Ultrastructure of cell wall and photosynthetic apparatus of the phycobilisome-less Synechocystis sp. strain BO 8402 and phycobilisome-containing derivative strain BO 9201

The ultrastructures of two closely related strains of a novel diazotrophic cyanobacterium, Synechocystis sp. BO 8402 and BO 9201, were examined using ultrathin sections and freeze-fracture electron microscopy. Cells of both strains were surrounded by an unusual thick peptidoglycan layer. Substructures in the layer indicated the presence of microplasmodesmata aligned perpendicular to the free cell surface and in the septum of dividing cells. Synechocystis sp. strain BO 8402 contained lobed, electronopaque, highly fluorescent inclusion bodies consisting of phycocyanin-linker complexes. The thylakoids lacked phycobilisomes and accommodated, in addition to randomly distributed exoplasmic freeze-fracture particles, patches of two-dimensionally ordered arrays of dimeric photosystem II particles in the exoplasmic fracture face. Determination of photosystem I and photosystem II suggested an increase of photosystem II in strain BO 8402. Strain BO 9201 performed phycobilisome-supported photosynthesis and showed rows of dimeric photosystem II particles in the exoplasmic fracture face. Corresponding particle-free grooves in the protoplasmic fracture face were lined by a class of large particles tentatively assigned as trimers of photosystem I. The different lateral organization of protein complexes in the thylakoid membranes and the fine structure of the cell wall are discussed with respect to absorption cross-section of photosynthesis and nitrogen fixation.Abbreviations EF Exoplasmic freeze-fracture face - P 700 Reaction centre chlorophyll of photosystem I - PF Protoplasmic freeze-fracture face - PS I Photosystem I - PS II Photosystem II     

An insertion element prevents phycobilisome synthesis in N2-fixing Synechocystis sp. strain BO 8402.

The unicellular diazotrophic cyanobacterium Synechocystis sp. strain BO 8402, isolated from Lake Constance, contains a novel insertion sequence, IS8402, in the apcA gene encoding a pigmented protein of phycobilisomes. IS8402 comprises 1,322 bp, flanked by two inverted repeats of 15 bp. Upon insertion in the target DNA, direct duplications of 8 nucleotides were generated. One open reading frame, potentially coding for a protein of 399 amino acids, was found. The deduced amino acid sequence shows homology to putative transposases of the IS4 family. Precise excision of the insertion element resulted in a spontaneous revertant, Synechocystis sp. strain BO 9201, that had regained the ability to form hemidiscoidal phycobilisomes. Apart from the unique insertion of IS8402 into apcA in strain BO 8402 both strains contain at least 12 further homologous insertion elements at corresponding sites in the genomes. The unique insertion in strain BO 8402 prevents the expression of apcABC operon and hence abolishes the formation of intact phycobilisomes. This decreases the quantum efficiency of photosystem II and promotes anaerobic N2 fixation in a unicellular cyanobacterium with a highly oxygen-sensitive nitrogenase.     

Utilization of Light for Nitrogen Fixation by a New Synechocystis Strain Is Extended by Its Low Photosynthetic Efficiency

Performance of photosynthesis and nitrogenase activity in a novel cyanobacterium, Synechocystis sp. strain BO 8402, isolated from Lake Constance, located at the northern fringe of the Alps in central Europe, and of a stable derivative, strain BO 9201, were examined. Strain BO 8402 is characterized by an extraordinarily high level of autofluorescence originating from paracrystalline phycobiliprotein-linker complexes located in inclusion bodies (W. Reuter, M. Westermann, S. Brass, A. Ernst, P. Böger, and W. Wehrmeyer, J. Bacteriol. 176:896-904, 1994). Energy transfer between paracrystalline phycobiliproteins and the photosystems is inefficient, resulting in a high oxygen compensation point and a decreased growth rate. The derivative strain BO 9201 exhibits hemidiscoidal phycobilisomes that support a high growth rate, even under low light intensities. Because of the differences in photosynthetic performance, anaerobic light-stimulated nitrogenase activity is maintained at higher light intensity in the original strain BO 8402 than in the derivative strain BO 9201. The results indicate that the formation of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 represents a hitherto-unknown means for a unicellular cyanobacterium to extend its capacity to fix nitrogen in the light.     

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.     

Accessory polypeptides in phycobilisomes of red algae and cyanobacteria

Phycobilisomes of red algae and cyanobacteria contain small amounts of nonpigmented polypeptides in addition to the major constituent biliprotein pigments. The localization of these polypeptides is analyzed by gel electrophoresis of phycobilisome fragments obtained by selective dissociation and subsequent separation. Five groups of biliprotein aggregates are determined, belonging to the 6, 11, 16, 18 and 23 S categories. Accessory nonpigmented high molecular weight proteins (80,000 MW) are exclusively bound to phycobilisome core fractions and thylakoids, thus apparently serving as links between the phycobilisomes and the photosynthetic units of the thylakoids. In contrast, smaller nonpigmented accessory polypeptides of 20,000 to 60,000 MW are preferably found in the peripheral biliprotein stacks. They may either form a compatible link between the phycobilisome core and periphery or bind and co-polymerize with hexameric biliproteins in the peripheral stacks to enhance or effect binding of the aggregates. Furthermore, they may determine the arrangement and composition of the phycobilisomes during development and chromatic adaptation.Abbreviations PE phycoerythrin - PEC phycoerythrocyanin - PC phycocyanin - APC allophycocyanin     

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.     

Characterization of the biliproteins of Gloeobacter violaceus chromophore content of a cyanobacterial phycoerythrin carrying phycourobilin chromophore

The biliproteins of the unicellular, thylakoid-less cyanobacterium Gleobacter violaceus were resolved by chromatography on hydroxylapatite and DEAE-cellulose into five components: phycoerythrin I and II, phycocyanin I and II, and allophycocyanin. Allophycocyanin B was not detected. Three of these components, phycoerythrin II, phycocyanin II, and allophycocyanin, were purified to homogeneity. Phycoerythrin II crystallized as hexagonal prisms. G. violaceus allophycocyanin crystallized as thin plates; unter similar conditions other cyanobacterial allophycocyanins crystallize as needles. The biliproteins in the phycoerythrin I and phycocyanin I components were present in polydisperse, high molecular weight aggregates, which may represent incompletely dissociated substructures of the phycobilisome.Both phycoerythrin components from G. violaceus carry phycoerythrobilin and phycourbilin groups in the ratio of 6:1. Separation of the and subunits of these biliproteins revealed that the phycoerythrobilins were equally distributed between the two subunits, and that the subunit alone carried the phycourobilin. These phycoerythrins are the first cyanobacterial phycobiliproteins found to carry a phycourobilin prosthetic group.Abbreviations used PE poycoerythrin - PC phycocyanin - AP allophycocyanin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - B Bangiophycean - R Rhodophytan - C Cyanobacterial     

A small multigene family encodes the rod-core linker polypeptides of Anabaena sp. PCC7120 phycobilisomes.

The cpc operon of Anabaena sp. PCC7120 is shown to encode ten genes: 5'-cpcB-cpcA-cpcC-cpcD-cpcE-cpcF- cpcG1-cpcG2-cpcG3-cpcG4-3'. The 3' portion of this operon includes four tandemly repeated genes encoding phycocyanin (PC)-associated, rod-core linker polypeptides of the phycobilisomes (PBS). The products of these four genes are most similar at their N termini, and overall are 50-61% identical and 68-76% similar to one another. The four CpcG proteins of Anabaena sp. PCC7120 are 41-47% identical and 62-65% similar to the single CpcG rod-core linker protein in Synechococcus sp. PCC7002. The N-terminal domains of the polypeptides are also more distantly related to the conserved domains of other types of rod-linker polypeptides associated with PC, phycoerythrin, and allophycocyanin (AP). Three of these rod-core linker proteins (CpcG1, CpcG2, and CpcG4) were demonstrated to occur in isolated PBS by N-terminal amino acid sequence analyses. These results indicate that previously proposed models for the PBS of Anabaena sp. are incorrect. It is suggested that the PBS of Anabaena sp. have eight peripheral rods, each of which interacts with the AP of the core via a specific rod-core linker (CpcG) polypeptide.     

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.     

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.     

Growth and Chromatic Adaptation of Nostoc sp. Strain MAC and the Pigment Mutant R-MAC

A spontaneous, stable, pigmentation mutant of Nostoc sp. strain MAC was isolated. Under various growth conditions, this mutant, R-MAC, had similar phycoerythrin contents (relative to allophycocyanin) but significantly lower phycocyanin contents (relative to allophycocyanin) than the parent strain. In saturating white light, the mutant grew more slowly than the parent strain. In nonsaturating red light, MAC grew with a shorter generation time than the mutant; however, R-MAC grew more quickly in nonsaturating green light.

When the parental and mutant strains were grown in green light, the phycoerythrin contents, relative to allophycocyanin, were significantly higher than the phycoerythrin contents of cells grown in red light. For both strains, the relative phycocyanin contents were only slightly higher for cells grown in red light than for cells grown in green light. These changes characterize both MAC and R-MAC as belonging to chromatic adaptation group II: phycoerythrin synthesis alone photocontrolled.

A comparative analysis of the phycobilisomes, isolated from cultures of MAC and R-MAC grown in both red and green light, was performed by polyacrylamide gel electrophoresis in the presence of 8.0 molar urea or sodium dodecyl sulfate. Consistent with the assignment of MAC and R-MAC to chromatic adaptation group II, no evidence for the synthesis of red light-inducible phycocyanin subunits was found in either strain. Phycobilisomes isolated from MAC and R-MAC contained linker polypeptides with relative molecular masses of 95, 34.5, 34, 32, and 29 kilodaltons. When grown in red light, phycobilisomes of the mutant R-MAC appeared to contain a slightly higher amount of the 32-kilodalton linker polypeptide than did the phycobilisomes isolated from the parental strain under the same conditions. The 34.5-kilodalton linker polypeptide was totally absent from phycobilisomes isolated from cells of either MAC or R-MAC grown in green light.

     

Allophycocyanin I and the 95 Kilodalton Polypeptide : The Bridge between Phycobilisomes and Membranes

Allophycocyanin was isolated from dissociated phycobilisomes from Nostoc sp. and was separated into allophycocyanin I, II, III, and B as described elsewhere. If the separation of the proteins following phycobilisome isolation is done in the presence of the protease inhibitor, phenylmethylsulfonylfluoride, associated with allophycocyanin I are two colored polypeptides of 95 kilodalton (kD) and 80 kD, belonging to the class of Group I polypeptides as defined by Tandeau de Marsac and Cohen-Bazire (Proc Natl Acad Sci USA 1977 74: 1635-1639). Allophycocyanin I has a fluorescence maximum of 680 nanometers as do intact phycobilisomes and has thus been suggested to be the final emitter of excitation energy in phycobilisomes. Thylakoid membranes washed in low ionic strength buffer containing phenylmethylsulfonylfluoride lose all biliproteins, but retain the 95 kD and 80 kD polypeptides. As suggested by Tandeau de Marsac and Cohen-Bazire, these are likely to be the polypeptides involved in binding the phycobilisome to the membrane. As these polypeptides are isolated with allophycocyanin I, structural evidence is provided for placing allophycocyanin I as the bridge between the phycobilisome and the membrane. These Group I polypeptides and the 29 kD polypeptide (involved in rod attachment to the APC core) are particularly susceptible to proteolytic breakdown. It is thought that in vivo the active protease may be selectively attacking these polypeptides to detach the phycobilisome from the membrane and release the phycoerythrin and phycocyanin containing rods from the allophycocyanin core for greater susceptibility of the biliproteins to protease attack.     

Linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus. I. Isolation and characterization of phycobiliprotein-linker-polypeptide complexes

Phycobilisomes from the cyanobacterium Mastigocladus laminosus cultured in white and red light were isolated and compared with respect to the phycoerythrocyanin (PEC) and linker polypeptide contents. It was verified that the production of PEC is induced by low light intensities. A PEC complex, (alpha PEC beta PEC)6LR34.5,PEC, and a phycocyanin (PC) complex, (alpha PC beta PC)6LR34.5,PC, were isolated from phycobilisomes by Cellex-D anion exchange chromatography and sucrose density gradient centrifugation. The absorption and fluorescence emission maxima of the PEC complex are at 575 and 620 nm and those of the PC complex are at 631 and 647 nm, respectively. The extinction coefficients of the two complexes were determined. From different experiments it was concluded that PEC is present as a hexameric complex, (alpha PEC beta PEC)6LR34.5,PEC, in the phycobilisome. The two linker polypeptides LR34.5,PEC and LR34.5,PC were isolated from their phycobiliprotein complexes by gel filtration on Bio-Gel P-100 in 50% formic acid. A 5-kDa terminal segment of both linker polypeptides was found to influence the hexamer formation of the phycobiliproteins. The same segments have been described to be responsible for the hexamer-hexamer linkage (Yu, M.-H. & Glazer, A.N. (1982) J. Biol. Chem. 257, 3429-3433). A 8.9-kDa linker polypeptide, LR(C)8.9, was isolated from a PEC fraction of the Cellex-D column by Bio-Gel P-100 gel filtration in 50% formic acid. Localisation of this protein within the phycobilisome was attempted. Its most probable function is to terminate the phycobilisomal rods at the end distal to the allophycocyanin core.     

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.     

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     

Acclimation processes in the light harvesting complex of the red alga Porphyridium purpureum (Bory) Drew et Ross, according to irradiance and nutrient availability

The time-course of acclimation (0-5h) of the red alga Porphyridium purpureum with respect to total proteins, phycoerythrin (PE) and phycobilisomes (PBS) has been studied at different N availability and different light regimes. After a high N input, acclimation takes place in two phases. The first one, which is photoindependent is characterized by simultaneous increase of proteins and PE. At low N input, this first phase is not detected. In the second phase the PE content increases only under low light together with an increase of the PBS size, followed probably by an increase in the number of PBS. The effectiveness of the energy transfer increases under these conditions. A rapid decrease in the PBS size correlated with a decrease of the energy transfer is observed at high irradiance. Free PE plays an important role in the organization-disorganization of the PBS at low N concentration (inverse correlation between free PE and PE attached to PBS). Free PE is not accumulated in the cell after a high N input at high irradiance. Independently of photoacclimation, two species of PBS appear with different PE content and different capacities to aggregate with other compounds. A clear correlation appears between the level of coupling of the PBS and the fluorescence ‘in vivo’ of the whole cells. The comparison between dissociated and undissociated PBS as well as between PBS obtained after the different acclimation processes allows the determination of the presence of two linker polypeptides probably associated with B-PE (37 and 32–5 kDa) and two associated with PC and APC (27 and 25 kDa). That suggests that acclimation of PBS requires a parallel stoichiometric response of biliproteins and the linker polypeptides involved in the efficiency of the energy transfer.     

Role of the colorless polypeptides in phycobilisome reconstitution from separated phycobiliproteins

A phycoerythrin (PE) and phycocyanin (PC) mixture was separated from allophycocyanin on calcium phosphate chromatography from completely dissociated phycobilisomes of the blue-green alga, Nostoc sp. After dialysis of the PE-PC mixture in 0.75 m potassium phosphate, pH 7, which allows reassociation of the dissociated pigment-proteins, complexes of PE and PC in a 2:1 m ratio (PE/PC complex) as well as complexes predominantly of PC (PC/PE complex) were then separated by sedimentation on linear sucrose gradients. These complexes resemble the rods of intact phycobilisomes and transfer energy efficiently from PE to PC. They contain the Group II colorless polypeptides described by Tandeau de Marsac and Cohen-Bazire (1977 Proc Natl Acad Sci USA 74: 1635 61639). Phycobilisomes can be reconstituted by combining the allophycocyanin pool with (a) the PE-PC mixture, (b) the PE/PC complex, or (c) the PC/PE complex. Successful reconstitution is measured by absorption, fluorescence, circular dichroism, and electron microscopy. The major requirement for reconstitution is the 29-kilodalton colorless polypeptide. In its absence, no phycobilisomes are formed. It is the only colorless polypeptide common to both the PE/PC complex and the PC/PE complex, and appears to be the polypeptide responsible for rod attachment to the allophycocyanin. In addition, high phosphate concentrations and 20 degrees C temperatures are needed for reconstitution.