Supplemental Information. Glycogen Synthesis and Metabolite Overflow. Contribute to Energy Balancing in Cyanobacteria

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1 Cell Reports, Volume 23 Supplemental Information Glycogen Synthesis and Metabolite Overflow Contribute to Energy Balancing in Cyanobacteria Melissa Cano, Steven C. Holland, Juliana Artier, Rob L. Burnap, Maria Ghirardi, John A. Morgan, and Jianping Yu

2 Supplemental information Figure S1. Energy charge and cell growth. Related to Figure 1. A. From left to right: Growth curves as followed by changes in A730nm, cell count (*10 7 ml -1 ), chlorophyll content (in μg ml -1 ), and energy charge. All cultures were inoculated to similar initial A730nm = 0.17 with inoculum of different ages (3 to 7 days-old inoculum, see color code in legend) under 50 μe m -2 s -1 illumination. Inoculation volumes varied from 1 to 4 ml in 50 ml fresh medium. Upper panels: WT; Lower panels: ΔglgC. B. Initial EC build-up (difference between the maximal EC measured, ECmax, and the EC measured initially, ECinitial) correlates with age of inoculum in WT. C. Duration of EC build-up correlates with age of inoculum in WT. D. Duration of lag phase correlates with initial EC build-up in WT.

3 Figure S2. Energy charge during light-dark-light transition. Related to Figure 2. A. Growth curves of WT Synechocystis sp. PCC 6803 (blue diamond) and ΔglgC mutant (red circles) monitored by A730nm before and after transition from non-saturating light to dark (grey area). B. EC values of cultures grown as in a. Values are expressed as % of the EC in the respective strains at time 24h. Vertical error bars represent standard deviation of biological replicates (n=3).

4 Figure S3. Energy charge and redox charge. Related to Figure 3. Upper panels: Growth curves of WT Synechocystis sp. PCC 6803 (blue diamond, left) and ΔglgC mutant (red circles, right) monitored by A730nm grown under 50 μe m -2 s-1 illumination (solid lines), 15 μe m -2 s-1 illumination (LL, dashed lines, open markers) and 200 μe m -2 s-1 illumination (HL, dashed line, closed markers). Lower panels: Redox charge ratio NADP + /(NADP + + NADPH) values measured throughout the growth presented in the upper panels. Assays were carried out using a bioluminescence assay kit (G9081, Promega, USA) according to the manufacturer s instruction. Vertical error bars represent standard deviation of biological replicates (n=3).

5 Figure S4. High light stress in WT and ΔglgC mutant. Related to Figure 3. A. Photoautotrophic growth curves of WT Synechocystis sp. PCC 6803 (blue diamond) and ΔglgC mutant (red circles) monitored by A730nm. The cultures were grown under atmospheric air supplemented with 5% CO2 and either 50 μe m -2 s-1-1 (closed markers) or 250 μe m-2 s illumination (open markers). Vertical error bars represent standard deviation of biological replicates (n=3). B. WT and ΔglgC cultures before high light exposure (HL 0h) and 6 days after continuous illumination at 250 μe m -2 s-1 (HL 144h).

6 Figure S5. Identification of 2-oxoglutarate and pyruvate in the extracellular growth medium of the ΔglgC strain after 24h cultivation under 250 μe m -2 s -1 illumination. Related to Figure 3. No organic acids were detected in the WT extracellular medium under similar growth conditions. Peak retention times and signal integrals were compared against known standard solutions prepared in the same growth medium.

7 Figure S6. Photosynthesis parameters in WT and ΔglgC mutant. Related to Figure 1. A & B. Fluorescence parameters of WT (blue) and ΔglgC (red) cultures. C. P700 absorbance kinetics with no inhibitors (left panels) and with addition of 10 μm DCMU (center) or DCMU + KCN (1 mm; right). Cells directly sampled from cultures with the same turbidity and chlorophyll content were used, and a high intensity actinic light was turned on for 10 s to fully oxidize P700, as shown by a decrease in absorbance. In the presence of DCMU, a slower rereduction in the subsequent darkness is observed, indicating a reduction in CEF (middle lower panel). In the absence of inhibitors, a transient re-reduction event is observed following the actinic illumination (left panels), eventually followed by a complete oxidation of PSI. This transient re-reduction event under actinic light is most probably due to a pool of electrons transitorily reducing P700 + and appeared delayed and shortened in ΔglgC compared to WT (see also (Holland et al., 2016) for further discussions about the P700 absorbance kinetics in those strains). The data are presented in uncorrected form (A750nm = 1; [Chl]=4 μg ml -1 ).

8 Figure S7. ATP-ADP bioluminescent assays. Related to experimental Procedures. A. Set-up for ATP-ADP luciferase assays under illumination. Cultures and microplates used for the assays are maintained under the desired growth conditions until readings are taken. B. Images of fluorescence microscopy of WT and ΔglgC cells permeabilization test with lysis buffer used in the ATP-ADP luciferase assays. 10 µl of cells in mid-exponential phase (A730nm 0.5) were sampled and 90 µl of BG11 medium (upper and middle panels) or lysis buffer (lower panels) were added. Cells were incubated with SYTOX Green Nucleic Acid Stain ((Molecular Probes, USA), a cell membrane impermeable nucleic acid stain, at a final concentration of 5µM (middle and lower panels). 5 µl of the cell suspensions were drop-cast onto un-modified glass slides and covered with glass cover slips (VWR, Randor, PA, USA). Images were captured using a Nikon C1 Plus microscope (Nikon, Tokyo, Japan), equipped with the Nikon C1 confocal system operated via Nikon s EZ-C1 software. Samples were irradiated with a tuneable Argon laser at a wavelength of 488 nm. Two signal channels consisting of fluorescence emission wavelengths of 650 nm (chlorophyll auto-fluorescence, shown on the left) and nm (SYTOX Green/DNA complex maximum emission range, shown on the right) were collected simultaneously. Images were analysed with ImageJ software (