Plant Molecular Biology 8: 317-326, (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
317
Sequencing and modification of psbB, the gene encoding the CP-47 protein of Photosystem II, in the cyanobacterium Synechocystis 6803 Wim E J. Vermaas*, John G. K. Williams & Charles J. Arntzen
E. I. du Pont de Nemours & Co., Inc., Central Research and Development Department, Experimental Station, E402, Wilmington, DE 19898, USA; * Present address." Department of Botany, Arizona State University, Tempe, A Z 85287, USA Received 28 August 1986; in revised form and accepted 5 January 1987
Keywords: cartridge mutagenesis, D N A sequencing, photosynthesis
Abstract
The Photosystem II protein CP-47 has been hypothesized to be involved in binding the reaction center chlorophyll. The psbB gene, encoding this protein, was cloned from the genome of the cyanobacterium Synechocystis 6803, and sequenced. The D N A sequence is 68% homologous with that of the psbB gene from spinach, whereas the predicted amino acid sequence is 76% homologous. The hydropathy patterns of Synechocystis and spinach CP-47 are almost indistinguishable, indicating the same general CP-47 folding pattern in the thylakoid membrane in the two species. There are five pairs of histidine residues in CP-47 that are spaced by 13 or 14 amino acids and that are located in hydrophobic regions of the protein; these histidine residues may be involved in chlorophyll binding. Interruption of the psbB gene by a D N A fragment carrying a gene conferring kanamycin resistance results in a loss of Photosystem II activity. This indicates that an intact CP-47 is required for a functional Photosystem II complex, but does not necessarily indicate that this protein would house the reaction center.
Introduction
Photosystem II (PS II) is a pigment-protein complex in the thylakoid membrane, consisting of at least five membrane-internal proteins (CP-47, CP-43, the '32 kDa' herbicide-binding protein D1, a 34 kDa D2 protein, and cytochrome b559) and several external proteins. Many of these proteins bind cofactors that are involved in photosynthetic electron transport (1, 22, 25). The determination of the function of the membrane-internal PS II 'core' proteins has often been based on circumstantial evidence. In particular, the role of CP-47 (a chlorophyll-binding protein
of 4 7 - 5 1 kDa) is still unclear. Evidence has been presented that CP-47 harbors the Photosystem II reaction center P680 (17, 31, 32). However, recent data have shown significant sequence homology between D1 and D2 and the reaction center proteins from purple photosynthetic bacteria (2, 15, 20). This fact, and similarities in cofactor function in PSII and the photosynthetic bacteria, have led to the suggestion that D1 and D2, not CP-47, create the binding environment of P680 (2, 15, 24). Our research goal is to develop an experimental system by which these opposing hypotheses can be tested. We have taken two different approaches in this study: in the first place, psbB (the gene encod-
318 ing CP-47) was cloned from a cyanobacterium, Synechocystis 6803, sequenced, and the sequence was compared with that ofpsbB from spinach (16). Since structurally or functionally important regions of the protein are likely to be conserved throughout evolution, whereas less stringent domains are not, sequence comparison will yield insight into which protein regions are likely to be important in the structure and function of CP-47. The second approach has been the generation of a well-defined CP-47 mutant. In the past, analysis o f mutants that were defective in PS II has been used to obtain correlative evidence on the possible function of the PS II core proteins (13, 14), using randomly generated and frequently pleiotropic mutations. However, techniques have recently been developed to introduce genetic material into cyanobacteria (6, 7, 29, 30). In both Synechocystis and Synechococcus species exogenous DNA can be incorporated into the chromosome by homologous recombination thus allowing directed mutagenesis ((6, 29), and J.G.K. Williams and L. Mclntosh, unpublished observations). However, since we are primarily interested in directed mutagenesis of PS II proteins, Synechocystis 6803 is the most suitable organism for our studies. Besides the availability of a genetic transformation system (8), Synechocystis 6803 also has the capability to grow in the absence of PS II activity (19). The present paper describes how the native psbB gene from Synechocystis 6803 can be interrupted by insertion of a gene encoding aminoglycoside 3'-phosphotransferase (conferring kanamycin (Km) resistance). This resistance marker allows selection of transformants, which subsequently can be characterized physiologically.
Materials and methods
Synechocystis 6803 was grown in BG-11 medium (19) at 30 °C. Liquid cultures were perfused with air that was humidified by bubbling through a 1o7o CuSO 4 solution and sterilized by passage through filters (Gelman #12123 and #4210). Solid medium was BG-11 supplemented with 1.50-/0 (w/v) agar, 0.3O7o sodium thiosulfate, and 10 mM T E S / K O H buffer, pH 8.2.
Synechocystis 6803 gene libraries were made by ligation of restriction-enzyme-cut Synechocystis 6803 DNA with pUC 18 (33) that was cut with the same enzymes. The ligation mix was used to transform E. coli HB101 to ampicillin resistance; plasmid DNA was isolated from the transformed bacterial culture. The Km-resistance cartridge used in constructions originated from the bacterial transposon Tn 903 (18). To prepare transformable Synechocystis 6803 ceils, an actively growing cyanobacterial culture was diluted in fresh medium to OD730 = 0.10, and was grown for 12-16 hrs. The cells were harvested and resuspended in BG-11 medium t o OD730 = 2.5. These cells were immediately used for transformation by adding 5 izl DNA (0.1-1 m g - m l - : in 10 mM Tris-HC1 pH 7.5 + 0.1 mM EDTA) to 500 #1 of the cell suspension. The DNA/cell mixture was incubated in a glass tube under standard growth conditions for 4 - 6 h r s . Subsequently, 200 izl was plated out on a membrane filter (Nucleopore, Pleasanton, CA) on solid medium in a petri dish. The plate was incubated for 1 8 - 2 0 hrs and the filter was transferred to solid medium containing 10 #g.ml ~ kanamycin. Colonies of transformed cells were visible in 4 - 5 days. For Southern hybridization analysis, DNA from agarose gels was transferred to Gene Screen Plus ® hybridization transfer membrane (NEN) and hybridized to 32p-nick translated probe as described in the Gene Screen Plus ® manual. DNA fragments to be sequenced were cloned into M13 mpl8 and M13 mpl9 (33). Single-stranded MI3 DNA was sequenced by the dideoxynucleotide chain termination method (21) with 'universal' (BRL) or custom-made oligonucleotides (15-18 mers) as primers, using standard techniques (MI3 sequencing manual; BRL). Long (55 cm) ultra-thin (0.2 mm) acrylamide gels (LKB), poured and handled according to the manufacturer's instructions, were used for DNA separation (3 000 V; 55 °C). For oxygen-evolution measurements, a fresh cyanobacterial culture was spun down, and resuspended in 10 mM tricine/NaOH pH 7.5 at 10 ~g.ml 1 chlorophyll. Light-dependent oxygen evolution was measured using a Hansatech O2-electrode. The electron acceptors were 0.1 mM
319 2,6-dimethyl-p-benzoquinone K3Fe(CN)6.
and
0.5 m M
ly 17 kb, with the EcoRI site about 600 bp upstream from the BamHI site in the psbB gene.
Results
DNA sequence analysis of psbB
Isolation of the psbB gene
In order to sequence the psbB gene from Synechocystis 6803, various fragments of the gene were
A 5.7 kb BamHI/BamHI fragment carrying most of the psbB gene was isolated from a Synechocystis 6803 genomic library; the fragment was identified by heterologous Southern hybridization using a psbB fragment from Cyanophora paradoxa (a kind gift from Dr Don Bryant) as a probe. Preliminary sequence analysis of the fragment, and comparison with the sequence from spinach (16) indicated that the 5 ; end of the psbB gene was not present in the BamHI/BamHI fragment. Searching gene libraries from Synechocystis 6803 D N A cut with other restriction enzymes was unsuccessful in finding the 5' end of the psbB gene. We suggest from this observation that a plasmid with the upstream region of the psbB gene is not replicated in E. coil without negative effects on bacterial cell growth. To recover the 5' end of the psbB gene without the use of genomic libraries, we followed an alternate strategy. This involved insertion of a Kmresistance cartridge into a Sinai site of the 5.7 kb BamHI/BamHI fragment that contained the incomplete psbB gene (Fig. 1A). Synechocystis 6803 was then transformed with this plasmid and a Kmresistant transformant was purified. Southern blots were used to show absence of wild-type D N A in the transformant and the insertion of the Kmresistance gene solely in psbB (Fig. 1B). The D N A of the transformant was then cut with combinations of restriction enzymes that do not cut between the site of Km resistance cartridge insertion and the upstream part of the psbB gene, and ligated into pUC19. The ligation mixtures were used to transform E. coli (HBI01). A plasmid containing the Km-resistance insert as well as the upstream segment of the original 5.7 kb BamHI/BamHI fragment was identified by restriction mapping and Southern blotting. An EcoRI/SalI fragment cloned by this method appeared to contain the 5' end of the psbB gene. The fragment size was approximate-
cloned into M13 mpl8 and M13 m p l 9 (33), namely the EcoRI/KpnI (1.1 kb) (with the upstream region of psbB), BamHI/SmaI (1.1 kb) and KpnI/KpnI (1.4 kb) fragments. The latter two fragments were obtained from the BamHI/BamHI fragment from the gene library. The nucleotide sequence obtained from these fragments is shown in Fig. 2; there is an open reading flame between nucleotides 363 and 1883. Although this publication does not provide information regarding the actual start of transcription, sequences can be found upstream from psbB that strongly resemble the consensus sequence for an Escherichia coli promoter: at nucleotides 160-165 and 191-196 the sequences are T G G A C A and TACAAT, respectively, whereas the E. coli - 35 and - 1 0 consensus sequences are T T G A C A and TATAAT, respectively. It is also possible that the sequence at nucleotides 168-173 (TTGCAG) could serve as - 3 5 sequence. In the upstream region of psbB we do not find sequences that strongly resemble - 1 0 or - 3 5 sequences of the promoter of the ribulose-l,5-bisphosphate carboxylase/oxygenase (rbc) or rrnA operons in the cyanobacterium Anacystis nidulans 6301 (11, 23). Figure 2 also provides the deduced amino acid sequence of the large open reading frame, and compares it with the CP-47 sequence from spinach (16). Because of the large homology (76% of the amino acids and 68% of the nucleotide are homologous), we postulate the large open reading flame to encode CP-47. The amino acid homology between the CP-47 proteins from spinach and Synechocystis 6803 appears to be almost complete in certain regions, but divergent in other domains. In order to check whether the differences are reflected in a difference of the overall hydrophobicity pattern of the proteins, we compared the CP-47 hydropathy plot (cal-
320
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culated according to Kyte and Doolittle (12)) from Synechocystis and spinach (Fig. 3). These data indicate that the hydropathy pattern of CP-47 from the two species is almost identical. This implies that the folding pattern of CPo47 within the thylakoid membrane in Synechocyst& is likely to be similar to that in spinach.
Cartridge mutagenesis of psbB The sequence in Fig. 2 shows that the SmaI site (nucleotide sequence recognized: CCCGGG) at which the Km-resistance cartridge was inserted (see
Fig. 1. A. Restriction m a p of the 5.7 kb BamHl/BamHl fragment carrying part of the psbB gene from Synechocystis 6803 (wild type, center). A Kin-resistance cartridge (Km R) was inserted at the Srnal site in the psbB gene. The corresponding restriction m a p of the transformed D N A is shown in the lower part. B. Southern blot of wild-type (lanes 1, 3 and 5) and mutant (lanes 2, 4 and 6) Synechocystis 6803 D N A cut with SmaI (lanes 1 and 2), KpnI (lanes 3 and 4) or NcoI (lanes 5 and 6), and probed with the 32P-nick-translated 2.8 kb Kpnl/Kpnl fragment containing the Km-resistance cartridge and part of psbB (see Fig. 1A, lower part).
321
GAATTCAGGTTCGCTCACCCCCATCATCAGGTCAGCAACAAAAGTGTAGTCCCTGGCCATTCCATCGTGG
70
CCGGCCATGGGCGATCGCCAGTTTCTTATGGGAGCAAGGTGGGGCTAACCGCACCGTCCCTAGGTCATTC
140
AAGCCCTTTGCCTATCCCCTGGACAGCTTGCAGAAATCCTGGCCGCTCGTTACAATCCTTCAAAATATTC -35? -35? -I0
210
TCACTTTGTAAGGGATAATGGATAAAACTTGACTCTGTCTGTCTTGTTCGGTTAACACAACCTATAGACA
280
AGGGTTTTATTTACCCAACGCAGAATAAAAATTAAAACGTCTTTAAGACACAAAACACTATTCGTTACTA GAAGGAGCGTCA
ATG MET
GGA Gly
CTA Leu
CCT Pro
TGG Trp
TAT Tyr
CGC Arg
GTT Val
CAT His
ACA Thr
GTT Val
GTC Val
CTG Leu
350 AAT Asn
404
GAT Asp
CCA Pro
GGG Gly
CGA Arg
CTC Leu
ATC Ile
TCT GTc Ser Val
CAT His
TTA ATG Leu Met Ile
CAC His
ACT Thr
GCC Ala
CTA Leu
GTG Val
GCT Ala
GGC Gly
458
TGG Trp
GCT Ala
GGA Gly
TCT ATG Ser Met
GCT Ala
CTC Leu
TAT Tyr
GAG Glu
TTG GCC Leu Ala
ATT Ile Val
TTT Phe
GAC Asp
TCC AGC Ser Ser Pro
GAT Asp
GCT Ala Pro
512
GTG Val
CTT Leu
AAC CCC Asn Pro Asp
ATG Met
TGG Trp
CGG Arg
CAA Gln
GGC Gly
ATG Met
TTT Phe
GTT Val
TTG Leu Ile
CCC Pro
TTC Phe
ATG Met
GCC Ala Thr
CGC Arg
566
CTC Leu
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GTC ACC AGT Val Thr Set Ile Asn
TCC Ser
TGG Trp
AAT GGC Asn Gly Gly
TGG Trp
AGC GTC Ser Val Ile
ACC Thr
GGA Gly
GAA Glu Gly
ACT Thr
GGT TTG Gly Leu Ile Thr
620
GAT Asp
CCC Pro
GGT TTC TGG Gly Phe Trp Ser Ile
TCC Ser
TTT GAA Phe Glu Tyr
GGG Gly
GTA Val
GCT Ala
GCT Ala Gly
GCC Ala
CAC His
ATC Ile
GTT CTA Val Leu Met Phe
TCT Set
674
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CTG Leu
TTG TTC Leu Phe Cys
CTA Leu
GCC Ala
GCC Ala
GTA Val Ile
TGG Trp
CAC His
TGG Trp
GTA Val
TTT Phe Tyr
TGG Trp
GAC Asp
CTG Leu
GAA Glu
TTA Leu Ile
728
TTT Phe
GTT GAC Val Asp Ser
CCC CGT Pro Arg Glu
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TTG Leu
CCC Pro
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782
ATT Ile
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CTC Leu
TTC Phe
TTG Leu
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TTG CTC Leu Leu Val Ala
TGC Cys
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GCT Ala
TTC Phe
CAC His
CTC Leu Val
836
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GGC Gly
ATG Met Ile
TGG Trp
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TCC GAC Set Asp
CCC Pro
TAT Tyr
GGT Gly
CTG Leu
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890
CAC GTA His Val Lys
CAG Gln
CCA Pro
GCA CCG GAA Ala Pro Glu Cys S e t Ala
TGG Trp
GGA Gly
CCA GCT Pro Ala Val Glu
GGC Gly
TTT Phe
AAC Asn Asp
CCG Pro
TTT Phe
AAC Asn Val
944
GTG Val
CCC Pro
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GTA GTG Val Val Ile Ala
GCT CAC Ala His Ser
CAC His
ATT GCT Ile A l a
GCT Ala
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ATT GTC Ile V a l Thr Leu
GGT Gly
ATC ATT Ile Ile Leu
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998
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CTT Leu
TTC Phe
CAC His
CTT Leu
ACG GTA Thr Val Ser
CGG Arg
CCC CCT GAG Pro Pro Glu Ser Gln
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AAA Lys
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1052
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1106
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GCC Ala
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1376
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1808
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GTGCTTCTTGCACAGCTTTTAACCACAGCTTAAGAGCGTG
1926
TTTGAAAAGCCTCCCTGGTCACCCAAGTTTGGGGGGAAACTAAGTCAAAGTCCCCCAGCATCGGGAGATT
1996
TAGGGAGCAGAGTCAGACTTTACAAACAGGTTCTAAGTCTTGGGAGTTATCCCTCATAATTCGAGCTAAC
2066
TCAATCAAAACTTAAAGTTTACAGAAACTATTTTTAAAGCGATTTCTTCTACAGGGGAAGTCGCTTTTTG
2136
TTGCTTTTTTGGAGTTATTAATAACCTTACCAGTGTGCTATGATGCCAGGGTCAAAGCCCCTGGTAAGCA
2206
ATTGAAATTAGTTGCTTTTACTAATTTTTAACATCCCTTGATTATCACCATGGTCGAATTAACTGAAAAT
2276
CACTACAACCTACAACAGGAACTGGCCGATGCCCTCCAATCCCTGGGCCGTAGTATTCATTCTTTGGAAA
2346
GGGACATAGTTATTAGCCATTGTGAAATTAATGCCGACCATGGGGTGGGGGTTTTACTGCAAAGGCTTTT
2416
TCCCAACTCAGATGAGCTATTGACAATTCGCTCCCATGATCTTTACGGCGGGCAACAGGAGTTTGGCGAT
2486
CGCCATTTTTTAGTGCAGGGAGGAAGTTTTGTCACCATTGCCCAACAGGTGCAGTGGCTGTTGCACTATT
2556
ATGCTCCCCGAAGATTTTTATCGGTACC
2584
Fig. 2. Nucleotide sequence of a 2584-base pair EcoRI/KpnI fragment from Synechocystis 6803 D N A , containing the psbB gene (open reading frame from 363 to 1886). The predicted amino acid sequence of CP-47 from Synechocystis 6803 is included under the bases of the open reading frame. A m i n o acids from spinach CP-47 (16) that differ from this sequence are indicated in italics, under the amino acid sequence of Synechocystis 6803; amino acids from spinach that are identical to that in the cyanobacterium have not been indicated. Putative - 3 5 and - 1 0 sequences are underlined.
323 4 2 0 a
-2
z -4 "1-
O >-/-
2 0 --2 --4 0
100
200
300
400
500
AMINO ACID SEQUENCE NUMBER
Fig. 3. Hydropathy profile of the predicted CP-47 amino acid sequence from Synechocystis 6803 (A) and spinach (B), in which amino acid residue number is plotted against the average hydrophobicity of 6 surrounding residues, according to the method of Kyte and Doolittle (12).
Fig. 1) is inside the psbB gene at nucleotides 1702-1707. This implies that the Km-resistance mutant generated has an interrupted psbB gene. This genetically well-defined mutant was recognized as being potentially useful in characterizing the function of CP-47 in Synechocystis 6803. However, before characterization of the mutant would be meaningful, it was necessary to establish that insertion of the kin-resistance cartridge into the psbB gene does not interfere with function of other genes possibly located downstream of psbB in the same operon. If transcription of other (downstream) genes would be affected by the Km-resistance cartridge insertion, a change in phenotype upon cartridge insertion might not be attributable to a change in the CP-47 protein. In order to check whether there were genes in the same operon as psbB affecting the phenotype of the organism, the Km-resistance cartridge was inserted at the NcoI site at nucleotides 2254-2259, about 370 bp downstream of the 3' end of the psbB gene. No open reading frame longer than 33 bp (nucleotides 2176-2208) can be identified between the end of the psbB gene and the insertion. Synechocystis 6803 was transformed with the plasmid having the Km-resistance cartridge at the NcoI site, and the resulting mutants were puri-
Fig. 4. Southern blot of wild-type (lane 1, 3 and 5) and mutant (having a Km-resistance insert at the NcoI site downstream of psbB) (lane 2, 4 and 6) Synechocystis 6803 D N A cut with SmaI (lanes 1 and 2), Kpnl (lanes 3 and 4) or Ncol (lanes 5 and 6), and probed with the 32p-nick-translated 2.8 kb Kpnl/Kpnl fragment containing part of psbB and the Km-resistance cartridge at the NcoI site. A restriction m a p o f p s b B and neighboring D N A in wild type and m u t a n t is included for identification of the bands.
324 fied and probed for photosynthetic growth. The Southern blot in Fig. 4 shows the absence o f wildtype DNA in the mutant, thus confirming that the desired mutant was obtained. This mutant grew as well as the wild type organism, with normal pigmentation, under both photoautotrophic and photoheterotrophic conditions (not shown), indicating that insertion of the Km-resistance cartridge at the NcoI site does not lead to any readily observable changes in the phenotype. Thus, any phenotypic changes observed in the mutant with Km-resistance cartridge insertion at the SmaI site are caused by a change in the CP-47 protein per se. Therefore, the properties of this mutant (referred to in this paper as CP-47 mutant) were analyzed.
grow photoautotrophically in BG-11 medium (requiring both PS II and P S I ) , but did grow photoheterotrophically in the presence of glucose (requiring PS I only). This strengthens the notion that an intact CP-47 is required for PS II reactions. This point was proven when measuring light-dependent oxygen evolution in wild-type and mutant cells using a combination of 2,6-dimethyl-p-benzoquinone and ferricyanide as PS II acceptor (Fig. 5B): no O2 evolution was monitored in the mutant, indicating an absence of Photosystem II activity. In a subsequent paper (26), the detailed characteristics of this mutant will be reported.
Discussion
Mutant phenotype analysis
Protein sequence analysis
Figure 5A shows that the CP-47 mutant did not
The predicted sequence of the 507-residue CP-47 protein from Synechocystis has a large homology to the 508-residue CP-47 from spinach. The homology is especially strong in certain regions o f the protein: amino acid 1 - 4 3 , 2 2 9 - 2 7 6 , and 458-481. From the hydropathy plots (Fig. 3) we predict the presence of 6 membrane-spanning regions, located approximately between the following residues: (1) 5 - 3 5 , (2) 95-120, (3) 138-162, (4) 198-220, (5) 232-255, and (6) 450-473. Therefore, the transmembrane regions 1, 5 and 6 appear to be part of the strongly conserved protein domains. According to Morris and Herrmann (16), CP-47 folds through the membrane 7 times. However, we think the interpretation of these authors that there is a membrane-spanning region between amino acid residues 55 and 75 (indicated as segment II in (16)) is unlikely, since Fig. 3 shows that there is only a significantly hydrophobic region between residues 60 and 72. This region is too short to span the 30 A. lipophilic membrane core, assuming that per residue an a-helix spans about 1.5 A, (3). Histidine is inferred to play a major role in binding bacteriochlorophyll (via the Mg atom) in pigment-protein complexes of purple photosynthetic bacteria (35). It is interesting to note that all histidines in spinach CP-47 are conserved in the Synechocystis sequence. One histidine residue in
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(hrs)
Fig. 5. A. G r o w t h o f w i l d t y p e ( o , n ) a n d C P - 4 7 m u t a n t ( o , u ) cultures, as reflected by light scattering measured at 730 nm. At time 0, cells grown in the presence of 5 m M glucose were resuspended at OD730=0.06 into fresh medium, o, o: cell growth in BG-11 medium under photoautotrophic conditions, requiring PS II and P S I activity, n, o : cell growth under photoheterotrophic conditions, in BG-I1 supplemented with 5 m M glucose (G) and 20 #M atrazine (A) (the latter being required to inhibit PS 11). Under these conditions only PS l activity is required for growth. B. Oxygen evolution reflecting PS II electron transport in wild-type and CP-47 mutant. As electron acceptors, 0.5 m M potassium ferricyanide and 0.1 m M 2,6-dimethyl-p-benzoquinone were present. 10 #g • ml I chlorophyll.
325 the cyanobacterial sequence, at position 77, does not occur in the spinach sequence. Out of a total of 15 histidine residues, 10 are separated from another histidine by 13 or 14 amino acid residues, and are located in the putative membrane-spanning segments 1, 2, 3, 4 and 6. Four pairs of histidine were already noted by Morris and Herrmann (16). The same spacing of his residues can be found in chlorophyll-proteins of PS I (4, 5) as well as in the CP-43 protein (9). The conserved putative bacteriochlorophyll binding sequence Ala-X-X-X-His (34), present in all but one antenna proteins of purple and green bacteria (27), is only seen twice in CP-47 (residues 110-114 and 212-216). At least 40 chlorophyll molecules are bound to one PS II core complex (10, 32). However, the total number of histidine residues in CP-47 and CP-43 combined is not more than 28. This suggests that histidine residues are not the only site where chlorophylls can bind. In this respect, a report of Wechsler et al. (28) is of particular interest: in the 5.6 kDa bacteriochlorophyll c-binding protein from Chloroflexus aurantiacus, glutamine and asparagine residues are suggested to bind the bacteriochlorophyll. The data presented here imply a cautionary note towards the interpretation of observed amino acid homologies. For example, the putative membraneinternal sequence his-leu-phe-leu-ser-gly-val-ala-cys from spinach CP-47 (residues 142-150), observed to be almost identical to a sequence from the D1 protein, was suggested to play a role in plastoquinone binding (16). However, in Synechocystis CP-47 this sequence is modified in two residues, suggesting the local sequence homology of spinach CP-47 with D1 to be merely fortuitous. Moreover, the sequence asp-pro-thr-thr-arg-arg, noted by Fish et al. (5) to occur both in the A2 polypeptide (from PS I) and in spinach CP-47, is not conserved in the cyanobacterial CP-47 (residues 500-505). As a third example, the sequence asp-pro, noted by Fish et al. (5) to be abundant in chlorophyll proteins, especially in CP-47 from spinach, is only conserved in 50% of the cases in Synechocystis CP-47. As shown in the Results section, the loss of PS II activity can be attributed to an interruption of the psbB gene. However, the fact that an uninterrupted psbB gene is indispensable for PS II activity does
not necessarily imply that CP-47 would bind P680. Other possibilities include (a) a CP-47 requirement for assembly of the PS II core complex, or (b) a structural change in the arrangement of the other PS II core complex proteins such that the reaction center chlorophyll(s) or another element in the electron transport chain can no longer bind properly. However, it has been shown that another chlorophyll-binding protein of PS II, CP-43, is still present in the mutant membranes (26). This renders the hypothesis that CP-47 would be required only for PS II assembly in the membrane unfavorable. One of the approaches to obtain further data concerning the role of CP-47 in PS II will be directed mutagenesis in the psbB gene. The primary target may be the D N A regions that encode amino acids in strongly conserved domains at or near presumed chlorophyll binding sites. In this way, the functions of regions within the CP-47 protein can be experimentally evaluated.
Acknowledgements We thank JoAnne Zeroka for technical assistance, Don Bryant for critical reading of the manuscript and for sending the psbB gene from Cyanophora paradoxa and we are grateful to John Pierce for his collegial assistance. This work has greatly benefitted from the synthetic oligonucleotides prepared by Ellen Doran and coworkers.
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