Carotenoid Formation by Staphylococcus Aureus RAY K

JOURNAL OF BACTERIOLOGY, July 1970, p. 191-198 Vol. 103, No. 1 Copyright a 1970 American Society for Microbiology Printed in U.S.A. Carotenoid Formation by Staphylococcus aureus RAY K. HAMMOND AND DAVID C. WHITE Department ofBiochemistry, University ofKentucky Medical Center, Lexington, Kentucky, 40506 Received for publication 26 January 1970 The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; c-carotene; 6-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-14C added to the culture was incorpo- rated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of 14C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of 14C for 2.5 bacterial doublings. The incorporation and turnover of 14C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids. Carotenoids are isoprenoid lipids containing 40 and Lincoln (11). In this study, the carotenoid carbon atoms (eight isoprene units) and are de- pigments of strain U-71 were characterized and tectable in the cell membranes of many micro- the general features of their metabolism are organisms (for a review see reference 4). The described. degree of unsaturation and conjugation of the double bonds ranges between colorless saturated MATERIALS AND METHODS polyenes to conjugated, hydroxylated carotenoids Materials. Commercially available, analytical grade which are intensely colored. organic solvents were used without further purifica- Microbial carotenogenesis has been reviewed by tion. Dibenzylethylenediamine-DL-mevalonate-2-14C Jensen (9) and more recently by Ciegler (4). was purchased from New England Nuclear Corp., Colonies of Staphylococcus aureus become deeply Boston, Mass. colored when allowed to grow to stationary phase. Growth of the bacteria. The strain and harvesting This color was attributed to the presence of carot- conditions of the bacteria have been described (7). A enoids by Zopf (18) in 1889, but no identification semi-synthetic growth medium used throughout this ofthe carotenoids ofS. aureus was published until study consisted of 0.3% (w/v) trypticase, 0.2% (w/v) 1933 when Chargaff (3) reported that zeaxanthin yeast extract, 50 mm sodium gluconate (15 mM glucose for anaerobic cultures), 25 mm phosphate, 0.4 mM was the only carotenoid detectable in lipid ex- MgC92, 15 j,M Fe(NH4)2(SO4)2, 35 mM NaCl, 4 nM tracts of S. aureus. However, later studies (14, 15) biotin, 0.7 ,M thiamine, 8 ,uM nicotinic acid, and 0.1 indicated that phytoene, ¢-carotene, b-carotene mM uracil, adenine, and xanthine. For the identifica- and rubixanthin were formed by most strains of tion of the pigments, 8 liters of medium was inoculated S. aureus. Other investigators (5) have indicated with 200 ml of exponentially growing S. aureus, and the presence of sarcinaxanthin and sarcinene in the culture was incubated at room temperature (24 to some strains of S. aureus. Since Chargaff's study 28 C) with vigorous stirring and aeration for 18 hr. (3), only one other report of zeaxanthin in S. The cells were harvested in the stationary phase and aureus has published (1). the lipids were extracted. For measurement of carot- been enoids during the growth cycle, 4 liters of medium Suzue (15, 16) has studied the synthesis of ca- was inoculated with 100 ml of an exponentially grow- rotenoids in S. aureus. He finds that the initial ing culture and incubated at 24 to 28 C. Periodically, formation of a C40 compound and sequential 400-ml portions were withdrawn, cells were harvested, desaturation to form other carotenoids is similar and the lipids were extracted. Bacteria were grown to that reported for tomato carotenoids by Porter anaerobically at 37 C in medium sparged with nitrogen 191 192 HAMMOND AND WHITE J. BACrERIOL. from just after autoclaving through the end of the Violet Products, Pasadena, Calif.). As soon as the growth cycle. The nitrogen was purified by passage paper dried, each band was cut out and eluted by over hot copper chips (7). Incorporation of '4C was soaking in 2% (v/v) acetone in hexane (5 ml per measured during growth in 1,500 ml of medium for ,umole of carotenoid). Ten per cent acetone in hexane 2.5 doublings in the presence of 50 ,uc of mevalonate- was used to elute the polar carotenoids. 2-'4C. To measure turnover, bacteria were grown at The polar mixture (RF 0.1 to 0.3 in first chromatog- 37 C in 250 ml of medium for three doublings in the raphy) was separated by descending chromatography presence of 25 1Ac of mevalonate-2-14C. The culture on alumina-impregnated paper as before with a sol- was centrifuged at 37 C; the cells were washed by vent of 2% acetone in hexane (v/v) into six compo- gently adding medium over the pellet, and were sus- nents as follows: RF 0.10 to 0.15, a yellow band; RF pended in 1,800 ml of nonradioactive medium for value 0.20 to 0.25, a red band; RF 0.40 to 0.45, an continued incubation. A 1,500-ml culture was grown orange band; RF 0.50 to 0.55, a yellow band; RF for 14 hr in the presence of 25 /Ac of mevalonate-2-14C 0.60 to 0.65, a colorless band detected as a dark-blue to provide radioactive carotenoids for autoradiog- band with ultraviolet (UV) light; and RF 0.80 to 0.85, a raphy. colorless band detected as a pale-blue band with UV Extraction of the lipids and isolation of the carot- light. Each of these carotenoids was extracted from enoids. The lipids were extracted by a modified Bligh the paper with 10% acetone in hexane (v/v). The and Dyer procedure (2). The modification was as radioautogram was prepared after ascending chroma- follows. Methanol and chloroform were added to the tography on alumina impregnated paper with a solvent bacteria suspended in 50 mm phosphate buffer, pH of 2% (v/v) acetone in hexane. 7.6 (10 ml of buffer per 30 mg, dry weight of cells), in Absorption spectra-The purified carotenoids were a separatory funnel such that the proportions were dissolved in hexane and the absorption spectra meas- 0.8:2.0: 1.0 (v/v), buffer-methanol-chloroform; this ured with the Cary Model 15 spectrophotometer. mixture was shaken vigorously. The one-phase mix- Carotenoid concentrations were calculated using molar ture was allowed to stand for a least 2 hr; then the extinction coefficients (cm-'m-1) of 6.65 X 104 for proportions were made to 0.9:1.0:1.0 (v/v), buffer- phytoene, 1.23 X 105 for c-carotene, 1.50 X 105 for methanol-chloroform by adding water and chloro- b-carotene, 1.25 X 105 for the rubixanthins, and 1.20 form. The mixture was shaken vigorously and the X 105 for the phytofluenols (8). layers were allowed to separate. The lower layer Assay of radioactivity. Radioactivity was measured (chloroform) was filtered through anhydrous sodium in a Packard Scintillation Spectrometer (model 2311) sulfate (about 1 g per 10 ml of chloroform) and in a scintillation fluid of 9.28 mm 2,5-bis-[2(5-tert- evaporated to dryness in vacuo. The residue was butyl-benzoxazoyl) ]-thiophene (BBOT) in toluene. dissolved in methanol containing 10% (v/v) water Radioactive samples were dried in the scintillation (approximately 1 ml per 3 mg, dry weight of cells vials, and a drop of Br2 was added to each vial. The extracted). Potassium hydroxide was added to the vials were then placed in a vacuum oven at 45 C for solution to a final concentration of 5% by weight. The 3 hr, after which 5 ml of scintillation fluid was added. lipids were saponified by stirring at room temperature Samples containing 14C were counted under conditions for 2 hr. Between 0.1 and 0.2 volumes of saturated, such that the efficiency of counting was 82%. Auto- aqueous sodium chloride was added, and the mixture radiograms were prepared by placing chromatograms was extracted three times with a volume of benzene- on Kodak No-Screen X-ray film (17). After 12 days, petroleum ether (1:1, v/v) equal to the volume of the the film was developed. saponification mixture. The combined benzene- petroleum ether extracts were washed once with an RESULTS equal volume of water. The organic phase was evapo- The extraction of the rated to dryness in vacuo. The nonsaponifiable lipid carotenoids in this study residue was dissolved in hexane (1 ml per 3 mg of cells involved a total lipid extraction with chloroform- extracted) and stored under nitrogen at 0 C. All these methanol rather than the usual methanol extrac- steps were carried out in a minimum of illumination tion. The methanol extraction method as de- and under nitrogen where possible. scribed by Suzue (15) yielded less than 10% of the Paper chromatography. Two cycles of paper chro- carotenoids recovered in the total lipid extract.

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