Cyanobacterial phytochrome2 regulates the heterotrophic metabolism and has a function in the heat and high-light stress response

Abstract

Cyanobacteria combine the photosynthetic and respiratory electron transport in one membrane system, the thylakoid membrane. This feature requires an elaborate regulation mechanism to maintain a certain redox status of the electron transport chain, hence allowing proper photosynthetic and respiratory energy metabolism. In this context, metabolic adaptations, as seen in the light-to-dark and dark-to-light transitions, are particularly challenging. However, the molecular basis of the underlying regulatory mechanisms is not well-understood. Here, we describe a function of cyanobacterial phytochrome2 (Cph2), a phytochrome of the cyanobacterial model system Synechocystis sp. PCC 6803, in regulation of the primary energy metabolism. When cells are shifted from photoautotrophic planktonic growth to light-activated heterotrophic growth and biofilm initiation, knockout of Cph2 results in impaired growth, a decrease in the activity of Glc-6-P dehydrogenase, a decrease of the transcript abundance/activity of cytochrome-c-oxidase, and slower phycocyanin degradation. Measurements of the plastoquinone reduction confirm an impaired heterotrophic metabolism in the cph2 knockout. When cells that were adapted to heterotrophic metabolism are shifted back to light conditions, the knockout of Cph2 results in an altered photosystem II chlorophyll fluorescence induction curve, which is indicative of an impaired redox balance of the electron transport chain. Moreover, Cph2 plays a role in the heat and high-light stress response, particularly under photomixotrophic conditions. Our results show a function of Cph2 in the adaptation of the primary energy metabolism to changing trophic conditions. The physiological role of Cph2 in biofilm formation is discussed.

Figure 1.

cph2 KO results in lower G6PDH activity, retarded growth, and impaired biofilm formation under LAHG. A, Photoautotrophic cells were supplied with 10 mm Glc and put in LAHG conditions for 4 d without agitation. Crude protein extracts were gained, and specific G6PDH activity was determined for the wild type (WT), cph2 KO, and four independent Cph2 OE lines. Data show the mean ± sem (n ≥ 3). Asterisks on top of columns indicate significant differences to WT; asterisks inside columns indicate significant differences to cph2 KO (Student’s t test: P < 0.05). B, Cells in the midexponential growth phase were set to an OD₇₅₀ of 0.3, supplied with 10 mm Glc, and incubated in 2-mL reaction tubes under LAHG conditions without agitation. At indicated time points, cells were harvested, and the OD₇₅₀ was determined. Data show the mean ± sem (n = 3). Asterisks indicate significant differences to WT (Student’s t test: P < 0.05). C, To check for the ability to form biofilms, 1 mL cells (OD₇₅₀ of 0.5) was applied in 24-well microtiter plates in the presence of 10 mm Glc and incubated for 4 d under LAHG without agitation. Then, the supernatant was removed, the biofilm attached to the bottom of the wells was washed off the plate, and the OD₇₅₀ was determined. Data show the mean ± sem (n = 4–5). Asterisks indicate significant differences to WT (Student’s t test: P < 0.05). D, Growth of corresponding strains on BG11 agar plates supplemented with 10 mm Glc under constant light or LAHG conditions. Pictures were taken 4 (Glc/light) or 7 d (Glc/LAHG) after transfer. [See online article for color version of this figure.]

Figure 2.

Cph2 regulates cyt-c-ox activity under LAHG. cyt-c-ox Activity was determined in a whole-cell spectroscopic assay using TMPD (Kovac reagent) as substrate. Cells were incubated under LAHG conditions without agitation for 1 (A) or 4 d (B). Then, cells were washed three times in BG11 medium and adjusted to an OD₇₅₀ of 0.2; 200 µL cells were provided in 96-well multititer plates, and oxidation of TMPD was followed at 610 nm. Relative activities with WT set to 100% ± sem are given (n = 4). Asterisks indicate significant differences to WT (Student’s t test: P < 0.05). Double asterisks indicate significant differences to WT (Student’s t test: P < 0.01). C, Gene expression studies of Zwf (G6PDH) and CtaDI. Cells were cultivated in the absence (photoautotrophic) or presence of 10 mm Glc (photomixotrophic) under permanent illumination or kept for 4 d in LAHG conditions without agitation. Total RNA was isolated; after reverse transcription, 500 ng cDNA was used as a template in PCR (32 cycles). PCR products were analyzed on a 1.5% (w/v) agarose gel and stained with ethidium bromide. The RNA subunit of ribonuclease P subunit B (RnpB) served as normalizer. The picture is representative for three independent experiments.

Figure 3.

cph2 KO results in an impaired modulation of relative variable chlorophyll fluorescence intensities. A, Cells were incubated for 1 d under LAHG conditions without agitation, adjusted to 1.5 µg chlorophyll mL⁻¹, and dark adapted for 1 min. Then, variable fluorescence intensities of PSII were determined. At 1 min, a saturating pulse was applied; 30 s thereafter, actinic light at an intensity of 270 µmol m⁻² s⁻¹ was applied for 4.5 min. Where indicated, CN at a final concentration of 0.01 mm was added. The relative variable fluorescence intensities were plotted over time. Gray arrows indicate the data points at 2.2 and 6 min as used for evaluations shown in B. B, Relative increase or decrease of relative variable fluorescence intensities between data points at 2.2 and 6 min. Data show the mean ± sem (n = 3).

Figure 4.

cph2 KO is impaired in PQ/PSI reduction in LAHG. A, Cells were incubated for 1 d under LAHG conditions without agitation; then, PQ reduction was determined. Cells at 1.5 µg chlorophyll mL⁻¹ were dark adapted for 1 min, inhibitors were added (1 mm KCN, 50 µm DBMIB, and 5 mm sodium ascorbate), and the increase in the fluorescence amplitude induced by weak measuring light was recorded. The relative variable fluorescence intensities were plotted over time. Data show the mean ± sem (n = 3). B, Measurement of P700⁺ reduction kinetics. Photoautotrophic cells were supplied with 10 mm Glc and incubated in the dark for 24 h in 2-mL reaction tubes without agitation. Reduction kinetics of P700⁺ were recorded in vivo with a dual PAM-100 measuring system. Prior measurement cells at 3 µg chlorophyll mL⁻¹ were dark incubated for 1 min in the presence or absence of 10 µm DCMU. Then, blue actinic illumination was provided to achieve complete oxidation of P700, and P700⁺ reduction kinetics were measured in the dark. Rate constants (k) were determined by fitting the decays with single exponential functions. The mean ± sem (n = 3) of the half-life of P700⁺ reduction is shown. Asterisk indicates significant differences to WT (Student’s t test: P < 0.05).

Figure 5.

cph2 KO is impaired in phycocyanin degradation in LAHG. The phycocyanin content was determined spectroscopically in photoautotrophic cells (0 d) and after 1, 2, and 4 d of incubation in LAHG without agitation. In another experiment, cells were incubated for 2 d in LAHG without agitation and then shifted to constant light (30 μmol m⁻² s⁻¹) for an additional 2 d. Data show the mean ± sem (n = 3). Asterisk indicates significant differences to WT (Student’s t test: P < 0.05). Double asterisk indicates significant differences to WT (Student’s t test: P < 0.01).

Figure 6.

cph2 KO is impaired in the heat stress response under photomixotrophic conditions. A, Cells in late exponential growth phase were diluted to an OD₇₅₀ of 0.4 and where indicated, supplied with 10 mm Glc; 10mL cells were incubated at 45°C under shaking (140 rpm) and permanent illumination (30 μmol m⁻² s⁻¹). At the indicated time points, the OD₇₅₀ was determined. Data show the mean ± sem (n = 3). B, The phycocyanin content of cells after 4 h of heat shock (as described in A) was determined. Data show the mean ± sem (n = 3). Asterisk indicates significant differences (Student’s t test: P < 0.05); triple asterisk indicates significant differences (Student’s t test: P < 0.001. C, Schematic illustration of the intersecting photosynthetic and respiratory electron transport (arrows) in the thylakoid membrane of Synechocystis sp. Inhibitors are shown, and dashed arrows indicate the location of their action. Cyt b₆f, cytochrome b₆f complex; PBS, phycobilisome; PC, plastocyanine. Figure 2 in Vermaas, 2001 with modifications.