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Two Distinct crt Gene Clusters for Two Different Functional Classes of Carotenoid in Bradyrhizobium

2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês

10.1074/jbc.m312113200

1083-351X

Éric Giraud, Laure Hannibal, Joël Fardoux, Marianne Jaubert, Philippe Jourand, B Dreyfus, James N. Sturgis, André Verméglio,

Protist diversity and phylogeny

Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids. In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain. Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene. We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress. Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome. Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors. Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer. This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated. Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids. In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain. Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene. We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress. Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome. Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors. Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer. This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated. Carotenoids comprise a large class of pigments that are widely distributed in living organisms. They are synthesized by all photosynthetic organisms from bacteria to plants where they play at least three essential functions (1Paulsen H. Frank H.A. Young A.J. Britton G. Cogdell R.J. The Photochemistry of Carotenoids. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 123-135Google Scholar). First, they act as accessory light-harvesting pigments by absorbing light in the 450–570 nm region. Second, they are important for the assembly and stability of some of these light-harvesting complexes. Finally they operate as photoprotectors by directly quenching both triplet excited (bacterio)chlorophylls and singlet oxygen. Carotenoids are also synthesized by a wide variety of nonphotosynthetic bacteria (2Armstrong G.A. J. Bacteriol. 1994; 176: 4795-4802Crossref PubMed Google Scholar). Less is known about their precise function in these bacteria, but it is well accepted that their strong antioxidant character may protect the organisms against (photo)oxidative damage. A remarkable feature of aerobic phototrophic bacteria, besides their ability to photosynthesize only under aerobic condition, is their carotenoid composition. Indeed most strains synthesize, in addition to the carotenoids involved in photosynthesis such as spirilloxanthin, a large amount of unusual carotenoid molecules (3Shimada K. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 105-122Google Scholar). The most striking complexity is observed for Erythrobacter species such as Erythrobacter longus or Erythrobacter ramosum, which have been reported to produce about 20 different carotenoids (4Takaichi S. Shimada K. Ishidsu J.-I. Arch. Microbiol. 1990; 153: 118-122Crossref Scopus (48) Google Scholar, 5Yurkov V.V. Beatty J.T. Microbiol. Mol. Biol. Rev. 1998; 62: 695-724Crossref PubMed Google Scholar). Another example comes from various strains of photosynthetic Bradyrhizobium, symbionts of Aeschynomene (6Fleischman D. Kramer D. Biochim. Biophys. Acta. 1998; 1364: 17-36Crossref PubMed Scopus (55) Google Scholar), which synthesize, in addition to spirilloxanthin, large amounts of canthaxanthin of unknown function (7Lorquin J. Molouba F. Dreyfus B.L. Appl. Environ. Microbiol. 1997; 63: 1151-1154Crossref PubMed Google Scholar, 8Molouba F. Lorquin J. Willems A. Hoste B. Giraud E. Dreyfus B. Gillis M. de Lajudie P. Masson-Boivin C. Appl. Environ. Microbiol. 1999; 6: 3084-3094Crossref Google Scholar). All carotenoids are synthesized from geranylgeranyl pyrophosphate. This compound is formed by the enzyme geranylgeranyl pyrophosphate synthase (CrtE), which catalyzes the condensation of farnesyl pyrophosphate with an isopentyl pyrophosphate moiety. The second step catalyzed by phytoene synthase (CrtB) is the formation of phytoene from the head-to-head condensation of two molecules of geranylgeranyl pyrophosphate. Subsequent dehydrogenations catalyzed by the phytoene desaturase (CrtI) convert the phytoene to neurosporene in three desaturation steps or to lycopene in four steps. After the action of these three enzymes (CrtE, CrtB, and CrtI), the biosynthetic pathways diverge depending on the species leading to the accumulation of various different carotenoids. The synthesis of canthaxanthin from lycopene necessitates two enzymes: CrtY, which catalyzes cyclization of lycopene leading to β-carotene, and CrtW, which oxygenates β-carotene to form canthaxanthin (see Fig. 1A) (9Misawa N. Satomi Y. Kondo K. Yokoyama A. Kajiwara S. Saito T. Ohtani T. Miki W. J. Bacteriol. 1995; 177: 6575-6584Crossref PubMed Scopus (368) Google Scholar). The sequence of the reactions from lycopene to spirilloxanthin includes the successive reactions of hydration, desaturation, and methylation catalyzed, respectively, by the three enzymes CrtC, CrtD, and CrtF (see Fig. 1A) (10Takaichi S. Frank H.A. Young A.J. Britton G. Cogdell R.J. The Photochemistry of Carotenoids. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 40-69Google Scholar, 11Pinta V. Ouchane S. Picaud M. Takaichi S. Astier C. Reiss-Husson F. Arch. Microbiol. 2003; 179: 354-362Crossref PubMed Scopus (21) Google Scholar). These reactions are performed first on one-half of the molecule and then on the other half. The genes encoding many carotenoid biosynthetic enzymes (crt genes) have been characterized in plants and in various bacteria (12Cunningham F.X. Gantt E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 557-583Crossref PubMed Scopus (778) Google Scholar, 13Armstrong G.A. Annu. Rev. Microbiol. 1997; 51: 629-659Crossref PubMed Scopus (190) Google Scholar). In bacteria, they are always found clustered, except in the Cyanobacteria. In purple photosynthetic bacteria, the genes involved in carotenoid biosynthesis are localized within the photosynthesis gene cluster, a 45-kb DNA region that contains the essential genes involved in the synthesis of the photosynthetic apparatus (14Alberti M. Burke D.H. Hearst J.E. Blankenship R.E. Madigan M.T. Bauer C.E. Anoxygenic Photosynthetic Bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands1995: 775-805Google Scholar, 15Choudhary M. Kaplan S. Nucleic Acids Res. 2000; 28: 862-867Crossref PubMed Scopus (56) Google Scholar, 16Yildiz F.H. Gest H. Bauer C.E. Mol. Microbiol. 1992; 6: 2683-2691Crossref PubMed Scopus (29) Google Scholar, 17Igarashi N. Harada J. Nagashima S. Matsuura K. Shimada K. Nagashima K.V. J. Mol. Evol. 2001; 52: 333-341Crossref PubMed Scopus (106) Google Scholar). In aerobic photosynthetic bacteria, a crt gene cluster has been characterized for the Bradyrhizobium ORS278 strain (18Hannibal L. Lorquin J. Angles d'Ortoli N. Garcia N. Chaintreuil C. Masson-Boivin C. Dreyfus B. Giraud E. J. Bacteriol. 2000; 182: 3850-3853Crossref PubMed Scopus (62) Google Scholar). This cluster contains the five crt genes, crtE, crtY, crtI, crtB, and crtW, necessary for the canthaxanthin biosynthesis. Neither photosynthesis genes nor specific genes of spirilloxanthin biosynthesis from lycopene (crtC, crtD, or crtF) have been identified in this cluster. Light stimulation of carotenoid biosynthesis has been reported in numerous organisms including plants, fungi, and bacteria (19Bramley P.M. Mackenzie A. Curr. Top. Cell. Regul. 1988; 29: 291-343Crossref PubMed Scopus (62) Google Scholar). In higher plants, regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression (20Von Lintig J. Welsch R. Bonk M. Giuliano A. Kleinig H. Plant J. 1997; 12: 625-634Crossref PubMed Scopus (200) Google Scholar). This expression is controlled by a phytochrome, a plant photoreceptor that mediates response to red and far-red light through photoconversion between two stable conformations, a red-absorbing form (Pr) and a far-red-absorbing form (Pfr). Biochemical and genetic studies have recently demonstrated the occurrence of phytochrome-like proteins in photosynthetic and non-photosynthetic bacteria (21Hughes J. Lamparter T. Mittmann F. Hartmann E. Gärtner W. Wilde A. Börner T. Nature. 1997; 386: 663Crossref PubMed Scopus (296) Google Scholar, 22Bhoo S.-H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (252) Google Scholar). Such a (bacterio)phytochrome appears to control the synthesis of the carotenoid deinoxanthin in the non-photosynthetic bacteria Deinococcus radiodurans (23Davis S.J. Vener A.V. Vierstra R.D. Science. 1999; 286: 2517-2520Crossref PubMed Scopus (294) Google Scholar). In this report, we describe the characterization of a second crt gene cluster, in the Bradyrhizobium ORS278 strain, coding the enzymes of spirilloxanthin synthesis. This second crt gene cluster contains all the genes necessary for the synthesis of spirilloxanthin from farnesyl pyrophosphate. Biochemical analysis and phenotypes of mutants deleted in specific genes of canthaxanthin and spirilloxanthin synthesis allow us to establish the involvement of spirilloxanthin in the photosynthesis activity and the protective role of canthaxanthin in response of the bacteria to oxidative stress. We also demonstrate that the spirilloxanthin crt genes are specifically regulated by light via the control of a bacteriophytochrome. These results provide the first demonstration of two independent and differently regulated crt gene clusters in a living organism. Bacterial Strains and Growth Conditions—Bradyrhizobium sp. strain ORS278 (wild-type strain) and isogenic mutants were grown in a modified yeast extract mannitol agar medium with addition of the appropriate antibiotic when required (24Giraud 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 cultured for 7 days in sealed Petri dishes at 35 °C in either complete darkness or different continuous illumination conditions 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. Pigment Analysis—Cells grown at the surface of the Petri dishes in the dark or under different light conditions were resuspended in 6 ml of water + 9 g/liter NaCl and centrifuged for 10 min at 4,000 × g. The pellets were extracted three times in the dark with 1 ml of cold acetone/methanol (7:2, v/v). The carotenoids in the pooled extracts were analysis by HPLC 1The abbreviations used are: HPLC, high pressure liquid chromatography; RC, reaction center; LH, light harvesting; WT, wild type; LDAO, lauryldimethylamine oxide. using a Waters Alliance 2690 system. The conditions were: 5-μm Hypersil C18 column (250 × 4.6 mm, Alltech), acetonitrile/methanol/isopropanol (40:50:10, v/v/v) as eluent, 0.8 ml/min flow rate. The eluted fractions were monitored using a Waters 996 photodiode array detector scanning from 270 to 600 nm every 2 s. Carotenoids were identified by their retention times and by comparison of the spectral features with those of pure compounds or with reported data. The amount of canthaxanthin was determined from the area of the peak detected at 480 nm using a calibration curve obtained with a canthaxanthin standard kindly provided by Aventis. The amount of spirilloxanthin was estimated from the area of the peak detected at 494 nm using the canthaxanthin correlation coefficient due to the lack of spirilloxanthin standard. The data represent the mean of three independent cultures. Light Action Spectrum of crt Gene Expression—The mutants harboring the various lacZ-crt fusions were grown under continuous illumination with low irradiance of different wavelengths as described previously (25Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (172) Google Scholar). After growth, the cells under the illuminated area were resuspended in 3 ml of water, and β-galactosidase activity was measured as described previously (24Giraud 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 crt Mutant Strains—Constructions of mutants deleted in the bacteriophytochrome (278ΔBrbphP) and in the transcriptional factor PpsR (278ΔppsR) have been described previously (25Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (172) Google Scholar). For the construction of a crtCD mutant (278ΔcrtCD), a region of about 3.5 kb containing crtC and crtD genes of ORS278 was amplified by PCR using the primers 5′-TAGTCGACGCAATGGCGCGCCACGATCTATC-3′ and 5′-ACAGTCGACCGGTCTTGGAGCGGTGATAATG-3′ and subsequently cloned in the pGEM-T vector (Promega, Madison, WI). A 2-kb region containing part of the crtC and crtD genes was deleted by XhoI digestion and replaced by the 4.7-kb SalI LacZ-Kmr cassette of pKOK5 (26Kokotek W. Lotz W. Gene (Amst.). 1989; 84: 467-471Crossref PubMed Scopus (158) Google Scholar). The resulting 6.2-kb SalI insert containing the mutated crtC and crtD genes was cloned into the pJQ200mp18 suicide vector (27Quandt J. Hynes M.F. Gene (Amst.). 1993; 127: 15-21Crossref PubMed Scopus (835) Google Scholar). To mutate the crtE.c gene of the canthaxanthin crt gene cluster, a region of about 1.2 kb containing crtE.c gene was amplified by PCR using the primers 5′-GGTAGATCTGGTCTGCATGCGCGGATGAAACAG-3′ and 5′-GGAAGATCTCGAAGGCAGGTTCAGAGTATG-3′, digested by BglII, and cloned into the BamHI sites of pJQ200mp18. The 4.7-kb BamHI LacZ-Kmr cassette of pKOK5 was then inserted into the unique BamHI site of crtE.c. To mutate the crtY gene, a 1.8-kb PstI fragment of pSTM78 (18Hannibal L. Lorquin J. Angles d'Ortoli N. Garcia N. Chaintreuil C. Masson-Boivin C. Dreyfus B. Giraud E. J. Bacteriol. 2000; 182: 3850-3853Crossref PubMed Scopus (62) Google Scholar) containing crtY gene was cloned into the PstI sites of pJQ200mp18. A 0.4-kb XhoI fragment containing part of crtY was then deleted and replaced by the 4.7 SalI LacZ-Kmr cassette of pKOK5. To mutate the crtI.s gene of the spirilloxanthin crt gene cluster, a region of 1.4 kb was amplified by PCR using the primers 5′-CGGGATCCTTGGCTGGCGAAAGCGTCAATTTC-3′ and 5′-CGGGATCCAGGACGACAGGCGCTGCTCGAAATC-3′, digested by BamHI, and cloned into the BamHI sites of pJQ200mp18. The 4.7-kb SalI LacZ-Kmr cassette of pKOK5 was then inserted into the unique XhoI site of crtI.s. For each construction, we verified by PCR and sequencing that the lacZ reporter gene was in the correct orientation. The pJQ200 derivatives obtained, which encoded a counterselective sacB marker, were transformed into E. coli S17-1 for mobilization into ORS278 as described previously (24Giraud 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. Preparation of Membranes and RC-LH1 Complexes—A 200-ml culture of Bradyrhizobium (wild type (WT) or mutant) was collected and resuspended in 10 ml of Tris-HCl buffer (50 mm, pH 8). The cells were disrupted by three passages through a French press at 50 megapascals. The suspension was centrifuged for 10 min at 4,000 × g to remove the unbroken cells and cells debris. The supernatant was loaded on a discontinuous sucrose gradient (0.6–1.2 m sucrose, 50 mm Tris-HCl buffer, pH 8) and centrifuged at 255,000 × g for 90 min. The membranes localized at the interface of the two sucrose layers constitute the chromatophore fraction, while the pellet contains the cytoplasmic membranes. Each fraction was diluted in 25 ml of Tris-HCl buffer (50 mm, pH 8), centrifuged at 255,000 × g for 90 min to removed the sucrose, and then resuspended in Tris-HCl buffer (10 mm, pH 8). RC-LH1 complexes were isolated by addition of 1.5% LDAO to purified chromatophores or cytoplasmic membranes whose optical density was adjusted to 5 OD/cm at 870 nm. After an incubation of 15 min at room temperature in the dark, the membrane suspension was loaded on a discontinuous sucrose gradient (0.1, 0.2, 0.3, 0.6 m sucrose, 50 mm Tris-HCl, pH 8, 0.02% LDAO) and centrifuged at 255,000 × g for 90 min. All canthaxanthin molecules do not enter the sucrose gradient. The RC-LH1 particles, collected at the interface between the 0.2 and 0.3 m sucrose layers, were diluted in Tris-HCl (50 mm, pH 8, 0.02% LDAO) and centrifuged at 255,000 × g for 180 min. They were resuspended in Tris-HCl (10 mm, pH 8, 0.02% LDAO). Further purification of the canthaxanthin-containing fraction and of the RC-LH1 particles was performed using a mono-Q column coupled to an FPLC (Amersham Biosciences) and submitted to a NaCl gradient. Absorption and Fluorescence Spectroscopy—Absorption spectra and light-induced absorption changes in intact cells were measured as described previously (24Giraud 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). Fluorescence excitation and emission spectra were recorded on a Spex Fluorolog 3 spectrofluorometer (Jobin Yvon). For excitation spectra, the excitation slits were 5 nm, and the emission was measured at 870 nm (on the blue side of the emission spectrum where the instrument sensitivity was highest) with 15-nm slits. For emission spectra excitation was through 10-nm slits, and emission was measured through 7-nm slits. Emission spectra were corrected for the wavelength dependence of the instrument response, and excitation spectra were corrected for variations in excitation intensity. For all fluorescence spectra the detector was protected from scattered excitation by a Wratten 88A gelatin filter. Isolation and Characterization of the Spirilloxanthin Biosynthesis Genes—In all photosynthetic bacteria studied so far, the carotenoid biosynthesis genes have been found linked to the photosynthesis gene cluster. We have previously isolated, from a genomic DNA library of the ORS278 strain, a cosmid (pSTM1) with an insert of ∼35 kb that contains some photosynthesis genes (24Giraud 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). Its partial sequencing reveals a gene arrangement close to that of purple photosynthetic bacteria with the superoperonal structure bchCXYZpufBALM conserved. This prompted us to check whether the upstream region of bchC gene contains some of the carotenoid synthesis genes as observed in other photosynthetic bacteria. The sequencing of this region reveals the presence of six open reading frames encoding proteins with similarities to known Crt enzymes assigned to crtE, crtF, crtC, crtD, crtI, and crtB genes (Fig. 1B). The orientation of these open reading frames suggests the existence of a minimum of three operons (crtEF, crtCD, and crtIB) as usually observed in purple photosynthetic bacteria (2Armstrong G.A. J. Bacteriol. 1994; 176: 4795-4802Crossref PubMed Google Scholar). The overlap observed between crtD and crtC (348 bp) was also reported in Rubrivivax gelatinosus (28Ouchane S. Picaud M. Vernotte C. Reiss-Husson F. Astier C. J. Biol. Chem. 1997; 272: 1670-1676Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The canthaxanthin crt gene cluster (Fig. 1C) was previously isolated from the cosmid pSTM73 (18Hannibal L. Lorquin J. Angles d'Ortoli N. Garcia N. Chaintreuil C. Masson-Boivin C. Dreyfus B. Giraud E. J. Bacteriol. 2000; 182: 3850-3853Crossref PubMed Scopus (62) Google Scholar). We showed by PCR that there is no overlap between this cosmid and the cosmid pSTM1 containing the spirilloxanthin gene cluster. Together these results demonstrate the presence of two distinct crt gene clusters in Bradyrhizobium ORS278, a spirilloxanthin gene cluster localized in the photosynthesis gene cluster region and a canthaxanthin gene cluster localized in a different part of the genome. Interestingly the genes crtE, crtI, and crtB that encode enzymes of lycopene biosynthesis, a common precursor of canthaxanthin and spirilloxanthin, are found in both clusters. However, they are obviously different and show little similarity (38% identity between the two CrtE proteins and 43 and 51.6% for the CrtB and CrtI pairs, respectively). To distinguish these genes, we call the genes from the spirilloxanthin crt cluster crtE.s, crtI.s, and crtB.s and those from the canthaxanthin crt cluster crtE.c, crtI.c, and crtB.c. To demonstrate that the canthaxanthin and the spirilloxanthin crt gene clusters we have identified are each responsible for the biosynthesis of the appropriate one of these two carotenoids, we constructed a mutant deleted in crtY gene (278ΔcrtY strain, canthaxanthin minus) and a mutant deleted in the crtCD genes (278ΔcrtCD strain, spirilloxanthin minus). The pigmentation of both mutants is clearly different from the WT strain (Fig. 2A). The 278ΔcrtCD mutant presents an orange color characteristic of canthaxanthin, while the 278ΔcrtY mutant deleted in a canthaxanthin gene is pink as is typical for spirilloxanthin. These variations in color are essentially due to the difference in absorption profiles of the carotenoids in the 400–600 nm region of the three strains (Fig. 2B). HPLC analysis of the carotenoids extracted from each mutant (Fig. 2C) confirmed the absence of spirilloxanthin in the 278ΔcrtCD mutant as well as the absence of canthaxanthin in the 278ΔcrtY mutant. Membrane Localization and Function of Canthaxanthin and Spirilloxanthin—Aerobic photosynthetic bacteria are known to possess a small amount of photosynthetic apparatus correlated with few invaginations of the cytoplasmic membrane (5Yurkov V.V. Beatty J.T. Microbiol. Mol. Biol. Rev. 1998; 62: 695-724Crossref PubMed Google Scholar). In a first attempt to clarify the function of the two carotenoids present in Bradyrhizobium ORS278, cytoplasmic and intracytoplasmic fractions of the membrane were separated on a sucrose gradient after breakage of the WT cells. The two types of membranes could be separated as a function of their density (see "Experimental Procedures"). The cytoplasmic membranes form a pellet, while the intracytoplasmic membrane fragments (chromatophores) sediment at the interface between 0.6 and 1.2 m sucrose layers. The absorption spectrum of the chromatophores reveals the presence of bacteriochlorophyll (873, 590, and 375 nm) and spirilloxanthin (548, 515, and 485 nm) molecules but no detectable canthaxanthin (Fig. 3A). In contrast the cytoplasmic membrane contains a large amount of canthaxanthin in addition to the photosynthetic pigments (Fig. 3B). From the relative amounts of bacteriochlorophyll molecules present in these two fractions, we deduce that about 40% of the photosynthetic units are present in the chromatophores, while the rest is localized in the cytoplasmic membrane. This experiment clearly shows that spirilloxanthin molecules are associated with the photosynthetic apparatus in agreement with previous observations on several other species (10Takaichi S. Frank H.A. Young A.J. Britton G. Cogdell R.J. The Photochemistry of Carotenoids. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 40-69Google Scholar). Another proof that only spirilloxanthin molecules are associated with the photosynthetic apparatus was obtained by the measurement of the excitation spectrum of intact cells of the bacteriochlorophyll fluorescence around 890 nm (Fig. 3C). Comparison between the absorption spectrum of intact cells (Fig. 2B) and the excitation spectrum associated with the fluorescence emission of the LH1 complexes (Fig. 3C) shows that only the spirilloxanthin (at 550, 515, and 485 nm) molecules are able to transfer energy to the photosynthetic units. There is no evidence of energy transfer from canthaxanthin. In addition, identical excitation spectra are measured for both the WT and the 278ΔcrtY mutant (canthaxanthin minus) (Fig. 3C). These results clearly demonstrate that spirilloxanthin, but not canthaxanthin, transfers light energy to the photosynthetic apparatus. An additional proof comes from the characteristics of the absorption spectrum of purified RC-LH1 complexes. These complexes can be easily purified from the cytoplasmic or the chromatophore membranes after addition of 1.5% LDAO (see "Experimental Procedures"). These particles do not contain any canthaxanthin as shown by both their absorption spectrum (Fig. 3D) and analysis of their carotenoid content (data not shown). The phenotypes of mutants deleted in one of the genes of the canthaxanthin (278ΔcrtY) or the spirilloxanthin (278ΔcrtCD) pathways also demonstrate that spirilloxanthin and not canthaxanthin is associated with the photosynthetic apparatus. Indeed a similar amount of photosynthetic apparatus is present in intact cells of the WT or of the canthaxanthin minus mutant (278ΔcrtY) (see Fig. 2B) demonstrating that the lack of canthaxanthin does not affect its formation. In contrast, the amount of photosynthetic apparatus is reduced by at least a factor of 3–5 in the spirilloxanthin minus mutant (278ΔcrtCD) compared with the WT as shown by the comparison of their absorption spectra (see Fig. 2B) or measurement of light-induced photooxidation of the cytochrome on intact cells (data not shown). In fact, the chemical analysis of the carotenoid content of this mutant reveals the presence of a significant amount of lycopene (0.05 mg/g of cells) (data not shown). This carotenoid is associated with the small fraction of photosystem present in the 278ΔcrtCD mutant as demonstrated by the absorption spectrum of the chromatophore fraction and of purified isolated RC-LH1 complexes (Fig. 3, A and D) and the fluorescence excitation spectrum (Fig. 3C), which all present the characteristic bands of lycopene around 520, 487, and 460 nm. Canthaxanthin and RC-LH1 complexes can be easily extracted from the cytoplasmic membranes. The fraction containing the canthaxanthin does not enter the sucrose gradient, while the RC-LH1 complexes sediment at the 0.6–1.2 m interface (not shown). Further purification of the canthaxanthin fraction on a Mono-Q column followed by gel electrophoresis shows that the canthaxanthin is not associated with protein. This suggests that, unless the detergent has destroyed a weak protein-canthaxanthin association, canthaxanthin is present in the lipid phase of the membrane and not associated with specific polypeptides. One putative function of canthaxanthin is to act as strong antioxidant protecting against (photo)oxidative damage. To test this hypothesis, we measured the survival of bacteria exposed to an oxidative stress caused by addition of methyl viologen for both the WT and the 278ΔcrtY and 278ΔcrtCD mutants. As seen on Fig. 4, the canthaxanthin minus mutant (278ΔcrtY) is more sensitive to the addition of increasing concentrations of methyl viologen than either the WT strain or the spirilloxanthin minus mutant (278ΔcrtCD). In addition, the canthaxanthin minus mutant is less resistant than the WT strain in patch assays with H2O2 as the oxidative stress inducer (data not shown). These results are a clear indication that canthaxanthin acts as a protective agent against oxidative stress in Bradyrhizobium ORS278 cells. Regulation of Canthaxanthin and Spirilloxanthin Synthesis by Light—We have previously shown that the photosynthetic activity in Bradyrhizobium is stimulated by far-red light through the action of the bacteriophytochrome BrbphP (25Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (172) Google Scholar). The BrbphP gene is localized close to the photosynthesis gene cluster and contiguous to an open reading frame homologous to the transcription factor PpsR. The PpsR protein is known to repress crt genes in Rhodobacter species at high oxygen tension or high light intensity (29Penfold R.J. Pemberton J.M. J. Bacteriol. 1994; 176: 2869-2876Crossref PubMed Google Scholar, 30Ponnampalam S.N. Buggy J.J. Bauer C.E. J. Bacteriol. 1995; 177: 2990-2997Crossref PubMed Google Scholar). The up-regulation of photosynthesis genes by far-red light illumination observed in Bradyrhizobium ORS278 strain results from the antirepressor effect of the Pr form of BrbphP on PpsR (25Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Verméglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (172) Google Scholar). This prompted us to clarify the role of both Brbp