JOURNAL OF BACTERIOLOGY, July 1970, p. 191-198 Vol. 103, No. 1 Copyright a 1970 American Society for Microbiology Printed in U.S.A. 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 phyto- ene; c-; 6-carotene; phytofluenol; a phytofluenol-like carotenoid, rubi- xanthin; and three rubixanthin-like 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 sa- ponification. 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, hy- droxylated 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 dou- blings. 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 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 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 , ¢-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. matography were used to separate the carotenoids. The Reextraction of the aqueous phase with chloro- carotenoid mixture was spotted as a series of adjacent form yielded no detectable carotenoids, indicating spots along the base of a sheet of alumina-impreg- that the modified Bligh and Dyer (2) extraction nated paper (SS-288, Carl Schleicher and Schuell, Co., removed all chloroform-extractable Keene, N.H.) with less than 15 nmoles per spot. carotenoids. Before chromatography, the paper was activated in The cell debris was white after extraction, indicat- a 102 C oven for 2 hr and stored in a dessicating ing that no appreciable amount of polar carote- cabinet for not longer than 3 days. Descending chro- noids remained in the residue. Figure 1 illustrates matography in a solvent of hexane (25 cm in approxi- the extraction and purification procedure used to mately 1 hr) separated the carotenoids as follows: RF detect nine carotenoid fractions. In the crude 0.1 to 0.3, the polar carotenoids; RF 0.40 to 0.45, a lipid preparation, the content of rubixanthins pale yellow band; RF 0.65 to 0.70, a deep yellow band; plus b-carotene, the two phytofluenols plus D-caro- and RF 0.85 to 0.90, a colorless band, detected as a tene, and the phytoene could be estimated from pale-green band with a longwave mineral light (Ultra the absorption spectrum. Comparison of the VOL. 103, 1970 CAROTENOID FORMATION BY S. AUREUS 193

S. aureus culture Centrifuge at 0 C

Supernatant medium Bacterial pellet Modified Bligh and Dyer extraction

Organic lower phase (crude Aqueous upper phase containing cell debris lipid extract) Filter and evaporate to dryness Dissolve in 5% methanolic KOH (containing 10% H2O) I Stir 2 hr Saponified lipids Add 0.1-0.2 volumes of saturated, aqueous NaCI Extract 3 times with benzene-petroleum ether, 1:1

Aqueous lower phase (fatty acid salts and Organic upper phase deacylated phospholipids and glycolipids) Wash with I volume of water Take to dryness I Dissolve in hexane Nonsaponifiable lipids Descending chromatography on alumina paper with hexane

I I I I Polar mixture, a-Carotene, yellow ¢-Carotene, yellow Phytoene, color- R,0 .1-0.3 Ry 0.4 R, 0.7 less RF 0.9 Descending chromatography on alumina paper with 2% acetone in hexane I I I I I Yellow Red Orange Yellow Phytofluenol-like, Phytofluenol, col- Rr 0.5 Ry 0.4 R, 0.2 R, 0.1 colorless, Rp 0.6 orless, R, 0.8 Rubixanthins

FIG. 1. Flow chart for isolationi atnd purification of' the caroteiioids of S. aureuis U-71.

estimates from the spectra of the lipid extract to trate was then used for chromatography. The ca- the yields after saponification indicated that about rotenoids were detected with the same mobilities 6% of the carotenoid.mixture was lost. As far as and in the same proportions as in preparations could be determined, there was no differential that had been saponified. This indicated that loss of any carotenoid. Comparison of the neither carotenoid esters nor carotenoid glyco- amounts of carotenoids from the spectrum of the sides were present in this strain as had been found non-saponifiable lipid mixture with the individual in other bacteria (13). carotenoids after chromatographic separation Absorption spectra of the carotenoids. The char- indicated that recovery after chromatography was acteristic absorption spectrum of carotenoids quantitative. could be demonstrated for each of the purified When the saponification step was omitted, the components of the non-saponifiable lipid extract recovery of carotenoids was quantitative. If the (Fig. 2). The components were identified as extract was not saponified, carotenoid separation follows. required removal of the phospholipids by an Phytoene. The RF 0.85 to 0.90 nonpolar com- acetone precipitation at room temperature (25 C). ponent had the chromatographic mobility and The crude lipid extract was dissolved in a minimal absorption spectrum (maxima at 277, 286, and volume of hexane (0.5 ml per 1.0 nmole of carote- 300 nm) of phytoene (8). noid), and acetone was added dropwise until a c-Carotene. The RF 0.65 to 0.70 nonpolar com- precipitate appeared. The mixture was filtered ponent had the chromatographic mobility and through Whatman no. 4 paper, and the residue absorption spectrum (maxima at 378, 398, and was washed with acetone until colorless. The fil- 425 nm) of i-carotene (8). 194 HAMMOND AND WHITE J. BACTERIOL.

SOLVENT FRONT - very similar to rubixanthin: RF 0.10 to 0.15 (maxima at and 499 0.40 to 0 4CPMC 438, 466, nm); RF 0.45 at and 482 and PHYTOENE 11 13,200 - (maxima 432, 458, nm); RF () 0.50 to 0.55 (maxima at 428, 456, and 478 nm). C-CAROTENE 393 Each of these components, when rechromato- graphed in the same system, migrated as a single spot. The rubixanthin will be referred to as ca- () rotenoid 2, the slower as 1, and the () 1,250 other two as 3 and 4. (y - CAROTENE) Autoradioautography. Once the nine principal carotenoids had been identified by chromato- 8-CAROTENE 210 graphic mobility and absorption spectra, trace ^ components were examined by autoradioautog- PHYTOFLUENOL 170 - raphy. Cells were grown with 25 puc of mevalon- 300 £ ate-2-'4C in 1,500 ml for 14 hr at 37 C, and the I ' 240 carotenoids were extracted, saponified, and sepa- rated on alumina-impregnated paper (Fig. 3). Examination of the chromatogram with UV light 1,400 showed the characteristic pale-green fluorescence U4 1 960 of phytoene at RF value 0.90; fluorescence ex- 550 tended below the spot on the autoradiogram. The pale-yellow c-carotene (RF 0.84) was clearly from the 03 A45,300 separated phytoene complex in this chromatogram. In most cases, preliminary chro- RUBIXANTHIN matography with hexane was necessary to sepa- # 1 446,200 rate phytoene and ¢-carotene. b-Carotene (RF 0.58) was yellow. Phytofluenol fluoresced pale FIG. 2. Autoradiogram after separrationI of the blue at RF 0.49 and carotenoid 5 fluoresced deep carotenoids from S. aureus U-71. Cells were grown with mevalonate-2-14C for 14 hr, and the carotenoids 201 were extracted, saponified, and separated by ascending PHYTOENE chromatography on1 alumina-impregnated paper with 0I 286 a solvent of 2% (v/v) acetone in hexane. Carotenoids m were identified by their color in visible light or fluo- rescence in ultraviolet light as inldicated by the dotted 300 400 500 lines. Solid lines indicate the major radioactive com- C-CAROTENE ponents; the 'IC (counts per minute) for each spot is 400 425 indicated at the right. Approximately 100 nmoles of in carotenoids containing 115,000 counts/mil of '14C was 300 400 500 applied to the origin. x 8-CAROTENE

b-Carotene. The RF 0.40 to 0.45 nonpolar com- 21 ~~~~~~~458 ponent had the chromatographic mobility and absorption spectrum (maxima at 430, 456, and 300 400 500 488 nm) of b-carotene (8). Phytofluenol. The RF 0.80 to 0.85 polar compo- nent had the chromatographic mobility and ab- D 348 PHYTOFLUENOL sorption spectrum (maxima at 333, 348, and 368 ;4\683 nm) of phytofluenol (8). The RF 0.65 to 0.70 ONUAA~NV polar component had a chromatographic mobility and on 300 400 500 absorption spectrum (maxima at 349, 373, .20 and 395 nm) similar to phytofluenol. This compo- 432 462 RUBIXANTHIN nent will be referred to as carotenoid 5. t10 494 Rubixanthin. The RF 0.20 to 0.25 polar compo- 'HO nent had the chromatographic mobility and ab- 300 400 S00 sorption spectrum (maxima at 437, 461, and 494 WAVELENGTH WIn) nm) similar to rubixanthin (8). This component FIG. 3. Absorption spectra and structures of the was red. Three other polar components had chro- major carotenoids of S. aureus U-71. Absorption matographic mobilities and absorption spectra spectra were\~~~~~~~~~~~.r-.measured in hexane. VOL. 103, 1970 CAROTENOID FORMATION BY S. AUREUS 195 blue at RF 0.38 in UV light. The xanthophyll 4 phytoene, ¢-carotene, and 6-carotene and the (RF 0.23) was yellow; 3 (RF 0.12) was orange; 2, polar phytofluenols predominate. However, later the rubixanthin (RF 0.05) was red; and 1, just in growth, the rubixanthins became predominant. above the origin was yellow. Some components Growth of cells in a lighted laboratory had no were detected only on the autoradiogram. At RF effect on the carotenoid levels when compared to 0.67, a complex spot with the mobility expected cells grown in total darkness. for neurosporene, lycopene, and 'y-carotene was The carotenoid concentrations in anaerobically detected. There were apparently three components growing S. aureus are illustrated in Fig. 5. The migrating with the phytofluenols: phytofluenol nonpolar carotenoids remained predominant (RF 0.49), an unknown radioactive component throughout the anaerobic growth cycle and the (RF 0.46), and component 5 (RF 0.38). Two com- rubixanthins reached approximately 1% of the ponents were detected on the autoradiogram with level found after aerobic growth. the xanthophyll component 4, one at RF 0.19 and Incorporation of mevalonate-2-'4C. The incor- one at RF 0.26. Rubixanthin and the xanthophyll poration of mevalonate-2-'4C into each carote- 3 were so heavily labeled that their separation did noid during aerobic growth is illustrated in Fig. 6. not show on the autoradiogram. These compo- The nonpolar carotenoids phytoene, c-carotene, nents account for 96% of 14C applied to the chro- and b-carotene and the polar phytofluenols matogram. The nine components that have been rapidly became labeled. The specific activities of identified by absorption spectra account for 97% the nonpolar carotenoids and phytofluenols of the 14C recovered from the chromatogram. reached a maximum and then began to decrease Carotenoid formation during aerobic and at about the time 14C appeared in the rubixan- anaerobic growth. The carotenoid concentration thins. in aerobically growing S. aureus is illustrated in Turnover of carotenoids labeled with mevalonate- Fig. 4. Early in growth, the nonpolar carotenoids 2-14C. Bacteria were grown with mevalonate-2-'4C for three bacterial doublings. The total radioac- Vil, 61 8-CAROTENE I tivity in each carotenoid during continued growth

,PHYTOENE in the absence of 14C is illustrated in Fig. 7. 0 Phytoene, c-carotene, and phytofluenol lost ..--CAROTENE radioactivity rapidly, whereas the total radioac- 1-2 -2 I -4~~ ICl I PHYTOFLUENOL

TOTAL

cr 20 40 60 80 20 40 60 80 20 40 60 80 3 -2 10 awJL waa. 1,2,3 64

-CAROTENE 10-3

0 4 12 20 28

'02I o. ,o E 4 '' U) s/ z GROWTH m gr cn m IC FIG. 4. Carotenoid formation by S. aureus during xX aerobic growth. Samples of400 ml were withdrawui from aerobically growing S. aureus, the lipids were extracted, 4 12 20 28 and the carotenoids were purified and assayed spectro- HOURS photometrically. The rubixanthins are designated as 1, 2, 3, and 4 and represent the polar carotenoids with FIG. 5. Carotenoid formation hy S. aureus during RF values of0.5, 0.4, 0.2, and 0.1 in Fig. 1. The plhyto- anaerobic growth. The carotenoids isolated from an fluenol-like carotenoid is designated as 5 (RF 0.6). anaerobic culture were treated as in Fig. 4. 196 HAMMOND AND WHITE J. BACrERIOL. tivity of the rubixanthins increased. The increase in radioactivity in the rubixanthins equaled the -PULSE-' CHASE- decrease in radioactivity in the precursors. During w 0.5 x growth in nonradioactive medium, the radio- C o ck4 activity of the total carotenoid did not change. cn m DISCUSSION 0.1

In this study, S. aureus U-71 has been shown to contain three nonpolar carotenoids and six xanthophylls as the primary pigments. Possibly 8-CAROTENE seven other unidentified components were pres- ent; these minor components accounted for less than 3% of the 14C in the carotenoid fraction. C., Phytoene, i-carotene, and b-carotene and rubi- /I w PHYTOFLUENOL xanthin have been reported previously in S. aureus z (14, 15). However, the presence of three other rubixanthin-like carotenoids and two phyto- iE a.w 102 fluenol-like carotenoids in S. aureus has not 25 50 75 100 Co previously been reported. , The carotenoid concentrations during aerobic t + C- TOTAL

0

105 105 I0 1 4

ROTENE lco4t TOENYT0EN~)0~ F _TM,,~-C 104 &.~~AROTENE PHYrTOFLuEi 1031 0 25 50 75 100 0 25 50 75 MINUTES FIG. 7. Turniover of caroten7oids labeled with meva- 103 103 lonate-2-'4C in S. aureus. S. aureus, grown for three doublings in the presence of 25 ,.c of mevalonate-2-14C 20 60 l00 20 60 100 per 250 ml of culture, were centrifuged at 35 C, the pellet was resuspended in 1,700 ml of nonradioactive medium, anid samples of 300 ml were withdrawn. lop 105 The lipids were extracted, and the carotenoids were purified and assayed for radioactivity as in Fig. 6. The upper graph illustrates the bacterial density dutring both periods of growth. The carotenoids are designated as in Fig. 4.

growth of S. aureus changed over wide limits. Aerobically, there was a 22-fold increase in the concentration of carotenoids per bacterium be- tween the exponential and stationary growth phases (Fig. 4). At the maximal 103 los level of carote- 20 60 100 20 60 100 MINUTES noid, the rubixanthins represented 75% of the FIG. 6. Incorporation of mevaloniate-2-14C into total compared to 35% in the exponential phase. carotenoids by S. aureus. Samples of 250 ml were In the anaerobic growth cycle the total carote- withdrawn from a culture of S. aureus growinig in the noids increased fourfold (Fig. 5). The rubixan- presence of S0 ,uc of mevaloniate-2-'4C per 1,500 ml, thins represented 8 % of the total in the stationary the lipids were extr acted, anid the carotenioids were phase and were undetectable (<10- ,moles per purified and assayed for radioactivity. Car-otenioids are g, dry weight) in the exponential phase. In the indicated as in Fig. 4. anaerobic stationary phase, the rubixanthins VOL. 103, 1970 CAROTENOID FORMATION BY S. AUREUS 197

represented 0.8% of the level in the aerobic Phytoene stationary phase. The considerably higher concentrations of 2H rubixanthins in the aerobic cells may reflect the Water (Phytofluene) Phytofluenol oxygen requirement for the hydroxylation of the Hydration carotenoids which apparently is common in bac- 2H 2H teria (6, 10). An alternate pathway to the rubixan- thins has been detected during anaerobic growth i-carotene Phytofluenol- (Fig. 5). Possibly the anaerobic pathway involves like the hydration of a terminal double bond of phy- toene to form phytofluenol. The reaction 2H I sequence postulated for carotenoid biosynthesis in S. aureus U-71 is illustrated in Fig. 8. Compo- (Neurosporene) Rubixanthins nents in parentheses in Fig. 8 were not character- j 2H ized, although these components could be de- tected by autoradioautography (Fig. 2). The (Lycopene) incorporation of mevalonate-2-'4C into the carot- enoids was consistent with the dehydrogenation cyclization of the nonpolar, relatively saturated phytoene to the more unsaturated i-carotene. The i-carotene (-a-Carotene) was then dehydrogenated and cyclized to b-caro- isomerization tene. The b-carotene was then hydroxylated to form the rubixanthins. a-Carotene The turnover of carotenoids labeled with 'IC (Fig. 7) was also consistent with the pathways 02j hydroxylation illustrated in Fig. 8. The loss of "4C from phy- toene, D-carotene, and b-carotene equaled 80% of the increase in radioactivity in the rubixanthins. 1u Presumably this represented the acLivity of the Rubixanthins aerobic hydroxylation pathway. The loss of "IC FIG. 8. Proposed scheme for the terminal reactions from the phytofluenols accounted for the other ofcarotenoid biosynthesis in S. aureus. The carotenoids 20% of the increase in 14C in the rubixanthins. in parentheses have not been characterized from this Presumably this represented rubixanthin synthesis organism, although evidence for their presence has been by hydration to phytofluenol and subsequent obtained (Fig. 2). cyclization. The hydration pathway appeared to function both anaerobically and aerobically. lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 3. Chargaff, E. 1933. Sur les carotenoides des bacteries. Compt. ACKNOWLEDGMENTS Rend. 197:946-948. 4. Ciegler, A. 1965. Microbial carotenogenesis. Advan. Appl. This investigation was supported by contract 12-14-100- Microbiol. 7:1-34. 9517(73) with the Agricultural Research Service, U.S. Depart- 5. Douglas, R. J., and J. T. Parisi. 1966. Chromogenesis by ment of Agriculture, administered by the Eastern Utilization and variants of Staphylococcus aureus. J. Bacteriol. 92:1578- Development Division, Philadelphia, Pa., and by Public Health 1579. Service grant GM-10285 from the National Institute of General 6. Eimhjellen, K. E., and S. L. Jensen. 1964. The biosynthesis Medical Sciences. of carotenoids in Rhodopseudomonas gelatinosa. Biochim. R. K. Hammond was supported by grant 5-TOI-GM-01026 Biophys. Acta 82:21-40. from the National Institutes of General Medical Sciences to the 7. Frerman, F. E., and D. C. White. 1967. Membrane lipid Department of Biochemistry, University of Kentucky Medical changes during formation of a functional electron transport Center. system in Staphylococcus aureus. J. Bacteriol. 94:1868-1874. 8. Goodwin, T. W. 1955. Carotenoids. Moderne Methoden der LITERATURE CITED Pflanzenanalyse. Springer Verlag, Berlin. 9. Jensen, S. L. 1965. Biosynthesis and function of carotenoid 1. Allegra, G., R. Nuitta, and G. Giuffrida. 1954. Studies on pigments in microorganisms. Annu. Rev. Microbiol. 19: carotenogenesis and on the role of carotenoids in Staphylo- 163-182. coccus aureus. I. Action of glucose on chromogenesis in 10. Olson, J. A. 1964. The biosynthesis and metabolism of carote- Staphylococcus aureus. Riv. Ist. Sieroterap. Ital. 29:263- noids and (vitamin A). J. Lipid Res. 5:281-299. 269. 11. Porter, J. W., and R. E. Lincoln. 1950. I. Lycopersicon selec- 2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total tions containing a high content of carotene and colorless 198 HAMMOND AND WHITE 3. BACTERIOL.

polyenes. 11. The mechanism of carotene biosynthesis. 15. Suzue, G. 1959. Studies of carotenogenesis by Micrococcus Arch. Biochem. Biophys. 27:390-403. pyogenes var aureus. J. Biochem. 46:1497-1504. 12. Rilling, H. 1962. Anaerobic carotenloid synthesis by an aerobic 16. Suzue, G. 1962. On the enzymatic synthesis of bacterial microorganism. Biochim. Biophys. Acta 65:156-158. phytoene from 2-'4C-mevalonic acid. J. Biochem. 51:246- 13. Smith, P. F. 1963. The carotenoid pigments of Mycoplasma. 252. J. Gen. Microbiol. 32:307-319. 17. White, D. C. 1968. Lipid composition of the electron trans- 14. Sobin, B. and G. L. Stahly. 1942. The isolation and absorption port imiemiibrane of Haelmiophilus paraiiiflielzae. J. Bacteriol. spectrum maxima of bacterial carotenoid pigments. J. 96:1159-1170. Bacteriol. 44:265-276. 18. Zopf, W. 1889. Ueber Pilzfarbstoffe. Botan. Ztg. 47:53-61.