(Received for publication, March 17, 1997, and in revised form, May 6, 1997)
From the Solar Energy Research Group and Photosynthesis Research Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan and the § Department of Biology, University of Tokyo, Komaba, Tokyo 153, Japan
The gene encoding the 12-kDa extrinsic protein of
photosystem II from Synechocystis sp. PCC 6803 was cloned
based on N-terminal sequence of the mature protein. This gene, named
psbU, encodes a polypeptide of 131 residues, the first 36 residues of which were absent in the mature protein and thus served as
a transit peptide required for its transport into the thylakoid lumen.
A psbU gene deletion mutant grew photoautotrophically in
normal BG11 medium at almost the same rate as that of the wild type
strain. This mutant, however, grew apparently slower than the wild type did upon depletion of Ca2+ or Cl from the
growth medium. Photosystem II oxygen evolution decreased to 81% in the
mutant as compared with that in the wild type, and the
thermoluminescence B- and Q-bands shifted to higher temperatures accompanied by an increase in the Q-band intensity. These results indicate that the 12-kDa protein is not essential for oxygen evolution but may play a role in optimizing the ion (Ca2+ and
Cl
) environment and maintaining a functional structure of
the cyanobacterial oxygen-evolving complex. In addition, a double
deletion mutant lacking cytochrome c-550 and the 12-kDa
protein grew photoautotrophically with a phenotype identical to that of
the single deletion mutant of cytochrome c-550. This
supports our previous biochemical results that the 12-kDa protein
cannot bind to photosystem II in the absence of cytochrome
c-550 (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832).
The oxygen-evolving system of cyanobacteria contains three extrinsic proteins, namely, a 33-kDa protein, cytochrome c-550, and a 12-kDa protein. The genes coding for the 33-kDa protein and cytochrome c-550 are psbO and psbV genes, respectively (for reviews see Refs. 1 and 2), whereas the gene for the 12-kDa protein has been tentatively named psbU (3, 4). The 33-kDa protein is commonly found in higher plant and cyanobacterial PSII,1 and its function has been studied extensively by both in vitro biochemical approaches and in vivo mutagenesis studies. Results from these studies suggested that the 33-kDa protein plays an important role in stabilizing the tetramanganese cluster, which directly catalyzes the water-splitting reaction. Loss of this protein by biochemical removal from isolated PSII (5-7) or genetic deletion (8, 9) from algal cells leads to a significant loss of the oxygen-evolving activity and in some conditions loss of manganese atoms from the tetramanganese cluster. The other two proteins, cytochrome c-550 and the 12-kDa protein, however, are present only in algal-type PSII but absent in higher plant PSII. Our previous in vitro biochemical (10) and in vivo genetic studies (11) have indicated that cytochrome c-550 is required for maintaining both the oxygen evolution and PSII stability in vivo. This cytochrome can bind to PSII essentially independent of the other extrinsic proteins (10). In accordance with this, a double deletion mutant of Synechocystis sp. PCC 6803 lacking both the 33-kDa protein and cytochrome c-550 showed a complete loss of photoautotrophic growth, which is caused by deactivation of oxygen evolution and destabilization of PSII in vivo (12). In contrast, both the single deletion mutant of the 33-kDa protein (9) or cytochrome c-550 (11) were able to grow autotrophically, albeit with reduced rates. These studies suggested that cytochrome c-550 binds to and functions in cyanobacterial PSII independent of the 33-kDa protein in maintaining the oxygen-evolving activity and PSII stability (12).
The 12-kDa protein was first found as a 9-kDa protein in PSII purified from a thermophilic Phormidium laminosum (13, 14). Dissociation of this protein from the isolated PSII caused a decrease in oxygen evolution that was partially restored upon rebinding of the protein (14, 15). The P. laminosum gene encoding the protein has been cloned and includes a leader sequence required for its transport into the thylakoid lumen (16). We have confirmed the presence of a homologous 12-kDa protein in PSII purified from another thermophilic cyanobacterium, Synechococcus vulcanus (17). Lack of the 12-kDa protein also led to a decrease in oxygen evolution of the Synechococcus PSII that was restored by rebinding of the protein (10). We further showed that binding of the 12-kDa protein in the isolated Synechococcus PSII requires presence of both the 33-kDa protein and cytochrome c-550; in the absence of either of the two proteins, the 12-kDa protein cannot bind to PSII efficiently (10). These results suggested that the 12-kDa protein is in close contact with both the 33-kDa protein and cytochrome c-550 at the lumenal side of cyanobacterial PSII; this has been confirmed by in vitro cross-linking studies showing that all of the three proteins were cross-linked together by 1-ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride (18). These studies were carried out in vitro with PSII purified from thermophilic cyanobacteria. In the present work, we demonstrated the presence of the 12-kDa protein in thylakoids of the mesophilic cyanobacterium, Synechocystis sp. PCC 6803 by N-terminal amino acid sequencing and cloned the gene coding for the 12-kDa protein based on its N-terminal sequence. The role of the 12-kDa protein was then studied by construction and characterization of the psbU gene deletion mutants.
The mesophilic, glucose-tolerant, transformable cyanobacterium,
Synechocystis sp. PCC 6803, was grown in BG11 medium at
30 °C at a light intensity of 25-30 µeinsteins m2
s
1. Thylakoid membranes were isolated from the
Synechocystis cells by glass beads according to Ref. 19, and
the isolated thylakoids were treated by sonication to release extrinsic
proteins as described in Ref. 20. The obtained sonication extract was
separated by a Mono Q column using a fast protein liquid chromatography
system (Pharmacia Biotech Inc.) (11). The fractions from column
chromatography were collected and electrophoresed in a gel containing
16% polyacrylamide and 6.0 M urea, and protein bands
around the 12-kDa molecular mass region were transferred to a
polyvinylidene difluoride membrane followed by N-terminal amino acid
sequencing. The 12-kDa protein was identified by comparing the obtained
sequences with the 9-kDa protein sequence from P. laminosum
(16) and the N-terminal sequence of the 12-kDa protein from
S. vulcanus (17).
Based on the N-terminal sequence, the gene coding for the 12-kDa protein was cloned from genomic DNA of Synechocystis PCC 6803 by a two-step, reversed PCR method (11). This gene was named psbU. DNA sequencing was performed with an ALFexpress DNA sequencer (Pharmacia) using Cy5 fluorescent primers, based on the Sanger dideoxy-sequencing method. PCR reaction was carried out using an ExpandTM High Fidelity PCR kit (Boehringer Mannheim), and each PCR product was sequenced from three independent clones to eliminate any possible inconsistencies. To obtain the clone containing the complete psbU gene, another PCR reaction was carried out with genomic DNA as template and primers flanking the whole gene. The resulted PCR product was cloned into pUC119 and used for subsequent mutagenesis studies.
To delete the 12-kDa protein from Synechocystis PCC 6803, a plasmid was constructed in which a 0.45-kb EcoT14 I-XbaI fragment containing the entire coding region of the gene was replaced by a kanamycin-resistant cassette. This plasmid was used to transform the glucose-tolerant Synechocystis PCC 6803 strain. Cyanobacterial transformation and selection of the kanamycin-resistant transformant were carried out according to published procedures (21). As a control, another plasmid was constructed in which the kanamycin-resistant cassette was inserted into an XbaI site downstream of the coding region of the 12-kDa protein gene, and this plasmid was also transformed to Synechocystis PCC 6803.
A double deletion mutant lacking cytochrome c-550 and the 12-kDa protein was constructed by introducing the psbU gene deletion plasmid into a host cell in which the psbV gene coding for cytochrome c-550 has been replaced by an erythromycin-resistant cassette as described previously (11). The transformants were selected and propagated in BG11 plates containing 25 µM atrazine, 5 mM glucose, 10 µg kanamycin/ml, and 20 µg/ml erythromycin.
Growth curves of the wild type and mutant strains in liquid BG11 were
recorded by measuring the light scattering of cells at 730 nm. For
depletion of Ca2+ or Cl from the BG11 medium,
0.24 mM CaCl2 in the original medium was replaced by either 0.48 mM NaCl or 0.24 mM
Ca(NO3)2. Synechocystis cells grown
in their mid-logarithmic phase were harvested, washed twice with
distilled water, and then transferred to the growth medium lacking
Ca2+ or Cl
. For electrophoresis, harvested
cells were broken by glass beads (100-150 µm), solubilized, and then
applied to a 16-22% polyacrylamide gradient gel containing 6.0 M urea. Cytochromes of c-type were detected by
monitoring peroxidase activity of the hemes on the gel with
3,3
,5,5
-tetramethylbenzidine and H2O2 as
described in Refs. 20 and 22.
Oxygen evolution of the Synechocystis cells was measured
with a Clark-type oxygen electrode under continuous, saturating yellow light at 30 °C in BG11 with 0.6 mM
2,6-dichlorobenzoquinone and 1 mM potassium ferricyanide as
acceptors. For thermoluminescence measurement, harvested
Synechocystis cells were suspended in BG11 in the absence or
the presence of 20 µM DCMU at 100 µg chlorophyll/ml, adapted in the dark for 5 min at room temperature, and then illuminated with single turn-over flashes at 0 °C for samples without DCMU or
with continuous light for 30 s at 5 °C for samples
supplemented with DCMU. Thermoluminescence glow curves were recorded as
described in Ref. 23, with a heating rate of 1.0 °C/s.
The 12-kDa protein was identified by N-terminal sequencing of several protein bands around 12-kDa molecular mass region obtained by column chromatography of sonication extracts from thylakoid membranes of Synechocystis PCC 6803. The sequence obtained is ELNAVDAKLTTDFGQKIDLNNSDIXDFXGLRGFYPXLAXXIIKN; this sequence is homologous to the 9-kDa protein of P. laminosum and the 12-kDa protein of S. vulcanus.
Based on the N-terminal sequence, the psbU gene encoding the
12-kDa protein was amplified and cloned by a two-step PCR method (11).
The obtained DNA sequence, together with its 5- and 3
-flanking regions, is shown in Fig. 1. The start codon for this
gene was assigned at the ATG codon of nucleotide number 253 in the DNA fragment shown in Fig. 1, because this codon is preceded by a typical
ribosome-binding sequence (Shine-Dalgarno sequence, AGGAG, underlined in Fig. 1) 7 base pairs upstream, as well as a
stop codon 39 base pairs upstream in the same reading frame (indicated by an asterisk in Fig. 1). According to this assignment, the
gene encodes a polypeptide of 131 amino acid residues with a total molecular mass of 14231 Da. Hydropathy analysis indicated that the
derived amino acid sequence is mostly hydrophilic, except the first 36 residues, which form a major hydrophobic loop (not shown). This is in
agreement with the previous results that the 12-kDa protein is a
hydrophilic one that is associated with lumenal surface of the
thylakoid membranes (10, 13-17). As upon PCR amplification of the
psbU gene, we detected only one band of the gene, we
consider that there is only one copy of the gene in
Synechocystis PCC 6803. This is consistent with the results
from computer analysis of the complete genome sequence of
Synechocystis PCC 6803 that was recently determined
(24).
The N-terminal sequence determined for the mature 12-kDa protein
corresponds to the sequence starting from residue number 37 of the
gene-derived amino acid sequence. This indicates the cleavage of the
first 36 residues after synthesis of the protein, which gave rise to a
mature 12-kDa protein of 95 residues with a calculated molecular mass
of 10490 Da. Thus, the first 36 residues served as a transit peptide
for the protein to be transferred across membranes. This is consistent
with the previous results that the 12-kDa protein is associated with
the lumenal side of thylakoid membranes (10). In fact, a similar
transit peptide has been found for the homologous 9-kDa protein from
P. laminosum (16), and recently it has been reported that
the same gene from Synechococcus sp. PCC 7002 also carried a
transit peptide (3). Although the three transit peptides for the 12-kDa
protein have virtually no similarity in their primary sequences, they
share some common features of bacterial-type transit peptides (25), e.g. there is a positive charge in the N terminus and a
hydrophobic cluster in the middle, and the residue immediately before
the cleavage site is Ala in all the three sequences (Fig.
2).
Fig. 2 compared the gene-derived 12-kDa protein sequence from Synechocystis PCC 6803 with those from Synechococcus sp. PCC 7002 (3) and P. laminosum (16) and also with the N-terminal partial sequence from S. vulcanus (17) and a red alga, Cyanidium caldarium (26). Among the three cyanobacteria for which the psbU gene has been cloned, an overall identity of 45.5-56.8% can be seen for the mature part of the sequence. When this comparison was made for the first 65 residues for which the sequence from S. vulcanus is known, the total homology lies between 49.2-57.1% among the four species of cyanobacteria compared. The sequence homology in the same N-terminal region, however, are only 30.3-36.5% between cyanobacteria and the red alga C. caldarium; this is significantly lower than those observed among different species of cyanobacteria. This indicates a remarkably higher divergence in the 12-kDa protein sequence between cyanobacteria and red algae than those found among cyanobacteria.
Mutant ConstructionsA deletion mutant psbU,
was constructed by replacing a 0.45-kb EcoT14I-XbaI fragment
containing the whole psbU gene with a 1.3-kb
kanamycin-resistant cassette. This replacement was confirmed by PCR
amplification of the DNA region containing the psbU gene. As
shown in Fig. 3, although PCR amplification with two
primers whose sequences correspond to the 5
- and 3
-sequences of the DNA fragment shown in Fig. 1 yielded a fragment of 0.92 kb from the
wild type cells, the same amplification yielded a fragment of 1.8 kb
from the targeted 12-kDa protein deletion mutant, which is exactly the
same as would be expected if the 0.45-kb psbU
gene-containing fragment was replaced by the 1.3-kb kanamycin cassette.
The original 0.92-kb fragment disappeared completely, indicating a
successful deletion of the only psbU gene in the mutant.
Fig. 3 also shows the successful construction of a double deletion
mutant,
psbU/
psbV, which is depleted of
both the 12-kDa protein and cytochrome c-550. In this
mutant, the psbU gene was replaced by the kanamycin cassette (Fig. 3, lane 4 in panel A) and a large part of
the psbV gene was replaced by an erythromycin-resistant
cassette (11) (Fig. 3, lane 4 in panel B). As a
control, a Synechocystis strain designated psbU/Km was constructed by inserting the 1.3-kb kanamycin
cassette into the XbaI site downstream of the
psbU gene. This insertion does not inactivate the
psbU gene and gives rise to a PCR fragment of 2.2 kb as
shown in lane 2 of panel A in Fig. 3, which thus indicates a successful construction of the psbU/Km
strain.
Characterization of the Mutants
Fig. 4 shows
the photoautotrophic growth curves and oxygen evolution of wild type
and mutant strains. Both the deletion mutant psbU and the
control strain psbU/Km showed a growth rate close to that of
the wild type, indicating that deletion of the 12-kDa protein had very
little, if any, effect on growth of the Synechocystis cells.
The PSII oxygen evolution decreased to 81% in the
psbU mutant as compared with that of the wild type strain. In contrast, the
psbU/Km strain had the same oxygen-evolving activity as that of the wild type. These results indicate a slight decrease in the
oxygen evolution upon deletion of the psbU gene. Apparently, this decrease in the PSII activity was not large enough to give rise to
a remarkable change in the growth rate of the mutant strain, presumably
because that the growth rate of the Synechocystis cells is
determined primarily by some photosynthetic steps other than the PSII
oxygen-evolving reaction. In fact, most of the mutants leading to an
impairment in PSII activity showed a smaller decrease in their growth
rates or overall photosynthetic electron transport activities than the
decrease observed in the PSII oxygen-evolving activity (see for example
Ref. 9).
The growth rate of the double deletion mutant
psbU/
psbV decreased to half of the wild
type strain (Fig. 4); this is comparable with that of the single
deletion mutant
psbV reported previously (11). The
activity of the double deletion mutant,
psbU/
psbV, is 41% of the wild type strain
(Fig. 4), which is also similar to the activity observed for the single
deletion mutant,
psbV (11). These results are in contrast
to the results observed with the single deletion mutant
psbU and indicates that further deletion of the
psbU gene from the
psbV mutant strain had no effect on its growth and oxygen evolution. This in turn suggests that
the 12-kDa protein did not function in oxygen evolution of the
psbV mutant, in agreement with the previous in
vitro results that the 12-kDa protein cannot bind to and function
in cyanobacterial PSII in the absence of both cytochrome
c-550 and the 33-kDa protein (10).
The effect of deletion of the 12-kDa protein on growth was also
examined in the absence of Ca2+ or Cl in the
growth medium. As shown in Fig. 5, the wild type strain was able to grow in the medium depleted of either Ca2+ or
Cl
with a slightly reduced rate in agreement with the
results reported previously (27, 28). A similar growth was observed for
the strain psbU/Km. The deletion mutant
psbU,
however, showed an apparent decrease in the growth rate in the absence
of either Ca2+ or Cl
. This is different from
the growth in the normal BG11 medium, where no effect was seen upon
deletion of the gene (Fig. 4). This suggests a role of the 12-kDa
protein in maintaining the optimum ion (Ca2+ and
Cl
) environment required for cyanobacterial oxygen
evolution. Presumably, deletion of the 12-kDa protein decreased the
affinity of PSII for Ca2+ and Cl
, which then
leads to a reduction of the growth in the absence of either one of
these two ions. In contrast, the double deletion mutant
psbU/
psbV cannot grow at all in the absence
of either Ca2+ or Cl
. This is caused
primarily by deletion of the psbV gene, because the single
deletion mutant
psbV is already unable to grow in the
absence of Ca2+ or
Cl
.2
The oxygen-evolving system in the mutant strains was further
investigated by thermoluminescence from cells excited with single turn-over flashes. The thermoluminescence B-band, which is obtained in
the absence of DCMU and arises principally from charge recombination between S2 and QB (29), had a
peak temperature of 24 °C for the wild type strain (Fig.
6). Its intensity becomes larger following two flashes
of illumination than after one flash of illumination, a typical feature observed for thermoluminescence from whole cyanobacterial cells (30).
The peak temperature and intensity of the B-band from the control
strain psbU/Km is very similar to those of the wild type
strain. The
psbU strain also showed a similar intensity of the B-band as that of the wild type or psbU/Km strains
following either one, two, or three flashes. Its peak temperature,
however, shifted to 28 °C; this is apparently higher than the peak
temperature of the B-band from wild type or psbU/Km strains.
This suggests a possible modification of the S2-state upon
deletion of the psbU gene. The peak temperature of B-band
from the double deletion mutant
psbU/
psbV
is around 30 °C, which is close to the single deletion mutant
psbU. The intensity of the B-band from the double deletion mutant
psbU/
psbV, however,
decreased significantly than that of the wild type or
psbU/Km strains. This implies that the shift of the B-band
peak temperature is caused by deletion of the 12-kDa protein, whereas
deletion of cytochrome c-550 mainly affected the overall
activity of PSII oxygen evolution but had essentially no effects on
properties of any single S-states.
The thermoluminescence Q-band, which is obtained in the presence of
DCMU and arises from charge recombination of
S2QA, showed a peak temperature
of 18 °C for the wild type and psbU/Km strains. This
temperature is slightly higher than that reported previously for
cyanobacterial cells (30), presumably because of the difference in the
growth conditions between the present and previous studies. The
intensity of the Q-band is similar to that of the B-band from the same
strains. The Q-band from the
psbU mutant strain showed a
peak temperature of 22 °C, which is higher than the Q-band from the
wild type or psbU/Km strains. This resembles the situation
observed for the B-band, thus confirming the modification of the
S2-state upon removal of the 12-kDa protein. The intensity
of the Q-band in the
psbU mutant strain, however, was
significantly higher than the intensity of the B-band from the same
strain. The cause for this increase is not clear at present but may be
due to a further modification on QA
upon
removal of the 12-kDa protein. In the double deletion mutant
psbU/
psbV, peak temperature of the Q-band
is around 24 °C, which is close to that of the Q-band observed for
the
psbU mutant. Its intensity also increased to be
slightly higher than that of the B-band from the same strain, although
this increase is not so large as that observed in the
psbU mutant. These features are also observed in the
single deletion mutant
psbV,2 suggesting that
the 12-kDa protein already lost its function in the
psbV
mutant. The smaller increase in the intensity of the Q-band in the
psbU/
psbV mutant is apparently due to the fact that the number of functional PSII reaction centers is decreased in the double deletion mutant, because deletion of psbV has
been shown to destabilize the PSII complex (11).
The psbU gene cloned in the present study represents the first gene from mesophilic cyanobacteria coding for the 12-kDa extrinsic protein of algal PSII. As in the thermophilic cyanobacteria (16), the gene obtained here had a leader sequence typical of a prokaryotic type. This suggests the transport of the protein after its synthesis, in support of the previous biochemical evidence that this protein is located in the lumenal side of the thylakoid membrane (10).
The 12-kDa protein, together with cytochrome c-550, are two PSII extrinsic proteins first found in prokaryotic algae cyanobacteria (17), and they recently have been confirmed in a eukaryotic red alga C. caldarium (26). The psbV gene for cytochrome c-550 has been found in the plastid genomes of eukaryotic algae Cyanophora paradoxa (31), Porphyra purpurea (32) and a diatom Odontella sinensis (33) for which the complete plastid genome sequences have been determined. The psbV genes from all the three types of algae had a leader sequence in their N terminus, consistent with a thylakoid lumenal location of this cytochrome. In the plastid genomes from these three species of eukaryotic algae, however, the psbU gene for the 12-kDa protein was not found. Although the presence of the 12-kDa protein in Cyanophora and diatom has not been confirmed, this protein has been found in the red alga C. caldarium (26), and therefore, the psbU gene must be present in nuclear genome of the red algae. In fact, we have recently cloned the psbU gene from the red alga C. caldarium, which shows features of a typical nuclear gene coding for thylakoid lumenal proteins, e.g. the presence of a bipartite transit peptide in its N terminus that is required for transport of the protein across both the envelope and thylakoid membranes of chloroplasts.3
The overall homology of the gene-derived amino acid sequence was 45.5-56.8% among three species of cyanobacteria P. laminosum, Synechocystis 7002, and Synechocystis 6803. The homology in the N-terminal region of the protein was 49.2-57.1% among four cyanobacteria including S. vulcanus. On the other hand, the homology in the same N-terminal region was 30.3-36.5% between the four species of cyanobacteria and the red alga C. caldarium (26). This is significantly lower than those observed among different species of cyanobacteria and therefore suggests an evolutionary divergence in the oxygen-evolving complex between prokaryotic cyanobacteria and eukaryotic red algae.
Function of the 12-kDa ProteinDeletion of the psbU gene slightly decreased the PSII oxygen evolution. This effect was apparently caused by loss of the 12-kDa protein, because insertion of the kanamycin cassette downstream of the psbU gene had no detectable effects. This agrees with the previous in vitro biochemical results that reconstitution with the 12-kDa protein to purified S. vulcanus PSII resulted in a small increase in the oxygen evolution (10). Similar results have been observed with PSII purified from P. laminosum, although in this case, the decrease observed after release of the 12-kDa protein was rather large presumably partly due to a concomitant loss of cytochrome c-550 from the PSII particles (14, 15). The decrease in oxygen evolution caused by loss of the 12-kDa protein, however, is rather small compared with that caused by loss of the other two extrinsic proteins, the 33-kDa protein and cytochrome c-550; loss of either of these two components resulted in a significant loss of oxygen evolution accompanied by an apparent reduction in the growth rate (9, 11, 27, 34, 35). In contrast, the 12-kDa protein deletion mutant grew photoautotrophically at a rate very similar to that of the wild type strain.
The growth rate of the psbU mutant, however, was
apparently lower than that of the wild type strain in the absence of
Ca2+ or Cl
. This suggests a role of the
12-kDa protein in maintaining the optimum ion (Ca2+ and
Cl
) environment required for cyanobacterial oxygen
evolution. In addition, a shift to higher temperature in the
thermoluminescence B- and Q-bands, as well as an increase in the
intensity of the Q-band, were observed upon deletion of the
psbU gene. These results may suggest a modification of the
S-state, in particularly the S2-state, upon depletion of
the 12-kDa protein, which thus suggests a role of the 12-kDa protein in
maintaining a functional structure of the algal PSII complex.
The double deletion mutant, psbU/
psbV,
showed a similar phenotype to that of the single deletion mutant
psbV in terms of its oxygen evolution, growth in the
presence or the absence of Ca2+ or Cl
, and
thermoluminescence properties.2 This indicates that
deletion of the psbU gene from the
psbV mutant
had essentially no further effect on the PSII oxygen-evolving complex,
thus suggesting that the 12-kDa protein did not function in the single
psbV deletion mutant lacking cytochrome c-550.
This agrees with the previous results that the 12-kDa protein cannot bind to and function in isolated PSII in the absence of either cytochrome c-550 or the 33-kDa protein. This double deletion
mutant
psbU/
psbV is thus very much
different from another double deletion mutant,
psbO/
psbV, which cannot grow
photoautotrophically in spite of the fact that either of the single
deletion mutants
psbO or
psbV can grow
autotrophically (12).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D84098.