ABSTRACT
The cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, PrhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited Photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased Photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.
INTRODUCTION
Cyanobacteria are oxygenic photosynthetic prokaryotes that can use light energy to fuel all intracellular biological processes. Cyanobacteria have been used as hosts to study photosynthetic processes due to their relative ease of genetic manipulation as compared to eukaryotic phototrophs. Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) has been used as a model cyanobacterial organism for decades,1 and many genetic tools and elements have been adapted and developed for this strain, which facilitate its genetic modification.2 Importantly, Synechocystis 6803 can use glucose as a carbon source to support heterotrophic growth, a desirable trait for the study of genes essential for photosynthesis.
Photosystem II (PSII) is a unique light-driven enzyme that oxidizes water into molecular oxygen, providing oxygen and the reducing equivalents to fix carbon, powering much of life on earth.3–4 PSII is an approximately 20 subunit membraneprotein complex. It binds numerous pigments and metal cofactors, as well as additional proteins that aid in assembly of the complex.5 PSII has a modular architecture, consisting of a reaction-center “core” module where primary charge separation occurs and two chlorophyll-binding “antenna” modules. The reaction center consists of the D1 and D2 protein subunits that bind the primary electron-transfer cofactors, along with several lower-molecular weight structural proteins. The antenna modules are based around the chlorophyll-binding protein subunits CP43 and CP47 and associated lower molecular weight structural proteins. A catalytic tetra-manganese cofactor is liganded primarily by the D1 protein, with one ligand supplied by the CP43 protein.
PSII, due to its demanding photochemistry, undergoes frequent photo-oxidative damage and must be assembled and repaired in an efficient and tightly regulated manner to maintain high photosynthetic activity.6–12 The D1 and D2 reaction center proteins are the most frequently damaged, as they are closest in proximity to the high-energy electron-transfer reactions taking place.12–14 To repair the damaged complex while maintaining the undamaged and expensive-to-synthesize chlorophyll-protein antenna modules, D1 and D2 are selectively removed and replaced by newly synthesized protein copies. This process of continual assembly and repair is called the PSII lifecycle, and has been extensively studied in mutants lacking key proteins in photosynthetic processes that are halted at various stages in PSII assembly.15–18 However, the lifecycle has been difficult to study as a complete cycle in the same organism due to the low abundance of intermediate complexes in photosynthetic cells, and the simultaneous presence of various PSII intermediate complexes at different stages of the lifecycle CRISPR/Cas (clustered regularly interspersed palindromic repeats/CRISPR-associated protein) technology has revolutionized the biotechnology field as a technique to precisely edit both eukaryotic and prokaryotic genomes, including cyanobacteria.19–22 As a complement to CRISPR/Cas technology, CRISPR interference (CRISPRi) utilizes a nuclease-deficient Cas protein (dCas protein) to bind to a target sequence but not introduce a double-stranded break. A CRISPRi system contains a dCas protein and a guide RNA (gRNA), which together form a dCas-gRNA ribonucleoprotein complex that binds to a genomic site complementary to the gRNA sequence. By binding to its target site, the dCas-gRNA complex blocks transcription and inhibits the expression of specific genes.23 CRISPRi, utilizing either dCas9 or ddCpf1 (DNase-dead Cpf1, also known as dCas12a), has been applied to several species of cyanobacteria.19, 24–28 However, only dCas9-CRISPRi systems have been tested for regulation of genes in Synechocystis 6803.24, 29
To dynamically and precisely regulate the expression of target genes, a CRISPRi system driven by a tightly controlled inducible promoter is of great interest. For Synechocystis 6803, many inducible promoters have been characterized,30–32 but only the anhydrotetracycline (aTc)-inducible promoter has been applied to CRISPRi in Synechocystis 6803.33 This TetR-regulated inducible system is challenging because aTc is light degradable, which leads to unreliable performance under phototrophic conditions. The rhamnose responsive promoter PrhaBAD has been shown to be a robust and inducible system in Synechocystis 6803. This promoter uses the E. Coli transcription factor RhaS as the regulator.32, 34 In this study, we used and modified the PrhaBAD promoter to drive expression of the ddCpf1 protein for CRISPRi, as the ddCpf1 protein has been shown to have lower toxicity in cyanobacteria than dCas9.21–22 We added a riboswitch to construct a tightly controlled promoter cassette that showed titratable behavior over a broad range of expression. When this inducible promoter is applied to our CRISPRi system, expression of the targeted gene psbD (encoding for D2 protein of Photosystem II) is reduced by over 95% with inducers added, while no repression was observed without inducers. Importantly, recovery of D2 expression was detected after release of CRISPRi repression by washing out inducers. We knocked down key PSII proteins through CRISPRi to almost eliminate formation of Photosystem II and then recovered the formation of the photosystem in its native milieu. Our system is an invaluable resource to study the PSII life cycle in a targeted manner and represents a new framework for studying de novo assembly of photosystems.
RESULTS AND DISCUSSION
Repression of psbD genes in Synechocystis 6803 by CRISPRi
The plasmid pCRISPR i-D2 was constructed, using PrhaBAD to drive transcription of both ddcpf1 and a gRNA targeted to the psbD genes (Figure 1A and Table S1). PsbD (D2) is a structural part of the core PSII reaction center,18 and essential for photoautotrophy. In Synechocystis 6803, there are two psbD genes, psbD1 and psbD2, which encode identical proteins but have slightly different nucleotide sequences. The site targeted on both psbD genes has the same sequence in both (the PAM sequence TTG is 7 nucleotides behind the start codon), so both psbD genes will be targeted by the same gRNA. Furthermore, psbD shares an operon with and is directly upstream of the psbC gene, which codes for the CP43 protein (Figure 1A), so with one gRNA, we expect to observe repression of both psbD genes and psbC.
After transfer of pCRISPR i-D2 (Figure S1) into wild type (WT) of Synechocystis 6803, we observed growth under autotrophic conditions (Figure 1B). WT and the CK01 control strain (CRISPRi plasmid with gRNA targeted to eyfp) grew similarly. However, growth of CRISPRi-D2 was impaired compared to WT, indicating that PSII is impaired in this strain even under un-induced conditions, and that PrhaBAD drives expression of ddCpf1 even without the addition of rhamnose. Conversely, in the cells induced with 2 g/L rhamnose, growth was not completely abolished, indicating that there is not full repression of PSII genes by CRISPRi in the induced strain, even though the growth rate is slower than the culture without rhamnose (Figure 1B). Intracellular protein levels of D2 and CP43 as measured by western blot were consistent with growth phenotypes. About 50% of WT D2 and CP43 protein levels were detected in the uninduced CRISPR i-D2 strain (Figure 1C) and about 20% of D2 and CP43 protein levels were observed in the CRISPR i-D2 strain with added rhamnose. These results show that the PrhaBAD promoter is not tightly regulated nor is it strong enough to support full repression.
To confirm that the PrhaBAD promoter has leaky expression, we placed the PrhaBAD cassette in front of the EYFP coding gene in Synechocystis 6803 to make the plasmid pRhaBAD-eyfp (Figure S2). EYFP fluorescence was compared to a control strain CK3068,35 in which the promoter in front of eyfp is removed. We observed a 2 to 2.5-fold higher level of fluorescence over the CK3068 strain in the uninduced culture (Figure S2). This directly confirmed that the PrhaBAD cassette as utilized here does not have tight control over expression of downstream genes. A reason might be the promoter PlacOI we use to drive the regulator protein RhaS in the cassette (Figure S1). RhaS is the regulator for the rhamnose responsive system, and its expression level is a key factor for the RhaBAD promoter to control downstream gene(s).36 The promoter used here might lead to an unbalanced intracellular level of the RhaS protein as compared with that used by Kelly et. al.34
Ideally, the CRISPRi system would maintain full photosynthetic activity under uninduced conditions, while being repressed nearly 100% in an induced condition, to become photoautotrophic and non-photoautotrophic in each state, respectively. This would allow us to observe complete de novo assembly of Photosystem II.
Construction of a chimeric promoter optimized the performance of the PrhaBAD promoter
To tighten the control and improve the strength of the promoter PrhaBAD, we switched out its ribosome binding sequence (RBS) for an RBS containing the theophylline responsive riboswitch (RSW), which has been effective in controlling the translation of downstream genes in cyanobacteria.37–38 We used the riboswitch sequence ‘E’ from Ma et. al.37 (Table S1). We call this new promoter PrhaBAD-RSW and placed the EYFP coding gene under its control to make the PrhaBAD-RSW-EYFP strain (Figure 2A).
Cell samples of PrhaBAD-RSW-EYFP strain were collected for fluorescence assay after 96 hours of growth in BG11 medium under various inducer concentrations. Fluorescence of the culture without inducers was not significantly different from that of the control strain CK3068 (Figure 2B), which indicated that there is not leaky expression from PrhaBAD-RSW. Fluorescence intensity increased with the concentration of inducers up to 5 g/L rhamnose and 2 mM theophylline, with a 120-fold difference between the uninduced culture and the culture with the highest concentrations of inducers (Figure 2B). Additionally, we tested the effect of adding either inducer at a time on the expression of the eyfp gene. We observed that adding only rhamnose achieved 90% of the fluorescence level as adding both inducers (Figure S3), indicating that activation by the chimeric promoter occurs mainly on the transcriptional level, and the riboswitch primarily prevents un-induced expression. All above results suggested that PrhaBAD-RSW is a tightly controllable and titratable promoter cassette with a wide induction range. We applied PrhaBAD-RSW to drive the expression of ddcpf1 in our CRISPRi system in subsequent experiments.
Tightly controlled expression of D2 and CP43 by the CRISPRiRSW system
We constructed a new plasmid applying the PrhaBAD-RSW promoter to control the expression of ddcpf1 and containing the same gRNA sequence targeting to psbD genes, named as the pCRISPR iRSW-D2 plasmid (Figure 3A). After transfer into WT Synechocystis 6803, the CRISPR iRSW-D2 strain grew similarly to WT under photoautotrophic conditions without inducers. When 5 g/L rhamnose and 2 mM theophylline were added, growth of the CRISPR iRSW-D2 strain was severely inhibited under autotrophic conditions (Figure 3B). This growth phenotype indicates that PSII is fully active in the un-induced strain, while has low to no activity when the strain is induced. To further analyze CRISPR iRSW-D2, subsequent experiments are performed with the addition of 10g/L glucose to the growth medium.
If PSII content is reduced by the CRISPRiRSW system, then we expect PSII activity to be lower in cells cultured with inducers than without inducers. We used a PAM fluorometer to assay the FV/FM, or PSII quantum yield, of cultures. The ratio FV/ FM is a relative measure of PSII activity in cells, and the higher the value, the more functional PSII is present. FV (variable fluorescence) represents the difference between Fm (maximal fluorescence) and F0 (background fluorescence) and can be attributed to fluorescence arising directly from active PSII reaction centers.39 The FV/FM of the CRISPR iRSW-D2 strain decreased with increased concentrations of inducers (Figure 3C). At the highest concentration of inducers, 5 g/L rhamnose and 2 mM theophylline, the FV/FM is 18% of that of the un-induced culture. The above results indicate that, consistent with control of EYFP by CRISPRiRSW, control of PSII content is titratable by our CRISPRiRSW system.
To check the specificity of CRISPR iRSW-D2 and quantify the level of repression, both semi-quantitative RT-PCR and quantitative PCR (q-PCR) were performed on WT, the CK02 control strain (containing the CRISPRiRSW plasmid with gRNA targeted to eyfp), and the CRISPR iRSW-D2 strain. In addition to the targeted genes psbD (D2) and psbC (CP43), the Photosystem I structural gene psaA and PSII reaction center genes psbA (D1) and psbB (CP47) were assayed as transcriptional controls. Both RT-PCR and q-PCR showed that no difference was observed between the three strains under photoautotrophic, uninduced conditions for all 5 genes (Figure S4 and S5). Because the CRISPR iRSW-D2 strain does not grow when induced under photoautotrophic conditions in BG11, samples were collected only from WT, CK02, and the un-induced CRISPR iRSW-D2 strain after 7 days growth under this condition.
However, when the strains were grown in BG11 medium with glucose, the psbD and psbC genes in the induced CRISPR iRSW-D2 strain showed 97% and 94% decrease in transcription, respectively (Figure 3D and Figure S4). Transcription of the psaA gene and the psbB gene (CP47) of PSII were unchanged, indicating a specific transcriptional response to induction of CRISPRi. Interestingly, psbA, which codes for the PSII protein D1, was up-regulated around 30% under induced conditions, indicating that its transcription is up-regulated in response to severely inhibited photosynthesis as a compensation mechanism, as the D1 protein is the most frequently damaged and replaced under normal and photo-inhibitory conditions.12 Intracellular protein levels of D2 and CP43 as measured by western blot are consistent with the mRNA levels. D2 and CP43 are reduced to 1% and 4% of un-induced levels, respectively, in the induced samples (Figure 3E).
The above results show that our CRISPRiRSW system is a powerful tool to tightly and specifically control the expression of target genes over a broad range and in a titratable way in Synechocystis 6803. Because there is no expression of ddCpf1 without inducers, and expression of target genes is reduced by over 90% with inducers added, it is an ideal system to study the recovery process after release of repression.
Release of CRISPRi repression allows recovery of the expression of D2 and CP43 proteins
The CRISPRiRSW system effectively knocks down D2 and PSII activity and is tightly regulated. However, to study Photosystem II assembly we require relief of this inhibition, to allow PSII to rebuild following repression. We did further experiments to observe the recovery of targeted genes after release of the CRISPRi induction. To test whether PSII can fully recover in the CRISPR iRSW-D2 strain, the CRISPR iRSW-D2 strain was cultured in BG11 medium with glucose and inducers. When the OD730 nm reached 2.0, cells were washed twice and re-suspended in fresh BG11 medium at a 20-fold dilution to OD730 nm of 0.1 (time point 0). During the recovery process, cells were collected to verify the de novo synthesis of D2 and CP43 proteins, as well as PSII.
After release of repression, we first checked the transcription of psbD and psbC through q-PCR. Samples were collected over a time course, and we observed the gradual increase of transcript levels with time, reaching around 40% of the WT mRNA level after 8 hours and 100% after 96 hours (Figure 4A). Two phases of recovery were observed. In the first phase, the initial 24 hours, transcript level increases from 5% to 50% of WT level. This is followed by a second, slower phase over the next 72 when transcript levels increase back to 100%. We also monitored the FV/FM increase over time (Figure 4B) following release of inhibition, which showed accumulation of active PSII in cells. Two phases of recovery were also observed for FV/FM. The first phase occurs over day 0 to day 3, during which the Fv/Fm value increased from 0.07 to 0.18, while in the second phase FV/FM increased slowly and linearly up to 0.35 over the following 13 days (Figure 4B).
Western blots (Figure 4C) showing protein recovery with increasing FV/FM levels demonstrate a slow increase in CP43 and D2 protein levels over time. At time point 0, corresponding to FV/FM of 0.07, D2 and C43 are less than 5% of the WT level. Interestingly, D2 protein levels increase to 200% of wild type as recovering cells approach the PSII activity of WT, while CP43 increases to just 85% of its wild type protein level. This creates a situation in which there are “extra” D2 copies. These copies of D2 could be in PSII assembly intermediate complexes, as de novo PSII assembly is thought to occur step-wise, first incorporating the D1 and D2 proteins, and then later the CP43 and CP47 proteins.40
Under photoautotrophic conditions, photosynthesis is the sole energy source for intracellular biological processes in Synechocystis 6803, including cell division. We expect cell division rate to be affected for the CRISPR iRSW-D2 strain over the recovery process. Cell numbers were counted every 24 hours during recovery for both WT and the CRISPR iRSW-D2 strain. As shown in Figure 4D, the two strains showed divergent cell division rate phenotypes. Compared to the WT strain, which showed more rapid division over the first 4 days before slowing, the CRISPR iRSW-D2 strain showed a lag phase before day 4, at which time point cells began to divide at a faster rate. This 4-day lag before division begins suggests that cell division was inhibited because of the insufficient energy generated from the low level of intracellular PSII, and correlates to the phases of the FV/FM curve (Figure 4B). The initial rapid increase in FV/FM can be explained by the fact that the FV (or the difference in fluorescence due to PSII activity compared to background chlorophyll fluorescence) increases due to the small new amount of active PSII, while the total background chlorophyll fluorescence (F0) does not start to increase until the cells begin to divide. Once cells begin to grow and divide, photosystem I-associated chlorophyll increases, as does synthesis of unassembled PSII subunits containing chlorophyll, which contribute to F0, making the subsequent increase in FV/FM slower.
D2 and CP43 are essential proteins in the core reaction center of PSII, and 18 inhibiting their biosynthesis eliminates the formation of PSII.18 As shown in Figure 4, a two-phase recovery was observed for all parameters measured, including transcription of psbD and psbC genes, FV/FM values, and cell numbers. This two-phase phenomenon is correlated with the PSII content, which affects the energy supply for biological processes, such as transcription, translation, and cell division. Because the CRISPRiRSW system constructed here knocks down the expression of targeted genes by over 90%, the intracellular PSII activity is very low, around 15% of WT based on FV/FM (Figure 3C and 4B), and less than 5% based on D2 and CP43 protein levels (Figure 3E and 4C). The CRISPR iRSW-D2 strain grew under near photoheterotrophic conditions when glucose and inducers were added to the medium. After resuspension in BG11 medium, cells must convert to autotrophic growth but with a very low initial PSII content. So, there is a rapid initial increase in mRNA and FV/FM, but energy stores are quickly used up, inhibiting cell division until sufficient energy derived from the small amount of initial PSII has allowed cells to divide and synthesize more PSII to make more energy. Therefore, there is a subsequent slow recovery of PSII activity, mRNA, and protein levels.
To study the process of PSII biogenesis, deletion of target genes is the classical genetic strategy. Using a deletion strategy, intermediate PSII assembly complexes form in the specific gene knockout mutant, and these complexes are analyzed to infer the PSII biogenesis process.41 The function of numerous proteins in PSII biogenesis has been identified through this strategy. However, deletion mutants cannot recover the expression of the deleted gene, so the biogenesis process cannot be systematically studied. The CRISPRi technology supplies an alternative and valuable way to uncover the biogenesis process. With this tightly controlled CRISPRiRSW system in Synechocystis 6803, we have developed a new platform to study not only PSII biogenesis, but also assembly of other protein complexes such as Photosystem I.
CONCLUSIONS
We built a chimeric promoter system, PrhaBAD-RSW, which exhibits tight control and a broad, titratable inducible range in Synechocystis 6803. Expression of ddCpf1 driven by this chimeric promoter in a CRISPRi system effectively and reversibly represses the expression of the targeted genes, psbD and psbC, and nearly inhibits the formation of PSII protein complex. Following release of the repression by removing inducers, PSII is re-built in the same cells. In contrast to a strategy of knocking-out to study gene function, our CRISPRiRSW system does not modify or edit the genome. Furthermore, we supply a new platform to study the biogenesis processes, during which protein complex intermediates can be tracked dynamically following release of inhibition.
METHODS AND MATERIALS
Strains and culture conditions
All cloning work was performed in E. coli strain XL1-Blue grown in LB medium in culture tubes or on agar plates at 37 °C, supplemented with 50 μg/ml kanamycin, 20 μg/ml chloramphenicol, or 100 μg/ml ampicillin, as needed. Synechocystis 6803 cells were cultured in BG11 medium supplemented with 30 μg/ml kanamycin as needed, under continuous white light at 30 μmol photons m-2 s-1 at 30 °C. Cultures were grown in 125-ml glass flasks or on agar plates. To monitor growth, 150 μL of cells suspension was loaded onto a 96-well plate and OD at 730 nm was measured on a plate reader (BioTek, VT).
Construction of recombinant plasmids and engineered strains
Plasmids and strains used in this study are listed in Table S2, and all primers used in this study are listed in Table S3. All plasmids were constructed by Gibson Assembly strategy,42 using linear fragments purified from PCR products. All plasmids used in this study are based on the broad host replicating plasmid pRSF1010.43 The DNA fragments used to assemble the CRISPRi plasmid were amplified from other plasmids in the Pakrasi lab.
A tri-parental conjugation method was used to transfer all pRSF1010 derivative plasmids to Synechocystis 6803 wild-type cells,44 using a helper strain of E. coli containing the pRL443 and pRL623 plasmids.45–46 Transformants were isolated on BG11 agar plates containing 20 μg/mL kanamycin, as needed. Isolated Synechocystis 6803 transformants were checked by PCR to confirm presence of the desired constructs.
All PCR amplifications were performed using Phusion High-fidelity DNA polymerase (Thermo Scientific). Plasmids and PCR products were purified using the GeneJET (Thermo Scientific) plasmid miniprep kit and gel extraction kit, respectively. Oligonucleotides were designed using the SnapGene software (GSL Biotech LLC) and synthesized by IDT (Coralville, IA). The sequences of all the plasmids constructed in this study were verified (Genewiz, NJ).
Measurement of EYFP fluorescence
EYFP fluorescence was measured directly from cells suspension in culture. The fluorescence intensity and the optical density of each culture were determined in 96-well black-walled clear-bottom plates on a BioTek Synergy Mx plate reader (BioTek, Winooski, VT). The excitation and emission wavelengths were set to 485 nm and 528 nm for EYFP. All measured fluorescence data were normalized by culture density.
Reverse transcription-PCR (RT-PCR) and quantitative PCR (q-PCR)
Total RNA samples for RT-PCR and q-PCR were extracted using the reagent RNAwiz (Ambion). After quantification of RNA, 200 ng of DNase-treated RNA samples, Superscript II reverse transcriptase, and random primers (Invitrogen) were used for reverse transcription according to the manufacturer’s instructions. cDNA generated after reverse transcription was used as the template for PCR to validate the transcription of genes.
QRT-PCR Sybr green dUTP mix (ABgene) was used for the q-PCR assay on an ABI 7500 system (Applied Biosystems). Each reaction was performed in triplicate, and the average threshold cycle (CT) value was used to calculate the relative transcriptional levels for the amounts of RNA. All primers used for RT-PCR and q-PCR are listed in Table S3.
Western blot analysis
Cyanobacterial cells were harvested and broken by bead beating as described previously47 with minor modifications. Cells were re-suspended in RB buffer (25% glycerol (wt/vol), 10mM MgCl2, 5mM CaCl2, 50 mM MES buffer pH 6.0) and broken by vortexing with 0.17 mm glass beads. Membrane fraction was isolated by centrifugation, re-suspended in RB, and solubilized by addition of β-D-dodecyl maltoside (DDM) to a final concentration of 0.8%. After incubation on ice in dark for 30 min, the solubilized membranes were separated from the insoluble material by centrifugation at gradually increasing speed from 120×g to 27,000×g at 4 °C for 20 min. The solubilized membranes were then stored at −80 °C for future use. The protein content was determined using bicinchoninic acid (BCA) protein assay reagent (Thermo Scientific).
SDS-PAGE was performed by loading the same amount of isolated membrane proteins on a 12.5% acrylamide resolving gel. After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), blocked using 5% bovine serum albumin (BSA) for 2 h at room temperature, and then separately incubated with the primary rabbit antibodies raised against D2 and CP43 proteins overnight at 4°C. The horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad) was diluted at 1:5,000 in 1.5% BSA. Bands were visualized using chemiluminescence reagents (EMD Millipore, Billerica, MA, USA) with an ImageQuant LAS-4000 imager (GE Healthcare).
Measurement of photosynthetic activity
PSII efficiency was analyzed in vivo using a double-modulation fluorometer, FL-200 (Photon System Instruments, Brno, Czech Republic). 1 mL sample was dark-adapted for 2 min before measurement. The instrument contains red LEDs for both actinic (20-s) and measuring (2.5-s) flashes and was used in the time range of 100 μs to 100 s. The maximal PSII quantum yield (FV/FM) was determined with the saturation pulse method.48
Cell number counting
20 μL of culture was sampled, and cells were counted with an automated cell counter (Cellometer Vision; Nexcelom). The counted images were manually curated to improve accuracy of the counts. The accompanying Cellometer software reported cell counts in cells per milliliter.
AUTHOR INFORMATION
°D.L. and V.M.J contributed equally to this work
Corresponding Author
*Himadri B. Pakrasi: pakrasi{at}wustl.edu
Author Contributions
D.L., V.M.J., and H.B.P. designed the experiments. D.L. and V.M.J. performed the experiments. D.L., V.M.J., and H.B.P. wrote the paper.
ACKNOWLEDGMENTS
We thank members of the Pakrasi lab for congenial discussion. This work was supported by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (DOE) (Grant DE-FG02-99ER20350 to H.B.P.) V.M.J was supported by a training grant T32 EB014855 from the National Institute of Biomedical Imaging and Bioengineering, NIH.