Vol. 173, No. 24

The Major Carotenoid Pigment of a PsychrotrophicMicrococcus roseus Strain: Purification, Structure, and

Interaction with Synthetic MembranesMEDICHARLA V. JAGANNADHAM,' V. JAYATHIRTHA RAO,2

AND S. SHIVAJI1*Centre for Cellular & Molecular Biology' and Indian Institute of Chemical Technology,2

Uppal Road, Hyderabad 500 007, India

Received 19 July 1991/Accepted 15 October 1991

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain was purified to homogeneityfrom methanol extracts of dried cells by reverse-phase liquid chromatography and was designated P-3. On thebasis of the UV-visible, infrared, mass, and 'H nuclear magnetic resonance spectra of P-3, it was identified as

bisdehydro-,-carotene-2-carboxylic acid. The pigment interacted with synthetic membranes of phosphatidyl-choline and dimyristoyl phosphatidylcholine and stabilized the membranes. These results also indicate that P-3is different from canthaxanthin, the major carotenoid pigment from a mesophilic M. roseus strain.

Carotenoid pigments are present in a wide variety ofbacteria, algae, fungi, and plants (1, 23). In photosyntheticorganisms, carotenoids are closely associated with the pho-tosynthetic membranes, and they help in harvesting andtransferring light energy to chlorophyll and also protect thephotosynthetic apparatus against photooxidation (34). Caro-tenoid pigments are also present in a wide variety of non-photosynthetic bacteria and serve as an important taxo-nomic marker for the identification of isolates of a particulargenus, such as Flavobacterium (11), or for the identificationof species of a particular genus, such as Micrococcus (15).Bacteria belonging to the genus Micrococcus may be yellow,yellowish green, or orange as in Micrococcus luteus; darkyellow as in M. varians; pink or red as in M. roseus and M.radiodurans; dark rose-red as in M. agilis; or cream or whiteas in M. lylae, M. kristinae, M. nishinomiyaensis, M.sedentarius, and M. halobius (15).

In the genus Micrococcus, the chemical nature of pig-ments has been determined so far only for two mesophilicspecies. For M. luteus, it was found to be a dihydroxy C50carotenoid (35-37), and for M. roseus the pigments werefound to be mainly a or 1B carotene derivatives, with can-

thaxanthin as the main pigment (3, 28, 38). Studies haveindicated that carotenoids in M. roseus do not protect thebacterium against photodynamic killing (29).

In our studies on the taxonomy of bacteria and yeasts ofSchirmacher Oasis, Antarctica, 10 Pseudomonas isolates(33) were not pigmented; isolates included seven Arthrobac-ter isolates (31), three Micrococcus isolates, two Planococ-cus isolates (32), two Sphingobacterium isolates (30a), andthree yeasts of the genus Rhodotorula (25). The predom-inance of pigmented psychrotrophic bacteria (14 of 30 iso-lates) in Antarctic soil may hint at a useful role for thepigment in cold adaptation. Further, little is known aboutwhether pigments are identical or different in bacteria thatbelong to the same species but that differ in that one is a

mesophile and the other a psychrotroph. This paper de-scribes the purification and structural elucidation of the

* Corresponding author.

major pigment from a psychrotrophic M. roseus strain (32),its localization in the bacterium, and its interaction withsynthetic membranes.

MATERIALS AND METHODS

Chemicals and reagents. All chemicals used for bacterialculture were obtained from Loba (Bombay, India). Metha-nol, hexane, and carbon disulfide used were of high-perfor-mance liquid chromatography (HPLC) grade and were ob-tained from Spectrochem, Bombay, India. Chloroform was

of analytical grade (BDH, Bombay, India). CDCI3 and(3-carotene were obtained from Sigma Chemical Co. (St.Louis, Mo.). N-Bromosuccinimide (NBS) and CCI4 were ofreagent grade. Preformed thin-layer chromatography platescoated on 0.2-mm-thick aluminum cards were from FlukaSG (Buchs, Switzerland).

Bacterial strains and growth conditions. The red-pigmentedpsychrotrophic bacterium, identified as M. roseus (MTCC678; IMTECH, Chandigarh, India) (32) was isolated fromsoil samples collected at Schirmacher Oasis, Antarctica. Theculture was maintained in a medium containing peptone(0.5%), yeast extract (0.2%), and soil extract (5%). Masscultures were set up in 2.5-liter flasks containing 1 liter ofthis medium, and the bacteria were grown with continuousshaking for 6 days in an environmental incubator at 25°C. Amesophilic type culture of M. roseus (NCTC 07523) was

obtained from the National Collection of Type Cultures.Nutrient broth was used as the culture medium, and the cellswere grown at 37°C.

Extraction of the pigment. The procedure used for theextraction of carotenoids from psychrotrophic M. roseus

was essentially a modification of published methods (16, 17).Cells were pelleted by centrifugation at 1,000 x g for 10 min,washed free of medium with distilled water, and freeze-driedto a powder. Methanol (100 ml) was added to about 1 g offreeze-dried cells, and the mixture was vortexed until themethanol layer turned red. The entire suspension of cellswas centrifuged (5,000 x g for 5 min), and the methanol layercontaining the crude pigment was recovered. The pale pink

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cell pellet was extracted once again with methanol, and themethanol layer was recovered as described above. Both ofthe methanol fractions were pooled and concentrated byvacuum evaporation.

Purification of the pigment. The crude concentrated pig-ment obtained above was purified to homogeneity by HPLC.The HPLC system (Hewlett-Packard 1090) consisted oftwo pumps, a Rheodyne injector fitted with a 250-,l loop,a photodiode array detector, an integrator (model 3392A),and a data system controller (model HP 85-B). Sampleswere analyzed on a Waters C-18 reverse-phase column (4.6by 250 mm; Dupont, Wilmington, Del.) which was elutedwith a gradient of 80 to 100% methanol at a flow rate of 1ml/min. The run lasted for 30 min, and the samples weremonitored at 470 nm. The absorption spectra of all relevantpeaks were recorded with the help of the on-line photo-diode array detector. ,8-Carotene, dehydro-1-carotene, andbisdehydro-1-carotenes were analyzed under identical con-ditions.

Preparation of cell walls from M. roseus. The method usedwas essentially that described by Work (42, 43), with minorchanges. About 100 mg of cells were suspended in 0.1 Msodium phosphate buffer (pH 7.8) and sonicated until 90% ofthe cells were disrupted (5 min); they were then heated for 10min at 60°C to inactivate lytic enzymes. The sonicated cellsuspension was centrifuged at 1,000 x g for 10 min, and thepellet, consisting of intact cells and lumps, was discarded.The supernatant was centrifuged at 25,000 X g for 15 min,and the sediment of cell walls was recovered. The cell wallfraction was resuspended in the buffer, spun once again at1,000 x g for 10 min, and then recovered from the superna-tant as a pellet by centrifugation as described above (25,000x g for 15 min). Cell walls thus obtained were once againsuspended in phosphate buffer and treated with DNase for 3h and then with trypsin for 12 h. The suspension was thencentrifuged at 1,000 x g for 10 min (pellet discarded), and thewalls in the resulting supernatant were sedimented at 25,000x g for 20 min and washed thrice with 0.9% NaCI. Thepigmented cell walls were lyophilized and stored as a pelletat -20°C.

Interaction of the pigment with membrane vesicles. Smallunilamellar vesicles (SUVs) were prepared by the sonication(Branson B-50 Sonifer) of an aqueous solution of the re-quired lipid in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer to clarity. The lipids used for thepreparation of the SUVs, phosphatidylcholine (PC) anddimyristoyl phosphatidylcholine (DMPC), were purchasedfrom Sigma. Interaction of the pigment with the SUVs wasmonitored with two fluorescent probes, pyrene and 8-anilino-1-naphthalenesulfonate (ANS). Fluorescence mea-surements were recorded on a Hitachi 650-1OS fluorescencespectrophotometer with 4-nm excitation and emission band-pass. A 2 mM aqueous solution of recrystallized ANS servedas a stock. ANS was excited at 370 nm, and the emissionspectra were recorded between 420 and 600 nm. Pyrene wasincorporated into the SUVs by rapidly mixing a stocksolution of 2 mM pyrene in methanol with the vesicles; thefinal concentration of methanol was not allowed to exceed1%. Pyrene was excited at 333 nm, and the emission spectrawere recorded from 360 to 520 nm.

Preparation of dehydro-f-carotene and bisdehydro- -caro-tene. Dehydro-13-carotene and bisdehydro-1-carotene wereprepared by using 1-carotene as the starting material (45).NBS (130 mg) was added to a solution of 200 mg of,B-carotene in 60 ml of carbon tetrachloride, and the solutionwas refluxed for 6 h in the dark under a nitrogen atmosphere.

Eo

1

u B

2

0 5 10 15 20 25 30

RETENTION TIME (min )

FIG. 1. HPLC profile of (A) the crude pigment of M. roseus andof (B) P-carotene (peak 1), dehydro-1-carotene (peak 2), and bisde-hydro-,B-carotene (peak 3), resolved by using a C-18 reverse-phasecolumn. P-1 to P-S refer to the five pigments in M. roseus. The insetshows the homogenous peak of P-3 (collected from panel A andrechromatographed).

The two carotenoids (dehydro-1-carotene and bisdehydro-1-carotene) which formed during the reaction were sepa-rated by HPLC, and their UV-visible absorption and massspectra were recorded. Carbon tetrachloride was removedprior to HPLC by purging with dry nitrogen.

Physical methods. UV-visible spectra of the pure pigmentin various solvents were recorded by using a Hitachi UV 330spectrophotometer. Infrared (IR) spectra were recorded byusing KBr pellets in a Bommem Michelson 100 FTIR instru-ment. Mass spectra of the pigment and of the other standardcarotenoids were recorded on VG Micromass 7070H at 70eV and at a probe temperature of 250°C. 1H nuclear magneticresonance ('H-NMR) was recorded on a Bruker AM 300MHz spectrometer by using CDCl3, with tetramethylsilaneused as an internal standard.

RESULTS

Extraction and purification of the pigment. The procedureused (13) in the present investigation was highly efficient andeffective in totally extracting the pigment from freeze-driedcells of M. roseus into methanol. The cell pellet and themethanol extract after the first cycle of extraction were palepink and red, respectively. Subsequent extraction withmethanol rendered the cell pellet white, but a little pigmentcould be detected spectrophotometrically in the methanollayer. Hence, pigment extraction from the cells with meth-anol was done twice. The procedure was carried out at roomtemperature, and all the glassware was covered with alumi-num foil to protect the pigment from light. Reverse-phaseHPLC of the crude pigment in methanol yielded five pig-ments, P-1 to P-5 (Fig. 1A), of which pigment 3 (P-3) was themajor one (73%) (Table 1). The five isolated pigments

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TABLE 1. Characteristics of pigments P-1 to P-S from M. roseusand of three synthetic carotenoids

Absorption No. of Mol % of totalPigment maximal double Mol pigment"

(X max) bonds t pigment

P-1 466, 493, 523 13 3P-2 466, 493, 523 13 13P-3 466, 493, 523 13 576 73P-4 466, 493, 523 13 9P-5 466, 493, 523 13 2p3-Carotened 425, 451, 482 11 536Dehydro-13-carotened 450, 474, 504 12 534Bisdehydro-p-carotened 465, 490, 522 13 532

" Absorption spectra were recorded by using an on-line diode arraydetector of HPLC.

b Molecular weights were determined from mass spectra.The percentage of the total pigment represented by each pigment was

determined by HPLC profiles by expressing the area of each pigment peak asa percentage of the total area of the five pigment peaks.

d Standard samples.

differed from P-carotene, dehydro-1-carotene, and bisdehy-dro-3-carotene in their polarities and retention times undersimilar conditions (Fig. 1B). The major pigment resolved asa single spot on thin-layer chromatography plates by usingeither ethyl acetate-methanol (9:1 [vol/vol]) or benzene-ethylacetate-methanol (50:40:10), with Rf values of 0.84 and 0.56,respectively.IR spectrum of P-3. The major pure pigment, P-3, isolated

by HPLC was used to obtain complete spectral data (IR,UV-visible absorption, mass, and 1H-NMR spectra) to de-termine its structure. The IR spectrum of P-3 indicated thepresence of a carboxylic group (18) (characterized by thepeaks at 3,500 and 1,735 cm-1) and showed bands due to CHstretching and C=C stretching and many other bands.

UV-visible absorption spectra of pigments P-1 to P-5. UV-visible absorption spectra of carotenoid pigments are ofimmense importance, since they aid a great deal in deter-mining the structure of carotenoids (8). Table 1 gives theabsorption maxima of the five isolated pigments, the pre-dicted number of double bonds in each of the pigments, andthe percentage of total pigment of each pigment. The UV-visible absorption spectra of all the pigments (P-i to P-5)appeared to be identical and exhibited a fine structure, withthree absorption maxima at 466, 493, and 523 nm. TheUV-visible absorption spectrum for the isolated pigment P-3is given in Fig. 2. The clear three-band shape of the absorp-tion spectrum of P-3 is characteristic of carotenoids andfurther reflects its purity. Comparison of the absorptionmaxima of P-3 with the absorption data available for variousother carotenoids suggested the presence of 13 conjugateddouble bonds in P-3; further comparison also suggested thepresence of ,B-cyclic end groups. The fine structure in theabsorption spectrum suggested that the carboxylic group(identified on the basis of IR and mass spectra) is not inconjugation with the 13-double-bond polyene chain but isprobably present on one of the cyclic ends of P-3. Thenonappearance of the "cis peak" in the UV region of theabsorption spectrum of the pigment allowed us to predictthat P-3 is in all trans configuration. The unequivocal iden-tification of the pigment as a carotenoid stems from thesolvent-induced bathochromic shift observed for the absorp-tion spectrum of P-3 (Table 2). Further, the spectra ofdehydro-3-carotene (12 double bonds) and bisdehydro-1-carotene (13 double bonds) synthesized from 13-carotene (11

W

z

(r0-50-0

0,25-

0

200 300 400 500 600

WAVELENGTH (nm)

FIG. 2. Absorption spectrum of P-3 from M. roseus in metha-nol.

double bonds) indicated that the absorption spectrum ofbisdehydro-1-carotene, with 13 conjugated double bonds,perfectly matched that of P-3 (Table 1), suggesting that P-3has the same number of double bonds as bisdehydro-,B-carotene and probably has the same skeleton.Mass spectrum of P-3. Figure 3 depicts the mass spectrum

and the proposed structure of P-3. The mass spectrum of thepigment displayed an [M'] at mlz 576, corresponding tomolecular formula C41H5202 with a high degree of unsatur-ation. A peak at m/z 470 (M-106) is characteristic of acarotenoid (5). Identification of a carboxylic group present inthe pigment stems from the fragments arising from M-44,M-68, and M-84 in the mass spectrum. The peak at mlz M-56represents one cyclic end of pigment which does not haveany substitution, whereas the peaks at m/z M-68 and M-84correspond to the other cyclic end carrying a carboxylicgroup at position 2. Similarly, the peaks at mlz M-56, M-121,and M-134 represent fragments originating from the noncar-

boxylic end of the pigment, whereas peaks at mlz M-84,M-165, and M-178 represent fragments originating from thecarboxylic end. The proposed tentative structure "bisdehy-dro-13-carotene-2-carboxylic acid" is consistent with mass

spectral and UV-visible data. Further, the proposed struc-ture is supported by NMR data.'H-NMR spectrum of P-3. The 'H-NMR spectrum of P-3

(300 MHz in CDCl3) showed olefinic protons between the 5and 7 8 regions and methyls and aliphatic protons in the

TABLE 2. Absorption characteristics" of P-3b in various solvents

Solvent X max (nm)

Methanol .......... 466, 493, 523Hexane .......... 465, 490, 522Chloroform .......... 478, 505, 538Carbon disulfide .......... 497, 525, 562

" Spectra were recorded on a Hitachi UV-visible UV-330 spectrophoto-meter. The absorption maxima given are accurate to within ± 2 nm.

b After exposure to light, samples develop a band in the UV region (around320 nm). indicative of a cis peak.

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U,)zW

I-

z

WIL.

340 380 420 460 500 540 580 - C U

m/eFIG. 3. Mass spectrum of P-3 recorded at 70 eV, indicating the fragmentation of P-3. The proposed structure of P-3 is shown above the

spectrum.

0.8 to 2.6 8 regions, respectively. The 16 olefinic hydro-gens fall in the very limited range of 1 8, making assign-ment complicated. The four sets of 18 olefinic hydrogens andtheir chemical shifts are as follows: 6.5 to 6.7 8 integratingfor six hydrogens, 6.3 to 6.45 8 for four hydrogens, 6.05 to6.28 8 for six hydrogens, and 5.35 to 5.5 8 for two hydrogens(Fig. 4).

Localization of pigment in M. roseus. The sonicated sus-

pension of M. roseus following differential centrifugationindicated the presence of the pigment in the low-speed 1,000x g pellet (which consisted of unbroken cells and lumps ofbroken or unbroken cells), in the 1,000 x g supernatant, andin the 25,000 x g pellet (which is likely to contain cell wallsand cell membranes). Trypsin treatment did not release thepigment from the 25,000 x g pellet. The pigment could beeasily extracted with methanol from the 25,000 x g pellet,and the UV-visible spectrum was identical to that of thecrude pigment extracted from intact cells and to those of P-1

to P-5. Hence, the pigment was directly extracted from theintact cells of M. roseus.

Interaction of P-3 with membrane vesicles. The binding ofP-3 to PC and DMPC vesicles was studied by using thefluorescent probes ANS and pyrene (Fig. 5). The binding ofANS to PC and DMPC was accompanied by large enhance-ments in the intensity of the fluorescence emission peak,with DMPC vesicles showing greater changes in intensitythan PC vesicles (Fig. 5A and B). Further, the emission peakof ANS was blue shifted from 515 nm to 485 and 480 nm inthe presence of PC and DMPC vesicles, respectively. Theaddition of P-3 to PC and DMPC vesicles in the presence ofANS quenched the fluorescence of ANS, and the quenchingwas observed to be dependent on the amount of pigmentadded (Fig. 5A and B). The pigment by itself did not alter thefluorescence intensity of ANS and did not shift the emissionmaximum. The emission spectra of pyrene incorporated intoPC and DMPC vesicles are shown in Fig. 5C and D. The

CHEMICAL SHIFT (ppm)

FIG. 4. 'H-NMR spectrum of the olefinic region of P-3 from M. roseus, recorded at 300 MHz in CDC13.

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VOL. 173, 1991 CAROTENOID PIGMENT OF MICROCOCCUS ROSEUS 7915

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WAVE LENGTH (mm)

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500 480 460 440 420 400 380 360 500 480 460 440 420 40

WAVELENGTH (nm) WAVELENGTH (nm)

FIG. 5. Interaction of P-3 from M. roseus with PC and DMPC vesicles. (A and B) Fluorescence emission spectra of ANS in the presenceof PC (A) and DMPC (B) vesicles. X (excitation), 370 nm; ANS concentration, 25 ,uM; lipid concentration, 150 ,uM. (A) a, free ANS; b, aplus 150 ,uM PC vesicles; c to g, successive additions of 1 ,ug of P-3 to b. (B) a, free ANS; b. a plus 150 ,uM DMPC vesicles; c to g, succes-sive additions of 1 ,ug of P-3 to b. (C and D) Fluorescence emission spectra of pyrene (4 ,uM) incorporated into PC (C) and DMPC (D) vesi-cles in the presence and absence of P-3 of M. roseus. X (excitation), 333 nm; lipid concentration, 150 ,uM. (C) a, free pyrene; b, a plus150 puM PC vesicles; c to 1, successive additions of 1 ,ug of P-3 to b. (D) a, free pyrene; b, a plus 150 puM DMPC; c to m, successive addi-tions of 1 ,ug of P-3 to b. Insets in panels C and D depict the decrease in the pyrene E/M (372 nm/470 nm) intensity ratio in the presence ofP-3.

excimer/monomer (E/M) emission intensity ratio of pyrenein DMPC was greater than in PC, indicating that DMPC ismore fluid. The pigment does not on its own decrease theE/M ratio of pyrene, but it decreased the E/M ratio in aconcentration-dependent manner when added to vesicles ofDMPC and PC (Fig. 5C and D).

DISCUSSION

Polar organic solvents such as acetone and methanol havebeen extensively used for the extraction of carotenoids frombacteria (2, 3, 14, 16, 24, 27). In the present investigation,two extractions with methanol liberated all the pigment from

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7916 JAGANNADHAM ET AL. J. BACTERIOL.

the psychrotrophic M. roseus strain. We chose this extrac-tion procedure and avoided the use of NaOH, KOH, andphenol because these agents are known to affect the stabilityof carotenoids (22). The present method of extracting pig-ment from the psychrotrophic M. roseus strain with metha-nol closely resembles the method described by Cooney et al.(3) for the mesophilic M. roseus strain but differs from that ofNelis and De Leenheer (22), who observed that only a smallfraction of the pigment of the mesophilic M. roseus strainwas extractable with methanol.HPLC has been used in the past for the purification of

carotenoids from bacteria by employing normal-phase chro-matography (7, 14, 16, 24, 27). However, since normal-phasechromatography is more variable (7) than reverse-phasechromatography, the latter method has now become morepopular (20-22) and has been used in conjunction with aphotodiode array detector. Reverse-phase chromatographywas used in the present investigation to purify the majorcarotenoid pigment from a psychrotrophic M. roseus strainfrom Antarctica (32). The carotenoids resolved into fivedistinct pigments, P-1 to P-5, and the absorption spectra ofthe pigments were simultaneously recorded with the help ofthe on-line photodiode array detector.

Earlier studies on the isolation and characterization ofcarotenoid pigments from a mesophilic M. roseus strain(ATCC 516) indicated the presence of at least nine pigments,of which six were identified as 3-hydroxy-4,4'-diketo-3-carotene (phoenicoxanthin), dihydroxy-3,4-dehydro-a-caro-tene, dihydroxy-ax-carotene, diketo-a-carotene, polyhydroxy-a-carotene, and 4,4'-diketo-,-carotene (canthaxanthin) (38).It was also observed that canthaxanthin was the majorpigment in M. roseus (3).The psychrotrophic M. roseus strain was also found to

contain a number of carotenoid pigments (at least five), butnone of them were similar to the pigments described above.All of them exhibited a fine structure in their absorptionspectra and were distinctly different from canthaxanthin. Itis difficult to understand the reasons for this total lack ofsimilarity between the carotenoids of strains of the samebacterial species that have adapted to different conditions.Our attempts to detect canthaxanthin, the major carotenoidof the mesophilic M. roseus strain, in the Antarctic strain hasbeen unsuccessful. Recently, in fact, Nelis and De Leenheer(22) also failed to find canthaxanthin in M. roseus CCM 839and reported that the major pigment exhibited a fine struc-ture, as observed by us, but they did not characterize it.Both of these earlier studies were of carotenoids of meso-philic M. roseus strains; the first group used M. roseusATCC 516, and the other used M. roseus CCM 839. How-ever, only Cooney and coworkers have characterized thecarotenoid pigments from M. roseus (3, 38). Hence, ourresults are compared only with their findings.On the basis of UV-visible, IR, mass, and 'H-NMR

spectrum data, our results indicate that P-3, which consti-tutes 73% of the total carotenoid of M. roseus, is bisdehydro-,-carotene-2-carboxylic acid. The characteristics of P-3 areas follows: (i) it is a polar carotenoid, with fine structure inits absorption spectrum, and exhibits solvent-inducedbathochromic shift; (ii) it has a polyene chain of 13 conju-gated double bonds with P-cyclic end groups; (iii) its geo-metrical configuration is all trans; (iv) it is a C41 carotenoidwith a molecular weight of 576 and with one end carboxy-lated; and (v) its 'H-NMR spectrum indicates that it hasolefinic, allylic, and aliphatic hydrogens.

All five pigments (P-i to P-5) exhibited identical peaks intheir absorption spectra at 466, 493, and 523 nm, indicating

that they all possess the same chromophore of 13 doublebonds and probably differ only in the type of functionalgroups they contain.

Carotenoids are known to act as photochemical buffersand thus protect photosynthetic and chemosynthetic organ-isms from photodynamic killing (9, 17). In M. roseus,however, carotenoids do not appear to protect the cells fromphotodynamic killing (29). In fact, cells of M. roseus inwhich carotenoid biosynthesis was stopped with dipheny-lamine showed very small amounts of carotenoids but ex-hibited greater resistance to photodynamic killing than didthe pigmented wild-type M. roseus. However, consideringthat carotenoids are generally associated with either the cellmembrane (26) or the cell wall (4, 42, 44), these pigmentsmay by their interactions alter these structures. Earlierstudies demonstrated that the M. roseus cell wall consists ofpolysaccharides (with galactosamine as the amino sugar),lipids, amino acids, and a peptidoglycan consisting of lysineand alanine (15, 32). Hence, the effect of P-3 on syntheticmembranes was studied by using the fluorescent probesANS and pyrene. The addition of P-3 to PC and DMPCvesicles in the presence of ANS quenched the fluorescenceofANS in a concentration-dependent manner. This would bepossible only if the pigment was binding to the vesicles andperturbing the membranes. ANS binding is known to dependon the nature of the membrane, with more disorderedmembranes accommodating greater amounts of ANS (19).

Binding of extraneous molecules like P-3 to membranevesicles may lead to changes in the fluidity of membraneswhich could be monitored by using pyrene. The fluorescenceemission intensity of the excimer peak of pyrene (470 nm)can be conveniently used to monitor membrane fluidity (30,41), since the formation of the excimer is related to thelateral mobility of pyrene molecules in the lipid phase (6, 40).The addition of pyrene to PC or DMPC vesicles decreasedthe E/M ratio of pyrene in a concentration-dependent man-ner. Hence, it appears that the pigment decreases the fluidityof PC and DMPC vesicles and thus stabilizes the mem-branes. Earlier studies also indicated that carotenoid-richmembranes are less fluid than carotenoid-poor membranes(12), but the physiological significance of this observation isstill unknown. Further, it is also not known whether caro-tenoids, by their ability to rigidify membranes, influence thesurvival of microorganisms at low temperatures. In fact,what is known is that when cells are shifted to temperaturesbelow their ambient temperature, they respond by increasingthe proportion of unsaturated fatty acids in the membrane(10), thereby increasing the fluidity of membranes by in-creasing the disorder in the lipid bilayer (39). Such fluiditychanges occur only at suboptimum temperatures, not attemperatures near the optimum, at which a more rigidmembrane is probably required. Hence, carotenoids, bytheir ability to rigidify membranes, may have an importantrole to play at temperatures near the optimum.

ACKNOWLEDGMENTS

We thank K. N. Ganesh, National Chemical Laboratory, Pune,India, and A. V. B. Shankaram, IICT, Hyderabad, India, for theuseful suggestions and discussions during the initial stages of thiswork. We are grateful also to M. Vairamani and G. K. ViswanadhaRao of IICT, Hyderabad, India, for their help in recording the massspectra.

REFERENCES1. Brambey, P. M., and A. Mackenzie. 1988. Regulation of caro-

tenoid biosynthesis. Curr. Top. Cell. Regul. 29:291-343.

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2. Britton, G., R. K. Singh, T. W. Goodwin, and A. Bon-Aziz. 1975.The carotenoids of Rhodomicrobiumn *'annielii (Rhodospiril-laceae) and the effect of diphenylamine on the carotenoidcomposition. Phytochemistry 14:2427-2433.

3. Cooney, J. J., H. W. Marks, and M. A. Smith. 1966. Isolationand identification of canthaxanthin from Micrococcus roseuis. J.Bacteriol. 92:342-345.

4. Eberhardt, U. 1971. The cell wall as the site of carotenoid in the'knallgas' bacterium, 12/60/X. Arch. Microbiol. 90:32-37.

5. Enzell, C. R., G. W. Francis, and S. Liaaen-Jensen. 1969. Massspectrometric studies of carotenoids. Acta Chem. Scand. 23:727-750.

6. Galla, H. J., and E. Sackmann. 1974. Lateral diffusion inhydrophobic regions of membranes: use of pyrene excimer asoptical probes. Biochim. Biophys. Acta 339:103-115.

7. Gillan, F. T., and R. B. Johns. 1983. Normal phase HPLCanalysis of microbial carotenoids and neutral lipids. J. Chro-matogr. Sci. 21:34-38.

8. Goodwin, T. W. 1976. Chemistry and biochemistry of plantpigments, vol. 1, p. 225-261. Academic Press, London.

9. Harrison, A. P., Jr. 1967. Survival of bacteria. Annu. Rev.Microbiol. 21:141-156.

10. Herbert, R. A. 1986. The ecology and physiology of psychro-philic microorganisms, p. 1-23. In R. Herbert and G. A. Codd(ed.), Microbes in extreme environments. Academic Press,London.

11. Holmes, B., R. J. Owen, and T. A. McMeekin. 1984. GenusFlavobacterium, p. 353-361. In N. R. Krieg and J. G. Holt (ed.),Bergey's manual of systematic bacteriology, vol. 1. TheWilliams & Wilkins Co., Baltimore.

12. Huang, L., and A. Haug. 1974. Regulation of membrane lipidfluidity in Acholeplasma laidlawii. Effect of carotenoid pigmentcontent. Biochim. Biophys. Acta 352:361-370.

13. Jensen, S. L., and A. Jensen. 1971. Quantitative determinationof carotenoids in photosynthetic tissues. Methods Enzymol.23:586-602.

14. Kester, A. S., and R. E. Thompson. 1984. Computer optimisednormal phase high performance liquid chromatographic separa-tion of Corynebacterium poinsettiae carotenoids. J. Chro-matogr. 310:372-378.

15. Kocur, M. 1984. Genus I. Micrococcus Cohn 1872, 151AL, p.1004-1008. In N. R. Krieg and J. G. Holt (ed.). Bergey's manualof systematic bacteriology, vol. 2. The Williams & Wilkins Co.,Baltimore.

16. Korthals, H. J., and C. L. M. Steenbergen. 1985. Separation andquantification of pigments from natural phototrophic microbialpopulations. FEMS Microbiol. Lett. 31:177-185.

17. Krinsky, N. I. 1971. Carotenoid function, p. 669-716. In 0. Isler(ed.), Carotenoids. Birkhauser Verlag, Basel, Switzerland.

18. Nakanishi, K., and P. H. Solomon. 1976. Infrared absorptionspectroscopy. Holden-Day Inc., Oakland, Calif.

19. Narayanan, R., R. Paul, and P. Balaram. 1980. Fluorescentprobe studies of mixed micelles of phospholipid and bile salts.Effect of cholesterol incorporation. Biochim. Biophys. Acta597:70-82.

20. Nelis, H. J. C. F., and A. P. De Leenheer. 1983. Isocraticnonaqueous reversed phase liquid chromatography of caro-tenoids. Anal. Chem. 55:270-275.

21. Nelis, H. J. C. F., and A. P. De Leenheer. 1988. Reversed phaseliquid chromatography of astacene. J. Chromatogr. 452:535-542.

22. Nelis, H. J. C. F., and A. P. De Leenheer. 1989. Profiling andquantitation of bacterial carotenoids by liquid chromatographyand photodiode array detection. Appl. Environ. Microbiol.55:3065-3071.

23. Nelis, H. J. C. F., and A. P. De Leenheer. 1991. Microbialsources of carotenoid pigments used in foods and feeds. J. Appl.

Bacteriol. 70:181-191.24. Palmisano, A. C., S. E. Cronin, and D. J. DesMorais. 1988.

Analysis of lipophilic pigments from a phototropic microbialmat community by high performance liquid chromatography. J.Microbiol. Methods 8:209-217.

25. Ray, M. K., S. Shivaji, N. Shyamala Rao, and P. M. Bhargava.1989. Yeast strains from the Schirmacher Oasis. Antarctica.Polar Biol. 9:305-309.

26. Salton, M. R. J., and A. F. M. Ehtisham-Ud-Din. 1965. Locali-sation of cytochromes and carotenoids in isolated bacterialmembranes. Aust. J. Exp. Biol. Med. Sci. 43:255-264.

27. Sandmann, G., W. S. Woods, and R. W. Tuveson. 1990. Identi-fication of carotenoids in Erwinia herbicola and in a transformedEscherichia coli strain. FEMS Microbiol. Lett. 71:77-82.

28. Schwartzel, E. H., and J. J. Cooney. 1970. Isolation and identi-fication of echinenone from Micrococcus roseus. J. Bacteriol.104:272-274.

29. Schwartzel, E. H., and J. J. Cooney. 1974. Isolation and char-acterisation of pigmentation mutants of Micrococcus roseus.Can. J. Microbiol. 20:1015-1021.

30. Shivaji, S. 1986. Binding of seminalplasmin to the plasma andacrosomal membranes of bovine spermatozoa. Fluorescencestudies on the changes in the lipid phase fluidity. FEBS Lett.196:255-258.

30a.Shivaji, S., et al. Unpublished data.31. Shivaji, S., N. Shyamala Rao, L. Saisree, G. S. N. Reddy, G.

Seshu Kumar, and P. M. Bhargava. 1989. Isolates of Arthrobac-ter from the soils of Schirmacher Oasis, Antarctica. Polar Biol.10:225-229.

32. Shivaji, S., N. Shyamala Rao, L. Saisree, V. Sheth, G. S. N.Reddy, and P. M. Bhargava. 1988. Isolation and identification ofMicrococcus roseus and Planococcus sp. from SchirmacherOasis, Antarctica. J. Biosci. 13:409-414.

33. Shivaji, S., N. Shyamala Rao, L. Saisree, V. Sheth, G. S. N.Reddy, and P. M. Bhargava. 1989. Isolation and identification ofPseuidomnonas spp. from Schirmacher Oasis, Antarctica. Appl.Environ. Microbiol. 55:767-770.

34. Siefirmann-Harms, D. 1987. The light harvesting and protectivefunctions of carotenoids in photosynthetic membranes. Physiol.Plant. 69:501-568.

35. Thirkell, D., and M. I. S. Hunter. 1969. Carotenoid-glycoproteinof Sarcina flai'a membrane. J. Gen. Microbiol. 58:289-292.

36. Thirkell, D., and M. I. S. Hunter. 1969. The polar carotenoidfraction from Sarcinaflava. J. Gen. Microbiol. 58:293-299.

37. Thirkell, D., and R. H. C. Strang. 1967. Analysis and compar-ison of the carotenoids of Sarcina flava and Sarcina lutea. J.Gen. Microbiol. 49:53-57.

38. Ungers, G. E., and J. J. Cooney. 1968. Isolation and character-ization of carotenoid pigments of Micrococcus roseus. J. Bac-teriol. 96:234-241.

39. Urano, S., K. Yano, and M. Matsuo. 1988. Membrane stabilisingeffect of vitamin E: effect of ox tocopherol and its modelcompounds on fluidity of lecithin liposomes. Biochem. Biophys.Res. Commun. 150:469-475.

40. Vanderkooi, J., and J. B. Callis. 1974. Pyrene. A probe of lateraldiffusion in hydrophobic region of membranes. Biochemistry13:4000-4006.

41. Vijayasarathy, S., S. Shivaji, and P. Balaram. 1982. Bull spermplasma and acrosomal membranes: fluorescence studies of lipidphase fluidity. Biochem. Biophys. Res. Commun. 108:585-591.

42. Work, E. 1964. Amino acids of walls of Micrococcus radiodu-rans. Nature (London) 201:1107-1109.

43. Work, E. 1971. Cell walls. Methods Microbiol. 5A:361-418.44. Work, E., and H. Griffiths. 1968. Morphology and chemistry of

cell walls of Micrococcus radiodurans. J. Bacteriol. 95:641-657.45. Zachmeister, L., and L. Wallcave. 1953. Action of N-bromosuc-

cinimide on ,B-carotene. J. Am. Chem. Soc. 75:4493-4495.

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