(Primary) (Strand Lighting Limited, Isleworth, UK), respectively. The photosynthetic photon flux density delivered through the filters was 20 pmol photon/m2/s.
The EMBO Journal vol.12 no.3 pp.997-1004, 1993
Transduction of the light signal during complementary chromatic adaptation in the cyanobacterium Calothrix sp. PCC 7601: DNA-binding proteins and modulation by phosphorylation Andre Sobczyk, Ghislain Schyns, Nicole Tandeau de Marsac and Jean Houmard1 Physiologie Microbienne (CNRS URA 1129), Ddpartement de Biochimie et G6edtique Moleculaire, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France 'Corresponding author Communicated by R.A.Dixon
The cyanobacterium Calothruc sp. PCC 7601 can adapt its pigment content in response to changes in the incident light wavelength. It synthesizes, as major light-harvesting pigments, either phycocyanin 2 (PC2, encoded by the cpc2 operon) under red light or phycoerythrin (PE, encoded by the cpeBA operon) under green light conditions. The last step of the signal transduction pathway is characterized by a transcriptional control of the expression of these operons. Partially purified protein extracts were used in gel retardation assays and DNase I footprinting experiments to identify the factors that interact with the promoter region of the cpeBA operon. We found that two proteins, RcaA and RcaB, only detected in extracts of cells grown under green light, behave as positive transcriptional factors for the expression of the cpeBA operon. Treatment of the fractions containing RcaA and RcaB with alkaline phosphatase prevents the binding of RcaA but not of RcaB to the cpeBA promoter region. A post-translational modification of RcaA thus modulates its affinity for DNA. Key words: photoregulation/phycoerythrin/post-translational modification/RcaA and RcaB/transcription factors
Introduction Adaptation to changes in the environment is of major importance for the survival of any living organism and a wide variety of mechanisms have already been described that allow cells to cope with these modifications. Cyanobacteria are photosynthetic prokaryotes that may exhibit a strict requirement for light and CO2 (obligate photoautotrophs) or can develop at the expense of alternative carbon sources (facultative heterotrophs). For most of them, however, light availability plays a major role for their growth in natural habitats where, besides the diurnal cycle, changes occurring over days and months also modify the wavelength of the light received by the cells. Some cyanobacteria are able to sense the light wavelength variations and to modify the composition of their light-harvesting antenna accordingly. The best known example of this kind of adaptation is the so-called 'complementary chromatic adaptation' phenomenon (for reviews, see Bogorad, 1975; Grossman, 1990; Tandeau de Marsac, 1991). Oxford University Press
In cyanobacteria able to undergo complementary chromatic adaptation (chromatic adapters), it has been shown that there must exist a photoreversible pigment which is modified in response to changes in light wavelength. Because this modification is a purely photochemical process, this pigment has been compared to the phytochrome molecule of eukaryotic plants (Fujita and Hattori, 1962; Diakoff and Scheibe, 1973). However, action spectra for Calothrix sp. PCC 7601 have shown that the most efficient wavelengths for the interconversion of the two forms of this photoreversible pigment are 540 nm (in the green) and 640 nm (in the red), instead of 660 nm (red) and 730 nm (farred) for the phytochrome (Haury and Bogorad, 1977; Vogelmann and Scheibe, 1978). At present, nobody has succeeded in isolating this cyanobacterial photoreceptor and very little is known about its mechanism of action. In our laboratory, we are investigating the various mechanisms that control gene expression in cyanobacteria, and especially in Calothnx PCC 7601 which is a facultatively heterotrophic filamentous strain that can fix nitrogen, differentiate proheterocysts and hormogonia (Damerval et al., 1991) and adapt its pigment content to the available light wavelengths (Tandeau de Marsac et al., 1988). To study complementary chromatic adaptation at a molecular level, most of the genes that encode the components of the cyanobacterial light-harvesting antenna, the phycobilisome, have been characterized for Calothrix PCC 7601 (for a review, see Tandeau de Marsac, 1991). Among them are the genes that encode the major phycobiliprotein subunits: the cpeBA operon which specifies the a and ,B subunits of phycoerythrin (PE) and is only expressed under green light; and the cpc2 operon which specifies the at and j3 subunits of phycocyanin-2 (PC2) and is only expressed under red light conditions (Conley et al., 1985; Mazel et al., 1986; Capuano et al., 1988; Tandeau de Marsac et al., 1988). Previous analyses have established that the transcription of the cpc2 operon, as well as that of the cpeBA and cpeCD operons which encode PE and its associated linker polypeptides (LRPE), respectively, depends on the prevailing light conditions (Conley et al., 1985; Tandeau de Marsac et al., 1988; Federspiel and Grossman, 1990). Thus, there must exist factors able to interact differentially with DNA sequences and/or with RNA polymerase to achieve this regulation. In this paper, we present evidence that at least two protein effectors specifically bind to the promoter region of the cpeBA operon of Calothrix PCC 7601, and therefore may act as transcriptional factors for its expression. These molecules have been partially purified and their DNAbinding sites characterized.
Results Evidence for the occurrence of DNA-binding proteins For the last few years, band shift experiments have been widely used to obtain evidence for the existence of
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interactions between effectors and DNA. To detect an alteration in their electrophoretic mobility, it is necessary to use DNA fragments of limited size in the binding assay. Figure 1 depicts the physical map of the promoter region of the cpeBA operon of Calothrix PCC 7601. Gel retardation experiments were performed using the whole 413 bp XbaI fragment and each of the two XbaI-AvaI fragments that can be derived from it. These DNA fragments were incubated with bovine serum albumin (BSA) or with fractions prepared from cells grown either under red (R) or green (G) light. Only fractions F40G and F60G were able to shift the electrophoretic mobility of the labelled 413 bp DNA fragment (Figure 2). These fractions contained molecules of the crude extract from cells grown in green light that had precipitated between 20 and 40% (F40G) or 40 and 60% (F60G) of saturation in ammonium sulphate. Under these conditions, altered mobility of the probe was not seen with any of the fractions originating from cells grown in red light (FiR samples). Similar results were obtained with the 191 bp AvaI-XbaI fragment while the electrophoretic 998
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mobility of the other XbaI -AvaI DNA fragment was unaffected whatever the extracts used (data not shown). All further experiments were performed with the 191 bp DNA fragment which was subcloned in pTE103 (Elliott and Geiduschek, 1984) to generate pPM125 (Figure 1). Chemical nature of the effectors To determine the chemical nature of the molecules able to bind to the DNA fragment, fractions F40G and F60G were treated with heat, RNase or proteinase K, before being incubated with the promoter region of the cpeBA operon. RNase had no effect on the gel shift while heat treatment and proteinase K completely prevented the formation of DNA complexes (Figure 3). We therefore concluded that there existed at least two proteins, only present in cells where the cpeBA genes were expressed, that were able to bind to the cpeBA promoter region. The corresponding proteins were designated RcaA and RcaB (Rca: regulator for complementary chromatic adaptation). Interaction of RcaA and RcaB with the cpeBA promoter region led to the formation of complex 1 (Cl) and complex 2 (C2),
respectively (Figure 3). Specificity of the binding The specificity of the interaction was demonstrated by means of competition experiments. Artificial DNA (poly [dI.dC].poly[dI.dC]), total Calothrix PCC 7601 DNA or the
unlabelled DNA probe were added to the reaction mixture before being loaded onto the acrylamide gel. The amount of label that stayed associated with the protein-DNA complex was inversely correlated with the molar ratio of unlabelled/labelled DNA fragments. A 50-fold excess of unlabelled versus labelled probe made radioactive Cl and C2 complexes far less abundant (Figure 4). Similar experiments demonstrated that at least 5000 times more Calothrix PCC 7601 total DNA must be added to challenge
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Fig. 5. Effect of alkaline phosphatase treatment on the DNA binding properties of the RcaA and RcaB proteins. The 191 bp DNA probe was incubated with the F4G or F60 protein fractions previously treated (+; lanes 3 and 5) or not (-; lanes 2 and 4) with calf intestine aLkaline phosphatase (Alk phosphatase). A control experiment was performed with a treated BSA fraction (lane 1). Cl and C2 indicate the RcaA-DNA and RcaB-DNA complexes, respectively.
the specific binding, while poly[dI.dC].poly[dI.dC] had no detectable effect in the same range of concentration. We also observed the expected dependence of the binding on protein concentration (data not shown).
Phosphorylation modulates the affinity of one of the effectors for its DNA target To characterize these effectors further, we looked for a putative role of phosphorylation in the transcriptional regulation that occurred during complementary chromatic
adaptation. Fractions were incubated in the presence of calf intestine alkaline phosphatase for time periods ranging from I to 15 min. Enzyme activity was then inhibited by increasing the phosphate concentration to 20 mM. Alkaline phosphatase treatment of fraction F40G, prior to the addition of the DNA probe, prevented the formation of the Cl DNA-protein complex (Figure 5). In contrast, the effector present in fraction F60G was unaffected by the same treatment. Phosphorylation thus modulated the affinity of RcaA, the effector present in F40G, for the cpeBA promoter region. Partial purification of the effectors Further purification of the putative transcription factors was performed in order to define precisely their targets on the DNA sequence. The two proteins could be co-precipitated using a 30-60% ammonium sulphate cut, and the resulting pellet was used as starting material instead of the two separate
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