Loading...
Reload document 
Open in new tab Article
DnaK3 Is Involved in Biogenesis and/or Maintenance
of Thylakoid Membrane Protein Complexes in the
Cyanobacterium Synechocystis sp. PCC 6803
Adrien Thurotte 1,2,†
, Ruven Jilly 1, Uwe Kahmann 3 and Dirk Schneider 1,*
1 Department of Chemistry, Biochemistry, Johannes Gutenberg University Mainz, 55128 Mainz, Germany;
, Tobias Seidel 1,†
2
adrienthurotte@netcourrier.com (A.T.); TobiasSeidel@gmx.net (T.S.); ruvenjilly@gmail.com (R.J.)
Institute of Molecular Biosciences, Goethe University Frankfurt, Max-von-Laue Straße 9,
60438 Frankfurt, Germany
3 Department of Molecular Cell Biology, Bielefeld University, 33615 Bielefeld, Germany; ZUD@gmx.de
* Correspondence: dirk.schneider@uni-mainz.de; Tel.: +49-6131-39-25833
† These authors contributed equally.
Received: 8 April 2020; Accepted: 28 April 2020; Published: 30 April 2020
Abstract: 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.
Keywords: chaperone; Hsp70; photosynthesis; thylakoid membrane biogenesis; photosystem
maintenance; Synechocystis sp. PCC6803
1. 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; doi:10.3390/life10050055
www.mdpi.com/journal/life
life(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7) Life 2020, 10, 55
2 of 16
as well as YidC, a membrane protein integrase [9–16]. Nevertheless, while some proteins are probably
more directly involved in TM formation, the structure and stability of TMs are also affected more
indirectly by pathways, which control the biogenesis of lipids and/or cofactors, and, e.g., mutants
defective in synthesis of chlorophyll or of the membrane lipid phosphatidylglycerol (PG) have severely
reduced TM systems [17–20].
Molecular chaperones of the Hsp70 family are involved in multiple cellular processes, such as
folding of newly synthesized proteins, protein disaggregation, prevention of protein misfolding,
protein transport, or the control of regulatory protein functions [21]. The thus far best characterized
Hsp70 chaperone is the DnaK protein of the bacterium Escherichia coli [22]. In cyanobacteria, at least two
DnaK proteins, DnaK2 and DnaK3, are highly conserved, and most cyanobacteria contain an additional
DnaK1 protein as well as further DnaK-like proteins [15,23,24]. While cyanobacterial genomes
typically encode several DnaK chaperones together with multiple DnaJ (Hsp40) proteins, which serve
as DnaK co-chaperones, the physiological function of this DnaK-DnaJ network in cyanobacteria is
essentially not understood. In recent years, the physiological roles of individual DnaK and DnaJ
proteins have been analyzed to some extent in the cyanobacteria Synechococcus sp. PCC 7942 and
Synechocystis [16,24–26]. In Synechocystis, three DnaK proteins are expressed together with at least
seven DnaJ proteins [15,25]. The two dnaK genes dnaK2 and dnaK3 are essential in Synechocystis,
but not dnaK1 [15]. The DnaK2 protein has been classified as the canonical DnaK protein involved in
cellular stress responses, and DnaK2 most likely functions together with Sll0897, the only type I DnaJ
protein expressed in Synechocystis [24,25]. In line with this, deletion of the sll0897 gene resulted in a
heat-sensitive phenotype [25]. However, interactions with other DnaJ proteins cannot be excluded,
and in fact, the DnaK2 protein interacts and cooperates with the type II J protein DnaJ2 in Synechococcus
sp. PCC 7942 [27].
In contrast to the remaining dnaJ genes, the dnaJ gene sll1933 (dnaJ3) could not be deleted in
Synechocystis, indicating that the encoded DnaJ3 protein is essential [25]. The dnaK3 and dnaJ3 genes
are organized in a conserved gene cluster in cyanobacteria, and a functional interaction of DnaK3
with DnaJ3 is assumed [28] DnaK3- and DnaJ3-homologs are encoded in essentially all cyanobacterial
genomes, except in Gloeobacter violaceus PCC 4721, a cyanobacterium that lacks TMs [29,30]. Based on
this observation it has been suggested that the physiological function of both proteins might be linked
to TMs, and consequently, DnaK3 and DnaJ3 were suggested to be involved in the biogenesis and/or
maintenance of TMs [16,25,31]. The DnaK3s of both Synechococcus and Synechocystis co-purify with
membranes, and the unique DnaK3 C-terminus has been implicated to mediate tight membrane
binding of DnaK3 in Synechocystis [15,31]. However, what might be the function of DnaK3 at TMs?
The function of a cyanobacterial DnaK3 has recently been linked to the PS II reaction center
protein D1 [16], the main target of stress-induced damage in the photosynthetic electron transport
chain, which is constantly degraded and replaced by newly synthesized proteins in a PS II repair
cycle [32,33]. Furthermore, a Hsp70 chaperone is involved in the biogenesis, protection and/or repair
of PS II complexes in chloroplasts [34,35]. Based on these observations we hypothesized that the
physiological functions of DnaK proteins might have diverged in cyanobacteria, and DnaK3 potentially
is specifically involved in biosynthesis/maintenance of TM complexes involved in photosynthesis.
In the present study, we have analyzed the role of the Hsp70 protein DnaK3 in TM maintenance
in the cyanobacterium Synechocystis sp. PCC 6803. Expression of DnaK3 is light-regulated. Reduction
of the cellular DnaK3 content resulted in decreased PS and phycobilisome (PBS) contents, a lowered
PS I-to-PS II ratio, a generally reduced photosynthetic activity as well as disturbed PS II activity at
elevated light conditions. The observation that the PS II activity is affected after photoinhibition in a
mutant strain, where the cellular DnaK3 content is reduced, and the comparison of the mutant strain
with Synechocystis wt suggests a specific function of DnaK3 in PS II protection and/or repair. However,
based on the here presented data its activity must be wider. Thus, our findings support the assumption
that DnaK3 is involved in biogenesis and/or maintenance of TM-localized electron transfer complexes
in cyanobacteria.
Life 2020, 10, 55
3 of 16
2. Materials and Methods
2.1. 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% CO2 and grown under fluorescent white light at
a light intensity of 20 (LL, low light) or 120 (HL, high light) µmol/m2 s, respectively. To determine
growth rates, the strains were initially adjusted to an OD750 of 0.05 in BG11 medium, containing 5 mM
glucose, and growth was followed by monitoring OD750. 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).
◦
2.2. SDS-PAGE and Immunoblot Analysis
Synechocystis cells were harvested in the exponential growth phase at an OD750 below 2.0.
Cell pellets were resuspended in buffer (50 mM HEPES, pH 7.0, 25 mM CaCl2, 5 mM MgCl2, 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.
◦
2.3. 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
−1.
step, the concentration of kanamycin was enhanced in the growth medium from 80 to 275 µg mL
To check whether the strain was completely segregated, genomic DNA was isolated and analyzed
by PCR using the primers NtdnaK3check (5’-gtttttagaagcggagaaagtgg-3´) and CtdnaK3check
(5´-cctttgggttggaaaccattgg-3´).
2.4. 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].
2.5. 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 KH2PO4, 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.
Life 2020, 10, 55
4 of 16
2.6. 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
−1. Ratios of cyanobacterial chromophores were determined using the
value of 300,000 cells mL
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
−1 in
Series 2 spectrofluorimeter. Cultures were adjusted to a chlorophyll concentration of 3 µg·mL
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.
2.7. 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
−1). Prior to the measurement, the cultures were adjusted to a chlorophyll concentration
photons m
−1
of 3 µg·mL
lincomycin was added prior to illumination (1500 µmol photons m
−2·s
−1 in BG11 medium. For experiments in presence of a protein synthesis inhibitor, 100 µg·mL
−2·s
−1).
2.8. 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 (F0) and
maximal (Fm and Fm´) 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.
2.9. P700 Re-Reduction Kinetics
Re-reduction kinetics were recorded using a Dual-PAM-100 measuring system. P700 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 (t1/2). Prior to the measurement, the different cultures were
adjusted to a chlorophyll concentration of 3 µg·mL
−1 in BG11 medium.
3. Results
3.1. 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.
Life 2020, 10, 55
5 of 16
Figure 1. Light-dependent accumulation of DnaK1, 2, and 3. Dark-adapted Synechocystis cultures were
shifted into the light (0–24 h). Cell extracts (20 µg protein) were analyzed at different time points via
immunodetection, using anti-DnaK1, 2, or 3 antibodies as well as antibodies directed against PS I
(PsaA/B) and PS II (PsbA) core subunits or the ribosomal protein L23 (loading control).
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
−1, a fragment corresponding in size to the wild type (wt) dnaK3
growth medium up to 275 µg·mL
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, x 5 of 17 Figure 1. Light-dependent accumulation of DnaK1, 2, and 3. Dark-adapted Synechocystis cultures were shifted into the light (0–24 h). Cell extracts (20 µg protein) were analyzed at different time points via immunodetection, using anti-DnaK1, 2, or 3 antibodies as well as antibodies directed against PS I (PsaA/B) and PS II (PsbA) core subunits or the ribosomal protein L23 (loading control). 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.