urobilin and phycoerythrobilin

Recombinant phycobiliprotein can be used as fluorescent label in immunofluorescence assay. In this study, pathway for phycocyanin beta subunit (CpcB) carrying noncognate chromophore phycoerythrobilin (PEB) and phycourobilin (PUB) was constructed in Escherichia coli. Lyase CpcS and CpcT could catalyze attachment of PEB to Cys84-CpcB and Cys155-CpcB, respectively. However, PEB was attached only to Cys84-CpcB when both CpcS and CpcT were present in E. coli. A dual plasmid expression system was used to control the expression of lyases and the attachment order of PEB to CpcB. The production of PEB-Cys155-CpcB was achieved by L-arabinose-induced expression of CpcS, CpcB, Ho1, and PebS, and then the attachment of PEB to Cys84-CpcB was achieved by IPTG-induced expression of CpcS. The doubly chromophorylated CpcB absorbed light maximally at 497.5 nm and 557.0 nm and fluoresced maximally at 507.5 nm and 566.5 nm. An amount of light energy absorbed by PUB-Cys155-CpcB is transferred to PEB-Cys84-CpcB in doubly chromophorylated CpcB, conferring a large stokes shift of 69 nm for this fluorescent protein. There are interactions between chromophores of CpcB which possibly together with the help of lyases lead to isomerization of PEB-Cys155-CpcB to PUB-Cys155-CpcB.

Topics: Bacterial Proteins; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Lyases; Phycobilins; Phycobiliproteins; Phycocyanin; Phycoerythrin; Plasmids; Urobilin

Marine

Topics: Acclimatization; Adaptation, Ocular; Bacterial Proteins; Color; Gene Expression Regulation; Genes, Bacterial; Lyases; Mutation; Phycobilins; Phycoerythrin; Recombinant Proteins; Seawater; Synechococcus; Urobilin

Marine Synechococcus thrive over a range of light regimes in the ocean. We examined the proteomic, genomic and physiological responses of seven Synechococcus isolates to moderate irradiances (5-80 μE m

Topics: Adaptation, Ocular; Carbon Cycle; Electron Transport; Light; Oxidative Stress; Photosynthesis; Phycobilins; Phycobilisomes; Phycoerythrin; Phylogeny; Proteomics; Synechococcus; Urobilin

The widespread unicellular cyanobacteria Synechococcus are major contributors to global marine primary production. Here, we report their abundance, phylogenetic diversity (as assessed using the RNA polymerase gamma subunit gene rpoC1) and pigment diversity (as indirectly assessed using the laterally transferred cpeBA genes, encoding phycoerythrin-I) in surface waters of the northwestern Pacific Ocean, sampled over nine distinct cruises (2008-2015). Abundance of Synechococcus was low in the subarctic ocean and South China Sea, intermediate in the western subtropical Pacific Ocean, and the highest in the Japan and East China seas. Clades I and II were by far the most abundant Synechococcus lineages, the former dominating in temperate cold waters and the latter in (sub)tropical waters. Clades III and VI were also fairly abundant in warm waters, but with a narrower distribution than clade II. One type of chromatic acclimater (3dA) largely dominated the Synechococcus communities in the subarctic ocean, while another (3dB) and/or cells with a fixed high phycourobilin to phycoerythrobilin ratio (pigment type 3c) predominated at mid and low latitudes. Altogether, our results suggest that the variety of pigment content found in most Synechococcus clades considerably extends the niches that they can colonize and therefore the whole genus habitat.

Topics: China; Ecosystem; Japan; Pacific Ocean; Phycobilins; Phycoerythrin; Phylogeny; Phylogeography; Pigments, Biological; Seawater; Synechococcus; Urobilin

The picocyanobacterium Synechococcus is a prominent primary producer in the marine environment. The marine Synechococcus strains are clustered into different clades representing ecologically distinct genotypes. In this study, we compared phylogeny, photophysiology and cell cycles of four novel phycoerythrin-containing Synechococcus strains (clade II of subcluster 5.1) isolated from different depths of the water column (surface and subsurface waters) in coastal and offshore regions of the eastern Arabian Sea. The surface water strains possessed a lesser number of thylakoid layers and had a higher zeaxanthin to chlorophyll a ratio than subsurface strains indicating possible influence of light intensity available at their niche. The DNA distribution pattern of the four strains was bimodal in optimal cellular physiology conditions with cell division restricted to the light period and synchronized with the light-dark cycle. The presence of phycourobilin or phycoerythrobilin and the ratio between these two chromophores in all four strains varied according to available spectral wavelength in situ This study indicates that the timing of cell division is conserved within these genotypically identical Synechococcus strains, despite their having different chromophore ratios. We conclude that the timing of cell division of the Synechococcus strains has a genetic basis rather than being determined by phenotypic characters, such as chromophore content and ratio.

Topics: Asia, Western; Cell Division; Chlorophyll; Chlorophyll A; DNA, Bacterial; Light; Microscopy, Electron, Transmission; Oceans and Seas; Phenotype; Photosynthesis; Phycobilins; Phycoerythrin; Phylogeny; Synechococcus; Urobilin; Water

Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift. The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

Topics: Cyanobacteria; Luminescent Proteins; Photochemical Processes; Phycobilins; Phycoerythrin; Temperature; Urobilin

Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610-660nm, and fluoresced at ~670nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540-575nm, and fluorescing at ~585nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly.

Topics: Absorption; Apoproteins; Cell Membrane; Escherichia coli; Lyases; Phycobilins; Phycobilisomes; Phycocyanin; Phycoerythrin; Recombinant Fusion Proteins; Solubility; Spectrometry, Fluorescence; Urobilin

Phylogenetic relationships among 33 Synechococcus strains isolated from the East China Sea (ECS) and the East Sea (ES) were studied based on 16S rRNA gene sequences and 16S-23S rRNA gene internal transcribed spacer (ITS) sequences. Pigment patterns of the culture strains were also examined. Based on 16S rRNA gene and ITS sequence phylogenies, the Synechococcus isolates were clustered into 10 clades, among which eight were previously identified and two were novel. Half of the culture strains belonged to clade V or VI. All strains that clustered into novel clades exhibited both phycoerythrobilin and phycourobilin. Interestingly, the pigment compositions of isolates belonging to clades V and VI differed from those reported for other oceanic regions. None of the isolates in clade V showed phycourobilin, whereas strains in clade VI exhibited both phycourobilin and phycoerythrobilin, which is in contrast to previous studies. The presence of novel lineages and the different pigment patterns in the ECS and the ES suggests the possibility that some Synechococcus lineages are distributed only in geographically restricted areas and have evolved in these regions. Therefore, further elucidation of the physiological, ecological, and genetic characteristics of the diverse Synechococcus strains is required to understand their spatial and geographical distribution.

Topics: DNA, Bacterial; DNA, Ribosomal Spacer; Genes, rRNA; Molecular Sequence Data; Pacific Ocean; Phycobilins; Phycoerythrin; Phylogeny; RNA, Ribosomal, 16S; Seawater; Sequence Analysis, DNA; Synechococcus; Urobilin; Water Microbiology

A preliminary study was carried out on a picocyanobacterial mixed culture harvested from the Gulf of Trieste (Northern Adriatic) and identified as Synechococcus spp. both by transmission electron microscopy observations, biliprotein composition and molecular analyses. Absorption and fluorescence spectra revealed phycourobilin and phycoerythrobilin chromophores, suggesting the presence of both CU- and C-phycoerythrin, besides phycocyanobilin chromophores typical for phycocyanins and allophycocyanins. Both biliprotein analyses and molecular identification indicated the presence of at least two Synechococcus subgroups presumably differing either in phycoerythrin type or in physiological traits. Among the exoenzymatic activities acting on different substrates, only aminopeptidase showed high hydrolysis rates and the uptake of organic molecules was positive for leucine but not for thymidine. The protein carbon mobilized was high compared with the leucine incorporation rates, resulting in low percentages of newly mobilized carbon utilized by cultures. The organic carbon incorporated as leucine was compared with the photosynthetically produced one, and the balance between the phototrophic- and heterotrophic-like processes was c. 3 : 1. Our findings suggest that the Synechococcus heterotrophy plays an important role in cell's metabolism, and that the photoheterotrophic behavior, together with their chromatic adaptation capability, might represent the key for the absolute dominance of this genus in the Adriatic Sea.

Topics: Aminopeptidases; Bacterial Proteins; DNA, Bacterial; DNA, Ribosomal; Electrophoresis, Polyacrylamide Gel; Light; Mediterranean Sea; Microscopy, Electron, Transmission; Nucleic Acid Denaturation; Organic Chemicals; Phycobilins; Phycocyanin; Phycoerythrin; Phylogeny; RNA, Ribosomal, 16S; Seawater; Sequence Analysis, DNA; Sequence Homology; Synechococcus; Urobilin

Chromatic adaptation (CA) in cyanobacteria has provided a model system for the study of the environmental control of photophysiology for several decades. All forms of CA that have been examined so far (types II and III) involve changes in the relative contents of phycoerythrin (PE) and/or phycocyanin when cells are shifted from red to green light and vice versa. However, the chromophore compositions of these polypeptides are not altered. Some marine Synechococcus species strains, which possess two PE forms (PEI and PEII), carry out another type of CA (type IV), occurring during shifts from blue to green or white light. Two chromatically adapting strains of marine Synechococcus recently isolated from the Gulf of Mexico were utilized to elucidate the mechanism of type IV CA. During this process, no change in the relative contents of PEI and PEII was observed. Instead, the ratio of the two chromophores bound to PEII, phycourobilin and phycoerythrobilin, is high under blue light and low under white light. Mass spectroscopy analyses of isolated PEII alpha- and beta-subunits show that there is a single PEII protein type under all light climates. The CA process seems to specifically affect the chromophorylation of the PEII (and possibly PEI) alpha chain. We propose a likely process for type IV CA, which involves the enzymatic activity of one or several phycobilin lyases and/or lyase-isomerases differentially controlled by the ambient light quality. Phylogenetic analyses based on the 16S rRNA gene confirm that type IV CA is not limited to a single clade of marine Synechococcus.

Topics: Adaptation, Physiological; Bacterial Proteins; Light; Light-Harvesting Protein Complexes; Lyases; Molecular Sequence Data; Phycobilins; Phycoerythrin; RNA, Bacterial; RNA, Ribosomal, 16S; Species Specificity; Synechococcus; Tetrapyrroles; Urobilin

Characterization of two genetically distinct groups of marine Synechococcus sp. strains shows that one, but not the other, increases its phycourobilin/phycoerythrobilin chromophore ratio when growing in blue light. This ability of at least some marine Synechococcus strains to chromatically adapt may help explain their greater abundance in particular ocean environments than cyanobacteria of the genus Prochlorococcus.

Topics: Adaptation, Physiological; Cyanobacteria; Light; Phycobilins; Phycoerythrin; Pyrroles; Seawater; Tetrapyrroles; Urobilin

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim. Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/PEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.

Topics: Atlantic Ocean; Cloning, Molecular; Cyanobacteria; DNA-Directed RNA Polymerases; Fresh Water; Genetic Variation; Movement; Pacific Ocean; Photosynthesis; Phycobilins; Phycoerythrin; Phylogeny; Polymerase Chain Reaction; Pyrroles; Seawater; Tetrapyrroles; Urobilin

Topics: Bile Pigments; Eukaryota; Phycobilins; Phycoerythrin; Pigments, Biological; Plant Proteins; Research; Spectrophotometry; Urobilin