Plant Cell Physiol. 42(6): 599–607 (2001)
JSPP © 2001
Functional Analysis of psbV and a Novel c-type Cytochrome Gene psbV2 of the
Thermophilic Cyanobacterium Thermosynechococcus elongatus Strain BP-1
Hiroshi Katoh 1, Suwako Itoh 1, Jian-Ren Shen 2 and Masahiko Ikeuchi 1, 3
1
2
Department of Life Sciences (Biology), University of Tokyo, Komaba 3-8-1, Meguro, Tokyo, 153-8902 Japan
RIKEN Harima Institute, Mikazuki-cho, Sayo-gun, Hyogo, 679-5148 Japan
;
used for reductive metabolism. Electrons are generated when
water is cleaved off and molecular oxygen is produced in the
water-splitting complex of PSII. Although the PSII complex is
highly conserved among all oxygenic photosynthetic organisms including cyanobacteria, eukaryotic algae and land plants,
extrinsic proteins in the water-splitting complex appear to
branch in two groups. Cytochrome c-550, 12 kDa and 33 kDa
proteins are involved in the S-state transition or stabilization of
the water-splitting complex in cyanobacteria, red algae and
some other lower eukaryotic algae (Shen and Inoue 1993a,
Enami et al. 1995), while 23–24 kDa, 16–18 kDa and 33 kDa
proteins are in green algae and higher plants.
Cytochrome c-550 has been a mysterious heme protein
with a very low potential (Em°¢= ca. –260 mV) as it is associated with the highly oxidative water-splitting complex of PSII
(Kerfeld and Krogman 1998, Shen et al. 1992, Shen and Inoue
1993a, Shen and Inoue 1993b). Biochemical analysis and
reconstitution experiments using the PSII complex from a thermophilic cyanobacterium have revealed that cytochrome c-550
is stoichiometrically bound to PSII and activates the O2evolving activity (Shen and Inoue 1993b). Mutational analysis
using the mesophilic cyanobacterium Synechocystis sp. PCC
6803 revealed that the psbV gene for cytochrome c-550 plays a
substantial role in maintaining the stability and function of the
manganese cluster (Shen et al. 1998). However, it is still not
clear how the low potential heme of cytochrome c-550 is
involved in the water-splitting reaction of PSII. The origin of
cytochrome c-550 may help explain the evolution of oxygenic
photosynthesis.
The thermophilic cyanobacterium Thermosynechococcus
(Synechococcus) elongatus became a new model organism for
photosynthesis research, since genetic engineering techniques
have been developed (Mühlenhoff and Chauvat 1996, Sugiura
and Inoue 1999, Katoh and Ikeuchi 2001). This cyanobacterium is very useful for biochemical and mutational studies on
the labile water-splitting system of PSII. It is also unique in
phylogeny. Based on the 16S rRNA sequence, it is thought to
have branched at a very early stage (Honda et al. 1999). In this
communication, we cloned psbV from the thermophilic T. elongatus and found a novel gene resembling psbV just downstream of psbV. By gene manipulation, we examined the physiological roles of these genes in PSII.
Cytochrome c-550 is an extrinsic protein associated
with photosystem II (PSII) in cyanobacteria and lower
eukaryotic algae and plays an important role in the watersplitting reaction. The gene (psbV) for cytochrome c-550
was cloned from the thermophilic cyanobacteria Thermosynechococcus (formerly Synechococcus) elongatus and T.
(formerly Synechococcus) vulcanus. In both genomes,
located downstream of psbV were a novel gene (designated
psbV2) for a c-type cytochrome and petJ for cytochrome c553. The deduced product of psbV2 showed composite similarities to psbV and petJ. Phenotype of psbV-disruptant in
Thermosynechococcus was practically the same as that
reported in Synechocystis sp. PCC 6803. Either psbV or
psbV2 gene of T. elongatus was expressed in the psbVdisruptant of Synechocystis sp. PCC 6803, which resulted in
recovery of the photoautotrophic growth. However, the
enhanced requirement of Ca2+ or Cl– ions in the psbVdisruptant of Synechocystis was suppressed by expression of
psbV but not by expression of psbV2. Thus, it is concluded
that psbV2 can partly replace the role of psbV in PSII. The
close tandem arrangement of psbV/psbV2/petJ implies that
psbV2 was created by gene duplication and intergenic
recombination during evolution.
Key words: Cytochrome — Photosystem II — psbV — psbV2
— Thermophilic cyanobacterium — Thermosynechococcus
(Synechococcus) elongatus.
Abbreviations: 2,6DCBQ, 2,6-dichloro-p-benzoquinone; 2,6DMBQ,
2,6-dimethyl-p-bezoquinone.
The authors propose to rename the thermophilic Synechococcus
elongatus to “Thermosynechococcus elongatus”, because it is distantly
related with the mesophilic Synechococcus elongatus as described in
Materials and Methods. The nucleotide sequences in this paper have
been registered in the EMBL, GenBank and DDBJ under accession
number AB052597 and AB052598 with the new organism names
“Thermosynechococcus elongatus strain BP-1” and “Thermosynechococcus vulcanus”.
Introduction
Oxygenic photosynthesis depends upon the ability of the
PSII complex to utilize water as a source of electrons to be
3
Corresponding author: E-mail, [email protected]; Fax, +81-3-5454-4337.
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psbV and a novel c-type cytochrome gene psbV2
Materials and Methods
Strain and standard culture conditions
The thermophilic cyanobacterium Thermosynechococcus (formerly Synechococcus) elongatus strain BP-1 was derived from a hot
spring in Beppu, Japan (Yamaoka et al. 1978). It has been identified as
Synechococcus elongatus based on cell morphology (Sonoike and
Katoh 1989). On the other hand, the 16S rRNA sequence of this
cyanobacterium is distantly diverged from all other Synechococcus
clusters (Honda et al. 1999). Thus, we tentatively renamed it as Thermosynechococcus elongatus BP-1 to avoid confusion with another
Synechococcus elongatus, which is defined for mesophilic species
derived from fresh water (Rippka and Herdman 1992). For convenience, we also rename Synechococcus vulcanus as Thermosynechococcus vulcanus, which was isolated from a hot spring in Yunomine,
Japan (Koike and Inoue 1983). These two thermophilic cyanobacteria
are closely related to each other at the nucleotide level (Katoh and
Ikeuchi 2001), although 16S rRNA of T. vulcanus has not yet been
determined.
Cells of T. elongatus were grown at 45°C in DTN medium
(Mühlenhoff and Chauvat 1996) under continuous illumination with
white fluorescent lamps (20 to 50 mE m–2 s–1). The psbV-disruptant of
T. elongatus was maintained with 7 mg ml–1 chloramphenicol but propagated in the absence of antibiotics for analytical experiments. The
glucose-tolerant substrain of the mesophilic cyanobacterium Synechocystis sp. PCC 6803 (Williams 1998) was grown at 31°C in BG11
medium (Stanier et al. 1971) supplemented with 20 mM HEPESNaOH (pH 7.0) under continuous illumination with white fluorescent
lamps (20 to 50 mE m–2 s–1). Synechocystis mutants were maintained in
the presence of 20 mg ml–1 erythromycin, 20 mg ml–1 spectinomycin
and/or 20 mg ml–1 chloramphenicol but propagated in the absence of
antibiotics for analytical experiments. The psbV-disruptant of Synechocystis, where a part of psbV was replaced with the erythromycinresistant cassette, was used as described previously (Shen et al. 1995).
Liquid cultures of T. elongatus and Synechocystis were bubbled with
air containing 1.0% (v/v) CO2. Growth of cells in liquid medium was
monitored as light scattering of cells at 730 nm.
For depletion of Ca2+ or Cl– in the DTN medium, 0.37 mM CaCl2
in the original DTN medium was replaced by either 0.74 mM NaCl or
0.37 mM Ca(NO3)2. In the case of Cl– depletion, FeCl3 and NH4Cl,
were also replaced with FeNH4(SO4)2 and NH4NO3, respectively. For
depletion of Ca2+ or Cl– in the original BG11 medium 0.24 mM CaCl2
was replaced with either 0.48 mM NaCl or 0.24 mM Ca(NO3)2.
Cyanobacterial cells at the mid log phase were harvested, washed
twice with CaCl2-depleted medium, and then transferred to the growth
medium depleted of Ca2+ or Cl–. For the low CO2 conditions, the DTN
medium, where 10 mM NaHCO3 was omitted, was bubbled with air
instead of CO2-enriched air.
Cloning of the gene coding for cytochrome c-550
Based on the N-terminal amino-acid sequence of cytochrome c550 (Shen et al. 1992), the psbV gene was cloned from genomic DNA
of the thermophilic cyanobacteria T. vulcanus and T. elongatus by a
two-step PCR method. The first PCR was done to amplify an internal
sequence with degenerate primers, 5¢-GC(A/G)(T/C)TNACNCCNGA(A/G)GT-3¢ and 5¢-GC(A/G)CANGC(A/G)TA(T/C)TG(A/G)AA3¢, which were based on the N-terminal amino acid sequence (Shen et
al. 1992). The PCR product of 110 bp was isolated, cloned into
pT7Blue-T vector (Novagen, Madison, U.S.A.) and sequenced. Based
on the internal sequence of the cloned psbV, the second inverse PCR
was designed with primers, 5¢-TTTTGCCCTCGCTGTTGA-3¢ and 5¢ACAATACCTAGAGGGTAA-3¢ and ApoI-digested and recirculized
genomic DNA. DNA fragments of the same 4.7 kbp were cloned from
Fig. 1 Gene arrangement of psbV and flanking regions and disruption of psbV in T. elongatus. (A) gene arrangement of psbV, psbV2 and
petJ on the genome and insertion of the chloramphenicol-resistant cassette (CmR). The fragment used as a probe for the Southern hybridization is shown by the arrow bar. (B) Southern blotting analysis of
genomic DNA of wild type (WT) and the mutant (DpsbV) digested
with EheI.
genomic DNA of T. elongatus and T. vulcanus. The nucleotide
sequence of a 2.3 kbp region carrying psbV from these fragments was
determined with a capillary DNA sequencer (model 310S, PE-biosystems, U.S.A.) using BigDyeTM terminator DNA sequencing kit (PEbiosystems). To eliminate PCR errors, at least four independent clones
were sequenced.
Construction of the psbV-disruptant
To inactivate psbV in T. elongatus, a chloramphenicol-resistant
cassette was inserted into PCR-cloned psbV as shown in Fig. 1A. The
resulting plasmid DNA was introduced into Thermosynechococcus cells
by electroporation basically according to Mühlenhoff and Chauvat
(1996). We designed a novel screening procedure for efficient isolation
of transformants as follows. After electroporation, cells were incubated
with 1 ml DTN medium by shaking for a day at 45°C. Then, cells were
mixed with three volumes of DTN medium containing 0.7% (w/v)
melted agar and spread on a chloramphenicol-containing agar plate.
Transformants emerged as green colonies after incubation under dim
illumination for about 10 d at 45°C.
Heterologous expression of Thermosynechococcus psbV or psbV2 in
Synechocystis psbV-disruptant
psbV or psbV2 gene of Thermosynechococcus was expressed
under two kinds of promoters in the psbV-disruptant of Synechocystis,
where a part of psbV had been replaced with an erythromycin-resistant
cassette (Shen et al. 1995) (Fig. 2). One was the strong psbAII promoter of Synechocystis to which the whole gene of psbV or psbV2 was
ligated. The 297 bp promoter region of psbAII was amplified with
primers, 5¢-CCCGACGTCATTATTTCATCTCCATTGTCCC-3¢ and
5¢- GTCGTTGTCATATGGTTATAATTCC-3¢, cloned into pT7Blue-T
vector and confirmed by nucleotide determination. The whole coding
regions of psbV and psbV2 were amplified with primers, 5¢-CATATGTTAAAAAAATGCGTT-3¢ and 5¢-AGGTTGGTACATGGGTGT-3¢,
or 5¢-CATATGTACCAACCTCACTTT-3¢ and 5¢-CAATCTTAGCCTGCCCAA-3¢, respectively, cloned and verified. The psbAII promoter
and psbV or psbV2 were ligated at the NdeI site, which was created at
the translation initiation site in the primers (underlined). As a selection marker, the chloramphenicol-resistant cassette derived from
pACYC184 was ligated with the psbAII promoter at the AatII site,
which was created in the first primer for the psbAII promoter. These
constructs were introduced into a neutral site of slr2031 in wild type
psbV and a novel c-type cytochrome gene psbV2
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structs was confirmed by nucleotide determination. These DNA constructs were introduced by homologous recombination into the psbV
locus, which had been partly replaced with the erythromycin-resistant
cassette in Synechocystis (Shen et al. 1995). Transformation of the
wild type and the psbV-disruptant of Synechocystis was done according to Hihara and Ikeuchi (1997).
Fig. 2 Diagrams of the constructs for heterologous expression of
Thermosynechococcus psbV or psbV2 in the psbV-disruptant of Synechocystis. (A) expression with the strong psbAII promoter. The chloramphenicol-resistant cassette (CmR), the promoter region of
Synechocystis psbAII (PpsbAII) and the whole coding regions of psbV or
psbV2 of T. elongatus were ligated to each other and inserted into the
slr2031 locus. (B) expression with the intrinsic promoter and the transit sequence of Synechocystis psbV. The mature coding regions of
psbV or psbV2 was ligated with the spectinomycin-resistant cassette
(SpR) and the DNA fragment carrying the intrinsic promoter and the
transit sequence of Synechocystis psbV. The host strain of this heterologous expression was the psbV-disruptant of Synechocystis, where a
part of psbV was replaced with the erythromycin-resistant cassette
(EryR). Note that only the part of the constructs, which was introduced by homologous recombination into the genome, is shown in this
figure, while the other parts of the constructs, which were recombined
with the genomic sequences but not incorporated in the genome, are
not shown.
(the glucose-tolerant strain) and the psbV-disruptant of Synechocystis
(Satoh et al. 2001). It is known that slr2031 is already inactivated by
deletion of a 154 bp segment in the glucose-tolerant strain of Synechocystis sp. PCC 6803 (Katoh et al. 1995), although it is active in regulation of motility and pigmentation in the original PCC strain (Kamei
et al. 1998).
Secondly, the putative mature region of psbV or psbV2 of Thermosynechococcus was expressed with the intrinsic promoter and presequence of Synechocystis psbV as a fusion at the deduced processing
site. The mature regions of psbV and psbV2 were amplified from T.
elongatus with primers, 5¢-CCGCGGAACTGACCCCTGA-3¢ and 5¢GCCGGCGTTGATAACTATAGGTTGGTACATGGGTGT-3¢, and 5¢-------------------------GTGATC-3¢ and 5¢-CAATCTTAGCCTGCCCAA-3¢, respectively,
cloned and verified. The DNA containing the promoter and the transit
sequence of Synechocystis psbV was amplified with primers, 5¢GGCAGTGGACAAGGTTGA-3¢ and 5¢-CCGCGGCATTGGCACTGCCGGCCGCCGCATTGGCACTGCCGA-3¢),
GCCGACCA-3¢ (or 5¢-------------------------cloned and verified. The transit of Synechocystis psbV and the mature
region of Thermosynechococcus psbV or psbV2 was ligated at SacII
(underlined) or NgoMI (double-underlined), respectively, without
changing any amino acid residues. The downstream DNA of Synechocystis psbV was prepared by excision with BglII of the PCR product, which was amplified with primers, 5¢-GGCAGTGGACAAGGTTGA-3¢ and 5¢-TGATCGGGAAATTGCTGA-3¢. These DNAs were
excised and ligated together with the spectinomycin-resistant cassette
derived from pRL453 as in Fig. 2. The correct ligation of these con-
Preparation of thylakoids
The T. elongatus cells at the late log phase were harvested by
centrifugation at 4,000´g for 10 min. After washing with 0.4 M sorbitol, 20 mM HEPES-NaOH (pH 7.0), 15 mM CaCl2 and 15 mM MgCl2,
cells were resuspended in 20 ml of the same buffer supplemented with
5 mM aminocaproic acid, 1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride and disrupted with zirconia/silica beads (0.1 mm
diameter, Biospec, Bartlesville, U.S.A.) in a bead-beater (Biospec)
with three cycles of 30 s homogenization and 2 min cooling. The
homogenate was centrifuged at 4,000´g for 10 min to remove cellular
debris and then centrifuged at 100,000´g for 1 h to precipitate thylakoid membranes. The membranes washed once with the buffer
described above, were resuspended in 1 M sucrose, 40 mM MESNaOH (pH 6.5), 15 mM CaCl2, 15 mM MgCl2 and 10 mM NaCl.
Assay of oxygen evolving activity
Oxygen evolving activity of thylakoids (final 30 mg Chl ml–1)
was measured in 1 M sucrose, 40 mM HEPES-NaOH (pH 7.0),
15 mM CaCl2, 15 mM MgCl2 and 10 mM NaCl at 25°C using a Clarktype oxygen electrode with saturating light of about 3,000 mE m–2 s–1
in the presence of 2,6DCBQ or 2,6DMBQ as electron acceptors.
SDS-urea-PAGE
For analysis of c-type cytochrome, a soluble cytochrome extract
was prepared by disruption of cells in a dilute buffer of 20 mM TrisHCl (pH 9.0) followed by centrifugation at 100,000´g for 30 min at
4°C. Proteins were solubilized with 2% (w/v) lithium dodecylsulfate,
60 mM dithiothreitol and 60 mM Tris-HCl (pH 8.8) and subjected to
SDS-urea-PAGE with the 16–22% (w/v) linear gradient of polyacrylamide gel containing 7.5 M urea as described by Ikeuchi and Inoue
(1988). Covalently-bound hemes were detected as a peroxidase activity with 3,3¢,5,5¢-tetramethylbenzidine and H2O2 according to Shen
and Inoue (1993b) and Thomas et al. (1976).
Results
Cloning of psbV gene
Based on the N-terminal amino acid sequence of cytochrome c-550 from T. vulcanus (Shen et al. 1992), the psbV
gene was cloned from T. vulcanus and T. elongatus (Fig. 1A).
Compared with N-terminal amino acid sequence of cytochrome c-550 from T. vulcanus (Shen et al. 1992), it was suggested that psbV codes for a presequence of 26 amino acid residues and a mature protein of 137 residues (Fig. 3). There was
no difference in nucleotide or amino acid sequence of psbV
between T. elongatus and T. vulcanus. Homology search of the
database revealed that psbV of the thermophilic Thermosynechococcus is more homologous to the algal homologs than
other cyanobacteria (details are presented in Discussion section)
Notably, a novel ORF encoding c-type cytochrome and
petJ gene encoding cytochrome c-553 were found just downstream of psbV (Fig. 1A). Homology search of the database
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psbV and a novel c-type cytochrome gene psbV2
Fig. 3 Sequence alignment of cytochrome c-550, PsbV2 and cytochrome c-553. Amino acid residues conserved between T. elongatus and other
organisms were indicated by black boxes. Asterisks on the top and at the end of sequences indicate the conserved residues of heme binding motif
and termination codons, respectively. The line above the N-terminal part indicates putative presequences. S. 6803, Synechocystis sp. PCC 6803 (c550, EMBL: D45178; c-553, EMBL: L25252), M. aeruginosa, Microcystis aeruginosa (c-550, EMBL: S03860; c-553, PIR: A00104), S. 7002:
Synechococcus sp. PCC 7002 (EMBL: D29788), S. lividus: Synechococcus lividus (PIR: A00106), A. 7120: Anabaena sp. PCC 7120 (EMBL:
M97009), C. paradoxa: Cyanophora paradoxa (EMBL: U30821), P. purpurea: Porphyra purpurea (EMBL: U38804).
revealed that this novel ORF has a c-type heme-binding motif
(Fig. 3, asterisks) and was most homologous to cytochrome c550 and to a lesser extent to cytochrome c-553. Hence, we
named this ORF psbV2. A likely processing site in the predicted product of psbV2 was searched by a program “SignalP”
(Nielsen et al. 1997). The product was suggested to consist of a
presequence of 34 amino acid residues and a mature part of 140
residues (Fig. 3). The sequence alignment revealed that PsbV2
protein was significantly homologous to cytochrome c-550
especially in the heme-binding region. It also showed limited
similarity to cytochrome c-553 in the heme-binding and C-terminal regions. The transit sequence prior to the putative
processing site was also conserved among the three groups of
proteins to some extent, while the N-terminal region of the
mature portion was most divergent.
Disruption of psbV gene in T. elongatus
The psbV gene of T. elongatus was inactivated by insertional mutagenesis and complete segregation was confirmed by
Southern blot analysis (Fig. 1B). Upon digestion with EheI, the
1.6 kbp fragment was detected in the wild type, whereas the
3.2 kbp fragment carrying the chloramphenicol-resistant cassette of 1.6 kbp was solely detected in the mutant. This indicates that the mutant was homozygous and ready for further
characterization. It is of note that psbV was dispensable for the
photoautotrophic growth in the thermophilic T. elongatus as
already reported in Synechocystis (Shen et al. 1995).
Disruption of psbV was also confirmed by heme-staining
(Fig. 4). In the soluble fraction from wild type, there were two
heme-stained bands corresponding to cytochromes c-550 and
c-553. Breakage of cells with dilute alkaline buffer successfully
extracted most of cytochrome c-550, which is tightly bound to
the active PSII. Expectedly, the psbV-disruptant completely
lacked the band of cytochrome c-550. We could not detect a
potential band of PsbV2.
Fig. 4 Heme-staining of wild type (WT) and psbV-disruptant
(DpsbV) of T. elongatus. Proteins of the soluble fraction were fractionated by SDS-PAGE. Soluble proteins from cells equivalent to 45 mg
Chl were loaded in each lane. Note that the phycobiliproteins were not
visualized by this method but were simply detected as covalently
bound chromophores.
psbV and a novel c-type cytochrome gene psbV2
603
Fig. 5 Growth of wild type (circles) and the psbV-disruptant (triangles) of T. elongatus as measured with A730 at 70 mE m–2 s–1 at 45°C with 1%
(v/v) CO2. Panel A, growth with (closed) or without (open) Ca2+; panel B, growth with (closed) or without Cl– (open).
Effects of psbV disruption on growth
Photoautotrophic growth of the psbV-disruptant was
slightly but reproducibly slower than that of wild-type cells
under various conditions of temperature or CO2. To examine
the possible role of the putative PsbV2 protein for substitution
of cytochrome c-550, we measured growth of the psbV-disruptant of T. elongatus in the absence of Ca2+ or Cl–. The psbVdisruptant of Synechocystis strictly requires a relatively high
concentration of both Ca2+ and Cl– in the growth medium for
photoautotrophic growth (Shen et al. 1998). Similarly, the
Thermosynechococcus mutant did not grow at all in the Cl–depleted DTN medium, while the wild-type cells grew to some
extent (Fig. 5B). This implies that PsbV2 protein does not
replace the role of cytochrome c-550 for high affinity to Cl–
under the experimental conditions. On the other hand, neither
the mutant nor wild-type cells could grow in the Ca2+-depleted
medium (Fig. 5A), implying that even the wild-type cells of T.
elongatus may not have a Ca2+ concentrating mechanism,
which exists in Synechocystis sp. PCC 6803 (Shen et al. 1998).
It is noteworthy that the psbV-disruptant of T. elongatus could
not grow at all under the condition of low CO2, at the wild-type
cells could grow (Fig. 6).
Effects of psbV disruption on oxygen evolution
We compared the O2 evolution activity in the psbVdisruptant of T. elongatus with that in the wild-type cells.
When 2,6DCBQ was added to cells, the rate of O2 evolution in
the psbV-disruptant was about 180–210 mmol O2 (mg Chl) –1 h–1,
while that in wild-type cells was about 250–290 mmol O2 (mg
Chl) –1 h–1. To avoid permeability barriers of cells, we tried to
isolate active thylakoid membranes according to Katoh and
Ikeuchi (2001). Fig. 7 shows the dependence of O2 evolution
on the concentration of 2,6DCBQ or 2,6DMBQ. The rate of O2
evolution in the mutant thylakoids was about 30% that in the
Fig. 6 Growth of wild type (circles) and the psbV-disruptant (triangles) of T. elongatus under conditions of low CO2 around 0.03%. Cells
were grown at 70 mE m–2 s–1 at 50°C.
wild-type thylakoids throughout a wide range of concentration
and the concentration dependence of the mutant appeared to be
similar to that of the wild-type thylakoids. This seems to agree
with the notion that cytochrome c-550 is a regulatory component of the water-splitting complex (Shen et al. 1995, Shen et
al. 1998). By contrast, the rate of O2 evolution was affected at
higher concentrations of 2,6DCBQ or 2,6DMBQ in the psbXdepleted PSII, suggestive of functioning of PSII-X protein at
QB site (Katoh and Ikeuchi 2001). We further isolated the PSII
complex by solubilization with dodecylmaltoside followed by
ion-exchange column chromatography. The PSII complex thus
isolated hardly retained O2-evolution activity or the 33 kDa
extrinsic protein (not shown). This indicates that even the thermostability of PSII is not sufficient for preservation of the
water-splitting complex depleted of cytochrome c-550.
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psbV and a novel c-type cytochrome gene psbV2
Fig. 7 Effects of 2,6DCBQ (panel A) and 2,6DMBQ (panel B) on the oxygen evolution activity of thylakoid membranes isolated from wild type
(circles) and psbV-disruptant (triangles) from T. elongatus.
Heterologous expression of Thermosynechococcus psbV and
psbV2 in the Synechocystis psbV-disruptant
As already reported (Shen et al. 1995, Shen et al. 1998),
the psbV-disruptant of Synechocystis sp. PCC 6803 grew photoautotrophically but at a significantly slower rate than the
wild-type cells even in the presence of CaCl2 (Fig. 8). When
psbV or psbV2 of T. elongatus was heterologously expressed
with the intrinsic psbV promoter or the strong psbAII promoter
in the psbV-disruptant of Synechocystis, the retarded growth of
the mutant was recovered to the level of the wild-type cells or
even higher level (Fig. 8). Introduction of the control construct
carrying the screening cassette and the promoter of psbAII at
the site of slr2031 did not affect the growth (not shown). These
findings suggest that not only psbV but also psbV2 from T.
elongatus may substitute, if not all, the role of the intrinsic
psbV to support the photoautotrophic growth of Synechocystis.
Heme-staining revealed that cytochrome c-550 of T. elongatus
was appreciably detected at the authentic position when
expressed in the psbV-disruptant of Synechocystis (not shown).
However, we could not detect a positive heme-stained band for
PsbV2 even under control of the strong promoter.
The functional substitution was further evaluated as
growth in the absence of Ca2+ or Cl–. The psbV-disruptant of
Synechocystis sp. PCC 6803 could not grow in the absence of
either Ca2+ or Cl–, as reported previously (Shen et al. 1998).
Expression of Thermosynechococcus psbV with either the
intrinsic promoter or the strong promoter clearly reversed the
suppression of growth (Fig. 9). The extent of the recovery was
reproducibly higher with the strong promoter than the intrinsic
promoter. On the other hand, expression of Thermosynechococcus psbV2 did not reverse the suppression of growth at all
regardless of the promoters (Fig. 10). These results suggest that
PsbV2 does not restore the high affinity to Ca2+ or Cl– ions,
which are essential for the water-splitting reaction of PSII.
Discussion
Fig. 8 Growth of wild type and mutants of Synechocystis in the presence of CaCl2. Wild type, closed circles; psbV-disruptant, opened circles; mutant with expression of T. elongatus psbV with intrinsic
promoter (open square) or the psbAII promoter (open triangle); mutant
with expression of T. elongatus psbV2 with intrinsic promoter (closed
square) or the psbAII promoter (closed triangle). Cells were grown
under illumination at 50 mE m–2 s–1 at 31°C with 1% (v/v) CO2.
We cloned and disrupted psbV in the thermophilic cyanobacterium T. elongatus and also expressed it heterologously in
the psbV-disruptant of Synechocystis sp. PCC 6803. Cytochrome c-550 encoded by psbV in T. elongatus was equivalent
to that in Synechocystis in that it enhanced the cellular affinity
to Ca2+ and Cl– for photoautotrophic growth. Notably, amino
acid sequences of the mature region of cytochrome c-550 from
T. elongatus and T. vulcanus were more homologous to those
from lower eukaryotic algae such as Porphyra purpurea (identity: 65%) and Cyanophora paradoxa (identity: 63%) than
other cyanobacteria Microcystis aeruginosa (identity: 45%),
psbV and a novel c-type cytochrome gene psbV2
605
Fig. 9 Effects of expression of Thermosynechococcus psbV on the growth of the Synechocystis psbV-disruptant in the absence of Ca2+ or Cl–.
Panel A, Ca2+-depleted conditions; panel B, Cl–-depleted conditions. Symbols are the same as in Fig. 8. Cells were grown under illumination at
50 mE m–2 s–1 at 31°C with 1% (v/v) CO2.
Synechocystis (identity: 44%) and Synechococcus PCC 7002
(identity: 42%) (Fig. 3). There is a tendency that negatively
charged residues are conserved in Thermosynechococcus and
algal cytochrome c-550, while positively charged residues are
found in other cyanobacteria. On the other hand, amino acid
sequences of cytochrome c-553 and nucleotide sequence of 16S
rRNA of the thermophilic cyanobacteria are less homologous
to those in algae than other cyanobacteria (Fig. 3, also see
Honda et al. 1999). At the moment, it is not clear why only
cytochrome c-550 of the thermophilic cyanobacteria is markedly homologous to that in the lower eukaryotic algae.
The reconstitution analysis of the water-splitting complex
has been extensively performed with PSII complexes and
extrinsic proteins, which had been separated beforehand by
treatments with high salts or chaotropic agents (Åkerlund et al.
1982, Miyao and Murata 1984, Shen and Inoue 1993a). However, the O2-evolving activity has never been fully restored by
such reconstitution experiments, probably due to inevitable
side effects of the extraction treatments. Using the active PSII
complex from mutants of the thermophilic cyanobacteria
would be a plausible alternative for reconstitution. Although
our initial attempt to isolate the active PSII complex from the
psbV-disruptant of T. elongatus was not so successful (Fig. 7),
further improvement of the isolation procedure or supplement
of other extrinsic proteins may provide a PSII preparation
devoid of cytochrome c-550 but competent for functional
reconstitution.
The novel c-type cytochrome encoded by psbV2 was significantly homologous to cytochrome c-550 in the region of the
heme binding motif and to cytochrome c-553 in the C-terminal
Fig. 10 Effects of expression of Thermosynechococcus psbV2 on the growth of the Synechocystis psbV-disruptant in the absence of Ca2+ or Cl–.
Panel A, Ca2+-depleted conditions; panel B, Cl–-depleted conditions. Symbols are the same as in Fig. 8. Cells were grown under illumination at
50 mE m–2 s–1 at 31°C with 1% (v/v) CO2.
606
psbV and a novel c-type cytochrome gene psbV2
region. It is not clear whether or not psbV2 is expressed in T.
elongatus. Even in the case of heterologous expression of
psbV2 with the strong promoter PpsbAII in Synechocystis, we
could not detect a positive heme-containing band of PsbV2.
Expression of PsbV2 may not be sufficient for heme-staining
or the band of PsbV2 may be masked by the intense band of
phycocyanin. It is necessary to know the exact position of the
PsbV2 protein by probing with a specific antibody or to purify
the protein.
The tandem arrangement and sequence homology of the
three cytochrome genes in Thermosynechococcus genome (Fig.
1, 3) suggests that psbV2 has evolved from psbV and petJ by
gene duplication and intergenic shuffling. Recent genome
sequencing has revealed that the psbV2 gene is also present
downstream of psbV in the draft genome sequence of Gloeobacter violaceus PCC 7421 (Tabata, S., personal communication). Based on the phylogenetic tree of cyanobacteria deduced
from 16S rRNA sequences, G. violaceus was branched almost
at the root and Thermosynechococcus was at the second,
although they are distantly related to each other (Honda et al.
1999). On the other hand, psbV2 is not retained in the complete genome of Synechocystis sp. PCC 6803 (Kaneko et al.
1996) or Anabaena sp. PCC 7120 (http://www.kazusa.or.jp/
cyano/anabaena/). These findings suggest that the origin of
psbV2 was very ancient and later lost in at least some branches
of cyanobacteria. In this context, it should be noted that psbV2
could substitute for psbV only partially for the photoautotrophic growth of Synechocystis (Fig. 8, 10). This may imply
that psbV2 plays a subsidiary role in PSII to psbV in T. elongatus, although nothing superior to psbV is known about psbV2 at
the moment. It is also of note that psbV and petJ are slightly
homologous to each other (Fig. 3), while they are barely
homologous to another soluble cytochrome cM (Malakhov et al.
1994). It is tempting to speculate that psbV was developed by
duplication of petJ or vice versa in the early evolution of the
oxygenic PSII. At that time, cytochrome c-550 may have had
capability of transferring electrons to PSII, as cytochrome c553 reduces PSI.
It is interesting to note that the psbV-disruptant of T. elongatus could not grow photoautotrophically under the low CO2
conditions (Fig. 6). The same phenotype was also observed for
psbU-disruptant of T. elongatus (Katoh, H. and Ikeuchi, M.,
unpublished results). One possible explanation is that cytochrome c-550 and PSII-U protein stabilize the labile watersplitting complex of PSII especially under stressed conditions
such as low CO2. These proteins confer tolerance to heat inactivation of the water-splitting machinery (Nishiyama et al. 1994,
Nishiyama et al. 1997). Secondly, HCO3– may directly interact
with the water-splitting complex. In higher plants, bicarbonate
ion has been reported to protect the donor side of PSII against
photoinhibition or other damages probably due to interaction
with the water-splitting complex (Klimov et al. 1997, Klimov
and Baranov 2001). Lastly, CO2 or HCO3– may be indirectly
linked to the Cl– demand of the water-splitting complex. We
recently identified non-Cl–-requiring mutants isolated from the
psbV-disruptant and found that inactivation of a novel transporter gene was responsible for this phenotype (Kobayashi, M.,
Katoh, H. and Ikeuchi, M., unpublished results). These findings
imply that this transporter exports Cl– ion in the wild-type
cyanobacteria at the expense of unidentified ions or energy.
One of the plausible candidates for this would be antiport of
HCO3–. Further studies on the uptake of HCO3– in the psbVdisruptant are needed to examine the novel phenotype under
the low CO2 conditions.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research (to M.I.), by the Program for Promotion of Basic Research
Activities for Innovative Biosciences of Japan (to M.I.) and by a Grant
for Scientific Research from the Human Frontier Science program (to
M.I.).
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(Received December 20, 2000; Accepted March 16, 2001)