Isolation and Identification of Echinenone from Micrococcus Roseus

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JOURNAL OF BACTERIOLoGY, Oct. 1970, p. 272-274 Vol. 104, No. 1 Copyright 0 1970 American Society for Microbiology Printed In U.S.A. Isolation and Identification of Echinenone from Micrococcus roseus

E. H. SCHWARTZEL AND J. J. COONEY Department ofBiology, University ofDayton, Dayton, Ohio 45409 Received for publication 22 June 1970 Downloaded from

An orange carotenoid from Micrococcus roseus was purified by solvent partition- ing followed by column and thin-layer chromatography. Absorption spectra, chromatographic mobility, and partition coefficient suggested that the pignent was echinenone (4-keto-ft-carotene). Reduction yielded a pigment with the spectral and polar properties of isocryptoxanthin (4-hydroxy-j3-carotene), the expected product. The orange pigment and its reduction product co-chromatographed with the respective authentic pigments, confirming the original pigment as echinenone. To our knowledge echinenone has not been identified previously as a bacterial pig-

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Masses of cells of Micrococcus roseus appear tone in petroleum ether. The orange-red band was orange-pink. The principal colored carotenoid is suspected of containing echinenone because of its canthaxanthin (4,4'-diketo-p-carotene) (3), and chromatographic mobility and because its absorption structures have been suggested for several other spectrum showed a single broad absorption maximum pigments (38). The monoketo compound echine- at 454 to 460 nm. The crude eluate was concentrated and applied to a none (4-keto-jS-carotene) is generally considered thin-layer chromatography (TLC) plate coated with an intermediate between and cantha- ,-carotene neutral alumina (AG 7, 100 to 200 mesh, BioRad on August 28, 2019 by guest xanthin in animals (14, 17, 20, 25, 29). Therefore, Laboratories, Richmond, Calif.). The plate was de- we sought this carotenoid in cells of M. roseus. veloped with 1 to 2% acetone in petroleum ether. Three colored zones were resolved: a yellow pigment, MATERIALS AND METHODS RF 0.9; an orange-red pigment, RF 0.5; and a red pig- In earlier work, M. roseus ATCC 516 was cultured ment, RF 0.1. Preparative TLC was then used to iso- on the surface of stock culture apr. In the present late the three pigments. work, larger yields of cells were desired so that trace Spectra were recorded in reagent grade solvents by pigments would not be overlooked. Therefore a liquid use of a Bausch & Lomb Spectronic 600 recording medium was used which approximated the composi- spectrophotometer. Partition coefficients were deter- tion of stock culture apr. Each 11-liter batch con- mined in a hexane:95% methanol system as described tained: beef heart infusion broth, 480 g; protease- byPetracekand Zechmeister (32). peptone, 10 g; gelatin, 10 g; tryptone, 10 g; isoelectric Keto groups were reduced to hydroxyl groups with casein, 5 g; sodium chloride, 5 g; disodium phosphate, sodium borohydride according to Krinsky and Gold- 4 g; sodium citrate, 2 g; and dextrose, 0.2 g. The pH smith (27). The products were then chromatographed was adjusted to 6.8. Cultures were incubated in a and the products were eluted as before. bench-top fermentor at 28 C aerated at 4,000 cc of air Authentic echinenone was a gift of 0. Isler, Hoff- per min. At 72 hr, stationary-phase cells were har- man-LaRoche, Basel, Switzerland. Isocryptoxanthin vested and washed three times with water. (4-hydroxy-jS-carotene) was prepared by reducing Cells were extracted repeatedly with methanol under echinenone with sodium borohydride. nitrogen in the dark until the pellet remaining after centrifugation was colorless. The combined methanol RESULTS AND DISCUSSION extracts were partitioned against several portions of petroleum ether (boiling point 30 to 60 C) until the Each of the three pigments eluted from alu- ether phase was colorless. The combined epiphasic mina TLC plates had a smooth absorption spec- petroleum ether phase was concentrated in a rotary trum with a single maximum. Absorption maxima evaporator. in several solvents are recorded in Table 1. Suffi- The pigment mixture was applied to a column of cient quantities of the yellow and red pigments Silica Gel G:Celite (3:1, w/w). Silica Gel G was ob- tained from7American Optical Corp., Richmond, Calif. have not been collected to allow adequate Two yellow bands were eluted with petroleum ether analyses, but the yellow pigment does not appear and an orange-red band was eluted with 3 to 5% ace- to have keto functions and the red pigment ap- 272 VOL. 104, 1970 ECHINENONE FROM M. ROSEUS 273 TABLE 1. Absorption maxima ofpigments eluted maxima typical of a ,B-carotene chromophore from alumina TLC plates (Fig. 1). The change in shape and the 7-nm de- crease in the principal absorption maximum Pigment Absorption maxima in indicate that one conjugated keto group was reduced. Similar properties were observed for Color RF Hexane CHCls CS2 Ethanol authentic echinenone and isocryptoxanthin. nm nm nm nm Moreover, the orange pigment and echinenone Yellow ... 0.9 454 466-468 482-484 456-458 co-chromatographed as one spot on alumina Orange 0.5 458 474 494 466 TLC plates developed with 1 to 2% acetone in Red . 0.1 464-466 476 496 470 petroleum ether. After reduction the products also co-chromatographed as one on alumina plates developed with 3 to 5% acetone in petroleum ether. Therefore, the orange pigment is identified as

echinenone, 4-keto-,8-carotene (Fig. 2). Keto Downloaded from carotenoids were isolated from other micrococci, including a radiation-resistant Micrococcus sp. (2), from M. radiodurans (35) and from M. lysodeikticus (33), but none has properties appropriate to echinenone. Echinenone has been identified in organisms as varied as blue-green algae (22), green algae (10, 12, 16, 21), euglenoids (27, 36), zooflagel- 0c .0a lates (31), hydra (28), polychaete worms (30),

water mites (4, 5, 7), water fleas (11, 20, 37), http://jb.asm.org/ 0 10 .0 various shrimps (9, 13, 14, 18, 25, 26), freshwater ox copepods (6), pill bugs (29), mosquitoes (8), starfish (15), sea urchins (19), and flamingos (17). It is generally believed that animals cannot synthesize carotenoids de novo. They can metabo- lize carotenoids, and can insert oxygen functions on carotenes to form xanthophylls. Therefore, echinenone isolated from animals was probably on August 28, 2019 by guest TABLE 2. Spectral and polar properties of authentic echinenone and orange pigment from M. roseus before and after 360 400 440 480 520 560 reduction Wavelength (nm) FIG. 1. Visible absorption spectra of the orange Pigment coefficientPartition Absorptionin hexanemaxima(nm) pigment eluted from TLC plates (solid line) and of the pigment after reduction (dashed line). Orange pigment...... 95% Epiphasic 458 Reduced orange pig- ...... 476 pears to be a monoketo carotenoid distinct from ment 91% Epiphasic (425)-, 451, Echinenone...... 95% Epiphasic 458 echinenone. Isocryptoxanthin.....1 91 % Epiphasic (425)a, 449, 476 The absorption spectrum of the orange pigment (Table 1, Fig. 1) was typical of a chromo- a Figures in parentheses indicate a shoulder rather than a phore containing 12 conjugated double bonds. distinct peak. Lack of fine structure and the slight asymmetry of the spectrum suggested that the chromophore was extended at one end. The pigment was 95% epiphasic in a hexane: 95% methanol system (Table 2), suggesting that it was a monoketo compound. After reduction the pigment was 51 more polar, typical of a monohydroxy carotenoid (Table 2). The absorption spectrum ofthe product 0 had considerable fine structure with absorption FiG. 2. Structure of echinenonte, 4-keto-f3-carotene. 274 SCHWARTZEL AND COONEY J. BACTERIOL ingested and stored, or it was formed by oxida- 16. DiAccadia, F., 0. Gribanovski-Sassu, A. Romagnoli, and tion of carotenoids in the diet. Plants and eu- L. Tuttobello. 1966. Isolation and identification of carotenoids produced by a green alga (Dictyococcus cinnabarinus) caryotes can synthesize carotenoids and convert in submerged culture. Biochem. J. 101:735-740. them to xanthophylls. Thus, animal carotenoids 17. Fox, D. L., V. E. Smith, and A. A. Wolfson. 1967. Carotenoid may originate far down the food chain. selectivity and feathers of lesser (African), Chilean and Among bacteria, micrococci (2), mycobacteria greater (European) flamingos. Comp. Biochem. Physiol. 23:225-232. (23, 24), and flexibacters (1) contain carotenoids 18. Gilchrist, B. M. 1968. Distribution and relative abundance with conjugated keto functions on a fB-ionone of carotenoid pigments in anostraca (Crustacea: Bran- ring. Canthaxanthin, with two such groups, was chiopoda). Comp. Biochem. Physiol. 24:123-147. isolated from Corynebacterium michagenense (34) 19. Griffiths, M. 1966. The carotenoids of the eggs and embryos of the sea urchin Strongylocentrotus purpuratus. Develop. and is the principal pigment of M. roseus (3, 38). Biol. 13:296-309. As far as we are aware, this is the first report of 20. Herring, P. J. 1968. The carotenoid pigments of Daphnia echinenone as a bacterial pigment. However, it magna Straus. I. The pigments of animals fed Chlorella may be fairly widespread among bacteria be- pyrenoidosa and pure carotenoids. Comp. Biochem. Phys- iol. 24:187-203. cause it is regarded as an intermediate in synthe- 21. Herrman, R. G. 1968. Die Plastidenpigmente einiger Desmi- Downloaded from sis of more oxygenated pigments (14, 17, 20, diaceen. Protoplasma 66:357-368. 25, 29). A pigment with appropriate chromato- 22. Hertzberg, S., and S. L. Jensen. 1966a. The carotenoids of graphic and spectral properties was isolated from blue-green algae. II. The carotenoids of Aphanizomenon flos-aquae. Phytochemistry 5:565-570. C. michagenense (34). 23. Hertzberg, S., and S. L. Jensen. 1966b. Bacterial carotenoids. LITERATURE CITED XIX. The carotenoids of Mycobacterium phki strain Vera. 1. The structures of the minor carotenoids. Acta Chem. 1. Aasen, A. J., and S. L. Jensen. 1966. Carotenoids of flexi- Scand. 20:1 187-1 194. bacteria. III. The structures of flexixanthin and deoxy- 24. Hertzberg, S., and S. L. Jensen. 1967. Bacterial carotenoids. flexixanthin. Acta Chem. Scand. 20:1970-1988. XX. The carotenoids of Mycobacterium phki strain Vera. 2. Bamji, M. S., and N. I. Krinsky. 1966. The carotenoid pig- 2. The structures of the phleixanthophylls-two novel

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