DnaK3 Is Involved in Biogenesis and/or Maintenance of Thylakoid Membrane Protein Complexes in the Cyanobacterium Synechocystis sp. PCC 6803

DnaK3, a highly conserved cyanobacterial chaperone of the Hsp70 family, binds to cyanobacterial thylakoid membranes, and an involvement of DnaK3 in the biogenesis of thylakoid membranes has been suggested. As shown here, light triggers synthesis of DnaK3 in the cyanobacterium Synechocystis sp. PCC 6803, which links DnaK3 to the biogenesis of thylakoid membranes and to photosynthetic processes. In a DnaK3 depleted strain, the photosystem content is reduced and the photosystem II activity is impaired, whereas photosystem I is regular active. An impact of DnaK3 on the activity of other thylakoid membrane complexes involved in electron transfer is indicated. In conclusion, DnaK3 is a versatile chaperone required for biogenesis and/or maintenance of thylakoid membrane-localized protein complexes involved in electron transfer reactions. As mentioned above, Hsp70 proteins are involved in photoprotection and repair of PS II in chloroplasts.


Introduction
In plants and cyanobacteria, the biogenesis and dynamics of thylakoid membranes (TMs) is light-controlled [1,2]. In plants, proplastids develop into chloroplasts, involving the de novo formation of an internal TM network [3], and a developed TM network dynamically reorganizes in the light [4]. When the cyanobacterium Synechocystis sp. PCC 6803 (from here on: Synechocystis) is grown in the dark under light-activated heterotrophic growth (LAHG) conditions, where glucose is the only available energy source, Synechocystis cells exhibit reduced or even just rudimentary TMs [5,6]. However, after shifting dark-adapted cells into the light, the Synechocystis cells quickly rebuild a TM network and recover photosynthetic activity [5,7]. While dark-adapted Synechocystis cells do not harbor active photosystem II (PS II) complexes, complete photosynthetic activity is regained within 24 h after transferring dark-adapted cells into the light, and reappearance of photosynthetic electron transfer processes is coupled to the formation of internal TMs [7]. However, it is still enigmatic how the formation of internal TMs is controlled, both in chloroplasts and cyanobacteria, although some proteins that might be involved in this process have already been described previously [8]. These proteins include the inner membrane-associated protein of 30 kDa (IM30, also known as Vipp1: The vesicle-inducing protein in plastids 1), Hsp70 (Heat shock protein 70) chaperones, dynamin-like proteins, a prohibitin-like protein, Life 2020, 10, 55 3 of 16

Growth Conditions
A glucose-tolerant Synechocystis sp. PCC 6803 wild type (wt) and the merodiploid dnaK3 (sll1932) knock-down (KD) strain [15] were cultivated photomixotrophically at 30 • C in liquid BG11 medium [36] supplemented with 5 mM glucose. Kanamycin (80 µg/mL) was added in case of the dnaK3KD strain. The cultures were aerated with air enriched with 2% CO 2 and grown under fluorescent white light at a light intensity of 20 (LL, low light) or 120 (HL, high light) µmol/m 2 s, respectively. To determine growth rates, the strains were initially adjusted to an OD 750 of 0.05 in BG11 medium, containing 5 mM glucose, and growth was followed by monitoring OD 750 . For LAHG cultures, Synechocystis cells were grown in a dark cabinet for at least two weeks, during which the cultures were diluted at least five times in fresh medium, as described previously (Barthel et al., 2013).

SDS-PAGE and Immunoblot Analysis
Synechocystis cells were harvested in the exponential growth phase at an OD 750 below 2.0. Cell pellets were resuspended in buffer (50 mM HEPES, pH 7.0, 25 mM CaCl 2 , 5 mM MgCl 2 , 10% (v/v) glycerol) and a proteinase inhibitor mix (Sigma Aldrich) was added at a 1:1000 dilution. Cells were broken with glass beads (0.25-0.5 mm diameter) in a beadbeater. Unbroken cells and glass beads were removed by centrifugation at 1600 g and the respective protein concentrations were determined by three independent Bradford assays. After addition of SDS sample buffer and heating at 65 • C for 15 min, cell extracts were loaded on an 8% polyacrylamide gel and proteins were separated by SDS gel electrophoresis. Subsequently, proteins were transferred to a polyvinylidene difluoride membrane, using a wet electroblotting system from Bio-Rad. The rabbit primary antibodies were used at 1:2000 (anti-L23 directed against the large ribosomal subunit protein L23 encoded by sll1801, Gramsch laboratories, Schwabhausen, Germany), 1:1000 (anti-DnaK1, anti-DnaK2 and anti-DnaK3 [15], anti-PsaA/PsaB [37]) or 1:100 (anti-PsbA [38]) dilutions, respectively, whereas the goat anti-rabbit secondary antibody (Sigma Aldrich) was diluted 1:10,000. PsbA/D1-HRP antibodies were obtained from Agrisera and used in 1:15,000 dilution. To visualize the protein bands, membranes were incubated with the enhanced chemiluminescence kit from Pierce. Each immunoblot analysis has been repeated at least three times.

Complete Deletion of DnaK3 in Synechocystis Cells Grown under LAHG Conditions
To test whether DnaK3 is dispensable in the dark, the dnaK3KD strain [15] was cultivated in liquid BG11 medium under LAHG conditions and diluted if necessary. During each dilution step, the concentration of kanamycin was enhanced in the growth medium from 80 to 275 µg mL −1 .

Cell Number and Chlorophyll Concentration Determination
Cell numbers were counted with a light microscope using a Thoma counting chamber. Chlorophyll concentrations were determined photometrically after methanol extraction [39].

Electron Microscopy
To study the cell morphology of the different Synechocystis strains, cell pellets obtained from a 10 mL cell suspension were washed and resuspended in buffer (50 mM KH 2 PO 4 , pH 7). Ultrastructural investigations were performed as described previously [37]. The number of thylakoid layers per cell was determined, evaluating more than 200 individual cells of wt and the DnaK3 depleted Synechocystis strain, respectively.

Absorbance and Low Temperature (77K) Fluorescence Spectra
Absorbance spectra of whole cells were recorded using a Perkin-Elmer Lambda 25 spectrophotometer equipped with an integrating sphere. Cell suspensions were adjusted to a constant value of 300,000 cells mL −1 . Ratios of cyanobacterial chromophores were determined using the absorption ratio at 625/680 (phycocyanin/chlorophyll) or at 490/440 (carotenoids/chlorophyll).
Low-temperature (77 K) fluorescence emission spectra were recorded using an Aminco Bowman Series 2 spectrofluorimeter. Cultures were adjusted to a chlorophyll concentration of 3 µg·mL −1 in BG11 medium and frozen in liquid nitrogen. Chlorophylls were excited at 435 nm and phycobilisomes (PBs) at 580 nm. Fluorescence emission was recorded from 630 to 760 nm.

Oxygen Evolution
Oxygen production of the cell suspensions was determined in the presence of 500 µM phenyl-p-benzoquinone (PPBQ) using a fiber-optic oxygen meter (PreSens) under actinic light (600 µmol photons m −2 ·s −1 ). Prior to the measurement, the cultures were adjusted to a chlorophyll concentration of 3 µg·mL −1 in BG11 medium. For experiments in presence of a protein synthesis inhibitor, 100 µg·mL −1 lincomycin was added prior to illumination (1500 µmol photons m −2 ·s −1 ).

Chlorophyll Fluorescence Induction Curves
Cultures were adjusted to a chlorophyll concentration of 3 µg·mL −1 in BG11 medium, and subsequently fluorescence induction curves were recorded at room temperature, using a Dual-PAM-100 measuring system equipped with Dual-E and DUAL-DR modules (Heinz Walz GmbH). During the initial dark phase, background fluorescence was probed by weak measuring light (0.024 µmol photons m -2 ·s -1 ) and after 40 s fluorescence was induced by switching on red actinic light (95 µmol photons m -2 ·s -1 ). Saturating pulses (600 ms, 10.000 µmol photons m -2 ·s -1 ) were applied once during the dark phase and at 30 s intervals during the light phase, to obtain minimal (F 0 ) and maximal (F m and F m´) fluorescence values [40,41]. The coefficient of photochemical quenching of the PS II Chl fluorescence (qP) was calculated using the software routine for light induction measurements (qP = (Fm-Fm')/(Fm-Fo')) after 250 s illumination with red actinic light.

P 700 Re-Reduction Kinetics
Re-reduction kinetics were recorded using a Dual-PAM-100 measuring system. P 700 was first reduced by 10 sec far-red and then oxidized by a 20 ms saturation light pulse (10.000 µmol photons m -2 ·s -1 ). 15 individual re-reduction curves were recorded, averaged, and fitted with single exponential functions to determine decay halftimes (t 1/2 ). Prior to the measurement, the different cultures were adjusted to a chlorophyll concentration of 3 µg·mL −1 in BG11 medium.

DnaK3 Synthesis is Light-Induced and Essential in the Dark
The Synechocystis dnaK2 and dnaK3 genes are essential in the light [15], and the DnaK1-3 proteins were detected by Western blot analyses in Synechocystis cells grown under constant illumination [15]. However, when Synechocystis cells were grown in the dark under LAHG conditions, the DnaK2 protein, but not DnaK1 and DnaK3, were detectable ( Figure 1, 0 h). Yet, when dark-adapted cells were shifted into the light, the DnaK2 level did not substantially alter, whereas the DnaK1 level quickly increased until two hours after shifting the cells into the light. DnaK3 was detectable already after one hour, and its cellular content increased steadily. Thus, the synthesis of DnaK1 and DnaK3 clearly is triggered by light in Synechocystis. Since DnaK1 is not essential for the viability of Synechocystis cells [15], we focused our subsequent analyses on DnaK3.
As DnaK3 is essential in the light [15], the observation of a light-induced DnaK3 synthesis indicated that DnaK3 might be dispensable in the dark. Therefore, we next attempted to completely delete the Synechocystis dnaK3 gene in cells grown in the dark under LAHG conditions. Yet, even after more than half a year of cultivation under LAHG conditions and increasing the kanamycin concentration in the growth medium up to 275 µg·mL −1 , a fragment corresponding in size to the wild type (wt) dnaK3 gene was always detected via PCR in the dnaK3 knock-down (KD) strain in addition to the dnaK3 gene disrupted by the kanamycin resistance (aphA) cassette (Figure 2A,B). As Synechocystis contains multiple identical genome copies, this result indicates that some, but not all, of the genomic dnaK3 copies were deleted in the mutant strain. Thus, DnaK3 likely is essential not only in the light but also in the dark under LAHG conditions. Yet, we recently showed that expression of dnaJ3 [25], which is organized in a gene cluster together with dnaK3, is essential in Synechocystis, and thus deletion of dnaK3 might have affected the expression of dnaJ3. To assess this potential polar effect, we also quantified the amount of the DnaJ3 protein in the dnaK3KD strain ( Figure 2A). Since the DnaJ3 level was not decreased compared to the wt, we concluded that insertion of the aphA cassette into the dnaK3 gene locus did not dramatically affect the expression of dnaJ3. Nevertheless, a polar effect on expression of dnaJ3 cannot be completely excluded.
To quantify the relative cellular DnaK3 content in the dnaK3KD strain, total cellular extracts of the wt and the KD strain were analyzed via Western blots ( Figure 2C). The intensity of each band was quantified using the Image J software and divided by the quantity of cellular extract loaded. Based on this analysis, the DnaK3 content was decreased by about 60% ± 10% in the dnaK3KD strain compared to the wt. Since DnaK1 is not essential for the viability of Synechocystis cells [15], we focused our subsequent analyses on DnaK3.
As DnaK3 is essential in the light [15], the observation of a light-induced DnaK3 synthesis indicated that DnaK3 might be dispensable in the dark. Therefore, we next attempted to completely delete the Synechocystis dnaK3 gene in cells grown in the dark under LAHG conditions. Yet, even after more than half a year of cultivation under LAHG conditions and increasing the kanamycin concentration in the growth medium up to 275 µg·mL −1 , a fragment corresponding in size to the wild type (wt) dnaK3 gene was always detected via PCR in the dnaK3 knock-down (KD) strain in addition to the dnaK3 gene disrupted by the kanamycin resistance (aphA) cassette (Figure 2A,B). As Synechocystis contains multiple identical genome copies, this result indicates that some, but not all, of the genomic dnaK3 copies were deleted in the mutant strain. Thus, DnaK3 likely is essential not only in the light but also in the dark under LAHG conditions. Yet, we recently showed that expression of dnaJ3 [25], which is organized in a gene cluster together with dnaK3, is essential in Synechocystis, and thus deletion of dnaK3 might have affected the expression of dnaJ3. To assess this potential polar effect, we also quantified the amount of the DnaJ3 protein in the dnaK3KD strain ( Figure 2A). Since the DnaJ3 level was not decreased compared to the wt, we concluded that insertion of the aphA cassette into the dnaK3 gene locus did not dramatically affect the expression of dnaJ3. Nevertheless, a polar effect on expression of dnaJ3 cannot be completely excluded.
To quantify the relative cellular DnaK3 content in the dnaK3KD strain, total cellular extracts of the wt and the KD strain were analyzed via Western blots ( Figure 2C). The intensity of each band was quantified using the Image J software and divided by the quantity of cellular extract loaded. Based on this analysis, the DnaK3 content was decreased by about 60% ± 10% in the dnaK3KD strain compared to the wt.
Life 2020, 10, 55 6 of 16 affect the expression of dnaJ3. Nevertheless, a polar effect on expression of dnaJ3 cannot be completely excluded.
To quantify the relative cellular DnaK3 content in the dnaK3KD strain, total cellular extracts of the wt and the KD strain were analyzed via Western blots ( Figure 2C). The intensity of each band was quantified using the Image J software and divided by the quantity of cellular extract loaded. Based on this analysis, the DnaK3 content was decreased by about 60% ± 10% in the dnaK3KD strain compared to the wt.  [15], the dnaK3 gene was disrupted by insertion of a kanamycin resistance cassette (aphA gene). (B) The dnaK3 gene locus of wt and dnaK3KD cells grown in the dark was analyzed via PCR using genomic DNA as a template, and the PCR products were loaded on a 1.5% agarose gel together with a molecular size marker (M). Fragments of about 500 bp and 1500 bp represent the wt and the dnaK3 gene interrupted by a kanamycin resistance cassette (aphA), respectively. (C) The relative DnaK3 content in the dnaK3KD strain was determined by immunoblot analysis. Cell extracts prepared from the wt and dnaK3KD strains, respectively, were loaded on a SDS-polyacrylamide gel in descending protein concentrations (15 µg to 5 µg) followed by a Western blot analysis using α-DnaK3, α-DnaJ3 and α-L23 (loading control) antibodies.

Reducing the DnaK3 Content Affects Cell Growth under Heat Stress Conditions
Next, we tested whether reducing the DnaK3 content affects growth of the mutant strain under low (LL) or high light (HL) growth conditions, respectively ( Figure 3A). The dnaK3KD and the wt cells had comparable doubling times of 11.2 h ± 0.1 (wt) and 11.1 h ± 0.2 (dnaK3KD), and of 8.1 h ± 0.5 (wt) and 9.4 ± 1.2 (dnaK3KD) under LL and HL growth conditions, respectively. Thus, reducing the cellular DnaK3 content does not severely affect the growth of Synechocystis cells, at least not under standard laboratory growth conditions. Subsequently, growth of the dnaK3KD strain was tested under various stress conditions (involving low pH, low temperature, oxidative and osmotic stress; data not shown), but solely increasing the temperature to 42 • C resulted in an obvious growth defect of the mutant strain, with doubling times of 26.4 ± 3.5 h (dnaK3KD) and 17.9 ± 0.4 h (wt) ( Figure 3B). This observation classifies DnaK3 as a traditional Hsp70 involved in heat-stress responses. The dnaK3 gene locus of wt and dnaK3KD cells grown in the dark was analyzed via PCR using genomic DNA as a template, and the PCR products were loaded on a 1.5% agarose gel together with a molecular size marker (M). Fragments of about 500 bp and 1500 bp represent the wt and the dnaK3 gene interrupted by a kanamycin resistance cassette (aphA), respectively. (C) The relative DnaK3 content in the dnaK3KD strain was determined by immunoblot analysis. Cell extracts prepared from the wt and dnaK3KD strains, respectively, were loaded on a SDS-polyacrylamide gel in descending protein concentrations (15 µg to 5 µg) followed by a Western blot analysis using α-DnaK3, α-DnaJ3 and α-L23 (loading control) antibodies.

Reducing the DnaK3 Content Affects Cell Growth under Heat Stress Conditions
Next, we tested whether reducing the DnaK3 content affects growth of the mutant strain under low (LL) or high light (HL) growth conditions, respectively ( Figure 3A). The dnaK3KD and the wt cells had comparable doubling times of 11.2 h ± 0.1 (wt) and 11.1 h ± 0.2 (dnaK3KD), and of 8.1 h ± 0.5 (wt) and 9.4 ± 1.2 (dnaK3KD) under LL and HL growth conditions, respectively. Thus, reducing the cellular DnaK3 content does not severely affect the growth of Synechocystis cells, at least not under standard laboratory growth conditions. Subsequently, growth of the dnaK3KD strain was tested under various stress conditions (involving low pH, low temperature, oxidative and osmotic stress; data not shown), but solely increasing the temperature to 42 °C resulted in an obvious growth defect of the mutant strain, with doubling times of 26.4 ± 3.5 h (dnaK3KD) and 17.9 ± 0.4 h (wt) ( Figure 3B). This observation classifies DnaK3 as a traditional Hsp70 involved in heat-stress responses.

The dnaK3KD Strain Has a Reduced Pigment Content
Photosynthesis is one of the most temperature-sensitive processes in phototrophic organisms and the photosynthetic activity is further impaired when heat-stress is combined with HL [42,43]. Thus, it was well possible that reducing the DnaK3 content affects photosynthetic processes in

The dnaK3KD Strain Has a Reduced Pigment Content
Photosynthesis is one of the most temperature-sensitive processes in phototrophic organisms and the photosynthetic activity is further impaired when heat-stress is combined with HL [42,43]. Thus, it was well possible that reducing the DnaK3 content affects photosynthetic processes in Synechocystis.
As expression of dnaK3 is light-controlled (Figure 1), we subsequently analyzed the pigment content of the wt and dnaK3KD strains after cultivation under LL and HL conditions, respectively. Adaptation of Synechocystis cells to HL conditions is typically accompanied by a reduction in the cellular amount of the two PSs, a decreased PS I-to-PS II ratio and a reduced chlorophyll (Chl) content per cell [44,45].
An overall reduction of the pigment content was observed under HL growth conditions when equal amounts of cells were analyzed ( Figure 4A). The Chl content was reduced to about half ( Figure 4B) and the relative content of plastocyanine (PC) ( Figure 4C) and carotenoids (Car) ( Figure 4D) were both increased. It has to be noted that while the contents of Chl, PC and Car were decreased under HL growth conditions ( Figure 4A), the PC/Chl as well as the Car/Chl ratios were increased in the wt strain, due to the more severely decreased Chl content ( Figure 4A,B). Even though light scattering could have contributed to some extent to the determined (absolute) absorbance values used in these analyses, these data clearly show the ability of the wt to reduce the overall pigment content and to adapt it to HL conditions. Life 2020, 10, x 7 of 17 4B) and the relative content of plastocyanine (PC) ( Figure 4C) and carotenoids (Car) ( Figure 4D) were both increased. It has to be noted that while the contents of Chl, PC and Car were decreased under HL growth conditions ( Figure 4A), the PC/Chl as well as the Car/Chl ratios were increased in the wt strain, due to the more severely decreased Chl content ( Figure 4A,B). Even though light scattering could have contributed to some extent to the determined (absolute) absorbance values used in these analyses, these data clearly show the ability of the wt to reduce the overall pigment content and to adapt it to HL conditions. Similarly, the dnaK3KD strain adapted to changing light conditions and reduced its pigment content as expected when grown under HL conditions. However, the dnaK3KD strain exhibited a severely reduced pigment content already when grown under LL conditions, and the Chl content as well as the pigment ratios were very similar to the ones observed when the wt was grown under HL conditions (Figure 4).
Besides the obvious differences in pigmentation, the TM structure was mostly unaffected, and we only observed a slightly reduced number of TM pairs in the mutant strain when the ultrastructure of Synechocystis grown under LL growth conditions was analyzed via electron microscopy ( Figure 5). Similarly, the dnaK3KD strain adapted to changing light conditions and reduced its pigment content as expected when grown under HL conditions. However, the dnaK3KD strain exhibited a severely reduced pigment content already when grown under LL conditions, and the Chl content as well as the pigment ratios were very similar to the ones observed when the wt was grown under HL conditions (Figure 4). Besides the obvious differences in pigmentation, the TM structure was mostly unaffected, and we only observed a slightly reduced number of TM pairs in the mutant strain when the ultrastructure of Synechocystis grown under LL growth conditions was analyzed via electron microscopy ( Figure 5). standard deviation from three independent experiments.
Similarly, the dnaK3KD strain adapted to changing light conditions and reduced its pigment content as expected when grown under HL conditions. However, the dnaK3KD strain exhibited a severely reduced pigment content already when grown under LL conditions, and the Chl content as well as the pigment ratios were very similar to the ones observed when the wt was grown under HL conditions (Figure 4).
Besides the obvious differences in pigmentation, the TM structure was mostly unaffected, and we only observed a slightly reduced number of TM pairs in the mutant strain when the ultrastructure of Synechocystis grown under LL growth conditions was analyzed via electron microscopy ( Figure 5).

Reducing the DnaK3 Content Results in an Altered PS I-to-PS II Ratio
Next, the relative amounts of PS II and PS I in the DnaK3 reduced strain were determined via 77 K fluorescence spectroscopy ( Figure 6A). Upon chlorophyll excitation at 435 nm, characteristic fluorescence emission maxima were detected at 721 nm (PS I), at 684 nm (CP43, PS II) and 693 nm (CP47, PS II).
Synechocystis wt cells grown under HL conditions showed a decreased PS I-to-PS II ratio compared to LL-adapted cells ( Figure 6A), which is a well-documented long-term adaptation to HL [44,46,47]. In contrast, dnaK3KD cells had a considerably decreased PS I-to-PS II ratio already under LL growth conditions. This finding is also supported by a Western blot analysis. When an identical quantity of protein was loaded, the Western blot shows that PS core subunits PsaA/B (PS I) and PsbA (PS II) are less abundant in the dnak3KD strain ( Figure 6C). When the cell extracts were normalized based on the Chl concentration ( Figure 6D), no difference in the band intensity was observed in case of PsaA/B, since in Synechocystis about 85% of the Chl is bound to PS I (assuming a PS I/PS II ratio of 2.5 [48], 96 chlorophylls per PS I [49], and 35 chl per PS II [50]). However, the PsbA band was more pronounced in the mutant strain when compared to the wt, which further supports the decreased PS I-to-PS II ratio in this strain. The decreased PS I-to-PS II ratio decreases even further when cells were shifted into HL ( Figure 6A).
However, at LL as well as at HL conditions, an increased relative fluorescence emission was observed at 684 nm (λ ex = 435nm) in the mutant strain ( Figure 6A). This fluorescence emission maximum originates from PS II as well as from the PBSs terminal emitter LCM [51], and thus indicates an increased relative phycobiliprotein content, as already observed in the absorbance measurements ( Figure 4A,C). Yet, the increased PBSs fluorescence emission at 684 nm is solely observed when PBSs are uncoupled and do not transfer the harvested light energy to the PSs [51,52]. Thus, to next assess energy transfer from PBSs to PS II, phycobiliproteins were excited at 580 nm and energy transfer to PS II was followed. When PBSs are coupled to PS II, light energy harvested by the PBS is transferred to PS II, resulting in quenching of the PBS fluorescence [53,54]. As can be seen in Figure 6B, upon PBS excitation an increased fluorescence emission at 684 nm (PBSs plus PS II) was observed but not at 693 nm (PS II) ( Figure 6B). Thus, the dnaK3KD strain indeed contains an increased amount of uncoupled PBSs compared to the wt. The decreased fluorescence emission at 721 nm results from the decreased PS I content ( Figure 6A) and most likely not from light-dependent energy distribution via state transitions, which is supposed to be physiologically important solely under LL growth conditions [55,56]. Taken together, the fluorescence spectra and the Western blot analyses reveal that the mutant has a generally decreased PS content, with a decreased PS I-to-PS II ratio and an increased amount of uncoupled phycobiliproteins. However, the mutant strain still adjusts the PS I-to-PS II ratio to changing light conditions, as observed for the wt strain.

The Photosynthetic Activity is Impaired in the DnaK3 Depleted Strain
Next, the photosynthetic activity of the mutant strain with a reduced DnaK3 content was studied in greater detail. By measuring oxygen evolution rates in presence of PPBQ, the activity of PS II can be specifically determined ( Figure 7A,B). When adapted to HL growth conditions, the O2 evolution rate per cell (OD750) was reduced in the wt strain compared to LL growth conditions ( Figure 7A), in line with the observation that the light-harvesting capacity is generally reduced in cyanobacterial cells under HL conditions [57]. In contrast to wt cells, the dnaK3KD strain showed a dramatically decreased O2 evolution rate already under LL conditions, when compared to the wt, and the activity decreased even further under HL conditions ( Figure 7A). However, when the O2 evolution rates were normalized to the Chl content, the O2 evolution rate remained essentially stable in the wt, regardless of the light conditions ( Figure 7B). In contrast, the O2 evolution rate was only marginally lower for the mutant strain under LL growth conditions than for the wt, but dramatically decreased under HL growth conditions. Thus, in contrast to the wt, the decreased O2 evolution in the mutant strain is not only a consequence of the decreased cellular Chl content ( Figure 4B), since the O2 evolution rate was also drastically decreased when the measurements were normalized to the Chl content ( Figure 7B). Taken together, the fluorescence spectra and the Western blot analyses reveal that the mutant has a generally decreased PS content, with a decreased PS I-to-PS II ratio and an increased amount of uncoupled phycobiliproteins. However, the mutant strain still adjusts the PS I-to-PS II ratio to changing light conditions, as observed for the wt strain.

The Photosynthetic Activity is Impaired in the DnaK3 Depleted Strain
Next, the photosynthetic activity of the mutant strain with a reduced DnaK3 content was studied in greater detail. By measuring oxygen evolution rates in presence of PPBQ, the activity of PS II can be specifically determined ( Figure 7A,B). When adapted to HL growth conditions, the O 2 evolution rate per cell (OD 750 ) was reduced in the wt strain compared to LL growth conditions ( Figure 7A), in line with the observation that the light-harvesting capacity is generally reduced in cyanobacterial cells under HL conditions [57]. In contrast to wt cells, the dnaK3KD strain showed a dramatically decreased O 2 evolution rate already under LL conditions, when compared to the wt, and the activity decreased even further under HL conditions ( Figure 7A). However, when the O 2 evolution rates were normalized to the Chl content, the O 2 evolution rate remained essentially stable in the wt, regardless of the light conditions ( Figure 7B). In contrast, the O 2 evolution rate was only marginally lower for the mutant strain under LL growth conditions than for the wt, but dramatically decreased under HL growth conditions. Thus, in contrast to the wt, the decreased O 2 evolution in the mutant strain is not only a consequence of the decreased cellular Chl content ( Figure 4B), since the O 2 evolution rate was also drastically decreased when the measurements were normalized to the Chl content ( Figure 7B). To test whether reducing the DnaK3 content somehow impairs PS II repair, we next determined O2 evolution rates under extreme HL conditions (1500 µmol photons m −2 ·s −1 ) in presence or absence of lincomycin, a protein synthesis inhibitor that has already been successfully used to block the PS II repair cycle in Synechocystis [58,59]. In absence of lincomycin, the wt strain did not show any changes in the PS II activity under constant extreme HL illumination, i.e., the wt cells harbor an effective PS II repair cycle ( Figure 7C). However, in presence of lincomycin, the PS II activity constantly decreased when cells were illuminated with extreme HL ( Figure 7D). The decreasing PS II activity, i.e., an impaired PS II protection and/or repair, can be observed for both the wt and the dnaK3KD strain in presence of lincomycin ( Figure 7D). However, in absence of lincomycin, the PS II activity was already lower in the dnaK3KD than in the wt strain after 10 min of illumination and constantly decreased further to about 50% after 1 h of illumination, whereas the wt activity remained about constant ( Figure 7C). Thus, PS II repair clearly is severely impaired in dnaK3KD cells, albeit the amount of expressed D1 protein did not alter (inlet in Figure 7C). Thus, PS II protection and/or repair is affected especially under light-stress conditions.
The photochemical efficiency of PS II can be specifically assessed using pulse amplitude modulated (PAM) fluorescence measurements. Therefore, dark/light induction curves were recorded ( Figure 8A). A minimal fluorescence (F0) is visible due to the measuring light, which, however, is not strong enough to stimulate photosynthetic electron transfer. Subsequently, a pulse of intense white light is given to reduce all PS II reaction centers, resulting in maximal fluorescence (Fm). The parameter Fv/Fm (Fv = Fm − Fo) is used to describe the maximal photochemical efficiency ( Figure 8B) [60]. To test whether reducing the DnaK3 content somehow impairs PS II repair, we next determined O 2 evolution rates under extreme HL conditions (1500 µmol photons m −2 ·s −1 ) in presence or absence of lincomycin, a protein synthesis inhibitor that has already been successfully used to block the PS II repair cycle in Synechocystis [58,59]. In absence of lincomycin, the wt strain did not show any changes in the PS II activity under constant extreme HL illumination, i.e., the wt cells harbor an effective PS II repair cycle ( Figure 7C). However, in presence of lincomycin, the PS II activity constantly decreased when cells were illuminated with extreme HL ( Figure 7D). The decreasing PS II activity, i.e., an impaired PS II protection and/or repair, can be observed for both the wt and the dnaK3KD strain in presence of lincomycin ( Figure 7D). However, in absence of lincomycin, the PS II activity was already lower in the dnaK3KD than in the wt strain after 10 min of illumination and constantly decreased further to about 50% after 1 h of illumination, whereas the wt activity remained about constant ( Figure 7C). Thus, PS II repair clearly is severely impaired in dnaK3KD cells, albeit the amount of expressed D1 protein did not alter (inlet in Figure 7C). Thus, PS II protection and/or repair is affected especially under light-stress conditions.
The photochemical efficiency of PS II can be specifically assessed using pulse amplitude modulated (PAM) fluorescence measurements. Therefore, dark/light induction curves were recorded ( Figure 8A). A minimal fluorescence (F 0 ) is visible due to the measuring light, which, however, is not strong enough to stimulate photosynthetic electron transfer. Subsequently, a pulse of intense white light is given to reduce all PS II reaction centers, resulting in maximal fluorescence (F m ). The parameter F v /F m (F v = F m − F o ) is used to describe the maximal photochemical efficiency ( Figure 8B) [60]. After switching on actinic light, an increased background fluorescence was detected, and PS II centers became photosynthetically active ( Figure 8A). Saturating light pulses resulted in a lowered Fm´ compared to the maximal fluorescence Fm measured in the dark, due to non-photochemical quenching processes [61]. An apparent increase of the absolute F0 background fluorescence was measured for wt cells grown under HL conditions compared to LL and for the dnaK3KD cells (grown under either condition), indicating a more reduced plastoquinone (PQ) pool ( Figure 8A). However, determining F0 and F0´ is somewhat problematic in cyanobacteria, as the PBS fluorescence can in part also contribute to the determined F0 fluorescence value [62], and thus discussion of solely F0 values is difficult. Hence, we also present the normalized coefficient of photochemical quenching of PS II Chl fluorescence (qP), which is not significantly biased by PBSs fluorescence [63]. qP is defined as 1 in the dark-adapted state and may decrease to 0 when all PS II centers are closed. In line with Figure  7B, in the wt strain slightly less PS II centers are open under HL conditions compared to LL. In the mutant strain, qP is similar to the wt under LL conditions, yet the value was dramatically decreased when dnaK3KD cells were grown under HL conditions, indicating an increased amount of closed PS II centers. Thus, the dnaK3KD strain can hardly cope with high light treatment. This observation is in excellent agreement with the determined O2 evolution rates (Figure 7), showing an impaired PS II protection and/or repair cycle. P700 + re-reduction measurements allow determining the time needed to re-reduce the oxidized PS I reaction center P700 + , which is not only affected by the PS I activity but also by the redox state of under LL conditions. A saturation pulse of 10,000 µmol photons was given for 20 ms to completely oxidize P 700 . The following fluorescence decrease illustrates re-reduction of P 700 + in the dark. At least ten traces were averaged and normalized. 1 represents completely oxidized and 0 completely reduced P 700 . (E) Re-reduction halftimes were determined via fitting the decay curves of the wt (black) and the dnaK3KD mutant strain (gray) with single exponential functions. Error bars represent standard deviation from at least three independent experiments.
After switching on actinic light, an increased background fluorescence was detected, and PS II centers became photosynthetically active ( Figure 8A). Saturating light pulses resulted in a lowered F mć ompared to the maximal fluorescence F m measured in the dark, due to non-photochemical quenching processes [61]. An apparent increase of the absolute F 0 background fluorescence was measured for wt cells grown under HL conditions compared to LL and for the dnaK3KD cells (grown under either condition), indicating a more reduced plastoquinone (PQ) pool ( Figure 8A). However, determining F 0 and F 0´i s somewhat problematic in cyanobacteria, as the PBS fluorescence can in part also contribute to the determined F 0 fluorescence value [62], and thus discussion of solely F 0 values is difficult. Hence, we also present the normalized coefficient of photochemical quenching of PS II Chl fluorescence (qP), which is not significantly biased by PBSs fluorescence [63]. qP is defined as 1 in the dark-adapted state and may decrease to 0 when all PS II centers are closed. In line with Figure 7B, in the wt strain slightly less PS II centers are open under HL conditions compared to LL. In the mutant strain, qP is similar to the wt under LL conditions, yet the value was dramatically decreased when dnaK3KD cells were grown under HL conditions, indicating an increased amount of closed PS II centers. Thus, the dnaK3KD strain can hardly cope with high light treatment. This observation is in excellent agreement with the determined O 2 evolution rates (Figure 7), showing an impaired PS II protection and/or repair cycle. P 700 + re-reduction measurements allow determining the time needed to re-reduce the oxidized PS I reaction center P 700 + , which is not only affected by the PS I activity but also by the redox state of the electron transport chain ( Figure 8D,E). The P 700 + absorbance signal increased when a saturating light pulse, which completely oxidized P 700 , was given and subsequently decreased due to P 700 + reduction by PC ( Figure 8D). The halftime of the re-reduction kinetic was determined by fitting the changes in the absorbance signal with a single exponential function ( Figure 8E). A faster re-reduction rate was observed for wt cells when cells were grown under HL compared to LL conditions, which likely originates from the reduced PS I content and the decreased PS I-to-PS II ratio (Figures 6 and 8).
In contrast, the dnaK3KD mutant strain had reduced re-reduction halftimes under both tested light conditions, and the halftimes were identical, regardless of the light condition ( Figure 8E). The reduced re-reduction halftimes can be explained by a more reduced PQ-pool and thus nicely support the conclusions drawn from the results shown in Figures 6 and 8. Together, these results demonstrate that the activity of PS II, but not of PS I, is impaired in the dnaK3KD mutant strain.

Discussion
Three different DnaK proteins are expressed in the cyanobacterium Synechocystis sp. PCC 6803. While two of the cyanobacterial DnaK proteins, DnaK2 and DnaK3, are essential, solely DnaK2 can be classified as a canonical Hsp70 protein, expression of which can largely alter under various stress conditions [24,64,65]. In contrast, the DnaK3 chaperone of Synechocystis has been suggested to be specifically involved in biogenesis and/or maintenance of TMs [16,25]. However, thus far this assumption was essentially exclusively based on the observations that (i) DnaK3 is attached to TMs and (ii) DnaK3 is encoded in all cyanobacterial genomes, except in Gloeobacter violaceus, the only cyanobacterium that does not contain an internal TM system [15,29].
Albeit the cellular DnaK3 content clearly is light-regulated (Figure 1), a basal DnaK3 level appears to be required for survival of Synechocystis cells not only in the light but also under LAHG conditions (Figure 2), where cells still have rudimentary TMs. Based on the CyanoExpress database the dnaK3 transcript level does not appear to adjust to changing light conditions in Synechocystis [66] or in Synechococcus sp. PCC 7942 [16]. Thus, (light-dependent) DnaK3 synthesis likely is post-transcriptionally regulated, as common in cyanobacteria [67,68].
A general decrease in the PS and PBS content per cell as well as a selective down-regulation of PS I is crucially involved in the adaptation of Synechocystis cells to HL conditions [46,67]. All these (expected) adjustments were observed when the Synechocystis wt strain was shifted from LL to HL growth conditions (Figures 6 and 7). Also, in case of the mutant strain, typical HL-adaptation processes were observed, although the light-induced changes were far less pronounced, since the mutant strain already exhibited characteristics of an HL-stressed strain under LL growth conditions. While in the dnaK3KD strain the relative PS I content was reduced and the PS I-to-PS II ratio per cell was lower than in the wt (Figure 6), PS I appears to function normally when DnaK3 was depleted, because re-reduction of PS I was even faster in the mutant strain ( Figure 8E), most likely due to the more reduced PQ-pool, which also results in a significant amount of the PBSs being detached from PS II ( Figure 6B).
Yet, the activity of PS II was clearly reduced in the mutant strain (Figures 7 and 8). A significant amount of PS II was inactive, potentially due to increased photodamage and/or impaired repair (Figures 7 and 8). Thus, DnaK3 likely is involved in PS II biogenesis and/or repair. In line with this assumption, expression of the Synechocystis dnaK3 gene was found being enhanced under UV-B stress [68]. Furthermore, the PS II core subunit D1 was proposed to be a substrate for DnaK3, which potentially guides the nascent polypeptide at the ribosome to the TM, where translation is completed [16]. The D1 protein is known to be especially susceptible to photodamage, and photodamaged D1 is rapidly degraded and replaced by newly synthesized protein to maintain a certain level of active PS II centers in cyanobacteria [33]. Thus, the here presented results clearly indicate that biogenesis and/or repair of PS II is impaired when the cellular DnaK3 content is reduced.
However, the observation that DnaK3 appears to be vital also in the dark (Figure 2), where PS II is inactive [7] and the finding that D1 is not essential for survival of Synechocystis under photoheterotrophic conditions [69] indicates that DnaK3 likely has additional physiological functions beyond PS II protection and/or repair. In cyanobacteria, TMs also contain the complexes of the respiratory e − -transfer chain [70], and the indications of an over-reduced PQ pool (Figures 6 and 8) suggests that other proteins and protein complexes are also affected when the DnaK3 content is reduced. A broader implication of DnaK3 in TM biogenesis and maintenance, involving biogenesis and/or repair of multiple TM complexes would be a convincing explanation.