©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Candidate Genes for the Phycoerythrocyanin Subunit Lyase
BIOCHEMICAL ANALYSIS OF pecE AND pecF INTERPOSON MUTANTS (*)

Linda J. Jung, Crystal F. Chan, and Alexander N. Glazer (§)

From the (1) Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The rod substructures of the Anabaena sp. PCC 7120 phycobilisome contain the light harvesting proteins C-phycocyanin and phycoerythrocyanin (PEC). Even at low light intensities, PEC represents no more than 5% of the phycobilisome protein. The subunits of both proteins carry thioether-linked phycocyanobilin (PCB) at -Cys-82 and -Cys-155; however, C-phycocyanin has PCB at -Cys-84 whereas PEC subunit carries phycobiliviolin at this position. The Anabaena sp. PCC 7120 pec operon is made up of five genes. PecB and pecA encode the and subunits of PEC, pecC encodes a linker polypeptide associated with PEC in the rod substructure, and pecE and pecF are genes of unknown function that show a high degree of homology to cpcE and cpcF, that encode a C-phycocyanin subunit PCB lyase (Fairchild, C. D., Zhao, J., Zhou, J., Colson, S. E., Bryant, D. A., and Glazer, A. N.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7017-7021). Insertional mutants in pecE and pecF, and an interposon mutant in which a portion of both pecE and pecF was deleted, were constructed. All three types of mutants grew 1.3 times slower than wild-type under limiting light conditions and showed a 20% reduction in the PCB content of whole cells relative to chlorophyll a. Holo-PEC was missing from the phycobilisomes of all three types of mutants and the level of the PEC linker polypeptide was reduced relative to the wild-type. However, 30% of the wild-type level of the PEC subunit was present in all of these phycobilisomes. In contrast, the PEC subunit was barely detectable in the pecE and pecF mutants, but was present in the pecEF deletion mutant as a PCB-adduct in a 1:1 ratio with the PEC subunit. The identity of this ``unnatural'' adduct was confirmed by isolation of the subunit and amino-terminal sequencing. These biochemical results support the inference that pecE and pecF encode a PEC subunit phycobiliviolin lyase, and, in conjunction with earlier findings, demonstrate that phycobiliprotein bilin lyases show high selectivity (rather than absolute specificity) for both the bilin and the polypeptide substrate.


INTRODUCTION

Phycobilisomes are macromolecular complexes of several million daltons that serve as important light-harvesting components of the photosynthetic apparatus of cyanobacteria and red algae. These complexes are made up of several homologous chromoproteins, the phycobiliproteins, whose assembly to form the phycobilisome is mediated by a set of ``linker'' polypeptides (1) . The major phycobiliproteins fall into three classes: the allophycocyanins, the phycocyanins, and the phycoerythrins. The fundamental building block of all these phycobiliproteins is an heterodimer. Their ability to absorb visible light is conferred by linear tetrapyrrole prosthetic groups (bilins) attached through thioether linkages to specific cysteinyl residues. Four isomeric bilins, differing only in the arrangement of double bonds, are found on cyanobacterial and red algal phycobiliproteins: phycocyanobilin (PCB),() phycobiliviolin, phycoerythrobilin, and phycourobilin (2) . The positions of the cysteinyl residues that serve as the bilin attachment sites are conserved among the different phycobiliproteins, e.g. -Cys-84 carries a bilin in allophycocyanins, phycocyanins, and phycoerythrins, but the total number of bilin attachment sites is the major distinguishing feature of phycobiliproteins belonging to different classes. Thus, allophycocyanins carry one bilin on each subunit, phycocyanins one on the and two on the subunit, and phycoerythrins carry either two or three on the and three on the subunit (2, 3) . The amino acid sequences of many phycobiliproteins have been determined either directly or by sequencing of the structural genes (4) . The crystal structures of several C-phycocyanins (5, 6) , phycoerythrocyanin (7) , and B-phycoerythrin (8) have been determined at high resolution. In spite of such a large base of structural information, the features that lead to the attachment of particular bilins at specific sites are not evident.

The in vitro uncatalyzed reaction of phytochromobilin with apophytochrome leads to a chromoprotein functionally indistinguishable from native phytochrome (9, 10) . The possibility of non-enzymatic addition has been investigated by analysis of the in vitro addition of PCB and phycoerythrobilin to apophycocyanin (11, 12) , and to the apophycoerythrin subunit (13) . With these apophycobiliproteins, spontaneous thioether bond formation does take place between the bilins and certain of the cysteinyl residues normally involved in bilin attachment. However, the products of in vitro addition differ from native phycobiliproteins. The predominant products are bilins, with an additional double-bond between the C-2 and C-3 carbons of ring A (11, 12, 13) . There is no discrimination in the addition of PCB and phycoerythrobilin at particular sites. Finally, addition takes place at some, but not all of the attachments sites. These results argue strongly against spontaneous bilin addition to apophycobiliproteins.

Studies of C-phycocyanin subunit phycocyanobilin lyase provide powerful support for the existence of multiple specific bilin lyases. The phycocyanin operon has been sequenced in several cyanobacteria (for references, see Ref. 14). This operon includes two genes, cpcE and cpcF. Insertional inactivation of either cpcE or cpcF in Synechococcus sp. PCC 7002 results in the same distinctive phenotype. These mutants produce a much lower amount of phycocyanin than the wild-type, and the phycocyanin that is produced lacks the PCB chromophore at -Cys-84 (15); however, the bilin-binding site is competent to react with PCB in vitro(16) . A minor fraction (20%) of the phycocyanin from the mutants does carry unnatural bilin adducts at the -84 site (16). The predominant adduct is mesobiliverdin (see Ref. 12). The two phycocyanin subunit PCB prosthetic groups are apparently unaffected in the mutants nor are the PCBs on allophycocyanin (16) . Direct biochemical studies of the catalytic activities of recombinant CpcE and CpcF decisively confirmed that these polypeptides together function as a specific phycocyanin subunit PCB lyase (17, 18) .

Analysis of a second-site revertant showed that a single amino acid substitution in the subunit of phycocyanin is sufficient to partially suppress the defect in PCB addition caused by the inactivation of cpcE(16) . Examination of the position of this substitution, -129-TyrCys, in the crystal structure of wild-type phycocyanin reveals that this residue is within 5 Å of the thioether linkage of the PCB to -Cys-84. This finding hints that the recognition of bilin-binding sites by bilin lyases involves three-dimensional features in the apopolypeptide and that the -129-TyrCys substitution may allow recognition of the -84 bilin attachment site by a protein bilin lyase specific for another site (16) .

In sum, the available information suggests that bilin addition at each site requires a specific lyase. In the organisms with phycobilisomes having the simplest phycobiliprotein composition, this would require eight such lyases; in those with the most complex phycobilisomes, 22 such enzymes might be needed (17) . However, this supposition rests solely on the studies of C-phycocyanin subunit PCB lyase referred to above. This paucity of information prompted us to search for other protein bilin lyases.

In addition to a single cpc operon that inter alia includes the C-phycocyanin -subunit PCB lyase genes cpcE and cpcF, Anabaena sp. PCC 7120 has an adjacent phycoerythrocyanin (pec) operon made up of five genes (19, 20, 21, 22) . PecB and pecA encode the and subunits of PEC, pecC encodes a linker polypeptide that functions in the assembly of PEC in the rod substructure, and pecE and pecF are genes of unknown function that show a high degree of homology to cpcE and cpcF (21, 22). We describe here the phenotypes and biochemical studies of pecE and pecF mutants. These studies support the inference that pecE and pecF encode a phycoerythrocyanin (PEC) subunit phycobiliviolin lyase.


EXPERIMENTAL PROCEDURES

Cultures and Strains

Anabaena sp. PCC 7120 was maintained in BG-11 medium (23) at room temperature with constant illumination at 40 µE m s. Plates of BG-11 contained washed Difco agar at 1%. One-liter cultures were constantly bubbled with 4% CO, 96% N and maintained at 30 °C. DH5 cells were used for transformation and plasmid maintenance and grown in Luria Broth medium with the appropriate antibiotics (kanamycin and streptomycin at 25 µg ml and ampicillin at 50 µg ml).

DNA Manipulations

Miniprep plasmid was isolated by the SDS-alkaline lysis method (24) . Larger scale plasmid preparations were prepared by a modified SDS-alkaline lysis method which included polyethylene glycol (average molecular weight 6,000) precipitation of the plasmid. Restriction endonucleases and other modifying enzymes were from Life Technologies, Inc. or New England Bio-Labs (Beverly, MA). DNA fragments were separated on agarose gels and eluted with Geneclean (Bio-101, La Jolla, CA). DH5 cells were transformed by the CaCl method or using the DNA transformation kit from Boehringer Mannheim. Southern analysis was employed on total DNA isolated from 10-ml cultures of the Anabaena strains as described by Cai and Wolk (25) . Southern blots were probed and visualized using the Luminescent Detection Kit (Genius 7, Boehringer Mannheim). In this method, the probe was made by random primed labeling of the DNA with a dNTP mixture that included digoxigenin-11-dUTP. The blot was incubated with the probe and then with anti-digoxigenin-alkaline phosphatase. Visualization was with Lumigen PPD chemiluminescent alkaline phosphatase substrate [4-methoxy-4-(3-phosphate-phenyl) spiro(1, 2-dioxetane-3, 2`-adamantane)].

Construction of Anabaena sp. PCC 7120 Mutant Strains

Plasmid pF500 (21, 22) containing the pec operon was digested with NheI and XhoI in order to excise pecB, pecA, and part of pecC. The ends were filled in with Klenow and the remaining plasmid containing the pBluescript KS vector (Stratagene, La Jolla, CA), part of pecC, pecE, and pecF was ligated to form pF500C`EF. PecE was interrupted with the C.S4 cassette (26) as follows. The C.S4 cassette, which confers resistance to streptomycin and spectinomycin, was excised with XbaI from pRL42 (26) . The cassette was partially filled in with dCTP and dTTP, and then ligated to a 4.5-kb fragment of pF500C`EF which had been partially digested with HindIII and then partially filled in with dATP and dGTP. A clone was isolated which contained the C.S4 cassette inserted into the HindIII site in pecE (Fig. 1A). The 4.5-kb fragment containing part of pecC, pecE interrupted with C.S4, and pecF was excised from pBluescript KS with XhoI and XbaI and ligated into pRL278 digested with XhoI and SpeI. The resulting plasmid was named pRL278:PECE. Plasmid pRL278 is a shuttle vector for transferring genes to Anabaena sp. PCC 7120 which contains the sacB gene (25, 27) . PecF was interrupted by the C.S4 cassette in a similar manner. C.S4 bounded by XbaI sites was ligated into pF500C`EF digested with SpeI (Fig. 1B). The 4.5-kb fragment containing part of pecC, pecE, and pecF interrupted with C.S4 was excised from pBluescript KSwith XhoI and XbaI and the resulting 4.5-kb fragment was ligated into pRL278 as described above and was named pRL278:PECF. The pecE and pecF genes were interrupted with the C.S4 cassette as follows. pF500C`EF was digested with HindIII, and the HindIII sites were partially filled in with dATP and dGTP. This deleted a 1.1-kb fragment (Fig. 1C) and replaced it with C.S4 bounded by XbaI sites and partially filled in with dCTP and dTTP. The 3.4-kb fragment containing part of pecC was excised with XhoI and XbaI and ligated into pRL278 as described above. This plasmid was designated as pRL278:PECEF (Fig. 1C).


Figure 1: Position of the interposon in the pecE and pecF mutant strains of Anabaena sp. PCC 7120. Shaded boxes indicate positions of the genes. Arrows indicate the direction of transcription. Selected restriction sites are indicated. A, the position of the interposon, C.S4, which interrupts pecE in the PECE strains. B, the position of the interposon, C.S4, which interrupts pecF in the PECF strains. C, the position of the interposon, C.S4, in strain PECEF-4 which replaces a portion of the pecE and pecF genes. The black boxes indicate portions of pecE and pecF used as probes for Southern analysis.



The interrupted gene(s) present in pRL278 were introduced into Anabaena sp.PCC 7120 by the triparental mating technique of Elhai and Wolk (28) . Strain J53(Rp4) was the conjugal strain, and HB101 cells containing the helper plasmid pRL528 were used in the mating. The cells were plated on 0.45-µm HATF filters (Millipore), placed on BG-11 agar plates for 2 days, and then transferred to plates containing spectinomycin and streptomycin each at 2.5 µg ml. After 2 weeks initial exconjugants appeared, several were grown in liquid BG-11 for 1 week and then plated on BG-11 plates containing 5% sucrose, spectinomycin, and streptomycin to select for potential double recombinants. After 2 weeks on this selective medium, colonies appeared which exhibited resistance to spectinomycin, streptomycin, and sucrose. Twenty of these colonies were replica-plated onto BG-11 plates containing 5% sucrose, spectinomycin, streptinomycin, and neomycin to confirm excision of pRL278 from the genome. Colonies unable to survive on this combination were then screened by Southern analysis. The mutant strains resulting from transformation with pRL278:PECE, pRL278:PECF, and pRL278:PECEF are referred to as PECE, PECF, and PECEF, respectively.

Whole Cell Difference Spectra (29

Two 1-ml aliquots of cells in mid to late log phase were taken. One sample was heated in an Eppendorf tube at 58 °C for 10 min and then immediately chilled on ice. The spectra of the untreated and heat-treated samples were taken using the frosted side of a quartz cuvette. To obtain the difference spectrum, the spectrum of the heat-treated sample was subtracted from that of the untreated one.

Quantitation of Chlorophyll and Phycocyanobilin

One ml of each strain at mid to late log phase was pelleted by centrifugation in a microfuge at 3,000 g for 5 min. One ml of 90% methanol was then added to the pellet, and the cells were sonicated two times for 5 min at 4 °C and then incubated in the dark for at least 30 min at 4 °C. The methanol extract was then centrifuged at full speed for 10 min in a microcentrifuge at 4 °C. The methanol supernatant was collected, and the reading at 663 nm was taken. The remaining pellet containing the phycobiliproteins was then resuspended in 0.9 ml of 9 M urea, pH 2.0, incubated for 15 min in the dark, at room temperature, with shaking and then centrifuged at full speed in a microcentrifuge for 10 min. The absorbance of the urea supernatants was measured at 665 nm. The concentration of chlorophyll was determined from the relationship Chl (µg ml) = OD 13.9 (30) ; an extinction coefficient of 35.4 mM cmat 665 nm was used for protein-bound PCB (31) .

Analysis of Phycobiliproteins from the Phycobilisomes

Phycobilisomes were isolated by the procedure of Yamanaka and Glazer (32) . Absorbance spectra were obtained with a Perkin-Elmer Lambda 6 UV/Vis spectrophotometer. Phycobiliprotein subunits were separated by high pressure liquid chromatography (HPLC) by the method of Swanson and Glazer (33) with a Waters 600E multisolvent delivery system and a Waters 991 photodiode array detector for data acquisition. Samples were collected as indicated, concentrated by evaporation with nitrogen gas, and aliquots subjected to amino-terminal sequencing on a gas-liquid phase sequencer (Perkin Elmer Applied Biosystems Division, Foster City, CA).

SDS-PAGE (34) was performed in a Bio-Rad Mini Protean II system using 14% acrylamide in the separating gel and 10% in the stacking gel and a monomer/bis ratio of 37.5:1. Two-dimensional gel electrophoresis was performed with the Bio-Rad Mini two-dimensional system. The first dimension was composed of 5% acrylamide (40% T, 5% C), 2% Triton X-100, 9.2 M urea, and 2% ampholytes (4:1 mixture of pH 5-7 and 3-10). The second dimension was a SDS-PAGE on a 14% separating gel and a 10% stacking gel. One-dimensional separations by horizontal isoelectric focusing were performed under the conditions given for vertical isoelectric focusing. These gels were then stained with Coomassie or 10 mM zinc acetate in 0.1 M glycine. The Coomassie-stained gels were scanned with an imaging system, and the intensity of the spots was calculated by integration with the program Scan Analysis (BioSoft; Cambridge, UK). Zinc-stained gels were scanned with a two-color, confocal fluorescence gel imaging system (35) , and the images were processed with the NIH image program Image 1.54 and IPLAB (Signal Analytics Corp; Vienna, VA). The fluorescence intensity of the spots was calculated by integration with Scan Analysis.


RESULTS

Interposon Mutagenesis of pecE and pecF

The pecE and pecF genes were individually interrupted with the C.S4 cassette at the sites shown in Fig. 1, A and B. A portion of the pecE and pecF genes was also deleted and replaced with the C.S4 cassette to inactivate both genes simultaneously (Fig. 1C). Initially, the ligation constructs were introduced into DH5 cells by electroporation. However, approximately 70% of the resulting transformants contained rearranged plasmids. Constructs in which the intervening sequence between pecE and pecF was deleted yielded a higher percentage (50%) of transformants with the correct plasmid. To avoid such complications, the ligation constructs were introduced by transformation of competent cells as this gave a higher percentage of transformants with the correct plasmid. The interrupted gene(s) were then ligated into the multiple cloning site in pRL278. As described in the ``Experimental Procedures,'' pRL278 contains the sacB gene which confers sucrose sensitivity as well as sequences which allow transfer of this plasmid into Anabaena sp. PCC 7120. Thus, interrupted genes ligated into pRL278 can be transferred to Anabaena sp. PCC 7120 for site-directed inactivation of the genomic copy and double recombinants are easily isolated by positive selection of sucrose-resistant clones.

Plasmids pRL278:PECE, pRL278:PECF, and pRL278:PECEF were introduced into Anabaena sp. PCC 7120 cells by the triple mating procedure of Elhai and Wolk (28) and double recombinants were selected as described under ``Experimental Procedures.'' These recombinants were subjected to Southern analysis (Fig. 2) in order to confirm the replacement of the genomic gene with the interrupted one. Two probes from the coding regions of pecE and pecF were used (Fig. 1C). Digestion with EcoRI yielded an 8 kb band from wild-type. Two bands from PECE-3, PECF-6, and PECEF-4 were detected due to internal EcoRI sites in the C.S4 cassette (Fig. 2A). A probe which contained the noncoding region between pecE and pecF was also used. Multiple bands were detected with this probe, indicating that the intervening sequence is present in multiple places in the genome (data not shown). The orientation of C.S4 was determined by digestion with HindIII (Fig. 2B), and mutant clones were selected in which the direction of transciption of the cassette was parallel with that of the gene. The mutants analyzed in this study are designated PECE-3, and PECE-5 for pecE mutants, PECF-6 and PECF-7 for pecF mutants, and PECEF-4 for the mutant in both pecE and pecF.


Figure 2: Southern blot of total DNA from wild-type and mutant strains to confirm isolation of double recombinants. Numbers to the left indicate the size of the DNA fragments in kilobase pairs. A, total DNA digested with EcoRI and probed with fragments from the coding regions of pecE and pecF (Fig. 1C). These probes hybridized to an 8-kb fragment in the wild-type, 4.5- and 3.3-kb fragments in strain PECE-3, two 4.5 kb bands in strain PECF-6, and 3.9- and 3.3-kb fragments for strain PECEF-4. B, total DNA digested with HindIII to confirm the direction of transcription of the C.S4 cassette. Probes used are the same as in A. These probes hybridized to 1.3- and 1.1-kb fragments in the wild-type, two 1.5-kb fragments in strain PECE-3, 2.2- and 1.1-kb fragments in strain PECF-6, and 3.7- and 1.5-kb fragments in strain PECEF-4.



Whole Cell Absorption Spectrum of Mutant Strains

The presence of PEC is revealed by a shoulder at 570 nm (due to the phycobiliviolin attached to the PEC subunit) in the wild-type absorption spectra of whole cells and phycobilisomes (36) . The contribution of the phycobiliproteins to the absorption spectrum of whole cells can be determined from a difference spectrum of untreated minus heated cells (29). Such difference spectra for the wild-type and mutant strains showed that the 570 nm shoulder was absent in all the mutant strains.

The growth rates of the mutant strains were compared to that of wild-type. Under limiting light conditions, the growth rate of the mutants was 30-50% lower than that of the wild-type, and the mutant strains had an average of 20% less PCB relative to chlorophyll a than wild-type (). The reduction in PCB content exceeds that expected for the loss of PEC alone and suggests a modest decrease in the mutants in the cell content of phycocyanin (and, possibly allophycocyanin) as well. For comparison, the cpcE and cpcF mutants of Synechococcus sp. PCC 7002 grew two times slower than wild-type (16) . Under high light, there was no significant difference between the growth rates of wild-type and mutant strains.

Characterization of the Phycobiliprotein Content of the Phycobilisomes

In phycobilisomes, PEC is present as a hexameric disc associated with a 34.5-kDa linker polypeptide encoded by the pecC gene (37, 38) . Whole cell difference spectra indicated a loss of PEC in strains PECE-3, PECF-6, and PECEF-4. To examine this further, we analyzed the phycobiliprotein composition and content in phycobilisomes from the mutant strains. The phycobilisomes were isolated on sucrose step gradients (32) and three major fractions were found at the 0.25, 0.5, and 0.75 M layers. Phycobilisomes isolated from wild-type and from strains PECE-3, PECE-5, PECF-6, PECF-7, and PECEF-4 sedimented primarily into the 0.75 M sucrose layer, with wild-type phycobilisomes sedimenting slightly further than those of the mutants. The shoulder at 570 nm that is present in wild-type phycobilisomes is absent in those from strains PECE-3, PECF-6, and PECEF-4. This indicates a loss of phycobiliviolin from the mutant phycobilisomes. When the absorption spectra are normalized at 650 nm the mutant strains exhibit a 22-25% decrease in the absorption at 615 nm indicative of a loss of C-phycocyanin as well as PEC, assuming that the content of allophycocyanin is unaffected. This observation correlates with the 20% decrease noted above in the PCB/chlorophyll a ratio in the mutants as compared to wild-type. The absorption spectra for the other two major fractions from the sucrose gradient were also examined. None of the slower sedimenting fractions from PECE-3, PECE-5, PECF-6, PECF-7, or PECEF-4 showed absorption spectra with a peak at 570 nm, which would indicate the presence of PEC or the PEC subunit.

SDS-PAGE of the wild-type, PECE-3, PECF-6, and PECEF-4 phycobilisomes is presented in Fig. 3. In this system the separation of the and subunits of PEC is incomplete, and PEC is seen as a fuzzy band located between the and subunits of C-phycocyanin. Coomassie staining shows a distinct reduction in the PEC polypeptides and the PEC linker polypeptide in phycobilisomes from strains PECE-3, PECF-6, and PECEF-4.


Figure 3: SDS-PAGE of the polypeptides of wild-type and mutant phycobilisomes. The abbreviations used are: WT, wild-type; PECE-3, pecE mutant; PECF-6, pecF mutant; PECEF-4, pecE and pecF mutant. Each lane was loaded with 25 µg of phycocyanin. Position of the PEC linker polypeptide (L), C-phycocyanin subunits (and ), and PEC are indicated at the left. Under these conditions the subunits of PEC are not well resolved.



The subunits of PEC are best resolved on two-dimensional gel separations, with isoelectric focusing in urea in the first dimension and SDS-PAGE in the second. Fig. 4A shows the separation of the phycobiliprotein polypeptides from wild-type phycobilisomes. In the PECEF-4 phycobilisomes, the quantities of the PEC and subunits were reduced relative to wild-type phycobilisomes (Fig. 4, A and B). The PECE-3 and PECF-6 phycobilisomes showed a decrease in the PEC subunit and absence of the PEC subunit (Fig. 4, C and D). Thus, the PEC subunit is still present in strain PECEF-4, but is not detectable by eye after Coomassie staining in strains PECE-3 and PECF-6. Coomassie-stained gels were scanned, and the densities of the PEC and subunits and the allophycocyanin subunit were obtained. The allophycocyanin subunit was used as the internal control and the densities of the PEC and subunits were expressed as a fraction of this subunit. In strains PECE-3 and PECF-6, 33 and 35% of the PEC subunit was present and 9 and 0% of the PEC subunit as compared to wild-type (A). In strain PECEF-4, the amounts of the PEC and subunits were 45 and 40%, respectively, relative to wild-type (A).


Figure 4: Two-dimensional separation of the polypeptides of wild-type and mutant phycobilisomes. For each phycobilisome sample, a load of 25 µg of phycocyanin was applied to the gel. Selected subunits are labeled in each panel. The abbreviations used are: ap, allophycocyanin subunit; cpc, C-phycocyanin subunit; cpc, C-phycocyanin subunit; pec, PEC subunit; pec, PEC subunit. Note the presence of a double spot for the PEC subunit in panel A.



To quantitate the and subunits on the basis of their bilin content, a duplicate set of gels was soaked in zinc acetate. Under these conditions polypeptides containing bilins fluoresce upon UV excitation. These gels were scanned to quantitate the fluorescence intensity of the polypeptide spots. For strains PECE-3 and PECF-6, respectively, 30 and 27% of the PEC subunit and 7 and 5% of the PEC subunit were present as compared to wild-type. In strain PECEF-4, 32 and 30% of the PEC and subunits were present relative to wild-type (see B).

The subunits of phycobiliproteins can be separated by HPLC on reverse-phase C-4 resin (33) . When phycobiliproteins from Anabaena sp.PCC 7120 are analyzed by this method, the PEC subunit elutes just before the C-phycocyanin subunit, but the subunits of PEC and C-phycocyanin coelute (33) . The wild-type and mutant strain phycobilisomes were analyzed with this chromatographic procedure. Chromatograms of these separations, monitored at 560 and 640 nm, are shown in Fig. 5, A and B. The wild-type phycobilisomes contain a major peak at 23.5 min which has a shoulder at 22.8 min (Fig. 5A). Spectroscopic analysis shows that C-phycocyanin subunit represents the major peak, and the PEC subunit gives rise to the shoulder. (Note that the spectrum at the center of the shoulder (Fig. 6A, 22.8 min) is characteristic of the PEC subunit.) The absorption spectrum at 22.8 min for the chromatogram of PECF-6 phycobilisomes showed that the phycobiliviolin-bearing subunit was absent (Fig. 6A). The chromatogram for PECE-3 phycobilisomes was virtually identical to that of PECF-6. Surprisingly, a new small peak was seen in the chromatogram monitored at 640 nm of PECEF-4 phycobilisomes, which also eluted at 22.8 min (Fig. 5, B and C) but had an absorption spectrum identical to that of C-phycocyanin subunit (Fig. 6B). To characterize this minor 640-nm peak from strain PECEF-4, samples from PECF-6, PECEF-4, and wild-type chromatograms (shown in Fig. 5A) which eluted between 22.5-23 min were collected. All three samples were subjected to amino-terminal sequencing. The sequences of the PECEF-4 and wild-type samples matched the sequence of the PEC subunit (I). The sample from PECF-6 gave no detectable sequence. Thus the 22.8 min peak from PECEF-4 has the absorption spectrum of C-phycocyanin subunit, but elutes at the position of the PEC subunit.


Figure 5: Separation of phycobiliprotein subunits from wild-type and mutant phycobilisomes. A, chromatogram of phycobilisomes from wild-type (-), PECF-6 (- - - -), and PECEF-4 (), monitored at 560 nm. The phycobilisomes were separated by HPLC on a C-4 reverse-phase resin, and 1.5 mg of phycocyanin was loaded for each type of phycobilisome. B, HPLC chromatogram monitored at 640 nm. Samples and conditions are the same as in A. C, HPLC chromatogram of wild-type and PECEF-4 phycobilisomes monitored at 640 nm. Sample load was 0.5 mg of phycocyanin each. At this lower load the minor peak containing the PCB-bearing PEC subunit at 23.7 min is resolved in the PECEF-4 phycobilisome sample. Note that at this lower load the polypeptides elute about 1 min later relative to the elution times seen in panel A.




Figure 6: Absorption spectra of early eluting components in the HPLC chromatograms of wild-type and mutant phycobilisomes. Separation was as described for Fig. 5 and under ``Experimental Procedures.'' Spectra were obtained with a diode array detector during subunit separation. A, the wild-type spectrum (-) at this time point (22.8 min; Fig. 5A) corresponds to that of the PEC subunit. The PECEF-4 spectrum (), corresponds to that of the C-phycocyanin subunit. No spectrum was detected from the PECF-6 (- - - -) sample. The sharp dip and spike at about 660 nm in the spectra obtained with the diode array detector are a feature of normal instrument operation. B, normalized absorption spectra acquired for the C-phycocyanin subunit (25 min; (-)) and for the component eluting at 23.7 min (- - - -) in the HPLC chromatogram of PECEF-4 (Fig. 5, panel C).



A portion of these samples was also analyzed by horizontal isoelectric focusing. A fluorescent band, which co-migrated with purified PEC subunit, was detected in the wild-type sample. Two bands were detected in the PECEF-4 sample, one which co-migrated with C-phycocyanin subunit and the other in between the C-phycocyanin and PEC subunits (Fig. 7). Both bands were also detected by Coomassie staining of the gel after it was scanned for the determination of fluorescence intensity.


Figure 7: Fluorescence scan of horizontal isoelectric focusing pattern of samples collected from HPLC chromatogram of wild-type and PECEF-4 phycobilisomes. PC, phycocyanin (2 µg); PEC, phycoerythrocyanin (1 µg); strain PECEF-4, component eluting from 22.5 to 23 min (Fig. 5B); and WT, wild-type, component eluting from 22.5 to 23 min (Fig. 5B).




DISCUSSION

Anabaena sp.PCC 7120 pecE and pecF genes are 47% identical at the amino acid sequence level to the cpcE and cpcF genes of the same organism (14) . Initial studies by Zhou et al.(15) using interposon mutagenesis showed that the homologous cpcE and cpcF genes from the cpc operon of Synechococcus sp. PCC 7002 are involved in the attachment of PCB to the C-phycocyanin subunit. The proof that these genes encode a heterodimeric phycocyanin subunit PCB lyase was provided by subsequent extensive biochemical studies and characterization of the CpcEF lyase (17, 18) . The function of the Anabaena sp.PCC 7120 pecE and pecF genes was investigated here by interposon mutagenesis.

Independently selected mutants in pecE (strains PECE-3 and PECE-5), or pecF (strains PECF-6 and PECF-7), all show loss of PEC and decrease of the PEC-associated linker polypeptide in phycobilisomes. The PEC subunit was barely detectable, and the PEC subunit was reduced to approximately 30% of wild-type level. Simultaneous deletion and interruption of the pecE and pecF genes (strain PECEF-4) also resulted in the loss of PEC and a reduction of the PEC linker polypeptide in phycobilisomes from this mutant. As in the pecE and pecF mutants, the PEC subunit was present at 33-40% of wild-type levels. In contrast, however, the PEC subunit was also present at 32-45% of wild-type level, but as an ``unnatural'' bilin adduct; the PEC subunit isolated from strain PECEF-4 contained PCB rather than phycobiliviolin and had a spectrum identical to that of C-phycocyanin subunit (Fig. 6B). In all three types of mutants, the spectra of isolated phycocyanin and allophycocyanin appeared normal (data not shown). Thus, the mutations in pecE and pecF had no apparent pleiotropic effects on the structure of other phycobiliproteins.

It is interesting to compare the consequences of mutations in cpcE and cpcF with those in pecE and pecF. Synechococcus sp. PCC 7002 mutants in cpcE and cpcF (or in both genes) show a much decreased level of phycocyanin (to 20% of wild-type), and this phycocyanin is modified. Whereas the subunit, with PCBs at -82 and -155, is normal, no wild-type phycocyanin holo- subunit is detectable. Phycocyanin subunit polypeptide in an amount similar to that of the is present in the mutants, but most of this subunit is present as the apopolypeptide, the rest (20%) carrying unnatural bilin adducts (16) characteristic of the in vitro addition of PCB to this apopolypeptide (11, 12) . A significant amount of the altered phycocyanin is associated with phycobilisomes. A parallel situation, but with some distinctive features, is seen in the Anabaena sp. PCC 7120 pecE and pecF mutants. Just as no wild-type C-phycocyanin is formed in the cpcE or cpcF mutants, no PEC is formed in the corresponding pec mutants. The presence in the phycobilisomes from the mutants of the PEC subunit at levels of 30% of wild-type, in the near absence of subunit polypeptide, suggests that in the absence of its normal partner, the PEC subunit can form hybrid monomers with the C-phycocyanin subunit, and that such monomers can be incorporated into phycobilisomes. This interpretation is plausible. The amino acid sequences of Anabaena sp.PCC 7120 C-phycocyanin and phycoerythrocyanin are 60% identical (19, 20, 21) . The three-dimensional structures of PEC and C-phycocyanin are likewise very similar as are the contacts at the / interface (5) . Glazer and Fang (39) showed that the and subunits of different C-phycocyanins, with primary structure divergence of up to 25%, formed stable hybrid monomers. Finally, substantial amounts of the PEC-associated linker polypeptide are present in the phycobilisomes from the pecE and pecF mutants.

A surprising difference is seen with respect to the PEC subunit in the PECEF mutant versus the PECE and PECF mutants. In the PECEF mutant, the subunit is present at a level similar to that of the PEC subunit (33-40% of wild-type); moreover, it carries the PCB chromophore. In the PECE and PECF mutants, the PEC subunit is either absent or present at barely detectable levels. What might account for this difference?

The average homology between the corresponding subunits of different C-phycocyanins is 74% while that between the corresponding and subunits of C-phycocyanins and PECs is 58 and 63%, respectively (14) . Hence, it is plausible that in the absence of PecE and PecF, the endogenous CpcEF lyase could attach PCB to the PEC apo- subunit. This would explain the high level of the PCB adduct of the PEC subunit in strain PECEF-4. It is unlikely that this PCB adduct is formed nonenzymatically. In vitro experiments have shown that the principal product of nonenzymatic addition of PCB to the C-phycocyanin (12) or to the C-phycoerythrin (13) apo- subunits is 3`-cysteinylmesobiliverdin, which has an absorption peak in 10 mM trifluoroacetic acid at 686 nm (12) . In 10 mM trifluoroacetic acid, the absorption spectrum of the PCB adduct of the PEC subunit peaks at 646 nm and is identical to that of the C-phycocyanin subunit. Consistent with the interpretation offered here, it is noteworthy that CpcEF from Synechococcus sp. PCC 7002 is able to use heterologous C-phycocyanin subunits as bilin donors in catalyzing the transfer of bilin from holo- subunit to apo- subunits (17) .

Phycocyanin subunit PCB lyase can add either PCB or phycoerythrobilin to the C-phycocyanin subunit. However, CpcEF has a 30-fold higher binding affinity for PCB and a turnover rate of 19 times that for PCB over phycoerythrobilin (18) . Thus the ratio of k/KPCB to k/K phycoerythrobilin is 570. This preference for PCB in binding affinity and in the rate of catalysis is sufficient to account for the selective attachment of PCB to the phycocyanin apo- subunit in the presence of phycoerythrobilin (18). The finding of a significant amount of PEC subunit carrying PCB in the PECEF mutant suggests that this is due to a similar selectivity (rather than absolute specificity) with regard to the polypeptide substrate in the lyase reaction. A testable hypothesis is that when the cognate lyase is missing, the phycocyanin subunit lyase ``rescues'' the PEC apo- subunit by adding PCB.

How is the near-absence of such a PCB adduct of the PEC subunit in the PECE and PECF mutants explained in the context of this hypothesis? When only PecE or PecF is present, either of these components (or a hybrid PecE/CpcF or PecF/CpcE lyase) may form a complex with the PEC apo- subunit which is thereby made unavailable to the CpcEF lyase. The formation of such a non-productive complex may also permit proteolytic degradation of the PEC apo- subunit. These possibilities will be tested in appropriately engineered Anabaena sp. PCC 7120 strains with deletions in cpcE and cpcF genes and ultimately by biochemical studies of the properties of recombinant PecE and PecF.

  
Table: Characteristics of Anabaena sp. PCC 7120 wild-type and mutant strains


  
Table: Quantitation of the amounts of PEC and subunits in phycobilisomes from wild-type and mutant strains of Anabaena sp. PCC 7120


  
Table: 1819897957p4in ND, not determined (no residue could be identified at this step).(119)


FOOTNOTES

*
This work was supported in part by National Institute of General Medical Sciences Grant GM28994 and by a grant from the Lucille P. Markey Charitable Trust. 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.

§
To whom correspondence should be addressed: MCB: Stanley/Donner ASU, 229 Stanley Hall 3206, University of California, Berkeley, CA 94720-3206. Tel.: 510-642-3126; Fax: 510-643-9290.

The abbreviations used are: PCB, phycocyanobilin; PEC, phycoerythrocyanin; PCC, Pasteur culture collection; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; kb, kilobase(s).


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