Molecular bases of an alternative dual-enzyme system for light color acclimation of marine Synechococcus cyanobacteria.

Marine Synechococcus cyanobacteria owe their ubiquity in part to the wide pigment diversity of their light-harvesting complexes. In open ocean waters, cells predominantly possess sophisticated antennae with rods composed of phycocyanin and two types of phycoerythrins (PEI and PEII). Some strains are specialized for harvesting either green or blue light, while others can dynamically modify their light absorption spectrum to match the dominant ambient color. This process, called type IV chromatic acclimation (CA4), has been linked to the presence of a small genomic island occurring in two configurations (CA4-A and CA4-B). While the CA4-A process has been partially characterized, the CA4-B process has remained an enigma. Here we characterize the function of two members of the phycobilin lyase E/F clan, MpeW and MpeQ, in Synechococcus sp. strain A15-62 and demonstrate their critical role in CA4-B. While MpeW, encoded in the CA4-B island and up-regulated in green light, attaches the green light-absorbing chromophore phycoerythrobilin to cysteine-83 of the PEII α-subunit in green light, MpeQ binds phycoerythrobilin and isomerizes it into the blue light-absorbing phycourobilin at the same site in blue light, reversing the relationship of MpeZ and MpeY in the CA4-A strain RS9916. Our data thus reveal key molecular differences between the two types of chromatic acclimaters, both highly abundant but occupying distinct complementary ecological niches in the ocean. They also support an evolutionary scenario whereby CA4-B island acquisition allowed former blue light specialists to become chromatic acclimaters, while former green light specialists would have acquired this capacity by gaining a CA4-A island.

Ecophysiological analysis reveals distinct environmental preferences in closely related Baltic Sea picocyanobacteria.

Cluster 5 picocyanobacteria significantly contribute to primary productivity in aquatic ecosystems. Estuarine populations are highly diverse and consist of many co-occurring strains, but their physiology remains largely understudied. In this study, we characterized 17 novel estuarine picocyanobacterial strains. Phylogenetic analysis of the 16S rRNA and pigment genes (cpcB and cpeBA) uncovered multiple estuarine and freshwater-related clusters and pigment types. Assays with five representative strains (three phycocyanin rich and two phycoerythrin rich) under temperature (10-30°C), light (10-190 µmol photons m-2 s-1 ), and salinity (2-14 PSU) gradients revealed distinct growth optima and tolerance, indicating that genetic variability was accompanied by physiological diversity. Adaptability to environmental conditions was associated with differential pigment content and photosynthetic performance. Amplicon sequence variants at a coastal and an offshore station linked population dynamics with phylogenetic clusters, supporting that strains isolated in this study represent key ecotypes within the Baltic Sea picocyanobacterial community. The functional diversity found within strains with the same pigment type suggests that understanding estuarine picocyanobacterial ecology requires analysis beyond the phycocyanin and phycoerythrin divide. This new knowledge of the environmental preferences in estuarine picocyanobacteria is important for understanding and evaluating productivity in current and future ecosystems.

Cyanobacterial phycobilisomes as a platform for the stable production of heterologous enzymes and other proteins.

Efforts to stably over-express recombinant proteins in cyanobacteria are hindered due to cellular proteasome activity that efficiently degrades foreign proteins. Recent work from this lab showed that a variety of exogenous genes from plants, humans, and bacteria can be successfully and stably over-expressed in cyanobacteria, as fusion constructs with the abundant ß-subunit of phycocyanin (the cpcB gene product) in quantities up to 10-15% of the total cell protein. The CpcB*P fusion proteins did not simply accumulate in a soluble free-floating form in the cell but, rather, they assembled as functional (α,ß*P)3CpcG1 heterohexameric light-harvesting phycocyanin antenna discs, where α is the CpcA α-subunit of phycocyanin, ß*P is the CpcB*P fusion protein, the asterisk denoting fusion, and CpcG1 is the 28.9 kDa phycocyanin disc linker polypeptide (Hidalgo Martinez et al., 2022). The present work showed that the CpcA α-subunit of phycocyanin and the CpcG1 28.9 kDa phycocyanin disc linker polypeptide can also successfully serve as leading sequences in functional heterohexameric (α*P,ß)3CpcG1 and (α,ß)3CpcG1*P fusion constructs that permit stable recombinant protein over-expression and accumulation. These were shown to form a residual light-harvesting antenna and to contribute to photosystem-II photochemistry in the cyanobacterial cells. The work suggested that cyanobacterial cells need phycocyanin for light absorption, photosynthesis, and survival and, therefore, may tolerate the presence of heterologous recombinant proteins, when the latter are in a fusion construct configuration with essential cellular proteins, e.g., phycocyanin, thus allowing their substantial and stable accumulation.

A hybrid type of chromatic acclimation regulated by the dual green/red photosensory systems in cyanobacteria.

Cyanobacteria are phototrophic bacteria that perform oxygenic photosynthesis. They use a supermolecular light-harvesting antenna complex, the phycobilisome (PBS), to capture and transfer light energy to photosynthetic reaction centers. Certain cyanobacteria alter the absorption maxima and/or overall structure of their PBSs in response to the ambient light wavelength-a process called chromatic acclimation (CA). One of the most well-known CA types is the response to green and red light, which is controlled by either the RcaEFC or CcaSR photosensory system. Here, we characterized a hybrid type of CA in the cyanobacterium Pleurocapsa sp. Pasteur Culture Collection (PCC) 7319 that uses both RcaEFC and CcaSR systems. In vivo spectroscopy suggested that strain PCC 7319 alters the relative composition of green-absorbing phycoerythrin and red-absorbing phycocyanin in the PBS. RNA sequencing and promoter motif analyses suggested that the RcaEFC system induces a gene operon for phycocyanin under red light, whereas the CcaSR system induces a rod-membrane linker gene under green light. Induction of the phycoerythrin genes under green light may be regulated through a yet unidentified photosensory system called the Cgi system. Spectroscopy analyses of the isolated PBSs suggested that hemidiscoidal and rod-shaped PBSs enriched with phycoerythrin were produced under green light, whereas only hemidiscoidal PBSs enriched with phycocyanin were produced under red light. PCC 7319 uses the RcaEFC and CcaSR systems to regulate absorption of green or red light (CA3) and the amount of rod-shaped PBSs (CA1), respectively. Cyanobacteria can thus flexibly combine diverse CA types to acclimate to different light environments.

Magnetic Fields as Inducers of Phycobiliprotein Production by Synechococcus elongatus PCC 7942.

This study aimed to analyze the effect of magnetic field (MF) application on the metabolism of Synechococcus elongatus PCC 7942. Concentrations of biomass, carbohydrate, protein, lipid, and photosynthetic pigments (chlorophyll-a, C-phycocyanin, allophycocyanin and phycoerythrin) were determined. In cultures with MF application (30 mT for 24 h d-1), there were increases of 47.5% in total protein content, 87.4% in C-phycocyanin, and 332.8% in allophycocyanin contents, by comparison with the control. Allophycocyanin is the most affected pigment by MF application. Therefore, its biosynthetic route was investigated, and four genes related to its synthesis were found. However, the analysis of the gene expression showed no statistical differences from the control culture, which suggests that induction of such genes may occur soon after MF application with consequent stabilization over time. MF application may be a cost-effective alternative to increase production of compounds of commercial interest by cyanobacteria.

Promoting Heme and Phycocyanin Biosynthesis in Synechocystis sp. PCC 6803 by Overexpression of Porphyrin Pathway Genes with Genetic Engineering.

Due to their unique biochemical and spectroscopic properties, both heme and phycocyanobilin are widely applied in the medical and food industries. Synechocystis sp. PCC 6803 contains both heme and phycocyanin, and is capable of synthesizing phycocyanin using heme as a precursor. The aim of this study was to uncover viable metabolic targets in the porphyrin pathway from Synechocystis sp. PCC 6803 to promote the accumulation of heme and phycocyanin in the recombinant strains of microalgae. A total of 10 genes related to heme synthesis pathway derived from Synechococcus elongatus PCC 7942 and 12 genes related to endogenous heme synthesis were individually overexpressed in strain PCC 6803. The growth rate and pigment content (heme, phycocyanin, chlorophyll a and carotenoids) of 22 recombinant algal strains were characterized. Quantitative real-time PCR technology was used to investigate the molecular mechanisms underlying the changes in physiological indicators in the recombinant algal strains. Among the 22 mutant strains, the mutant overexpressing the haemoglobin gene (glbN) of strain PCC 6803 had the highest heme content, which was 2.5 times higher than the wild type; the mutant overexpressing the gene of strain PCC 7942 (hemF) had the highest phycocyanin content, which was 4.57 times higher than the wild type. Overall, the results suggest that genes in the porphyrin pathway could significantly affect the heme and phycocyanin content in strain PCC 6803. Our study provides novel crucial targets for promoting the accumulation of heme and phycocyanin in cyanobacteria.

A far-red cyanobacteriochrome lineage specific for verdins.

Cyanobacteriochromes (CBCRs) are photoswitchable linear tetrapyrrole (bilin)-based light sensors in the phytochrome superfamily with a broad spectral range from the near UV through the far red (330 to 760 nm). The recent discovery of far-red absorbing CBCRs (frCBCRs) has garnered considerable interest from the optogenetic and imaging communities because of the deep penetrance of far-red light into mammalian tissue and the small size of the CBCR protein scaffold. The present studies were undertaken to determine the structural basis for far-red absorption by JSC1_58120g3, a frCBCR from the thermophilic cyanobacterium Leptolyngbya sp. JSC-1 that is a representative member of a phylogenetically distinct class. Unlike most CBCRs that bind phycocyanobilin (PCB), a phycobilin naturally occurring in cyanobacteria and only a few eukaryotic phototrophs, JSC1_58120g3's far-red absorption arises from incorporation of the PCB biosynthetic intermediate 181,182-dihydrobiliverdin (181,182-DHBV) rather than the more reduced and more abundant PCB. JSC1_58120g3 can also yield a far-red-absorbing adduct with the more widespread linear tetrapyrrole biliverdin IXα (BV), thus circumventing the need to coproduce or supplement optogenetic cell lines with PCB. Using high-resolution X-ray crystal structures of 181,182-DHBV and BV adducts of JSC1_58120g3 along with structure-guided mutagenesis, we have defined residues critical for its verdin-binding preference and far-red absorption. Far-red sensing and verdin incorporation make this frCBCR lineage an attractive template for developing robust optogenetic and imaging reagents for deep tissue applications.

Artificial complementary chromatic acclimation gene expression system in Escherichia coli.

BACKGROUND: The development of multiple gene expression systems, especially those based on the physical signals, such as multiple color light irradiations, is challenging. Complementary chromatic acclimation (CCA), a photoreversible process that facilitates the control of cellular expression using light of different wavelengths in cyanobacteria, is one example. In this study, an artificial CCA systems, inspired by type III CCA light-regulated gene expression, was designed by employing a single photosensor system, the CcaS/CcaR green light gene expression system derived from Synechocystis sp. PCC6803, combined with G-box (the regulator recognized by activated CcaR), the cognate cpcG2 promoter, and the constitutively transcribed promoter, the PtrcΔLacO promoter. RESULTS: One G-box was inserted upstream of the cpcG2 promoter and a reporter gene, the rfp gene (green light-induced gene expression), and the other G-box was inserted between the PtrcΔLacO promoter and a reporter gene, the bfp gene (red light-induced gene expression). The Escherichia coli transformants with plasmid-encoded genes were evaluated at the transcriptional and translational levels under red or green light illumination. Under green light illumination, the transcription and translation of the rfp gene were observed, whereas the expression of the bfp gene was repressed. Under red light illumination, the transcription and translation of the bfp gene were observed, whereas the expression of the rfp gene was repressed. During the red and green light exposure cycles at every 6 h, BFP expression increased under red light exposure while RFP expression was repressed, and RFP expression increased under green light exposure while BFP expression was repressed. CONCLUSION: An artificial CCA system was developed to realize a multiple gene expression system, which was regulated by two colors, red and green lights, using a single photosensor system, the CcaS/CcaR system derived from Synechocystis sp. PCC6803, in E. coli. The artificial CCA system functioned repeatedly during red and green light exposure cycles. These results demonstrate the potential application of this CCA gene expression system for the production of multiple metabolites in a variety of microorganisms, such as cyanobacteria.

Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling.

Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

Metabolic engineering of Escherichia coli for efficient biosynthesis of fluorescent phycobiliprotein.

BACKGROUND: Phycobiliproteins (PBPs) are light-harvesting protein found in cyanobacteria, red algae and the cryptomonads. They have been widely used as fluorescent labels in cytometry and immunofluorescence analysis. A number of PBPs has been produced in metabolically engineered Escherichia coli. However, the recombinant PBPs are incompletely chromophorylated, and the underlying mechanisms are not clear. RESULTS AND DISCUSSION: In this work, a pathway for SLA-PEB [a fusion protein of streptavidin and allophycocyanin that covalently binds phycoerythrobilin (PEB)] biosynthesis in E. coli was constructed using a single-expression plasmid strategy. Compared with a previous E. coli strain transformed with dual plasmids, the E. coli strain transformed with a single plasmid showed increased plasmid stability and produced SLA-PEB with a higher chromophorylation ratio. To achieve full chromophorylation of SLA-PEB, directed evolution was employed to improve the catalytic performance of lyase CpcS. In addition, the catalytic abilities of heme oxygenases from different cyanobacteria were investigated based on biliverdin IXα and PEB accumulation. Upregulation of the heme biosynthetic pathway genes was also carried out to increase heme availability and PEB biosynthesis in E. coli. Fed-batch fermentation was conducted for the strain V5ALD, which produced recombinant SLA-PEB with a chromophorylation ratio of 96.7%. CONCLUSION: In addition to reporting the highest chromophorylation ratio of recombinant PBPs to date, this work demonstrated strategies for improving the chromophorylation of recombinant protein, especially biliprotein with heme, or its derivatives as a prosthetic group.

PhiReX: a programmable and red light-regulated protein expression switch for yeast.

Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana. Robust gene expression and high protein levels are achieved by combining genome integrated red light-sensing components with an episomal high-copy reporter construct. The gene of interest as well as the synTALE DNA-binding domain can be easily exchanged, allowing the flexible regulation of any desired gene by targeting endogenous or heterologous promoter regions. To allow low-cost induction of gene expression for industrial fermentation processes, we engineered yeast to endogenously produce the chromophore required for the effective dimerization of PhyB and PIF3. Time course experiments demonstrate high-level induction over a period of at least 48 h.

Orange carotenoid protein burrows into the phycobilisome to provide photoprotection.

In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS), the cyanobacterial antenna, induced by the orange carotenoid protein (OCP). A single photoactivated OCP bound to the core of the PBS affords almost total energy dissipation. The precise mechanism of OCP energy dissipation is yet to be fully determined, and one question is how the carotenoid can approach any core phycocyanobilin chromophore at a distance that can promote efficient energy quenching. We have performed intersubunit cross-linking using glutaraldehyde of the OCP and PBS followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) to identify cross-linked residues. The only residues of the OCP that cross-link with the PBS are situated in the linker region, between the N- and C-terminal domains and a single C-terminal residue. These links have enabled us to construct a model of the site of OCP binding that differs from previous models. We suggest that the N-terminal domain of the OCP burrows tightly into the PBS while leaving the OCP C-terminal domain on the exterior of the complex. Further analysis shows that the position of the small core linker protein ApcC is shifted within the cylinder cavity, serving to stabilize the interaction between the OCP and the PBS. This is confirmed by a ΔApcC mutant. Penetration of the N-terminal domain can bring the OCP carotenoid to within 5-10 Å of core chromophores; however, alteration of the core structure may be the actual source of energy dissipation.

Complementary chromatic and far-red photoacclimations in Synechococcus ATCC 29403 (PCC 7335). I: The phycobilisomes, a proteomic approach.

Synechococcus ATCC 29403 (PCC 7335) is a unicellular cyanobacterium isolated from Puerto Peñasco, Sonora Mexico. This cyanobacterium performs complementary chromatic acclimation (CCA), far-red light photoacclimation (FaRLiP), and nitrogen fixation. The Synechococcus PCC 7335 genome contains at least 31 genes for proteins of the phycobilisome (PBS). Nine constitutive genes were expressed when cells were grown under white or red lights and the resulting proteins were identified by mass spectrometry in isolated PBS. Five inducible genes were expressed under white light, and phycoerythrin subunits and associated linker proteins were detected. The proteins of five inducible genes expressed under red light were identified, the induced phycocyanin subunits, two rod linkers and the rod-capping linker. The five genes for FaRLiP phycobilisomes were expressed under far-red light together with the apcF gene, and the proteins were identified by mass spectrometry after isoelectric focusing and SDS-PAGE. Based on in silico analysis, Phylogenetic trees, and the observation of a highly conserved amino acid sequence in far-red light absorbing alpha allophycoproteins encoded by FaRLiP gene cluster, we propose a new nomenclature for the genes. Based on a ratio of ApcG2/ApcG3 of six, a model with the arrangement of the allophycocyanin trimers of the core is proposed.

Specific Chemical and Genetic Markers Revealed a Thousands-Year Presence of Toxic Nodularia spumigena in the Baltic Sea.

In the Baltic Sea, diazotrophic cyanobacteria have been present for thousands of years, over the whole brackish water phase of the ecosystem. However, our knowledge about the species composition of the cyanobacterial community is limited to the last several decades. In the current study, the presence of species-specific chemical and genetic markers in deep sediments were analyzed to increase the existing knowledge on the history of toxic Nodularia spumigena blooms in the Baltic Sea. As chemical markers, three cyclic nonribosomal peptides were applied: the hepatotoxic nodularin, which in the sea was detected solely in N. spumigena, and two anabaenopeptins (AP827 and AP883a) characteristic of two different chemotypes of this species. From the same sediment samples, DNA was isolated and the gene involved in biosynthesis of nodularin, as well as the phycocyanin intergenic spacer region (PC-IGS), were amplified. The results of chemical and genetic analyses proved for the first time the thousands-year presence of toxic N. spumigena in the Baltic Sea. They also indicated that through all this time, the same two sub-populations of the species co-existed.

Biosynthesis of Fluorescent ß Subunits of C-Phycocyanin from Spirulina subsalsa in Escherichia coli, and Their Antioxidant Properties.

Phycocyanin, which covalently binds phycocyanobilin chromophores, is not only a candidate fluorescent probe for biological imaging, but also a potential antioxidative agent for healthcare. Herein, a plasmid harboring two cassettes was constructed, with cpcB from Spirulina subsalsa in one cassette and the fusion gene cpcS::ho1::pcyA in the other, and then expressed in Escherichia coli. PCB-CpcB(C-82), a fluorescent phycocyanin ß subunit, was biosynthesized in E. coli, exhibiting an absorption maximum at 620 nm and fluorescence emission maximum at 640 nm. When cpcS was replaced by cpcT, PCB-CpcB(C-153), another fluorescent phycocyanin ß subunit, was produced, exhibiting an absorption maximum at 590 nm and fluorescence emission maximum at 620 nm. These two fluorescent biliproteins showed stronger scavenging activity toward hydroxyl and DPPH free radicals than apo-CpcB. The IC50 values for hydroxyl radical scavenging by PCB-CpcB(C-82), PCB-CpcB(C-153), and apo-CpcB were 38.72 ± 2.48 µg/mL, 51.06 ± 6.74 µg/mL, and 81.82 ± 0.67 µg/mL, respectively, and the values for DPPH radical scavenging were 201.00 ± 5.86 µg/mL, 240.34 ± 4.03 µg/mL, and 352.93 ± 26.30 µg/mL, respectively. The comparative antioxidant capacities of the proteins were PCB-CpcB(C-82) > PCB-CpcB(C-153) > apo-CpcB, due to bilin binding. The two fluorescent biliproteins exhibited a significant effect on relieving the growth of E. coli cells injured by H2O2. The results of this study suggest that the fluorescent phycocyanin ß subunits of S. subsalsa were reconstructed by one expression vector in E. coli, and could be developed as potential antioxidants.

Adapting photosynthesis to the near-infrared: non-covalent binding of phycocyanobilin provides an extreme spectral red-shift to phycobilisome core-membrane linker from Synechococcus sp. PCC7335.

Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation.

Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes.

Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.

Resolving the contribution of the uncoupled phycobilisomes to cyanobacterial pulse-amplitude modulated (PAM) fluorometry signals.

Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1-1000 s). The saturation pulse method allows determination of the quantum yields of maximal (F(M)) and minimal fluorescence (F(0)), parameters related to the activity of the photosynthetic apparatus. Also, when the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined. In the case of cyanobacteria, however, the recorded fluorescence does not exclusively stem from the chlorophyll a in photosystem II (PSII). The phycobilins, the pigments of the cyanobacterial light-harvesting complexes, the phycobilisomes (PB), also contribute to the PAM signal, and therefore, F(0) and F(M) are no longer related to PSII only. We present a functional model that takes into account the presence of several fluorescent species whose concentrations can be resolved provided their fluorescence quantum yields are known. Data analysis of PAM measurements on in vivo cells of our model organism Synechocystis PCC6803 is discussed. Three different components are found necessary to fit the data: uncoupled PB (PB(free)), PB-PSII complexes, and free PSI. The free PSII contribution was negligible. The PB(free) contribution substantially increased in the mutants that lack the core terminal emitter subunits allophycocyanin D or allophycocyanin F. A positive correlation was found between the amount of PB(free) and the rate constants describing the binding of the activated orange carotenoid protein to PB, responsible for non-photochemical quenching.

Combinatorial biosynthesis of Synechocystis PCC6803 phycocyanin holo-α-subunit (CpcA) in Escherichia coli and its activities.

In order to investigate the feasibility for the biosynthetic pathway of CpcA conjugated protein to be reconstituted in Escherichia coli and its antioxidant ability and protective effect on the growth of E. coli, the minimal biosynthetic pathway in cyanobacteria leading from heme to the formation of the cysteinyl residue of phycocyanobilin with deprosthetic CpcA was reconstituted in E. coli using a relatively simple and effective method. When the constructed plasmid pETDuet-6 bearing five genes involved in the biosynthesis of CpcA was transformed into E. coli, the screened transformant acquired a pronounced blue color. Visualization of proteins on SDS-PAGE gel showed a 29 kDa distinct band, corresponding to the theoretically calculated molecular weight of CpcA. Upon exposure to Zn(2+) and UV illumination, the CpcA band was fluorescent. Western blot analysis using His-tag monoclonal antibody confirmed the expression of CpcA in the recombinant E. coli. After the optimization of critical medium components by response surface methodology, the recombinant cells produced 22.29 mg/l of CpcA. The recombinant CpcA displayed a strong ability to scavenge three free radicals ·OH, ·DPPH, and O2 (-) to protect against the oxidation of linoleic acid and to restore the growth of E. coli cells injured by DPPH and H2O2 at a relatively low concentration. These results lay a good foundation for the production and future use of CpcA.