Rod substructure in cyanobacterial phycobilisomes: phycoerythrin assembly in synechocystis 6701 phycobilisomes

Synechocystis 6701 phycobilisomes consist of a core of three cylindrical elements in an equilateral array from which extend in a fanlike manner six rods, each made up of three to four stacked disks. Previous studies (see Gingrich, J. C., L. K. Blaha, and A. N. Glazer, 1982. J. Cell Biol. 92:261-268) have shown that the rods consist of four disk-shaped complexes of biliproteins with "linker" polypeptides of 27-, 33.5-, 31.5-, and 30.5-kdaltons, listed in order starting with the disk proximal to the core: phycocyanin (alpha beta)6-27 kdalton, phycocyanin (alpha beta)6-33.5 kdalton, phycoerythrin (alpha beta)6- 31.5 kdalton, phycoerythrin (alpha beta)6-30.5 kdalton, where alpha beta is the monomer of the biliprotein. Phycoerythrin complexes of the 31.5- and 30.5-kdalton polypeptides were isolated in low salt. In 0.05 M K-phosphate-1 mM EDTA at pH 7.0, these complexes had the average composition (alpha beta)2-31.5 and (alpha beta)-30.5 kdalton polypeptide, respectively. Peptide mapping of purified 31.5- and 30.5- kdalton polypeptides showed that they differed significantly in primary structure. In 0.65 M Na-K-phosphate at pH 8, these phycoerythrin complexes formed rods of stacked disks of composition (alpha beta)6- 31.5 or (alpha beta)6-30.5 kdaltons. For the (alpha beta)-30.5 kdalton complex, the yield of rod assemblies was variable and the self- association of free phycoerythrin to smaller aggregates was an important competing reaction. Complementation experiments were performed with incomplete phycobilisomes from Synechocystis 6701 mutant strain CM25. These phycobilisomes are totally lacking in phycoerythrin and the 31.5- and 30.5-kdalton polypeptides, but have no other apparent structural defects. In high phosphate at pH 8, the phycoerythrin-31.5- kdalton complex formed disk assemblies at the end of the rod substructures of CM25 phycobilisomes whereas no interaction with the phycoerythrin-30.5 kdalton complex was detected. In mixtures of both the phycoerythrin-31.5 and -30.5 kdalton complexes with CM25 phycobilisomes, both complexes were incorporated at the distal ends of the rod substructures. The efficiency of energy transfer from the added phycoerythrin in complemented phycobilisomes was approximately 96%. The results show that the ordered assembly of phycoerythrin complexes seen in phycobilisomes is reproduced in the in vitro assembly process.     

Cyanobacterial phycobilisomes. Particles from Synechocystis 6701 and two pigment mutants

The phycobilisomes of the unicellular cyanobacterium Synechocystis 6701, grown in white light, contain C-phycoerythrin, C-phycocyanin, and allophycocyanin in a molar ration of approximately 2:2:1, and in addition, polypeptides of 99, 46, 33.5, 31.5, 30.5, and 27 x 10(3) Daltons, as well as a trace of a approximately 9 x 10(3)-dalton component. Two nitrosoguanidine-induced mutants of this organism produce aberrant phycobilisomes. Crude cell extracts of these mutants, 6701-NTG25 and NTG31, contain phycoerythrin, phycocyanin, and allophycocyanin in a molar ration of 1:5:1:1 and 0.55:0.3:1.0, respectively. The phycobilisomes from both mutants lack the 33.5 x 10(3)-dalton polypeptide. Wile-type phycobilisomes consist of a core composed of an equilateral array of three cylindrical elements surrounded by six rods in a fanlike arrangement. The rods are made up of stacked disks, 11 nm in diameter and 6 nm thick. In phycobilisomes of mutant 6701-NTG25, numerous particles with fewer than six rods are seen. Mutant 6701-NTG31 produces incomplete structures that extend from triangular core particles, through cores with one or two attached rods, to cores with as many as five rods. The structure of the core appears unaltered throughout. The amount of phycocyanin (relative to allophycocyanin) appears to determine the number of rods per core. A common assembly form seen in 6701-NTG31 is the core with two rods attached at opposite sides. From observations of this form, it is concluded that the core elements are cylindrical, with a height of 14 nm and a diameter of 11 nm. No consistently recognizable structural details are evident within the core elements.     

Heterologous assembly and rescue of stranded phycocyanin subunits by expression of a foreign cpcBA operon in Synechocystis sp. strain 6803.

Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membrane-associated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin beta subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin alpha and beta subunits assemble with endogenous phycobilisome components. The phycocyanin alpha subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.     

Role of the Colorless Polypeptides in Phycobilisome Assembly in Nostoc sp

We have identified the function of the `extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrin- and phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70: 1055-1059); the 29 kilodalton polypeptide attaches the phycoerythrin- and phycocyanin-containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redlight-adapted cells; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination.     

Mutations that affect structure and assembly of light-harvesting proteins in the cyanobacterium Synechocystis sp. strain 6701.

The unicellular cyanobacterium Synechocystis sp. strain 6701 was mutagenized with UV irradiation and screened for pigment changes that indicated genetic lesions involving the light-harvesting proteins of the phycobilisome. A previous examination of the pigment mutant UV16 showed an assembly defect in the phycocyanin component of the phycobilisome. Mutagenesis of UV16 produced an additional double mutant, UV16-40, with decreased phycoerythrin content. Phycocyanin and phycoerythrin were isolated from UV16-40 and compared with normal biliproteins. The results suggested that the UV16 mutation affected the alpha subunit of phycocyanin, while the phycoerythrin beta subunit from UV16-40 had lost one of its three chromophores. Characterization of the unassembled phycobilisome components in these mutants suggests that these strains will be useful for probing in vivo the regulated expression and assembly of phycobilisomes.     

Dynamic aspects of phycobilisome structure: Modulation of phycocyanin content of Synechococcus phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain the phycobiliproteins phycocyanin, allophycocyanin, and allophycocyanin B, and four major non pigmented polypeptides of 75, 33, 30, and 27 kdaltons. The molar ratio of phycocyanin to allophycocyanin in wild type phycobilisomes can be varied over about a two-fold range by alterations in culture conditions with parallel changes in the amounts of the 33 and 30 kdalton polypeptides whereas the levels of the 27 and 75 kdalton polypeptides do not vary. Two nitrosoguanidine-induced mutants, AN112 and AN135, produce abnormally small phycobilisomes, containing only 35 and 50% of the wild type level of phycocyanin. AN135 phycobilisomes contain less 33 kdalton polypeptide than wild type and the 30 kdalton polypeptide is only detected in phycobilisomes from cultures grown under conditions favoring high levels of phycocyanin. AN112 lacks both the 30 and 33 kdalton polypeptides and produces phycobilisomes of constant size and composition, independent of growth conditions. Both mutant phycobilisomes have wild type levels of 27 and 75 kdalton polypeptides relative to allophycocyanin and have normal energy transfer properties. These results indicate that modulation of phycobilisome size involves concurrent regulation of the levels of phycocyanin and of both the 30 and 33 kdalton polypeptides with no change in the composition of the allophycocyanin-containing core.Abbreviations LP cells cells grown under conditions favoring low p phycobiliprotein levels - HP cells cells grown under conditions favoring high phycobiliprotein levels - SDS sodium dodecylsulfate - EDTA ethylenediamine tetraacetic acid - NaK-PO₄ NaH₂PO₄ titrated with K₂HPO₄ to a given pH A preliminary report of some of this work was presented at the 81st Annual Meeting of the American Society for Microbiology, Dallas, Texas, March 1981     

Cyanobacterial phycobilisomes. Role of the linker polypeptides in the assembly of phycocyanin

The phycocyanin-containing segments of the rod substructures of Anabaena variabilis phycobilisomes consist of complexes of phycocyanin with "linker" polypeptides of 27,000 and 32,500 daltons (Yu, M.-H., Glazer, A. N., and Williams, R. C. (1981) J. Biol. Chem. 256, 13130-13136). Complexes (alpha beta)3.27,000, (alpha beta)3.32,500, (alpha beta)6.27,000, [(alpha beta)6.32,500]n, (alpha beta)6.27,000 - (alpha beta)6.32,500 were prepared, where alpha beta represents a monomer of phycocyanin, and 27,000 and 32,500 represent the 27,000- and 32,500-dalton polypeptides, respectively. Tryptic digestion of (alpha beta)3.32,500 leads to a stable (alpha beta)3.28,000 complex which does not form higher aggregates. The 32,500 polypeptide is stable to trypsin in the [(alpha beta)6.32,500]n and (alpha beta)6.27,000 - [(alpha beta)6.32,500]n=1.2 aggregates. Upon trypsin treatment of all 27,000 still assembled into higher aggregates, (alpha beta)6.21,0900 and (alpha beta)6.21,000 - (alpha beta)6.32,500. The spectroscopic properties of phycocyanin-linker polypeptide complexes were not modified by the tryptic cleavages. These results show that the 32,500 polypeptide has two distinct functional domains, a 28,000 portion necessary to the stabilization of a trimeric phycocyanin complex and a 4,500 domain which links consecutive phycocyanin hexamers in the rod substructure. The 27,000 polypeptide likewise has two distinct functional domains: a 21,000 domain stabilizes a trimeric phycocyanin complex, a 6,000 domain is exposed in all of the assembly forms examined. From these and earlier studies, it is concluded that the 6,000 domain functions in the attachment of the rod substructures to the core of the phycobilisome.     

Core substructure in cyanobacterial phycobilisomes

The tricylindrical core of Synechocystis 6701 phycobilisomes is made up of four types of allophycocyanin-containing complexes: A, (alpha AP beta AP)3; B, (alpha AP beta AP)3 .10K; C, (alpha APB1 alpha AP2 beta AP3).10K; D, (alpha AP beta AP)2.18.5K.99K; where AP is allophycocyanin, APB is allophycocyanin B, and 10K, 18.5K, and 99K are polypeptides of 10,000, 18,500, and 99,000 daltons, respectively. The 18.5K polypeptide is a hitherto unrecognized biliprotein subunit with a single phycocyanobilin prosthetic group. The tricylindrical core is made up of 12 subcomplexes in the molar ratio of A:B:C:D: of 4:4:2:2. Complexes C and D act as terminal energy acceptors. From these results and previous analysis of the bicylindrical core of Synechococcus 6301 phycobilisomes [14,15] it is proposed that the two cylinders of the Synechocystis 6701 core, proximal to the thylakoid membrane, each have the composition ABCD, and that the distal cylinder has the composition A2B2.     

Structure of the genes encoding the rod-core linker polypeptides of Mastigocladus laminosus phycobilisomes and functional aspects of the phycobiliprotein/linker-polypeptide interactions.

The 3' portion of the cpc operon in Mastigocladus laminosus encloses the genes 5'-cpcF-cpcG1-cpcG2-cpcG3 3'. The three cpcG genes encode different phycocyanin-associated rod-core linker polypeptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N-terminal domains (190 amino acids) and an overall identity of 47-53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66-81% identity). It is suggested that these pairs of rod-core linker polypeptides mediate the same specific type of phycocyanin----allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod-core linker polypeptide of Synechococcus sp. PCC 7002 (36-41% identity) is lower. The rod-core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N-terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the beta subunits of phycocyanin, phycoerythrin and phycoerythrocyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein-hexamer/rod and rod-core linker interactions.     

Cyanobacterial phycobilisomes. Phycocyanin assembly in the rod substructures of anabaena variabilis phycobilisomes

Phycocyanin complexes with "linker" polypeptides (Lundell, D. J., Williams, R. C., and Glazer, A. N. (1981) J. Biol. Chem. 256, 3580-3592) of 27 and 32.5 kilodaltons have been isolated from dissociated Anabaena variabilis phycobilisomes. In 0.05 M phosphate at pH 7.0, these "trimeric" complexes have the molar composition (alpha beta)3 . 27,000 and (alpha beta)3 . 32,500, where alpha and beta are the subunits of phycocyanin and 27,000 and 32,500 denote single copies of the linker polypeptides. The (alpha beta)3 . 27,000 and (alpha beta)3 . 32,500 complexes have lambda max at 638 and 629 nm and fluorescence emission maxima at 651 and 646 nm, respectively. In 0.6 M phosphate at pH 8.0, the (alpha beta)3 . 27,000 complex forms an (alpha beta)6 . 27,000 disc-shaped aggregate as seen in the electron microscope, whereas the (alpha beta)3 . 32,500 complex forms discs, (alpha beta)6 . 32,500, and stacked disc rods of varying lengths. The former material, containing the 27,000 polypeptide, when mixed with the (alpha beta)6 . 32,500 discs, limits their assembly into rods. The spectroscopic properties of the discs and rods assembled in vitro indicate that energy transfer in phycobilisome rod substructures proceeds from (alpha beta)6 . 32,500 discs to the (alpha beta)6 . 27,000 disc proximal to the core and thence to the core.     

Molecular architecture of a light-harvesting antenna. Comparison of wild type and mutant Synechococcus 6301 phycobilisomes

Phycobilisomes of the cyanobacterium Synechococcus 6301 contain C-phycocyanin and allophycocyanin in a molar ratio of approximately 3.8:1, a minor biliprotein, allophycocyanin B, and nonpigmented polypeptides of 75, 33, 30, and 27 kilodaltons. A nitrosoguanidine-induced mutant AN112 produces altered phycobilisomes with the molar ratio of C-phycocyanin to allophycocyanin reduced to approximately 1.4:1 and without any of the 33- and 30-kilodalton polypeptides. The mutant and wild type phycobilisomes contain the same molar amount of the 75- and 27-kilodalton polypeptides relative to allophycocyanin. As seen by electron microscopy, the allophycocyanin-containing core of the mutant and of the wild type phycobilisomes appears the same. In some views of the core, each of the two core units in the mutant particles can be seen to consist of four discs approximately 3 nm thick. In wild type phycobilisomes five or six rods, made up of two to six stacked discs (11.5 X 6 nm) are attached to the core. In the mutant, no such rods are seen; rather, single disc-shaped elements, ranging from two to six in number, are found attached. Spectroscopic measurements show that the assembly form of phycocyanin in the mutant phycobilisomes differs from that in the wild type particles but reveal no difference in the organization of the core elements. These results indicate that the portions of the rod substructures of wild type phycobilisomes, beyond the disc proximal to the core, are made up of phycocyanin and the 33- and 30-kilodalton polypeptides. Emission from phycocyanin is a significant component in the fluorescence from isolated Synechococcus 6301 phycobilisomes and indicates an upper limit of 94% for the efficiency of energy transfer from phycocyanin to allophycocyanin and allophycocyanin B in these particles.     

Ultra-violet mutagenesis of Synechocystis sp. 6701: mutations in chromatic adaptation and phycobilisome assembly

Mutations affecting pigmentation of the cyanobacterium Synechocystis sp. 6701 were induced with ultraviolet light. Two mutants with phycobilisome structural changes were selected for structural studies. One mutant, UV08, was defective in chromatic adaptation and incorporated phycoerythrin into phycobilisomes in white or red light at a level typical of growth in green light. The other mutant, UV16, was defective in phycobilisome assembly: little phycocyanin was made and none was attached to the phycobilisome cores. The cores were completely free of any rod substructures and contained the major core peptides plus the 27,000 M_r linker peptide that attaches rods to the core. Micrographs of the core particles established their structural details. Phycoerythrin in UV 16 was assembled into rod structures that were not associated with core material or phycocyanin. The 30,500 M_r and 31,500 M_r linker peptides were present in the phycoerythrin rods with the 30,500 M_r protein as the major component. Phycobilisome assembly in vivo is discussed in light of this unusual mutant.Abbreviations PE phycoerythrin - PC phycocyanin - AP allophycocyanin - W white light - G green light - R red light - SDS sodium dodecyl sulfate - Na–K–PO₄ equimolar solutions of NaH₂PO₄ · H₂O and K₂HPO₄ · 3 H₂O titrated to the desired pH     

Purification and characterization of phycocyanin from the blue-green alga Aphanizomenon flos-aquae

Aphanizomenon flos-aquae (AFA) is a blue-green alga and represents a nutrient-dense food source. In this study the presence of phycocyanin (PC), a blue protein belonging to the photosynthetic apparatus, has been demonstrated in AFA. An efficient method for its separation has been set up: PC can be purified by a simple single step chromatographic run using a hydroxyapatite column (ratio A620/A280 of 4.78), allowing its usage for health-enhancing properties while eliminating other aspecific algal components. Proteomic investigation and HPLC analysis of purified AFA phycobilisomes revealed that, contrary to the well-characterized Synechocystis and Spirulina spp., only one type of biliprotein is present in phycobilisomes: phycocyanins with no allo-phycocyanins. Two subunit polypeptides of PC were also separated: the beta subunit containing two bilins as chromophore and the alpha subunit containing only one.     

Molecular architecture of a light-harvesting antenna. Structure of the 18 S core-rod subassembly of the Synechococcus 6301 phycobilisome

The 18 S subassembly particles obtained by partial dissociation of phycobilisomes from Synechococcus 6301 (Anacystis nidulans) strain AN 112 contain approximately one-half of the mass of the phycobilisome and include core-rod junctions (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086). The polypeptide composition of 18 S complexes, determined by analysis of uniformly 14C-labeled phycobilisomes, gave the following stoichiometry: 75K:27K:18.3K:alpha beta allophycocyanin monomer: alpha beta phycocyanin monomer of 1:2:1:5:6; where 75K, 27K, etc. represent polypeptides of 75, 27 kilodaltons, etc. The 18.3K polypeptide is a hitherto underscribed biliprotein bearing a single phycocyanobilin. The NH2-terminal sequence of this subunit was determined to be homologous to that of the beta subunit of allophycocyanin. Chromatography of products resulting from limited trypsin treatment of the 18 S complex led to the isolation of three subcomplexes: a mixture of (alpha beta)3 . 22K and (alpha beta)3 . 24K phycocyanin complexes, an (alpha beta)3 allophycocyanin trimer, and an (alpha beta)2 . 18.3K.40K.11K allophycocyanin-containing complex. The 22K and 24K components were products of the degradation of the 27K polypeptides, whereas the 40K and 11K components were derived from the 75K polypeptide. The subcomplexes accounted for the composition of the 18 S complex. Determination of the composition, stoichiometry, and spectroscopic properties of the subcomplexes has led to a model of the polypeptide arrangement within the 18 S complex and of the pathway of energy transfer among these polypeptides.     

Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301.

A procedure is described for the preparation of stable phycobilisomes from the unicellular cyanobacterium Synechococcus sp. 6301 (also known as Anacystis nidulans). Excitation of the phycocyanin in these particles at 580 nm leads to maximum fluorescence emission, from allophycocyanin and allophycocyanin B, at 673 nm. Electron microscopy shows that the phycobilisomes are clusters of rods. The rods are made up of stacks of discs which exhibit the dimensions of short stacks made up primarily of phycocyanin (Eiserling, F. A., and Glazer, A. N. (1974) J. Ultrastruct. Res. 47, 16-25). Loss of the clusters, by dissociation into rods under suitable conditions, is associated with loss of energy transfer as shown by a shift in fluorescence emission maximum to 652 nm. Synechococcus sp. 6301 phycobilisomes were shown to contain five nonpigmented polypeptides in addition to the colored subunits (which carry the covalently bound tetrapyrrole prosthetic groups) of the phycobiliproteins. Evidence is presented to demonstrate that these colorless polypeptides are genuine components of the phycobilisome. The nonpigmented polypeptides represent approximately 12% of the protein of the phycobilisomes; phycocyanin, approximately 75%, and allophycocyanin, approximately 12%. Spectroscopic studies that phycocyanin is in the hexamer form, (alpha beta)6, in intact phycobilisomes, and that the circular dichroism and absorbance of this aggregate are little affected by incorporation into the phycobilisome structure.     

Two novel phycoerythrin-associated linker proteins in the marine cyanobacterium Synechococcus sp. strain WH8102

The recent availability of the whole genome of Synechococcus sp. strain WH8102 allows us to have a global view of the complex structure of the phycobilisomes of this marine picocyanobacterium. Genomic analyses revealed several new characteristics of these phycobilisomes, consisting of an allophycocyanin core and rods made of one type of phycocyanin and two types of phycoerythrins (I and II). Although the allophycocyanin appears to be similar to that found commonly in freshwater cyanobacteria, the phycocyanin is simpler since it possesses only one complete set of alpha and beta subunits and two rod-core linkers (CpcG1 and CpcG2). It is therefore probably made of a single hexameric disk per rod. In contrast, we have found two novel putative phycoerythrin-associated linker polypeptides that appear to be specific for marine Synechococcus spp. The first one (SYNW2000) is unusually long (548 residues) and apparently results from the fusion of a paralog of MpeC, a phycoerythrin II linker, and of CpeD, a phycoerythrin-I linker. The second one (SYNW1989) has a more classical size (300 residues) and is also an MpeC paralog. A biochemical analysis revealed that, like MpeC, these two novel linkers were both chromophorylated with phycourobilin. Our data suggest that they are both associated (partly or totally) with phycoerythrin II, and we propose to name SYNW2000 and SYNW1989 MpeD and MpeE, respectively. We further show that acclimation of phycobilisomes to high light leads to a dramatic reduction of MpeC, whereas the two novel linkers are not significantly affected. Models for the organization of the rods are proposed.     

Structure, composition, and assembly of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 and of phycobilisomes in the derivative strain BO 9201.

The phycobiliproteins of the unicellular cyanobacterium Synechocystis sp. strain BO 8402 and its derivative strain BO 9201 are compared. The biliproteins of strain BO 8402 are organized in paracrystalline inclusion bodies showing an intense autofluorescence in vivo. These protein-pigment aggregates have been isolated. The highly purified complexes contain phycocyanin with traces of phycoerythrin, corresponding linker polypeptides LR35PC and LR33PE (the latter in a small amount), and a unique colored polypeptide with an M(r) of 55,000, designated L55. Allophycocyanin and the core linker polypeptides are absent. The substructure of the aggregates has been studied by electron microscopy. Repetitive subcomplexes of hexameric stacks of biliproteins form extraordinary long rods associated side by side in a highly condensed arrangement. Evidence that the linker polypeptides LR35PC and LR33PE stabilize the biliprotein hexamers is presented, while the location and function of the colored linker L55 remain uncertain. The derivative strain BO 9201 contains established hemidiscoidal phycobilisomes comprising phycoerythrin, phycocyanin, and allophycocyanin as well as the corresponding linker polypeptides. The core-membrane linker protein (LCM), and two polypeptides with M(r)s of 40,000 and 45,000 which are present in small amounts, exhibit strong cross-reactivity in Western blot (immunoblot) analysis using an antibody directed against the colored LCM of a Nostoc sp. In contrast, strain BO 8402 exhibits no polypeptide with a significant immunological cross-reactivity in Western blot analysis. Physiological and genetic implications of the unusual pigment compositions of both strains are discussed.     

Changes in polypeptide composition of Synechocystis sp. strain 6308 phycobilisomes induced by nitrogen starvation.

Phycobilisomes isolated from actively growing Synechocystis sp. strain 6308 (ATCC 27150) consist of 12 polypeptides ranging in molecular mass from 11.5 to 95 kilodaltons. The phycobilisome anchor and linker polypeptides are glycosylated. Nitrogen starvation causes the progressive loss of phycocyanin and allophycocyanin subunits with molecular masses between 16 and 20 kilodaltons and of two linker polypeptides with molecular masses of 27 and 33 kilodaltons. Nitrogen starvation also leads to enrichment of four additional polypeptides with molecular masses of 46, 53, 57, and 61 kilodaltons and a transient enrichment of 35- and 41-kilodalton polypeptides in isolated phycobilisomes. The 57-kilodalton additional polypeptide was identified by immunoblotting as the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proteins with the same molecular weights as the additional polypeptides were also coisolated with the 12 phycobilisome polypeptides in the supernatant of nitrogen-replete Synechocystis thylakoid membranes extracted in high-ionic-strength buffer and washed with deionized water. These observations suggest that the additional polypeptides in phycobilisomes from nitrogen-starved cells may be soluble or loosely bound membrane proteins which associate with phycobilisomes. The composition and degree of association of phycobilisomes with soluble and adjacent membrane polypeptides appear to be highly dynamic and specifically regulated by nitrogen availability. Possible mechanisms for variation in the strength of association between phycobilisomes and other polypeptides are suggested.     

Regulation of Nostoc sp. phycobilisome structure by light and temperature.

Nostoc sp. strain MAC cyanobacteria were green in color when grown in white light at 30 degrees C and contained phycobilisomes that had phycoerythrin and phycocyanin in a molar ratio of 1:1. Cells grown for 4 to 5 days in green light at 30 degrees C or white light at 39 degrees C turned brown and contained phycoerythrin and phycocyanin in a molar ratio of greater than 2:1. In addition to the change in pigment composition, phycobilisomes from brown cells were missing a 34.5-kilodalton, rod-associated peptide that was present in green cells. The green light-induced changes were typical of the chromatic adaptation response in cyanobacteria, but the induction of a similar response by growth at 39 degrees C was a new observation. Phycobilisomes isolated in 0.65 M phosphate buffer (pH 7) dissociate when the ionic strength or pH is decreased. Analysis of the dissociation products from Nostoc sp. phycobilisomes suggested that the cells contained two types of rod structures: a phycocyanin-rich structure that contained the 34.5-kilodalton peptide and a larger phycoerythrin-rich complex. Brown Nostoc sp. cells that lacked the 34.5-kilodalton peptide also lacked the phycocyanin-rich rod structures in their phycobilisomes. These changes in phycobilisome structure were indistinguishable between cells cultured at 39 degrees C in white light and those cultured at 30 degrees C in green light. A potential role is discussed for rod heterogeneity in the chromatic adaptation response.