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• W2024073408 abstract "The two closely related bacteria Bradyrhizobium and Rhodopseudomonas palustris show an unusual mechanism of regulation of photosystem formation by light thanks to a bacteriophytochrome that antirepresses the regulator PpsR. In these two bacteria, we found out, unexpectedly, that two ppsR genes are present. We show that the two Bradyrhizobium PpsR proteins exert antagonistic effects in the regulation of photosystem formation with a classical repressor role for PpsR2 and an unexpected activator role for PpsR1. DNase I footprint analysis show that both PpsR bind to the same DNA TGTN12ACA motif that is present in tandem in the bchC promoter and the crtED intergenic region. Interestingly, the cycA and aerR promoter regions that contain only one conserved palindrome are recognized by PpsR2, but not PpsR1. Further biochemical analyses indicate that PpsR1 only is redox sensitive through the formation of an intermolecular disulfide bond, which changes its oligomerization state from a tetramer to an octamer under oxidizing conditions. Moreover, PpsR1 presents a higher DNA affinity under its reduced form in contrast to what has been previously found for PpsR or its homolog CrtJ from the Rhodobacter species. These results suggest that regulation of photosystem synthesis in Bradyrhizobium involves two PpsR competing for the binding to the same photosynthesis genes and this competition might be modulated by two factors: light via the antagonistic action of a bacteriophytochrome on PpsR2 and redox potential via the switch of PpsR1 oligomerization state. The two closely related bacteria Bradyrhizobium and Rhodopseudomonas palustris show an unusual mechanism of regulation of photosystem formation by light thanks to a bacteriophytochrome that antirepresses the regulator PpsR. In these two bacteria, we found out, unexpectedly, that two ppsR genes are present. We show that the two Bradyrhizobium PpsR proteins exert antagonistic effects in the regulation of photosystem formation with a classical repressor role for PpsR2 and an unexpected activator role for PpsR1. DNase I footprint analysis show that both PpsR bind to the same DNA TGTN12ACA motif that is present in tandem in the bchC promoter and the crtED intergenic region. Interestingly, the cycA and aerR promoter regions that contain only one conserved palindrome are recognized by PpsR2, but not PpsR1. Further biochemical analyses indicate that PpsR1 only is redox sensitive through the formation of an intermolecular disulfide bond, which changes its oligomerization state from a tetramer to an octamer under oxidizing conditions. Moreover, PpsR1 presents a higher DNA affinity under its reduced form in contrast to what has been previously found for PpsR or its homolog CrtJ from the Rhodobacter species. These results suggest that regulation of photosystem synthesis in Bradyrhizobium involves two PpsR competing for the binding to the same photosynthesis genes and this competition might be modulated by two factors: light via the antagonistic action of a bacteriophytochrome on PpsR2 and redox potential via the switch of PpsR1 oligomerization state. In anoxygenic photosynthetic bacteria, the conversion of light energy into chemical energy requires several specialized membranous complexes, the light-harvesting complexes, the photosynthetic reaction center, and the cytochrome bc1 complex. After absorption of light by the bacteriochlorophyll and carotenoid molecules associated with the light harvesting complexes, the energy is transferred to the reaction center where a charge separation occurs, leading to an electron transfer that generates a transmembrane proton-motive force ultimately used for ATP synthesis. The synthesis of this photosynthetic apparatus is highly regulated in response to oxygen tension and light intensity (for reviews, see Refs. 1Oh J.I. Kaplan S. Mol. Microbiol. 2001; 39: 1116-1123Crossref PubMed Google Scholar, 2Bauer C.E. Elsen S. Swem L.R. Swem D.L. Masuda S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 147-154Crossref PubMed Scopus (88) Google Scholar, 3Zeilstra-Ryalls J.H. Kaplan S. Cell. Mol. Life Sci. 2004; 61: 417-436Crossref PubMed Scopus (71) Google Scholar). Under aerobic conditions, the expression of photosynthesis genes is low, and the bacteria develop by drawing on the energy derived from aerobic respiration, whereas under semiaerobic or anaerobic conditions and in the light, the photosynthetic apparatus is synthesized in large amounts permitting growth by utilization of light energy.The genes necessary for the formation of the photosynthetic apparatus are clustered in a 45-kb region designated as the photosynthesis gene cluster (PGC). 1The abbreviations used are: PGC, photosynthesis gene cluster; DTT, dithiothreitol; TMPD, N,N,N′,N′-tetramethylphenylenediamine dihydrochloride; BrBphP, Bradyrhizobium bacteriophytochrome photoreceptor; Rps., Rhodopseudomonas.1The abbreviations used are: PGC, photosynthesis gene cluster; DTT, dithiothreitol; TMPD, N,N,N′,N′-tetramethylphenylenediamine dihydrochloride; BrBphP, Bradyrhizobium bacteriophytochrome photoreceptor; Rps., Rhodopseudomonas. The PGC includes several operons involved in 1) the photopigment synthesis (bacteriochlorophyll (bch), and carotenoid (crt)); 2) the light-harvesting polypeptides (pucBAC and pufBA); 3) the reaction center subunits (puhA, pufLM); and 4) various regulators (ppsR, tspO, and aerR) (4Alberti M. Burke D.E. Hearst J.E. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Nederlands1995: 1083-1106Google Scholar, 5Choudhary M. Kaplan S. Nucleic Acids Res. 2000; 28: 862-867Crossref PubMed Scopus (56) Google Scholar). The fine control of photosystem synthesis by oxygen relies on the combined action of several transcription factors that activate or repress the expression of photosynthesis genes in response to redox conditions (2Bauer C.E. Elsen S. Swem L.R. Swem D.L. Masuda S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 147-154Crossref PubMed Scopus (88) Google Scholar, 6Gregor J. Klug G. FEMS Microbiol. Lett. 1999; 179: 1-9Crossref PubMed Google Scholar). One well characterized regulator is the aerobic repressor PpsR in Rhodobacter sphaeroides or its homolog CrtJ in Rhodobacter capsulatus (7Penfold R.J. Pemberton J.M. J. Bacteriol. 1994; 176: 2869-2876Crossref PubMed Google Scholar, 8Gomelsky M. Kaplan S. J. Bacteriol. 1995; 177: 1634-1637Crossref PubMed Google Scholar). Both proteins share the same mode of action: under oxidizing conditions, PpsR/CrtJ blocks transcription by binding as a tetramer to a palindromic (TGTN12ACA) motif, which is found in tandem in the bch, crt, and puc promoters (9Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Elsen S. Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1998; 273: 30762-30769Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Gomelsky M. Horne I.M. Lee H. Pemberton J.M. McEwan A.G. Kaplan S. J. Bacteriol. 2000; 182: 2253-2261Crossref PubMed Scopus (44) Google Scholar). In addition, biochemical studies have recently shown that CrtJ/PpsR have redox sensing capabilities thanks to the formation of an intramolecular disulfide bond promoted by oxygen, which stimulates their binding to target promoters (12Masuda S. Dong C. Swem D. Setterdahl A.T. Knaff D.B. Bauer C.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7078-7083Crossref PubMed Scopus (73) Google Scholar, 13Masuda S. Bauer C.E. Cell. 2002; 110: 613-623Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar).Light intensity also controls the expression of photosynthesis genes with repressive effects less drastic than oxygen. The molecular mechanism of this regulation, described in R. sphaeroides, implicates the antagonistic actions of the repressor PpsR and the flavoprotein AppA (14Braatsch S. Gomelsky M. Kuphal S. Klug G. Mol. Microbiol. 2002; 45: 827-836Crossref PubMed Scopus (144) Google Scholar). Masuda and Bauer (13Masuda S. Bauer C.E. Cell. 2002; 110: 613-623Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) have recently shown that AppA is a blue light photoreceptor that modulates the DNA binding activity of PpsR in response to oxygen and light signals, using two distinct mechanisms: it may break the disulfide bond in oxidized PpsR, or form an inactive AppA-PpsR2 complex.A new type of light regulation has been described for the two closely related species Bradyrhizobium strain ORS278 and Rhodopseudomonas palustris (15Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (171) Google Scholar). In these two bacteria, depending on the oxygen tension, far-red light is necessary to trigger the expression of photosynthesis genes. The mechanism of this light regulation has been partially elucidated and involves a bacteriophytochrome. This chromoprotein exerts its regulatory activity by switching between a red light absorbing form (Pr) and a far-red light absorbing form (Pfr) (16Quail P.H. Boylan B.T. Parks B.M. Short T.W. Xu Y. Wagner D. Science. 1995; 268: 675-680Crossref PubMed Scopus (650) Google Scholar). It is noteworthy that the bacteriophytochrome genes (bphP) responsible for this regulation are found in the PGC region contiguous to a ppsR gene in both bacteria. Mutation in bphP of Bradyrhizobium strain ORS278 results in the absence of photosystem formation irrespective of the light conditions, whereas mutation in ppsR leads to the opposite phenotype. Based on phenotypes of these bphP and ppsR mutants and the action spectrum of the photosystem synthesis, it was proposed that the Pr form of the bacteriophytochrome antagonizes the repressive effect of PpsR. More recently, similar results have been obtained for Rps. palustris strain CEA001 (17Giraud E. Zappa S. Jaubert M. Hannibal L. Fardoux J. Adriano J.M. Bouyer P. Genty B. Pignol D. Verméglio A. Photochem. Photobiol. Sci. 2004; 3: 587-591Crossref PubMed Google Scholar).In this study, we reveal the presence of a second ppsR gene linked to the PGC of Bradyrhizobium strain ORS278. Examination of the genome sequence of Rps. palustris (available at http://spider.jgi-psf.org/JGI-microbial/html/) also reveals the presence of a second ppsR gene located in the PGC region. The unexpected presence of two ppsR genes in the same bacterium raises several questions. Why two different PpsR? Do both of them exert the same repressive effect as usually observed in purple bacteria? On the same photosynthesis genes? Are both PpsR involved in redox and light regulation circuit? Here, we specify the roles and the mechanisms of action of both PpsR from Bradyrhizobium, by combining genetic and biochemical approaches.EXPERIMENTAL PROCEDURESBacterial Strains and Growth Conditions—Bradyrhizobium strain ORS278 (wild type strain) and isogenic mutants were grown in a modified YM-agar medium with addition of appropriate antibiotics when required (18Giraud E. Hannibal L. Fardoux J. Verméglio A. Dreyfus B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14795-14800Crossref PubMed Scopus (75) Google Scholar). All the strains were grown at 35 °C for 7 days under either semiaerobic (Petri dishes filled at 40% with YM medium and sealed with a double adhesive tape) or aerobic conditions (Petri dishes with ventilated ergot). Illumination of the cultures was provided by light emitting diodes of different wavelengths between 590 and 870 nm with an irradiance of 6.6 μmol of photons/m2/s. Escherichia coli was grown in Luria-Bertani (LB) medium supplemented with the appropriate antibiotics.Determination of Photosynthetic Activity—The amount of the photosynthetic apparatus in intact cells was estimated as previously described (18Giraud E. Hannibal L. Fardoux J. Verméglio A. Dreyfus B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14795-14800Crossref PubMed Scopus (75) Google Scholar).Construction of ppsR1, ppsR2, and ppsR2/BrbphP Mutant Strains— Construction of the ppsR2 mutant has been previously described (15Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (171) Google Scholar). For the construction of a ppsR1 mutant, a region of about 2.3 kb containing the ppsR1 gene was amplified by PCR using the primers 5′-TCAGGATCCTGACCGAACGGCGATAGCTTTAG-3′ and 5′-ATCG-GATCCTGCGCCGGACGGCCCCACACAATGAG-3′ and subsequently cloned in the pGEM-T vector (Promega, Madison, WI). The 4.7-kb BamHI lacZ-Kmr cassette of pKOK5 (19Kokotek W. Lotz W. Gene (Amst.). 1989; 84: 67-471Crossref Scopus (157) Google Scholar) was then inserted in the unique BglII site of ppsR1. The resulting 7-kb BamHI insert containing the mutated ppsR1 gene was cloned into the pJQ200mp18 suicide vector linearized by BamHI digestion (20Quandt J. Hynes M.F. Gene (Amst.). 1993; 127: 15-21Crossref PubMed Scopus (828) Google Scholar). For the construction of the double mutant ppsR2/BrbphP, a region of about 5 kb containing the contiguous ppsR2 and BrbphP genes was amplified by PCR using the primers 5′-TGCGGATCCGCACCCCGTCCTGTGCCAGCGTATC-3′ and 5′-TAGGGATCCATAACCACCGCCGCCTGTGATGATAAAAC-3′ and subsequently cloned into the pGEM-T vector. A 3.2-kb region containing a part of the ppsR2 and BrbphP genes was deleted by XhoI digestion and replaced by the 4.7-kb SalI lacZ-Kmr cassette of pKOK5. The resulting 6.6-kb BamHI insert containing the mutated ppsR2 and Brb-phP genes was cloned into the pJQ200mp18 suicide vector linearized by BamHI digestion. The pJQ200 derivatives obtained, which encoded a counter selective sacB marker, were transformed into E. coli S17-1 for mobilization into ORS278 as previously described (18Giraud E. Hannibal L. Fardoux J. Verméglio A. Dreyfus B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14795-14800Crossref PubMed Scopus (75) Google Scholar). Double recombinants were selected on sucrose and the insertion was confirmed by PCR.β-Galactosidase Assay—For analysis of ppsR1 and ppsR2 expression, β-galactosidase activity was assayed as previously described (15Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (171) Google Scholar).Cloning, Protein Expression, and Purification—The ppsR1 and ppsR2 genes from Bradyrhizobium strain ORS278 were amplified by PCR using Pfu DNA polymerase and the following pairs of primers: PpsR1.pBAD.f, 5′-GGATCCGTGAGGGCGTTCAGAGCTCCGAAAGA-G-3′ and PpsR1.pBAD.r, 5′-AAGCTTCTATTCCAACTGACTGTCTTCT-TCGCTTG-3′; PpsR2.pBAD.f, 5′-ATGGATCCATGGCCGAGTTTCAC-GGTCCACGA-3′ and PpsR2.pBAD.r, 5′-ATGGATCCCTAGCTCCCCT-TTTCGGTTTCCT-3′. The PCR products were digested (BamHI/HindIII for ppsR1; BamHI for ppsR2) and ligated into the expression vector pBAD/HisB (Invitrogen). The recombinant PpsR proteins were overexpressed in E. coli LMG194 (Invitrogen) by induction with l-arabinose as previously described (17Giraud E. Zappa S. Jaubert M. Hannibal L. Fardoux J. Adriano J.M. Bouyer P. Genty B. Pignol D. Verméglio A. Photochem. Photobiol. Sci. 2004; 3: 587-591Crossref PubMed Google Scholar). Purification of His6-appended PpsR proteins was performed using nickel chelate affinity chromatography as described (17Giraud E. Zappa S. Jaubert M. Hannibal L. Fardoux J. Adriano J.M. Bouyer P. Genty B. Pignol D. Verméglio A. Photochem. Photobiol. Sci. 2004; 3: 587-591Crossref PubMed Google Scholar). The purified protein were quantified using the Bradford assay (Bio-Rad) and then aliquoted and stored at –80 °C in the presence of 20% (v/v) glycerol.Reduction and oxidation of both PpsR were tested using various reagents: 10 mm dithiothreitol (DTT), 0.1 and 1 mm H2O2, 0.5 and 5 mm K3Fe(CN)6 (potassium ferricyanide), or a mixture of 0.5 and 5 mm K3Fe(CN)6 with 10 μm TMPD (N,N,N′,N′-tetramethylphenylenediamine dihydrochloride) diluted in water, and air by exposing the sample to a constant flow of air gas during 3 h. After addition of the oxidizing or reducing reagents, the proteins were incubated overnight on ice and subsequently analyzed by nonreducing SDS-PAGE and gel filtration chromatography, or used for DNase I footprint experiments.Site-directed Mutagenesis—Single mutations were introduced in PpsR1 and PpsR2 amino acid sequences using the QuikChange™ site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations. To construct the PpsR1-C429S mutant, the plasmid pBAD::ppsR1 was used as a template with the following primers: PpsR1-C429S sense (5′-GTGATCGAGCGGATGTCGATCGAGACCGC-GCTC-3′) and PpsR1-C429S antisense (5′-GAGCGCGGTCTCGATCG-ACATCCGCTCGATCAC-3′). The Y407C mutation on the PpsR2 sequence was generated using the plasmid pBAD::ppsR2 as a template and the primers PpsR2-Y407C sense (5′-GTCGAGCAGCATTGTGTCC-GCGCCGCG-3′) and PpsR2-Y407C antisense (5′-CGCGGCGCGG-ACACAATGCTGCTCGAC-3′).DNase I Footprint Analysis—Four probes corresponding to the promoter regions of bchC, crtE, aerR, and cycA were used for studying the DNA binding of PpsR1 and PpsR2. Probes were prepared by PCR using 32P5′-end labeled oligonucleotide primers, as previously described (17Giraud E. Zappa S. Jaubert M. Hannibal L. Fardoux J. Adriano J.M. Bouyer P. Genty B. Pignol D. Verméglio A. Photochem. Photobiol. Sci. 2004; 3: 587-591Crossref PubMed Google Scholar).The bchC promoter region was obtained using the primers: 5′-CTC-GAGGATCCGCGGAATTCAAGCTTCTACGACGACCCGCTGCCTA-C-3′ and 5′-CTCGAGGATCCGCGGAATTCAAGCTTCTACGACGACC-CGCTGCCTAC-3′; the crtE promoter region was obtained using the primers: 5′-GCCGATTGCGATCTGGCGCATCTTG-3′ and 5′-AGGGA-TTTTTCGATCCGGGCCATCAC-3′; the cycA promoter region was obtained using the primers: 5′-GAGAGTCTTGAGAATGTCGTTAG-3′ and 5′-GTCGCCGCCGTCCAGCGCGAATG-3′; the aerR promoter region was obtained using the primers: 5′-CGGGCACGCACTCGGGGA-TTTG-3′ and 5′-TGTCGCGCCGGCGCTTTTCCTC-3′. DNase I footprint experiments were performed as previously described (17Giraud E. Zappa S. Jaubert M. Hannibal L. Fardoux J. Adriano J.M. Bouyer P. Genty B. Pignol D. Verméglio A. Photochem. Photobiol. Sci. 2004; 3: 587-591Crossref PubMed Google Scholar). PpsR1, PpsR1-C429S, or PpsR2 DNA binding isotherms to the bchC promoter were generated by DNase I titration assays using different protein dilutions treated by 10 mm DTT or a mixture of 0.5 mm K3Fe(CN)6 with 10 μm TMPD. The gel was then analyzed for PpsR binding isotherms using a PhosphorImager (Storm, Amersham Biosciences) to quantify the level of PpsR protection of a single band in the upstream and downstream palindromes. Values were corrected for loading by normalization with a band from an unprotected region.Gel Filtration Chromatography—The oligomerization states of PpsR1 and PpsR2 were estimated by gel filtration chromatography. The Superdex 200 26/60 column (Amersham Biosciences) was equilibrated with 20 mm HEPES (pH 8.5), 50 mm NaCl in the presence of either 1 mm DTT or 1 mm K3Fe(CN)6. Before injection of the samples, the purified proteins were previously incubated under different redox conditions as described above. Each experiment was carried out in duplicate. The column was previously size calibrated using commercial gel filtration standards (Amersham Biosciences).RESULTSppsR1 and ppsR2: Identification and Sequence Analysis—We have previously isolated the PGC region of Bradyrhizobium strain ORS278 and revealed the presence of a ppsR gene contiguous to a bacteriophytochrome gene named BrbphP. The sequencing of the downstream region revealed the presence of a second ppsR gene located around 5 kb from the first one (Fig. 1A). Examination of the PGC region of Rps. palustris also revealed the presence of two ppsR genes, one of them being also contiguous to a bacteriophytochrome gene (Fig. 1A). In the genome annotation of Rps. palustris, the ppsR gene contiguous to bphP is named ppsR2 and the other one is called ppsR1. As the gene organization is similar in both bacteria, we propose to use the same nomenclature for Bradyrhizobium and Rps. palustris. Interestingly PpsR1 and PpsR2 from both bacteria show a relatively weak amino acid sequence identity (32% between the two PpsR identified in Bradyrhizobium and 33% between PpsR1 and PpsR2 from Rps. palustris). It is noteworthy that a stronger similarity is observed between the two PpsR1 (54% of identity) and the two PpsR2 (44%). Furthermore, phylogenetic analysis using the available PpsR or CrtJ sequences clearly shows that the two PpsR1 and the two PpsR2 belong to two distinct clusters (Fig. 1B). These data suggest functional relationships between the two PpsR1 and between the two PpsR2 identified in Bradyrhizobium ORS278 and Rps. palustris.Despite their low sequence similarity, PpsR1 and PpsR2 of both organisms present a similar architecture to the one predicted for Rhodobacter PpsR/CrtJ (11Gomelsky M. Horne I.M. Lee H. Pemberton J.M. McEwan A.G. Kaplan S. J. Bacteriol. 2000; 182: 2253-2261Crossref PubMed Scopus (44) Google Scholar), with the presence of two PAS domains and a helix-turn-helix DNA binding motif at the carboxyl-terminal region. One significant feature of both PpsR2 sequences is the absence of the Cys residue. In contrast, the PpsR1 of Bradyrhizobium strain ORS278 contains only one Cys residue, whereas three are present in the PpsR1 of Rps. palustris. The absence of the Cys residue in both PpsR2 strongly suggests a mode of action different from the “classical” one described in Rhodobacter species.Role of PpsR1 and PpsR2 in Bradyrhizobium Strain ORS278—To specify the role of PpsR1 and PpsR2 identified in Bradyrhizobium strain ORS278, we constructed isogenic mutants (278ΔppsR1, 278ΔppsR2) and compared their photosynthetic phenotypes to the wild type strain after growth under different light and oxygen conditions.The synthesis of the photosynthetic apparatus is highly enhanced by far-red light when the WT strain is grown under semiaerobic conditions (Fig. 2A). When the oxygen tension is increased (aerobic conditions), the stimulatory effect of far red light is not observed anymore (Fig. 2B) and the photosystem synthesis remains very low irrespective of the light conditions. This suggests that oxygen exerts a repressive effect that dominates the activation by far-red light.Fig. 2Effect of illumination on photosynthetic activity of Bradyrhizobium ORS278 wild type strain and mutants 278ΔppsR1 and 278ΔppsR2. A, under semiaerobic conditions. B, under aerobic conditions. C, image of the long-wavelength fluorescence emission of the bacteriochlorophyll of the 278ΔppsR2 mutant cultivated under aerobic conditions in the presence of light of different wavelengths (see “Experimental Procedures”). D, wavelength dependence of photosystem synthesis of the 278ΔppsR2 mutant cultivated in aerobic conditions. The data represent the mean of three experiments (error bars indicate ± S.D.)View Large Image Figure ViewerDownload (PPT)In contrast with the WT strain, we previously observed that the 278ΔppsR2 mutant constitutively synthesized the photosystem under semiaerobiosis irrespective of the light conditions (Fig. 2A). We therefore concluded that PpsR2 acts as a repressor and that far-red light antagonizes its repressive effect under semiaerobic conditions via the action of the bacteriophytochrome BrBphP (15Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (171) Google Scholar). Under aerobic conditions, the phenotype of the 278ΔppsR2 mutant is puzzling. Indeed, we observed that the synthesis of the photosystem is high in darkness and low under far-red light illumination (Fig. 2B). To specify the nature of the pigment involved in this light inhibition, we cultivated the 278ΔppsR2 mutant in aerobiosis under a series of light-emitting diodes of different wavelengths (between 590 and 870 nm). Bacteriochlorophyll fluorescence was used as a specific marker of the presence of the photosynthetic apparatus (Fig. 2C). We observed a clear negative effect of light in the 700–770 nm region, with a maximum close to 750 nm (Fig. 2D). The action spectrum of this repressive effect of light under aerobic conditions is very similar to the absorption of the Pfr form of a bacteriophytochrome. To test whether this effect of light under aerobic conditions was controlled by BrBphP, we constructed a PpsR2 and BrBphP double mutant. The same repressive effect of far red light was observed in this mutant (data not shown) demonstrating that BrBphP is not involved in this light inhibition. Therefore, a second bacteriophytochrome is likely involved in this light regulatory circuit. Such an hypothesis makes sense because sequence analysis of the closely related Rps. palustris genome revealed the presence of 6 genes encoding putative bacteriophytochromes, four of them being located close to photosynthesis genes (21Larimer F.W. Chain P. Hauser L. Lamerdin J. Malfatti S. Do L. Land M.L. Pelletier D.A. Beatty J.T. Lang A.S. Tabita F.R. Gibson J.L. Hanson T.E. Bobst C. Torres y Torres J.L. Peres C. Harrison F.H. Gibson J. Harwood C.S. Nat. Biotechnol. 2004; 22: 55-61Crossref PubMed Scopus (551) Google Scholar).The phenotype of the 278ΔppsR1 mutant was also unexpected as we observed a very low photosystem synthesis whatever the light and oxygen conditions (Fig. 2, A and B). This clearly shows that contrary to PpsR2 and PpsR/CrtJ of the Rhodobacter species, PpsR1 is an activator of transcription that plays a key role because its presence appears essential for the synthesis of the photosynthetic apparatus in Bradyrhizobium.Oligomerization States of PpsR1 and PpsR2 in Response to Redox Conditions—The His-tagged versions of both PpsR were successfully overexpressed in E. coli LMG194 using the pBAD-HisB vector. They were subsequently isolated to a high level of purity (>95% as judged by Coomassie Blue staining) (Fig. 3, A and B) by affinity binding of the His6 tag to a Ni2+ column.Fig. 3Redox dependence of PpsR1 and PpsR2. SDS-PAGE analysis of PpsR after reduction or oxidation using various reagents. A, PpsR2. B, PpsR1. Lane a shows the effect of exposure of PpsR to 10 mm DTT; lane b shows untreated PpsR; lane c shows the effect of exposure to air; lanes d and e show the effect of 0.1 and 1 mm H2O2, respectively; lanes f and g show the effect of 0.5 and 5 mm ferricyanide, respectively; lanes h and i show the effect of 0.5 and 5 mm ferricyanide, respectively, with 0.01 mm TMPD. C, examination of disulfide bond formation in PpsR1, PpsR1-C429S, PpsR2, and PpsR2-Y407C by SDS-PAGE analysis after reduction with DTT (10 mm) or oxidation with 5 mm ferricyanide and 0.01 mm TMPD. D, elution profiles of oxidized or reduced PpsR1 that was chromatographed in Superdex 200 column. E and F, standard curves drawn according to the peak elution volumes (Ve, elution volume; V0, column volume) of the molecular mass standards (A, 440 kDa; B, 232 kDa; C, 108 kDa; D, 67 kDa; E, 43 kDa) as detected by absorption at 280 nm. The estimated position of elution of native PpsR1, PpsR2, and PpsR1-C429S are shown after reduced (E) or oxidized (F) treatments.View Large Image Figure ViewerDownload (PPT)PpsR/CrtJ from Rhodobacter species were shown to be redox sensitive via the formation of an intramolecular disulfide bond between two conserved cysteine residues, which modulates the DNA binding affinity (12Masuda S. Dong C. Swem D. Setterdahl A.T. Knaff D.B. Bauer C.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7078-7083Crossref PubMed Scopus (73) Google Scholar, 13Masuda S. Bauer C.E. Cell. 2002; 110: 613-623Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). The absence of the Cys residue in PpsR2 as well as the presence of a unique Cys residue in PpsR1 suggest fundamental differences in their response to redox conditions. To assess this hypothesis, the purified proteins were incubated overnight at 4 °C under various oxidizing or reducing conditions and subsequently analyzed by SDS-PAGE under non-reducing conditions. As expected from the sequence analysis, the variation of the redox potential does not influence the oligomerization state of PpsR2. Indeed, a single band around 50 kDa, corresponding to its calculated molecular mass, is observed whatever the redox conditions (Fig. 3A). In contrast, the oligomerization state of PpsR1 depends on the redox conditions. Under reducing conditions, PpsR1 migrates as a monomer (Fig. 3B, lane a), whereas in appropriate oxidizing conditions, an additional band corresponding to a protein of 100 kDa is revealed (Fig. 3B, lanes e, h, and i). This molecular mass corresponds to a dimer of PpsR1, suggesting that oxidizing conditions lead to the formation of an intermolecular disulfide bond. Contrary to CrtJ or PpsR, which are oxidized after 5 min of air exposure (9Ponnampalam S.N. Bauer C.E. J. Biol. Chem. 1997; 272: 18391-18396Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 13Masuda S. Bauer C.E. Cell. 2002; 110: 613-623Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), PpsR1 remains reduced even after several hours of aeration (Fig. 3B, lane c). The maximal effect on the PpsR1 oligomerization state was only reached when ferricyanide was added in the presence of TMPD (a mediator that facilitates the equilibrium of electrons from the oxidizing reagent to the protein) (see Fig. 3B, lanes h and i). About equal amounts of protein were present in the bands of 50 and 100 kDa when PpsR1 had been incubated with 0.5 mm ferricyanide + TMPD. Higher concentrations of ferricyanide and TMPD up to 10 mm did not increase this ratio. This result suggests that the quaternary structure of the fully oxidized form of PpsR1 corresponds to a complex in which half" @default.
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• W2024073408 title "Light and Redox Control of Photosynthesis Gene Expression in Bradyrhizobium" @default.
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• W2024073408 cites W1900934458 @default.
• W2024073408 cites W1967668674 @default.
• W2024073408 cites W1968366930 @default.
• W2024073408 cites W1969472583 @default.
• W2024073408 cites W1973683804 @default.
• W2024073408 cites W1986123070 @default.
• W2024073408 cites W1987282101 @default.
• W2024073408 cites W1987604802 @default.
• W2024073408 cites W1988591380 @default.
• W2024073408 cites W2007180283 @default.
• W2024073408 cites W2011553700 @default.
• W2024073408 cites W2012610509 @default.
• W2024073408 cites W2019121099 @default.
• W2024073408 cites W2035959381 @default.
• W2024073408 cites W2060904314 @default.
• W2024073408 cites W2069245610 @default.
• W2024073408 cites W2074283351 @default.
• W2024073408 cites W2093560200 @default.
• W2024073408 cites W2101500463 @default.
• W2024073408 cites W2125455480 @default.
• W2024073408 cites W2131350123 @default.
• W2024073408 cites W2136716836 @default.
• W2024073408 cites W2141494560 @default.
• W2024073408 cites W2164471904 @default.
• W2024073408 cites W2167557351 @default.
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