The cpcE and cpcF Genes of Synechococcus sp. PCC 7002 CONSTRUCTION AND PHENOTYPIC CHARACTERIZATION OF INTERPOSON MUTANTS*

The 3’ region of the cpc operon of Synechococcus sp. PCC 7002 has been sequenced, transcriptionally characterized, and analyzed by interposon mutagenesis. The cpc operon contains six genes, 5’ cpcB-cpcA-cpcC-cpcD-cpcE-cpF 3’, and gives rise to at least eight (more likely ten) discrete mRNA transcripts. The steady-state levels of transcripts for the cpcE and cpcF genes are very low and are estimated to represent only about 1-2% of the total transcripts arising from the cpc locus. The cpcE gene predicts a protein of 268 amino acid residues, whereas the cpcF gene predicts a protein of 205 amino acid residues. The deduced amino acid sequences of these proteins are about 50% identical and 70% similar to the predicted products of ho- mologous genes which have been identified in other cyanobacterial cpc operons. Interposon insertion mu- tations were constructed in the cpcE and cpcF genes, and an interposon deletion mutation affecting both genes was constructed. The phenotypes of all mutant strains were similar. These strains were yellow-green in color, had doubling times approximately twice that of the wild-type strain, and failed to accumulate normal levels of phycocyanin. Further analyses indicated that these strains contained substantial amounts of apparently normal phycocyanin B subunits; however the majority of the phycocyanin a subunit (about 90%) did not carry a phycocyanobilin

The 3' region of the cpc operon of Synechococcus sp. PCC 7002 has been sequenced, transcriptionally characterized, and analyzed by interposon mutagenesis. The cpc operon contains six genes, 5' cpcB-cpcA-cpcC-cpcD-cpcE-cpF 3', and gives rise to at least eight (more likely ten) discrete mRNA transcripts. The steady-state levels of transcripts for the cpcE and cpcF genes are very low and are estimated to represent only about 1-2% of the total transcripts arising from the cpc locus. The cpcE gene predicts a protein of 268 amino acid residues, whereas the cpcF gene predicts a protein of 205 amino acid residues. The deduced amino acid sequences of these proteins are about 50% identical and 70% similar to the predicted products of homologous genes which have been identified in other cyanobacterial cpc operons. Interposon insertion mutations were constructed in the cpcE and cpcF genes, and an interposon deletion mutation affecting both genes was constructed. The phenotypes of all mutant strains were similar. These strains were yellow-green in color, had doubling times approximately twice that of the wild-type strain, and failed to accumulate normal levels of phycocyanin. Further analyses indicated that these strains contained substantial amounts of apparently normal phycocyanin B subunits; however the majority of the phycocyanin a subunit (about 90%) did not carry a phycocyanobilin chromophore. During serial subculturing of the mutant strains, suppressor mutations, which allowed cells to regain the ability to synthesize phycocyanin, arose at significant frequency. Based upon the results reported here, as well as those presented in the accompanying paper (Swanson, R. V., Zhou, J., Leary, J. A., Williams, T., de Lorimier, R., Bryant, D. A., and Glazer, A. N. (1992) J. Biol. Chem. 267, 16146-16154), we propose that the CpcE and CpcF polypeptides are the two subunits of a phycocyanobilin lyase specifically required for chromophorylation of the phycocyanin a subunit.
Phycobilisomes serve as the major light-harvesting antennae for photosynthesis in cyanobacteria, red algae, cryptomonads, and phylogenetically ambiguous cyanelle-containing * This work was supported by National Institutes of Health Grant GM-31625 (to D. A. B.). The costs of Publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted M93569.
to the GenBankTM/EMBL Data Bank with accession number(s) $ T o whom correspondence and reprint requests should be addressed: s-231 Frear Bldg., Dept. of Molecular and Cell Biology, Penn %ate University, University Park, PA 16802. Tel.: 814-865-1992;Fax: 814-863-7024. flagellates such as Cyanophora paradona (for reviews, see Glazer, 1985Glazer, , 1987Glazer, , 1989Bryant, 1991). Phycobilisomes are composed of the brightly colored phycobiliproteins (PBP),' which account for approximately 85% of the protein in these structures, and a small number of typically nonchromophorylated linker polypeptides, which are responsible for directing the assembly of the phycobilisome and modulating the spectroscopic properties of the constituent PBPs. The PBPs have been extensively characterized biochemically, and the structures for three phycocyanins (PC; Schirmer et al., 1985Schirmer et al., ,1986Schirmer et al., , 1987Duerring et al., 1991) and a phycoerythrocyanin (Duerring et al., 1990) have been determined by x-ray crystallography. Protein sequences for all types of PBPs have been determined, and these and other studies have shown that PBPs are an homologous family of proteins derived from a single ancestral gene by gene duplication and divergence processes (see Zuber, 1987;Bryant, 1991).
Although much is known about the structure af PBPs and their chromophore contents (see Glazer, 1989), much less is known about how the phycobilin chromophores are synthesized and how these linear tetrapyrroles become attached to the PBP apoproteins. Beale and co-workers (1991aBeale and co-workers ( , 1991bBeale and co-workers ( , 1991c have recently proposed a biosynthetic pathway leading from heme to the presumed precursor of polypeptide-bound phycocyanobilin, 3(E)-phycocyanobilin, in the acidothermophilic red alga Cyanidium caldarium. In vitro studies performed with apoPC subunits demonstrated that a variety of linear tetrapyrroles could be added by simple chemical addition to the a-82 and p-84 positions, but no addition to the p-155 position was observed in these studies (Arciero et al., 1988a. The chemical addition of 3(E)-phycocyanobilin resulted in a mixture of products with differing stereochemistry and reduction states. These observations suggested that the chromophore attachment reactions must be enzymatic, much like the attachment of heme to apocytochromes c and cl.
In characterizing the cpc operon of Synechococcus sp. PCC 7002, two genes, denoted cpcE and cpcF, were identified which did not encode structural components of the phycobilisomes. Nonetheless, these two genes were cotranscribed with the other four genes of the operon (cpcB, cpcA, cpcc, and cpcD (see de Lorimier et al., 1984;de Lorimier et al., 1990ade Lorimier et al., , 1990b. Similar genes have been localized downstream from the genes encoding the subunits of PC in Anabaena sp. PCC 7120 (Belknap and Haselkorn, 1987), Culothrix sp. PCC 7601 (Mazel et ul., 1988), and Pseudunabaena sp. PCC 7409 (Dubbs, 1990 Bryant, unpublished observations). The thickness of the arrows indicates the relative abundance of the transcripts. The six shorter transcripts could be directly detected under appropriate electrophoretic conditions; two different mRNAs are also assumed to occur for the cpcBACDE and cpc-RACDEF transcript classes, although only mRNAs of a single size for each class could be directly observed after electrophoresis and hybridization (see Fig. 4). The position of the interposon in strain PR6230 (Swanson et al., 1992) which replaces the cpcB, cpcA, and a portion of the cpcC gene is shown. E , restriction map showing the positions of the aphll and erm gene interposons in strains PR6240, PR6250, and PR6280. C , the position of the aphII interposon in strain PR6054 and strain PR6260.

RESULTS
Nucleotide Sequence and Deduced Amino Acid Sequences for the cpcE and cpcF Genes-A physical map of the region surrounding the cpcBACDEF operon of Synechococcus sp. PCC 7002 is shown in Fig. 1A. In the present work the nucleotide sequence of the region extending from the HindIII site 3' to the cpcD gene to the right-most PstI site was Portions of this paper (including "Experimental Procedures" and Table 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. completely determined on both strands. The nucleotide sequence data obtained have been deposited in GenBank under the accession number M93569. The cpcE gene initiates 137 bp downstream from the cpcD gene (de Lorimier et al., 1990a) and predicts a protein of 268 amino acids (Fig. 2) with a predicted molecular mass of 29,172 Da and a calculated isoelectric point of 4.6. The predicted protein contains about 49% nonpolar amino acids, and secondary structure algorithms suggest that the protein should be largely composed of a-helical secondary structure. The start codon is preceded by a polypurine motif 5'-AAAGGAGAA-3' which strongly resembles typical prokaryotic ribosome-binding (Shine-Dalgarno) sites. The cpcF gene initiates 54 bp downstream from the cpcE gene and predicts a protein of 205 amino acids with a calculated molecular mass of 22,303 Da and a calculated isoelectric point of 5.4. The start codon is preceded only by the purine trinucleotide 5' GAA 3' which might play a role in ribosome binding. The protein contains 51% nonpolar amino acids and is also predicted to consist of predominantly a-    (Dubbs, 1990), Calothrk sp. PCC 7601 , and Anabaena sp. PCC 7120 (Belknap andHaselkorn, 1987, Bryant et al., 1991). Only residues which differ from those of the Synechococcus sp. PCC 7002 proteins are shown. Hyphens indicate insertions/deletions included t.o optimize the homology. The Anabaena sp. PCC 7120 CpcF sequence is not complete; the region not sequenced occurs between the slashes. helical segments. Two imperfect inverted repeat structures, which have the potential to form energetically favorable stemloop structures, occur downstream from the cpcF gene. One or both of these structure could play a role in transcription termination or mRNA stabilization. Analyses of the 2472-bp region downstream from the cpcF gene revealed several potential ORFs on both strands. The largest of these ORFs occur on the strand opposite the cpcBACDEF operon (see Fig.  1A). However, database searches revealed no significant homology to any of these potential ORFs. An insertion mutation constructed at the XhoI restriction site within the first ORF downstream from cpcF (see Fig. 1) did not appear to affect P B P levels in the cell and produced no other discernable phenotype (data not shown). Fig. 2 shows a comparison of the deduced CpcE protein sequences for Synechococcus sp. PCC 7002, Pseudanabaena sp. PCC 7409 (Dubbs, 1990), Calothrix sp. PCC 7601 , and Anabaena sp. PCC 7120 (Belknap and Haselkorn, 1987). The sequences are approximately 50% identical and 65-70% similar in sequence. Several regions of the predicted proteins are invariant, or nearly so, in sequence, and all of the CpcE proteins are similar in size (268-276 amino acids). Fig. 2 also shows a comparison of the CpcF proteins of the same cyanobacterial species. In constrast to the situation observed with CpcE, the sizes of the CpcF proteins are quite variable; Synechococcus sp. PCC 7002 and probably Anabaena sp. PCC 7120 have CpcF polypeptides of slightly more than 200 amino acids; however the CpcF proteins of Pseudanabaena sp. PCC 7409 and Calothrix sp. PCC 7601 are considerably larger (282 and 260 amino acids, re-spectively). Nonetheless, all of these sequences are homologous in their NH2-terminal regions and are about 53% identical and 70% similar overall.

MNEETGIPDDNLL I
Transcript Analyses for the cpcBACDEF Operon- Fig. 3 shows the results of Northern blot hybridization experiments using a variety of DNA fragments from the cpc operon as probes. In each panel, lane 1 contains total RNA isolated from cells grown in nitrogen-replete medium and lane 2 contains an equivalent amount of total RNA isolated from cells which had been starved for nitrogen for 5 h. When the 3.01kbp HindIII fragment encoding the cpcB, cpcA, and cpcC genes was used as the hybridization probe, at least eight transcripts could be detected (Fig. 3, A and B ) ; the levels of all transcripts were approximately 50-to 100-fold lower in cells starved for nitrogen. The smallest pair of transcripts, approximately 1350 and 1500 nucleotides in length, encode only the cpcB and cpcA genes and differ in length by 161 nucleotides at their 5' The pair of transcripts of approximately 2350 and 2500 nucleotides encode the cpcB, cpcA, and cpcC genes; when the 0.884-kbp XhoI-Hind111 fragment specific for cpcC was used as probe, only transcripts of this size were detected (Fig. 3C). Less abundant transcripts of approximately 2900, 3900, and 4600 nucleotides were also detected with the cpcBAC probe. The 2900-nt transcripts, which in some experiments could be resolved into two species, hybridized specifically to the 0.364-kbp HindIII fragment encoding cpcD (Fig. 30). This result indicates that these transcripts encode the cpcB, cpcA, cpcC, and cpcD genes. Hybridization with the 1.998-kbp HindIII-XhoI fragment en-  . 4). Fig. 4A shows a comparison of the hybridization patterns for strains PR6000 (wild type) and PR6240 (cpcE-).
-23 S ing the cpcE and cpcF genes (1.998-kbp HindIII-XhoI fragment, see Fig. lA) is 1.3 kbp larger than in the wild type as -16 s expected. After digestion with HindIII and BglII, two fragments of 1.6 and 3.27 kbp hybridized to this probe (Fig. 4A,  lane 4 ) since the interposon has a single BglII site. These results confirm the insertion of the aphll gene into the EcoRI site and indicate that the segregation of the mutant allele HindIII fragment hybridizing to a probe fragment encod- from the wild-type allele is complete (Fig. 4A, lanes 1 and 2).

b-1.46
).98 coding and cpcF (Fig* 3E) demonstrated that the exstrain PR6000 and lanes 2 and 4 contain total DNA isolated from the panel, lanes I and 3 contain"tota1 DNA isolated from the wild-type tremelY hv-dx~ndance transcripts OfaPProximatelY 3900 and mutant strain. The DNAs in lanes I and 2 were digested with HindIII, 4600 nucleotides encoded the cpcBACDE and cpcBACDEF genes, respectively. A smear of hybridization for this latter probe fragment to transcripts of approximately 1400-1600 nucleotides could indicate that a secondary promoter might exist somewhere in the 3' region of the cpc operon. Alternatively, primary transcripts encoding the cpcBACDEF genes might be endonucleolytically processed to produce low-abundance transcripts encoding only the cpcE and cpcF genes. Hybridization experiments with probe fragments derived from the region 3' to cpcF did not identify transcripts from this region (data not shown). Construction of Interposon Mutants in the cpcE and cpcF Genes-A previous study (Bryant et al., 1990) had shown that the phycobilisomes of Synechococcus sp. PCC 7002 did not contain the polypeptide products of the cpcE and cpcF genes. However, the results described above indicate that the cpcE and cpcF genes are cotranscribed with the cpcBACD genes, a result which suggested that these genes might encode proteins functionally related to PC. In order to reveal the function of the products of these two genes, insertional mutations within each of the genes and a deletion mutation inactivating both genes were constructed (see "Experimental Procedures"). The mutant constructions are shown diagramatically in Fig. 1, B and C. The constructions indicated were introduced into Synechococcus sp. PCC 7002 by transformation; homozygous and the DNAs in lanes 3 and 4 were digested with HindIII and BglII. The probe fragment employed in all cases was the 1.998-kbp HindIII-XhoI fragment encoding both the cpcE and cpcF genes (see Fig. 1). A , confirmation of strain PR6240. The probe hybridized to the 3.56kbp HindIII fragment in the wild-type ( l a n e I ) and a 4.88-kbp HindIII fragment in the mutant (lane 2). The 1.3-kbp increase in size is due to the insertion of the aphll gene in the EcoRI site of the cpcE gene (see Fig. 1R). Correspondingly, after digestion with HindIII and BglII, t.he snme rcsults arc obtained with the wild-t.ype strain, but two fragments (1.6 and 3.27 kbp) hybridize in the mutant DNA because of the presence of a single BglII site in the aphll interposon. B, confirmation of strain PR6260. The probe hybridized to a 3.56-kbp HindIII fragment in the wild-type strain PR6000 (lane I ) and hybridized to a 4.88-kbp HindIII fragment in the mutant (lane 2) due to the interposon inserted in the RclI site of the cpcF gene. After digestion with HindIII and BglII, hybridization of a single 3.56-kbp HindIII fragment was still observed in the wild-type strain (lane 3), whereas in the mutant strain two fragments of 3.4-and 1.46-kbp hybridized because of the introduction of the BglII site in the aphll interposon (see Fig. 1). C, confirmation of strain PR6260. In the wildtype strain, the probe hybridizes to two HindIII-Sal1 fragments of 0.98 and 2.58 kbp (lane 1 ), whereas in the mutant strain only a single fragment of 4.35 kbp hybridizes, since the Sal1 site was deleted during this construction (see Fig. 1). Digestion of the wild-type DNA with HindIII and BglII produced a single hybridizing fragment of 3.56 kbp, whereas digestion of strain PR6260 DNA with HindIII and BglII produced two hybridizing fragments of 2.74 and 1.6 kbp hybridize because of the introduction of the BglII site in the aphII interposon (see Fig. 1).

FIG. 5. Absorption spectra of whole cells and fractions ob-
tained from phycobilisome isolation. A, whole-cell absorption spectra for the wild-type strain PR6000 and the mutant strains PR6240, PR6250, and PR6260. R, the absorption spectra of fractions 1 obtained from the tops of sucrose gradients employed for the isolation of phycobilisomes from strains PR6240, PR6250, and PR6260 (see text for details). The spectra have been normalized at their absorption maxima (607 nm) to facilitate comparison. C, absorption spectra of phycobilisomes isolated from the wild-type strain PR6000 and the three mutant strains PR6240, PR6250, and PR6260. Similar results were also obtained with mutant strain PR6054 (data not shown). The three mutant spectra were normalized a t 670 nm, a wavelength a t which PC does not absorb, to facilitate their comparison. The wild-type spectrum has not been similarly normalized for this presentation.
Similar results were obtained with strains PR6250 and PR6260, as shown in Fig. 4, B and C, and for strain PR6054 (data not shown). In each case the hybridization experiments confirmed the complete segregation of the mutant allele from the wild type and the configurations shown in Fig. 1. Table l3 presents genotype and growth-rate data at high and moderate light intensities for strains constructed in these and related studies (see Swanson et al., 1992). Strains carrying mutations in either the cpcE or cpcF genes, or a deletion affecting both genes, were similar in appearance. All strains were yellow-green in color, suggesting that they do not accumulate normal levels of PC, and had doubling times which were approximately twice that of the wild type. Nonetheless, the relative growth rates were significantly faster than that of strain PR6230, which does not produce PC because the structural genes for its two subunits have been deleted (Swanson et al., 1992). As shown in Fig. 5, the whole-cell absorption spectra of the mutants confirmed that most of the 630-nm absorption due to PC was absent in the mutant strains. When phycobilisomes were isolated from the three mutant strains, two major blue-colored fractions, denoted fraction 1 and fraction 2, were observed on the sucrose gradients. Fraction 1 was found at the top of the sucrose gradient at the interface between the sample layer and the 0.25 M sucrose layer. This fraction for all three mutants had an absorption maximum at 607 nm (Fig. 5B); the spectrum was similar to that observed for the PC /3 subunit (Glazer et al., 1973). Fractions 2 for the three mutants were isolated from the 0.75 M sucrose zone, indicating that these phycobilisomes were significantly smaller than those of the wild-type strain, which are typically recovered from the interface of the 1.0 and 2.0 M sucrose layers. The absorption spectra of these fractions suggested the presence of a small amount of PC in the mutant cells. As shown in Fig. 5C, varying amounts of absorbance at 630 nm, characteristic of PC, could be observed (also see below) but did not exceed about 10% the level found in wild-type cells.

Properties of Strains Carrying Znterposon Mutations in the cpcE and cpcF Genes-
Further analyses of the gradient fractions by SDS-PAGE indicated that fractions 1 contained a blue-colored polypeptide which comigrated with authentic PC /3 subunit (Fig. 6).
In addition, the three fractions 1 contained a polypeptide which migrated slightly more rapidly than the wild-type PC CY subunit. The behavior of this polypeptide upon SDS-PAGE was identical to that observed for the apoPC CY subunit expressed in Escherichia coli (Bryant et al., 1985), and immunoblotting experiments with antibodies directed against the CY subunit of Synechococcus sp. PCC 6301 PC confirmed that this polypeptide was the PC CY subunit apoprotein (data not shown). This identification has also been confirmed by isolation and tryptic peptide mapping of the polypeptide (Swanson et al., 1992). A sensitive method for detecting polypeptides carrying linear tetrapyrrole chromophores takes advantage of the fluorescence of these polypeptides after the formation of the Zn-bilin chelate (Raps, 1990). No fluorescence could be detected from the apoPC CY subunit, but the fluorescence of the /3 subunit and the subunits of allophycocyanin appeared to be normal (Fig. 6A, lane 1 ). Interestingly, the phycobilisome-containing fractions 2 from all three mutants also contained some apoPC CY subunit. In addition, a small amount of a polypeptide comigrating with the chromophorylated form of the CY subunit could be detected (Fig. 6, B, lane 2, and C, lanes   2 and 4 ) .
During serial subculturing of the three mutant strains, the color phenotypes of the strains became noticeably more bluegreen than those of the original mutant strains. Whole-cell  1 and 2 contain fractions 1 and 2, respectively, from a phycobilisome preparation from strain PR6250, and lanes 3 and 4 contain fractions 1 and 2, respectively, from a phycobilisome preparation from strain PR6260. Lane 5 contains phycobilisomes isolated from the wild-type strain PR6000. Selected phycobilisome polypeptides are identified.
absorption spectra of such cultures indicated that the PC levels were substantially increased (compare Figs. 5A and  7 A ) . As much as 50% of the absorbance lost at 630 nm was recovered in some subculturing experiments (Fig. 7 A ) . Phycobilisomes isolated from such strains had substantially greater absorbance at 630 nm as well (Fig. 7 B ) . Low light intensity (~1 0 0 microeinsteins m-' s-') and elevated temperature (239 "C) favored increased PC content, whereas high light intensity (2200 microeinsteins m-' s") and lower temperature (24-28 "C) favored the original mutant phenotype (lower apparent PC content).

DISCUSSION
The cpc operon of Synechococcus sp. PCC 7002 is shown to contain six genes which are transcribed into a t least eight, but more likely ten (see Fig. 1A) transcript species. The steady-state levels of the transcripts which include the cpcE and cpcF genes, which occur at the 3' end of the operon, are quite low and are estimated to account for only about 1-2% of the total transcripts arising from this locus. Previous studies have demonstrated that the CpcE and CpcF products are not structural components of the phycobilisomes of Synechococcus sp. PCC 7002 (Bryant et al., 1990). The low steadystate levels of the transcripts encoding these proteins are consistent with this observation and suggest that these proteins are only required in catalytic amounts. The transcription patterns observed here differ from those reported for Calothrix sp. PCC 7601 (Maze1 et al., 1988) and Anabaena sp. PCC 7120 (Belknap and Haselkorn, 1987; also see Bryant et al., 1991).

FIG. 7. Absorption spectra of whole cells and fractions obtained from phycobilisome isolation.
A, whole-cell absorption spectra normalized at 681 nm of of mutant strains PR6240, PR6250, and PR6260 after many serial subculturings. B , phycobilisome fractions isolated from the serially subcultured mutant cells whose spectra are shown in A. The wild-type and mutant spectra were normalized at 650 nm to facilitate their comparison.
In Calothrix sp. PCC 7601, the cpcE gene is cotranscribed with the cpcB1 and cpcA1 genes, but the cpcF gene is transcribed as a monocistronic mRNA. Transcription of the cpc operon in Anabaena sp. PCC 7120 is more complex; a t least some transcripts occur in which cpcE and cpcF are cotranscribed with the cpcBACD structural genes as well as the cpcGlGZG3G4 genes (Belknap and Haselkorn, 1987;Bryant et al., 1991). At present it is not known how the complex family of transcripts observed for Synechococcus sp. PCC 7002 arises. Sequences capable of forming energetically stable stem-loop structures occur between the cpcA-cpcC, cpcC-cpcD, cpcD-cpcE, and cpcE-cpcF gene pairs as well as downstream from the cpcF gene, and such sequences could play a role in mRNA stabilization or transcription termination or both processes. Transcription might partially terminate after the cpcA, cpcC, cpcD, cpcE, and cpcF genes in a sequential and additive fashion such that the longest transcripts would therefore be least abundant. Alternatively, primary transcripts might initially encode all six genes but could be rapidly processed by endonucleolytic or exonucleolytic processing of the full-length primary transcripts to the shorter species. Transcripts stabilized by the largest number of secondary structures (i.e. those encoding the cpcB and cpcA genes) would thus be most abundant as observed. Additional experiments will be required to distinguish between these two possibilities.
The deduced amino acid sequences of the CpcE and CpcF proteins did not exhibit significant homology to other proteins in databases which could suggest their function, and a retrogenetics approach was thus adopted for examining their possible functions. The mutations in the cpcE gene reported here are the first mutations identified or constructed in this gene. However, Tandeau de Marsac and co-workers ) have previously isolated a spontaneous deletion mutant as well as mutants in which insertion elements (IS701 and IS703) inactivated the cpcF gene. These mutants are phenotypically similar to those reported here; they are defective in PC accumulation and have levels that are only about 10% those of the wild-type strain. Although it was initially proposed that these mutations affected the translation of the cpcBA mRNA, a reinvestigation of the properties of these mutants, based upon preliminary results from observations in Synechococcus sp. PCC 7002, revealed that these mutants are also defective in the chromophorylation of the PC a subunit (Tandeau de Marsac et al., 1990). Interestingly, the Calothrix sp. PCC 7601 mutants are also defective in their ability to transcribe the genes encoding phycoerythrin when the cells are grown in green light.
Insertional inactivation of either the Synechococcus sp. PCC 7002 cpcE or cpcF genes, or a deletion affecting both genes, produced strains with identical phenotypes. The yellow-green color phenotypes of these mutants suggested that the cells did not accumulate normal levels of PC. However, biochemical studies revealed that significant levels of normally chromophorylated PC ( 3 subunit were present in all three mutant strains (Swanson et al., 1992). However, only small amounts of chromophorylated PC a subunit along with substantial amounts of PC a apoprotein were found. All other PBPs appeared to be normally chromophorylated with phycocyanobilin; this indicates that these mutations did not affect phycocyanobilin synthesis, since phycocyanobilin is the only chromophore found in this cyanobacterium (Bryant et al., 1990). These observations strongly suggest that the CpcE and CpcF proteins are involved in the specific attachment of chromophores to the PC a subunit. Other gene products are apparently involved in chromophore attachment to other PBP subunits. It could be argued that only the CpcF polypeptide is required and that the phenotype of strain PR6240 strain arises from a polarity effect of the interposon insertion in the cpcE gene. However, the chromophorylation of the PC CY subunit is normal in mutant strains PR6009, PR6011, and PR6014 which have the same interposon inserted in the cpcC or cpcD genes which occur 5' to the cpcE and cpcF genes (see Fig. 1 and de Lorimier et al., 1990ade Lorimier et al., , 1990b. Hence, it is unlikely that the inability to chromophorylate the PC CY subunit in strain PR6240 is due to a polarity effect on the cpcF gene; therefore, it is probable that both the CpcE and CpcF product are specifically required for this process. The small amounts of chromophorylated PC 01 subunit detected in strains PR6240, PR6250, and PR6260 could arise from a spontaneous chemical reaction between phycocyanobilin and the apoPC CY subunit (Arciero et al., 1988a(Arciero et al., , 1988b. Alternatively, the apoPC 01 subunit could be a poor substrate for a chromophore attachment enzyme normally acting on another PBP substrate (e.g. an enzyme involved in the chromophore attachment to the PC p subunit or an allophycocyanin subunit). Studies presented in the accompanying manuscript (Swanson et al., 1992) suggest both of these processes can occur. The plasticity of the phenotypes of serial subcultures of these mutants is consistent with the notion that secondary mutations can arise in the cells which can partially or even completely restore the ability to chromophorylate the PC 01 subunit (Swanson et al., 1992).
In summary, results presented here and in the accompanying paper (Swanson et al., 1992) suggest that the CpcE and CpcF proteins form a lyase which specifically attaches phycocyanobilin to the PC 01 subunit. Recently, these proteins have been individually overproduced in E. coli5 The availability of large amounts of the CpcE and CpcF proteins allows in vitro experiments to test this hypothesis and possibly reveal details of the mechanism of this process.