Bacterial strains and general growth conditions.
The Synechocystis strains used in this study were slr0688i, whose outer membrane is deprived due to conditional repression of slr0688 by the CRISPRi system regulated by both dCas9 and sgRNA, and dCas9, a control strain (serving as the wild-type strain in this study) whose chromosomal DNA contains the same genetic construct as slr0688i without the sgRNA25. These strains were grown photoautotrophically at 30 °C, 100 µmol photons m− 2 s− 1, with constant shaking at 140 rpm in BG11 medium consisting of the following ingredients (per L): 2 mL of Solution I (0.5 g/L Na2EDTA·2H2O, 3 g/L ammonium iron (III) citrate, 3 g/L citric acid), 25 mL of Solution II-a (60 g/L NaNO3, 3 g/L MgSO4), 25 mL of Solution II-b (1.56 g/L K2HPO4), 2 mL of Solution III (14.3 g/L CaCl2), 1 mL of A6 Solution (2.86 g/L H3BO3, 1.81 g/L MnCl2·4H2O, 0.22 g/L ZnSO4·7H2O, 0.08 g/L CuSO4·5H2O, 0.021 g/L Na2MoO4·H2O, 0.0494 g/L Co(NO3)2·6H2O, 1 droplet of H2SO4), and 20 mL of 1 M TES-KOH (pH 7.5). Deprivation of the outer membrane of slr0688i was induced by treating precultured cells dilluted to OD730 = 0.1 with 1 µg/mL anhydrotetracycline dissolved in DMSO, as described in Kojima and Okumura, 202025. Synechocystis cells at log phase were used in all the following experiments.
Electrochemical measurements. The electrochemical setup was composed of a cylindrical glass chamber (Φ 20 × 30 mm; geometrical surface area, 3.14 cm2), an ITO-coated glass (GEOMATEC) as a working electrode placed at the bottom of the chamber, a platinum wire as a counter electrode, and a Ag/AgCl reference electrode (HOKUTO DENKO). All electrochemical measurements were conducted at 30 °C with either a potentiostat/galvanostat HA-1510 (HOKUTO DENKO) or electrochemical analyzer Model 760C (ALS).
Common procedures for electrochemical measurements performed in the current study were as follows: Synechocystis cell suspensions of 4 mL were separated into sedimented cells and supernatant by centrifugation at 2,500 × g for 10 min, and the supernatant was first injected into the electrochemical chamber. The sedimented cells were then resuspended with the rest of the supernatant, drawn up into a syringe, injected by gravity onto the ITO glass at the bottom of the chamber, and incubated to settle the cells down.
To avoid detection of a pseudo-photocurrent, which is explained in detail below, the pH of Synechocystis cell suspensions was always confirmed and adjusted when necessary prior to electrochemical measurements. The pH of the suspension preferably should be below approximately 8.0, and should never be above 8.5. Otherwise, the buffering capacity of TES (pH 6.8–8.2) is outcompeted by drastic changes (increases) in the pH of the medium due to photosynthesis61–63, resulting in detection of a pseudo-photocurrent of the following reaction:
Mn2+ + 2H2O ⇌ Mn3+(OOH) + 3H+ + e−,
where Mn2+ comes from MnSO4, a component of the BG11 medium. Some examples of detected pseudo-photocurrents of manganese are shown and described in detail in Supplementary Fig. 10. Therefore, before electrochemical measurements, the pH of cell suspensions was always confirmed to be below approximately 8.0, and when necessary, fresh BG11 or HCl was added not only to adjust the optical density, but also the pH of cell suspensions below approx. 8.0. In chronoamperometry, the applied voltage was fixed to + 0.25 V vs. Ag/AgCl, at which sufficient current generation occurred (Supplementary Fig. 11) and a psedo-photocurrent was never detected under an appropriate pH (Supplementary Fig. 10).
When the supernatant needed to be replaced, it was removed after centrifugation and substituted with the supernatant of interest, which was prepared by filtration with an Ultrafree-MC-GV, 0.22 µm (Millipore), or with fresh BG11.
Size-fractionated supernatants were prepared as follows: the supernatant was first filtrated from a cell suspension of slr0688i with a Millex-GV Syringe Filter Unit, 0.22 µm (Millipore) and freeze-dried with a freeze dryer (TOKYO RIKAKIKAI). The obtained powder was suspended with water to yield a 10× concentrated supernatant and size fractionated by ultrafiltration with Amicon Ultra Centrifugal Filters (Millipore) to yield a supernatant containing compounds with MW > 50,000, > 30,000, > 3,000 and < 3,000. Each fraction was then diluted to 0.1× concentration with fresh BG11, and mixed with slr0688i cells to measure photocurrent generation.
For experiments using pCMB, DCMU, and KCN, cell suspensions adjusted to OD730 = 1.5 and pH 7.6 by adding fresh BG11 were incubated with 100 µM pCMB, 10 µM DCMU, or 5 mM KCN for 1.5 h in the dark on ITO in the electrochemical chambers prior to electrochemical measurements. Additional experiments using 50 µM PMA were performed with cell suspensions whose chlorophyll concentration was 12 µg Chl/mL and pH was adjusted to 7.6 by adding HCl.
Ferricyanide assay. The rate of ferricyanide reduction was measured using 2.1 mL of Synechocystis cell suspensions adjusted to OD730 = 1.0. Then, 1 mM potassium ferricyanide was added to the cell suspensions and they were incubated at 30 °C with constant shaking at 140 rpm either under illumination (50 µmol photons m− 2 s− 1) or in the dark. Changes in the concentration of ferricyanide (extinction coefficient, 1.052 mM cm− 1) were monitored up to approx. 140 min by measuring absorbance at 420 nm with a UV/VIS spectrophotometer UV-1850 (SHIMADZU).
Steady-state oxygen evolution and uptake measurements. The photosynthetic activity of Synechocystis cells was evaluated with a Clark-type electrode (Hansatech) at 25 °C. To measure steady state photosynthetic activity, 2 mL of cell suspension (12 µg Chl/mL) containing 5 mM NaHCO3 was illuminated with a CoolLED pE-100 LED at PPFD of 750 µmol photons m− 2 s− 1. Dark respiration was evaluated from the oxygen consumption rate before switching on the light, and photosynthetic activity was calculated by adding the rate of dark respiration to that of oxygen evolution under illumination.
The same oxygen electrode setup was used to analyze the effects of KCN and pCMB on photosynthetic activity. In the case of KCN, Synechocystis cells were collected and resuspended with fresh BG11 to a final concentration of 12 µg Chl/mL, and mixed with 5 mM NaHCO3 and 5 mM KCN, immediately after which oxygen evolving activity was measured. In the case of pCMB, Synechocystis cells collected and resuspended with fresh BG11 (24 µg Chl/mL) were incubated either with 100 µM pCMB or with its solvent DMSO for 1.5 h in the dark. The cell suspensions were then mixed with 5 mM NaHCO3 and diluted to 12 µg Chl/mL with fresh BG11, followed by immediate measurement with an oxygen electrode.
NADPH fluorescence was measured with a DUAL-PAM-100 (Walz) instrument as described previously64,65. Slr0688i cells collected and resuspended with fresh BG11 (24 µg Chl/mL) were incubated either with 100 µM pCMB or with its solvent DMSO for at least 1.5 h in the dark. The cell suspensions were then injected and diluted to 2.4 µg Chl/mL in a 1 × 1 cm cuvette installed in the DUAL-PAM. The measuring light of 365 nm was set at an intensity of 10 on the DUAL-PAM software, and the measuring frequency was programmed to increase from 500 to 5000 Hz during illumination with actinic light of 635 nm at 785 µmol photons m− 2 s− 1.
NADP + /NADPH quantification assay.
The amount of NADP+/NADPH in Synechocystis cells was quantified using an NADP+/NADPH Assay Kit-WST (DOJINDO), following the manufacturer’s manual.
Fluorometric measurement of membrane potential of Bacillus cells. Bacillus cereus (hereafter Bacillus) cells were cultured at 30 °C with constant shaking at 140 rpm in Luria-Bertani (LB) medium. An overnight preculture was diluted 100-fold in fresh LB medium and grown for another 5 h before analysis. Bacillus cells were then collected by centrifugation at 10,000 × g for 4 min and resuspended in fresh BG11 medium, whose OD600 was adjusted to approx. 0.3. CCCP dissolved in DMSO was added to a final concentration of 30 µM, and the cell suspension was incubated for 30 min at 30 °C with constant shaking at 140 rpm. The cells were then collected and washed once with BG11 to remove the CCCP, followed by final resuspension with BG11, in which the cells were allowed to rest for about 40 min (here, the OD600 of the cell suspention was supposed to be approx. 0.6). A voltage-sensitive dye, DiSC3(5), was used to monitor changes in the membrane potential, following Winkel et al.66; depolarization of the membrane is reflected as an increase in fluorescence originating from the dye, whereas repolarization leads to a decrease in fluorescence67. Initial fluorescence levels were first recorded with the cell suspension incubated with 2 µM DiSC3(5) for about 5 min prior to the measurement. After the addition of supernatant of either slr0688i or dCas9 of the same volume as the cell suspension, making the concentration of DiSC3(5) 1 µM, the fluorescence emission spectra of the cell suspension, excited at 610 nm, were recorded from 630 to 700 nm time-dependently with an FP-8500 spectrofluorometer (JASCO). Changes in the fluorescence intensity at 674 nm were plottted to analyze changes in membrane potential across the cytoplasmic membrane of Bacillus cells.
Casamino acid and glucose dissolved in fresh BG11 were used as positive controls, and fresh BG11 was used as a negative control.
Supernatants were prepared by filtration from cell suspensions of slr0688i or dCas9 with a Millex-GV Syringe Filter Unit, 0.22 µm (Millipore). Concentration and size fractionation were done by filtration with Amicon Ultra Centrifugal Filters (Millipore). All the supernatants and control samples were confirmed to be at the same pH (within approx. pH 7.6–7.8) before the measurements, and when necessary, were adjusted to pH 7.6–7.8 by addition of HCl.
Membrane vesicles free of intracellular components were prepared from Bacillus subtilis according to the method described in Konings et al., 197368.
Note that because the supernatant of slr0688i contained phycocyanins25, it yielded unignorable fluorescence in the tested range from 630 to 700 nm when excited at 610 nm (Supplementary Fig. 12). The fluorescence from phycocyanins disappeared when purged with oxygen, which leads to self-sensitized bleaching of phycocyanins69, or treated with heat (Supplementary Fig. 12). It was confirmed that phycocyanins did not interefere with changes in fluorescence from DiSC3(5) (data not shown).