Chromatic adaptation in a mutant of Fremyella diplosiphon incapable of phycoerythrin synthesis

BIOCIIIMIE, 1985, 67, 109-117

Chromatic adaptation in a mutant of Fremyella diplosiphon incapable of phycoerythrin synthesis.

Simone BEGUIN, G6gard GUGLIELMI, Rosmarie RIPPKA, Germaine COHEN-BAZIRE.

Unitd de Physiologie Microbienne, Ddpartement de Biochhnie et Gdndtique Moldculaire - - C.N.R.S-E.R.A. 398 - - Institut Pasteur, 28, rue da Docteur Roux, 75724 Paris Cedex 15.

R~sum~ - - Fremyella diplosiphon est une o'anobactdrie soumise h l'adaptation chromatique. Un mutant en a ~td isold qui ne synthdtise phts la phycodrythrhze mais qui est to l l jo t trs capable de moduler son contenu en biliprotdines suivant la qualitd de la htmidre regue pendant sa croissance.

Aprds croissance en htmi~re rouge, la composition en biliprotdines du mutant et du tjTe sauvage est identique. Par contre, la htmidre verte hlduit, chez le mutant, la synthdse d'un chromophore de t)Te bilivioline que l'on trouve lid ~ la sous-unitd ct de sa phycocyanhle. Ces rdsultats sont discutds en relation avec la rdgulation et les voies de biosynthOse possibles des biliprotdines.

Mots-cl~s : cyanobact~rie / adaptation chromatique / phycnbiliprot~ines / chromophores.

Summary m A mutant o f the chromatically adapting cyanobacterium Fremyella diplosiphon, hwapable o f phycoerythrin synthesis but responding to wavelength modulation o f its biliprotehl content, was isolated. The biliprotein composition o f the mutant and o f the wild type were identical after growth in red light, but green light hldaced, hi the mutant, the synthesis o f a biliviolin-type chromophore bound to some o f the eL subunits o f its phycoo,anin.

Implications o f the results on the regulation and possible pathways o f biliprotehl biosynthesis are discussed.

Key-words : cyanobacterium / chromatic adaptation / phycobiliproteins / chromophores.

Introduction

Cyanobacteria are the only group of photosynthetic procaryotes that perform an oxygenic type of photosynthesis [1]. As in the eucaryotic red algae, phycobiliproteins are the major light har-

Abbreviations :

AP : allophycooanin; PC : phycocyanin;" PE : phycoerythrin; PEC : phycoerythrocyanin; PCB : phycoo'anobilin;

vesting pigments. These brightly coloured soluble proteins are organized in multimolecular structures, the phycobilisomes [2], that are attached to the stromal surfaces of the photosynthetic lamel- lae (thylakoids), where the photochemical reactions of photosynthesis take place.

PEB : phycoerythrobilin; PXB : phycobilivioBn-type chromophore o f unknown stntc-

lure; SDS : sodium dodecyl sulfate; ~-SH : [~-mercaptoethanol.

110 S. Beguhl and coll.

All cyanobacteria synthesize at least two types of phycobiliproteins : allophycocyanin (AP) and phycocyanin (PC) which absorb light energy between 600 and 650 nm. Many species synthesize, in addition, a third type of biliprotein, phycoerythrin (PE) which extends their light absorbing capacity in the green region of the visible spectrum to around 560 nm. A few species, which do not form PE, synthesize a fourth type of biliprotein, phycoerythrocyanin (PEC) which absorbs maximally around 568 nm [1, 3]. All these biliproteins are multimeric proteins whose monomers are made up of two dissimilar polypeptide chains et and [3 of molecular masses (Mr) between 16 and 22 Kd, each containing one or more open chain tetrapyrrolic chromophores covalently bound to the polypeptide chain by at least one thioether linkage [4]. They are the major components of phycobilisomes and represent approximately 85 to 88 per cent of the total phycobilisomal protein content. The remaining 12 to 15 per cent are constituted by a number of linker polypeptides that allow the proper assem- bly of biliproteins, the channeling of light energy, and the binding of these organelles to the photosynthetic membranes [51.

Intact phycobilisomes can be isolated from the cells [6] and their structure, as observed in the electron microscope reveals two distinct do- mains : a core made up of two or three small cylinders, and six rods composed of stacked discs which diverge, in a hemidiscoidal array, around the core [4]. The composition of the core is complex [4]: it contains all the allophycocyanin of the phycobilisome as well as minor biliproteins that function as terminal energy acceptors in the phycobilisomes [51. The rods are made up of stacked hexameric molecules of PC, to whose periphery are added hexameric molecules of PE or of PEC in strains which synthesize these latter biliproteins.

In terms of macromolecular composition, phycobiliproteins represent a large fraction (30 to 60 per cent) of the cyanobacteriai cellular proteins. Some PE synthesizing strains have evolved a regulatory mechanism which enables them to modulate the rate of synthesis of one or both of their major phycobiliproteins in response to the quality of light received by. the c611s during growth : this phenomenon is known as corn~le- mentaty chromatic adaptation [7, 8]. Two types of chromatically adapting strains have been described [9] : the first regulates only the synthesis of PE, whereas the second type photoregulates the synthesis of bot.h PE and PC.

Freno'ella diplosiphon (Calothrix PCC 7601) shows the characteristic features of the second group of chromatic adapters. Growth in green light elicits the synthesis of PE and of its specific linker polypeptides. Growth in red light abolishes the synthesis of PE and associated linker polypeptides, whereas the PC content of the cells in- creases due to synthesis of a second type of PC (PC2) in addition to PC~. In green light, only PC~ is synthesized, at a rate equivalent to that in red light [10, 11]. Studies of phycobilisome structure and composition in chromatic adapting strains [12, 13] have shown that chromatic regulation is achieved by a modification of the composition of the peripheral portion of the rods while the allophycocyanin containing core is unaffected.

Studies of action spectra for chromatic adaptation [8] have led to the hypothesis that this process is under the control of a photoreversible pigment analogous to, but spectrally distinct from, phyto- chrome, the photoregulatory biliprotein of plants [14]. Whereas chromatic regulation appears to operate at the transcriptional level and requires synthesis of both mRNA and protein [15], the photocontrol itself seems to he photochemical in nature [16].

Underlying this macroregulatory mechanism, there exists a general and finer one: under normal physiological conditions, the synthesis of phycobiliproteins themselves is a well coordinated process : it involves not only the synthesis of the apoproteins of the et and 13 subunits in equimolar amounts, but the concommitant pro- duction and attachment of the appropriate bilin chromophores (phycocyanobilin, phycoerythrobilin or phycobiliviolin-type) to the proper cysteinyl residues on the apoproteins.

In an attempt to gain some insight into these regulatory processes, a number of spontaneous or induced mutants affected in the biosynthesis of biliproteins or their photocontrol have beer~ isolated in our laboratory. Several mutants which-are affected in the photocontrol of PE or PC synthesis are similar to some of the mutants described by Cobley et al. [17]; others seem to be impaired in the synthesis of phycobiliproteins. One such mutant appears to have a lesion in the normal pathway of PE synthesis: this paper describes its properties.

PhycoerythrhT-less mutant of Fremye l l a d i p l o s i p h o n 111

M a t e r i a l s and methods

Biological material and growth conditions

Fremyella diplosiphon, a facultative photoheterotro- phic filamentous cyanobacterium, was kindly provided by L. Bogorad. This strain has been maintained in the Pasteur Culture Collection as Calothrix PCC 7601 (Rippka, unpublished). Liquid cultures were grown as described by Rippka et al. [18] either in BGI 1 medium or in BGII medium supplemented with 0.5% w/v glucose. Chromatic illumination was provided by red or green plastic filters interposed between the culture vessels and the fluorescent tubes (Osram Universal cool white); their transmittance has been published by Tandeau de Marsac [9]. Light intensity was approximately 5.!.tE.m-'s -~ after inoculation and was gradually increased to 20.1aE.m-2s -~ as the turbidity of the cultures increased. The cells were collected by centrifugation towards the end of the exponential growth. Solid media were prepared as described by Alien [19]. To obtain colonies derived from a minimum number of ceils, the long filaments were broken by several pas- sages through a fine needle (20 gauge) attached to a syringe. Plates were incubated in transparent plastic boxes covered, when desired, by red or green plastic filters, and illuminated from above.

Colonies were examined under a dissecting microscope, and mutant sectors or filaments of unusual colour were isolated with a finely drawn-out Pasteur pipette. They were purified on fresh solid medium through at least three successive transfers.

protease inhibitor and no ILmercaptoethanol (~-SH). After breakage of the cells in the French pressure cell, the clarified extract was treated with 1% w/vol strep- tomycin sulphate for 1 h at 4 °C to remove most of the membrane material.

Isolation of the c~ and [I subunits of phycoo,anhl

Phycocyanin subunits were separated by chromato- graphy on Bio-Rex 70 at pH 2.8 with increasing concentrations of urea, as described by Glazer and Fang [20]. The ct subunits were eluted with 7.5 M urea, 10mM ~-SH; the I~ subunits with 9 M urea, 10mM 13-SH.

Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis in the presence of 8 M urea at pH 7.8 was performed as described by Cohen-Bazire et al. [21], in 15 cm tubes with an inside diameter of 0.5 cm. Approximately 100 I.tg of protein was deposited on each gel. The gels were photographed without staining immediately after electrophoresis. Polyacrylamide electrophoresis in the presence of SDS was performed on slab gels 1.5 mm thick, using the discontinuous buffer of Laemmli [22]. The running gels, containing 15 % w/v acrylamide (acrylamide : bis-acrylamide : 30 : 0.8), were stained for i h with Coomassie brilliant blue (0.25 % w/v) in a mixture of acetic acid : methanol : H20 (10 vol : 25 vol : 65 vol) and destained with a solution of acetic acid : methanol : H20 (15 vol : 30 vol : 55 vol).

Mutagenesis

Exponentially growing cells were collected by centrifugation, transferred to BGII medium containing 300 I~g/ml of N-methyl-W-nitro N-nitrosoguanidine at a density of approximately 5.10 s filaments per ml and incubated for 2 h in the light. The cell suspension was washed three times with sterile medium, transferred into fresh medium (10 s filaments per ml) and incubated in the light. After two divisions, the filaments were collected by centrifugation, broken by passage through a syringe and plated at appropriate dilutions. Colonies appeared after two to three weeks of incubation under white light.

Preparation of phycobilisomes

Phycobilisomes were isolated and purified by centrifugation on sut:rose step gradients following the procedure of Bryant et aL [12].

Purification of phycoo,anhl

Phycocyanin was purified from 40g o1" cell paste following the procedure described by Glazer and Fang ~20] for Anabaena PCC 6411, with the following modifications : all 'buffers contained 1 mM EDTA as

Speclroscoplc measurements

Absorption spectra were measured on a Cary 17 spectrophotometer. The molar amount of individual biliproteins in either extracts or in phycobilisome preparations was estimated using the simultaneous equations of Bryant et al. [12]. For the estimations of polypeptide bound chromophores, the following molar absorption coefficients were used : 35 500 M -~ cm -~ at 662 nm for phycocyanobilin, [20] and 43 000 M -~ cm- ' at 590 nm for the phycobiliviolin-type chromophore, [23] both in 8 M urea at pH 3.

Results

After mutagenes i s o f F. diplosiphon, colonies o r sectors o f co lours d i f fe ren t f rom the o l ive-green wi ld type co lon ies a p p e a r e d on pla tes exposed to white light. Some were ex t r eme ly pale green o r ye l low green, some blue , a n d some a p p e a r e d more in tense ly red than the wi ld type. The i r p h e n o t y p i c cha rac t e r i za t ion will be p u b l i s h e d e lsewhere . One o f the ye l low green colonies , G9, was se lec ted for the p resen t s tudy.

112 S. Beguh7 and coll.

Phycobiliproteh7 composition of wild O'pe and mutant G9 ceils after growth hi red and green light

The absorption spectra of soluble extracts of G9 and wild type ceils are shown in Figures IA and 1B.

After growth in red light, the biliprotein content and composition of the mutant and of the wild type were identical; their biliprotein content approximated 40 % of the total cell proteins and consisted of phycocyanin (PC) and allophycocyanin (AP) in a molar ratio close to 3:1. The pattern of biliprotein subunits revealed by electrophoresis in 8 M urea at pH 7.80 was also identical (Fi- gure 2, gels 1 and 2) : two different ct subunits, cq and ct2 of a colour typical for PC, were visible on these gels, showing that both the inducible and the constitutive phycocyanin were synthesized under these conditions, the most basic subunit being ct~ of the inducible phycocyanin (PC2) [11] that is synthesized only in red light*.

The difference between the mutant and the wild type, although visible in white light, became most apparent after growth in green light. Under these conditions, the biliprotein content of the mutant was reduced to 20% of the total cell proteins, whereas it represented 47 % in the wild type cells.

As shown in Figure IB, this difference in the mutant could be accounted for by the absence of the large amount of phycoerythrin (PE) (absorbing maximally at 563 nm) present in the wild type extract, since the amounts of allophycocyanin (AP) and of phycocyanin (PC) were very similar in both cell types. The calculated molar ratio of the three major biliproteins in the wild type extract (PE'PC:AP) was 3"1:1; in the mutant the molar ratio of PC to AP was 0.7. Immuno- precipitin reactions carried out with antibodies raised against pure PE confirmed the absence of this biliprotein or of any cross-reacting material in the mutant "extracts (data not shown). The absorption spectrum of the mutant extract showed the presence of a new pigment with an absorption maximum around 580 nm. The synthesis of this new pigment was under chromatic control since it appeared only in cells grown in green light.

* The denomination used for the subunits of the two PC types, constitutive (PC0 and inducible (PC2) is that used by Bryant and Cohen-Bazire [10] and Bryant [ l l l : PC~ : ct_, l~z, PC2 : eft ~:.

The pattern of coloured bands on gels after electrophoresis in the presence of 8 M urea of an extract of mutant cells grown in green light confirmed the absence of PE subunits (Figure 2, gel 3) which, as evidenced by partially purified PE from a red mutant (Figure 2, gel 4), are normally located just below the I~ subunit of AP. However, contrary to extracts from cells grown in red light, the band corresponding to the ct, subunit of PC was not blue but slightly purple. Thus, the new photoregulated pigment is carried by a polypeptide with the same charge as that of the ct2 subunit of PC~.

Phycobilisomes of wild O,pe and mutant G9 grown hi red and green light : polypeptide composition

The phycobilisomes from G9 grown in green light sedimented more slowly than those of the wild type in sucrose step gradients, and accumu- lated in the region of the boundary between the 0.5 M and 0.75 M sucrose layers. The wild type phycobilisomes, and those extracted from the mutant grown in red light, sedimented in the region of the gradient overlapping the 0.75 M and 1 M sucrose layers. The polypeptide composition of phycobilisomes was analyzed by polyacrylamide slab gel electrophoresis in the presence of SDS. The photographs of the stained gels are shown in Figures 3a and 3b.

As expected, the phycobilisomes of wild type and G9 grown in red light were of identical composition (Fig. 3a, lanes 1 and 2). In addition to the bilin carrying subunits ofAP, PC~ and PC2, four linker polypeptides of Mr 30, 38, 39 and 92 K were present. The polypeptide of Mr 92 K has been shown to carry a bilin-chromophore [unpublished observatiofi, 24].

In the type of gel presented here, some of the subunits of the different biliproteins a~e not separated: their position is indicated on the margin of the gels.

The pattern of polypeptides of phycobilisomes extracted from green light grown wild type and mutant cells is shown in Figure 3b (lanes 3 and 4).

The phycobiliprotein subunits of the wild type are relatively well resolved : the ~ subunit of PC~ is visible above the two subunits of AP; the 1~ subunit of PC~ migrates below the two heavily stained subunits of PE. Four linker polypeptides are visible : the 30 K and 92 K polypeptides that are found in red light phycobilisomes, and two new polypeptides of Mr35 K and 36 K that

Phycoerythrin-less mutant of Fremyella diplosiphon 113

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FIG. 1. - - A and B. Absorption spectra o f ph)cobiliproteins in extracts ofFremye l l a d ip los iphon wild type and mutant G9 after growth in.

A = red light; B : grenn light. A = dotted line : wild type; solid line : mutant . B = dotted line : wild type; solid line : mutant .

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Fits. 2. - - Polyacrylamide gel electrophoresis in 8 M urea at pH LS, o f biliproteins in extracts o f red and green light-grown F. d ip los iphon wild type and mutant G9.

The gels have been photographed without stain. Red light g rown : wild type ' ( l ) ; mutan t (2). Green light g rown mutan t (3). Partly purified E diplosiphon phycoerythr in (4).

replace the 38 K and 39 K polypeptides normally present in red light. Synthesis of these new polypeptides is correlated with that of PE. In the mutant phycobilisomes (Fig. 3b, lane 4), the two subunits of PE are absent; a polypeptide (ttz PXB), purple before colouration of the gel, is visible above the Ctz subunit of PCI. These phycobilisomes show only the two linker polypeptides of Mr30 K and 92 K that are not

photoregulated in the wild type. The two polypeptides of Mr35 and 36 K associated with PE synthesis are completely absent. Trace amounts o f a 48 K polypeptide are also visible on the gel : a polypeptide of similar molecular mass has been observed in many preparations of phycobilisomes; since its concentration varies from batch to batch, it may not be an integral component of these organelles.

The phycoo,anhl of G9 grown hi green light

The separation of the pigment absorbing around 580 nm, synthesized by G9 grown in green light, could not be achieved by standard methods of protein purification. This pigment was associated with the phycocyanin fraction through all purification steps. The spectrum of the purified phycocyanin fraction in 10 mM K PO4 buffer at pH 7 and of an equivalent amount in 8 M urea at pH 3 are shown in Figure 4. The native protein shows two absorption maxima in the red, at 608 a n d - 5 9 0 n m . The spectrum of the denatured protein shows a pronounced shoulder around 605 nm, a maximum at 662 nm in the red, and a broad absorption band between 330 and 35 nm in the blue. Whereas the 662 nm absorption band can be ascribed to polypeptide bound phycocyanobilin (PCB) [20], the shoulder at 605 nm and the absorbance in the blue is indicative of a chromo- l~hore similar to the phycobiliviolin-type chromophore carried by the (z subunit of phycoerythrocyanin [25].

114

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FIG. 3, -- Polyacr)'lamide gel electrophoresis hi the presence o f SDS, o f phycobilisomes fronl red light : 3a and green light : 3b - - grown wild type and mutan t cells, The gels have been stained with Coomassie brilliant blue.

3a • wild type (1); mutan t (2); 3b : wild type (3); mutan t (4). The identifications of biliprotein subuni ts and the Mr of the linker polypeptides are indicated on the Figures et2 PXC identifies the ctPC~ subuni t which carries the PXB chromophore.

The subunit carrying exclusively the biliviolin- type chromophore (PXB) eluted from a Bio-Rex70 column at pH 2.8 and 7.5 M urea slightly ahead of the ct subunits that were enri-

ched in PCB but never entirely free from the biliviolin type chromophore. This could be chec- ked easily by measuring the absorbance of all fractions at 662 nm and at 590 nm.

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,0.1 FtG. 4. -- Absorption spectra o f purified phyco- c)'anin from the mutant G9 ofF. diplosiphon grown in green light : purified phycocyanin in 10 mM K PO~ buffer pH 7 : solid line; equivalent amount of phycocyanin in 8 M urea at pH 3 : dotted line. The absorbance scale for the spect rum in urea is twice that of the spectrum in 10 m M K PO~.

Phycoerythrht-less mtttant of Fremyella diplosiphon 115

The 13 subunit of G9 PC was eluted with 9 M acid urea : its spectrum was that of polypeptide- bound PCB [26].

Calculations, based on the molar extinction coefficient of polypeptide bound PCB at 662 nm (35,500 M -~ cm-~), allow the conclusion that hal f of the ct subunits carry a PCB type and half of them carry a biliviolin-type chromophore (PXB), provided that each et subunit carries only one type of chromophore.

The pooled fractions which did not show any absorbance at 662 nm were brought to pH 7 with 1.5 M K PO4 buffer and renaturated by dialysis in 10 mM K PO4 buffer at pH 7. The spectra before and after renaturation are shown in Figure 5. The spectrum in acid urea is very similar to that o f the denatured c~ subunit of phycoerythrocyanin with its broad absorption between 550 and 600 nm in the red and a sharp absorption maximum at 330 nm. However, upon renaturation, the two spectra differ substantially. With the exception of the absorption band at 330 nm, the entire spectrum of the G9 PC et subunit carrying the bill- violin-type chromophore (PXB) is shifted towards the red : it shows increasing absorption from 500 to 590 nm and a sharp absorption band at 608 nm, and is therefore different from the cL subunit o f phycoerythrocyanin which has a broad absorption peak extending from 500 to a maximum at 560 nm [25].

D i s c u s s i o n and conc lus ions

The mutation affecting G9 is not apparent when the cells are grown in red light but is evidenced best after growth in green light. In this

mutant the synthesis of the major biliprotein is still photoregulated and thus the photoregulatory pigment must be active.

After growth in red light, extracts of both the wild type and the mutant, as well as their phycobilisomes, have the same biliprotein composition ~. the molar ratio of PC to AP is 3 to 1. Based on previous studies [12], we can deduce that the phycobilisome rods contain 3 hexamers of PC • one hexamer of PC, and two of PC2. The two linker polypeptides of Mr 38 and 39 K appear coordinately synthesized with the hexamers of PC2.

After growth in green light, the molar ratio of biliproteins in the wild type is 3 PE:IPC~ :lAP. The rods of the phycobil isomes contain 3 hexamers of PE and one of PCj. The synthesis of the two linker polypeptides of Mr35 and 36 K is coordinated with the synthesis of three hexamers of PE. The linker polypeptides of Mr 38 and 39 K synthesized in red light are either absent, or their synthesis is strongly diminished.

The mutant G9 grown in green light does not synthesize any PE. This biliprotein can neither be detected spectroscopically nor by fluorescence emission. Immunoprecipi ta t ion test performed on whole extracts with antibodies raised against purified PE showed no detectable cross reacting material. Green light phycobilisomes of mutant G9 are devoid of PE and of PE specific linkers, and of course of PC2 and its specific linker polypeptides. The rods of green light phycobilisomes are short, consisting of only one hexamer of an abnormal PC. This PC contains 13 subunits with phycocyanobilin (PCB) chromophores attached to them. These subunits are linked to a mixture of a subunits with two different types of chromophores : one fraction carries the normal

FIG. 5. - - Absorption spectra o f the a-PC subunit carrying the PXB chromophore isolated from the green light grown mutant : in 7.5 M urea at pH 2.8 : dotted line; after renaturat ion in 10 mM K PO~ buffer at pH 7 : solid line. The absorbance scale of the spectrum in urea is twice that of the' spectrum in 10 m K PO~.

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PCB chromophore, the other carries a chromophore of unknown structure, PXB. There is a remarkable similarity between this PXB and the biliviolin type chromophore carried by the ct subunits of PEC, but only in the denatured state of these subunits. In the renatured state, the absorption spectra of these two et subunits are quite different, possibly reflecting different inte- ractions of the two types of polypeptide chains on the absorption spectra of the chromophores.

Several questions arise from our s tudies: where is the precise site of the mutation in G9 ? Is it affecting the synthesis of the PE apoprotein subunits or the synthesis of the chromophore of PE, phycoerythrobilin (PEB)?

The chemical structures of PC and PE chromophores, phycocyanobilin (PCB) and phycoerythrobilin (PEB), respectively, have been establis- hed in addition to their peptide bound forms [27, 28] (see Fig. 6) : PCB and PEB are isomers. The exact chemical structure of the biliviolin-type chromophore carried by the a subunit of PEC and, afortiori, the structure of the PXB chromo-

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phore in mutant G9, are unknown. In a recent review, A.N. Glazer [4] speculated on the bio- synthetic pathway of phycobiliproteins and drew attention to an unanswered quest ion: "What determines which bilin is present at a given position on a polypeptide chain of a biliprotein ?" One hypothesis proposed by Glazer was the following : a phycocyanobilin (PCB) binds, in an enzyme catalyzed reaction, to the available cysteinyl residues in the polypeptide chain. The other bilins, PEB and PUB (the phycourobilin chromophore present in red algal PE), are formed by modifications of the PCB bound to the polypeptide, either by specific isomerases or, non enzymatically, by the folding energy of the polypeptide chain. Our results seem to support these hypotheses.

The phenotype of the F. diplosiphon mutant can be explained if one assumess that the mutation has abolished the capacity to synthesize the apopolypeptides of PE. Green light activates or induces an isomerase which, in the wild type, acts on PCB after it is fixed on the correct polypeptide chains, ke. those o f the PE subunits. In the mutant the polypeptides are absent. It is probable that the folding of the c~ polypeptide chains of PC is such that the PCB chromophore bound to it is accessi- ble to the isomerase. The isomerase transforms some PCB bound to cx PC into PXB which, probably for steric reasons, cannot be further transformed into PEB. This last assumption im- plies that the biliviolin-type PXB chromophore is an intermediate stage in the isomerization of protein bound PCB into PEB. Another possibility could be that the biliviolin-like properties of the chromophore are the result of its interaction with a polypeptide chain that is not its natural accep- tor, i.e. the ct subunit o f PC~. Only when attached to the apoproteins of PE would it present its PEB configuration. This last point will be resolved only when the chemical structure of the PY~B in G9 and of the biliviolin-type chromophore of PEC have been determined.

Acknowledgements

One of us (G. CB.) shouM like to thank Pr. A.N. Glazer (University o f California, Berkeley, USA)for stimulating discussions. IVe are gratefid to Air. Jean-Marc Panaud (Service de Photographie. Institut Pasteur) for his skillful help with the photographic work and to Dr. M. Herdman for reading and correcthlg the manuscript.

Phycoeo,thrhl-less mutant o f Fremyella diplosiphon 117

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