Carotenogenesis diversification in phylogenetic lineages of Rhodophyta.

Carotenoid composition is very diverse in Rhodophyta. In this study, we investigated whether this variation is related to the phylogeny of this group. Rhodophyta consists of seven classes, and they can be divided into two groups on the basis of their morphology. The unicellular group (Cyanidiophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae) contained only ß-carotene and zeaxanthin, "ZEA-type carotenoids." In contrast, within the macrophytic group (Bangiophyceae, Compsopogonophyceae, and Florideophyceae), Compsopogonophyceae contained antheraxanthin in addition to ZEA-type carotenoids, "ANT-type carotenoids," whereas Bangiophyceae contained α-carotene and lutein along with ZEA-type carotenoids, "LUT-type carotenoids." Florideophyceae is divided into five subclasses. Ahnfeltiophycidae, Hildenbrandiophycidae, and Nemaliophycidae contained LUT-type carotenoids. In Corallinophycidae, Hapalidiales and Lithophylloideae in Corallinales contained LUT-type carotenoids, whereas Corallinoideae in Corallinales contained ANT-type carotenoids. In Rhodymeniophycidae, most orders contained LUT-type carotenoids; however, only Gracilariales contained ANT-type carotenoids. There is a clear relationship between carotenoid composition and phylogenetics in Rhodophyta. Furthermore, we searched open genome databases of several red algae for references to the synthetic enzymes of the carotenoid types detected in this study. ß-Carotene and zeaxanthin might be synthesized from lycopene, as in land plants. Antheraxanthin might require zeaxanthin epoxydase, whereas α-carotene and lutein might require two additional enzymes, as in land plants. Furthermore, Glaucophyta contained ZEA-type carotenoids, and Cryptophyta contained ß-carotene, α-carotene, and alloxanthin, whose acetylenic group might be synthesized from zeaxanthin by an unknown enzyme. Therefore, we conclude that the presence or absence of the four enzymes is related to diversification of carotenoid composition in these three phyla.

A conformational change of the γ subunit indirectly regulates the activity of cyanobacterial F1-ATPase.

The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30-40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α(3)ß(3)γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ε inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ε inhibition.

Opposite chirality of α-carotene in unusual cyanobacteria with unique chlorophylls, Acaryochloris and Prochlorococcus.

Among all photosynthetic and non-photosynthetic prokaryotes, only cyanobacterial species belonging to the genera Acaryochloris and Prochlorococcus have been reported to synthesize α-carotene. We reviewed the carotenoids, including their chirality, in unusual cyanobacteria containing diverse Chls. Predominantly Chl d-containing Acaryochloris (two strains) and divinyl-Chl a and divinyl-Chl b-containing Prochlorococcus (three strains) contained ß-carotene and zeaxanthin as well as α-carotene, whereas Chl b-containing Prochlorothrix (one strain) and Prochloron (three isolates) contained only ß-carotene and zeaxanthin but no α-carotene as in other cyanobacteria. Thus, the capability to synthesize α-carotene seemed to have been acquired only by Acaryochloris and Prochlorococcus. In addition, we unexpectedly found that α-carotene in both cyanobacteria had the opposite chirality at C-6': (6'S)-chirality in Acaryochloris and normal (6'R)-chirality in Prochlorococcus, as reported in some green algae and land plants. The results represent the first evidence for the natural occurrence and biosynthesis of (6'S)-α-carotene. All the zeaxanthins in these species were of the usual (3R,3'R)-chirality. Therefore, based on the identification of the carotenoids and genome sequence data, we propose a biosynthetic pathway for the carotenoids, particularly α-carotene, including the participating genes and enzymes.

Characterization of carotenogenesis genes in the cyanobacterium Anabaena sp. PCC 7120.

Cyanobacteria produce many kinds of carotenoids for light harvesting and light protection in photosynthesis. To elucidate the biosynthetic pathways of carotenoids in Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), we have produced gene-disruption mutants lacking selected proposed carotenoid biosynthetic enzymes. Here we describe the construction of mutants by triparental mating. A cargo plasmid, bearing a target gene interrupted by an antibiotic-resistant cassette, is transformed to E. coli donor containing a helper plasmid, and is introduced into Anabaena cells by conjugation. Double-reciprocal recombination replaces the target genes in Anabaena genome with mutated ones on the plasmid. Carotenoids in the selected double recombinants are identified using high-performance liquid chromatography.

α-Carotene and its derivatives have a sole chirality in phototrophic organisms?

Carotenoids in eukaryotic phototrophic organisms can be classified into two groups; ß-carotene and its derivatives, and α-carotene and its derivatives. We re-examined distribution of α-carotene and its derivatives among various taxa of aquatic algae (17 classes) and land plants. α-carotene and its derivatives were found from Rhodophyceae (macrophytic type), Cryptophyceae, Euglenophyceae, Chlorarachniophyceae, Prasinophyceae, Chlorophyceae, Ulvophyceae, Charophyceae, and land plants, while they could not be detected from Glaucophyceae, Rhodophyceae (unicellular type), Chryosophyceae, Raphidophyceae, Bacillariophyceae, Phaeophyceae, Xanthophyceae, Eustigmatophyceae, Haptophyceae, and Dinophyceae. We also analyzed the chirality of α-carotene and/or its derivatives, such as lutein and siphonaxanthin, and found all of them had only (6'R)-type, not (6'S)-type.

Genomic structure of an economically important cyanobacterium, Arthrospira (Spirulina) platensis NIES-39.

A filamentous non-N(2)-fixing cyanobacterium, Arthrospira (Spirulina) platensis, is an important organism for industrial applications and as a food supply. Almost the complete genome of A. platensis NIES-39 was determined in this study. The genome structure of A. platensis is estimated to be a single, circular chromosome of 6.8 Mb, based on optical mapping. Annotation of this 6.7 Mb sequence yielded 6630 protein-coding genes as well as two sets of rRNA genes and 40 tRNA genes. Of the protein-coding genes, 78% are similar to those of other organisms; the remaining 22% are currently unknown. A total 612 kb of the genome comprise group II introns, insertion sequences and some repetitive elements. Group I introns are located in a protein-coding region. Abundant restriction-modification systems were determined. Unique features in the gene composition were noted, particularly in a large number of genes for adenylate cyclase and haemolysin-like Ca(2+)-binding proteins and in chemotaxis proteins. Filament-specific genes were highlighted by comparative genomic analysis.

Unique carotenoids in the terrestrial cyanobacterium Nostoc commune NIES-24: 2-hydroxymyxol 2'-fucoside, nostoxanthin and canthaxanthin.

Cyanobacteria produce some carotenoids. We identified the molecular structures, including the stereochemistry, of all the carotenoids in the terrestrial cyanobacterium, Nostoc commune NIES-24 (IAM M-13). The major carotenoid was beta-carotene. Its hydroxyl derivatives were (3R)-beta-cryptoxanthin, (3R,3'R)-zeaxanthin, (2R,3R,3'R)-caloxanthin, and (2R,3R,2'R,3'R)-nostoxanthin, and its keto derivatives were echinenone and canthaxanthin. The unique myxol glycosides were (3R,2'S)-myxol 2'-fucoside and (2R,3R,2'S)-2-hydroxymyxol 2'-fucoside. This is only the second species found to contain 2-hydroxymyxol. We propose possible carotenogenesis pathways based on our identification of the carotenoids: the hydroxyl pathway produced nostoxanthin via zeaxanthin from beta-carotene, the keto pathway produced canthaxanthin from beta-carotene, and the myxol pathway produced 2-hydroxymyxol 2'-fucoside via myxol 2'-fucoside. This cyanobacterium was found to contain many kinds of carotenoids and also displayed many carotenogenesis pathways, while other cyanobacteria lack some carotenoids and a part of carotenogenesis pathways compared with this cyanobacterium.

Survey of the distribution of different types of nitrogenases and hydrogenases in heterocyst-forming cyanobacteria.

As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.

Substrate specificities and availability of fucosyltransferase and beta-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp. strain PCC 7120 compared with Synechocystis sp. strain PCC 6803.

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.

Presence of free myxol and 4-hydroxymyxol and absence of myxol glycosides in Anabaena variabilis ATCC 29413, and proposal of a biosynthetic pathway of carotenoids.

We identified the molecular structures of all carotenoids in Anabaena variabilis ATCC 29413 (= IAM M-204). The major carotenoids were beta-carotene, echinenone and canthaxanthin. Myxol glycosides were absent, while free forms of myxol and 4-hydroxymyxol were present. The 4-hydroxyl group of the latter was a mixture of (4R) and (4S) configurations, which is a rare mixture in carotenoids. Thus, this strain was the first cyanobacterium found to have free myxol and not myxol glycosides, and seemed to lack the gene for or activity of glycosyl transferase. In another strain of A. variabilis IAM M-3 (= PCC 7118), we recently identified (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, and hence the strain ATCC 29413 might be useful for investigating the characteristics of myxol glycosides in cyanobacteria. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in A. variabilis ATCC 29413, we proposed a biosynthetic pathway of the carotenoids and the corresponding genes and enzymes. The homologous genes were searched by sequence homology only from the functionally confirmed genes.

The cyanobacterium Anabaena sp. PCC 7120 has two distinct beta-carotene ketolases: CrtO for echinenone and CrtW for ketomyxol synthesis.

Two beta-carotene ketolases, CrtW and CrtO, are widely distributed in bacteria, although they show no significant sequence homology with each other. The cyanobacterium Anabaena sp. PCC 7120 was found to have two homologous genes. In the crtW deleted mutant, myxol 2'-fucoside was present, but ketomyxol 2'-fucoside was absent. In the crtO deleted mutant, beta-carotene was accumulated, and the amount of echinenone was decreased. Therefore, CrtW catalyzed myxol 2'-fucoside to ketomyxol 2'-fucoside, and CrtO catalyzed beta-carotene to echinenone. This cyanobacterium was the first species found to have both enzymes, which functioned in two distinct biosynthetic pathways.

Myxol and 4-ketomyxol 2'-fucosides, not rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and proposal for the biosynthetic pathway of carotenoids.

We identified the molecular structures of carotenoids in some Anabaena and Nostoc species. The myxoxanthophyll and ketomyxoxanthophyll in Anabaena (also known as Nostoc) sp. PCC 7120, Anabaena variabilis IAM M-3, Nostoc punctiforme PCC 73102 and Nostoc sp. HK-01 were (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, respectively. The glycoside moiety of the pigments was fucose, not rhamnose. The major carotenoids were beta-carotene and echinenone, and the minor ones were beta-cryptoxanthin, zeaxanthin, canthaxanthin and 3'-hydroxyechinenone. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, we proposed a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. Since only zeta-carotene desaturase (CrtQ) from Anabaena sp. PCC 7120 and beta-carotene ketolase (CrtW) from N. punctiforme PCC 73102 have been functionally identified, the other genes were searched by sequence homology only from the functionally confirmed genes. Finally, we investigated the phylogenetic relationships among some Anabaena and Nostoc species, including some newly isolated species.