First published online September 24, 2003; 10.1105/tpc.015016
The Plant Cell, Vol. 15, 2448-2463,
October 2003, Copyright © 2003,
American Society of Plant Biologists
Lesions in Phycoerythrin Chromophore Biosynthesis in Fremyella diplosiphon Reveal Coordinated Light Regulation of Apoprotein and Pigment Biosynthetic Enzyme Gene Expression
Richard M. Alveya,
Jonathan A. Kartyb,
Elicia Roosa,
James P. Reillyb and
David M. Kehoe1,a
a Department of Biology, Indiana University, Bloomington, Indiana 47405
b Department of Chemistry, Indiana University, Bloomington, Indiana 47405
1 To whom correspondence should be addressed. E-mail dkehoe{at}bio.indiana.edu; fax 812-855-6705
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ABSTRACT
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We have characterized the regulation of the expression of the pebAB operon, which encodes the enzymes required for phycoerythrobilin synthesis in the filamentous cyanobacterium Fremyella diplosiphon. The expression of the pebAB operon was found to be regulated during complementary chromatic adaptation, the system that controls the light responsiveness of genes that encode several light-harvesting proteins in F. diplosiphon. Our analyses of pebA mutants demonstrated that although the levels of phycoerythrin and its associated linker proteins decreased in the absence of phycoerythrobilin, there was no significant modulation of the expression of pebAB and the genes that encode phycoerythrin. Instead, regulation of the expression of these genes is coordinated at the level of RNA accumulation by the recently discovered activator CpeR.
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INTRODUCTION
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Photosynthetic organisms use light-harvesting antennae to maximize their use of ambient light for photosynthesis. Phycobilisomes (PBS), the light-harvesting structures used by cyanobacteria and red algae, are macromolecular structures composed primarily of two protein classes, chromophorylated proteins called phycobili- proteins and nonpigmented structural proteins called linkers (Tandeau de Marsac and Cohen-Bazire, 1977 ; Gantt, 1981; Glazer, 1985; Bryant, 1991; MacColl, 1998). The number, size, and composition of PBS within a cell are dynamic and responsive to many environmental parameters, including nutrient availability, light quantity, and light quality (Grossman et al., 2001). The syntheses of the different proteins that make up the PBS must be regulated tightly and also must be coordinated closely with the production of the chromophores that are attached to phycobili- proteins.
Decades of research have provided insight into the molecular mechanisms involved in the acclimation of PBS to light quality in cyanobacteria, a process known as chromatic adaptation (Gaidukov, 1903; Bogorad, 1975; Tandeau de Marsac, 1977; Grossman and Kehoe, 1997; Palenik, 2001). Although several types of chromatic adaptation exist, the best studied occurs in the filamentous species Fremyella diplosiphon (also called Calothrix sp strain PCC 7601) and is referred to as complementary chromatic adaptation (CCA). Action spectra have demonstrated that CCA in this species responds to green light (GL; 540 to 550 nm) and red light (RL; 650 to 660 nm) (Haury and Bogorad, 1977; Vogelmann and Scheibe, 1978). During growth in GL, the red-colored phycobiliprotein phycoerythrin (PE) and its associated linker proteins are produced and incorporated into PBS. When grown in RL, blue-colored phycocyanin (PC), called inducible phycocyanin (PCi), and its linkers are synthesized and added to PBS (reviewed by Kehoe and Grossman, 1994). Other components of the PBS, such as constitutive PC (PCc) and allophycocyanin, do not significantly change in abundance during CCA (Conley et al., 1986; Houmard et al., 1988a, 1988b; Mazel et al., 1988). PE maximally absorbs GL ( 560 nm) and PCi maximally absorbs RL (620 nm), which closely correspond to the maxima of the CCA action spectrum. Thus, CCA appears to allow this organism to maximize photosynthetic efficiency by spectrally tuning the absorption characteristics of its PBS to the predominant wavelength of ambient light (Campbell, 1996).
The expression of the genes that encode the  - and  -subunits of PE (cpeBA), the PE linkers (cpeCDE), and the - and -subunits of PCi and its associated linkers (cpcB2A2H2I2D2; abbreviated cpcB2A2) is regulated by light quality; where it has been examined, this regulation occurs primarily at the level of transcription (Conley et al., 1985, 1986; Mazel et al., 1986; Lomax et al., 1987; Oelmüller et al., 1988; Federspiel and Grossman, 1990; Federspiel and Scott, 1992). Genetic approaches have led to the isolation of several components of the regulatory system that controls CCA (Chiang et al., 1992a; Kehoe and Grossman, 1996, 1997; Cobley et al., 2002; Noubir et al., 2002; Seib and Kehoe, 2002). Three of these appear to be part of a complex phosphorelay system that includes a phytochrome-class sensor, RcaE, and two response regulators. Mutants lacking functional RcaE are black (FdBk mutants) regardless of the light conditions in which they are grown. This is the result of the accumulation of intermediate levels of PE and PCi (Kehoe and Grossman, 1996; K. Terauchi and D. Kehoe, unpublished data). Another regulatory component is CpeR, which has sequence similarity to PP2C-class protein phosphatases and is an activator of cpeBA but not cpeCDE expression (Cobley et al., 2002; Seib and Kehoe, 2002). cpeR appears to be cotranscribed with the cpeCDE operon and has been proposed to assist in coordinating the expression of cpeBA with cpeCDE (Cobley et al., 2002). Finally, there is also strong evidence that an additional CCA light-sensing system controls cpeBA and cpeCDE light responsiveness, although no components of this pathway have been identified (Kahn et al., 1997; Seib and Kehoe, 2002).
The - and -subunits of PE and PCi have covalently attached linear tetrapyrroles, or bilins, that absorb light in the visible region of the spectrum to collect energy for photosynthesis. In cyanobacteria, phycoerythrobilin (PEB) is attached to PE, whereas phycocyanobilin (PCB) is attached to PC (Ó 1963>Heocha, 1963; Ó Carra et al., 1964; Chapman et al., 1967; Cole et al., 1967; Crespi et al., 1967). The syntheses of PEB, PCB, and phytochromobilin (P B), the chromophore of plant phytochrome photoreceptors, are initiated by the conversion of heme to biliverdin IX (BV) by the heme oxygenases HY1 (in plants) and ho1 (in cyanobacteria) (Cornejo et al., 1998; Davis et al., 1999; Muramoto et al., 1999). In plants, the synthesis of PB is completed by the reduction of BV to 3Z-PB by HY2, which is a 3Z-PB synthase (Figure 1) (Kohchi et al., 2001). In cyanobacteria, PCB is produced by the reduction of BV to 3Z-PCB by 3Z-PCB:ferredoxin oxidoreductase, whereas PEB is synthesized from BV through a two-step process: the production of the intermediate 15,16-dihydrobiliverdin (DHBV) by 3Z-15,16-DHBV:ferredoxin oxidoreductase (PebA) and the subsequent conversion of DHBV to 3Z-PEB by 3Z-PEB:ferredoxin oxidoreductase (PebB) (Figure 1) (Cornejo and Beale, 1997; Frankenberg et al., 2001).
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Figure 1. Biosynthesis Pathways Leading to the Synthesis of PEB, PCB, and PB from BV.
Information regarding the bilin biosynthetic enzymes listed is provided in the text. Dashed arrow indicates step of the pathway that has been shown to exist in red algae but that has not yet been identified in cyanobacteria.
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Tetrapyrroles are increasingly being recognized as important plant signaling molecules. The tetrapyrrole biosynthetic intermediate Mg-protoporphyrin IX is a key molecule in chloroplast–nucleus communication in plants (Strand et al., 2003). Photosynthetic organisms may use heme to inhibit glutamyl-tRNA reductase and are known to coordinate chlorophyll synthesis with the production of chlorophyll binding proteins (Ryberg and Terry, 2002; reviewed by Vavilin and Vermaas, 2002; Cornah et al., 2003). In cyanobacteria, the influence of tetrapyrroles on protein accumulation is well documented. Numerous studies have shown that phycobiliproteins that do not attach bilins fail to accumulate, although there is no obvious effect on transcript accumulation levels (Anderson and Toole, 1998).
At present, little is known about the mechanisms by which genes that encode bilin biosynthetic enzymes, such as HY1, ho1, HY2, pcyA, pebA, and pebB, are regulated. In cyanobacteria, it is not clear if there is coordinated expression of these genes and cpeBA, cpeCDE, and cpcB2A2, and if so, how such regulation occurs. Is the absence of a specific chromophore a signal that regulates the expression of genes that encode the corresponding phycobiliproteins, or is coordination achieved through a shared transcriptional regulatory pathway? Here, we demonstrate that in F. diplosiphon, expression of the pebAB operon is upregulated by GL. We have determined that this occurs, at least in part, by the same mechanism that controls the GL induction of cpeBA during CCA. We also establish that the absence of PEB does not significantly affect cpeBA, cpeCDE, or pebAB expression. Thus, in F. diplosiphon, the coordination of pebAB and cpeBA expression appears to operate primarily through a shared transcriptional regulatory mechanism rather than through a feedback control system that senses low PEB levels.
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RESULTS
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A Turquoise Mutant Contains Two Mutations That Lead to the Loss of Detectable CCA, the Accumulation of holoPC, and the Absence of holoPE
The turquoise (FdTq) mutant FdTq26 was generated in a FdBk mutant background. The wild-type CCA phenotype, in which PE accumulated in GL and PC accumulated in RL (Figure 2A), was nearly abolished in FdBk cells, in which PE and PC levels were largely constant regardless of ambient light quality (Figure 2B). Pigment accumulation in FdTq26 differed dramatically from that in the FdBk mutant, with PC levels high and PE undetectable regardless of ambient light wavelength (Figure 2C). This phenotype was identical to that of the previously described FdTq1 mutant, which was unable to accumulate cpeBA RNA as a result of a lesion in cpeR (Cobley et al., 2002; Seib and Kehoe, 2002).
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Figure 2. Whole-Cell Absorption Spectra of F. diplosiphon Wild-Type and Mutant Lines Grown in GL or RL.
(A) Wild type.
(B) FdBk mutant.
(C) FdTq26 mutant.
(D) FdTq26 mutant transformed with pDK4 (containing rcaE).
(E) FdG13 mutant.
Solid lines indicate cells grown in GL, and dotted lines indicate cells grown in RL. The scan shown for each line is representative of at least three independent replications. Maximum PE and PC absorption peaks are indicated. Chlorophyll a absorption peaks are at 430 and 680 nm.
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The rcaE mutation and impairment of CCA in FdTq26 made it unclear whether its pigmentation phenotype resulted from an overaccumulation of PC, a lack of PE, or both. FdTq26 cells transformed with wild-type rcaE displayed normally regulated PC levels in GL and RL but failed to accumulate spectrally detectable PE (Figure 2D). Thus, the FdTq26 phenotype appeared to be the result of two separate mutations. The first, in rcaE, led to the accumulation of intermediate levels of PC in RL and GL, and the second resulted in the lack of holo-PE under both light conditions. Collectively, these mutations made the phenotype of the FdTq26 mutant similar to that of RL-grown wild-type cells. This second mutation was not in cpeR, cpeBA, cpeCDE, or cpeYZ, which encodes a putative PE lyase responsible for catalyzing the attachment of chromophore to PE apoprotein (Kahn et al., 1997) (data not shown).
Mutation of a Gene That Encodes a Bilin Reductase Causes the FdTq26 Mutant Phenotype
The mutation that led to the absence of detectable holo-PE in FdTq26 was identified by complementation using a plasmid library containing wild-type F. diplosiphon genomic DNA (Kehoe and Grossman, 1998). Approximately 6000 FdTq26 transformants were grown in white light under kanamycin selection and screened for the FdBk mutant phenotype. One such colony was isolated that, when removed from selective pressure, clearly reverted to the FdTq26 phenotype (data not shown). The complementing plasmid, pPLER2, was rescued and found to contain a 4.1-kb fragment of F. diplosiphon genomic DNA. Retransformation of FdTq26 with pPLER2 led to the restoration of the FdBk mutant phenotype (Figure 3A).
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Figure 3. Whole-Cell Absorption Spectra of FdTq26 and FdG13 Cells Transformed with Different Plasmids and Grown in GL or RL.
(A) and (D) FdTq26 (A) and FdG13 (D) transformed with pPLER2.
(B) and (E) FdTq26 (B) and FdG13 (E) transformed with pRA-4.
(C) and (F) FdTq26 (C) and FdG13 (F) transformed with pRA-45.
Solid lines indicate cells grown in GL, and dotted lines indicate cells grown in RL. The scan shown for each line is representative of at least three independent replications. Maximum PE and PC absorption peaks are indicated. Chlorophyll a absorption peaks are at 430 and 680 nm.
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Sequencing revealed that this genomic DNA fragment contained five major open reading frames (ORFs) (Figure 4A). ORF1 was most similar to a gene that encodes a hypothetical protein in Nostoc punctiforme. ORF2 was most closely related to ORF200 in the marine cyanobacterium Synechococcus sp WH 8020, which was located near the cpeBA and pebAB operons in that organism (Wilbanks and Glazer, 1993; Frankenberg et al., 2001). ORF3 was most similar to a gene that encodes a hypothetical protein in N. punctiforme. ORF4 and ORF5 encoded proteins with sequences closely related to bilin reductases (Frankenberg et al., 2001). The ORF4 product was most similar to cyanobacterial PebA proteins, and ORF5 was most closely related to cyanobacterial PebB proteins (Figures 4C and 4D). ORF4 and ORF5 were encoded on the same strand in the F. diplosiphon genome, and their positions relative to each other were similar to those of pebA and pebB genes found in other cyanobacterial species (see Discussion). The ORF4 and ORF5 proteins were most closely related to PebA and PebB in the filamentous cyanobacterium N. punctiforme (Figures 4C and 4D). F. diplosiphon ORF4 and N. punctiforme PebA were 81% identical and 89% similar over 243 amino acids, and ORF5 and PebB were 84% identical and 91% similar over 245 amino acids.
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Figure 4. The F. diplosiphon Genomic DNA Fragment within pPLER2 Contains Five Significant ORFs.
(A) Relative positions and orientations of the five ORFs. The total number of amino acids encoded by each ORF and the number of nucleotides between each ORF, or between the ORF and the end of the DNA fragment, are indicated. The translation start site used was at the first Met for each ORF except ORF5, for which the first Ile was used. The locations of the DNA insertions are indicated for FdG13 (open arrowhead) and FdTq26 (closed arrowhead).
(B) Regions of pPLER2 used to construct the subclones pRA-4 (top) and pRA-45 (bottom).
(C) Amino acid sequence of ORF4 and alignment with PebA sequences from two other cyanobacterial species. The locations of the lesions within the PebA sequences of FdG13 and FdTq26 are noted by the open and closed circles, respectively.
(D) Amino acid sequence of ORF5 and alignment with PebB sequences from two other cyanobacterial species.
In (C) and (D), dark gray blocks denote identical amino acid residues, and light gray blocks denote similar amino acid residues as follows: M = I = L = V, W = Y = F, N = Q, D = E, and K = R. N. punc., N. punctiforme; Syn 8020, Synechococcus sp WH 8020.
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The next most closely related PebA and PebB were in Synechococcus WH 8020 (40% identical and 58% similar over 232 amino acids [PebA] and 50% identical and 64% similar over 241 amino acids [PebB]). Despite the sequence divergence of PebA and PebB in F. diplosiphon and cyanobacterial species other than N. punctiforme, all residues >70% conserved in this family (Frankenberg et al., 2001) were present in F. diplosiphon ORF4 and ORF5. In addition, there were two principal areas of sequence conservation among the six sequences presented in Figures 4C and 4D. The first was a region of 60 amino acids starting nearly 60 residues from the N termini of the F. diplosiphon proteins, and the second was a region of 40 amino acids at the C termini of these proteins. Highly conserved residues within these conserved regions have been identified for this class of enzymes and may contain active-site residues (Frankenberg et al., 2001).
PCR amplification of this 4.1-kb region in the FdTq26 genome revealed an additional 2 kb of DNA that was not present in the wild-type genome at this location (data not shown). Further PCR amplification and sequencing analysis showed that the inserted DNA was within ORF4, located in the DNA corresponding to codon 86 of the protein (Figure 4C).
To determine if the lesion in ORF4 was responsible for the FdTq26 phenotype, two fragments were amplified by PCR from wild-type genomic DNA and cloned into the shuttle vector pPL2.7 (Chiang et al., 1992b) (Figure 4B). pRA-4 contained ORF4 and pRA-45 contained both ORF4 and ORF5. These plasmids were each transformed into FdTq26. Transformants were grown in RL and GL and assayed spectrophotometrically for the accumulation of holo-PE and the reestablishment of the FdBk mutant phenotype (the background from which FdTq26 was generated). FdTq26 cells transformed with pRA-4 accumulated relatively low levels of PE and tended only slightly toward the FdBk mutant phenotype in GL and RL (Figure 3B). Cells transformed with pRA-45 contained moderate levels of PE and showed a very weak FdBk mutant phenotype after growth in RL but were phenotypically identical to the FdBk mutant when grown in GL (Figure 3C). These results demonstrated that the insertion in ORF4 was responsible for the FdTq26 phenotype and strongly suggested that this insertion also had a polar effect on ORF5 expression that was more pronounced in RL than in GL.
HPLC–Mass Spectrometry Analysis Demonstrates That ORF4 Encodes PebA and ORF5 Encodes PebB
To determine whether ORF4 and ORF5 were actually pebA and pebB, free bilins (i.e., bilins not covalently attached to a peptide) were extracted from cells grown in GL and analyzed using HPLC–mass spectrometry (MS). As part of this effort, we isolated a new member of the green (FdG) mutant class (Cobley and Miranda, 1983; Bruns et al., 1989), FdG13, which was generated in a wild-type background and contained a DNA insertion in codon 69 of ORF4 (data not shown). FdG13 properly regulated PCi in RL (Figures 2E and 3D to 3F) but, like FdTq26, failed to accumulate holo-PE in GL (cf. Figures 2C and 2E). The absence of a lesion within rcaE in FdG13 resulted in higher levels of spectrally detectable PE in this mutant, compared with FdTq26, after transformation with pRA-45 and growth in GL (cf. Figures 3C and 3F). Also, like FdTq26, FdG13 mutant cells transformed with either pRA-4 or pRA-45 and grown in GL (Figures 3E and 3F) had a partially rescued phenotype, whereas transformation of FdG13 cells with pPLER2 completely rescued the phenotype (Figure 3D), suggesting a polar effect on ORF5 expression.
The four bilins used as standards were PEB, PCB, DHBV, and BV. All four gave strong, singly protonated pseudomolecular ions (M+H)+ that were within 0.1 D of their predicted monoisotopic masses (583.2 for BV, 585.3 for DHBV, and 587.3 for PCB and PEB). In all cases, the dominance of the pseudomolecular ion allowed the data to be analyzed using extracted ion chromatograms, in which the intensity at any time point represents the sum of the ion current from mass-to-charge ratio 583.0 to 584.0, 585.0 to 586.0, and 587.0 to 588.0. The chromatogram obtained from the injection of PEB, PCB, DHBV, and BV is presented in Figure 5A (top). The elution profiles were very similar to those reported previously (Frankenberg et al., 2001), and the mass spectra (Figure 5A, bottom) show the dominance of each bilin's pseudomolecular ion. HPLC-MS analysis of free bilin extracts from FdG13 cells grown in GL revealed no detectable PEB or DHBV, but a small amount of BV and a larger amount of PCB were present, as determined by elution time (Figure 5B, top), mass spectra (Figure 5B, bottom), and UV/visible light spectra (data not shown).
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Figure 5. HPLC-MS Analyses of Free Bilins Present within F. diplosiphon.
Extracted ion chromatograms (top) and mass spectra (bottom) of bilin standards (A), the FdG13 mutant (B), the FdG13 mutant transformed with pRA-4 (C), and the FdG13 mutant transformed with pRA-45 (D). In the top panels, the x axis indicates retention time and the y axis indicates ion current sums (in arbitrary units) from mass/charge ratios of 583.0 to 584.0, 585.0 to 586.0, and 587.0 to 588.0; in the bottom panels, integrated mass spectra are provided. Retention times for the labeled peaks are as follows: for FdG13, + = 11.5 min,  = 13.8 min, and  = 16.5 min; for FdG13/pRA-4,  = 8.2 min and # = 9.1 min; for FdG13/pRA-45, * = 8.3 min. Data shown are representative of at least three independent experiments (including transformations, as applicable) for each sample.
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We predicted that if ORF4 encoded PebA, extracts from GL-grown FdG13 transformed with pRA-4 would contain DHBV as a result of pebA expression from the introduced plasmid and reduced pebB expression as a result of the polar effect from the pebA insertion (Figure 4A). We found that FdG13 cells transformed with pRA-4 did contain both free DHBV and PEB but no detectable free PCB or BV (Figure 5C). We also expected that if ORF5 encoded PebB, transformation of FdG13 with pRA-45 and growth in GL would lead to the production of equal amounts of PebA and PebB and result in the accumulation of free PEB but not DHBV or BV, which was the result we obtained (Figure 5D). It is noteworthy that of the lines examined here, only the FdG13 mutant accumulated any detectable BV. It also produced an unidentified species with a retention time of 13.8 min and a mass of 585.3 (Figure 5B) with a UV/visible light absorption spectrum similar to that of BV (data not shown).
pebAB Expression Is Responsive to RL and GL and Requires CpeR
The cpeBA operon is transcriptionally regulated by GL and RL in F. diplosiphon (Oelmüller et al., 1988). This, together with the fact that FdTq26 transformed with pRA-45 was complemented more completely to the FdBk mutant phenotype when grown in GL than in RL (Figure 3C), led us to investigate whether pebAB transcript accumulation also was regulated by GL and RL. RNA gel blot analyses were conducted initially with probe 1, which covered most of the pebA gene and the 5' flanking region (Figure 6A). This probe detected multiple RNA species ranging from slightly >3.0 kb to 0.6 kb, including an RNA of 1.6 kb, that were abundant in cells grown in GL and nearly undetectable in RL-grown wild-type cells (Figure 6B). The same RNA patterns were detected in the FdBk mutant, although the differential regulation by both GL and RL was reduced relative to that in the wild type (Figure 6B). In FdTq26 cells, two RNAs of 0.4 and 0.6 kb were detected, and these were expressed in both GL and RL at slightly below the levels of the RNAs in FdBk mutant cells (Figure 6B). For all three mutants, the transcripts appeared to be unstable and partially degraded, despite the fact that rRNAs (Figure 6) and cpeBA, cpeCDE, and cpcB2A2 transcripts (data not shown) on the same blots showed no signs of degradation.
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Figure 6. RNA Gel Blot Analyses Demonstrate That the Expression of pebAB Is Light Regulated.
(A) Map showing the coverage (thick lines) of each of the three probes (1, 2, and 3) used in (B) to (E). The approximate location of the DNA insertion within pebA in FdTq26 is denoted by the black arrowhead.
(B) Autoradiogram of a blot after hybridization of probe 1 to RNA isolated from RL- and GL-grown wild-type (WT), FdBk mutant (FDBK), and FdTq26 (TQ26) cells.
(C) Autoradiogram of a blot after hybridization of probe 2 to RNA isolated as in (B).
(D) Autoradiogram of a blot after hybridization of probe 3 to RNA isolated as in (B).
(E) Autoradiogram of a blot after hybridization of probe 1 to RNA isolated from RL- and GL-grown FdTq1 (cpeR-) and FdTq31 (cpeA-) cells.
The autoradiograms shown for each sample are representative of three independent experiments. Approximate sizes (in kb) are provided at left, and an autoradiogram of each blot after hybridization to a ribosomal probe is provided below each gel. The 1.6-kb RNA species is marked with an asterisk.
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We hypothesized that the shorter RNA detected by probe 1 in FdTq26 resulted from premature termination of transcription caused by the insertion in pebA (Figure 6A). This notion was tested by conducting RNA gel blot analyses with probe 2, which extended from the 3' end of the inserted DNA to the 3' end of pebA (Figure 6A). For wild-type and FdBk mutant cells, the results obtained were essentially identical to those obtained using probe 1 (cf. Figures 6B and 6C). However, there was no detectable hybridization of probe 2 to RNA from FdTq26 (Figure 6C). Thus, although the 5' end of pebA is transcribed in FdTq26, the 3' end does not appear to be, suggesting that transcription termination occurs within the inserted DNA located in pebA.
The 1.6-kb RNA detected by probe 1 in wild-type and FdBk mutant cells was the correct size to encompass either ORF3 and pebA or pebA and pebB, whereas the larger RNA species were capable of encoding all three. The question of whether or not ORF3 was cotranscribed with pebA and pebB was resolved by RNA gel blot analyses using probe 3 to examine the ORF3 transcript size and expression pattern (Figure 6A). An RNA between 500 and 550 bp in length was detected with this probe in wild-type, FdBk, and FdTq26 cells in both GL and RL (Figure 6D), demonstrating that ORF3 is not cotranscribed significantly with pebA and pebB. Collectively, the data in Figure 6 demonstrate that in F. diplosiphon wild-type cells, pebA must be cotranscribed with pebB and the expression of this operon is highly upregulated by GL.
In F. diplosiphon, both cpeBA and cpeCDE are induced by GL (Mazel et al., 1986; Oelmüller et al., 1988; Federspiel and Grossman, 1990; Federspiel and Scott, 1992). However, recent studies have shown that the regulation of cpeBA expression requires the activator CpeR, whereas cpeCDE expression does not (Cobley et al., 2002; Seib and Kehoe, 2002). In a cpeR mutant background, cpeBA RNA is undetectable in GL, whereas cpeCDE RNA still accumulates. The mechanism of action of CpeR is unknown. It has limited sequence similarity to members of the PP2C class of protein phosphatases (Seib and Kehoe, 2002), and similar proteins are present in other cyanobacterial species that produce PE (Cobley et al., 2002). To determine if pebAB expression was regulated by CpeR, we used RNA gel blots to examine its expression in FdTq1, a mutant that lacks functional CpeR (Seib and Kehoe, 2002). pebAB RNA failed to accumulate in GL or RL in FdTq1 (Figure 6E). This requirement for CpeR suggested a shared regulatory mechanism for pebAB and cpeBA expression.
We also wished to determine whether pebAB RNA levels were influenced by the absence of cpeBA RNA and PE. Thus, pebAB RNA levels were examined in FdTq31, a mutant that fails to accumulate spectrally detectable PE in GL and contains insertions in both rcaE and cpeBA (R.M. Alvey and D.M. Kehoe, unpublished data). RNA accumulation patterns for pebAB were similar in FdTq31 (Figure 6E) and the mutant class from which it was derived, the FdBk mutant (Figure 6B). This finding demonstrated that pebAB expression was not affected by the lack of normal accumulation of cpeBA RNA or PE itself. It also eliminated the possibility that the lack of pebAB expression in FdTq1 was an indirect effect of the failure to accumulate either cpeBA RNA or PE.
The requirement of CpeR for the expression of both pebAB and cpeBA suggested that these operons might be regulated transcriptionally using a common mechanism. If, as for cpeBA, the light regulation of the pebAB operon occurs transcriptionally, then corresponding cis-acting control elements might be expected to reside in the region upstream of pebA. Therefore, we examined the regions upstream of these operons for shared sequence elements that might regulate their activity. We first analyzed the cpeBA promoter regions from several chromatically adapting species for shared sequence motifs (Figure 7A). Several areas of strong sequence conservation were found in the proximal 70 bp of the promoters in N. punctiforme, Pseudanabaena sp PCC 7409, and F. diplosiphon (Figure 7A). These results confirm and extend the findings of previous studies (Anderson and Grossman, 1990; Dubbs and Bryant, 1991; Sobczyk et al., 1993). Within this region, three subregions were identified: the most highly conserved was subregion 1 of the cpeBA promoter, which was identical at 24 of 29 bases with the region from -94 through -66 (relative to the putative translation start site) of pebAB (Figure 7B). The two additional subregions of similarity in the cpeBA promoters were subregion 2 (identical at 5 of 7 bp with -63 through -57 of pebAB) and subregion 3 (identical at 11 of 22 bp with -47 through -26 of pebAB). Although subregion 3 was highly conserved between cpeBA promoters (Figure 7A), it was the least conserved between pebAB promoter regions from N. punctiforme and F. diplosiphon (Figure 7C).
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Figure 7. Promoters of the pebAB and cpeBA Operons Contain Regions of Conserved Sequence.
(A) Sequence comparison of cpeBA promoter elements from three cyanobacterial species.
(B) Sequence comparison of the pebAB and cpeBA promoter elements from F. diplosiphon.
(C) Sequence comparison of the pebAB promoters from two cyanobacterial species.
Shaded positions denote shared sequence identity, and boxed sequences encompass the pentanucleotide direct repeat. F. dip., F. diplosiphon; Nostoc, N. punctiforme; Pseud., Pseudanabaena sp PCC 7409. Fractions in parentheses indicate comparisons between species of the numbers of identical nucleotides divided by the total number of nucleotides for that subregion (indicated at bottom): F, F. diplosiphon; N, N. punctiforme; P. Pseudanabeana. Distances from the Pseudanabaena sp PCC 7409 cpeBA transcription start site (Dubbs and Bryant, 1991) are shown above the sequences, and binding sites for the DNA binding proteins RcaA (solid line) (Sobczyk et al., 1993) and PepB (dashed line) (Schmidt-Goff and Federspiel, 1993) are shown below the sequences.
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Expression Patterns of Several Major Phycobiliprotein Genes Are Not Altered Extensively in Cells Lacking PEB
RNA gel blots were used to determine if the absence of PEB in FdTq26 and FdG13 affected the expression patterns of cpeBA, cpeCDE, and cpcB2A2. Representative autoradiograms for these three operons and a ribosomal loading control in the wild type, the FdBk mutant, FdTq26, and FdG13 cells are shown in Figure 8A, and histograms of data from at least three independent experiments, normalized to ribosomal values, are shown in Figures 8B to 8D. In the wild type, cpeBA RNA levels were 20 times higher in GL-grown cells compared with RL-grown cells, whereas the FdBk mutant retained only 2.4-fold higher cpeBA RNA levels in GL- versus RL-grown cells (Figure 8B). The decrease in responsiveness in the FdBk mutant was attributable to increased levels in RL and slightly reduced levels in GL relative to the wild type.
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Figure 8. RNA Gel Blot Analyses of cpeBA, cpeCDE, and cpcB2A2 Expression in RL- and GL-Grown Wild-Type, FdBk Mutant, FdTq26, and FdG13 Cells.
(A) Representative autoradiograms of the data presented in (B) through (D) showing hybridization to the probes listed at left. WT, wild type.
(B) and (C) Mean values from at least three independent experiments of cpeBA (B) or cpeCDE (C) RNA levels in these four lines expressed as a percentage of the wild-type GL value, which was set to 100%.
(D) Mean values from at least three independent experiments of cpcB2A2 RNA levels in these four lines expressed as a percentage of the wild-type RL value, which was set to 100%.
In (B) to (D), standard errors are shown, and selected P values are provided in the text. All measurements were normalized using relative ribosomal hybridization values before calculation of the means.
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In FdTq26, cpeBA RNA levels also were less highly induced by GL (1.8 fold) than in the wild type. However, overall, there appeared to be more cpeBA RNA present in FdTq26 than in the FdBk mutant in both GL and RL, although these differences were not statistically significant (GL, P = 0.21; RL, P = 0.18) (Figure 8B). For cpeCDE expression, neither the reduced degree of GL-RL responsiveness in the FdBk mutant nor the overall lower levels measured relative to wild-type cells was significantly different from that of FdTq26 in either GL (P = 0.53) or RL (P = 0.81) (Figure 8C). Finally, the FdBk mutant and FdTq26 showed an equivalent loss of CCA responsiveness for expression of the cpcB2A2 operon (Figure 8D). In FdG13 cells, cpeBA RNA levels were the same as in the wild type during growth in RL and were 1.7 times higher in FdG13 than in wild-type cells during growth in GL (Figure 8B). cpeCDE RNA levels in RL-grown FdG13 and wild-type cells were essentially the same and were 60% of wild-type levels during growth in GL (Figure 8C), mirroring the pattern observed for FdTq26 (Figure 8C). Finally, cpcB2A2 RNA levels in FdG13 during both RL and GL growth were similar to those measured for wild-type cells under the same conditions (Figure 8D). Overall, the absence of PEB in both FdTq26 and FdG13 had no major effects on RNA accumulation patterns for these three operons.
Both apoPE and PE Linker Peptides Fail to Accumulate in the Absence of PEB
The accumulation of cpeBA RNA in cells that failed to produce PEB, such as FdTq26 and FdG13 (Figure 8B), raised the possibility that apoPE also accumulated in these mutants. Therefore, we isolated total protein from wild-type, FdBk mutant, and FdTq26 cells grown in GL and RL. We used whole-cell extracts instead of purified PBS to examine the entire cellular pool of phycobiliproteins rather than only those incorporated into PBS. Protein samples were separated by SDS-PAGE, and gels were examined for the presence of PE, PE, and the linker peptides CpeC, CpeD, and CpeE. As demonstrated previously for this species, PE and the PE linkers were present in wild-type cells at low levels in RL but were very abundant in GL (PE could not be resolved from PCc) (Figure 9) (Bryant, 1981; Lomax et al., 1987; Federspiel and Grossman, 1990; Federspiel and Scott, 1992). These proteins also were present at moderate levels in FdBk mutant cells in both GL and RL (Figure 9), which paralleled the partial accumulation of cpeBA and cpeCDE RNA (Figures 8B and 8C) and spectroscopically detectable PE (Figure 2B) under these light conditions. In FdTq26, however, PE, CpeC, CpeD, and CpeE were undetectable in both GL and RL (Figure 9). Therefore, although cpeBA and cpeCDE transcripts accumulated to relatively high levels in this mutant (Figures 8B and 8C), the corresponding proteins either were not made or were degraded rapidly after synthesis as a result of the absence of PEB.
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Figure 9. SDS-PAGE of Total Protein Extracted from Wild-Type, FdBk Mutant, and FdTq26 Cells Grown in RL or GL.
Five micrograms of purified PBS proteins extracted from wild-type cells grown in RL or GL was used as a control. Components of the PBS are labeled as identified previously (Bryant, 1981; Federspiel and Grossman, 1990). WT, wild type.
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DISCUSSION
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This investigation has identified and characterized two F. diplosiphon mutants that fail to produce PEB as a result of a lesion in pebA. pebA was found to be cotranscribed with pebB. The regulation of pebAB expression and the effects of the absence of PEB on cpeBA and cpeCDE expression were analyzed to explore the mechanism(s) by which the synthesis of PE, PE linkers, and PEB are coordinated. We determined that, as for cpeBA, pebAB expression was regulated by GL and RL and required CpeR. In addition, the pebAB promoter contained a sequence that was similar to a 69-bp region in the cpeBA promoter that was determined previously to contain a protein binding site (Schmidt-Goff and Federspiel, 1993; Sobczyk et al., 1993). Our results have revealed the fourth CCA-responsive operon in this species and suggest that coordinated regulation of cpeBA and pebAB expression occurs, at least in part, via a transcriptional mechanism. In addition, during growth in GL, these mutants continued to accumulate high levels of cpeBA and cpeCDE RNAs but failed to accumulate normal levels of PE or PE linkers. This finding indicates that feedback mechanisms exist to reduce PE and PE linker levels in the absence of PEB but not to significantly control cpeBA and cpeCDE RNA levels. pebAB RNA also accumulated normally in a cpeBA mutant, demonstrating that no strong feedback mechanism operated to reduce pebAB expression in the absence of PE.
In Vivo Dynamics of the PEB Biosynthetic Pathway
The data presented here demonstrate that both the FdTq26 and FdG13 mutants contained genetic lesions in pebA and that this gene was immediately upstream of and cotranscribed with pebB. In both mutants, the insertion within pebA appeared to have a strong, albeit partial, polar effect on pebB expression (Figures 3B, 3C, 3E, and 3F). Alternatively, the polar effect of the pebA insertion on pebB could be complete and pebB could be expressed at a low level from its own promoter. In multiple independent experiments, pPLER2 also complemented both of these mutants more completely than either pRA-4 or pRA-45 (Figure 3). This finding suggests that either pebA and pebB were more highly expressed from pPLER2 than from pRA-4 and pRA-45 or the spatial separation of the synthesis of PebA/PebB and the proteins encoded by ORF1, ORF2, or ORF3 decreased the amount of functional PebA and PebB produced. We favor the latter possibility, because the entire promoter region between ORF3 and pebA was included in the pRA-4 and pRA-45 plasmids, there was no measurable coexpression of ORF3 and pebAB (Figure 6), and there is no obvious reason why pebAB would be more highly expressed from pPLER2 than from pRA-4 and pRA-45.
Our HPLC-MS data (Figure 5) demonstrate that ORF4 is pebA and ORF5 is pebB and explain the absence of holo-PE in the FdTq26 and FdG13 mutants. They also provide in vivo support for the two-step biosynthetic pathway from BV to PEB in cyanobacteria (Cornejo and Beale, 1997; Frankenberg et al., 2001). Furthermore, the accumulation of PCB in FdG13 cells (Figure 5B) demonstrates that cyanobacteria are capable of synthesizing PCB without a PEB intermediate, as occurs in red algae (Figure 1) (Beale and Cornejo, 1991a).
Previous studies conducted with cell extracts or in vitro led to the proposal that the conversion of DHBV to PEB by PebB is not the rate-limiting step in the pathway from BV to PEB (Beale and Cornejo, 1991a, 1991b; Rhie and Beale, 1992; Frankenberg et al., 2001). The presence of PEB and the absence of DHBV in FdG13 cells transformed with pRA-45 (Figure 5D) provides in vivo data to support this hypothesis. In addition, the accumulation of DHBV, but not BV, in FdG13 cells transformed with pRA-4 (Figure 5C) suggests that the reduction of BV to DHBV by PebA in vivo is not reversible to any measurable extent. The fact that FdG13 cells contained free PCB and BV during growth in GL (Figure 5B) implies that blocking the pathway from BV to PEB under these growth conditions leads to an accumulation of BV and spillover into the PCB biosynthetic pathway. The absence of measurable BV in FdG13 transformed with pRA-45 (Figure 5D) demonstrates that during growth in GL, BV does not accumulate if the cells are capable of synthesizing PEB. High BV levels might not be tolerated by these cells for several reasons. Excess BV could attach to both PE and PC, as has been shown to occur in vitro (Arciero et al., 1988; Fairchild and Glazer, 1994), although we did not observe any shifts in the absorption maxima for FdG13 (Figure 2E) despite the accumulation of measurable free BV (Figure 5B). Alternatively, the BV level could normally be kept low to regulate the production of bilirubin, which then could either control the flux of BV into the bilin biosynthetic pathway or regulate phycobilisome gene expression (Schluchter and Glazer, 1997).
It also might be hypothesized that the GL activation of pebAB expression could shunt part of the cellular pool of BV into the PEB biosynthetic pathway, thus decreasing PCB production and perhaps indirectly downregulating cpcB2A2 RNA accumulation. However, our data do not support this possibility, because in GL-grown FdG13 cells, not only is there a lack of PEB synthesis but free PCB also accumulates (Figure 5B). Yet, under these conditions, cpcB2A2 RNA levels in FdG13 are just as low as in wild-type cells (Figure 8D). Thus, the shutdown of cpcB2A2 expression in GL-grown wild-type cells does not appear to be caused by the increased production of PEB.
Finally, these data demonstrate that PEB is not a chromophore required for CCA, because normal CCA regulation of cpcB2A2 expression occurred in FdTq26 cells transformed with rcaE (Figure 2D) and FdG13 cells (Figure 2E) and proper cpeBA expression occurred in FdG13 cells (Figure 8), which lacked PEB (Figure 5B). This result was not unexpected, because CCA is controlled in part by RcaE, a phytochrome-class sensor, and PEB has not been found to act as a functional chromophore for prokaryotic phytochromes (Yeh et al., 1997; Bhoo et al., 2001; Lamparter et al., 2002) as a result of its inability to photoisomerize at the C15-C16 double bond (Li and Lagarias, 1992).
RcaE Does Not Regulate Phycobiliprotein Gene Expression by Sensing the Absence of PEB
Phytochrome-related molecules have been proposed as possible sensors of bilins, including those that control chromatic adaptation (Montgomery and Lagarias, 2002). The analysis of light-responsive phycobiliprotein gene expression in both FdTq26 and FdG13 cells (Figure 8) allowed us to test the hypothesis that RcaE played a role in controlling the RNA levels of these genes by sensing and responding to PEB levels. Both of these lines carry a mutation in pebA, but only FdTq26 also has a secondary mutation in rcaE. Thus, any effect of RcaE sensing of PEB levels on gene expression would be manifested by a difference in RNA accumulation patterns, relative to background, between these two lines. However, no significant differences could be detected between the two lines. For cpeBA RNA, the levels during growth in RL were the same in wild-type and FdG13 cells and were 1.7 times higher in FdG13 than in wild-type cells during growth in GL, results that were similar to those obtained with the FdBk mutant and FdTq26 (Figure 8B). cpeCDE RNA levels were slightly lower in both RL and GL in FdG13 than in the wild type, a trend that was mirrored in FdTq26 and FdBk mutant cells (Figure 8C). Finally, cpcB2A2 RNA levels in FdG13 and wild-type cells were comparable, just as they were in FdTq26 and FdBk mutant cells (Figure 8D). Collectively, these data demonstrate that RcaE has no significant role in controlling transcript levels from these phycobiliprotein genes by sensing the absence of PEB.
Structure of the Region That Contains the pebAB Operon
The arrangement of the pebAB operon of F. diplosiphon is similar to that found in all other cyanobacterial genomes in which this operon has been identified (Frankenberg et al., 2001): Synechococcus WH 8020 (Wilbanks and Glazer, 1993), Synechococcus WH 8102 (Joint Genome Initiative [JGI] contig 72), N. punctiforme (JGI contig 472, genes 70 and 71), Prochlorococcus MED4 (JGI genes 320 and 321), and Prochlorococcus SS120. pebA is immediately 5' of pebB, and both are transcribed from the same DNA strand. pebA and pebB are cotranscribed in F. diplosiphon (Figure 6), and we predict that this will be the case for other cyanobacterial species with a similar operon structure. This would provide important control of the stoichiometry and the location of PebA and PebB production if, as has been proposed, they form a dual-enzyme complex (Frankenberg et al., 2001). In the cyanobacterial species Synechococcus WH 8020 (Wilbanks and Glazer, 1993), Synechococcus WH 8102 (JGI contig 72), and N. punctiforme (JGI contig 472), there is clustering of pebAB with genes that encode putative PE apoproteins, linkers, and lyases. This arrangement presumably allows the coordination of gene expression and increases the efficiency of interactions between the gene products during PE biosynthesis. In F. diplosiphon, although such genes were not found immediately upstream of pebAB, they may be present downstream of and cotranscribed with pebAB. We could not test this possibility here, because the F. diplosiphon genomic DNA in pPLER2 included only 240 bp downstream of the 3' end of pebB (Figure 4A). However, this possibility is strengthened by the fact that RNA species of >1.6 kb were detected on our pebA RNA gel blots (Figures 6B and 6C).
Regulation of pebAB Expression
We are not aware of any previous reports on the regulation of the expression of nonmammalian bilin reductase genes. pebAB is also the only CCA-regulated operon to be identified that does not encode a component of the PBS. Our data show that the expression of pebAB is not controlled significantly by any feedback mechanism that responds to the lack of accumulation of the RNAs or proteins made from cpeBA and pebAB (Figures 6B and 6E). In addition, because PEB was not made in FdTq26, pebAB expression was not influenced significantly by the absence of this chromophore (Figure 6B).
The pebAB operon also showed the residual light responsiveness observed previously for the cpeBA and cpeCDE operons in an FdBk mutant (Figures 6B and 6C) (Seib and Kehoe, 2002). As for cpeBA and cpeCDE, the impairment of the CCA regulation of pebAB in the FdBk mutant involved both the loss of GL-mediated RNA accumulation and RL-mediated RNA reduction. This finding suggests that the second light-responsive system controlling the cpe operons also controls pebAB expression. Because the CCA regulation of cpeBA expression has been shown to be primarily transcriptional (Oelmüller et al., 1988), a significant part of the coordination of cpeBA and pebAB expression, as well as the subsequent PE and PEB synthesis, is likely to occur at the transcriptional level.
The expression of cpeBA and pebAB (but not cpeCDE) required the presence of CpeR (Figure 6E) (Cobley et al., 2002; Seib and Kehoe, 2002). This finding, along with the evidence that cpeR is cotranscribed with cpeCDE and thus appears to coordinate the expression of cpeBA with cpeCDE during growth in GL (Cobley et al., 2002), suggests that CpeR plays a role in coordinating the expression of at least two GL-induced operons with cpeCDE. Because genes that encode proteins with similarity to CpeR are present in PE-producing cyanobacteria that do not undergo CCA (Cobley et al., 2002), the sole function of CpeR in F. diplosiphon may be to coordinate the expression of the genes that encode PE apoproteins, linker peptides, and PEB biosynthetic enzymes rather than to broadly regulate the overall GL response during CCA. We recently isolated a number of additional GL-induced genes from this organism (E. Stowe-Evans and D. Kehoe, unpublished data). It will be interesting to determine if the expression of these new genes requires CpeR as well. If so, it would suggest that CpeR is an important component in the coordination of many GL-induced genes during CCA in F. diplosiphon.
In addition to their common requirement for CpeR, the F. diplosiphon cpeBA and pebAB operons also had several regions of strong sequence similarity in the proximal regions of their promoters (Figure 7B). This finding further supports the idea that there is coordinated transcriptional regulation of these operons. These sequences, which are present close to the transcription start site in the cpeBA promoter, appear to be similarly positioned upstream of pebAB, because the 3' end of this region is 26 bp upstream of the predicted PebA translation start site.
The sequence conservation within this region of cpeBA promoters of three cyanobacterial species, including F. diplosiphon, has been analyzed previously (Anderson and Grossman, 1990; Dubbs and Bryant, 1991; Sobczyk et al., 1993). Two highly conserved domains were identified in this 70-bp region (from -2 to -22 and from -29 to -69). The first contained an Escherichia coli–like -10 promoter element, whereas the second contained an E. coli–like -35 element and, upstream, a direct repeat contained within the sequence 5'-TGTTAN5TGTTA-3'. We found that the cpeBA promoter structure was similar in the closely related species N. punctiforme throughout the majority of the 70-bp region, although the sequence of the direct repeat was somewhat degenerate (8 of 10 identical) relative to the other species examined (Figure 7A) (Anderson and Grossman, 1990; Dubbs and Bryant, 1991; Sobczyk et al., 1993). DNA-footprinting studies have demonstrated that in F. diplosiphon, this repeat was within the binding site of the DNA binding proteins RcaA and PepB, which extended from -45 to -67 (Figure 7B) (Schmidt-Goff and Federspiel, 1993; Sobczyk et al., 1993).
The finding that pebAB expression is regulated by CCA in F. diplosiphon prompted us to compare the cpeBA and pebAB promoter regions and to predict possible roles of the three subregions we have designated within it (Figure 7). The sequence within subregion 1 was as conserved between these promoters as between cpeBA promoters from different species (Figures 7A and 7B); this also was true for the sequence within subregion 2. However, the sequence contained within subregion 3 was less conserved between the F. diplosiphon cpeBA and pebAB promoters than between cpeBA promoters from different species (Figures 7A and 7B). Interestingly, there are similar relationships between these subregions in pebAB promoter sequences from F. diplosiphon and N. punctiforme, with high sequence conservation in subregions 1 and 2 and more limited consensus within subregion 3 (Figure 7C). These data, along with the facts that both cpeBA and pebAB promoters are regulated through a common mechanism (Figures 6B and 6E) and that the pebAB promoter is significantly weaker than the cpeBA promoter (R. Alvey and D. Kehoe, unpublished data), provide support for the following model. The sequence within subregion 1 has been highly conserved to maintain strong interaction with a trans-acting component(s) that may act within the CCA signal transduction pathway, perhaps RcaA/PepB and/or RcaB (Sobczyk et al., 1993), whereas the variation that exists between subregion 3 sequences leads to differences in the binding affinity of DNA-dependent RNA polymerase that are manifested by differences in promoter strength. This model is an extension of previous proposals that subregion 1 is critical for the binding of a factor involved in CCA responsiveness and that subregions 2 and 3 make up a portion of the binding site for RNA polymerase (Anderson and Grossman, 1990; Dubbs and Bryant, 1991; Schmidt-Goff and Federspiel, 1993; Sobczyk et al., 1993).
Effect of PEB Absence on cpeBA and cpeCDE RNA and Protein Accumulation
The effect of bilin loss on the accumulation of its associated apoprotein and corresponding RNA in vivo has not been examined previously in cyanobacteria. Although the lack of PEB did not significantly change the levels of cpeBA, cpeCDE, or cpcB2A2 transcripts in FdTq26 in RL or GL (Figures 8B to 8D), the PE subunit apoprotein and linker polypeptides in this mutant were reduced to very low levels (Figure 9). These data match most results obtained in previous studies that have analyzed protein and RNA accumulation patterns in lyase mutants as well as phycobiliprotein site-directed and truncation mutants (Swanson et al., 1992; Bhalerao and Gustafsson, 1994; Jung et al., 1995; reviewed by Anderson and Toole, 1998). One exception was the finding that, in Synechococcus PCC 7002, the cpcE and cpcF mutants (which encode the PC subunit lyase) still contained substantial amounts of apoPC subunit within their PBS (Zhou et al., 1992). The data presented here demonstrate that in F. diplosiphon, the accumulation of PE and PE linkers is regulated tightly by the degree of conversion of apoprotein to holoprotein (Anderson and Toole, 1998), whereas cpeBA and cpeCDE RNA accumulation is not affected significantly by the absence of PEB.
It remains to be determined whether there are any similarities in the mechanisms that control the coordination of bilin and apoprotein synthesis in cyanobacteria and plants. Several different species of plants with deficiencies in PB biosynthesis have been examined for their ability to accumulate phytochrome mRNA and Phy apoprotein. In the tomato aurea mutant, which contains very little spectrally detectable holophytochrome, phytochrome RNA accumulates to wild-type levels but the plants contain very little immunologically detectable Phy apoprotein (Parks et al., 1987; Sharrock et al., 1988). These results parallel our data for PE synthesis in the absence of PEB (Figures 8 and 9). However, the consequences of failing to accumulate PB are different in other plant species. Arabidopsis hy1 and hy2 mutants, which contain lesions in genes that encode heme oxygenase and PB synthase (Davis et al., 1999; Muramoto et al., 1999; Kohchi et al., 2001), are not affected in Phy apoprotein accumulation despite their reduced levels of PB chromophore (Chory et al., 1989; Parks et al., 1989). Phy apoprotein also accumulated in pea after treatment with the transaminase inhibitor gabaculine, which severely reduced PB levels (Jones et al., 1986; Konomi and Furuya, 1986). Thus, for both plants and cyanobacteria, loss or reduction in the amount of a bilin does not appear to impair the accumulation of the RNA that encodes the corresponding apoprotein, but the stability of the apoprotein in the absence of chromophore varies among species. It will be interesting to determine if light-regulated transcriptional control, which is likely to coordinate the expression of the cpeBA and pebAB operons in F. diplosiphon, also is a mechanism by which phy, hy1, and hy2 RNA accumulation is coordinated in plants.
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METHODS
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Strains
The wild type is a short-filament strain of Fremyella diplosiphon designated Fd33 (previously SF33; Cobley et al., 1993). The FdBk mutant was isolated from the wild type as described previously (Kehoe and Grossman, 1996). All FdTq mutants (FdTq1, FdTq26, and FdTq31) were isolated as pigmentation mutants from plates of FdBk mutant cells after heat-shock treatment, which mobilizes endogenous insertion elements in F. diplosiphon (Seib and Kehoe, 2002; K. Shockley and D. Kehoe, unpublished data), whereas FdG13 was isolated in a similar manner from wild-type cells (R. Alvey and D. Kehoe, unpublished data).
Growth Conditions
Strains were cultured and analyzed spectrophotometrically as described previously (Seib and Kehoe, 2002).
Transformation
Transformations used electroporation, and the genetic lesion in FdTq26 was identified by complementation with an F. diplosiphon wild-type genomic library as described previously (Kehoe and Grossman, 1998). Transformants exhibiting the FdBk mutant phenotype were analyzed as described previously (Seib and Kehoe, 2002).
DNA Cloning, Sequencing, and Analysis
DNA size polymorphisms in cpeBAYZ and cpeCDE in the FdTq26 mutant were checked using the PCR-based approach described previously (Seib and Kehoe, 2002). The region around cpeR, cpeS, and cpeT was examined using the same approach and primers tqPCR1 (5'-CTTACT- ACGACCCCAAATCAAG-3') and tqsp16 (5'-AGTATTGCGGAAATAGCTTAACG-3'). The presence of an insertion element within pebA in FdTq26 and FdG13 was established via PCR using genomic DNA from each mutant as a template and the primers ER2B4 (5'-CCAATACATTCACTA- TCGCACC-3') and ER2A3 (5'-TAATGGCACAGCATCTTCAAACA-3'). The precise location of each insertion and the sequence of the F. diplosiphon genomic DNA contained within pPLER2 were determined by DNA sequencing using an ABI 3700 (Perkin-Elmer, Foster City, CA).
The primers ER2ORF236a (5'-CGCGGATCCTTAGAGTTGACCGTAGGTGAG-3') and ER2ORF236b (5'-CGCGGATCCCTGATAGAGAGTCAACTTCGCT-3') were used to PCR amplify the region of pebA for the construction of pRA-4. BamHI restriction sites (underlined) were incorporated into each primer. The amplified DNA was cut with BamHI and ligated into the BamHI site in pPL2.7 (Chiang et al., 1992b). A similar strategy was used to make pRA-45, except that the primers used were ER2ORF236a and ER2ORF257 (5'-CGCGGATCCCGTTTGCAGTGAGTT- CTGTAGT-3').
Sequence alignments were made using CLUSTAL W (http://
ebi.ac.uk/clustalw/) with minor adjustments using protein Basic Local Alignment Search Tool (BLAST) analysis information (http://www.ncbi.nlm.nih.gov/BLAST).
RNA Analysis
RNA extraction and analysis using probes for cpeBA, cpeCDE, and cpcB2A2 were performed as described previously (Seib and Kehoe, 2002). The two probes used for the detection of pebA and pebB transcripts were created by PCR amplification. The primers used for probe 1 synthesis were ER2ORF236a and ER2A3 (5'-TAATGGCACAGCATCTTCAAACA-3'), and the primers used for probe 2 were ER2B5 (5'-CACTATGATTTGCCCTTACTCG-3') and ER2A3 (5'-TAATGGCACAGCATCTTCAAACA-3'). Probe 3, which was used to analyze ORF3 expression, was amplified by PCR using ER2B3 (5'-TGTAAGCACGGTAAGAATGATGA-3') and ER2B4RC (5'-GGTGCGATAGTGAATGTATTGG-3').
Protein Analysis
F. diplosiphon cells were grown in 50 mL of BG-11 to midlogarithmic-growth phase (A750 = 0.6) in either red light or green light at 15 µmol·m-2·s-1. Cells were quick-cooled to 4°C by swirling in flasks submerged in liquid nitrogen and then centrifuged at 4000g at 4°C for 10 min. The resulting pellet was resuspended in an equal volume of ice-cold extraction buffer A (50 mM Tris, pH 7.5, 300 mM NaCl, 0.05% Tween 20, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoeth- anol, 0.2 mM benzamidine, and 50 µM 6-amino-n-hexanoic acid) and recentrifuged as described above. The cell pellet then was suspended in 3 mL of extraction buffer A and lysed at 4°C by two passages through a French pressure cell at 18,000 p.s.i. Protein concentrations were estimated using Bradford reagent (Bio-Rad, Hercules, CA) with BSA as a standard. Protein samples were combined with an equal volume of 5x SDS loading buffer, boiled for 3 min, and then centrifuged at 15,000g for 1 min. Approximately 50 µg of protein per lane were loaded onto a 16% SDS-PAGE gel and run for 3.5 h at 300 V. The gel was stained with Coomassie Brilliant Blue R 250 and then destained before imaging with a UMAX PowerLook 3 flatbed scanner (UMAX Technologies, Taipei, Taiwan).
Free Bilin Extraction and HPLC–Mass Spectrometry Analysis
Free bilins were isolated from F. diplosiphon by growing cells in 100 mL of liquid culture to an OD750 of 1.0 and then centrifuged at 3600g for 10 min at 4°C. The supernatant was discarded, and the pellet was frozen rapidly in liquid nitrogen. Thirty milliliters of acetone was added to the frozen pellet and vortexed briefly. The suspension was centrifuged, the supernatant decanted, and the pellet frozen as described above. This acetone wash was performed twice more, and then the pellet was washed two more times as described above with methanol instead of acetone. All subsequent steps were performed with the room lights off. The pellet was resuspended in 10 mL of methanol containing 2 mg/mL HgCl2. The mixture was passed through a sintered glass filter, and the filter was washed once with 10 mL of methanol. The filtered liquid was diluted 10-fold with 0.1% aqueous trifluoroacetic acid (TFA).
This solution was applied to a Sep-Pak C18 cartridge (Waters, Milford, MA) that had been serially equilibrated with the following: 3 mL of CH3CN, 3 mL of water, 3 mL of 10% methanol and 0.1% TFA, 3 mL of CH3CN and 0.1% TFA (20:80, v/v), 3 mL of CH3CN and 0.1% TFA (60:40, v/v), 3 mL of CH3CN, 3 mL of water, and finally 3 mL of 10% methanol and 0.1% TFA (Frankenberg et al., 2001). The sample was loaded onto the cartridge, washed once with 3 mL of 0.1% TFA and once with 3 mL of CH3CN and 0.1% TFA (20:80, v/v), and then eluted with 3 mL of CH3CN and 0.1% TFA (60:40, v/v). Eluted samples were dried down in a Speed-Vac concentrator (Savant Instruments, Holbrook, NY) without heat and stored in the dark at -20°C before HPLC–mass spectrometry analysis.
Bilins were resuspended in 1.5 µL of DMSO. After thorough mixing, 13.5 µL of HPLC mobile phase were added to the extracts. Phycoerythrobilin and phycocyanobilin standards were a gift from Beronda Montgomery (Indiana University, Bloomington), and the 15,16-dihydrobiliverdin standard was a gift from Nicole Frankenberg in the laboratory of Clark Lagarias (University of California, Davis). Commercial biliverdin IX hydrogen chloride (Frontier Scientific, Logan, UT) was also used as a standard. Standards were prepared at 5 µM in HPLC mobile phase. The chromatographic conditions were similar to those described previously (Frankenberg et al., 2001). Briefly, 12.5-µL injections of the bilin extracts were separated isocratically on a 2.1- x 250-mm Ultracarb 5-µm ODS20 column (Phenomenex, Torrence, CA) using a 1:1 acetone (Optima grade; Fisher):20 mM aqueous formic acid (Aldrich, St. Louis, MO) solution. A constant flow rate of 200 µL/min was provided by a Surveyor MS pump (ThermoElectron Corp., Waltham, MA). The eluent was analyzed by a UV-visible light photodiode array (Surveyor PDA; ThermoElectron Corp.) and an ion trap mass spectrometer (LCQ DecaXP Plus; ThermoElectron Corp.). UV-visible light spectra from 350 to 700 nm were recorded at 5 Hz. The flow was split after the photodiode array immediately before the ion trap, and eluent was directed into its microelectrospray source at 5.5 µL/min. The source needle was maintained at +2.9 kV relative to the grounded skimmer, and a small flow of nitrogen was used to assist desolvation in the 180°C ion transfer tube. Positive ion mass spectra between mass-to-charge ratios of 400 and 1000 were recorded at 1 Hz. All chromatographic and mass spectrometric data were analyzed using the XCalibur 1.3 software package (ThermoElectron Corp.). The identities of the eluted bilins were confirmed by their elution times, mass spectra, and UV-visible light spectra.
Genome Analysis
All sequences from the Department of Energy's JGI can be located at http://spider.jgi-psf.org/JGI_microbial/html/. National Center for Biotechnology Information Entrez nucleotide data are located at http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?CMD=searchandDB=nucleotide.
Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact David M. Kehoe, dkehoe{at}bio.indiana.edu.
Accession Numbers
The GenBank accession number for the entire F. diplosiphon genomic DNA fragment contained within pPLER2 is AY363679. Accession numbers for the other sequences mentioned in this article are as follows: ZP_00109076 and ZP_00111776 (hypothetical proteins in N. punctiforme); AAK38598.1 and Q93TM8 (N. punctiforme PebA and PebB); Q02189 and Q02190 (Synechococcus WH 8020 PebA and PebB); NZ_AABC01000017 (cpeBA promoter in N. punctiforme); X63073 (cpeBA promoter in Pseudanabaena sp PCC 7409); X04592 (cpeBA promoter in F. diplosiphon); and AJ278499 (Prochlorococcus SS120).
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Acknowledgments
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We thank Nicole Frankenberg for the gift of DHBV and Beronda Montgomery for the gift of PEB and PCB. We also thank Sam Beale, Clark Lagarias, and Nicole Frankenberg for helpful discussions and Beronda Montgomery, Emily Stowe-Evans, Barbara Balabas, James Ford, and Lina Li for thoughtful comments on the manuscript. This material is based on work supported by the National Science Foundation under Grant MCB-0084297 to D.M.K. and Grant CHE-0094579 to J.P.R. We also gratefully acknowledge support from the Indiana Genomics Initiative, which is funded in part by the Lilly Endowment, Inc.
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Footnotes
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Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.015016.
Received June 25, 2003;
accepted August 15, 2003.
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ABSTRACT
INTRODUCTION