Cloning of the cpcE and cpcF genes from Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Two open reading frames denoted as cpcE and cpcF were cloned and sequenced from Synechococcus sp. PCC 6301. The cpcE and cpcF genes are located downstream of the cpcB2A2 gene cluster in the phycobilisome rod operon and can be transcribed independently of the upstream cpcB2A2 gene cluster. The cpcE and cpcF genes were separately inactivated by insertion of a kanamycin resistance cassette in Synechococcus sp. PCC 7942 to generate mutants R2EKM and R2FKM, respectively, both of which display a substantial reduction in spectroscopically detectable phycocyanin. The levels of - and -phycocyanin polypeptides were reduced in the R2EKM and R2FKM mutants although the phycocyanin and linker genes are transcribed at normal levels in the mutants as in the wild type indicating the requirement of the functional cpcE and cpcF genes for normal accumulation of phycocyanin. Two biliprotein fractions were isolated on sucrose density gradient from the R2EKM/R2FKM mutants. The faster sedimenting fraction consisted of intact phycobilisomes. The slower sedimenting biliprotein fraction was found to lack phycocyanin polypeptides, thus no free phycocyanin was detected in the mutants. Characterization of the phycocyanin from the mutants revealed that it was chromophorylated, had a _max similar to that from the wild type and could be assembled into the phycobilisome rods. Thus, although phycocyanin levels are reduced in the R2EKM and R2FKM mutants, the remaining phycocyanin seems to be chromophorylated and similar to that in the wild type with respect to phycobilisome rod assembly and energy transfer to the core.     

Biliprotein assembly in the hemidiscoidal phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus Cohn. Characterization of dissociation products with special reference to the peripheral phycoerythrocyanin-phycocyanin complexes

The dissociation products of isolated phycobilisomes of Mastigocladus laminosus were separated and analyzed by ultracentrifugation and, in part, by isoelectric focusing. With the exception of the allophycocyanin core, the sedimentation constants of peripheral phycocyanin- and phycoerythrocyanin-phycocyanin complexes lay in the range of 6 to 17S. The latter was represented by a 17S aggregate of two hexameric phycocyanins (dodecamer, dipartite unit). A complex with an absorption maximum at 610 nm (phycocyanin) and a shoulder at 580 nm (phycoerythrocyanin), a fluorescence emission maximum at 645 nm and a sedimentation constant of 11 S is described as a heterogeneously composed hexamer of ()₃-phycoerythrocyanin-()₃-phycocyanin. It was stable under extended dissociation in the cold and under isoelectric focusing. An aggregate of 14 S with an absorption maximum at 576 nm and a shoulder in the fluorescence emission spectrum at 625 nm (phycoerythrocyanin) in addition to the maximum at 645 nm (phycocyanin) is interpreted as a polar phycoerythrocyanin/ phycoerythrocyanin-phycocyanin complex. Combining these complexes with phycocyanin dodecamers creates peripheral rods of the phycobilisome. A proposal of the phycobiliprotein distribution within the phycobilisome of M. laminosus is presented.Abbreviations APC allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin     

Energy transfer and trapping in <Emphasis Type="Italic">Synechococcus</Emphasis> WH 7803

Excitation energy transfer (EET) and trapping in Synechococcus WH 7803 whole cells and isolated photosystem I (PSI) complexes have been studied by time-resolved emission spectroscopy at room temperature (RT) and at 77 K. With the help of global and target analysis, the pathways of EET and the charge separation dynamics have been identified. Energy absorbed in the phycobilisome (PB) rods by the abundant phycoerythrin (PE) is funneled to phycocyanin (PC645) and from there to the core that contains allophycocyanin (APC660 and APC680). Intra-PB EET rates have been estimated to range from 11 to 68/ns. It was estimated that at RT, the terminal emitter of the phycobilisome, APC680, transfers its energy at a rate of 90/ns to PSI and at a rate of 50/ns to PSII. At 77 K, the redshifted Chl a states in the PSI core were heterogeneous, with maximum emission at 697 and 707 nm. In 72% of the PSI complexes, the bulk Chl a in equilibrium with F697 decayed with a main trapping lifetime of 39 ps.     

Cobalt chloride induced stimulation of Photosystem II electron transport in Synechocystis sp. PCC 6803 cells

Synechocystis sp. PCC 6803 when grown in the presence of sublethal (M) levels of cobalt chloride shows an enhancement of Photosystem II (PS II) catalyzed Hill reaction. This stimulation seems to be induced by cobalt ions as other metal ions inhibit para-benzoquinone catalyzed Hill reaction. At saturating white light intensity, this enhancement is two times over that of the control cells on unit chlorophyll basis. Analysis of the PS II electron transport rate at varying intensities of white, blue or yellow light suggests an increased maximal rates but no change in the quantum yield or effective antenna size of CoCl₂-grown cells. There were no structural and functional changes in the phycobilisome as judged by the absence of changes in the phycocyanin/allophycocyanin ratio, fluorescence emission spectra, second derivative absorption spectra at 77 K and SDS-PAGE analysis of isolated phycobilisomes. The 77 K fluorescence emission spectra of the cells showed a decrease in the ratio of Photosystem I emission (F725) to Photosystem II emission (F685) in CoCl₂-grown cells compared to the control cells. These observations indicate three possibilities: (1) there is an increase in the number of Photosystem II units; (2) a faster turnover of Photosystem II centers; or (3) an alteration in energy redistribution between PS II and PS I in CoCl₂-grown cells which causes stimulation of Photosystem II electron transport rate.Abbreviations APC allophycocyanin - Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - EDTA ethylene diamine tetraacetic acid - PBS phycobilisome - PC phycocyanin - PSI Photosystem I - PS II Photosystem II - pBQ p-benzoquinone - PMSF phenyl methyl sulfonyl fluoride     

Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation

Summary The phycobilisome rod linker genes in the two closely related cyanobacteria Synechococcus sp. PCC 6301 and Synechococcus sp. PCC 7942 were studied. Southern blot analysis showed that the genetic organization of the phycobilisome rod operon is very similar in the two strains. The phycocyanin gene pair is duplicated and separated by a region of about 2.5 kb. The intervening region between the duplicated phycocyanin gene pair was cloned from Synechococcus sp. PCC 6301 and sequenced. Analysis of this DNA sequence revealed the presence of three open reading frames corresponding to 273, 289 and 81 amino acids, respectively. Insertion of a kanamycin resistance cassette into these open reading frames indicated that they corresponded to the genes encoding the 30, 33 and 9 kDa rod linkers, respectively, as judged by the loss of specific linkers from the phycobilisomes of the insertional mutants. Amino acid compositions of the 30 and 33 kDa linkers derived from the DNA sequence were found to deviate from those of purified 33 and 30 kDa linkers in the amounts of glutamic acid/glutamine residues. On the basis of similarity of the amino acid sequence of the rod linkers between Synechococcus sp. PCC 6301 and Calothrix sp. PCC 7601 we name the genes encoding the 30, 33 and 9 kDa linkers cpcH, cpcI and cpcD, respectively. The three linker genes were found to be co-transcribed on an mRNA of 3700 nucleotides. However, we also detected a smaller species of mRNA, of 3400 nucleotides, which would encode only the cpcH and cpcI genes. The 30 kDa linker was still found in phycobilisome rods lacking the 33 kDa linker and the 9 kDa linker was detected in mutants lacking the 33 or the 30 kDa linkers. Free phycocyanin was found in the mutants lacking the 33 or the 30 kDa linkers, whereas no free phycocyanin could be found in the mutant lacking the 9 kDa linker.Abbreviations PCC Pasteur Culture Collection - UTEX University of Texas Culture Collection The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank Nucleotide Sequence Databases under the accession number M94218     

A high molecular weight terminal pigment (“anchor polypeptide”) and a minor blue polypeptide from phycobilisomes of the cyanobacterium Nostoc sp. (MAC): Isolation and characterization

A 94 kD pigment-polypeptide, which is presumed to be involved in anchoring the phycobilisomes to the thylakoids, was isolated from Nostoc phycobilisomes by gel filtration in 63 mM formic acid. The isolation condition did not require detergents or denaturating reagents, as in previous procedures, and enzymatic degradation was not observed at the low pH of 2.5. The anchor polypeptide thus obtained had absorption (Abs) and fluorescence maxima (Em) at 658 and 673 nm, respectively, in 63 mM formic acid at room temperature. The maxima shifted to longer wavelengths in 100 mM potassium phosphate (pH 6.8), Abs 665 and Em 683 nm at room temperature, and Abs 665 and Em 684 nm at liquid nitrogen temperature. The fluorescence maxima at both temperatures correspond to the longest wavelength component resolved in phycobilisomes from second derivative spectra. A minor blue polypeptide was also found by this isolation method. The molecular weight of this polypeptide was ca. 18,000 and is probably similar to a polypeptide which has been found in the phycobilisome core of other cyanobacteria.Abbreviations used -APB -subunit of allophycocyanin B - APC allophycocyanin - kD kilodalton - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PBS phycobilisome - PE phycoerythrin - PC phycocyanin - PS pigment system     

Pigment orientation changes accompanying the light state transition in Synechococcus sp. PCC 6301

Low temperature (77 K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. PCC 6301 and those accompanying chromatic acclimation in Porphyridium cruentum in samples stabilized by glutaraldehyde fixation. In light state 2 compared to light state 1 intact cells of Synechococcus showed an increased alignment of allophycocyanin parallel to the cells' long axis whereas the phycobilisomethylakoid membrane fragments exhibited an increased allophycocyanin alignment parallel to the membrane plane. The phycobilisome-thylakoid membrane fragments showed less alignment of a short wave-length chlorophyll a (Chl a) Q_y transition dipole parallel to the membrane plane in state 2 relative to state 1.To aid identification of the observed Chl a orientation changes in Synechococcus, linear dichroism spectra were obtained from phycobilisome-thylakoid membrane fragments isolated from red light-grown (increased number of PS II centres) and green light-grown (increased number of PS I centres) cells of the red alga Porphyridium cruentum. An increased contribution of short wavelength Chl a Q_y transition dipoles parallel to the long axis of the membrane plane was directly correlated with increased levels of PS II centres in red light-grown P. cruentum.Our results indicate that the transition to state 2 in cyanobacteria is accompanied by an increase in the orientation of allophycocyanin and a decrease in the orientation of Chl a associated with PS II with respect to the thylakoid membrane plane.Abbreviations APC - allophycocyanin - Chl a - chlorophyll a - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LD - linear dichroism - LD/A - linear dichroism divided by absorbance - LHC - light-harvesting complex - PBS - phycobilisome - PC - phycocyanin - PS - Photosystem     

Phycobilisome structure and function

Phycobilisomes are aggregates of light-harvesting proteins attached to the stroma side of the thylakoid membranes of the cyanobacteria (blue-green algae) and red algae. The water-soluble phycobiliproteins, of which there are three major groups, tetrapyrrole chromophores covalently bound to apoprotein. Several additional protiens are found within the phycobilisome and serve to link the phycobiliproteins to each other in an ordered fashion and also to attach the phycobilisome to the thylakoid membrane. Excitation energy absorbed by phycoerythrin is transferred through phycocyanin to allophycocyanin with an efficiency approximating 100%. This pathway of excitation energy transfer, directly confirmed by time-resolved spectroscopic measurements, has been incorporated into models describing the ultrastructure of the phycobilisome. The model for the most typical type of phycobilisome describes an allophycocyanin-containing core composed of three cylinders arranged so that their longitudinal axes are parallel and their ends form a triangle. Attached to this core are six rod structures which contain phycocyanin proximal to the core and phycoerythrin distal to the core. The axes of these rods are perpendicular to the longitudinal axis of the core. This arrangement ensures a very efficient transfer of energy. The association of phycoerythrin and phycocyanin within the rods and the attachment of the rods to the core and the core to the thylakoid require the presence of several linker polypeptides. It is recently possible to assemble functionally and structurally intact phycobilisomes in vitro from separated components as well as to reassociate phycobilisomes with stripped thylakoids. Understanding of the biosynthesis and in vivo assembly of phycobilisomes will be greatly aided by the current advances in molecular genetics, as exemplified by recent identification of several genes encoding phycobilisome components.Combined ultrastructural, biochemical and biophysical approaches to the study of cyanobacterial and red algal cells and isolated phycobilisome-thylakoid fractions are leading to a clearer understanding of the phycobilisome-thylakoid structural interactions, energy transfer to the reaction centers and regulation of excitation energy distribution. However, compared to our current knowledge concerning the structural and functional organization of the isolated phycobilisome, this research area is relatively unexplored.     

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     

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.     

Diversity and evolution of phycobilisomes in marine <Emphasis Type="Italic">Synechococcus</Emphasis>spp.: a comparative genomics study

Background  

Marine Synechococcus owe their specific vivid color (ranging from blue-green to orange) to their large extrinsic antenna complexes called phycobilisomes, comprising a central allophycocyanin core and rods of variable phycobiliprotein composition. Three major pigment types can be defined depending on the major phycobiliprotein found in the rods (phycocyanin, phycoerythrin I or phycoerythrin II). Among strains containing both phycoerythrins I and II, four subtypes can be distinguished based on the ratio of the two chromophores bound to these phycobiliproteins. Genomes of eleven marine Synechococcus strains recently became available with one to four strains per pigment type or subtype, allowing an unprecedented comparative genomics study of genes involved in phycobilisome metabolism.     

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.     

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin and subunits; allophycocyanin and subunits; subunit of allophycocyanin B; the allophycocyanin -subunit-like polypeptide of M_r 18 000; the linker phycobiliprotein of M_r 99 000; and non-chromophore-carrying linker polypeptides of M_r 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin and subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.Abbreviations used SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate - 2D-PAGE two-dimensional gel electrophoresis in which the first dimension consisted of isoelectric focusing in the presence of 8.0 M urea in the pH range 4–6 and the second dimension consisted of electrophoresis in the presence of sodium dodecylsulfate. The nomenclature employed for the phycobiliprotein subunits and linker polypeptides is that defined by Glazer (1985)     

Pigment protein complexes and the concept of the photosynthetic unit: Chlorophyll complexes and phycobilisomes

The photosynthetic unit includes the reaction centers (RC 1 and RC 2) and the light-harvesting complexes which contribute to evolution of one O₂ molecule. The light-harvesting complexes, that greatly expand the absorptance capacity of the reactions, have evolved along three principal lines. First, in green plants distinct chlorophyll (Chl) a/b-binding intrinsic membrane complexes are associated with RC 1 and RC 2. The Chl a/b-binding complexes may add about 200 additional chromophores to RC 2. Second, cyanobacteria and red algae have a significant type of antenna (with RC 2) in the form of phycobilisomes. A phycobilisome, depending on the size and phycobiliprotein composition adds from 700 to 2300 light-absorbing chromophores. Red algae also have a sizable Chl a-binding complex associated with RC 1, contributing an additional 70 chromophores. Third, in chromophytes a variety of carotenoid-Chl-complexes are found. Some are found associated with RC 1 where they may greatly enhance the absorptance capacity. Association of complexes with RC 2 has been more difficult to ascertain, but is also expected in chromophytes. The apoprotein framework of the complexes provides specific chromophore attachment sites, which assures a directional energy transfer whithin complexes and between complexes and reaction centers. The major Chl-binding antenna proteins generally have a size of 16–28 kDa, whether of chlorophytes, chromophytes, or rhodophytes. High sequence homology observed in two of three transmembrane regions, and in putative chlorophyll-binding residues, suggests that the complexes are related and probably did not evolve from widely divergent polyphyletic lines.Abbreviations APC allophycocyanin - B phycoerythrin-large bangiophycean phycoerythrin - Chl chlorophyll - L_CM linker polypeptide in phycobilisome to thylakoid - FCP fucoxanthin Chl a/c complex - LHC(s) Chl-binding light harvesting complex(s) - LHC I Chl-binding complex of Photosystem I - LHC II Chl-binding complex of Photosystem II - PC phycocyanin - PCP peridinin Chl-binding complex - P700 photochemically active Chl a of Photosystem I - PS I Photosystem I - PS II Photosystem II - RC 1 reaction center core of PS I - RC 2 reaction center core of PS II - R phycoerythrin-large rhodophycean phycoerythrin - sPCP soluble peridinin Chl-binding complex     

Distinct roles of CpcG1 and CpcG2 in phycobilisome assembly in the cyanobacterium Synechocystis sp. PCC 6803

Structural role of the second copy of the rod–core linker CpcG, which was found by genome analysis, was studied in Synechocystis sp. PCC 6803 by gene disruption and fractionation of phycobilisome (sub)complexes. Disruption of cpcG2 (sll1471) resulted in a marked decrease in phycocyanin content both in the background of wild-type and cpcG1 (slr2051)-disruptant. The unique phycocyanin rod–CpcG2 complex without the major allophycocyanin components was isolated from the cpcG1-disruptant. By fluorescence analysis, it was proposed that CpcG2 protein connects the rods with a minor allophycocyanin component, to support energy transfer to Photosystem I.     

The structure of cyanobacterial phycobilisomes: a model

Phycobilisomes, supramolecular complexes of water-soluble accessory pigments, serve as the major light-harvesting antennae in cyanobacteria and red algae. Regular arrays of these organelles are found on the surface of the thylakoid membranes of these organisms. In the present study, the hemi-discoidal phycobilisomes of several species of cyanobacteria were examined in thin sections of cells and by negative staining after isolation and fixation. Their fundamental structures were found to be the same. Isolated phycobilisomes possessed a triangular core assembled from three stacks of disc-shaped subunits. Each stack contained two discs which were 12 nm in diameter and 6–7 nm thick. Each of these discs was probably subdivided into halves 3–3.5 nm thick. Radiating from each of two sides of the triangular core were three rods 12 nm in diameter. Each rod consisted of stacks of 2 to 6 disc-shaped subunits 6 nm thick. These discs were subdivided into halves 3 nm thick.The average number of discs of 6 nm thickness forming the peripheral rods varied among the strains studied. For certain chromatically adapting strains, the average rod length was dependent upon the wavelength of light to which cells were exposed during growth. Analyses of phycobilisomes by spectroscopic techniques, polyacrylamide gel electrophoresis, and electron microscopy were compared. These analyses suggested that the triangular core was composed of allophycocyanin and that the peripheral rods contained phycocyanin and phycoerythrin (when present). A detailed model of the hemi-discoidal phycobilisome is proposed. This model can account for many aspects of phycobiliprotein assembly and energy transfer.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin - AP-B allophycocyanin B - C- cyanobacterial - R- rhodophytan - B- Bangiophycean - SDS sodium dodecyl sulfate - LPP Lyngbya-Plectonema-Phormidium group - Na-KPO₄ buffers NaH₂PO₄ titrated with a solution of KH₂PO₄ of equivalent molarity to a given pH     

Changes in the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6714 following light-to-dark and dark-to-light transitions

The photosynthetic apparatus of Synechocystis sp. PCC 6714 cells grown chemoheterotrophically (dark with glucose as a carbon source) and photoautotrophically (light in a mineral medium) were compared. Dark-grown cells show a decrease in phycocyanin content and an even greater decrease in chlorophyll content with respect to light-grown cells. Analysis of fluorescence emission spectra at 77 K and at 20 °C, of dark- and light-grown cells, and of phycobilisomes isolated from both types of cells, indicated that in darkness the phycobiliproteins were assembled in functional phycobilisomes (PBS). The dark synthesized PBS, however, were unable to transfer their excitation energy to PS II chlorophyll. Upon illumination of dark-grown cells, recovery of photosynthetic activity, pigment content and energy transfer between PBS and PS II was achieved in 24–48 h according to various steps. For O₂ evolution the initial step was independent of protein synthesis, but the later steps needed de novo synthesis. Concerning recovery of PBS to PS II energy transfer, light seems to be necessary, but neither PS II functioning nor de novo protein synthesis were required. Similarly, light, rather than functional PS II, was important for the recovery of an efficient energy transfer in nitrate-starved cells upon readdition of nitrate. In addition, it has been shown that normal phycobilisomes could accumulate in a Synechocystis sp. PCC 6803 mutant deficient in Photosystem II activity.Abbreviations APC allophycocyanin - CAP chloroamphenicol - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - CP-47 chlorophyll-binding Photosystem II protein of 47 kDa - EF exoplasmic face - PBS phycobilisome - PC phycocyanin - PS Photosystem     

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     

Organization and nucleotide sequence of genes encoding core components of the phycobilisomes from Synechococcus 6301

Summary Cyanobacteria possess specialized organelles, called phycobilisomes, which collect and transfer light energy to the reaction centres of photosystem II, in the photosynthetic membrane. Phycobilisomes consist of a central core, mainly composed of allophycocyanin, from which six rods radiate. We report here the isolation, for the first time, of three genes that encode core components of cyanobacterial phycobilisomes. The genes coding for the -and -subunit apoproteins of allophycocyanin (apcA and apcB) were cloned from Synechococcus PCC 6301 and subjected to nucleotide sequence analysis. Dowstream of apcB, we found a third open reading frame (apcC) which, by comparison with known amino acid sequences, was assigned to L _c ^7.8 , a linker polypeptide associated with phycobiliproteins within the core of the phycobilisomes. Homologies between amino acid sequences deduced from the nucleotide sequence of the Synechococcus PCC 6301 apc genes and the amino acid sequences published for corresponding proteins either from cyanobacteria or chloroplast-like organelles of eukaryotic organisms, are 75% or more. The genetic organization of this photosynthetic gene cluster relative to that observed in the cyanelle genome of the flagellate Cyanophora paradoxa is discussed.     

Changes in the antenna size of Photosystem I and Photosystem II in Synechococcus sp. strain PCC 7942 grown in the presence of SANDOZ 9785 — a Photosystem II inhibitor

SANDOZ 9785, also known as BASF 13.338, is a pyridazinone derivative that inhibits Photosystem II (PS II) activity leading to an imbalance in the rate of electron transport through the photosystems. Synechococcus sp. strain PCC 7942 cells grown in the presence of sublethal concentration of SANDOZ 9785 (SAN 9785) for 48 hours exhibited a 20% decrease in Chl a per cell. However, no changes were observed in the content of phycocyanin per cell, the size of the phycobilisomes or in the PS II:PS I ratio. From an estimate of PS II electron transport rate under varying light intensities and spectral qualities and analysis of room temperature Chl a fluorescence induction, it was deduced that growth of Synechococcus PCC 7942 in the presence of SAN 9785 leads to a redistribution of excitation energy in favour of PS II. Though the redistribution appears to be primarily caused by changes affecting the Chl a antenna of PS II, the extent of energetic coupling between phycobilisomes and PS II is also enhanced in SAN 9785 grown Synechococcus PCC 7942 cells. There was a reduction in the effective size of PS I antenna based on measurement of P700 photooxidation kinetics. These results indicate that when PS II is partially inhibited, the structure of photosynthetic apparatus alters to redistribute the excitation energy in favour of PS II so that the efficiency of utilization of light energy by the two photosystems is optimized. Our results suggest that under the conditions used, drastic structural changes are not essential for redistribution of excitation energy between the photosystems.Abbreviations APC Allophycocyanin - Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophyenyl)-1,1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - F_o fluorescence when all the reaction centres are open - f_m fluorescence yield when all the reaction centres are closed - F_v variable chlorophyll fluorescence - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic Acid - I₅₀ concentration that causes 50% inhibition in activity - MV methyl viologen - pBQ para benzoquinone - PBS phycobilisome - PC phycocyanin - PS I, PS II Photosystem I, Photosystem II - P700 reaction centre Chl a of PS I - SAN 9785 SANDOZ 9785 i.e. 4-chloro-5-dimethylamino-2-phenyl-3 (2H) pyridazinone, also known as BASF 13.338