(Received for publication, November 21, 1994; and in revised form, December 22, 1994)
From the
The gene coding for cytochrome c-550 in Synechocystis sp. PCC 6803 was cloned based on the N-terminal sequence of the mature polypeptide. Using the most probable translation start codon, the gene is expected to code for 160 amino acid residues. This includes a cleavable N-terminal leader sequence of 25 residues. This leader sequence has an Arg-Asn-Arg sequence immediately before the cleavage site; this is characteristic for transit peptides in prokaryotes. Comparison of this sequence with the leader sequence of the photosystem II-associated extrinsic 33-kDa protein from the same cyanobacterium showed an identity of 13 out of 25 residues. These results suggest that after synthesis of the apoprotein, cytochrome c-550 is transported into the thylakoid lumen. Using the cloned gene, insertion and deletion mutants of Synechocystis sp. PCC 6803 were constructed. In the absence of cytochrome c-550, both mutants were capable of photoautotrophic growth but at a significantly reduced rate. Atrazine binding and Western blot analysis showed that these mutants on a per-chlorophyll basis contained 53-67% of the amount of photosystem II as compared with wild type. The photosystem II-specific oxygen-evolving activity at saturating light intensity was reduced to about 40% of that in the wild type strain. Taken together, these results indicate that the cytochrome c-550 is transported into the thylakoid lumen and contributes to optimal functional stability of photosystem II in cyanobacteria. This supports our biochemical evidence that cytochrome c-550 is associated with the lumenal side of photosystem II as one of the extrinsic proteins enhancing oxygen evolution (Shen, J.-R., Ikeuchi, M., and Inoue, Y.(1992) FEBS Lett. 301, 145-149; Shen, J.-R., and Inoue, Y.(1993) Biochemistry 32, 1825-1832). Based on these results, the gene for cytochrome c-550 was named psbV. The possible evolutionary relationship among extrinsic proteins of the photosystem II donor side is discussed.
Cytochrome (cyt) ()c-550 is one of the major c-type cytochromes found in cyanobacterial cells when whole
cell extracts are viewed by heme
staining(1, 2, 3) . This cyt is water soluble
and has a low redox potential (-260 mV). Since it can accept
electrons from ferredoxin II in cell extracts in the presence of
dithionite, it has been proposed that cyt c-550 functions in
removing excess electrons generated in cells grown under anaerobic
conditions by reducing ferredoxin(4, 5) . This
proposal, however, was based on results obtained in vitro.
We recently have found that cyt c-550 is associated stoichiometrically with PSII particles purified from a thermophilic cyanobacterium, Synechococcus vulcanus(6) . We showed that this cyt is an extrinsic protein of PSII as it can be released from PSII particles by washing with high concentrations of divalent cations or Tris. Removal of cyt c-550 caused a decrease in oxygen-evolving activity that could be restored by rebinding of the cyt to PSII(7) . Furthermore, cyt c-550, together with the extrinsic 33-kDa protein, was found to be required for binding of another extrinsic protein, the 12-kDa protein, to PSII(7) . These results suggest a close contact of cyt c-550 with both the extrinsic 12- and 33-kDa proteins and hence imply a lumenal location of cyt c-550.
This functional evidence was obtained with the thermophilic cyanobacterium, S. vulcanus, which grows at 55-60 °C. The in vitro evidence for a reaction between ferredoxin II and cyt c-550 was obtained on cell extracts from a mesophilic bacterium(4, 5) . The apparent discrepancy might be due to a possible difference of the water-splitting system in thermophilic cyanobacteria as compared with that in mesophilic ones. Another possible source of difference may be that there are different forms of cyt c-550 in cyanobacterial cells (one is a soluble form, and another is a membrane-bound form), as suggested by Kang et al.(8) . Although we have shown that this is unlikely and all cyt c-550 is found in its membrane-bound form associated with PSII(3) , again this was demonstrated for the thermophilic S. vulcanus. To clarify the function of cyt c-550 in vivo in mesophilic cyanobacteria, we have determined the N-terminal sequence of the protein and studied the in vivo effects of inactivation of the cyt c-550 gene in Synechocystis sp. PCC 6803. Our results indicate that cyt c-550 is synthesized with a leader sequence that has a high level of homology with the transit peptide of the PSII-associated extrinsic 33-kDa protein from the same cyanobacterium. We made insertion and deletion mutants lacking cyt c-550 and observed that the oxygen evolution and stability of PSII was affected upon deletion of the cyt. This indicates a lumenal or periplasmic location of mature cyt c-550 and infers a role of cyt c-550 in normal function and stabilization of the cyanobacterial PSII complex.
To measure growth rates, cells of wild type and
mutant strains were inoculated in liquid BG11 medium either in the
presence or absence of 5 mM glucose at 30 °C. The cell
density was determined by measuring light scattering of the culture at
730 nm. The amount of PSII reaction center in intact cells was
determined by [C]atrazine binding according to
the procedures described in (15) . Cells of wild type or mutant
strains were incubated with 100-1000 nM [
C]atrazine (specific activity, 25
mCi/mmol) (Sigma) for 30 min at room temperature in the growth medium
at 50 µg of chlorophyll/ml, followed by a brief centrifugation to
pellet the cells. The radioactivity in the supernatants was counted by
a liquid scintillation counter, and the amount of bound
[
C]atrazine was calculated. For each
concentration of [
C]atrazine, duplicate samples
were measured, and the obtained values were averaged. The nonspecific
binding of [
C]atrazine to cells was determined
in the presence of 20 µM unlabeled
3-(3,4-dichlorophenyl)-1,1-dimethylurea.
To measure photosynthetic
electron transport activities, cells were harvested in mid-logarithmic
growth phase and resuspended in the growth medium. Whole-chain electron
transport activity was measured by oxygen evolution with 10 mM NaHCO as acceptor at 30 °C at 10 µg of
chlorophyll/ml. Oxygen-evolving activity of PSII was measured with 0.5
mM 2,6-dichloro-p-benzoquinone and 1 mM potassium ferricyanide as acceptor. Activity of PSI was measured
as oxygen uptake, using 1 mM ascorbic acid and 1 mM 2,3,5,6-tetramethyl-p-phenylenediamine as electron donor,
2 mM methyl viologen as acceptor, and 20 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea as inhibitor.
Figure 1: Nucleotide sequence of a 0.86-kb fragment containing the coding region of the gene encoding cyt c-550 in Synechocystis sp. PCC 6803. The deduced amino acid sequence encoded by the gene is shown below the nucleotide sequence. The downwardarrowhead indicates the cleavage site generating the mature cyt c-550 apoprotein. Underlinednucleotides indicate the ribosome binding sequence (Shine-Dalgarno sequence); * indicates an upstream stop codon in the same reading frame as the gene.
Two other potential translation start sites are apparent before nucleotide 133 and after the termination codon in the same reading frame (indicated with an asterisk in Fig. 1). First of all, a GTG is present at nucleotides 58-60, preceded at nucleotides 46-50 by a sequence (AGGAG) closely resembling a Shine-Dalgarno sequence. Another possible translation start site is the ATG codon at nucleotides 112-114; however, this is not preceded by a Shine-Dalgarno sequence. Therefore, we strongly favor the assignment of nucleotides 58-60 to serve as translation start site for cyt c-550. This assignment makes the gene 480 base pairs in length, encoding a polypeptide of 160 amino acids. The deduced amino acid sequence, as shown in Fig. 1, is homologous to the known sequence of cyt c-550 from other cyanobacteria(16) . The sequence of the mature polypeptide is identical to the sequence deduced from the cyt c-550 gene recently cloned from the same cyanobacterium (8) except for differences caused by the DNA sequence of the GC-rich region in nucleotides 328-339. In our sequence, amino acid residues 91, 92, and 95 are Arg, Arg, and Val, respectively, whereas in the sequence reported by Kang et al.(8) the corresponding residues are Pro, Ser, and Arg, respectively. A possible cause for this discrepancy may be related to some sequencing errors; however, it should be noted that our sequence was confirmed from three independent clones.
As noted above, cyt c-550 must have a cleavable leader sequence to give rise to the N-terminal sequence that was determined. If indeed the start codon is located at nucleotides 58-60 as suggested from the presence of a putative Shine-Dalgarno sequence, the leader sequence would be 25 residues long. This putative leader sequence has all of the attributes of a transit peptide. There is a net positive charge adjacent to the N terminus (Lys and Arg) and a hydrophobic core of 13 residues in the middle of the sequence starting from residue 4 (Phe). These are common features found in transit peptides of both prokaryotic and eukaryotic origin(17) . In particular, the residue sequence immediately before the cleavage site is Arg-Asn-Arg, which is typical for transit peptides in prokaryotes(17) . In Fig. 2, this putative leader sequence has been compared with the transit sequence for the 33-kDa PSII extrinsic protein of the same cyanobacterium(18) . With introduction of two small gaps, 13 out of 25 residues of the putative transit peptide for cyt c-550 were identical with that of the transit sequence for the 33-kDa protein. These results strongly suggest that the translation start site of cyt c-550 is 25 codons upstream of the codon corresponding to the first amino acid in the mature protein. The striking homology of this transit peptide with that of the lumenal 33-kDa PSII extrinsic protein from the same cyanobacterium also suggests that cyt c-550 is targeted to the lumenal side of thylakoid membranes.
Figure 2: Comparison of the transit peptide sequences between cyt c-550 and the extrinsic 33-kDa protein of PSII in Synechocystis sp. PCC 6803. * indicates identical residues; aa, amino acids.
Figure 3:
Construction of insertion and deletion
mutants of cyt c-550 in Synechocystis sp. PCC 6803.
The 0.86-kb fragment containing the cyt c-550 gene was cloned
into the plasmid pUC18. Insertional inactivation of the gene was
achieved by inserting a kanamycin resistance cassette into a HincII site within the gene, and a deletion mutant was
constructed by replacing the gene by an erythromycin resistance
cassette. The reconstitution mutant c-550 was
constructed by inserting the kanamycin resistance cassette into the XbaI site located at the stop codon of the cyt c-550
gene. WT, wild type; ORF, open reading
frame.
At this moment, no
information is available regarding the genome organization around the
cyt c-550 gene in Synechocystis 6803. Therefore, we
wanted to determine whether insertion of an antibiotic resistance
cassette just outside the coding region affected the phenotype of the
organism. To do so, another plasmid was constructed in which the
kanamycin resistance cassette was inserted at the XbaI site
located at the very end of the gene. This insertion should not alter
the coding sequence of the gene and thus should not affect the
synthesis of cyt c-550. This construct was introduced into the
deletion mutant, and the resulting kanamycin-resistant transformant was
designated c-550.
To confirm that upon
transformation the desired recombination had taken place between
plasmid DNA and genomic DNA from Synechocystis, genomic DNA
was isolated from the transformants after allowing segregation of
mutant and wild type genome copies by propagation for 2-3 months
in the presence of antibiotics. Using this genomic DNA as a template,
PCR amplification was carried out with primers flanking the coding
region of the cyt c-550 gene. As a control, wild type genomic
DNA was amplified as well. As shown in Fig. 4A, the PCR
product obtained from the wild type strain was 0.86 kb in length (lane1), and this product was entirely absent upon
PCR amplification using template DNA from any of the three mutants. In
c-550I and c-550
strains, the
PCR product was 2.2 kb long, and no other bands could be detected on
the gel (lanes2 and 4). A 2.2-kb band
indeed would be as expected if insertion of the kanamycin cassette into
the cyt c-550 gene of the Synechocystis genome was
successful. Similarly, PCR amplification of DNA from the
c-550D mutant gave rise to a single band of 1.8 kb,
indicating the complete replacement of wild type gene by the mutant
plasmid containing the erythromycin-resistant cassette.
Figure 4:
A,
PCR amplification of Synechocystis sp. PCC 6803 genomic DNA
with primers flanking the cyt c-550 gene. PCR was performed
using 35 cycles of 93 °C (denaturation, 45 s), 54 °C
(annealing, 1 min), and 73 °C (DNA polymerization, 3 min). 5 µl
out of a 50-µl reaction mixture was analyzed using a 1% agarose
gel. Lane1, wild type; lane2,
insertion mutant (c-550I); lane3,
deletion mutant (
c-550D); and lane4,
reconstitution mutant (c-550
). B,
heme staining of whole cell extracts with
TMBZ-H
O
. Cells were disrupted by sonication as
described under ``Materials and Methods,'' and samples
equivalent to 15 µg of chlorophyll were loaded in each lane. Numbers of lanes correspond to those
in panelA. PB,
phycobiliproteins.
In Fig. 4B, we determined the composition of c-type cytochromes in cells of wild type and mutant strains by
means of heme staining. In cells of wild type and c-550 strains, two distinctive bands were
detected, of which the upper band is cyt f, and the lower band
is cyt c-550 (3) (the dense, diffuse band below cyt c-550 is due to phycobiliproteins, which retain their blue
color upon heme staining; see also (3) ). In contrast, in cells
of the
c-550I and
c-550D mutants, only a
single band (corresponding to cyt f) could be detected upon
heme staining; the band corresponding to cyt c-550 was
completely absent. These results confirm the lack of cyt c-550
in the insertion and deletion mutants and also indicate that insertion
of the kanamycin resistance cassette at a site just outside the coding
region apparently has little or no effect on the accumulation of this
cyt.
Figure 5:
Growth rates of wild type and cyt c-550 mutant strains in BG11 at 30 °C. A, in the
absence of glucose; B, in the presence of 5 mM glucose. , wild type strain;
,
c-550I
mutant;
,
c-550D mutant;
, c-550
mutant.
To determine the primary effect of
the lack of cyt c-550 on photosynthetic electron transport,
electron transport activities in the wild type and mutant strains were
determined for PSI and PSII separately, as well as for whole-chain
electron transport. As shown in Table 1, on a per-chlorophyll
basis, the c-550I and
c-550D mutant strains
showed a 30-40% decrease in the whole electron transfer chain
activity compared with that of the wild type strain. This decrease
became more significant if PSII electron transfer activity was measured
with an artificial quinone as acceptor, implying that the decrease
observed in the whole electron transfer chain activity of the two cyt c-550-less mutants was caused by a decrease in the PSII
activity upon deletion of cyt c-550. On the other hand, the
mutant strains lacking cyt c-550 showed a small increase in
the PSI activity as compared with wild type or the c-550
strain. As will be shown later, this
most likely is due to a decrease in the PSII abundance in these
mutants, resulting in an increase in the PSI content on a chlorophyll
basis and thus an increase in the PSI activity in the mutants. These
results strongly suggest that cyt c-550 is required for normal
function or stability of the PSII complex. As biochemical(7) ,
functional, and genetic results indicate the involvement of cyt c-550 in PSII activity, we propose to name the cyt c-550 gene psbV.
Figure 6:
Atrazine binding assays of Synechocystis sp. PCC 6803 wild type and cyt c-550
mutant cells. , wild type strain;
,
c-550I
mutant;
,
c-550D mutant;
, c-550
mutant. chl,
chlorophyll.
Figure 7:
Immunoblot analysis of cell extracts of Synechocystis sp. PCC 6803 wild type and cyt c-550
mutant strains. Antibodies used were anti-D1 (A), anti-D2 (B), and anti-CP43 (C). Cells were disrupted as
described under ``Materials and Methods,'' and samples
equivalent to 4 µg of chlorophyll were loaded in each lane. Lane1, wild type; lane2, c-550 mutant; lane3,
c-550I mutant; lane4,
c-550D mutant.
Using the mesophile Synechocystis 6803, the present study extends the experimental basis for the concept that cyt c-550 is a PSII component in cyanobacteria. We obtained two lines of evidence indicating that cyt c-550 is associated with PSII at the lumenal side of thylakoid membranes and involved in stabilization of the PSII complex in the mesophilic Synechocystis 6803. First, the gene for cyt c-550 bears a leader sequence encoding a transit peptide that has been cleaved off in the mature cyt apoprotein. This peptide has features that are typical for transit peptides in both prokaryotes and eukaryotes, i.e. it has a net positive charge directly adjacent to the N terminus and a hydrophobic core in the middle. The sequence immediately before the cleavage site is Arg-Asn-Arg, which is typical for transit peptides of prokaryotic membranes(17) ; moreover, the transit peptide sequence as a whole showed significant homology with the transient peptide found in the PSII 33-kDa extrinsic protein of the same cyanobacterium(18) . Secondly, the loss of cyt c-550 by genetic inactivation of the gene coding for this cyt significantly decreased the in vivo PSII oxygen-evolving activity, which can be attributed to both an impairment in the normal function of PSII electron transfer and a decrease in the amount of PSII on a chlorophyll basis. The latter effect suggests a destabilization of PSII in the absence of the cyt. These effects were caused directly by inactivation of the cyt c-550 gene because insertion of a kanamycin resistance cassette just beyond the coding region did not yield a phenotype different from that of wild type, thus excluding the possibility that the results observed upon loss of cyt c-550 are caused by secondary effects, such as by proteins coded for by genes in the same operon and downstream of the cyt c-550 gene. These results, combined with our previous results, indicate that cyt c-550 is an extrinsic protein associated with PSII.
The gene for cyt c-550 tentatively had been named petK (8) based on a presumed involvement of the cyt in secondary electron transport. Based on the results obtained in the present study, this cyt is a component of cyanobacterial PSII, and therefore the name of petK seems inappropriate. We propose here to rename the cyt c-550 gene as psbV.
The N-terminal sequence of the mature polypeptide we deduce from the gene sequence is very similar to the sequence reported by Kang et al.(8) , although three amino acid residues were different between the two sequences, presumably due to sequencing errors. A major difference between the two studies is that Kang et al.(8) did not realize the presence of a leader sequence in cyt c-550, even though they report to have obtained an N-terminal sequence of the mature polypeptide. As upon deletion of the cyt c-550 gene no other cyt c-type proteins other than cyt f were detected; we interpret the results to indicate that there is only a single type of cyt c-550 in the cyanobacterial cells. This protein is present in the thylakoid lumen, and at least one of the functions of this cyt is to stabilize PSII and oxygen evolution in vivo. These conclusions are also supported by a recent observation that in another cyanobacterium, Synechococcus sp. PCC 7002, the low potential cyt c-550 carries a transit peptide and increases heat stability of oxygen evolution(21) .
It should be pointed out, however, that Krogmann (4) and Krogmann and Smith (5) have observed a specific reduction of ferredoxin II by cyt c-550 in isolated membrane fractions in the presence of dithionite. While this phenomenon could be due to the relatively low redox potential of cyt c-550 enabling this cyt to reduce a host of substances of higher redox potential, the cause for specific reduction of ferredoxin II by cyt c-550 remains to be clarified. Nonetheless, an apparently lumenal localization of cyt c-550 precludes a physiological involvement of cyt c-550 in reduction of cytoplasmic ferredoxin II.
The
phenotype of the cyt c-550-less mutants obtained in the
present study to some extent resembles that of psbO mutants previously
reported(22, 23, 24) . Both types of mutants
showed a decrease in their photoautotrophic growth rate. As has been
reported(25, 26) , in the psbO
mutant this decrease was caused by impairment of PSII function
and stability. A similar situation was also observed in the cyt c-550-less mutants, although in this case, the stability of
PSII appeared to be decreased more significantly as indicated by three
lines of evidence, i.e. an increase in the PSI activity on a
per-chlorophyll basis, an increase in the numbers of chlorophylls per
herbicide-binding site, and a reduction in the band intensities of D1,
D2, and CP43 probed with immunoblotting in the mutants. Drastic effects
of the loss of extrinsic components on the stability of the PSII
complex have been seen mostly in eukaryotes(27, 28) .
The finding of cyt c-550 as an extrinsic protein functioning at the donor side of cyanobacterial PSII may also suggest a possible evolutionary linkage at the donor side of reaction centers between photosynthetic purple bacteria and cyanobacteria, since in purple bacteria, another cyt, cyt c-2(30) , is known to present and function at the donor side of the reaction center. Although there is no significant sequence homology between cyt c-2 and cyt c-550, both of them are soluble, c-type, monoheme cytochromes and are located at the donor side of the reaction center. In higher plant PSII, the monoheme cyt may have been replaced by (or may have evolved to) the non-heme extrinsic 23-kDa protein. A similar loose but apparent evolutionary linkage could also be seen at the donor side of PSI. The reaction center and acceptor side of PSI of both cyanobacteria and higher plants are homologous to the reaction center of green sulfur bacteria(31, 32) . In green sulfur bacteria, a membrane-bound c-type cyt, cyt c-551(553), functions as the electron donor to the reaction center(33, 34) , whereas in cyanobacteria either cyt c-553 or plastocyanin is the functional donor to the PSI reaction center(35, 36, 37) . cyt c-551 in green bacteria and cyt c-553 in cyanobacteria are not homologous in their sequence, but again they have a similar function. Moreover, cyt c-553 was further replaced in higher plants by a significantly different copper-containing protein, plastocyanin. In this respect, it is also interesting to note that cyt c-553 and cyt c-550 share a significant regional homology (not shown; see (8) ), and both cytochromes are present in cyanobacteria but not in higher plants.
In summary, cyt c-550 is a component that is translocated out of the cytoplasm in cyanobacteria and is involved in maintaining PSII oxygen-evolving activity and stabilization of the PSII complex. Even though at this moment no redox function can be ascribed to cyt c-550, it is possible that it is a ``molecular appendix'' remaining from an evolutionary ancestor in which a c-type cyt could serve as electron donor to the photosynthetic reaction center. Its properties seem to preclude redox involvement in photosynthetic electron flow, but a role in maintaining normal function of PSII electron transfer and stabilization of the PSII complex has been established.
The nucleotide sequence reported in this paper has been registered in the GSDB, DDBJ, EMBL, and NCBI data bases with the accession number D45178[GenBank].