©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Cytochrome c-550 as Studied through Reverse Genetics and Mutant Characterization in Synechocystis sp. PCC 6803 (*)

(Received for publication, November 21, 1994; and in revised form, December 22, 1994)

Jian-Ren Shen (§) Wim Vermaas (1) Yorinao Inoue

From the Solar Energy Research Group, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan and the Department of Botany and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1601

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Cytochrome (cyt) (^1)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.


MATERIALS AND METHODS

Purification and N-terminal Sequencing of cyt c-550

Synechocystis sp. PCC 6803 was grown in BG11 medium at 30 °C at a light intensity of 25-30 µeinsteins m s provided by white fluorescent tubes. Thylakoid membranes were extracted from the cells according to (9) , with glass beads of 0.1 mm in diameter. The isolated thylakoids were suspended in 30 mM Hepes (pH 7.5) and 20 mM NaCl and were disrupted by sonication in the presence of 1 M CaCl(2)(3) . The sonication extract was collected by centrifugation and dialyzed against 20 mM Mes-NaOH (pH 6.5), followed by separation with a Mono-Q anion-exchange column (Pharmacia Biotech Inc.) using a 0-0.5 M NaCl gradient in 20 mM Mes-NaOH (pH 6.5). The fraction containing cyt c-550 was identified by electrophoresis and heme staining with TMBZ-H(2)O(2)(3, 10) . This fraction was collected, electrophoresed in a gel containing 16% polyacrylamide and 7.5 M urea, and then transferred to polyvinylidene difluoride membrane. The band of cyt c-550 was cut from the membrane and used for N-terminal sequencing as described in (11) .

Cloning of the Gene Coding for cyt c-550

To clone the gene coding for cyt c-550, a two-step PCR method was employed. First, PCR was conducted to amplify the DNA fragment corresponding to the N-terminal part of the protein using genomic DNA from Synechocystis 6803 as template and mixed primers synthesized based on the N-terminal amino acid sequence obtained. The resulting PCR product was cloned into a TA-cloning vector (Invitrogen) and sequenced. The second PCR step involved inverse PCR. The template for this second step was genomic DNA from Synechocystis 6803 that was digested with HindIII and religated to form circularized DNA fragments. Primers were designed on the basis of sequence information obtained from the first PCR product. The inverse PCR reaction yielded a 3.1-kb product, which was cloned into the TA-cloning vector and sequenced to confirm that it contained the cyt c-550 gene. This sequence was combined with the partial sequence corresponding to the N-terminal part of the gene to yield the whole sequence of the gene. DNA sequencing was performed using an ALF DNA Sequencer (Pharmacia) with fluorescent primers, based on the Sanger dideoxy-sequencing method. Each PCR product was sequenced from at least three independent clones to eliminate any inconsistency due to misincorporation of nucleotides during the PCR reaction. To obtain a complete clone of the gene, PCR was conducted using primers corresponding to sequences upstream and downstream of the gene and with genomic DNA as template. The obtained product (858 base pairs in length) was cloned into the TA vector and then subcloned into pUC18. The presence of the desired insert was confirmed by sequencing.

Construction of Deletion and Insertion Mutants

To remove cyt c-550 from Synechocystis 6803, insertion and deletion mutants were constructed. Insertional inactivation of the gene was carried out by inserting a 1.3-kb kanamycin resistance cassette from pUC4K into the HincII site in the gene that is located about 80 base pairs down from the presumed translational start site. For deletion mutagenesis, a plasmid was constructed in which a gene-internal 0.4-kb HincII-XbaI fragment was replaced by a 1.4-kb erythromycin-resistant cassette from pRL425(12) . Cyanobacterial transformation was performed as described in (13) , and transformants were selected and propagated on BG11 plates supplemented with 25 µM atrazine, 5 mM glucose, and 5 µg/ml kanamycin or 20 µg/ml erythromycin. As a control, the cyt c-550 deletion mutant was transformed with a cyt c-550 plasmid in which the kanamycin resistance cassette was inserted at the XbaI site at the stop codon of the gene. This insertion did not alter the coding region of the cyt c-550 gene; the resulted mutant is referred to as the c-550 strain.

Characterization of cyt c-550 Mutants

The wild type and the cyt c-550 strains were grown in BG11. Except when determining growth rates, the insertion and deletion mutant strains lacking cyt c-550 were grown in BG11 supplemented with 5 mM glucose. Cells used in the following characterization experiments were harvested in their mid-logarithmic growth phase. For electrophoresis, harvested cells were resuspended in BG11 medium supplemented with 2% (w/v) lithium dodecyl sulfate, 60 mM dithiothreitol, and 60 mM Tris-HCl (pH 8.5) at 0.25 mg of chlorophyll/ml and disrupted by sonication for 3 min with a 20-kHz ultrasonic disrupter (Tomy Seiko, model UR-200P). SDS-PAGE was carried out at room temperature with a 16-22% polyacrylamide gradient gel containing 6.0 M urea(14) . Cytochromes of c-type were detected by monitoring peroxidase activity of the hemes on the gel upon incubation of the gel with 6.3 mM TMBZ in 50% methanol and 0.5 M acetate (pH 5.0) for 1 h followed by an addition of H(2)O(2) to 1%(3, 10) . For immunoblotting, proteins on the gel were transferred to a nitrocellulose membrane (Schleicher & Schuell, 0.2 µm thick), incubated with antibodies, and visualized by alkaline phosphatase-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.). Antibodies against the spinach PSII reaction center proteins, D1 and D2, and against CP43 were kind gifts from Dr. M. Ikeuchi.

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 [^14C]atrazine binding according to the procedures described in (15) . Cells of wild type or mutant strains were incubated with 100-1000 nM [^14C]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 [^14C]atrazine was calculated. For each concentration of [^14C]atrazine, duplicate samples were measured, and the obtained values were averaged. The nonspecific binding of [^14C]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(3) 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.


RESULTS

The N-terminal Sequence of the Mature cyt c-550 Polypeptide

The first step in our research was to isolate the cyt c-550 protein from Synechocystis 6803 and to determine its N-terminal sequence. The sequence obtained was VELTESTRTIPLDEAGGTTXLTARQFTNGQKIFVDTXTQXXLQGKKT. This N-terminal sequence is homologous to the sequence of cyt c-550 reported from other cyanobacteria (not shown), strongly suggesting that the sequence obtained indeed represents the sequence of mature cyt c-550 from Synechocystis 6803.

Cloning and Sequence Analysis of the Gene for cyt c-550

The cyt c-550 gene from Synechocystis sp. PCC 6803 was cloned using the two-step PCR protocol described under ``Materials and Methods.'' The DNA sequence of the gene, together with its 5`- and 3`-flanking regions, is shown in Fig. 1. The N-terminal sequence determined biochemically corresponds to the region starting at nucleotide 133. Kang et al.(8) presumed that the GTG codon starting at nucleotide 133 corresponded to the translation start site of the gene. However, this is not possible because in that case a methionine should have been found in the first position. Even though GTG can function as start codon in prokaryotes, as a start codon it codes for N-formyl Met rather than for Val; the N-terminal sequence of mature cyt c-550 indicates the presence of Val at this position, thus excluding the possibility that the translated region of the gene starts here.


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.



Deletion Mutagenesis of cyt c-550

To create mutants lacking cyt c-550, two plasmids were constructed (Fig. 3). In one plasmid, a kanamycin resistance cassette was inserted into the HincII site within the cyt c-550 gene (insertional inactivation). Another plasmid was made by replacing a HincII-XbaI fragment representing most of the coding region of the cyt c-550 gene with an erythromycin resistance cassette (deletion inactivation). These plasmids were used to transform the wild type strain of Synechocystis 6803. After selection in plates containing respective antibiotics, kanamycin- and erythromycin-resistant transformants were obtained, which were designated Deltac-550I and Deltac-550D, respectively (with I and D referring to insertion and deletion).


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 Deltac-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 Deltac-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 (Deltac-550I); lane3, deletion mutant (Deltac-550D); and lane4, reconstitution mutant (c-550). B, heme staining of whole cell extracts with TMBZ-H(2)O(2). 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 Deltac-550I and Deltac-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.

Effects of Deletion of cyt c-550 on Growth and Photosynthetic Activity

In Fig. 5, growth curves of wild type and mutant strains in the presence or absence of glucose have been presented. In the presence of glucose in the growth medium, supporting photoheterotrophic and photomixotrophic growth, no significant differences were found in the growth rates between wild type and any of the mutant strains (Fig. 5B). In the absence of glucose, however, mutants Deltac-550I and Deltac-550D grew more slowly than wild type, with a 2-fold increase in their doubling time as compared with that of the wild type or c-550 strains (Fig. 5A and Table 1). These results indicate that the disruption or deletion of the cyt c-550 gene affected the photoautotrophic growth rate of the cyanobacterium and that this effect was correlated directly with loss of cyt c-550.


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. circle, wild type strain; , Deltac-550I mutant; bullet, Deltac-550D mutant; up triangle, 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 Deltac-550I and Deltac-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.

Abundance of PSII Reaction Centers in the cyt c-550 Mutants

In many PSII mutants analyzed thus far, a decrease in oxygen-evolving activity (on a chlorophyll basis) is often correlated with a decrease in the relative amount of PSII in thylakoids of these mutants. To check this possibility in the case of cyt c-550-less mutant, the abundance of PSII reaction centers on a chlorophyll basis was determined in wild type and mutant strains. This was done by two different methods: one utilizing binding of PSII-specific herbicides and another employing immunoblot analysis with antibodies against the D1, D2, and CP43 subunits of PSII. Fig. 6depicts the results of herbicide binding analysis of wild type and mutant strains using ^14C-labeled atrazine. Assuming that there is one herbicide-binding site per PSII reaction center, there was one PSII per 580 chlorophylls in wild type strain (Table 1). This value is consistent with values previously reported for the same cyanobacterium(15, 19) . A similar value was obtained for the c-550 mutant. In the Deltac-550I mutant, however, the ratio of chlorophyll to PSII increased to 860 (Table 1). This ratio was even higher(1100) in the deletion mutant Deltac-550D (Table 1). Although the cause for this slight difference in chlorophyll to PSII ratios of the two cyt c-550-less mutants was not clear, the present results apparently indicate a decrease in the PSII content upon loss of cyt c-550. This decrease of PSII abundance on a chlorophyll basis is also confirmed by immunoblot analysis with antibodies against PSII D1, D2, and CP43 polypeptides. As shown in Fig. 7, upon Western blotting and immunodetection, the cross-reactivity of D1, D2, and CP43 with their respective antibodies was weaker for the two cyt c-550-less mutants than for the wild type or cyt c-550-reconstituted strains. This effect was similar for all of the D1, D2, and CP43 polypeptides detected, suggesting that the loss of cyt c-550 did not affect the synthesis or accumulation of a specific PSII component but affected the stability of PSII complex in the Synechocystis cells, which thus resulted in a decrease in PSII content in the mutant cells. In the immunoblot analysis, we also noticed that the band intensities of D1, D2, and CP43 were slightly weaker in the deletion mutant than in the inactivation mutant. In any case, the decrease in the PSII content in turn caused a relative increase in the PSI content in the cyt c-550-less mutant cells, which then gave rise to an increase in PSI activity, as was shown in the previous section. It should be pointed out, however, that the decrease in PSII content of the two cyt c-550-less mutants was always less than 50%, but we consistently observed a larger decrease in the PSII oxygen-evolving activity in these two mutants. This suggests that the loss of cyt c-550 not only affected the stability but also impaired normal function of PSII electron transfer. The latter effect is consistent with our previous in vitro release-reconstitution results with PSII complex purified from thermophilic S. vulcanus(7) .


Figure 6: Atrazine binding assays of Synechocystis sp. PCC 6803 wild type and cyt c-550 mutant cells. circle, wild type strain; , Deltac-550I mutant; bullet, Deltac-550D mutant; up triangle, 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, Deltac-550I mutant; lane4, Deltac-550D mutant.




DISCUSSION

Location and Function of cyt c-550

We have shown previously that cyt c-550 is associated with a purified PSII complex from the thermophilic cyanobacterium S. vulcanus as one of the extrinsic proteins involved in oxygen evolution(6, 7) . Based on the requirement of both the extrinsic 33-kDa protein and cyt c-550 for binding of another extrinsic protein, the 12-kDa protein, to the PSII complex, we proposed that cyt c-550 associates with PSII at the lumenal side of thylakoid membranes(7) . By means of chemical cross-linking, we obtained evidence that, in addition to the association of cyt c-550 with the extrinsic 33-kDa protein, the intrinsic D2 reaction center protein may provide a binding site for this cyt(20) . Release-reconstitution studies have indicated that cyt c-550 is required for optimal rates of oxygen evolution in the purified PSII complex from thermophilic cyanobacteria (7) .

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) .

Possible Evolutionary Relationship of PSII Donor Side Components

Our present and previous results indicated that in addition to the well known 33-kDa protein, there are two extrinsic proteins, cyt c-550 and a 12-kDa protein, associated with cyanobacterial PSII. These two extrinsic proteins, however, are absent in higher plant PSII. Instead, higher plant PSII contains two other proteins of 23 and 17 kDa, which are also absent in cyanobacterial PSII. Although the 23- and 17-kDa proteins in higher plants and cyt c-550 and the 12-kDa protein in cyanobacteria are not homologous in their primary sequence, they share some common features with respect to their binding and function(7, 20, 29) .

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.


FOOTNOTES

*
This work was supported in part by a grant provided to The Institute of Physical and Chemical Research (RIKEN) by The Science and Technology Agency of Japan and by National Science Foundation Grant MCB-9058279 (to W. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
To whom correspondence should be addressed. Tel.: 81-48-462-1111 (ext. 5543); Fax: 81-48-462-4685; shen{at}rkna50.riken.go.jp.

(^1)
The abbreviations used are: cyt, cytochrome; CP43, an intrinsic chlorophyll-binding protein associated with photosystem II reaction center; D1 and D2, reaction center proteins of photosystem II; PCR, polymerase chain reaction; PSI and PSII, photosystem I and photosystem II; TMBZ, 3,3`,5,5`-tetramethylbenzidine; kb, kilobase(s); Mes, 4-morpholineethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. Masahiko Ikeuchi for help in determining the N-terminal sequence and also for providing antibodies against D1, D2, and CP43.


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