A turquoise mutant genetically separates expression of genes encoding phycoerythrin and its associated linker peptides.
ABSTRACT: During complementary chromatic adaptation (CCA), cyanobacterial light harvesting structures called phycobilisomes are restructured in response to ambient light quality shifts. Transcription of genes encoding components of the phycobilisome is differentially regulated during this process: red light activates cpcB2A2, whereas green light coordinately activates the cpeCDE and cpeBA operons. Three signal transduction components that regulate CCA have been isolated to date: a sensor-photoreceptor (RcaE) and two response regulators (RcaF and RcaC). Mutations in the genes encoding these components affect the accumulation of both cpcB2A2 and cpeBA gene products. We have isolated and characterized a new pigmentation mutant called Turquoise 1. We demonstrate that this mutant phenotype is due to a dramatic decrease in cpeBA transcript abundance and results from a lesion in the cpeR gene. However, in this mutant cpeCDE RNA levels remain near those found in wild-type cells. Our results show that the coordinate regulation of cpeBA and cpeCDE by green light can be uncoupled by the loss of CpeR, and we furnish the first genetic evidence that different regulatory mechanisms control these two operons. Sequence analysis of CpeR reveals that it shares limited sequence similarity to members of the PP2C class of protein serine/threonine phosphatases. We also demonstrate that cpeBA and cpeCDE retain light quality responsiveness in a mutant lacking the RcaE photoreceptor. This provides compelling evidence for the partial control of CCA through an as-yet-uncharacterized second light quality sensing system.
Project description:Complementary chromatic adaptation appears to be controlled by a complex regulatory system with similarity to four-step phosphorelays. Such pathways utilize two histidine and two aspartate residues for signal transduction. Previous studies of the signaling system controlling complementary chromatic adaptation have uncovered two elements of this pathway, a putative sensor, RcaE, and a response regulator, RcaC. In this work, we describe a second response regulator controlling complementary chromatic adaptation, RcaF, and identify putative DNA binding and histidine phosphoacceptor domains within RcaC. RcaF is a small response regulator with similarity to SpoOF of Bacillus subtilis; the latter functions in the four-step phosphorelay system controlling sporulation. We have also determined that within this phosphorelay pathway, RcaE precedes RcaF, and RcaC is probably downstream of RcaE and RcaF. This signal transduction pathway is novel because it appears to use at least five, instead of four, phosphoacceptor domains in the phosphorelay circuit.
Project description:Complementary chromatic acclimation (CCA) allows many cyanobacteria to change the composition of their light-harvesting antennae for maximal absorption of different wavelengths of light. In the freshwater species Fremyella diplosiphon, this process is controlled by the ratio of red to green light and allows the differential regulation of two subsets of genes in the genome. This response to ambient light color is controlled in part by a two-component system that includes a phytochrome class photoreceptor and a response regulator with an OmpR/PhoB class DNA binding domain called RcaC. During growth in red light, RcaC is able to simultaneously activate expression of red light-induced genes and repress expression of green light-induced genes through binding to the L box promoter element. Here we investigate how the L box functions as both an activator and a repressor under the same physiological conditions by analyzing the effects of changing the position, orientation, and sequence of the L box. We demonstrate that changes in the local sequences surrounding the L box affect the strength of its activity and that the activating and repressing functions of the L box are orientation dependent. Also, the spacing between the L box and the transcription start site is critical for it to work as an activator, while its repressing role during light regulation requires additional upstream and downstream DNA sequence elements. The latter result suggests that the repressing function of RcaC requires it to operate in association with multiple additional DNA binding proteins, at least one of which is functioning as an activator.
Project description: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 ?rcaE 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 ?rcaE 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 ?rcaE 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.
Project description:Many biological processes in photosynthetic organisms can be regulated by light quantity or light quality or both. A unique example of the effect of specific wavelengths of light on the composition of the photosynthetic apparatus occurs in cyanobacteria that undergo complementary chromatic adaptation. These organisms alter the composition of their light-harvesting organelle, the phycobilisome, and exhibit distinct morphological features as a function of the wavelength of incident light. Fremyella diplosiphon, a filamentous cyanobacterium, responds to green light by activating transcription of the cpeBA operon, which encodes the pigmented light-harvesting component phycoerythrin. We have isolated and determined the complete nucleotide sequence of another operon, cpeCD, that encodes the linker proteins associated with phycoerythrin hexamers in the phycobilisome. The cpeCD operon is activated in green light and expressed as two major transcripts with the same 5' start site but differing 3' ends. Analysis of the kinetics of transcript accumulation in cultures of F. diplosiphon shifted from red light to green light and vice versa shows that the cpeBA and cpeCD operons are regulated coordinately. A common 17-base-pair sequence is found upstream of the transcription start sites of both operons. A comparison of the predicted amino acid sequences of the phycoerythrin-associated linker proteins CpeC and CpeD with sequences of other previously characterized rod linker proteins shows 49 invariant residues, most of which are in the amino-terminal half of the proteins.
Project description: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.
Project description:Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation.
Project description:Here, we have demonstrated visible light-emitting diode light-driven selective and efficient aerobic oxidation of primary/secondary alcohols to aldehydes/ketones and oxidative azo-coupling of anilines using biomass rice husk-derived chemically activated carbon sheet-supported copper-iron bimetallic hybrid nanomaterials (Cu x Fe1-x @RCAC) under oxidant and additive-free conditions. The catalytic activity of the Cu x Fe1-x @RCAC materials has been investigated for the oxidation of alcohols and anilines, and Cu0.9Fe0.1@RCAC was established as the best catalyst. Moreover, a tandem one-pot protocol has been developed for the sequential oxidation of alcohols followed by condensation to functionalized imidazole and imine derivatives in high isolated yields. The hybrid materials were highly robust and stable under the reaction conditions and were recovered simply by filtration and recycled up to 12th run without considerable loss in catalytic activity.
Project description:The genomes of many photosynthetic and nonphotosynthetic bacteria encode numerous phytochrome superfamily photoreceptors whose functions and interactions are largely unknown. Cyanobacterial genomes encode particularly large numbers of phytochrome superfamily members called cyanobacteriochromes. These have diverse light color-sensing abilities, and their functions and interactions are just beginning to be understood. One of the best characterized of these functions is the regulation of photosynthetic light-harvesting antenna composition in the cyanobacterium Fremyella diplosiphon by the cyanobacteriochrome RcaE in response to red and green light, a process known as chromatic acclimation. We have identified a new cyanobacteriochrome named DpxA that maximally senses teal (absorption maximum, 494 nm) and yellow (absorption maximum, 568 nm) light and represses the accumulation of a key light-harvesting protein called phycoerythrin, which is also regulated by RcaE during chromatic acclimation. Like RcaE, DpxA is a two-component system kinase, although these two photoreceptors can influence phycoerythrin expression through different signaling pathways. The peak responsiveness of DpxA to teal and yellow light provides highly refined color discrimination in the green spectral region, which provides important wavelengths for photosynthetic light harvesting in cyanobacteria. These results redefine chromatic acclimation in cyanobacteria and demonstrate that cyanobacteriochromes can coordinately impart sophisticated light color sensing across the visible spectrum to regulate important photosynthetic acclimation processes.The large number of cyanobacteriochrome photoreceptors encoded by cyanobacterial genomes suggests that these organisms are capable of extremely complex light color sensing and responsiveness, yet little is known about their functions and interactions. Our work uncovers previously undescribed cooperation between two photoreceptors with very different light color-sensing capabilities that coregulate an important photosynthetic light-harvesting protein in response to teal, green, yellow, and red light. Other cyanobacteriochromes that have been shown to interact functionally sense wavelengths of light that are close to each other, which makes it difficult to clearly identify their physiological roles in the cell. Our finding of two photoreceptors with broad light color-sensing capabilities and clearly defined physiological roles provides new insights into complex light color sensing and its regulation.
Project description: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.
Project description:A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene. Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues). Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence. On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein. A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1). In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751. The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons. The two remaining ORFs on Tn5469 encode proteins of unknown function. Southern blot analysis showed that wild-type F. diplosiphon harbors five genomic copies of Tn5469. In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene. Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.