Complementary Chromatic Acclimation Research Articles

Green/red light-sensing mechanism in the chromatic acclimation photosensor.

Certain cyanobacteria alter their photosynthetic light absorption between green and red, a phenomenon called complementary chromatic acclimation. The acclimation is regulated by a cyanobacteriochrome-class photosensor that reversibly photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. Here, we elucidated the structural basis of the green/red photocycle. In the Pg state, the bilin chromophore adopted the extended C15-Z,anti structure within a hydrophobic pocket. Upon photoconversion to the Pr state, the bilin is isomerized to the cyclic C15-E,syn structure, forming a water channel in the pocket. The solvation/desolvation of the bilin causes changes in the protonation state and the stability of π-conjugation at the B ring, leading to a large absorption shift. These results advance our understanding of the enormous spectral diversity of the phytochrome superfamily.

Environmental Tuning of Homologs of the Orange Carotenoid Protein-Encoding Gene in the Cyanobacterium Fremyella diplosiphon.

The orange carotenoid protein (OCP) family of proteins are light-activated proteins that function in dissipating excess energy absorbed by accessory light-harvesting complexes, i.e., phycobilisomes (PBSs), in cyanobacteria. Some cyanobacteria contain multiple homologs of the OCP-encoding gene (ocp). Fremyella diplosiphon, a cyanobacterium studied for light-dependent regulation of PBSs during complementary chromatic acclimation (CCA), contains several OCP homologs – two full-length OCPs, three Helical Carotenoid Proteins (HCPs) with homology to the N-terminus of OCP, and one C-terminal domain-like carotenoid protein (CCP) with homology to the C-terminus of OCP. We examined whether these homologs are distinctly regulated in response to different environmental factors, which could indicate distinct functions. We observed distinct patterns of expression for some OCP, HCP, and CCP encoding genes, and have evidence that light-dependent aspects of ocp homolog expression are regulated by photoreceptor RcaE which controls CCA. RcaE-dependent transcriptional regulator RcaC is also involved in the photoregulation of some hcp genes. Apart from light, additional environmental factors associated with cellular redox regulation impact the mRNA levels of ocp homologs, including salt, cold, and disruption of electron transport. Analyses of conserved sequences in the promoters of ocp homologs were conducted to gain additional insight into regulation of these genes. Several conserved regulatory elements were found across multiple ocp homolog promoters that potentially control differential transcriptional regulation in response to a range of environmental cues. The impact of distinct environmental cues on differential accumulation of ocp homolog transcripts indicates potential functional diversification of this gene family in cyanobacteria. These genes likely enable dynamic cellular protection in response to diverse environmental stress conditions in F. diplosiphon.

Artificial complementary chromatic acclimation gene expression system in Escherichia coli

BackgroundThe 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.ResultsOne 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.ConclusionAn 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.

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.

RcaE-Dependent Regulation of Carboxysome Structural Proteins Has a Central Role in Environmental Determination of Carboxysome Morphology and Abundance in Fremyella diplosiphon.

Carboxysomes are central to the carbon dioxide-concentrating mechanism (CCM) and carbon fixation in cyanobacteria. Although the structure is well understood, roles of environmental cues in the synthesis, positioning, and functional tuning of carboxysomes have not been systematically studied. Fremyella diplosiphon is a model cyanobacterium for assessing impacts of environmental light cues on photosynthetic pigmentation and tuning of photosynthetic efficiency during complementary chromatic acclimation (CCA), which is controlled by the photoreceptor RcaE. Given the central role of carboxysomes in photosynthesis, we investigated roles of light-dependent RcaE signaling in carboxysome structure and function. A ΔrcaE mutant exhibits altered carboxysome size and number, ccm gene expression, and carboxysome protein accumulation relative to the wild-type (WT) strain. Several Ccm proteins, including carboxysome shell proteins and core-nucleating factors, overaccumulate in ΔrcaE cells relative to WT cells. Additionally, levels of carboxysome cargo RuBisCO in the ΔrcaE mutant are lower than or unchanged from those in the WT strain. This shift in the ratios of carboxysome shell and nucleating components to the carboxysome cargo appears to drive carboxysome morphology and abundance dynamics. Carboxysomes are also occasionally mislocalized spatially to the periphery of spherical mutants within thylakoid membranes, suggesting that carboxysome positioning is impacted by cell shape. The RcaE photoreceptor links perception of external light cues to regulating carboxysome structure and function and, thus, to the cellular capacity for carbon fixation. IMPORTANCE Carboxysomes are proteinaceous subcellular compartments, or bacterial organelles, found in cyanobacteria that consist of a protein shell surrounding a core primarily composed of the enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) that is central to the carbon dioxide-concentrating mechanism (CCM) and carbon fixation. Whereas significant insights have been gained regarding the structure and synthesis of carboxysomes, limited attention has been given to how their size, abundance, and protein composition are regulated to ensure optimal carbon fixation in dynamic environments. Given the centrality of carboxysomes in photosynthesis, we provide an analysis of the role of a photoreceptor, RcaE, which functions in matching photosynthetic pigmentation to the external environment during complementary chromatic acclimation and thereby optimizing photosynthetic efficiency, in regulating carboxysome dynamics. Our data highlight a role for RcaE in perceiving external light cues and regulating carboxysome structure and function and, thus, in the cellular capacity for carbon fixation and organismal fitness.

Additional families of orange carotenoid proteins in the photoprotective system of cyanobacteria

The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Using phylogenomic analysis, we have revealed two new paralogous OCP families, each distributed among taxonomically diverse cyanobacterial genomes. Based on bioinformatic properties and phylogenetic relationships, we named the new families OCP2 and OCPx to distinguish them from the canonical OCP that has been well characterized in Synechocystis, denoted hereafter as OCP1. We report the first characterization of a carotenoprotein photoprotective system in the chromatically acclimating cyanobacterium Tolypothrix sp. PCC 7601, which encodes both OCP1 and OCP2 as well as the regulatory fluorescence recovery protein (FRP). OCP2 expression could only be detected in cultures grown under high irradiance, surpassing expression levels of OCP1, which appears to be constitutive; under low irradiance, OCP2 expression was only detectable in a Tolypothrix mutant lacking the RcaE photoreceptor required for complementary chromatic acclimation. In vitro studies show that Tolypothrix OCP1 is functionally equivalent to Synechocystis OCP1, including its regulation by Tolypothrix FRP, which we show is structurally similar to the dimeric form of Synechocystis FRP. In contrast, Tolypothrix OCP2 shows both faster photoconversion and faster back-conversion, lack of regulation by the FRP, a different oligomeric state (monomer compared to dimer for OCP1) and lower fluorescence quenching of the phycobilisome. Collectively, these findings support our hypothesis that the OCP2 is relatively primitive. The OCP2 is transcriptionally regulated and may have evolved to respond to distinct photoprotective needs under particular environmental conditions such as high irradiance of a particular light quality, whereas the OCP1 is constitutively expressed and is regulated at the post-translational level by FRP and/or oligomerization.

Identification of Cyanobacteriochromes Detecting Far-Red Light.

The opacity of mammalian tissue to visible light and the strong attenuation of infrared light by water at ≥900 nm have contributed to growing interest in the development of far-red and near-infrared absorbing tools for visualizing and actuating responses within live cells. Here we report the discovery of cyanobacteriochromes (CBCRs) responsive to light in this far-red window. CBCRs are linear tetrapyrrole (bilin)-based light sensors distantly related to plant phytochrome sensors. Our studies reveal far-red (λmax = 725-755 nm)/orange (λmax = 590-600 nm) and far-red/red (λmax = 615-685 nm) photoswitches that are small (<200 amino acids) and can be genetically reconstituted in living cells. Phylogenetic analysis and characterization of additional CBCRs demonstrated that far-red/orange CBCRs evolved after a complex transition from green/red CBCRs known for regulating complementary chromatic acclimation. Incorporation of different bilin chromophores demonstrated that tuning mechanisms responsible for red-shifted chromophore absorption act at the A-, B-, and/or C-rings, whereas photoisomerization occurs at the D-ring. Two such proteins exhibited detectable fluorescence extending well into the near-infrared region. This work extends the spectral window of CBCRs to the edge of the infrared, raising the possibility of using CBCRs in synthetic biology applications in the far-red region of the spectrum.

Competition-based phenotyping reveals a fitness cost for maintaining phycobilisomes under fluctuating light in the cyanobacterium Fremyella diplosiphon

Phycobilisomes (PBSs) are pigment-rich super-complexes required for efficient harvest and transfer of light energy to photosynthetic reaction centers of cyanobacteria. The model cyanobacterium Fremyella diplosiphon is able to adjust PBS pigmentation and size in response to the prevailing light spectrum through a process called complementary chromatic acclimation to optimize spectral light absorption, concomitantly optimizing photosynthesis and growth. We explored the fitness costs versus advantages of modulating antennae size and composition under sinusoidal continuous and fluctuating light conditions in F. diplosiphon by comparing growth of wild-type (WT) cells with a mutant strain deficient in PBSs in both monoculture and polyculture conditions. Comparative analyses of WT and the PBS-deficient FdCh1 strain under continuous vs. fluctuating sinusoidal light suggest a potential fitness advantage for maintaining PBSs in WT cells during continuous light and a fitness cost during transitions to and acclimation under fluctuating light. We explored the physiological changes correlated with the observed differential growth to understand the dynamics and biochemical bases of comparative fitness of distinct strains under defined growth conditions. Wild-type F. diplosiphon appears to accumulate longer PBS rods and exhibits higher oxidative stress under fluctuating light conditions than continuous sinusoidal light, which may impact responses and the fitness of cells that do not adapt to rapid changes in external light.

Regulation of BolA abundance mediates morphogenesis in Fremyella diplosiphon.

Filamentous cyanobacterium Fremyella diplosiphon is known to alter its pigmentation and morphology during complementary chromatic acclimation (CCA) to efficiently harvest available radiant energy for photosynthesis. F. diplosiphon cells are rectangular and filaments are longer under green light (GL), whereas smaller, spherical cells and short filaments are prevalent under red light (RL). Light regulation of bolA morphogene expression is correlated with photoregulation of cellular morphology in F. diplosiphon. Here, we investigate a role for quantitative regulation of cellular BolA protein levels in morphology determination. Overexpression of bolA in WT was associated with induction of RL-characteristic spherical morphology even when cultures were grown under GL. Overexpression of bolA in a ΔrcaE background, which lacks cyanobacteriochrome photosensor RcaE and accumulates lower levels of BolA than WT, partially reverted the cellular morphology of the strain to a WT-like state. Overexpression of BolA in WT and ΔrcaE backgrounds was associated with decreased cellular reactive oxygen species (ROS) levels and an increase in filament length under both GL and RL. Morphological defects and high ROS levels commonly observed in ΔrcaE could, thus, be in part due to low accumulation of BolA. Together, these findings support an emerging model for RcaE-dependent photoregulation of BolA in controlling the cellular morphology of F. diplosiphon during CCA.

Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation

To explore the variation of the light-regulated genes during complementary chromatic acclimation (CCA), we determined the complete genome sequence of the cyanobacterium Geminocystis sp. strain NIES-3708. Within the light-regulated operon for CCA, we found genes for phycoerythrin but not phycocyanin, suggesting that this cyanobacterium modulates phycoerythrin composition only (type II CCA).

Morphogenes bolA and mreB mediate the photoregulation of cellular morphology during complementary chromatic acclimation in Fremyella diplosiphon.

Photoregulation of pigmentation during complementary chromatic acclimation (CCA) is well studied in Fremyella diplosiphon; however, mechanistic insights into the CCA-associated morphological changes are still emerging. F. diplosiphon cells are rectangular under green light (GL), whereas cells are smaller and spherical under red light (RL). Here, we investigate the role of morphogenes bolA and mreB during CCA using gene expression and gene function analyses. The F. diplosiphon bolA gene is essential as its complete removal from the genome was unsuccessful. Depletion of bolA resulted in slow growth, morphological defects and the accumulation of high levels of reactive oxygen species in a partially segregated ΔbolA strain. Higher expression of bolA was observed under RL and was correlated with lower expression of mreB and mreC genes in wild type. In a ΔrcaE strain that lacks the red-/green-responsive RcaE photoreceptor, the expression of bolA and mre genes was altered under both RL and GL. Observed gene expression relationships suggest that mreB and mreC expression is controlled by RcaE-dependent photoregulation of bolA expression. Expression of F. diplosiphon bolA and mreB homologues in Escherichia coli demonstrated functional conservation of the encoded proteins. Together, these studies establish roles for bolA and mreB in RcaE-dependent regulation of cellular morphology.

Temporal dynamics of changes in reactive oxygen species (ROS) levels and cellular morphology are coordinated during complementary chromatic acclimation in Fremyella diplosiphon.

Fremyella diplosiphon alters the phycobiliprotein composition of its light-harvesting complexes, i.e., phycobilisomes, and its cellular morphology in response to changes in the prevalent wavelengths of light in the external environment in a phenomenon known as complementary chromatic acclimation (CCA). The organism primarily responds to red light (RL) and green light (GL) during CCA to maximize light absorption for supporting optimal photosynthetic efficiency. Recently, we found that RL-characteristic spherical cell morphology is associated with higher levels of reactive oxygen species (ROS) compared to growth under GL where lower ROS levels and rectangular cell shape are observed. The RL-dependent association of increasedROS levels with cellular morphology was demonstrated by treating cells with a ROS-scavenging antioxidant which resulted in the observation of GL-characteristic rectangular morphology under RL. To gain additional insights into the involvement of ROS in impacting cellular morphology changes during CCA, we conducted experiments to study the temporal dynamics of changes in ROS levels and cellular morphology during transition to growth under RL or GL. Alterations in ROS levels and cell morphology were found to be correlated with each other at early stages of acclimation of low white light-grown cells to growth under high RL or cells transitioned between growth in RL and GL. These results provide further general evidence that significant RL-dependent increases in ROS levels are temporally correlated with changes in morphology toward spherical. Future studies will explore the light-dependent mechanisms by which ROS levels may be regulated and the direct impacts of ROS on the observed morphology changes.

Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330-680 nm). Two CBCR subfamilies detect near-UV to blue light (330-450 nm) via a "two-Cys photocycle" that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilin-cysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state ((15Z)P(g)) and a red-absorbing 15E state ((15E)P(r)). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as (15Z)P(g) but protonated as (15E)P(r). This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a "protochromic photocycle," in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.

DIFFERENTIAL RESPONSES OF ANABAENA SP. PCC 7120 (CYANOPHYCEAE) CULTURED IN NITROGEN-DEFICIENT AND NITROGEN-ENRICHED MEDIA TO ULTRAVIOLET-B RADIATION(1).

Stratospheric ozone depletion increases the amount of ultraviolet-B radiation (UVBR) (280-320 nm) reaching the surface of the earth, potentially affecting phytoplankton. In this work, Anabaena sp. PCC 7120, a typically nitrogen (N)-fixing filamentous bloom-forming cyanobacterium in freshwater, was individually cultured in N-deficient and N-enriched media for long-term acclimation before being subjected to ultraviolet-B (UVB) exposure experiments. Results suggested that the extent of breakage in the filaments induced by UVBR increases with increasing intensity of UVB stress. In general, except for the 0.1 W · m(-2) treatment, which showed a mild increase, UVB exposure inhibits photosynthesis as evidenced by the decrease in the chl fluorescence parameters maximum photochemical efficiency of PSII (Fv /Fm ) and maximum relative electron transport rate. Complementary chromatic acclimation was also observed in Anabaena under different intensities of UVB stress. Increased total carbohydrate and soluble protein may provide some protection for the culture against damaging UVB exposure. In addition, N-deficient cultures with higher recovery capacity showed overcompensatory growth under low UVB (0.1 W · m(-2) ) exposure during the recovery period. Significantly increased (∼830%) ATPase activity may provide enough energy to repair the damage caused by exposure to UVB.