Two ORFs, cphA and cphB, encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp. PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp. PCC7601. While CphA carries a cysteine within a highly conserved amino-acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine. Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two-component signal transduction. In Calothrix, all four genes are expressed under white light irradiation conditions, albeit in low amounts. For heterologous expression and convenient purification, the cloned genes were furnished with His-tag encoding sequences at their 3¢ end and expressed in Escherichia coli. The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the P r and P fr forms (red and far-red absorbing forms, respectively) of the plant phytochromes and Cph1. A red shift in the absorption maxima of the CphB/PCB complex (k max ¼ 685 and 735 nm for P r and P fr , respectively) is indicative for a noncovalent incorporation of the chromophore (k max of P r , P fr of CphA: 663, 700 nm). A CphB mutant generated at the chromophore-binding position (Leu246 fi Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB. The kinetics of the light-induced P fr formation of the CphA-PCB chromoprotein, though similar to that of its ortholog from Synechocystis, showed differences in the kinetics of the P fr formation. The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator. In contrast, the light-induced kinetics of the CphB-PCB complex was markedly different, clearly due to the noncovalently bound chromophore.Keywords: Calothrix sp. strain PCC 7601; flash photolysis; heterologous expression; phytochrome-like apoproteins CphA and CphB; response regulators RcpA and RcpB.A precise qualitative and quantitative measurement of the surrounding light is essential to photosynthetic organisms in order to either adapt to the environmental light conditions or, in the case of motility, to proceed towards a favorable habitat. Higher plants have developed the phytochromes, a chromoprotein family, which absorb light in the longwavelength region and regulate numerous photomorphogenetic processes [1][2][3]. In cyanobacteria, the presence and molecular structure of comparable sensory system(s) have long been debated. It has been suggested that the photosynthetic apparatus itself or blue-light-and/or other noncharacterized light-absorbing photoreceptors might fulfil these functions [4][5][6][7].The complete s...

The genome of the filamentous cyanobacterium Calothrix sp. PCC7601 contains two genes, cphA and cphB, encoding proteins with similarity to plant phytochromes and bacterial histidine kinases. In vitro, CphA and CphB readily attach a tetrapyrrole chromophore to develop spectrally active holoproteins that are photointerconvertible between a red light‐absorbing and a far‐red light‐absorbing form. Together with the putative response regulators, RcpA and RcpB, the putative histidine kinases, CphA and CphB, are suggested to constitute two two‐component systems of light‐dependent signal transduction. In this report, we demonstrate the kinase activity of both CphA and CphB. In vitro experiments carried out on the purified proteins show that CphA and CphB are autophosphorylated in the presence of ATP and that phospho‐CphA is capable of efficient phosphotransfer to RcpA as is phospho‐CphB towards RcpB. The autophosphorylation and the phosphorelay are dependent on light. Both activities are reduced under red light vs. far‐red light irradiation. No phosphoryl transfer occurred between phospho‐CphA and RcpB or between phospho‐CphB and RcpA. The response regulators RcpA and RcpB can receive a phosphoryl moiety also from the small phospho‐donor acetyl phosphate. The stability of the phosphorylated regulators is not affected by CphA and CphB or light.

The transcription factor, DeltaFosB, a splice isoform of fosB, accumulates in rodents in a brain-region-specific manner in response to chronic administration of drugs of abuse, stress, certain antipsychotic or antidepressant medications, electroconvulsive seizures, and certain lesions. Increasing evidence supports a functional role of such DeltaFosB induction in animal models of several psychiatric and neurologic disorders. Fos family proteins, including DeltaFosB, are known to heterodimerize with Jun family proteins to create active AP-1 transcription-factor complexes, which bind to DNA specifically at AP-1 consensus sites. We show here, using a range of biochemical and biophysical means, that recombinant, purified DeltaFosB forms homodimers as well, at concentrations less than 500 nM, and that these homodimers specifically bind to DNA oligonucleotides containing AP-1 consensus sequences in the absence of any Jun partner. Our results suggest that, as DeltaFosB accumulates to abnormally elevated protein levels in highly specific regions of the brain in response to chronic stimulation, functional homodimers of DeltaFosB are formed with the potential to uniquely regulate patterns of gene expression and thereby contribute to the complex processes of neural and behavioral adaptation.

Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner. For both eyanobacterial phytochromes, red and far-red light-induced photochemistry has been reported. Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome. Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phytochrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3'). The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red-absorbing forms of phytochrome [P(r) and P(fr) forms]). However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P(r)-P(fr) photochemistry can be initiated and to which the system relaxes in the dark after illumination. The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog. A comparison of the spectral properties (P(r) and P(fr) forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry. Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light-induced physiological response.