Lesions in Phycoerythrin Chromophore Biosynthesis …...Biosynthesis Pathways Leading to the Synthesis of PEB, PCB, and P B from BV. Information regarding the bilin biosynthetic enzymes

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Lesions in Phycoerythrin Chromophore Biosynthesis in

Fremyella diplosiphon

Reveal Coordinated Light Regulation of Apoprotein and Pigment Biosynthetic Enzyme Gene Expression

Richard M. Alvey,

a

Jonathan A. Karty,

b

Elicia Roos,

a

James P. Reilly,

b

and David M. Kehoe

a,1

a

Department of Biology, Indiana University, Bloomington, Indiana 47405

b

Department of Chemistry, Indiana University, Bloomington, Indiana 47405

We have characterized the regulation of the expression of the

pebAB

operon, which encodes the enzymes required for phy-coerythrobilin synthesis in the filamentous cyanobacterium


. The expression of the

pebAB

operonwas found to be regulated during complementary chromatic adaptation, the system that controls the light responsivenessof genes that encode several light-harvesting proteins in

F. diplosiphon

. Our analyses of

pebA

mutants demonstrated thatalthough the levels of phycoerythrin and its associated linker proteins decreased in the absence of phycoerythrobilin, therewas no significant modulation of the expression of

pebAB

and the genes that encode phycoerythrin. Instead, regulation ofthe expression of these genes is coordinated at the level of RNA accumulation by the recently discovered activator CpeR.

INTRODUCTION

Photosynthetic organisms use light-harvesting antennae to maxi-mize their use of ambient light for photosynthesis. Phycobilisomes(PBS), the light-harvesting structures used by cyanobacteria andred 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, andcomposition of PBS within a cell are dynamic and responsive tomany environmental parameters, including nutrient availability,

light quantity, and light quality (Grossman et al., 2001). The syn-theses of the different proteins that make up the PBS must beregulated tightly and also must be coordinated closely with theproduction of the chromophores that are attached to phycobili-proteins.

Decades of research have provided insight into the molecu-lar mechanisms involved in the acclimation of PBS to lightquality in cyanobacteria, a process known as chromatic adapta-tion (Gaidukov, 1903; Bogorad, 1975; Tandeau de Marsac, 1977;Grossman and Kehoe, 1997; Palenik, 2001). Although severaltypes of chromatic adaptation exist, the best studied occurs in the

filamentous species


(also called

Calothrix

sp strain PCC 7601) and is referred to as complementary chro-matic adaptation (CCA). Action spectra have demonstrated thatCCA 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. Whengrown in RL, blue-colored phycocyanin (PC), called induciblephycocyanin (PCi), and its linkers are synthesized and added toPBS (reviewed by Kehoe and Grossman, 1994). Other compo-nents of the PBS, such as constitutive PC (PCc) and allophyco-cyanin, 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 maximallyabsorbs RL (

�

620 nm), which closely correspond to the max-ima of the CCA action spectrum. Thus, CCA appears to allowthis organism to maximize photosynthetic efficiency by spec-trally tuning the absorption characteristics of its PBS to the pre-dominant 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

�

-sub-units of PCi and its associated linkers (

cpcB2A2H2I2D2

; abbrevi-ated

cpcB2A2

) is regulated by light quality; where it has been

examined, this regulation occurs primarily at the level of tran-scription (Conley et al., 1985, 1986; Mazel et al., 1986; Lomax etal., 1987; Oelmüller et al., 1988; Federspiel and Grossman, 1990;Federspiel and Scott, 1992). Genetic approaches have led to theisolation of several components of the regulatory system thatcontrols 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 phosphore-lay system that includes a phytochrome-class sensor, RcaE,and two response regulators. Mutants lacking functional RcaEare black (FdBk mutants) regardless of the light conditions inwhich they are grown. This is the result of the accumulation ofintermediate levels of PE and PCi (Kehoe and Grossman, 1996;K. Terauchi and D. Kehoe, unpublished data). Another regulatorycomponent 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

1

To whom correspondence should be addressed. E-mail [email protected]; fax 812-855-6705.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.015016.

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and has been proposed to assist in coordinating the expressionof

cpeBA

with

cpeCDE

(Cobley et al., 2002). Finally, there isalso strong evidence that an additional CCA light-sensing sys-tem controls

cpeBA

and

cpeCDE

light responsiveness, althoughno components of this pathway have been identified (Kahn etal., 1997; Seib and Kehoe, 2002).

The

�

- and

�

-subunits of PE and PCi have covalently attachedlinear tetrapyrroles, or bilins, that absorb light in the visible regionof the spectrum to collect energy for photosynthesis. In cyano-bacteria, phycoerythrobilin (PEB) is attached to PE, whereas phy-cocyanobilin (PCB) is attached to PC (Ó Heocha, 1963; Ó Carraet 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 ini-tiated by the conversion of heme to biliverdin IX

�

(BV) by theheme 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 P

�

B is completed by the reduction ofBV to 3Z-P

�

B by HY2, which is a 3Z-P

�

B synthase (Figure 1)(Kohchi et al., 2001). In cyanobacteria, PCB is produced by thereduction of BV to 3Z-PCB by 3Z-PCB:ferredoxin oxidoreduc-tase, whereas PEB is synthesized from BV through a two-stepprocess: the production of the intermediate 15,16-dihydrobiliverdin(DHBV) by 3Z-15,16-DHBV:ferredoxin oxidoreductase (PebA) andthe subsequent conversion of DHBV to 3Z-PEB by 3Z-PEB:ferre-doxin oxidoreductase (PebB) (Figure 1) (Cornejo and Beale, 1997;Frankenberg et al., 2001).

Tetrapyrroles are increasingly being recognized as importantplant signaling molecules. The tetrapyrrole biosynthetic inter-mediate Mg-protoporphyrin IX is a key molecule in chloroplast–nucleus communication in plants (Strand et al., 2003). Photo-synthetic organisms may use heme to inhibit glutamyl-tRNA

reductase and are known to coordinate chlorophyll synthesiswith the production of chlorophyll binding proteins (Ryberg andTerry, 2002; reviewed by Vavilin and Vermaas, 2002; Cornah etal., 2003). In cyanobacteria, the influence of tetrapyrroles on pro-tein accumulation is well documented. Numerous studies haveshown that phycobiliproteins that do not attach bilins fail to accu-mulate, although there is no obvious effect on transcript accu-mulation levels (Anderson and Toole, 1998).

At present, little is known about the mechanisms by whichgenes that encode bilin biosynthetic enzymes, such as

HY1

,

ho1

,

HY2

,

pcyA

,

pebA

, and

pebB

, are regulated. In cyanobacteria, it isnot clear if there is coordinated expression of these genes and

cpeBA

,

cpeCDE

, and

cpcB2A2

, and if so, how such regulationoccurs. Is the absence of a specific chromophore a signal thatregulates the expression of genes that encode the correspond-ing phycobiliproteins, or is coordination achieved through a sharedtranscriptional regulatory pathway? Here, we demonstrate that in

F. diplosiphon

, expression of the

pebAB

operon is upregulated byGL. We have determined that this occurs, at least in part, by thesame mechanism that controls the GL induction of

cpeBA

duringCCA. We also establish that the absence of PEB does not signifi-cantly affect

cpeBA

,

cpeCDE

, or

pebAB

expression. Thus, in

F.diplosiphon

, the coordination of

pebAB

and

cpeBA

expressionappears to operate primarily through a shared transcriptionalregulatory mechanism rather than through a feedback controlsystem that senses low PEB levels.

RESULTS

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 FdBkmutant background. The wild-type CCA phenotype, in whichPE accumulated in GL and PC accumulated in RL (Figure 2A),was nearly abolished in FdBk cells, in which PE and PC levelswere largely constant regardless of ambient light quality (Figure2B). Pigment accumulation in FdTq26 differed dramatically fromthat in the FdBk mutant, with PC levels high and PE undetectableregardless of ambient light wavelength (Figure 2C). This pheno-type was identical to that of the previously described FdTq1 mu-tant, which was unable to accumulate

cpeBA

RNA as a result of alesion in

cpeR

(Cobley et al., 2002; Seib and Kehoe, 2002).The

rcaE

mutation and impairment of CCA in FdTq26 made itunclear whether its pigmentation phenotype resulted from anoveraccumulation of PC, a lack of PE, or both. FdTq26 cells trans-formed with wild-type

rcaE

displayed normally regulated PC levelsin GL and RL but failed to accumulate spectrally detectable PE(Figure 2D). Thus, the FdTq26 phenotype appeared to be theresult of two separate mutations. The first, in

rcaE

, led to the ac-cumulation of intermediate levels of PC in RL and GL, and thesecond resulted in the lack of holo-PE under both light condi-tions. Collectively, these mutations made the phenotype of theFdTq26 mutant similar to that of RL-grown wild-type cells. Thissecond mutation was not in

cpeR

,

cpeBA

,

cpeCDE

, or

cpeYZ

,which encodes a putative PE lyase responsible for catalyzing

Figure 1. Biosynthesis Pathways Leading to the Synthesis of PEB,PCB, and P�B from BV.

Information regarding the bilin biosynthetic enzymes listed is provided inthe text. Dashed arrow indicates step of the pathway that has been shownto exist in red algae but has not yet been identified in cyanobacteria.

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Regulation of Bilin Synthesis 3 of 16

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 inFdTq26 was identified by complementation using a plasmid li-brary containing wild-type

F. diplosiphon

genomic DNA (Kehoeand Grossman, 1998). Approximately 6000 FdTq26 transformantswere grown in white light under kanamycin selection and screenedfor the FdBk mutant phenotype. One such colony was isolatedthat, when removed from selective pressure, clearly reverted tothe FdTq26 phenotype (data not shown). The complementingplasmid, pPLER2, was rescued and found to contain a 4.1-kbfragment of

F. diplosiphon

genomic DNA. Retransformation ofFdTq26 with pPLER2 led to the restoration of the FdBk mutantphenotype (Figure 3A).

Sequencing revealed that this genomic DNA fragment con-tained five major open reading frames (ORFs) (Figure 4A). ORF1was most similar to a gene that encodes a hypothetical proteinin

Nostoc punctiforme

. ORF2 was most closely related to ORF200in the marine cyanobacterium

Synechococcus

sp WH 8020,which was located near the

cpeBA

and

pebAB

operons in that or-ganism (Wilbanks and Glazer, 1993; Frankenberg et al., 2001).ORF3 was most similar to a gene that encodes a hypotheticalprotein in

N. punctiforme

. ORF4 and ORF5 encoded proteins withsequences closely related to bilin reductases (Frankenberg et al.,2001). The ORF4 product was most similar to cyanobacterialPebA proteins, and ORF5 was most closely related to cyanobac-terial PebB proteins (Figures 4C and 4D). ORF4 and ORF5 wereencoded on the same strand in the

F. diplosiphon

genome, andtheir positions relative to each other were similar to those of

pebA

and

pebB

genes found in other cyanobacterial species (see Dis-cussion). The ORF4 and ORF5 proteins were most closely relatedto PebA and PebB in the filamentous cyanobacterium

N. puncti-forme

(Figures 4C and 4D).

F. diplosiphon

ORF4 and

N. puncti-forme

PebA were 81% identical and 89% similar over 243 aminoacids, and ORF5 and PebB were 84% identical and 91% similarover 245 amino acids.

The next most closely related PebA and PebB were in

Syn-echococcus

WH 8020 (40% identical and 58% similar over 232amino acids [PebA] and 50% identical and 64% similar over 241amino acids [PebB]). Despite the sequence divergence of PebAand PebB in

F. diplosiphon

and cyanobacterial species otherthan

N. punctiforme

, all residues

�

70% conserved in this family(Frankenberg et al., 2001) were present in

F. diplosiphon

ORF4and ORF5. In addition, there were two principal areas of se-quence conservation among the six sequences presented inFigures 4C and 4D. The first was a region of

�

60 amino acidsstarting nearly 60 residues from the N termini of the

F. diplosi-

Solid lines indicate cells grown in GL, and dotted lines indicate cellsgrown in RL. The scan shown for each line is representative of at leastthree independent replications. Maximum PE and PC absorption peaksare indicated. Chlorophyll

a

absorption peaks are at 430 and 680 nm.

Figure 2.

Whole-Cell Absorption Spectra of

F. diplosiphon

Wild-Typeand 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.

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proteins, and the second was a region of 40 amino acidsat the C termini of these proteins. Highly conserved residueswithin these conserved regions have been identified for this classof enzymes and may contain active-site residues (Frankenberget al., 2001).

PCR amplification of this 4.1-kb region in the FdTq26 ge-nome revealed an additional 2 kb of DNA that was not presentin the wild-type genome at this location (data not shown). Fur-ther PCR amplification and sequencing analysis showed thatthe inserted DNA was within ORF4, located in the DNA corre-sponding to codon 86 of the protein (Figure 4C).

To determine if the lesion in ORF4 was responsible for theFdTq26 phenotype, two fragments were amplified by PCR fromwild-type genomic DNA and cloned into the shuttle vector pPL2.7(Chiang et al., 1992b) (Figure 4B). pRA-4 contained ORF4 andpRA-45 contained both ORF4 and ORF5. These plasmids wereeach transformed into FdTq26. Transformants were grown in RLand GL and assayed spectrophotometrically for the accumula-tion of holo-PE and the reestablishment of the FdBk mutant

phenotype (the background from which FdTq26 was gener-ated). FdTq26 cells transformed with pRA-4 accumulated rela-tively low levels of PE and tended only slightly toward the FdBkmutant phenotype in GL and RL (Figure 3B). Cells transformedwith pRA-45 contained moderate levels of PE and showed avery weak FdBk mutant phenotype after growth in RL but werephenotypically identical to the FdBk mutant when grown in GL(Figure 3C). These results demonstrated that the insertion inORF4 was responsible for the FdTq26 phenotype and stronglysuggested that this insertion also had a polar effect on ORF5expression 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 newmember 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 ofORF4 (data not shown). FdG13 properly regulated PCi in RL(Figures 2E and 3D to 3F) but, like FdTq26, failed to accumulateholo-PE in GL (cf. Figures 2C and 2E). The absence of a lesionwithin

rcaE

in FdG13 resulted in higher levels of spectrally de-tectable PE in this mutant, compared with FdTq26, after trans-formation with pRA-45 and growth in GL (cf. Figures 3C and3F). Also, like FdTq26, FdG13 mutant cells transformed with ei-ther pRA-4 or pRA-45 and grown in GL (Figures 3E and 3F) hada partially rescued phenotype, whereas transformation of FdG13cells with pPLER2 completely rescued the phenotype (Figure3D), 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 pseudomolecu-lar ions (M

�H)� that were within 0.1 D of their predicted monoiso-topic masses (583.2 for BV, 585.3 for DHBV, and 587.3 for PCBand PEB). In all cases, the dominance of the pseudomolecular ionallowed the data to be analyzed using extracted ion chromato-grams, in which the intensity at any time point represents thesum 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 obtainedfrom the injection of PEB, PCB, DHBV, and BV is presented inFigure 5A (top). The elution profiles were very similar to those re-ported previously (Frankenberg et al., 2001), and the mass spec-tra (Figure 5A, bottom) show the dominance of each bilin’spseudomolecular ion. HPLC-MS analysis of free bilin extractsfrom FdG13 cells grown in GL revealed no detectable PEB orDHBV, but a small amount of BV and a larger amount of PCBwere present, as determined by elution time (Figure 5B, top),mass spectra (Figure 5B, bottom), and UV/visible light spectra(data not shown).

We predicted that if ORF4 encoded PebA, extracts from GL-grown FdG13 transformed with pRA-4 would contain DHBV asa result of pebA expression from the introduced plasmid and re-duced pebB expression as a result of the polar effect from thepebA insertion (Figure 4A). We found that FdG13 cells trans-

Figure 3. Whole-Cell Absorption Spectra of FdTq26 and FdG13 CellsTransformed 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 cellsgrown in RL. The scan shown for each line is representative of at leastthree independent replications. Maximum PE and PC absorption peaksare indicated. Chlorophyll a absorption peaks are at 430 and 680 nm.

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formed with pRA-4 did contain both free DHBV and PEB but nodetectable free PCB or BV (Figure 5C). We also expected that ifORF5 encoded PebB, transformation of FdG13 with pRA-45 andgrowth in GL would lead to the production of equal amounts ofPebA and PebB and result in the accumulation of free PEB but notDHBV or BV, which was the result we obtained (Figure 5D). It isnoteworthy that of the lines examined here, only the FdG13 mu-tant accumulated any detectable BV. It also produced an un-identified species with a retention time of 13.8 min and a massof 585.3 (Figure 5B) with a UV/visible light absorption spectrumsimilar 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 RLin F. diplosiphon (Oelmüller et al., 1988). This, together with thefact that FdTq26 transformed with pRA-45 was complementedmore completely to the FdBk mutant phenotype when grown inGL than in RL (Figure 3C), led us to investigate whether pebABtranscript accumulation also was regulated by GL and RL. RNAgel blot analyses were conducted initially with probe 1, whichcovered most of the pebA gene and the 5� flanking region (Fig-ure 6A). This probe detected multiple RNA species ranging

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 be-tween 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 eachORF except ORF5, for which the first Ile was used. The locations of the DNA insertions are indicated for FdG13 (open arrowhead) and FdTq26 (closedarrowhead).(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 thePebA 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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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 undetect-able in RL-grown wild-type cells (Figure 6B). The same RNA pat-terns were detected in the FdBk mutant, although the differentialregulation by both GL and RL was reduced relative to that in thewild type (Figure 6B). In FdTq26 cells, two RNAs of �0.4 and 0.6kb were detected, and these were expressed in both GL and RL atslightly below the levels of the RNAs in FdBk mutant cells (Figure6B). For all three mutants, the transcripts appeared to be unstableand partially degraded, despite the fact that rRNAs (Figure 6) andcpeBA, cpeCDE, and cpcB2A2 transcripts (data not shown) onthe same blots showed no signs of degradation.

We hypothesized that the shorter RNA detected by probe 1in FdTq26 resulted from premature termination of transcriptioncaused by the insertion in pebA (Figure 6A). This notion wastested by conducting RNA gel blot analyses with probe 2, whichextended from the 3� end of the inserted DNA to the 3� end ofpebA (Figure 6A). For wild-type and FdBk mutant cells, the re-sults obtained were essentially identical to those obtained usingprobe 1 (cf. Figures 6B and 6C). However, there was no detect-able hybridization of probe 2 to RNA from FdTq26 (Figure 6C).Thus, although the 5� end of pebA is transcribed in FdTq26, the3� end does not appear to be, suggesting that transcription ter-mination occurs within the inserted DNA located in pebA.

The �1.6-kb RNA detected by probe 1 in wild-type and FdBkmutant cells was the correct size to encompass either ORF3 andpebA or pebA and pebB, whereas the larger RNA species werecapable of encoding all three. The question of whether or notORF3 was cotranscribed with pebA and pebB was resolved byRNA gel blot analyses using probe 3 to examine the ORF3 tran-script size and expression pattern (Figure 6A). An RNA between500 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 withpebA and pebB. Collectively, the data in Figure 6 demonstratethat in F. diplosiphon wild-type cells, pebA must be cotranscribedwith pebB and the expression of this operon is highly upregulatedby GL.

In F. diplosiphon, both cpeBA and cpeCDE are induced byGL (Mazel et al., 1986; Oelmüller et al., 1988; Federspiel andGrossman, 1990; Federspiel and Scott, 1992). However, recentstudies have shown that the regulation of cpeBA expression re-quires the activator CpeR, whereas cpeCDE expression doesnot (Cobley et al., 2002; Seib and Kehoe, 2002). In a cpeR mu-tant background, cpeBA RNA is undetectable in GL, whereascpeCDE RNA still accumulates. The mechanism of action of CpeRis unknown. It has limited sequence similarity to members of thePP2C class of protein phosphatases (Seib and Kehoe, 2002), and

Figure 5. HPLC-MS Analyses of Free Bilins Present within F. diplosi-phon.

Extracted ion chromatograms (top) and mass spectra (bottom) of bilinstandards (A), the FdG13 mutant (B), the FdG13 mutant transformedwith pRA-4 (C), and the FdG13 mutant transformed with pRA-45 (D). Inthe top panels, the x axis indicates retention time and the y axis indi-cates 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 pan-els, integrated mass spectra are provided. Retention times for the la-beled 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 in-dependent experiments (including transformations, as applicable) foreach sample.

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similar proteins are present in other cyanobacterial species thatproduce PE (Cobley et al., 2002). To determine if pebAB expres-sion was regulated by CpeR, we used RNA gel blots to examineits expression in FdTq1, a mutant that lacks functional CpeR (Seiband Kehoe, 2002). pebAB RNA failed to accumulate in GL or RL inFdTq1 (Figure 6E). This requirement for CpeR suggested a sharedregulatory mechanism for pebAB and cpeBA expression.

We also wished to determine whether pebAB RNA levels wereinfluenced by the absence of cpeBA RNA and PE. Thus, pebABRNA levels were examined in FdTq31, a mutant that fails to ac-cumulate spectrally detectable PE in GL and contains insertionsin both rcaE and cpeBA (R.M. Alvey and D.M. Kehoe, unpub-lished data). RNA accumulation patterns for pebAB were simi-lar in FdTq31 (Figure 6E) and the mutant class from which itwas derived, the FdBk mutant (Figure 6B). This finding demon-strated that pebAB expression was not affected by the lack of

normal accumulation of cpeBA RNA or PE itself. It also eliminatedthe possibility that the lack of pebAB expression in FdTq1 was anindirect effect of the failure to accumulate either cpeBA RNA or PE.

The requirement of CpeR for the expression of both pebABand cpeBA suggested that these operons might be regulatedtranscriptionally using a common mechanism. If, as for cpeBA,the light regulation of the pebAB operon occurs transcription-ally, then corresponding cis-acting control elements might beexpected to reside in the region upstream of pebA. Therefore,we examined the regions upstream of these operons for sharedsequence elements that might regulate their activity. We first an-alyzed the cpeBA promoter regions from several chromaticallyadapting species for shared sequence motifs (Figure 7A). Sev-eral areas of strong sequence conservation were found in theproximal 70 bp of the promoters in N. punctiforme, Pseudana-baena sp PCC 7409, and F. diplosiphon (Figure 7A). These re-

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 insertionwithin 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 anautoradiogram 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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sults 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 wereidentified: the most highly conserved was subregion 1 of thecpeBA promoter, which was identical at 24 of 29 bases with theregion from 94 through 66 (relative to the putative translationstart site) of pebAB (Figure 7B). The two additional subregions ofsimilarity in the cpeBA promoters were subregion 2 (identical at 5of 7 bp with 63 through 57 of pebAB) and subregion 3 (identi-cal at 11 of 22 bp with 47 through 26 of pebAB). Although sub-region 3 was highly conserved between cpeBA promoters (Fig-ure 7A), it was the least conserved between pebAB promoterregions from N. punctiforme and F. diplosiphon (Figure 7C).

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 inFdTq26 and FdG13 affected the expression patterns of cpeBA,cpeCDE, and cpcB2A2. Representative autoradiograms for thesethree operons and a ribosomal loading control in the wild type,the FdBk mutant, FdTq26, and FdG13 cells are shown in Figure8A, and histograms of data from at least three independent ex-periments, normalized to ribosomal values, are shown in Figures8B to 8D. In the wild type, cpeBA RNA levels were 20 timeshigher in GL-grown cells compared with RL-grown cells, whereasthe FdBk mutant retained only �2.4-fold higher cpeBA RNA lev-els in GL- versus RL-grown cells (Figure 8B). The decrease in re-sponsiveness in the FdBk mutant was attributable to increasedlevels in RL and slightly reduced levels in GL relative to the wildtype.

In FdTq26, cpeBA RNA levels also were less highly inducedby GL (�1.8 fold) than in the wild type. However, overall, thereappeared to be more cpeBA RNA present in FdTq26 than in the

FdBk mutant in both GL and RL, although these differenceswere not statistically significant (GL, P � 0.21; RL, P � 0.18)(Figure 8B). For cpeCDE expression, neither the reduced de-gree of GL-RL responsiveness in the FdBk mutant nor the over-all lower levels measured relative to wild-type cells was signifi-cantly different from that of FdTq26 in either GL (P � 0.53) orRL (P � 0.81) (Figure 8C). Finally, the FdBk mutant and FdTq26showed an equivalent loss of CCA responsiveness for expres-sion of the cpcB2A2 operon (Figure 8D). In FdG13 cells, cpeBARNA levels were the same as in the wild type during growth inRL and were �1.7 times higher in FdG13 than in wild-type cellsduring growth in GL (Figure 8B). cpeCDE RNA levels in RL-grownFdG13 and wild-type cells were essentially the same and were60% of wild-type levels during growth in GL (Figure 8C), mirroringthe pattern observed for FdTq26 (Figure 8C). Finally, cpcB2A2RNA levels in FdG13 during both RL and GL growth were simi-lar to those measured for wild-type cells under the same condi-tions (Figure 8D). Overall, the absence of PEB in both FdTq26and FdG13 had no major effects on RNA accumulation patternsfor 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 producePEB, such as FdTq26 and FdG13 (Figure 8B), raised the possi-bility that apoPE also accumulated in these mutants. Therefore,we isolated total protein from wild-type, FdBk mutant, and FdTq26cells grown in GL and RL. We used whole-cell extracts instead ofpurified PBS to examine the entire cellular pool of phycobiliproteinsrather than only those incorporated into PBS. Protein sampleswere separated by SDS-PAGE, and gels were examined for thepresence of PE�, PE�, and the linker peptides CpeC, CpeD,and CpeE. As demonstrated previously for this species, PE�

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; Nos-toc, N. punctiforme; Pseud., Pseudanabaena sp PCC 7409. Fractions in parentheses indicate comparisons between species of the numbers of iden-tical nucleotides divided by the total number of nucleotides for that subregion (indicated at bottom): F, F. diplosiphon; N, N. punctiforme; P. Pseudan-abeana. 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) areshown below the sequences.

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and the PE linkers were present in wild-type cells at low levelsin RL but were very abundant in GL (PE� could not be resolvedfrom PCc�) (Figure 9) (Bryant, 1981; Lomax et al., 1987; Federspieland Grossman, 1990; Federspiel and Scott, 1992). These pro-teins also were present at moderate levels in FdBk mutant cells

in both GL and RL (Figure 9), which paralleled the partial accu-mulation of cpeBA and cpeCDE RNA (Figures 8B and 8C) andspectroscopically detectable PE (Figure 2B) under these lightconditions. In FdTq26, however, PE�, CpeC, CpeD, and CpeEwere undetectable in both GL and RL (Figure 9). Therefore, al-though cpeBA and cpeCDE transcripts accumulated to relativelyhigh levels in this mutant (Figures 8B and 8C), the correspondingproteins either were not made or were degraded rapidly aftersynthesis as a result of the absence of PEB.

DISCUSSION

This investigation has identified and characterized two F. diplo-siphon mutants that fail to produce PEB as a result of a lesionin pebA. pebA was found to be cotranscribed with pebB. Theregulation of pebAB expression and the effects of the absenceof PEB on cpeBA and cpeCDE expression were analyzed to ex-plore the mechanism(s) by which the synthesis of PE, PE link-ers, and PEB are coordinated. We determined that, as for cpeBA,pebAB expression was regulated by GL and RL and requiredCpeR. In addition, the pebAB promoter contained a sequence thatwas similar to a 69-bp region in the cpeBA promoter that wasdetermined previously to contain a protein binding site (Schmidt-Goff and Federspiel, 1993; Sobczyk et al., 1993). Our results have

Figure 8. RNA Gel Blot Analyses of cpeBA, cpeCDE, and cpcB2A2 Ex-pression in RL- and GL-Grown Wild-Type, FdBk Mutant, FdTq26, andFdG13 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 ofcpeBA (B) or cpeCDE (C) RNA levels in these four lines expressed as apercentage of the wild-type GL value, which was set to 100%.(D) Mean values from at least three independent experiments ofcpcB2A2 RNA levels in these four lines expressed as a percentage ofthe wild-type RL value, which was set to 100%.In (B) to (D), standard errors are shown, and selected P values are pro-vided in the text. All measurements were normalized using relative ribo-somal hybridization values before calculation of the means.

Figure 9. SDS-PAGE of Total Protein Extracted from Wild-Type, FdBkMutant, and FdTq26 Cells Grown in RL or GL.

Five micrograms of purified PBS proteins extracted from wild-type cellsgrown in RL or GL was used as a control. Components of the PBS arelabeled as identified previously (Bryant, 1981; Federspiel and Grossman,1990). WT, wild type.

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revealed the fourth CCA-responsive operon in this species andsuggest that coordinated regulation of cpeBA and pebAB ex-pression occurs, at least in part, via a transcriptional mechanism.In addition, during growth in GL, these mutants continued to ac-cumulate high levels of cpeBA and cpeCDE RNAs but failed toaccumulate normal levels of PE or PE linkers. This finding indi-cates that feedback mechanisms exist to reduce PE and PElinker levels in the absence of PEB but not to significantly con-trol cpeBA and cpeCDE RNA levels. pebAB RNA also accumu-lated normally in a cpeBA mutant, demonstrating that no strongfeedback mechanism operated to reduce pebAB expression inthe absence of PE.

In Vivo Dynamics of the PEB Biosynthetic Pathway

The data presented here demonstrate that both the FdTq26 andFdG13 mutants contained genetic lesions in pebA and that thisgene was immediately upstream of and cotranscribed with pebB.In both mutants, the insertion within pebA appeared to have astrong, albeit partial, polar effect on pebB expression (Figures 3B,3C, 3E, and 3F). Alternatively, the polar effect of the pebA inser-tion on pebB could be complete and pebB could be expressedat a low level from its own promoter. In multiple independentexperiments, pPLER2 also complemented both of these mutantsmore completely than either pRA-4 or pRA-45 (Figure 3). Thisfinding suggests that either pebA and pebB were more highlyexpressed from pPLER2 than from pRA-4 and pRA-45 or thespatial separation of the synthesis of PebA/PebB and the pro-teins encoded by ORF1, ORF2, or ORF3 decreased the amountof functional PebA and PebB produced. We favor the latter pos-sibility, because the entire promoter region between ORF3 andpebA was included in the pRA-4 and pRA-45 plasmids, therewas no measurable coexpression of ORF3 and pebAB (Figure6), and there is no obvious reason why pebAB would be morehighly expressed from pPLER2 than from pRA-4 and pRA-45.

Our HPLC-MS data (Figure 5) demonstrate that ORF4 is pebAand ORF5 is pebB and explain the absence of holo-PE in theFdTq26 and FdG13 mutants. They also provide in vivo supportfor the two-step biosynthetic pathway from BV to PEB in cyano-bacteria (Cornejo and Beale, 1997; Frankenberg et al., 2001).Furthermore, the accumulation of PCB in FdG13 cells (Figure5B) demonstrates that cyanobacteria are capable of synthesiz-ing PCB without a PEB intermediate, as occurs in red algae (Fig-ure 1) (Beale and Cornejo, 1991a).

Previous studies conducted with cell extracts or in vitro ledto the proposal that the conversion of DHBV to PEB by PebB isnot the rate-limiting step in the pathway from BV to PEB (Bealeand Cornejo, 1991a, 1991b; Rhie and Beale, 1992; Frankenberget al., 2001). The presence of PEB and the absence of DHBV inFdG13 cells transformed with pRA-45 (Figure 5D) provides invivo data to support this hypothesis. In addition, the accumula-tion of DHBV, but not BV, in FdG13 cells transformed with pRA-4 (Figure 5C) suggests that the reduction of BV to DHBV by PebAin vivo is not reversible to any measurable extent. The fact thatFdG13 cells contained free PCB and BV during growth in GL (Fig-ure 5B) implies that blocking the pathway from BV to PEB underthese growth conditions leads to an accumulation of BV andspillover 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 accumu-late if the cells are capable of synthesizing PEB. High BV levelsmight not be tolerated by these cells for several reasons. Ex-cess BV could attach to both PE and PC, as has been shown tooccur in vitro (Arciero et al., 1988; Fairchild and Glazer, 1994),although we did not observe any shifts in the absorption max-ima for FdG13 (Figure 2E) despite the accumulation of measur-able free BV (Figure 5B). Alternatively, the BV level could nor-mally be kept low to regulate the production of bilirubin, whichthen could either control the flux of BV into the bilin biosyntheticpathway or regulate phycobilisome gene expression (Schluchterand Glazer, 1997).

It also might be hypothesized that the GL activation of pebABexpression could shunt part of the cellular pool of BV into thePEB biosynthetic pathway, thus decreasing PCB productionand perhaps indirectly downregulating cpcB2A2 RNA accumu-lation. However, our data do not support this possibility, becausein GL-grown FdG13 cells, not only is there a lack of PEB synthe-sis but free PCB also accumulates (Figure 5B). Yet, under theseconditions, cpcB2A2 RNA levels in FdG13 are just as low asin wild-type cells (Figure 8D). Thus, the shutdown of cpcB2A2expression in GL-grown wild-type cells does not appear to becaused by the increased production of PEB.

Finally, these data demonstrate that PEB is not a chromophorerequired for CCA, because normal CCA regulation of cpcB2A2expression occurred in FdTq26 cells transformed with rcaE (Fig-ure 2D) and FdG13 cells (Figure 2E) and proper cpeBA expres-sion occurred in FdG13 cells (Figure 8), which lacked PEB (Figure5B). This result was not unexpected, because CCA is controlledin part by RcaE, a phytochrome-class sensor, and PEB has notbeen found to act as a functional chromophore for prokaryoticphytochromes (Yeh et al., 1997; Bhoo et al., 2001; Lamparter etal., 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 possi-ble sensors of bilins, including those that control chromatic ad-aptation (Montgomery and Lagarias, 2002). The analysis of light-responsive phycobiliprotein gene expression in both FdTq26 andFdG13 cells (Figure 8) allowed us to test the hypothesis thatRcaE played a role in controlling the RNA levels of these genesby sensing and responding to PEB levels. Both of these linescarry a mutation in pebA, but only FdTq26 also has a second-ary mutation in rcaE. Thus, any effect of RcaE sensing of PEBlevels on gene expression would be manifested by a differencein RNA accumulation patterns, relative to background, betweenthese two lines. However, no significant differences could be de-tected between the two lines. For cpeBA RNA, the levels duringgrowth in RL were the same in wild-type and FdG13 cells andwere �1.7 times higher in FdG13 than in wild-type cells duringgrowth in GL, results that were similar to those obtained with theFdBk mutant and FdTq26 (Figure 8B). cpeCDE RNA levels wereslightly lower in both RL and GL in FdG13 than in the wild type,a trend that was mirrored in FdTq26 and FdBk mutant cells

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(Figure 8C). Finally, cpcB2A2 RNA levels in FdG13 and wild-type cells were comparable, just as they were in FdTq26 andFdBk mutant cells (Figure 8D). Collectively, these data demon-strate that RcaE has no significant role in controlling transcriptlevels from these phycobiliprotein genes by sensing the absenceof PEB.

Structure of the Region That Contains the pebAB Operon

The arrangement of the pebAB operon of F. diplosiphon is sim-ilar to that found in all other cyanobacterial genomes in whichthis operon has been identified (Frankenberg et al., 2001): Syn-echococcus WH 8020 (Wilbanks and Glazer, 1993), Synechoc-occus WH 8102 (Joint Genome Initiative [JGI] contig 72), N.punctiforme (JGI contig 472, genes 70 and 71), ProchlorococcusMED4 (JGI genes 320 and 321), and Prochlorococcus SS120.pebA is immediately 5� of pebB, and both are transcribed fromthe same DNA strand. pebA and pebB are cotranscribed in F.diplosiphon (Figure 6), and we predict that this will be the casefor other cyanobacterial species with a similar operon structure.This would provide important control of the stoichiometry andthe location of PebA and PebB production if, as has been pro-posed, 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 con-tig 72), and N. punctiforme (JGI contig 472), there is clustering ofpebAB with genes that encode putative PE apoproteins, linkers,and lyases. This arrangement presumably allows the coordina-tion of gene expression and increases the efficiency of interac-tions between the gene products during PE biosynthesis. In F.diplosiphon, although such genes were not found immediatelyupstream of pebAB, they may be present downstream of andcotranscribed with pebAB. We could not test this possibilityhere, because the F. diplosiphon genomic DNA in pPLER2 in-cluded only 240 bp downstream of the 3� end of pebB (Figure4A). However, this possibility is strengthened by the fact thatRNA species of �1.6 kb were detected on our pebA RNA gelblots (Figures 6B and 6C).

Regulation of pebAB Expression

We are not aware of any previous reports on the regulation ofthe expression of nonmammalian bilin reductase genes. pebABis also the only CCA-regulated operon to be identified that doesnot encode a component of the PBS. Our data show that theexpression of pebAB is not controlled significantly by any feed-back mechanism that responds to the lack of accumulation ofthe RNAs or proteins made from cpeBA and pebAB (Figures 6Band 6E). In addition, because PEB was not made in FdTq26,pebAB expression was not influenced significantly by the ab-sence of this chromophore (Figure 6B).

The pebAB operon also showed the residual light respon-siveness observed previously for the cpeBA and cpeCDE oper-ons in an FdBk mutant (Figures 6B and 6C) (Seib and Kehoe,2002). As for cpeBA and cpeCDE, the impairment of the CCAregulation of pebAB in the FdBk mutant involved both the lossof GL-mediated RNA accumulation and RL-mediated RNA re-duction. This finding suggests that the second light-responsive

system controlling the cpe operons also controls pebAB expres-sion. Because the CCA regulation of cpeBA expression has beenshown to be primarily transcriptional (Oelmüller et al., 1988), asignificant part of the coordination of cpeBA and pebAB expres-sion, as well as the subsequent PE and PEB synthesis, is likely tooccur at the transcriptional level.

The expression of cpeBA and pebAB (but not cpeCDE) re-quired the presence of CpeR (Figure 6E) (Cobley et al., 2002;Seib and Kehoe, 2002). This finding, along with the evidence thatcpeR is cotranscribed with cpeCDE and thus appears to coordi-nate the expression of cpeBA with cpeCDE during growth in GL(Cobley et al., 2002), suggests that CpeR plays a role in coordi-nating the expression of at least two GL-induced operons withcpeCDE. Because genes that encode proteins with similarity toCpeR are present in PE-producing cyanobacteria that do notundergo CCA (Cobley et al., 2002), the sole function of CpeR inF. diplosiphon may be to coordinate the expression of the genesthat encode PE apoproteins, linker peptides, and PEB biosyn-thetic enzymes rather than to broadly regulate the overall GL re-sponse during CCA. We recently isolated a number of additionalGL-induced genes from this organism (E. Stowe-Evans and D.Kehoe, unpublished data). It will be interesting to determine ifthe expression of these new genes requires CpeR as well. Ifso, it would suggest that CpeR is an important component inthe 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 re-gions of strong sequence similarity in the proximal regions oftheir promoters (Figure 7B). This finding further supports the ideathat there is coordinated transcriptional regulation of these oper-ons. These sequences, which are present close to the transcrip-tion start site in the cpeBA promoter, appear to be similarly posi-tioned upstream of pebAB, because the 3� end of this region is 26bp upstream of the predicted PebA translation start site.

The sequence conservation within this region of cpeBA pro-moters of three cyanobacterial species, including F. diplosi-phon, has been analyzed previously (Anderson and Grossman,1990; Dubbs and Bryant, 1991; Sobczyk et al., 1993). Twohighly conserved domains were identified in this �70-bp region(from 2 to 22 and from 29 to 69). The first contained anEscherichia coli–like 10 promoter element, whereas the sec-ond contained an E. coli–like 35 element and, upstream, a di-rect repeat contained within the sequence 5�-TGTTAN5TGTTA-3�. We found that the cpeBA promoter structure was similar inthe closely related species N. punctiforme throughout the ma-jority of the �70-bp region, although the sequence of the directrepeat was somewhat degenerate (8 of 10 identical) relative to theother species examined (Figure 7A) (Anderson and Grossman,1990; Dubbs and Bryant, 1991; Sobczyk et al., 1993). DNA-foot-printing studies have demonstrated that in F. diplosiphon, this re-peat was within the binding site of the DNA binding proteinsRcaA 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 pebABpromoter regions and to predict possible roles of the three sub-regions we have designated within it (Figure 7). The sequence

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within subregion 1 was as conserved between these promotersas between cpeBA promoters from different species (Figures7A and 7B); this also was true for the sequence within subre-gion 2. However, the sequence contained within subregion 3was less conserved between the F. diplosiphon cpeBA andpebAB promoters than between cpeBA promoters from differ-ent species (Figures 7A and 7B). Interestingly, there are similarrelationships between these subregions in pebAB promoter se-quences from F. diplosiphon and N. punctiforme, with high se-quence conservation in subregions 1 and 2 and more limitedconsensus within subregion 3 (Figure 7C). These data, alongwith the facts that both cpeBA and pebAB promoters are regu-lated through a common mechanism (Figures 6B and 6E) andthat the pebAB promoter is significantly weaker than the cpeBApromoter (R. Alvey and D. Kehoe, unpublished data), providesupport for the following model. The sequence within subre-gion 1 has been highly conserved to maintain strong interactionwith a trans-acting component(s) that may act within the CCAsignal transduction pathway, perhaps RcaA/PepB and/or RcaB(Sobczyk et al., 1993), whereas the variation that exists be-tween subregion 3 sequences leads to differences in the bind-ing affinity of DNA-dependent RNA polymerase that are mani-fested by differences in promoter strength. This model is anextension of previous proposals that subregion 1 is critical forthe binding of a factor involved in CCA responsiveness and thatsubregions 2 and 3 make up a portion of the binding site forRNA polymerase (Anderson and Grossman, 1990; Dubbs andBryant, 1991; Schmidt-Goff and Federspiel, 1993; Sobczyk etal., 1993).

Effect of PEB Absence on cpeBA and cpeCDE RNA and Protein Accumulation

The effect of bilin loss on the accumulation of its associatedapoprotein and corresponding RNA in vivo has not been exam-ined previously in cyanobacteria. Although the lack of PEB didnot significantly change the levels of cpeBA, cpeCDE, or cpcB2A2transcripts in FdTq26 in RL or GL (Figures 8B to 8D), the PE� sub-unit apoprotein and linker polypeptides in this mutant were re-duced to very low levels (Figure 9). These data match most resultsobtained in previous studies that have analyzed protein and RNAaccumulation patterns in lyase mutants as well as phycobilipro-tein site-directed and truncation mutants (Swanson et al., 1992;Bhalerao and Gustafsson, 1994; Jung et al., 1995; reviewedby Anderson and Toole, 1998). One exception was the findingthat, in Synechococcus PCC 7002, the cpcE and cpcF mutants(which encode the PC� subunit lyase) still contained substan-tial amounts of apoPC� subunit within their PBS (Zhou et al.,1992). The data presented here demonstrate that in F. diplosi-phon, the accumulation of PE and PE linkers is regulatedtightly by the degree of conversion of apoprotein to holoprotein(Anderson and Toole, 1998), whereas cpeBA and cpeCDE RNAaccumulation is not affected significantly by the absence of PEB.

It remains to be determined whether there are any similaritiesin the mechanisms that control the coordination of bilin and ap-oprotein synthesis in cyanobacteria and plants. Several differ-ent species of plants with deficiencies in P�B biosynthesishave been examined for their ability to accumulate phytochrome

mRNA and Phy apoprotein. In the tomato aurea mutant, whichcontains very little spectrally detectable holophytochrome, phy-tochrome RNA accumulates to wild-type levels but the plantscontain very little immunologically detectable Phy apoprotein(Parks et al., 1987; Sharrock et al., 1988). These results parallelour data for PE synthesis in the absence of PEB (Figures 8 and9). However, the consequences of failing to accumulate P�Bare different in other plant species. Arabidopsis hy1 and hy2mutants, which contain lesions in genes that encode heme ox-ygenase and P�B synthase (Davis et al., 1999; Muramoto etal., 1999; Kohchi et al., 2001), are not affected in Phy apopro-tein accumulation despite their reduced levels of P�B chro-mophore (Chory et al., 1989; Parks et al., 1989). Phy apoproteinalso accumulated in pea after treatment with the transaminaseinhibitor gabaculine, which severely reduced P�B levels (Joneset al., 1986; Konomi and Furuya, 1986). Thus, for both plantsand cyanobacteria, loss or reduction in the amount of a bilindoes not appear to impair the accumulation of the RNA that en-codes the corresponding apoprotein, but the stability of theapoprotein in the absence of chromophore varies among spe-cies. It will be interesting to determine if light-regulated tran-scriptional control, which is likely to coordinate the expressionof the cpeBA and pebAB operons in F. diplosiphon, also is amechanism by which phy, hy1, and hy2 RNA accumulation iscoordinated in plants.

METHODS

Strains

The wild type is a short-filament strain of Fremyella diplosiphon desig-nated Fd33 (previously SF33; Cobley et al., 1993). The FdBk mutant wasisolated from the wild type as described previously (Kehoe andGrossman, 1996). All FdTq mutants (FdTq1, FdTq26, and FdTq31) wereisolated as pigmentation mutants from plates of FdBk mutant cells afterheat-shock treatment, which mobilizes endogenous insertion elementsin F. diplosiphon (Seib and Kehoe, 2002; K. Shockley and D. Kehoe, un-published data), whereas FdG13 was isolated in a similar manner fromwild-type cells (R. Alvey and D. Kehoe, unpublished data).

Growth Conditions

Strains were cultured and analyzed spectrophotometrically as describedpreviously (Seib and Kehoe, 2002).

Transformation

Transformations used electroporation, and the genetic lesion in FdTq26was identified by complementation with an F. diplosiphon wild-type ge-nomic library as described previously (Kehoe and Grossman, 1998).Transformants exhibiting the FdBk mutant phenotype were analyzed asdescribed previously (Seib and Kehoe, 2002).

DNA Cloning, Sequencing, and Analysis

DNA size polymorphisms in cpeBAYZ and cpeCDE in the FdTq26 mu-tant were checked using the PCR-based approach described previously(Seib and Kehoe, 2002). The region around cpeR, cpeS, and cpeT was

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examined using the same approach and primers tqPCR1 (5�-CTTACT-ACGACCCCAAATCAAG-3�) and tqsp16 (5�-AGTATTGCGGAAATAGCT-TAACG-3�). The presence of an insertion element within pebA in FdTq26and FdG13 was established via PCR using genomic DNA from each mu-tant 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. diplo-siphon genomic DNA contained within pPLER2 were determined byDNA sequencing using an ABI 3700 (Perkin-Elmer, Foster City, CA).

The primers ER2ORF236a (5�-CGCGGATCCTTAGAGTTGACCGTA-GGTGAG-3�) and ER2ORF236b (5�-CGCGGATCCCTGATAGAGAGT-CAACTTCGCT-3�) were used to PCR amplify the region of pebA for theconstruction of pRA-4. BamHI restriction sites (underlined) were incor-porated into each primer. The amplified DNA was cut with BamHI and li-gated into the BamHI site in pPL2.7 (Chiang et al., 1992b). A similar strat-egy was used to make pRA-45, except that the primers used wereER2ORF236a 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, andcpcB2A2 were performed as described previously (Seib and Kehoe,2002). The two probes used for the detection of pebA and pebB tran-scripts were created by PCR amplification. The primers used for probe 1synthesis were ER2ORF236a and ER2A3 (5�-TAATGGCACAGCATC-TTCAAACA-3�), and the primers used for probe 2 were ER2B5 (5�-CAC-TATGATTTGCCCTTACTCG-3�) and ER2A3 (5�-TAATGGCACAGCATC-TTCAAACA-3�). Probe 3, which was used to analyze ORF3 expression,was amplified by PCR using ER2B3 (5�-TGTAAGCACGGTAAGAAT-GATGA-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

mol·m2·s1. Cells were quick-cooled to 4�C by swirling in flasks sub-merged in liquid nitrogen and then centrifuged at 4000g at 4�C for 10min. The resulting pellet was resuspended in an equal volume of ice-coldextraction 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 M 6-amino-n-hexanoic acid) and re-centrifuged as described above. The cell pellet then was suspended in3 mL of extraction buffer A and lysed at 4�C by two passages through aFrench pressure cell at 18,000 p.s.i. Protein concentrations were esti-mated using Bradford reagent (Bio-Rad, Hercules, CA) with BSA as astandard. Protein samples were combined with an equal volume of 5�SDS loading buffer, boiled for 3 min, and then centrifuged at 15,000g for1 min. Approximately 50 g 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 Coo-massie Brilliant Blue R 250 and then destained before imaging with aUMAX 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 mLof liquid culture to an OD750 of �1.0 and then centrifuged at 3600g for 10min at 4�C. The supernatant was discarded, and the pellet was frozen

rapidly in liquid nitrogen. Thirty milliliters of acetone were added to thefrozen pellet and vortexed briefly. The suspension was centrifuged, thesupernatant decanted, and the pellet frozen as described above. Thisacetone wash was performed twice more, and then the pellet waswashed two more times as described above with methanol instead ofacetone. All subsequent steps were performed with the room lights off.The pellet was resuspended in 10 mL of methanol containing 2 mg/mLHgCl2. The mixture was passed through a sintered glass filter, and the fil-ter was washed once with 10 mL of methanol. The filtered liquid was di-luted 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 and0.1% TFA (20:80, v/v), 3 mL of CH3CN and 0.1% TFA (60:40, v/v), 3 mL ofCH3CN, 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 and0.1% 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 at20�C before HPLC–mass spectrometry analysis.

Bilins were resuspended in 1.5 L of DMSO. After thorough mixing, 13.5

L of HPLC mobile phase were added to the extracts. Phycoerythrobilin andphycocyanobilin standards were a gift from Beronda Montgomery (IndianaUniversity, Bloomington), and the 15,16-dihydrobiliverdin standard was a giftfrom Nicole Frankenberg in the laboratory of Clark Lagarias (University ofCalifornia, Davis). Commercial biliverdin IX� hydrogen chloride (Frontier Sci-entific, Logan, UT) was also used as a standard. Standards were prepared at�5 M in HPLC mobile phase. The chromatographic conditions were similarto those described previously (Frankenberg et al., 2001). Briefly, 12.5-L in-jections of the bilin extracts were separated isocratically on a 2.1- � 250-mmUltracarb 5-m ODS20 column (Phenomenex, Torrence, CA) using a 1:1 ac-etone (Optima grade; Fisher):20 mM aqueous formic acid (Aldrich, St. Louis,MO) solution. A constant flow rate of 200 L/min was provided by a SurveyorMS pump (ThermoElectron Corp., Waltham, MA). The eluent was analyzedby a UV-visible light photodiode array (Surveyor PDA; ThermoElectron Corp.)and an ion trap mass spectrometer (LCQ DecaXP Plus; ThermoElectronCorp.). 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 L/min. Thesource 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 iontransfer tube. Positive ion mass spectra between mass-to-charge ratios of400 and 1000 were recorded at �1 Hz. All chromatographic and mass spec-trometric data were analyzed using the XCalibur 1.3 software package (Ther-moElectron Corp.). The identities of the eluted bilins were confirmed by theirelution times, mass spectra, and UV-visible light spectra.

Genome Analysis

All sequences from the Department of Energy’s JGI can be located athttp://spider.jgi-psf.org/JGI_microbial/html/. National Center for Biotech-nology 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 pub-lication will be made available in a timely manner to all investigators onsimilar terms for noncommercial research purposes. To obtain materials,please contact David M. Kehoe, [email protected].

Accession Numbers

The GenBank accession number for the entire F. diplosiphon genomicDNA fragment contained within pPLER2 is AY363679. Accession num-

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bers for the other sequences mentioned in this article are as follows:ZP_00109076 and ZP_00111776 (hypothetical proteins in N. puncti-forme); 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 pro-moter in F. diplosiphon); and AJ278499 (Prochlorococcus SS120).

ACKNOWLEDGMENTS

We thank Nicole Frankenberg for the gift of DHBV and Beronda Montgomeryfor the gift of PEB and PCB. We also thank Sam Beale, Clark Lagarias, andNicole Frankenberg for helpful discussions and Beronda Montgomery,Emily Stowe-Evans, Barbara Balabas, James Ford, and Lina Li forthoughtful comments on the manuscript. This material is based onwork supported by the National Science Foundation under Grant MCB-0084297 to D.M.K. and Grant CHE-0094579 to J.P.R. We also gratefullyacknowledge support from the Indiana Genomics Initiative, which isfunded in part by the Lilly Endowment, Inc.

Received June 25, 2003; accepted August 15, 2003.

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DOI 10.1105/tpc.015016; originally published online September 24, 2003;Plant Cell

Richard M. Alvey, Jonathan A. Karty, Elicia Roos, James P. Reilly and David M. KehoeLight Regulation of Apoprotein and Pigment Biosynthetic Enzyme Gene Expression

Reveal CoordinatedFremyella diplosiphonLesions in Phycoerythrin Chromophore Biosynthesis in

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