Microbiology (2014), 160, 228–241
DOI 10.1099/mic.0.073080-0
Essential roles of iron superoxide dismutase in
photoautotrophic growth of Synechocystis sp.
PCC 6803 and heterogeneous expression of
marine Synechococcus sp. CC9311 copper/zinc
superoxide dismutase within its sodB knockdown
mutant
Wen-Ting Ke, Guo-Zheng Dai, Hai-Bo Jiang, Rui Zhang
and Bao-Sheng Qiu
Correspondence
Bao-Sheng Qiu
[email protected]
Received 7 September 2013
Accepted 6 November 2013
School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, PR China
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal
University, Wuhan 430079, Hubei, PR China
Synechocystis sp. PCC 6803 possesses only one sod gene, sodB, encoding iron superoxide
dismutase (FeSOD). It could not be knocked out completely by direct insertion of the kanamycin
resistance cassette. When the promoter of sodB in WT Synechocystis was replaced with the
copper-regulated promoter PpetE, a completely segregated PpetE–sodB strain could be
obtained. When this strain was cultured in copper-starved BG11 medium, the chlorophyll a
content was greatly reduced, growth was seriously inhibited and the strain was nearly dead during
the 8 days of growth, whilst the WT strain grew well under the same growth conditions. These
results indicated that sodB was essential for photoautotrophic growth of Synechocystis. The
reduction of sodB gene copies in the Synechocystis genome rendered the cells more sensitive to
oxidative stress produced by methyl viologen and norflurazon. sodB still could not be knocked out
completely after active expression of sodC (encoding Cu/ZnSOD) from Synechococcus sp.
CC9311 in the neutral site slr0168 under the control of the psbAII promoter, which means the
function of FeSOD could not be complemented completely by Cu/ZnSOD. Heterogeneously
expressed sodC increased the oxidation and photoinhibition tolerance of the Synechocystis sodB
knockdown mutant. Membrane fractionation followed by immunoblotting revealed that FeSOD
was localized in the cytoplasm, and Cu/ZnSOD was localized in the soluble and thylakoid
membrane fractions of the transformed Synechocystis. Cu/ZnSOD has a predicted N-terminal
signal peptide, so it is probably a lumen protein. The different subcellular localization of these two
SODs may have resulted in the failure of substitution of sodC for sodB.
INTRODUCTION
Reactive oxygen species (ROS) are inevitable byproducts of
aerobic metabolism and potent agents that cause oxidative
damage. ROS usually include singlet-state oxygen (1O2), the
superoxide ion (O22), hydrogen peroxide (H2O2) and the
highly destructive hydroxyl radical (OH.). The reaction
centres of photosystem (PS) I and II in thylakoids are the
major sites of ROS generation (Asada, 2006). Within PSII,
1
O2 is produced by energy input to oxygen from photosensitized chlorophyll, and univalent reduction of O2 using
Abbreviations: MV, methyl viologen; NF, norflurazon; PS, photosystem;
ROS, reactive oxygen species; SOD, superoxide dismutase.
Five supplementary figures are available with the online version of this
paper.
228
electrons from PSII generates O22 at the reducing side of PSI
(Latifi et al., 2009).
Low-concentration ROS play an important role in defence
against infection, cell signalling and apoptosis (Valko et al.,
2007). However, when ROS are formed in excess, the
oxidation of cellular biomolecules by ROS initiates oxidative
damage of proteins, lipids and nucleic acids (Polle, 2001).
Living organisms have developed various defences to protect
against ROS damage (Imlay, 2013). Whilst some of these
defences are non-enzymic (such as carotenoid, glutathione,
vitamin A, C, E, etc.), others are enzymic [superoxide
dismutases (SODs), catalases and peroxidases] (Apel & Hirt,
2004; Latifi et al., 2009). SODs are the first line of defence to
alleviate oxidative stress in virtually all living organisms that
survive in oxic environments (McCord & Fridovich, 1969),
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SODs and Synechocystis
and catalyse the transformation of O22 to O2 and H2O2. The
scavenging of H2O2 differs between cyanobacteria and
higher plant chloroplasts (Asada, 2006). Cyanobacteria do
not contain an ascorbate peroxidase, and they use catalase
and/or thioredoxin peroxidase (2-cysteine peroxiredoxin)
activities to catalyse H2O2 to H2O and O2 (Regelsberger
et al., 2002).
To date, all SODs have been found to be metalloproteins
with a redox-active metal. SOD isoforms are classified
according to the redox metal within the active site, which
includes iron, manganese, copper (with a structural zinc
ion) and nickel. In addition, a cambialistic form that uses
iron/manganese in its active site also exists (Lancaster et al.,
2004). The existence of multiple SODs may result from the
fact that cells are divided into compartments by internal
membranes. Since O22 ions are negatively charged and
cannot readily cross a phospholipid bilayer, they are
effectively trapped within the compartment where they
were generated. This may have selected for the evolution of
multiple SODs in compartmentalized cells (Takahashi &
Asada, 1983). Herbert et al. (1992) carried out the first
genetic analysis of SODs in a unicellular cyanobacterium
(Synechococcus elongatus PCC 7942) and suggested that
multiple SODs (FeSOD in soluble extracts and other SODs
in membrane fractions) exist to protect the multiple cell
compartments against oxidative stress. Cyanobacteria
represent the first oxygenic photoautotroph and they bear
a particular risk of oxygen toxicity, because molecular O2
can be photoreduced to O22 by electrons from PSI
(Mehler, 1951), and highly conserved A-type flavoproteins
Flv1 and Flv3 are essential for this process in vivo (Helman
et al., 2003). Therefore, cyanobacterial SODs play very
important roles in preventing the oxidative damage
associated with photosynthesis.
Among the four kinds of SODs, the structure, phylogenetic
diversity, regulation and cellular location of FeSOD and
MnSOD have been studied intensively in the past decades
(Miller, 2004; Wolfe-Simon et al., 2005). However, there is
rather limited information regarding Cu/ZnSOD and
NiSOD in cyanobacteria. Cu/ZnSOD is rare among cyanobacteria, and genome sequence analyses of 64 cyanobacterial SODs confirmed its existence only in six Synechococcus
and one Lyngbya strains, including Synechococcus sp.
CC9311 (hereafter called Synechococcus 9311) (Priya et al.,
2007). Cu/ZnSOD has different primary and tertiary
structures from FeSOD and MnSOD, and almost certainly
evolved independently (Wolfe-Simon et al., 2005). Chadd
et al. (1996) first identified Cu/ZnSOD within the marine
cyanobacterium Synechococcus sp. WH 7803 according to
different sensitivities of in-gel activity staining bands to
5 mM H2O2, 2 mM cyanide or 2 mM copper chelator
diethyldithiocarbamate. Two different signatures (G-F-H[ILV]-H-x-[NGT]-[GPDA]-[SQK]-C and G-[GA]-G-G-[AEG]R-[FIL]-[AG]-C-G) were found in the cyanobacterial Cu/
ZnSOD protein sequence (Priya et al., 2007), and protein
sequences (GFHLHAGDQC and GGGGARIACG) of the Cu/
ZnSOD putative encoding gene (sync_1771, sodC) in
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Synechococcus 9311 (Palenik et al., 2006) fitted well with
them. Combined with in-gel activity staining and immunoblotting results, we concluded that sync_1771 in Synechococcus
9311 encoded Cu/ZnSOD (unpublished data).
Based on 64 cyanobacterial SODs available in public
databases, an analysis found that cyanobacteria harbour
either FeSOD or NiSOD alone, or a combination of FeSOD
and MnSOD, FeSOD and Cu/ZnSOD or NiSOD and Cu/
ZnSOD (Priya et al., 2007). All cyanobacteria have a
cytosolic sod gene encoding either soluble FeSOD or
soluble NiSOD, and these could be the housekeeping SODs
(Regelsberger et al., 2004; Priya et al., 2007). Some cyanobacteria have additional SODs, e.g. filamentous species
with nitrogenase activity appear to need one or more
supplementary membrane-bound MnSODs to effectively
lower O22 levels (Regelsberger et al., 2004). When MnSOD
was knocked out in nitrogen-fixing cyanobacterium
Anabaena sp. PCC 7120, it was more susceptible to
photoinhibition, and both PSI and II were damaged more
severely by the photoinhibitory light, suggesting that
MnSOD plays important roles in the protection of both
PSs (Zhao et al., 2007). Furthermore, their protective roles
during oxidative stress are dependent on nitrogen status:
under nitrogen-fixing conditions, overexpression of MnSOD
enhanced oxidative stress tolerance, whilst FeSOD overexpression was detrimental; under nitrogen-supplemented
conditions, overexpression of either SOD protein, especially
FeSOD, conferred significant tolerance against oxidative
stress (Raghavan et al., 2011). Some Synechococcus and all
Prochlorococcus genomes sequenced or assembled from
metagenomic data contain a sodN gene coding for NiSOD
(Palenik et al., 2003; Rusch et al., 2010; Dupont et al., 2012).
Several attempts to inactivate insertionally the sole sod gene,
sodN, in Synechococcus WH8102 were unsuccessful and its
knockout appeared to be lethal (Dupont et al., 2012). When
sodN of Synechococcus WH8102 was interrupted with sodB
from Synechococcus sp. WH7803, the knockout strains
exhibited an impaired ability to grow at low iron concentrations (Dupont et al., 2012). Except for NiSOD, Synechococcus
sp. CC9311, CC9902 and CC9605 have an additional Cu/
ZnSOD (Priya et al., 2007). However, attempts to interrupt
their sodN genes were also unsuccessful (Dupont et al., 2012),
suggesting that cytosolic NiSOD was necessary for the
survival of these strains. Synechococcus elongatus PCC 7942
possesses only one kind of SOD (FeSOD), which is encoded
by two genes (Priya et al., 2007). Knocking out one of them
had no influence on photoautotrophic growth of
Synechococcus elongatus PCC 7942, while double mutation
was not conducted, which could be lethal (Herbert et al.,
1992; Thomas et al., 1998).
So far, little information is available on whether different
kinds of SODs from various cyanobacterial species
function differently. Synechocystis sp. PCC 6803 (hereafter
called Synechocystis 6803) is used widely to study
fundamental processes of oxygenic photosynthesis. There
is only one sod gene (slr1516, sodB, encoding FeSOD) in
Synechocystis 6803 (Kaneko et al., 1996), which makes it
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W.-T. Ke and others
convenient for comparing the function of SODs derived
from different species. In order to investigate whether Cu/
ZnSOD from marine Synechococcus 9311 can substitute the
function of FeSOD in freshwater Synechocystis 6803, we
attempted to knock out sodB and heterologously express
sodC in Synechocystis 6803. However, sodB was found to be
essential for photoautotrophic growth of Synechocystis 6803
in this study. It could not be knocked out completely under
antibiotic pressure even after heterologous expression of
active sodC. Heterologous expression of sodC increased the
tolerance of Synechocystis sodB2 to oxidation and photoinhibition stress. Different localizations of these two SODs
may determine their inability to interchange in the
transformed cyanobacterium.
METHODS
Cyanobacterial strains, culture conditions and general methods. A glucose-tolerant Synechocystis 6803 strain (hereafter referred to
as WT) was cultured in BG11 medium at 30 uC under continuous
illumination of 30 mmol photons m22 s21; for copper-regulated
growth experiments, illumination of 45 mmol photons m22 s21 was
used. BG11 plates were prepared by adding 1.4 % (w/v) agar, 8 mM
TES (pH 8.2) and 0.3 % (w/v) Na2S2O3. According to culture
requirements of the mutants or complemented strains, kanamycin
(25 mg ml–1), spectinomycin (20 mg ml–1) or both were added into
BG11 medium. Synechococcus 9311 was cultured in SN medium based
on artificial seawater at 22 uC under continuous illumination of
15 mmol photons m22 s21. The preparation of SN medium was as
described by Waterbury & Willey (1988). For complete segregation of
the sodB2 mutant, cultures were grown under conditions of decreased
illumination of 5 mmol photons m22 s21 in the presence of 0.1 % (w/
v) glucose, 10 mM NaHCO3, 100 mg BSA ml21 and 20 mg leucine
ml21. For the analysis of Synechocystis 6803 growth in copper-starved
medium, cupric sulfate was omitted from the BG11 medium.
Glassware used for copper starvation experiments was soaked with
10 % (v/v) HCl to remove residual copper and then rinsed five times
with MilliQ water (Millipore) prior to use. For copper starvation
experiments of Synechocystis 6803, exponential phase WT and PpetE–
sodB strains grown in BG11 medium were inoculated into copperstarved BG11 medium at an OD730 of 0.02 and cultured for 4 days.
Before transferring and cultivating in fresh copper-starved BG11
medium, the cells were collected by centrifugation and washed twice
with fresh copper-starved medium. After transferring to copperstarved BG11 medium for three times as above, the strains in the
exponential phase were used for growth comparison experiments.
After transferring to copper-starved BG11 medium once as above, the
strains in the exponential phase were used for whole-cell absorbance
spectra comparison.
The growth of Synechocystis 6803 was monitored by turbidity (OD730)
or chlorophyll a content from at least three parallel cultures. The
specific growth rates (day21) were calculated as follows:
m5(lnX42lnX0)/4, where X4 and X0 are the OD730 values of the day
4 and initial inocula, respectively. A blank was prepared using growth
medium. Chlorophyll a content was determined spectrophotometrically in 95 % ethanol extracts (Lichtenthaler & Buschmann, 2001).
A blank was prepared using 95 % ethanol. Whole-cell absorbance
spectra from 400 to 800 nm were recorded on a Cary 300 UV/visible
spectrophotometer (Varian). A blank was prepared using BG11
medium.
Construction of plasmids. Molecular manipulations were per-
formed following standard protocols. Enzymes were used according
230
to the instructions provided by the manufacturers. Sticky ends of
DNA fragments were blunted using T4 DNA polymerase (Promega).
Restriction enzymes and T4 DNA ligase were purchased from Takara.
Plasmids and primers used are listed in Table 1.
Construction of pHS168 for inactivating sodB. The DNA fragment
containing the full-length sodB gene (slr1516) and its flanking
sequences was generated by PCR using primers slr1516 up and
slr1516 down with Synechocystis 6803 chromosome DNA as template,
cloned into pMD18-T vector (Takara) (Yang et al., 2008), and
confirmed by sequencing. The kanamycin-resistant cassette (C.K2)
excised from pRL446 (GenBank accession no. EU346690) (Elhai &
Wolk, 1988) by PvuII was inserted into the HpaI site of the abovementioned sequenced plasmid, resulting in pHS168 for the
inactivation of sodB in Synechocystis 6803. The physical map of the
Synechocystis 6803 genome region containing sodB, indicating the
insertion position of C.K2 into sodB, is shown in Fig. 1(a). The
resulting pHS168 was used to transform WT Synechocystis 6803, in
which sodB was inactivated by insertion of C.K2.
Construction of pHS203 for complementing the Synechocystis
sodB” mutant. The copper-regulated petE promoter (PpetE) (Zhang
et al., 1992) fragment (436 bp, corresponding to –436 to –1, relative
to the ATG of the petE gene) was generated by PCR using primers
PpetE-1 and PpetE-2, with Synechocystis 6803 chromosome DNA as
template, cloned into pMD18-T, and confirmed by sequencing,
resulting in pHS179. A spectinomycin resistance cassette (Omega)
excised by DraI from pRL57 (Black et al., 1993) was inserted into the
XbaI site of pHS179, resulting in pHS185. The DNA fragment
containing the full-length sodB was generated by PCR using primers
slr1516 exp-1 and slr1516 exp-2 with Synechocystis 6803 chromosome
DNA as template, cloned into pMD18-T, and confirmed by
sequencing, resulting in pHS193. The Omega–PpetE fragment was
excised by SalI/BamHI from pHS185 and cloned into the SalI sites of
pHS193, resulting in pHS194. The Omega–PpetE–sodB fragment was
excised by PstI/BamHI from pHS194 and cloned into the EcoRI sites
of plasmid pKW1188 (Williams, 1988), resulting in pHS203. The
main construction steps are shown in Fig. S1 (available in
Microbiology Online). pKW1188 contained a slr0168 gene, which
was regarded as a platform for inserting the exogenous gene by
homologous recombination. The resulting pHS203 was used to
transform the Synechocystis 6803 sodB2 mutant, in which sodB was
expressed under the control of the petE promoter.
Construction of pHS198 for regulating the expression of FeSOD
with the petE promoter in WT Synechocystis 6803. The DNA
fragment containing the upstream sequences of sodB (the putative
promoter region, 700 bp, corresponding to –735 to –34, relative to
the ATG of the sodB gene) was generated by PCR using primers
slr1516 up and slr1516-3 with Synechocystis 6803 chromosome DNA
as template, cloned into pMD18-T, and confirmed by sequencing,
resulting in pHS197. The Omega–PpetE–sodB fragment was excised by
PstI/BamHI from pHS194 and cloned into the PstI sites of pHS197,
resulting in pHS198. The main construction steps of pHS198 are
shown in Fig. S2. The physical map of sodB in the Synechocystis 6803
genome with genetic modifications is shown in Fig. S3(a). pHS198
was used to transform WT Synechocystis 6803, in which sodB was
expressed under the control of the petE promoter.
Construction of pHS304 for expressing sodC (sync _ 1771,
encoding Cu/ZnSOD) using a PpsbAII expression platform
vector within the Synechocystis sodB” mutant. sodC (sync_1771)
from Synechococcus 9311 genome was generated by PCR using
primers sync_1771 up and sync_1771 down. The PCR products were
inserted into the NdeI site (blunted with T4 DNA polymerase) of the
expression platform plasmid pHS298 (Jiang et al., 2012), making the
gene in the same orientation as the psbAII promoter (PpsbAII).
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SODs and Synechocystis
pHS298 was constructed based on pKW1188 by inserting an Omega–
PpsbAII fragment into the EcoRI site of slr0168, which was also regarded
as a platform for inserting the exogenous gene by homologous
recombination (Jiang et al., 2012). The main construction steps of
pHS304 are shown in Fig. S4. The resulting pHS304 was used to
transform the Synechocystis 6803 sodB2 mutant, in which sodC was
expressed under the control of the psbAII promoter.
Construction of the mutant and complementation strains of
Synechocystis 6803. Transformation of Synechocystis 6803 was
performed as described by Williams (1988). Homologous doublecross-over recombinants were generated between the plasmid and
genomic DNA under positive selection of corresponding antibiotics
(Labarre et al., 1989). The degree of segregation of the sodB2 mutant
was examined by PCR using the primer pair slr1516 up and slr1516
down with PCR of chromosomal DNA from WT Synechocystis 6803
and plasmid pHS168 as controls. The degree of segregation of the
PpetE–sodB strain was examined by PCR using the primer pair slr1516
up and slr1516 exp-2 with PCR of chromosomal DNA from WT
Synechocystis 6803 and plasmid pHS198 as controls. To complement
the sodB2 mutant, pHS203 and pHS304 were introduced, respectively.
During growth in BG11 medium with both 25 mg kanamycin ml21 and
20 mg spectinomycin ml21, sodB and sodC were expressed constitutively, respectively. Details of the generation of mutant and
complementation strains are shown in Table 1, and Figs S1, S2 and S4.
Detection of active SOD. Riboflavin can be reduced photochemically
in the presence of the oxidizable substance N,N,N9,N9-tetramethylethylenediamine (TEMED) and generates O22, and the superoxide radical
in turn will reduce colourless nitro blue tetrazolium (NBT) to a blue
formazan that is insoluble practically. SOD inhibited the formation of
the blue formazan and signalled its location on acrylamide gels by
causing achromatic zones on otherwise uniformly blue gels (Beauchamp
& Fridovich, 1971). The in-gel SOD activity staining procedure was
performed according to the method of Beauchamp & Fridovich (1971)
as follows. The proteins were first separated by non-denaturing PAGE
(native PAGE). The gel was then soaked in 2.5 mM NBT in phosphate
buffer (20 mM NaH2PO4, 20 mM K2HPO4, pH 7.8) for 30 min before
it was transferred to phosphate buffer containing 28 mM TEMED and
28 mM flavin for another 20 min. The gel was then rinsed twice with
phosphate buffer before being illuminated with white light at an
intensity of 100 mmol photons m22 s21.
Measurement of chlorophyll fluorescence. PSII activity was
analysed in vivo with a WATER-PAM chlorophyll fluorometer
(Walz). All samples were dark-adapted for 10 min before measurement.
The maximum PSII quantum yield (Fv/Fm) was determined with the
saturation pulse method (Schreiber et al., 1995; Campbell et al., 1998).
Antibody production against FeSOD and Cu/ZnSOD. sodB and
sodC genes from Synechocystis 6803 and Synechococcus 9311 were
obtained by PCR amplification with their corresponding chromosomal DNA template in the presence of the high-fidelity enzyme
Pfu DNA polymerase (Promega), by using the primers sets: A 1516-1/
A 1516-2 and sync_1771 exp-1/sync_1771 exp-2, respectively. The
amplified fragments were first cloned into pMD18-T vector and
sequenced. The fragments obtained by digestion with NdeI and XhoI
from the resultant sequenced plasmid were cloned into pET41a
(Novagen) and transformed into Escherichia coli strain BL21(DE3) for
overexpression. The overexpressed proteins were purified by His-bind
resin (Novagen) and then used to generate two kinds of polyclonal
antibodies in rabbits that, respectively, recognized FeSOD and Cu/ZnSOD
in WT Synechocystis 6803 and sodC-transformed Synechocystis 6803.
Membrane isolation and immunoblotting analysis. The trans-
formed Synechocystis 6803 cells were collected by centrifugation at
6000 g, resuspended in 20 mM potassium phosphate (pH 7.8) with
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100 mM PMSF and ruptured with glass beads by vortexing at
2500 r.p.m. for 30 s per time, with 30 s interval of cooling on ice.
This process was repeated 10 times and the total time for breaking
cells was 5 min. Observation under a microscope revealed most cells
to be broken. The cell debris was removed by centrifugation at 4 uC
and 4000 g for 10 min. The supernatant was then centrifuged at 4 uC
and 100 000 g for 30 min to separate membrane proteins from
soluble proteins; the pellet was the membrane fraction. The
membranes were washed three times with 20 mM potassium
phosphate (pH 7.8) and named crude membranes. Cell fractions
containing the thylakoid membrane or the plasma membrane were
prepared according to the procedures described by Huang et al.
(2002) and Srivastava et al. (2005). Equal amounts of membrane and
soluble proteins were loaded, separated by 12 % SDS-PAGE,
transferred to nitrocellulose filters (Millipore), detected with antiFeSOD, anti-Cu/ZnSOD, anti-NrtA or anti-CP47 rabbit antiserum,
and visualized with goat anti-rabbit alkaline phosphatase antibody
(Invitrogen) with NBT and 5-bromo-4-chloro-3-indolylphosphate
(Amresco) as substrates. SDS-PAGE and immunoblotting were
performed using standard methods. Antibodies against NrtA and
CP47 of Synechocystis 6803 (used at 1 : 6000 dilution) were provided
kindly by Professor Xudong Xu (Institute of Hydrobiology, Chinese
Academy of Sciences, Wuhan, PR China).
RESULTS
sodB is essential for photoautotrophic growth of
Synechocystis 6803
sodB is the sole sod gene in the Synechococcus 6803
chromosome (see Cyanobase at http://genome.microbedb.
jp/cyanobase/). We tried to inactivate it by insertion of a
kanamycin resistance cassette. As cyanobacteria contain 10–
20 copies of their chromosome in individual cells (Labarre
et al., 1989), in order to segregate the mutant allele of sodB to
a homozygous condition, they were selected under antibiotic
pressure in solid or liquid BG11 medium many times. Many
attempts to segregate the mutant allele of sodB to a
homozygous condition were unsuccessful, even though the
BG11 medium was supplemented with glucose (0.1 %),
NaHCO3 (10 mM), BSA (100 mg ml21) and leucine (20 mg
ml21), and the strains were cultured under a decreased
illumination intensity of 5 mmol photons m22 s21, according to Herbert et al. (1992) and Nefedova et al. (2003). PCR
analysis of transformant’s chromosomal DNA with genespecific primers indicated that these colonies were still
heterozygous (Fig. 1b). Although nearly all of the WT sodB
copies in the Synechocystis genome were missed (Fig. 1b),
FeSOD could still be detected at the protein level by
immunoblotting and in-gel activity staining (Fig. 1c, d).
These results indicated that sodB in Synechocystis 6803 could
not be knocked out completely by insertion of the
kanamycin resistance cassette directly. The Synechocystis
6803 sodB2 mutant was complemented with pHS203 carrying
the WT sodB through homologous double-cross-over
recombinants with the genome. In the complemented
strain, sodB was expressed under the control of PpetE and
the growth was restored to the WT level (Fig. 2).
Furthermore, a fragment containing Omega–PpetE was
integrated upstream of sodB in the genome of WT
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W.-T. Ke and others
Table 1. Cyanobacterial strains, plasmids, and primers used in this study
Strain, plasmid or primer
Synechocystis PCC 6803 strains
WT
sodB : C.CK2 (sodB2)
slr0168 : : Omega–PpetE–sodB
(sodB2 com)
Omega–PpetE–sodB (PpetE–sodB)
Omega–PpsbAII–sodC (sodB2
–sodC com)
Plasmids
pMD18-T
Derivation and/or relevant characteristics
Glucose tolerant, without antibiotic resistance
Kmr, Synechocystis 6803 mutant
Kmr Spr, Omega–PpetE–sodB integrated into the EcoRI site of slr0168 in the
genome of mutant Synechocystis sodB2
Kmr Spr, Omega–PpetE integrated upstream of sodB in the genome of
Synechocystis WT
Kmr Spr, Omega–PpsbAII–sodC integrated into slr0168 in the genome
of mutant Synechocystis sodB2
This study
This study
This study
Apr, cloning vector
Yang et al.
(2008)
This study
pKW1188
pRL446
Apr Kmr, PCR fragment containing sodB and its flanking sequences cloned
into pMD18-T and C.K2 inserted in its HpaI site
Apr, PCR fragment containing the petE promoter region cloned into pMD18-T;
the size of the petE promoter is 436 bp, corresponding to –436 to –1, relative
to the ATG of the petE gene
Apr Spr, spectinomycin resistance cassette Omega from pRL57 inserted into
pHS179 at the XbaI site
Apr, PCR fragment containing full-length sodB cloned into pMD18-T
Apr Spr, fragment Omega–PpetE from pHS185 which was excised by SalI/BamHI
was inserted into SalI of pHS193
Apr, PCR fragment containing upstream sequences of sodB (700 bp, corresponding
to –735 to –34, relative to the ATG of sodB gene) cloned into pMD18-T
Apr Spr, fragment Omega–PpetE–sodB from pHS194 which was excised by
PstI/BamHI was inserted into pHS197 at the PstI site
Apr Spr, fragment Omega–PpetE–sodB from pHS194 which was excised by
PstI/BamHI was inserted into pKW1188
Apr Spr, PCR fragment containing full-length sodC from Synechococcus cloned
in the PpsbAII expression vector pHS298 at the NdeI site
Apr Spr, PpsbAII expression vector, bearing an integrative platform slr0168 for
Synechocystis 6803; the size of the psbAII promoter in pHS298 is 318 bp,
corresponding to –331 to –1 (without the high-light-responsive region 230
to 216) relative to the ATG of the psbAII gene
Apr Kmr, plasmid bearing an integrative platform slr0168 for Synechocystis 6803
Kmr, cloning vector with the kanamycin resistance cassette (C.K2)
pRL57
Spr, cloning vector with the spectinomycin resistance cassette Omega
Primers
PpetE-1
PpetE-2
slr1516 up
slr1516 down
slr1516 exp-1
slr1516 exp-2
slr1516-3
PpsbAII oe-1
59-AAGGATTCATAGCGGTTGCC-39
59-ACTTCTTGGCGATTGTATC-39
59-TACCGAGGAGCAACAGGACT-39
59-CAGGCTGGAAGCGTCAATCA-39
59-TGGAATCCCCTATTGAGTAGAG-39
59-CTAGGCCGCTGCTAAGTTAG-39
59-TGGCTCTCACTTTACCCTA-39
59-GCGTGCAAGGCCCAGTGATC-39
PpsbAII oe-2
59-CATATGGTTATAATTCCTTAGTTCAGATTGGAACTGACTAAACTTAGTC-39
sync_1771
sync_1771
sync_1771
sync_1771
59-ATGTACCGACTTGGTGCACTCTTGG-39
59-TCAAGACCCCACACCGCAGG-39
59-CATATGAGCACGATTGAGGTCACGATTAATAG-39
59-CTCGAGAGACCCCACACCGCAGGCA-39
pHS168
pHS179
pHS185
pHS193
pHS194
pHS197
pHS198
pHS203
pHS304
pHS298
232
up
down
exp-1
exp-2
Reference or
source
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This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Jiang et al.
(2012)
Williams (1988)
Elhai & Wolk
(1988)
Black et al.
(1993)
This study
This study
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Jiang et al.
(2012)
Jiang et al.
(2012)
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Microbiology 160
SODs and Synechocystis
Table 1. cont.
Strain, plasmid or primer
A 1516-1
A 1516-2
Derivation and/or relevant characteristics
Reference or
source
59-CATATGATGGCTTACGCACTACCTAACTTAC-39
59-CTCGAGGGCCGCTGCTAAGTTAG-39
Synechocystis 6803 through homologous recombination
(Fig. S3a) to produce the PpetE–sodB strain. PCR analysis
of transformant’s chromosomal DNA with gene-specific
primers indicated that these colonies were homozygous
(Fig. S3b). The Omega cassette upstream of the petE promoter has stem–loop structures at both ends to terminate
background transcription. When the strain acclimated to
copper starvation was transferred to BG11 medium
containing the indicated amounts of supplementary copper,
the growth photograph on day 4 showed clearly that the
chlorophyll a content was regulated by the Cu2+ concentration (Fig. S5a). When the Cu2+ concentration was
reduced to 9.9 nM (in normal BG11 medium with 316 nM
Cu2+), no FeSOD signal was detected with immunoblotting
(Fig. S5b) and trace FeSOD activity could be determined
with in-gel activity staining (Fig. S5c). When the cells were
cultured in copper-starved medium under illumination of
45 mmol photons m22 s21, the PpetE–sodB strain exhibited
remarkable etiolation symptoms compared with that
cultured in normal BG11 medium and WT cultured in
copper-starved medium (Fig. 3a, b). Chlorophyll a content
of the PpetE–sodB strain cultured in copper-starved medium
remained constant during 8 days of growth (Fig. 3c) and the
ratio of chlorophyll a to OD730 on day 4 was decreased
significantly compared with the same strain cultured in
normal BG11 medium (Fig. 3d). These results indicated that
sodB was essential for photoautotrophic growth of
(a)
(b)
300 bp
This study
This study
Synechocystis 6803. The whole-cell absorption spectra of
the PpetE–sodB strain and WT Synechocystis 6803 cultured in
normal BG11 and copper-starved medium are shown in Fig.
3(e, f). The pigment contents showed no significant
differences between WT Synechocystis 6803 cultured under
normal and copper-starved BG11 medium conditions (Fig.
3e). For the PpetE–sodB strain, when it was cultured under
copper starvation conditions, the ratio of carotenoid to
chlorophyll a increased compared with that cultured in
normal BG11 medium, and the ratio of phycobilin to
chlorophyll a did not differ significantly between the two
culture conditions (Fig. 3f).
Synechococcus 9311 sodC transformed into the
Synechocystis 6803 sodB” mutant
To investigate whether sodC from Synechococcus 9311 was
functionally complementary to sodB, pHS304 for expressing sodC was transformed into the sodB2 mutant. In
order to obtain homozygous mutants, the transformants
were transferred many times on BG11 plates or in liquid
BG11 medium supplemented with both kanamycin and
spectinomycin. PCR analysis of these kanamycin- and
spectinomycin-resistant cells showed that sodB was not
disrupted fully by the kanamycin resistance cassette (Fig.
4a). Although Cu/ZnSOD was expressed actively, obvious
FeSOD activity was still determined in the complemented
Marker 1
2
3
4
C.K2
slr1516 up
5
3435 bp
HpaI slr1516 down
1980 bp
sodB
(c)
(d)
WT
sodB–
WT
sodB–
Anti-FeSOD
Fig. 1. Construction and detection of the Synechocystis 6803 sodB” mutant. (a) Physical map of the Synechocystis 6803
genome region containing sodB, with indications of the insertion position of the kanamycin resistance cassette (C.K2) into sodB
at the HpaI site. (b) Detection of degree of segregation of the sodB” mutant by PCR amplification. PCR was carried out using
primers slr1516 up and slr1516 down with the genomic DNA of the WT (land 1), plasmid pHS168 (lane 2) and mutants (three
clones, lanes 3–5) as templates. The primer sequences are listed in Table 1. Molecular markers are shown on the left side of the
gel, and represent 6000, 5000, 4000, 3500, 3000, 2500, 2000 and 1500 bp, respectively (from top to bottom). (c, d)
Detection of the amount of FeSOD in the WT and sodB” mutant by immunoblotting (c) and in-gel activity staining (d); 40 mg
total protein was loaded in each lane for immunoblotting and 120 mg total protein was loaded in each lane for in-gel staining.
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233
W.-T. Ke and others
(a)
WT
sodB–
sodB– com
(b) 2.0
0d
WT
sodB–
sodB– com
OD730
1.5
3d
1.0
0.5
5d
0.0
0
1
2
3
Time (day)
4
5
Fig. 2. Growth characteristics and curves of the WT, sodB” mutant and its complementary strain (sodB” com) grown in normal
BG11 medium under illumination of 30 mmol photons m”2 s”1 at 30 6C. (a) Photographs of the three strains grown in normal
BG11 medium: 0 d, 3 d and 5 d represent the initial day, third day and fifth day, respectively. (b) Growth curves (determined by
OD730) of the three strains cultured in normal BG11 medium. Data are mean±SD (n53). Error bars indicate SD.
strain (Fig. 4b). It suggested that the function of FeSOD
could not be substituted by Cu/ZnSOD. However, when
sodC was introduced into the WT Synechocystis genome,
sodB was disrupted in this background by insertion of a
kanamycin resistance cassette, and a fully segregated mutant
of sodB expressing actively Synechococcus 9311 sodC was
attempted. Nevertheless, PCR analysis and SOD activity
staining showed the same results as above (data not shown).
These results indicated that sodC could not complement
completely the function of sodB.
In order to study the function of Cu/ZnSOD in oxidative
stress, the growth rates of three Synechocystis 6803 strains
(WT, sodB2 mutant and sodC complement strain) were
compared in the presence or absence of methyl viologen
(MV) or norflurazon (NF) (Fig. 5). MV is a cell-dependent
O22-producing agent and NF can promote the formation
of 1O2 within the thylakoid membrane (Thomas et al.,
1998). Upon 0.5 mM MV treatment, the relative growth
rates of WT and the sodB2 mutant decreased by 7.2 and
20.7 %, respectively, whilst the sodC complement strain
showed little effect, compared with their corresponding
controls (Fig. 5a). Thus, the relative growth rate of the
sodB2 mutant was reduced significantly, whilst that of the
sodC complement strain was increased significantly compared with the WT strain (P,0.05, Tukey multiple
comparison) (Fig. 5a, b). Treatment with 40 mM NF
caused the relative growth rates of the WT, sodB2 mutant
and sodC complement strains to decrease by 14.0, 22.2 and
12.2 % respectively, compared with their corresponding
controls (Fig. 5c). Thus, the relative growth rate of the sodC
complement strain showed no difference from the WT,
while that of the sodB2 mutant was decreased significantly
(P,0.05, Tukey multiple comparison) (Fig. 5c, d).
Photoinhibition of PSII by strong light has been proposed
to occur by several hypothetical mechanisms, some of
which include the role of O22 (Richter et al., 1990;
Nishiyama et al., 2001; Tjus et al., 2001; Song et al., 2006).
234
To test whether FeSOD and Cu/ZnSOD were involved in
protecting PSII against photoinhibition, samples of the
WT, sodB2 mutant and sodC complement strain were
subjected to photoinhibitory conditions of 1000 mmol
photons m22 s21 at 30 uC in the absence or presence of
lincomycin (100 mg ml21), an antibiotic that blocks the
repair of PSII by inhibiting de novo protein synthesis. PSII
activity was estimated by measuring Fv/Fm with the
WATER-PAM chlorophyll fluorometer. In the presence
of lincomycin, PSII activity of the three strains declined at
the same rate with exposure to strong light (Fig. 6), which
indicated Cu/ZnSOD and FeSOD were not involved in
protecting PSII against photodamage. Upon exposure to a
strong light in the absence of lincomycin, PSII activity of
the sodC complement strain decreased slower than for the
WT and sodB2 mutant (Fig. 6). After 30 min strong light
exposure, the Fv/Fm values of the WT, sodB2 mutant and
sodC complement strain were decreased to 16.2, 20.4 and
32.9 % of the corresponding values at time zero, respectively. The sodB2 mutant and WT strain had a similar
response pattern to photoinhibition during 90 min exposure to strong light (Fig. 6), suggesting that FeSOD was
not involved in protecting PSII against strong light.
Synechocystis 6803 transformed with Cu/ZnSOD was more
resistant to strong light than the WT and sodB2 mutant, which
indicated that Cu/ZnSOD played a role in alleviating
photoinhibition. Combined with these results, it appeared that
the expression of Cu/ZnSOD enhanced the repair of photodamaged PSII, but did not protect PSII from photodamage.
Subcellular localization of FeSOD and Cu/ZnSOD
in sodC-transformed Synechocystis 6803
Analysis of the protein sequences of slr1516 (encoding
FeSOD) and sync_1771 (encoding Cu/ZnSOD) with
the SignalP program 4.1 server (http://www.cbs.dtu.dk/
services/SignalP/) predicted that no signal peptide existed
in FeSOD, whilst an evident N-terminal signal peptide
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Microbiology 160
SODs and Synechocystis
(a)
WT +Cu
PpetE–sodB +Cu
(b)
WT –Cu
PpetE–sodB –Cu
0d
4d
8d
(d) 4.8
WT control
PpetE–sodB control
WT –Cu BG11
PpetE–sodB -Cu BG11
10
Control
–Cu BG11
4.0
Chlorophyll a/OD730
_
Chlorophyll a content (mg ml 1)
(c) 12
8
6
4
2
3.2
2.4
1.6
0.8
0
0.0
0
2
4
Time (day)
6
(e)
WT +Cu
WT –Cu
Chl a
1.4
8
WT
(f)
1.4
PpetE–sodB
PpetE–sodB +Cu
PpetE–sodB -Cu
Chl a
Absorption
Car
1.2
PC
Chl a
1.0
0.8
Absorption
Car
1.2
PC
Chl a
1.0
0.8
400 450 500 550 600 650 700 750
Wavelength (nm)
400 450 500 550 600 650 700 750
Wavelength (nm)
Fig. 3. Growth characteristics and chlorophyll a content of the WT and PpetE–sodB strain grown in normal BG11 or copperstarved BG11 medium. (a, b) Photographs of the WT (a) and PpetE–sodB strain (b) grown in BG11 medium under normal
copper concentrations (+Cu) or copper starvation conditions (–Cu). Three photographs are shown for each strain: 0 d, 4 d
and 8 d represent the initial day, fourth day and eighth day of growth at the third transfer, respectively. (c) Growth curves
(determined by the chlorophyll a content) of the WT and PpetE–sodB strain cultured in normal BG11 or copper-starved BG11
medium. (d) Chlorophyll a/OD730 ratios of the WT and PpetE–sodB strain on the fourth day of growth at the third transfer. Data
are mean±SD (n53). Error bars indicate SD. (e, f) The whole-cell absorption spectra of the WT (e) and PpetE–sodB strain (f)
after 4 days of cultivation in normal BG11 and copper-starved BG11 medium. The spectra were normalized to the chlorophyll a
maximal absorption peak at 440 nm. Car, carotenoid; Chl, chlorophyll; PC, phycocyanin.
existed in Cu/ZnSOD. To examine the subcellular localization of these two SODs, plasma membrane, thylakoid
membrane or soluble fractions were separated from cell
http://mic.sgmjournals.org
extracts as described by Huang et al. (2002) and Srivastava
et al. (2005). Detection of FeSOD and Cu/ZnSOD was
performed with each subcellular fraction by immunoblotting
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235
W.-T. Ke and others
(a) Marker 1
3
2
4
sodB– sodB––sodC com
WT
(b)
3435 bp
1980 bp
Fig. 4. Detection of degree of segregation of sodB in the sodC-complemented sodB” mutant by PCR amplification and in-gel
activity staining. (a) PCR detection. PCR was carried out using the primers slr1516 up and slr1516 down with the plasmid
pHS168 (lane 1), and genomic DNA of the WT (land 2), sodB” mutant (lane 3) and its sodC complement strain (lane 4) as
templates. The primers sequences are listed in Table 1. Molecular markers are shown on the left side of the gel, and represent
4000, 3500, 3000, 2500, 2000 and 1500 bp, respectively (from top to bottom). (b) In-gel activity staining; 120 mg total protein
was loaded in each lane for this detection. The additional SOD band in the complement strain compared with WT and mutant
represented Cu/ZnSOD.
with their corresponding antibodies. FeSOD was only
detected in the soluble fraction (Fig. 7a), whilst Cu/ZnSOD
was detected in both the soluble fraction and thylakoid
membrane fraction (Fig. 7b, c), and no detectable signal
existed in the plasma membrane fraction (Fig. 7c). NrtA, a
nitrate/nitrite transport system substrate-binding protein,
was used as a marker for the plasma membrane. CP47, a
chlorophyll-binding protein of PSII, was used as a marker for
Relative growth rate (% control)
c
a
WT
b
_
80
+
sodB––sodC com
sodB–
_
_
+
+
60
40
20
0
WT
sodB– sodB––sodC com
(c) 120
Relative growth rate (% control)
+/– MV
(b)
(a) 120
100
the thylakoid membrane. As expected, NrtA was found
mainly in the plasma membrane fraction and CP47 was
found almost exclusively in the thylakoid membrane fraction
(Fig. 7d, e), indicating that these two membrane fractions
were purified. These results indicated that FeSOD was
localized in the soluble fraction, and Cu/ZnSOD was
localized in both the soluble fraction and thylakoid
membrane of transformed Synechocystis 6803.
100
+/– NF
(d)
a
a
b
80
WT
_
+
sodB––sodC com
sodB–
_
_
+
+
60
40
20
0
WT
sodB– sodB––sodC com
Fig. 5. Effects of 0.5 mM MV and 40 mM NF on the growth of the WT, sodB” mutant and its sodC-complemented strain,
respectively. The WT, sodB” mutant and its sodC-complemented strain were grown under normal BG11 medium, and (a, b)
0.5 mM MV or (c, d) 40 mM NF was added into BG11 medium for 4 days. The growth rate of each strain grown in normal BG11
medium was used as control. All data are presented as the per cent of corresponding controls. Columns with different letters
(a–c) on the error bars are significantly different (P,0.05, Tukey multiple comparison). Data are mean±SD (n53–6).
236
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Microbiology 160
SODs and Synechocystis
80
Relative Fv/Fm
6803 could be knocked out by insertion inactivation under
decreased illumination conditions with the addition of
10 mM NaHCO3, 100 U catalase ml21 (or 100 mg BSA
ml21) and 20 mg leucine ml21, and catalase required for the
growth of mutants could be substituted successfully with
BSA. However, in the present study, the mutants were still
heterozygous after transferring for .1 year under conditions
of decreased illumination of 5 mmol photons m22 s21 in the
presence of 0.1 % glucose, 10 mM NaHCO3, 100 mg BSA
ml21 and 20 mg leucine ml21. Immunoblotting and in-gel
activity staining results showed that these mutants were still
knockdown strains and sodB could not be knocked out by
insertion of an antibiotic cassette (Fig. 1). The discrepancy is
probably due to the different strains used. The basic
ROS level between the glucose-tolerant (in this study) and
glucose-intolerant WT (Nefedova et al., 2003) Synechocystis
6803 cells could be distinct, which may lead to their different
requirements of SOD.
WT, –Lin
sodB–, –Lin
sodB––sodC com, –Lin
WT, +Lin
sodB–, +Lin
sodB––sodC com, +Lin
100
60
40
20
0
0
20
40
60
Time (min)
80
100
Fig. 6. Effect of strong light (1000 mmol photons m”2 s”1) on Fv/
Fm of the WT, sodB” mutant and its sodC-complemented strain in
the absence (open symbols) or presence (closed symbols) of
lincomycin (Lin; 100 mg ml”1). Exponential growth cultures with
OD730 0.4 were exposed to the strong light and withdrawn at the
indicated times for Fv/Fm measurement. The 100 % values
corresponded to the values at time point zero. Data are
mean±SD (n53–6). Error bars indicate SD.
When the PpetE–sodB strain was cultured in copper-starved
medium under illumination of 45 mmol photons m22 s21,
the chlorophyll a content decreased significantly and the
strain nearly died during 8 days of growth (Fig. 3), and
chlorophyll a content declined modestly when the culture
light intensity was decreased to 30 mmol photons m22 s21
(data not shown). These results indicated that a decrease in
sodB expression led to a remarkable decrease in chlorophyll a
content and light tolerance. A previous study had reported
that there are three FeSODs (FSD1, FSD2, and FSD3) in
Arabidopsis thaliana (Myouga et al., 2008). A heteromeric
protein complex formed by FSD2 and FSD3 defended
chloroplast nucleoids against oxidative stress, and was
essential for chloroplast development in Arabidopsis
(Myouga et al., 2008). Hence, FeSOD could probably
influence the synthesis or degradation of chlorophyll a, and
this effect is regulated by light intensity in Synechocystis 6803.
DISCUSSION
Genome sequence data have shown that Synechocystis 6803
contains only one sod gene, sodB (Kaneko et al., 1996). This
suggests that FeSOD in Synechocystis 6803 scavenges all
O22, in contrast to other cyanobacterial species containing
multiple SODs (Kim & Suh, 2005). The expression of
FeSOD in WT Synechocystis 6803 was light dependent and
was more active in cultures incubated under continuous
light (50 mmol photons m22 s21) compared with those
incubated under continuous dark (Kim & Suh, 2005).
Nefedova et al. (2003) reported that sodB of Synechocystis
T
S
(a) Anti-FeSOD
CM
Most chlorophyll a in photosynthetic organisms serves as
light-harvesting antennae, and some chlorophyll a in the PS
T
S
(b) Anti-Cu/ZnSOD
PM
TM
PM
(d) Anti-NrtA
(c) Anti-Cu/ZnSOD
PM
(e) Anti-CP47
http://mic.sgmjournals.org
TM
TM
CM
Fig. 7. Subcellular localization of FeSOD and
Cu/ZnSOD in the Synechocystis 6803 sodCcomplemented sodB” mutant. (a, b) Total protein
extract (T) from transformed Synechocystis 6803
was separated into soluble (S) and crude
membrane (CM) fractions by ultracentrifugation,
and detected using antibodies against FeSOD
(a) and Cu/ZnSOD (b); 40 mg total protein was
loaded in each lane. (c–e) The crude membrane
fraction was separated into the plasma membrane (PM) fraction and the thylakoid membrane
(TM) fraction by sucrose density gradient centrifugation in combination with aqueous polymer
two-phase partitioning and examined by immunoblotting using antibodies against Cu/ZnSOD
(c), NrtA (d) and CP47 (e); 6 mg total protein was
loaded in each lane.
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237
W.-T. Ke and others
reaction centres is indispensable for energy transduction.
When sodB was knocked down in Synechocystis 6803, the
activity of PSI and II would be compromised, especially PSI,
because most of chlorophyll a was bound with PSI (Shen
et al., 1993; Jordan et al., 2001; Zouni et al., 2001). The
damage to PSI in the Synechococcus sp. PCC 7942 sodB2
mutant was more sensitive to oxidative stress than that of
PSII (Herbert et al., 1992). In the present study, Fv/Fm values
of the PpetE–sodB strain were also decreased significantly
when it was cultured in low copper (9.9 nM) medium
compared with the same strain cultured in normal BG11 or
the WT strain cultured in low copper (9.9 nM) medium
(data not shown). This suggested that FeSOD in Synechocystis 6803 is involved in protecting both PSs against oxidative
stress, as with MnSOD in Anabaena sp. PCC 7120 (Zhao et al.,
2007). Carotenoids also act as light-harvesting pigments
involved in photosynthesis, and can dissipate excess energy of
excited chlorophyll and eliminate ROS. Thus, they are
efficient ROS scavengers (Latifi et al., 2009). In this study,
the ratio of carotenoid to chlorophyll a was increased when
the PpetE–sodB strain was transferred to the copper-starved
BG11 medium for 4 days, compared with that cultured in
normal BG11 medium. This increased ratio of carotenoid to
chlorophyll a could be a strategy to cope with oxidative stress
under conditions of decreased sodB expression.
MV is a very-low-potential redox compound that accepts
primarily electrons from the FA and FB iron–sulfur clusters of
PSI centres at the expense of ferredoxin reduction (Fujii et al.,
1990; Blot et al., 2011). By reacting very quickly with molecular
oxygen, the reduced MV?+ radical is reoxidized spontaneously
back to MV with the formation of the membrane-impermeable O22. The vast majority of O22 is generated at the FA
and FB centres of PSI in photosynthetic organisms after MV
addition in light (Fujii et al., 1990). NF is a carotenoid
synthesis inhibitor, which inhibits phytoene desaturase
specifically, and thus blocks the synthesis of b-carotene and
other carotenoids (Ben-Aziz & Koren, 1974; Kümmel &
Grimme, 1975). Phytoene desaturase is a key enzyme in
carotenoid biosynthesis, responsible for the conversion of
phytoene into b-carotene (Martı́nez-Férez & Vioque, 1992).
Carotenoids (b-carotene in particular) normally quench 1O2
in the chlorophyll antenna and thus the addition of NF
promotes the formation of 1O2 within the thylakoid
membrane. Both FeSOD and heterologously expressed Cu/
ZnSOD played important roles in protecting Synechocystis
6803 against oxidative stress induced by MV or NF (Fig. 5).
The different localization of MnSOD and FeSOD in
Anabaena sp. PCC 7120 suggests that they have different
roles in protecting cellular molecules against O22 damage
(Li et al., 2002; Regelsberger et al., 2002, 2004). In
Arabidopsis thaliana, chloroplastic Cu/ZnSOD is located
mainly in the stromal face of thylakoid membranes where
the PSI complex is located (Ogawa et al., 1995). So far, Cu/
ZnSOD found in cyanobacteria is encoded by one sodC gene
and is predicted to be membrane associated. Using isolated
thylakoid membranes and plasma membranes, we demonstrated that Cu/ZnSOD was localized in the soluble fraction
238
and thylakoid membrane fraction of sodC-transformed
Synechocystis 6803, although in lower quantities in the latter
(Fig. 7b, c). The presence of Cu/ZnSOD in the thylakoid
membrane fraction cannot be due to cross-contamination of
the soluble fraction, since total membranes were washed
three times prior to isolating different membrane fractions.
Cu/ZnSOD has a predicted evident N-terminal signal
peptide as shown by using the SignalP program 4.1 server,
so it is likely on the lumen side of thylakoid membrane.
FeSOD was detected only in the soluble fraction (Fig. 7a)
and it does not contain any predicted signal peptide or
transmembrane region, so it should be in the cytoplasm.
This spatial separation of Cu/ZnSOD and FeSOD in cells
may result in the observed lack of complementation of
sodB2 mutants by the heterologously expressed sodC.
Inhibition of the activity of PSII under strong light is referred
to as photoinhibition. This phenomenon is due to the
imbalance between the rate of photodamage to PSII and the
rate of the repair of damaged PSII (Nishiyama et al., 2006).
Nishiyama and co-workers have suggested that the PSII
repair cycle is sensitive to oxidative damage (Nishiyama et al.,
2004; Kojima et al., 2007; Ejima et al., 2012), but PSII
damaging reaction is not sensitive to oxidative damage
(Nishiyama et al., 2001). In the present study, when cells were
exposed to strong light in the presence of lincomycin, PSII
activity of the WT, sodB2 mutant and sodC complement
strain declined at the same rate (Fig. 6). This result indicated
that Cu/ZnSOD and FeSOD were not involved in protecting
PSII against photodamage, which corroborated the conclusion that PSII damage was not sensitive to oxidative stress
(Nishiyama et al., 2001). In the absence of lincomycin, the
sodC complement strain was more tolerant against strong
light than the WT and sodB2 mutant, whilst the WT and
sodB2 mutant did not show significantly different tolerances
against strong light (Fig. 6). Combined with the effects of
strong light on PSII activity of the three strains in the
presence and absence of lincomycin, we concluded that Cu/
ZnSOD plays some role in PSII repair by alleviating oxidative
damage produced by strong light, whilst FeSOD was not
involved in the repair of photodamaged PSII. A previous study
reported that FeSOD provided little protection in photoinhibition (Herbert et al., 1992), which is consistent with the present
results. The different roles played by FeSOD and Cu/ZnSOD in
photoinhibition may result from their different subcellular
localization in the transformed Synechocystis 6803. Under
illumination of PSII, O22 is generated at the acceptor side by
reduction of molecular oxygen (Ananyev et al., 1994; Song
et al., 2006). This O22 generated on the stromal side of PSII
may migrate to the lumenal surface through the equilibrium
between the charged O22 and the uncharged protonated
radical HO2 (Asada, 1999), resulting in O22 being conveniently
scavenged by membrane-associated Cu/ZnSOD (Henmi et al.,
2004; Zhang et al., 2011). This was similar to previous findings
that the membrane-associated MnSOD protects the photosynthetic apparatus of Anabaena sp. PCC 7120 (Zhao et al., 2007).
In conclusion, as the sole SOD, FeSOD located in the
cytoplasm of Synechocystis 6803 has fluid characteristics
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SODs and Synechocystis
and should be involved in scavenging all O22 generated
within the whole cell. FeSOD is essential for photoautotrophic growth of Synechocystis 6803. However, membrane-associated Cu/ZnSOD has not been found to exist as
the sole SOD in any cyanobacterial species so far and it
may function as an auxiliary SOD with high efficiency to
remove O22 generated around the thylakoid membrane.
Hence, the coexistence of multiple SODs within one cell is
advantageous for organisms to cope with various stresses
and adapt to their complex niches.
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ACKNOWLEDGEMENTS
Herbert, S. K., Samson, G., Fork, D. C. & Laudenbach, D. E. (1992).
This study was funded by the National Natural Science Foundation of
China (30970212) and the Program for New Century Excellent
Talents in University (NCET-08-0786).
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