Deletion of the transcriptional regulator cyAbrB2 deregulates primary carbon metabolism in Synechocystis sp. PCC 6803

Abstract

cyAbrB is a transcriptional regulator unique to and highly conserved among cyanobacterial species. A gene-disrupted mutant of cyabrB2 (sll0822) in Synechocystis sp. PCC 6803 exhibited severe growth inhibition and abnormal accumulation of glycogen granules within cells under photomixotrophic conditions. Within 6 h after the shift to photomixotrophic conditions, sodium bicarbonate-dependent oxygen evolution activity markedly declined in the ΔcyabrB2 mutant, but the decrease in methyl viologen-dependent electron transport activity was much smaller, indicating inhibition in carbon dioxide fixation. Decreases in the transcript levels of several genes related to sugar catabolism, carbon dioxide fixation, and nitrogen metabolism were also observed within 6 h. Metabolome analysis by capillary electrophoresis mass spectrometry revealed that several metabolites accumulated differently in the wild-type and mutant strains. For example, the amounts of pyruvate and 2-oxoglutarate (2OG) were significantly lower in the mutant than in the wild type, irrespective of trophic conditions. The growth rate of the ΔcyabrB2 mutant was restored to a level comparable to that under photoautotrophic conditions by addition of 2OG to the growth medium under photomixotrophic conditions. Activities of various metabolic processes, including carbon dioxide fixation, respiration, and nitrogen assimilation, seemed to be enhanced by 2OG addition. These observations suggest that cyAbrB2 is essential for the active transcription of genes related to carbon and nitrogen metabolism upon a shift to photomixotrophic conditions. Deletion of cyAbrB2 is likely to deregulate the partition of carbon between storage forms and soluble forms used for biosynthetic purposes. This disorder may cause inactivation of cellular metabolism, excess accumulation of reducing equivalents, and subsequent loss of viability under photomixotrophic conditions.

Figure 1.

Growth properties and pigment contents of the wild type and the ΔcyabrB2 mutant under photomixotrophic conditions. At time 0, the wild type and the ΔcyabrB2 mutant grown under photoautotrophic conditions (20 μmol photons m^–2 s^–1, ambient CO₂) were transferred to photomixotrophic conditions with 5 mm Glc, and changes at OD₇₃₀ (A), chlorophyll content (B), and phycocyanin content (C) were monitored. Data and error bars were calculated from the results of three independent experiments. WT, Wild type.

Figure 2.

Electron micrographs of ultrathin sections of wild-type and ΔcyabrB2 mutant cells before and after incubation under photomixotrophic conditions for 12 h. Magnified images of one cell are shown in the lower panels. WT, Wild type; Ca, carboxysomes; G, glycogen granules; Cy, cyanophycins; P, residual holes of polyphosphate bodies. Bars = 1 μm.

Figure 3.

Changes in photosynthetic activities of wild-type and ΔcyabrB2 mutant cells upon a shift to photomixotrophic conditions. A, The light saturation curves of photosynthesis in wild-type and ΔcyabrB2 mutant cells incubated under photomixotrophic conditions for 0, 6, or 12 h. Rates of CO₂-dependent O₂ evolution on a per OD₇₃₀ basis were measured as a function of actinic light intensity. Data and error bars were calculated from the results of at least three independent experiments. B, Comparison of CO₂-dependent photosynthetic activity and methyl viologen-dependent whole-chain electron transport activity before and after incubation under photomixotrophic conditions for 6 h. Measurements were performed under saturated light conditions (200 μmol photons m^–2 s^–1). Methyl viologen-dependent electron transport activity was measured as O₂ consumption rate on a per OD₇₃₀ basis. Data and error bars were calculated from the results of at least three independent experiments. WT, Wild type; MV, methyl viologen.

Figure 4.

Changes in transcript levels of the wild type and the ΔcyabrB2 mutant upon the shift to photomixotrophic conditions as determined by RNA gel-blot analysis. The amount of total RNA loaded per lane was as follows: 2 μg for rbcL, urtA, and glnB, 4 μg for gnd, and 6 μg for sigE, ppc, pyk1, pyk2, and icd. Total RNA was stained with methylene blue to compare within each blot. WT, Wild type.

Figure 5.

Metabolome analysis of the wild type and ΔcyabrB2 mutant under different trophic conditions. The wild type and ΔcyabrB2 mutant were grown photoautotrophically (Glc–) or photomixotrophically (Glc+) for 12 h, and then metabolites were extracted and analyzed by CE/MS. Data and error bars were calculated from the results of four independent experiments (nmol g^–1 fresh weight). WT, Wild type.

Figure 6.

Effects of addition of metabolites to the culture medium on growth of the ΔcyabrB2 mutant under photomixotrophic conditions. At time 0, the ΔcyabrB2 mutant grown under photoautotrophic conditions (20 μmol photons m^–2 s^–1, ambient CO₂) was supplemented with 5 mm Glc and 40 mm metabolites. Growth profiles of the mutant under photoautotrophic conditions (no addition), under photomixotrophic conditions without addition of any metabolites (+Glc), under photomixotrophic conditions with 2OG (+Glc, +2OG), with pyruvate (+Glc, +pyruvate), and with Glu (+Glc, +Glu) were determined. Data and error bars were calculated from the results of three independent experiments.

Figure 7.

Effects of addition of 2OG to the culture medium on the photosynthetic activity of the ΔcyabrB2 mutant under photomixotrophic conditions. The light saturation curve of photosynthesis in ΔcyabrB2 mutant cells incubated under photomixotrophic conditions with or without 2OG for 0, 6, or 12 h is presented. Rates of CO₂-dependent O₂ evolution on a per OD₇₃₀ basis were measured as a function of actinic light intensity. Data and error bars were calculated from the results of at least three independent experiments.

Figure 8.

Effects of addition of 2OG to the culture medium on cellular metabolism of the ΔcyabrB2 mutant under photomixotrophic conditions. The ΔcyabrB2 mutant was incubated under photomixotrophic conditions with or without 40 mm 2OG for 24 h, and then metabolites were extracted and analyzed by CE/MS. Extent of induction or repression upon addition of 2OG is shown as a fold-change value. Values are means ± sd of three independent and duplicate experiments (n = 6). Increased (more than 2-fold, P < 0.05) and decreased (less than one-half, P < 0.05) metabolite levels are indicated by boxes and shading, respectively.