Long-term response toward inorganic carbon limitation in wild type and glycolate turnover mutants of the cyanobacterium Synechocystis sp. strain PCC 6803

Abstract

Concerted changes in the transcriptional pattern and physiological traits that result from long-term (here defined as up to 24 h) limitation of inorganic carbon (C(i)) have been investigated for the cyanobacterium Synechocystis sp. strain PCC 6803. Results from reverse transcription-polymerase chain reaction and genome-wide DNA microarray analyses indicated stable up-regulation of genes for inducible CO(2) and HCO(3)(-) uptake systems and of the rfb cluster that encodes enzymes involved in outer cell wall polysaccharide synthesis. Coordinated up-regulation of photosystem I genes was further found and supported by a higher photosystem I content and activity under low C(i) (LC) conditions. Bacterial-type glycerate pathway genes were induced by LC conditions, in contrast to the genes for the plant-like photorespiratory C2 cycle. Down-regulation was observed for nitrate assimilation genes and surprisingly also for almost all carboxysomal proteins. However, for the latter the observed elongation of the half-life time of the large subunit of Rubisco protein may render compensation. Mutants defective in glycolate turnover (DeltaglcD and DeltagcvT) showed some transcriptional changes under high C(i) conditions that are characteristic for LC conditions in wild-type cells, like a modest down-regulation of carboxysomal genes. Properties under LC conditions were comparable to LC wild type, including the strong response of genes encoding inducible high-affinity C(i) uptake systems. Electron microscopy revealed a conspicuous increase in number of carboxysomes per cell in mutant DeltaglcD already under high C(i) conditions. These data indicate that an increased level of photorespiratory intermediates may affect carboxysomal components but does not intervene with the expression of majority of LC inducible genes.

Figure 1.

Semiquantitative RT-PCR of C_i responsive genes sbtA, bicA, rbcL, ccmK2, and flv3. Wild-type cells were grown in BG11 medium at pH 7 and bubbled with air supplemented with 5% CO₂. Samples were taken 0, 1, 2, 3, 12, and 24 h after transfer to pure air. The transcript level of the constitutive expressed gene rnpB served as control.

Figure 2.

Characterization of relative PSI content by fluorescence emission spectra and PSI activity by P700 absorbance changes of wild-type cells grown in BG11 medium at pH 7 and bubbled with air supplemented with 5% CO₂ (HC) or for 24 h with pure air (LC). A, 77 K fluorescence emission spectra of HC (thick line) and LC (thin line) acclimated wild-type cells at 440 nm excitation. Mean values of four independent samples of each culture are shown, which were standardized between 780 and 688 nm. sd is indicated by the vertical bars. B and C, Effects of different far-red (FR) irradiance on P700 redox state in the presence (black circles) and absence (white circles) of strong white light (+WL), exciting PSII. Mean values of five independent samples of wild-type cells grown at HC (B) or LC (C) conditions are shown (bars indicate sd).

Figure 3.

Semiquantitative RT-PCR and immunoblotting to determine effects of mutation in gcvT and glcD on the expression of rbcL. RNA was isolated from cells of wild type, ΔgcvT, or ΔglcD grown in BG11 medium at pH 7 and bubbled with air supplemented with 5% CO₂. The transcript level of the constitutively expressed gene rnpB served as control.

Figure 4.

Effect of long-term C_i limitation on the transcript and protein level of RbcL (A) and CcmK (B). Wild-type cells were grown in BG11 medium at pH 7 and bubbled with air supplemented with 5% CO₂. Samples were taken 0, 12, and 24 h after transfer to pure air. The rbcL and ccmK2 transcript levels were compared by semiquantitative RT-PCR (top sections) and the amount of RbcL or CcmK proteins were checked by immunoblotting using specific antibodies (bottom sections). In the immunoblotting experiments the same amount of total protein (5 μg) was applied to each lane.

Figure 5.

Effect of long-term C_i limitation on the half-life of RbcL. Wild-type cells were grown in BG11 medium at pH 7 and bubbled with air supplemented with 5% CO₂ (HC) or with pure air (LC), as indicated. At time point zero the translational inhibitor lincomycin (final concentration 250 μg mL⁻¹) was added. Samples were taken 0, 3, 6, 9, 12, and 24 h after application of lincomycin. Same amount of total protein (5 μg) was applied to each lane. RbcL was detected by immunoblotting using a specific antibody.

Figure 6.

Changes in cell morphology during acclimation toward LC conditions. A, Representative electron micrographs of wild type (a + d), ΔgcvT (b + e), and ΔglcD (c + f) cells, respectively, are shown. Cells were pregrown in BG11 medium at pH 7 and 5% CO₂. Samples were taken before (top section) and 24 h after transfer to pure air (bottom row). Carboxysomes (C), polyphosphate bodies (P), and glycogen granules (G) are marked by arrows. B, Carboxysomes per cell were counted from 50 thin sections per strain and treatment, presumably cut in the middle of the cell. Means and confidence intervals are shown. *, Statistically significant difference in the carboxysome number compared to wild-type cells grown with 5% CO₂ (P ≤ 0.05, n = 50).