independent measurements. (E and F) Histogram of genes differentially expressed 1.5-fold or more (FDR = 0.05). (E) Genes differentially regulated in the wild type at L7 compared to D1 and D16. (F) Genes differentially regulated in the ΔsigB ΔsigE strain compared to the wild type at L1 and D1. The genes were divided into functional categories as described for panels B and C.

FIG. 2.

Effect of sigma mutations on heterotrophic growth. Cultures of the wild type (WT) and sigma mutants were grown photoautotrophically and were spotted (at the optical density at 750 nm indicated) onto BG-11 plates containing 5 mM glucose. Plates were incubated in the light for 96 h (left gels) and then were transferred to the dark for 96 h (right gels).

FIG. 3.

Relationship between sigma factor abundance and the impact of SigB or SigD removal on global regulation of gene expression in light-to-dark and dark-to-light transitions. (A) Wild-type Synechocystis sp. strain PCC 6803. The relative amounts of the sigma factor proteins are represented by the numbers above the sigma factors, based on the calculations of Imamura et al. (19). A twofold increase in SigB and a twofold decrease in SigE were observed following transfer to the dark for 1 h. Functional groups containing differentially regulated genes at 1 h of dark and 1 h of light are shown in boxes; the up arrow indicates that transcripts were up-regulated in the light compared to their status in the dark, and the two-headed arrow indicates that there were both up- and down-regulated genes in these groups. The rpl and rps genes encode ribosome protein large subunits and small subunits, respectively. (B) Differentially regulated genes in ΔsigB compared to regulation in the wild type at 1 h of dark and 1 h of light. The up arrow indicates functional categories that contained up-regulated transcripts in the mutant compared to the wild type. (C) Differentially regulated genes in the ΔsigD strain compared to the wild type at 1 h of dark and 1 h of light. The up arrow indicates functional categories that contain up-regulated transcripts in the mutant, and the two-headed arrow indicates both increased and decreased transcript abundance in genes belonging to these functional categories.

FIG. 4.

Model of sigma factor and histidine kinase interactions in the light and the dark. Interactions are based on transcript abundance in the Δhik8, ΔsigB, ΔsigD, and ΔsigE mutants compared to that of the wild type. The data are from Sherman and Singh (43), this study, and Osanai et al. (35) for the Δhik8 strain, ΔsigB and ΔsigD strains, and ΔsigE strain, respectively. The light/dark conditions at which the results were obtained are shown in the key.