Phycobiliproteins Phycoerythrin Research Articles

Optimization of spectral light quality for growth and product formation in different microalgae using a continuous photobioreactor

Many parameters for microalgae cultivation are not fully optimized so far. Even though light is one important process parameter, little research is done regarding its spectral composition. In contrast to other published data, mostly with monochromatic or dichromatic illumination, the objective of this study was to investigate the influence of a broad range of different light qualities to the productivity of cell dry weight as well as the productivity of their phycobiliproteins phycoerythrin or phycocyanin, by keeping the overall light intensity constant. Instead of cultivating in batch mode, cultivations were done in continuous mode in closed, thermal sterilizable photobioreactors. A total of 37 light quality conditions are shown in this work. In case of Chlamydomonas reinhardtii a composition of αred:αgreen:αblue of 80:10:10 resulted in the highest biomass productivity, with a value of 252mg·L−1·d−1. A maximum biomass productivity is achieved for Galdieria sulphuraria at pure red light (304mg·L−1·d−1), whereas the highest phycocyanin productivity is reached at a ratio of αred:αgreen:αblue of 60:0:40 (4.99mg·L−1·d−1). The highest biomass and phycoerythrin productivity was achieved for Porphyridium purpureum at αred:αgreen:αblue of 40:40:20, with values of 312mg·L−1·d−1 and 16.93mg·L−1·d−1, respectively. The collected data show clearly the significance to investigate the right mixture of light for every microalgal strain and the desired product of interest individually.

Phycobiliproteins in Prochlorococcus marinus: Biosynthesis of pigments and their assembly into proteins

Prochlorococcus sp. is a very unique and highly abundant class of organisms within the cyanobacteria. Found in the world's oceans Prochlorococcus is very small in size and possesses the smallest genome of a photosynthetic autotroph. Prochlorococcus is characterized by a special chlorophyll antenna for light harvesting and the absence of classical cyanobacterial phycobilisomes. Despite the lack of phycobilisomes Prochlorococcus possesses remnants thereof which is the phycobiliprotein phycoerythrin (PE) encoded in a PE operon as well as genes encoding enzymes of phycobilin biosynthesis. The size of this PE operon varies depending on the light-adapted ecotype. While high-light strains only possess a β-subunit of PE, low-light adapted strains possess both, an α- and a β-subunit. α-/β-subunits are also present in functional phycobilisomes. Consistent with the number of subunits is also the varying number of putative lyase genes, involved in the transfer and attachment of phycobilins (open-chain tetrapyrroles) to the PE subunits. This minireview summarizes the only sparely available data on the biosynthesis and assembly of Prochlorococcus PE. On one hand the quite well understood biosynthesis of pigments will be reviewed but also new data on the phycobiliprotein lyase-mediated transfer of the phycobilins to the PE subunits will be discussed.

Distinctive organization of genes for light-harvesting proteins in the cryptophyte alga Rhodomonas

Cryptophyte algae contain two kinds of light-harvesting protein, phycobiliproteins and chlorophyll a,c-binding proteins. The β subunit of the phycobiliprotein phycoerythrin (PE) is encoded in the chloroplast. Genes for the other PE polypeptides are located in the nucleus but little is known of their organization. We cloned and sequenced six cpeA genes encoding the phycoerythrin α subunit from a genomic library of the cryptophyte Rhodomonas CS24. Derived peptide sequences of the cpeA genes show that α subunits occur in at least two forms, a longer α 1 form and a shorter α 2 form. Remarkably, all six cpeA genes occur in divergent pairs encoding one α 1 and one α 2 subunit. Four cac genes encoding chlorophyll a,c-binding proteins were cloned and sequenced and also found to occur in divergent pairs comprising one cac1 and one cac2 gene. Inspection of the predicted targeting sequences of the α 1 and α 2 phycoerythrin polypeptides shows that only the α 1 polypeptides have a thylakoid lumen targeting sequence, corresponding to the TAT pathway. Given the previously reported lack of a lumen-targeting sequence on the β subunit, we propose a novel import mechanism in which the entire α 1α 2ββ phycoerythrin complex is assembled in the stroma and transported into the thylakoid under the direction of the single targeting sequence on the α 1 protein. The FAP motif implicated in plastid targeting in diatoms appears to be conserved in this cryptophyte.

Field spectrometry: New methods to investigate epilithic micro-algae on rocky shores

The use of field reflectance spectrometry to detect and quantify chlorophyll- a in intertidal epilithic micro-algae is evaluated. Sandstone disks were placed into intertidal areas for different amounts of time to accumulate micro-algae. Field reflectance spectra (350–1050 nm) were acquired from each of the disks before and after they were collected from the intertidal zone. Pigments were extracted from the sandstone disks using methanol and chlorophyll- a concentration was determined spectrophotometrically. Other pigments were identified using High Performance Liquid Chromatography (HPLC). Different ratios of reflectance were tested. A ratio of reflectances at 750 and 670 nm ( R 750 / R 670) had the tightest correlation with chlorophyll- a ( R 2 = 0.93; SE = 1.14; P = < 0.001; n = 25). Data from the field were used to predict values of chlorophyll- a; predictions were strongly and linearly related to observed values ( R 2 = 0.95; SE = 0.15; P < 0.001; n = 8). The average difference and average absolute difference between observed and predicted values of chlorophyll- a was 0.03 μg cm − 2 (1.79% of the measured range) and 0.14 μg cm − 2 (8.38% of the measured range), respectively. Absorptions due to chlorophylls a, b and c, carotenoids and the phycobiliproteins phycoerythrin, phycocyanin and allophycocyanin were identified. The techniques described here enable estimation of amounts of chlorophyll- a and the detection of several other pigments. Thus, investigations of microflora in studies of succession, biofilms or grazing in intertidal habitats can be enhanced by field spectrometry.

Direct observation of ligand recognition by T cells.

The activation of T cells through interaction of their T-cell receptors with antigenic peptide bound to major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a crucial step in adaptive immunity. Here we use three-dimensional fluorescence microscopy to visualize individual peptide-I-E(k) class II MHC complexes labelled with the phycobiliprotein phycoerythrin in an effort to characterize T-cell sensitivity and the requirements for forming an immunological synapse in single cells. We show that T cells expressing the CD4 antigen respond with transient calcium signalling to even a single agonist peptide-MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present. This sensitivity is highly dependent on CD4, because blocking this molecule with antibodies renders T cells unable to detect less than about 30 ligands.

Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity.

Prochlorococcus is a major photosynthetic prokaryote in nutrient-limited, open ocean environments and an important participant in the global carbon cycle. This phototroph is distinct from other members of the cyanobacterial lineage to which it belongs because it utilizes a chlorophyll a2/b(2) light-harvesting complex as its major antenna, instead of phycobilisomes. Recently, genes encoding the phycobiliprotein phycoerythrin were identified in several Prochlorococcus isolates, thus making it the only extant photosynthetic prokaryote to possess a chlorophyll a/b antenna as well as phycobiliprotein genes. In order to understand the evolution of phycobiliproteins in this genus, the authors have sequenced the phycoerythrin genes of two isolates that are the most deeply branching in the Prochlorococcus lineage and share the highest degree of 16S rDNA sequence similarity to phycobilisome-containing marine SYNECHOCOCCUS: Sequence analyses suggest that within the Prochlorococcus lineage, the selective forces shaping the evolution of the phycoerythrin gene set have not been uniform. Although strains that are most closely related to marine Synechococcus possess genes (cpeB, cpeA) encoding both subunits of phycoerythrin, a more recently evolved strain is shown to lack cpeA and to possess a degenerate form of cpeB. Differences in phycoerythrin gene sequences between Prochlorococcus and Synechococcus appear to be consistent with a model of elevated mutation rates rather than relaxed selection. This suggests that although phycoerythrin is not a major constituent of the light-harvesting apparatus in Prochlorococcus, as it is in Synechococcus, the cpeB and cpeA genes are still under selection, albeit a different type of selection than in Synechococcus. The evolution of the Prochlorococcus light-harvesting antenna complex provides an important system for understanding the origins and scope of phylogenetic diversity in ocean ecosystems.

Two-photon-induced fluorescence from the phycoerythrin protein

Two-photon-induced fluorescence is observed from the photodynamic phycobiliprotein phycoerythrin. Temporal, spectral, and intensity-dependent properties of the two-photon-induced fluorescence emission from phycoerythrin excited by a 1.06-mum laser beam are reported. The measured two-photon absorption cross section of phycoerythrin is an order of magnitude larger than that of Rhodamine 6G. The potential applications of phycobiliproteins for two-photon-induced fluorescence for microscopy of three-dimensional biological samples and three-dimensional optical memory are discussed.

A role for cpeYZ in cyanobacterial phycoerythrin biosynthesis.

Pigment mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon is characterized by constitutive synthesis of the phycobiliprotein phycoerythrin due to insertional inactivation of the rcaC regulatory gene by endogenous transposon Tn5469. Whereas the parental strain Fd33 harbors five genomic copies of Tn5469, cells of strain FdR1 harbor six genomic copies of the element; the sixth copy in FdR1 is localized to the rcaC gene. Electroporation of FdR1 cells yielded secondary pigment mutant strains FdR1E1 and FdR1E4, which identically exhibited the FdR1 phenotype with significantly reduced levels of phycoerythrin. In both FdR1E1 and FdR1E4, a seventh genomic copy of Tn5469 was localized to the cpeY gene of the sequenced but phenotypically uncharacterized cpeYZ gene set. This gene set is located downstream of the cpeBA operon which encodes the alpha and beta subunits of phycoerythrin. Complementation experiments correlated cpeYZ activity to the phenotype of strains FdR1E1 and FdR1E4. The predicted CpeY and CpeZ proteins share significant sequence identity with the products of homologous cpeY and cpeZ genes reported for Pseudanabaena sp. strain PCC 7409 and Synechococcus sp. strain WH 8020, both of which synthesize phycoerythrin. The CpeY and CpeZ proteins belong to a family of structurally related cyanobacterial proteins that includes the subunits of the CpcE/CpcF phycocyanin alpha-subunit lyase of Synechococcus sp. strain PCC 7002 and the subunits of the PecE/PecF phycoerythrocyanin alpha-subunit lyase of Anabaena sp. strain PCC 7120. Phycobilisomes isolated from mutant strains FdR1E1 and FdR1E4 contained equal amounts of chromophorylated alpha and beta subunits of phycoerythrin at 46% of the levels of the parental strain FdR1. These results suggest that the cpeYZ gene products function in phycoerythrin synthesis, possibly as a lyase involved in the attachment of phycoerythrobilin to the alpha or beta subunit.

Cy7PE and Cy7APC: bright new probes for immunofluorescence.

We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a "brightness" (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a "brightness" comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC.

Cy7PE and Cy7APC: Bright new probes for immunofluorescence

We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a “brightness” (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a “brightness” comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC. © 1996 Wiley-Liss, Inc.

Fine structure and immunolocalisation of proteins inAphanizomenonsp. from the Baltic Sea

The subcellular arrangement of vegetative cells in the colony-forming cyanobacterium Aphanizomenon sp., collected from blooms in the Baltic Sea in the summer of 1991 and 1992, was characterised by a loose network of thylakoids with intrathylakoid spaces. The phycobiliprotein phycoerythrin (PE) was found in vegetative cells at locations corresponding to thylakoids and similar relative quantities of PE were found in heterocysts; however, the subcellular location was ambiguous in heterocysts. Cellular inclusion bodies common in cyanobacteria, such as polyhedral bodies, cyanophycin and polyphosphate granules, were present in most vegetative cells but not in heterocysts. A large polyhedral crystal was present in both cell types. Antibodies against Rubisco did not bind to this inclusion but to the smaller polyhedral bodies (i.e. carboxysomes) and free Rubisco in the cytoplasm. In addition, gas vesicles were found to be exclusive to vegetative cells and occasionally microtubule-like inclusions and spherical memb...

Occurrence and Localization of Phycoerythrin in Symbiotic Nostoc of Cycas revoluta and in the Free-Living Isolated Nostoc 7422

The phycobiliprotein phycoerythrin was localized in symbiotic and free-living Nostoc of the cycad Cycas using immunocytochemistry. In symbiotic Nostoc, phycoerythrin was associated with the thylakoid membranes of vegetative cells and absent from heterocysts. Similar cellular/subcellular localization was observed between symbiotic Nostoc and the free-living Cycas isolate Nostoc 7422.

Cell morphology and growth characteristics ofPorphyridium aerugineum (Rhodophyta)

Four unialgal strains of the freshwater coccoid red algaPorphyridium aerugineumGeitler were cultivated under laboratory conditions. Cell morphology was studied with the light microscope. The cell surface was examined by means of electron microscopy in order to contribute to the knowledge of polysaccharide sheaths and cytoplasmic membranes. Optimum growth conditions were determined. The range of cell sizes and the average dry masses of single cells were compared in all four strains cultivated at exactly defined temperatures and irradiances. Photosynthetic pigment maxima were measured in intact cells. The red-coloured phycobiliprotein phycoerythrin was not found in any of the examined strains.