Vol. 149, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1982, p. 831-8390021-9193/82/030831-09$02.00/0

Rhodopseudomonas sphaeroides Membranes: Alterations inPhospholipid Composition in Aerobically and Phototrophically

Grown CellsJANET C. ONISHIt AND ROBERT A. NIEDERMAN*

Department of Biochemistry, Bureau of Biological Research, Rutgers University, Piscataway, New Jersey08854

Received 24 August 1981/Accepted 23 October 1981

The effects of growth conditions on phospholipid composition in Rhodopseudo-monas sphaeroides have been reexamined. The levels of phosphatidylethanol-amine (27 to 28%), phosphatidylglycerol (23 to 24%), and phosphatidylcholine (11to 18%) were very similar in cells grown aerobically or phototrophically at a highlight intensity, consistent with findings for another member of Rhodospirillaceae.In addition, an unknown phospholipid species was detected which comprised 20to 30% of the total phospholipid in these cells. In cells growing phototrophically atlow-intensity illumination, the level of phosphatidylethanolamine increased byabout 1.6-fold and that of the unknown phospholipid markedly decreased.Although the synthesis of photosynthetic pigments, light-harvesting protein, andintracytoplasmic photosynthetic membranes also increased markedly, the ratiosof individual phospholipid species were essentially identical in photosyntheticmembrane and cell wall fractions purified from these cells. Since a significantexchange of lipids apparently did not occur during the isolation of these fractions,it was suggested that the changes in cellular phospholipid accumulation were notdue to a unique composition within the photosynthetic membrane. Instead, thesephosphoglyceride changes were found to be related to overall phospholipidmetabolism and could be accounted for principally by differences in biosyntheticrates. These results, together with studies in nutrient-restricted aerobic cells,suggested that the mechanism by which phospholipid levels are regulated may berelated to radiant energy flux rather than cellular energy limitation.

The facultative photoheterotrophic bacteriumRhodopseudomonas sphaeroides has been auseful model system for the study of membranebiogenesis because alterations in oxygen tensionand incident illumination levels result in dramat-ic changes in membrane structure, function, andprotein composition (22, 27, 28). In aerobically(chemotrophically) grown cells, the cell enve-lope consists of outer and cytoplasmic mem-brane layers typical of gram-negative bacteria(5). When the oxygen tension is reduced suffi-ciently, an extensive system of intracytoplasmicmembranes is elaborated which in R. sphaer-oides appears to consist of a continuous vesicu-lar network (16, 28, 34). Upon mechanical dis-ruption, this structure gives rise to an essentiallyuniform (10, 25) population of sealed vesicles(chromatophores) which contain several majorprotein components not found in other cellularmembranes (14, 32, 40). These chromatophore-specific proteins can be accounted for largely bythe polypeptide components of pigment-protein

t Present address: Merck & Co., Inc., Rahway, NJ 07065.

complexes that function in the harvesting of lightenergy and primary photochemical events (3, 9,30). The formation of these complexes and of thechromatophore membrane is related inversely toincident light intensity.Although a role for phospholipid in the assem-

bly of pigment-protein complexes has been es-tablished recently (4, 15), it is not yet clearwhether the lipid composition of the chromato-phore membrane is also different from that of thecell envelope. Despite early reports which sug-gested that the chromatophore membrane of R.sphaeroides does not have a unique phospholip-id composition (18, 24), significantly elevatedlevels of phosphatidylglycerol have been report-ed for this organism in a more recent study (35).Furthermore, enrichment of both phosphatidyl-glycerol and phosphatidylethanolamine has beensuggested for the chromatophore membrane ofthe closely related organism R. capsulata (37).In the case of R. sphaeroides, discrepancies mayhave been related to variations in growth condi-tions, methods of lipid analysis, or the lack ofrepresentative membrane preparations.

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832 ONISHI AND NIEDERMAN

The alterations in total membrane phospholip-id ratios reported for R. capsulata (37) werecorrelated with conditions of lowered light inten-sity known to derepress (22) chromatophoremembrane formation. Although it was suggestedthat this could be a consequence of a uniquechromatophore phospholipid composition (37),the altered ratios could also reflect environmen-tal effects on overall phospholipid metabolism.In this case, the phospholipid composition ofboth the chromatophores and the cell envelopecould be essentially the same. In the presentstudy, the composition and levels of phospholip-id species have been critically assessed in mem-branes purified from R. sphaeroides grown aero-bically and under phototrophic conditions atdifferent levels of incident illumination. An un-known phospholipid species designated tenta-tively as PX was detected under some growthconditions. Alterations in cellular phospholipidcomposition at decreased light intensities ofphototrophic growth were shown also to occurin R. sphaeroides. Despite the accumulation ofconsiderable intracytoplasmic photosyntheticmembrane under these circumstances, no signif-icant differences in the ratios of phosphoglycer-ide species were detected between chromato-phore and envelope fractions isolated from suchcells. Studies of phospholipid metabolism sug-gested that differences in overall biosyntheticrates could largely account for the alterations inthe phospholipid ratios.

MATERIALS AND METHODSGrowth of organism. R. splhaeroides NCIB 8253 was

grown in the MS-S medium of Lascelles (23). Cellswere grown aerobically at 30°C in the dark in Fernbachflasks filled to 0.25 volume on a Gyrotory shaker(model G-25; New Brunswick Scientific Co., NewBrunswick, N.J.) at 350 rpm. Cells were grown photo-heterotrophically at 30°C under 95% nitrogen-5% CO,in 1-liter reagent bottles filled to capacity. Constantincandescent illumination was adjusted to provide 215,2,690, and 21,520 lx, measured at the vessel surfacewith a Weston model 750 light meter. These illumina-tion levels were found to support minimal, intermedi-ate, and maximal growth rates, respectively. Growthwas monitored from an optical density at 680 nmmeasured with a 1-cm light path on a Gilford spectro-photometer (Gilford Instrument Laboratories, Inc.,Oberlin, Ohio). Cells were harvested at optical densi-ties of 0.4 to 0.8.

Continuous culture studies at a submaximal growthrate were conducted in a Bioflo benchtop chemostat(model C-30; New Brunswick Scientific Co.) kindlyprovided by A. C. St. John. A generation time of 10 hwas obtained by adjusting of the flow rate to 20 ml/hand limiting the supply of malate and glutamate in MS-S medium to 10 and 7 mM, respectively. The 300-mlchemostat culture was inoculated with exponentiallygrowing aerobic cells and maintained at 30°C withaeration and stirring at 0.6 ml/min and 500 rpm,respectively.

Lipid analyses. Exponentially growing cells adaptedto a medium containing 1 mM phosphate were trans-ferred to fresh medium containing 32Pi (0.5 ,uCi/ml;specific radioactivity, 0.5 p.Ci/umol) and adjusted toan optical density of 0.1. Growth was continued for atleast three generations. Cells were harvested by cen-trifugation at 8,000 x g and washed twice in 0.01 MTris buffer, pH 7.2. Lipids were extracted by a modifi-cation of the method described in reference 1. Aque-ous suspensions of whole cells and membranes wereextracted at concentrations of 20 and 8 mg of proteinper ml, respectively. The final respective proportionsof methanol, chloroform, and water were 2:1:0.8 (vol/vol). Lipid extracts were separated from the precip-itated residue after centrifugation at 10,000 x g for 10min. One volume each of chloroform and 50 mMMgCl2 was added to the extract, and the chloroformphase was washed three times with 0.25 volume of0.15 M NaCl to remove unincorporated 32p,.

Lipids were separated by a two-dimensional thin-layer chromatography procedure (33). The lipids werelocated by exposing developed plates to iodine vapors.32P-labeled phospholipids were detected by radioau-tography after exposure of plates to Kodak XRP film.Radioactivity was determined by scraping appropriateareas from the chromatogram and liquid scintillationcounting (4). Phospholipid species were identified bytheir mobilities relative to those of standards (SigmaChemical Co., St. Louis, Mo.; Supelco, Bellefonte,Pa.) and reactions with molybdenum blue (13), ninhy-drin, and periodate-benzidine (8) sprays.The total fatty acid content and acyl composition of

the phosphoglycerides were determined by gas-liquidchromatography after preparation of methyl esters bymild alkaline methanolysis. Methyl esters were sepa-rated from photosynthetic pigments by thin-layerchromatography on Silica Gel G plates (Analtech Inc.,Newark, Del.) predeveloped in hexane-diethyl ether(1:1, vol/vol) and developed in hexane-diethyl ether(95:5, vol/vol). The desired bands were located withrhodamine 6G spray (0.05% [wt/vol] in 95% ethanol),and methyl esters were eluted with the predevelop-ment solvent and concentrated under nitrogen. Themethyl esters were separated on a Hewlett-Packardgas chromatograph (model 7106A; Hewlett-Packard,San Diego, Calif.) equipped with a hydrogen flameionization detector. Fatty acid composition was ex-pressed as weight percent of total fatty acids.

For detection of water-soluble deacylated products,phospholipids (1.5 to 2.0 mg) were evaporated todryness and solubilized in an ultrasonic bath with 1 mlof 0.1 N methanolic KOH. Incubation at room tem-perature was continued for 15 to 25 min, and thedeacylated products were recovered from the upperaqueous phase after the addition of 1 ml of water, 0.6ml of chloroform, and 0.3 ml of methanol. The upperphase was neutralized by stirring with 0.5 ml ofAmberlite IR-200 (H+ form) and desalted by passagethrough a 2-ml column prepared with the same resin.The deacylated products were eluted with methanol-water (9:1, vol/vol) and dried under reduced pressurewith a Savant concentrator (Savant Instruments, Inc.,Hicksville, N.Y.). The samples were dissolved inmethanol-ammonia (9:1, vol/vol) and chromato-graphed on an Eastman cellulose chromatogram (no.6065; Eastman Kodak Co., Rochester, N.Y.) with thesolvent system of reference 31. Glycerol phosphate

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PHOSPHOLIPIDS OF R. SPHAEROIDES 833

esters were detected with a phosphate ester-specificspray (20).Membrane isolation. Chromatophores and a cell

wall-enriched fraction were purified from phototrophi-cally grown cells by differential and rate-zone sedi-mentation (27). For the isolation of cytoplasmic andouter membrane fractions from aerobically growncells, French pressure cell extracts (27) in 0.01 M Tris-hydrochloride buffer (pH 7.2) were treated with 100 ,ugof lysozyme per ml for 30 min on ice, brought to 10mM EDTA, and centrifuged at 250,000 X gav for 60min in a Beckman 60 Ti rotor (Beckman Instruments,Inc., Palo Alto, Calif.). The resuspended pellet waslayered onto a gradient prepared with equal volumesof 20, 40, and 60% (wt/wt) sucrose (12) in 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-0.01 M EDTA (pH 7.5) and centrifuged at 96,000 X gavfor 15 h in a Beckman SW27 rotor. The gradient profileof succinate dehydrogenase (succinate: phenazinemethosulfate oxidoreductase [EC 1.3.99.1]) activity(32) and the polypeptide profiles in sodium dodecylsulfate-polyacrylamide gel electrophoresis (32) of thecytoplasmic and outer membrane fractions indicatedthat the separation was essentially complete.

Chemical analyses. The procedures used for determi-nation of protein, bacteriochlorophyll a (Bchl), carot-enoids, and phospholipids were those described previ-ously (4).

RESULTSCellular phospholipid composition. An im-

proved two-dimensional thin-layer chromatogra-phy procedure (33) proved to be a satisfactorymethod for the direct separation of the phospho-lipid components of R. sphaeroides. The autora-diogram in Fig. 1 shows a representative separa-tion of 32P-labeled phospholipids of aerobicallygrown cells. The phospholipids were identifiedby comparison with standard preparations andby the specific color reactions described above.Neither the single- nor the two-dimensional pro-cedures described previously (18, 35) resolvedan unknown phospholipid component (PX; seeAddendum) from phosphatidylglycerol andphosphatidylethanolamine, respectively. In ad-dition to PX, phosphatidylglycerol, phosphati-dylcholine, phosphatidylethanolamine, and car-diolipin, as well as three minor unidentifiedspots, were separated widely.Although the structure of PX has not yet been

elucidated completely, the following evidenceindicates that this component is an endogenousphospholipid. The 32p labeling characteristics(see below) and the similarity in fatty acid com-position of PX to that of the other phospholipidsisolated from R. sphaeroides (J.C. Onishi, Ph.D.thesis, Rutgers University, New Brunswick,N.J., 1980) suggest that the unknown compo-nent is a phosphoglyceride synthesized in acommon pathway. Despite the comigration ofPX with phosphatidate and lysophosphatidyleth-anolamine, the unknown is not a product ofphospholipase degradation since it stained spe-

cifically with periodate-benzidine (8), indicatingthe presence of ox-glycol groups. Furthermore,determinations of ester content as describedpreviously (38) gave an ester-to-phosphate ratioof 2.1:1 rather than the 1:1 expected of lysophos-pholipids. The unknown phospholipid does notappear to be an artifact of extraction or chroma-tography since no iodine staining spot corre-sponding to PX developed from a mixture ofphosphatidylcholine, phosphatidylglycerol,phosphatidylethanolamine, and cardiolipin stan-dards subjected to these procedures. Thus, it isunlikely that PX represents a lipid-salt complex(21). Chromatographic analyses of the organicsolvent (J.C. Onishi, Ph.D. thesis) and water-soluble products of mild alkaline hydrolysis arealso consistent with the identity of PX as aphosphoglyceride. Similar to other 0-acyl phos-phoglycerides, PX was rendered quantitativelywater soluble on the basis of 32P radioactivity.The deacylated product of PX detected with aphosphate ester spray (20) did not cochromato-graph with the glycerophosphate, glycerylphos-phorylglycerol, or diglycerylphosphorylglycerolresulting from the hydrolysis of phosphatidicacid, phosphatidylglycerol, and cardiolipin stan-dards, respectively. On the basis of all of theabove evidence, radioactivity associated withPX was included in the calculated phospholipidcompositions.

Estimates of phospholipid composition wereperformed on cells labeled uniformly with 32p;.

FIG. 1. Autoradiograms of two-dimensional thin-layer chromatographic separation (33) of 32P-labeledlipids from aerobically grown cells. PX, Unknownphospholipid; PG, phosphatidylglycerol; PC, phos-phatidylcholine; PB, phosphatidylethanolamine; CL,cardiolipin.

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834 ONISHI AND NIEDERMAN

TABLE 1. Phospholipid composition of aerobically and phototrophically grown cells

Composition'Growth condition Light intensity (Ix) Specific Bchl Bchl".-

PX PG PC PE CL

Aerobic 31 24 11 28 0.2

Aerobic chemostat'

Phototrophic

64 3.4 4.1 6.1 14

2152,690

21,520

34 7.1 27 16 43 1.814 5.4 28 16 43 3.03.1 21 23 18 27 2.6

a Micrograms of Bchl per milligram of protein.Abbreviations defined in the legend to Fig. 1. Expressed as percent 32P in lipid extract.

" Generation time increased from 2.3 to 10 h by nutrient limitation.

Lipid composition was shown in aerobic cells tobe dependent upon the growth medium. Al-though significant levels of PX were demonstrat-ed in cells grown on a medium containing 1 mMphosphate (23), no PX was detected when thecells were grown as described previously (32);instead, the phosphatidylethanolamine andphosphatidylglycerol levels were elevated by1.7- and 1.4-fold, respectively. Even though thephosphate concentration was 20-fold higher in

the latter medium, significant levels of PX were

also produced when the phosphate level was

increased to 20 mM in MS-S medium (23).Because cells could be labeled with 32Pp that wasmaintained at higher specific radioactivities,MS-S medium at the low phosphate concentra-tion was used in subsequent experiments.

In phototrophically grown R. sphateroides, thephospholipid composition was shown to be de-pendent on the incident light intensity (Table 1).The distribution of radioactivity within the phos-pholipid species of the cells maintained at thehighest illumination level most closely resem-bled the aerobically grown cells; the phosphati-dylglycerol and phosphatidylethanolamine lev-

els were essentially identical, although some

decrease in PX was observed in the former. Incontrast, upon lowering illumination levels 8- to100-fold in the phototrophically grown cells, thePX level was markedly diminished, and thatof phosphatidylethanolamine increased by 1.6-fold.

Lipid composition of purified membranes. Fora determination of whether the light-dependentquantitative phospholipid alterations were dueto the synthesis of a chromatophore membranedifferentiated in phospholipid content, repre-sentative membranes were purified (27) from thevarious cells. In general, the phospholipid com-

positions of the chromatophore and cell wall-enriched fractions were quite similar, indepen-dent of the incident illumination levels duringgrowth (Table 2). When the membranes fromphototrophically grown cells are compared, it isnoteworthy that chromatophore and cell wallfractions each comprised about 28% of the lipidphosphorus in cells grown under low-intensityillumination (1, 828 lx), with much of the remain-der present in small membrane fragments (C. W.Radcliffe, and R. A. Niederman, unpublished

TABLE 2. Phospholipid composition of purified membrane fractions

Growth 3/2 Composition"cronditio Fraction Specific Bchl" XH/2P pG sPCi PEcondition PX PG PC PE

Aerobic Cytoplasmic membrane 26 26 14 29Outer membrane 23 28 19 22

Phototrophic215 lx Chromatophore 102 0.015 6.2 26 21 36

Cell wall 8.1 0.215 4.5 27 19 40

21,520 lx Chromatophore 57 (0.061 18 30 21 23Cell wall 19 0.186 14 26 22 33

' Micrograms of Bchl per milligram of protein.h Contamination of chromatophores by cell envelope phospholipids (details in the text).Abbreviations defined in the legend to Fig. 1. Expressed as percent 32P in lipid extract.

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PHOSPHOLIPIDS OF R. SPHAEROIDES 835

data). In contrast to the suggestion that theincreased phosphatidylethanolamine levels in R.capsulata grown at a low light intensity may bedue to the formation of a chromatophore mem-

brane with an elevated content of this phospho-lipid (37), no such enrichment was observedhere in the chromatophore fraction from R.sphaeroides grown under this condition. More-over, the phospholipid composition of the cyto-plasmic and the outer membranes from aero-

bically grown cells did not differ greatly (Table2).The previous demonstration that the mem-

branes of aerobically grown cells and the non-

chromatophore membranes of phototrophiccells exhibit similar sedimentation properties(27) was used to test the possibility that signifi-cant randomization of phospholipids may haveoccurred during the membrane isolation proce-

dures. Aerobic cells were labeled to equilibriumwith [2-3H]glycerol (0.27 [.Ci/[Lmol) and mixedwith an equivalent amount of phototrophic cellslabeled with 32P, (0.25 p.Ci/pLmol). The cells werepassed through the French press, and the chro-matophore and cell wall were fractionated (27).Since only the aerobic cell envelope phospholip-ids were labeled with [2-3H]glycerol, the degreeof contamination of the isolated chromatophorefractions was estimated by the 3H/32P ratio. The3H/32P ratio of the lipid extracts (Table 2) was

consistent with the possibility that in the cellsgrown at a low light intensity, the isolated chro-matophores were contaminated with phospho-lipids of nonchromatophore origin by less than10%, whereas the figure approached 30% in thehigh-light-intensity chromatophores.

In agreement with previous reports (19, 35),the total cellular fatty acid composition (J. C.Onishi, Ph.D. thesis) did not depend upon thegrowth mode. In general, cis-vaccenic acid(C18:1 cis-A1l) comprised about 80 to 90% of thetotal fatty acids, with stearic acid (C180) ac-

counting for most of the remainder (6 to 11%),independent of both oxygen tension and lightintensity during growth. With regard to the fattyacid composition of the isolated membrane frac-tions, cis-vaccenic acid again comprised the vastmajority of fatty acyl species, but some enrich-ment in palmitic acid (C16:0) levels of outermembrane-enriched fractions was observed; thisfatty acid accounted for 4.4 and 6.1% of the totalin the aerobic and phototrophic fractions, re-

spectively, compared with 2.3% in the cytoplas-mic membrane and 3.5% in chromatophores.Palmitic acid enrichment has also been reportedin the outer membranes of Rhodospirillum ru-

brum (11, 29) and Escherichia coli (41). For thephospholipids purified from the cytoplasmic andchromatophore membranes, the composition offatty acyl moieties was essentially identical (J.

C. Onishi, Ph.D. thesis), in agreement withresults reported for the phospholipids obtainedfrom R. sphaeroides Ga cells (26). This is consis-tent with the theory that the various phospholip-id species originated from a common pool ofphosphatidic acid.

Phospholipid metabolism. Although changes inlevels of individual phospholipid species as afunction of growth conditions were found here,no major differences were observed betweentheir levels in the chromatophore and their lev-els in the other cellular membranes. This sug-gested that these changes were due to differ-ences in overall phospholipid metabolism. Therelative rates of phospholipid biosynthesis anddegradation were therefore examined in cellsgrown aerobically and under phototrophic con-ditions at a low light intensity. In Fig. 2, theincorporation of 32p, into each phospholipid spe-cies is plotted as a function of changes in cellmass in the respective exponentially growingcultures. Therefore, the various slopes representthe biosynthetic rates. After an initial lag in eachculture, the rates of phosphatidylethanolamineand phosphatidylglycerol biosyntheses were in-creased about 2.4- and 2.6-fold, respectively, inthe phototrophic cells relative to those grownaerobically. In contrast, the biosynthetic rate ofPX was decreased about 6.6-fold in the photo-trophic culture. In addition to these differencesin the relative rates of biosynthesis, the stabilityof the 32P-labeled phospholipids also differedunder the two growth conditions. For compari-sons of phospholipid stability, homogeneouslylabeled cells were shifted to unlabeled media,and the phosphoglycerides were analyzed peri-odically for about 0.5 generation during thischase. 32P, that was incorporated into each phos-pholipid species of the aerobic culture and phos-phatidylcholine and PX in the phototrophic cellsremained quite stable despite a small initialdilution (Fig. 3). In the slowly growing photo-trophic culture, however, the initial turnover ofphosphatidylethanolamine and phosphatidylgly-cerol was more marked, amounting to about 30and 40%, respectively, during the first 0.08generation. Thereafter, these phospholipids alsoremained quite stable, and the small increase intheir radioactivity during prolonged phototro-phic growth may reflect reincorporation of theisotope from degraded phospholipid.Although no detailed analysis of phospholipid

turnover was performed, the overall differencesin biosynthesis rates, rather than changes inturnover, are generally sufficient to account forthe observed alterations in the relative levels ofthe phospholipid species. The initial decrease inthe level of phosphatidylethanolamine in thephototrophic cells is most likely accounted forby phosphatidylcholine biosynthesis. The more

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836 ONISHI AND NIEDERMAN

5.0

4.0

3.0

2.0 _

1.0 _

0

C-)

3.0 - PG

2.01-

1.0 _

02.0

I.0 _

0 L -- -*,4 -

1.00 1.05 1.10 1.15 1.20 1.

energy fluxes. As an approach to this problem,the nutrient supply was limited to an aerobicculture in a chemostat such that the generationtime was increased to 10 h. This doubling timewas essentially equivalent to that of the photo-trophic cells grown at 215 lx. Although thenutritional states of both of these slowly growingcultures are not necessarily equivalent, the ener-gy supply of the aerobic cells should also bereduced significantly.

After the optical density of the chemostatculture had reached a plateau (33 h), 32p; wasadded and labeling was continued for an addi-tional 32 h. These results are also shown inTable 1. Instead of the decrease in PX and theincrease in phosphatidylethanolamine levels ob-

30 1 X X l

X AEROBIC

U

10

.25 0CELL MASS

FIG. 2. Rates of phospholipid biosynthesis in aero-bically and phototrophically grown cells. Phototrophicgrowth was at 215 lx. The increase in 32P-labeledphospholipid is plotted as a function of cell massmeasured from the optical density at 680 nm, with thatat the start of the labeling set arbitrarily at 1.0. Thegeneration times were 2.2 and 11.8 h for the aerobicand phototrophic cultures, respectively. Cells werelabeled as described in the text with 0.5 kCi of 32Pi perml of medium. No radioactivity was detected in car-diolipin in either culture, and phosphatidylcholinebecame labeled only after approximately 1.2 dou-blings. The lines through the points were calculated bylinear regression analyses. Abbreviations are definedin the legend to Fig. 1.

0 20 40 60 80

(Min)

10

8

6Q.

4

substantial turnover of phosphatidylglycerol inthe slowly growing phototrophic culture may

also be reflected in the apparent decrease in therate of 32p, incorporation into this phospholipidafter 1.1 generations (Fig. 2, dotted portion).This high turnover rate may also account for thelack of accumulation of anionic phospholipidsunder these circumstances (see below).

It had been suggested (37) that qualitativealterations in phospholipid composition in R.capsulata were due to changes in adenosinenucleotide levels. An alternative theory is thatsuch changes are related more directly to radiant

2

0

0 2 4(Hr)

6

FIG. 3. Phospholipid turnover in aerobically andphototrophically grown cells. Phototrophic growthwas at 215 lx. Cells were labeled with 0.5 1jCi of 32p;per ml of medium. Abbreviations are defined in thelegend to Fig. 1.

PE

PX ~~-." 0

\X-X -x x

PE

A--

*PC-

I

X ANAEROBIC (20 ft-c)

I

x

x x X~x P-~ E

PXI

, ., P

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PHOSPHOLIPIDS OF R. SPHAEROIDES 837

served for the cells grown phototrophically at a

low light intensity, a two-fold increase in PXlevels and a marked decrease in the other phos-pholipid species were observed. In addition,significantly elevated levels of cardiolipin were

also seen in this energy-limited culture.

DISCUSSIONThe evidence presented here has confirmed

that the phospholipid composition of R. sphaer-oides is dependent upon growth conditions. Inaccord with results obtained with R. capsulata(37), both the qualitative and the quantitativephospholipid compositions were essentiallyidentical in cells grown aerobically and photo-trophically at a high light intensity. This isconsistent with the suggestion that in Rhodospir-illaceae, differences in levels of phospholipidspecies do not result from changes in oxygentension (37), but further studies under conditionsof varying oxygen levels are necessary to estab-lish this point. Furthermore, the relation be-tween individual phosphoglyceride levels andincident light intensity during phototrophicgrowth observed for R. capsulata (37) has alsobeen generally corroborated by the present re-

sults. In addition, the improved resolution of thechromatographic procedure (33) used here haspermitted the detection of an unknown phospho-lipid species, designated PX, which accountedfor more than 30% of the total phospholipid inaerobically grown cells. In thin-layer chroma-tography, this component behaved as an acidicand amphipathic compound. It was shown thatthe content of PX was dependent upon thegrowth medium and that the levels of this com-

ponent were markedly decreased at low lightintensities in phototrophically growing cells.Under these circumstances, phosphatidyletha-nolamine levels increased about 1.6-fold com-

pared with cells grown aerobically or photo-trophically at high incident illumination levels.Although on a protein basis, the decrease in lightintensity was accompanied by a 10-fold increasein Bchl levels, the differentiation in phospholipidcomposition is apparently not due to the synthe-sis of chromatophore membrane with a uniquephospholipid composition, as had been suggest-ed for both R. sphaeroides (35) and R. capsulata(37). This was shown here for R. sphaeroides bythe similarity in phospholipid composition be-tween purified chromatophore and cell wall frac-tions in which neither significant exchange ofphospholipids nor hybrid membrane formationduring isolation apparently occurred. The recentfinding that an apparent in vivo intermembranephospholipid transfer in R. sphaeroides is notspecific for phospholipid species (6) is also inaccord with our results.Changes in biosynthetic rates for individual

phospholipid species were generally sufficient toaccount for the different cellular phospholipidcompositions. This may be the basis for in-creases in PX in the aerobic cells and phosphati-dylethanolamine in cells grown phototrophicallyat 215 lx. Although phosphatidylglycerol synthe-sis is also accelerated under the latter circum-stances, the extensive and prolonged turnovermay account for the lack of excessive accumula-tion of this phospholipid in these slowly growingphototrophic cells. Substantial phosphatidyl-glycerol turnover is also observed in E. coli andthis is thought to be related mainly to the mobili-zation of the head group for membrane-derivedoligosaccharide synthesis (36). In R. sphaer-oides grown at 2,690 lx in which the generationtime was decreased more than twofold, a 1.3-fold increase in total levels of phospholipidscontaining glycerophosphate head groups (phos-phatidylglycerol plus cardiolipin) was observedin comparison with aerobically grown cells (Ta-ble 1); increases as high as 1.5-fold have alsobeen observed (Radcliffe and Niederman, un-published data). Although the total levels ofthese phosphoglycerides do not approach thosereported previously (35), the magnitude of thedifferences between the aerobic and these pho-totrophic cells was similar. This provides somesupport for the suggestion from spin-label stud-ies (2) that negatively charged phospholipidspreferentially associate with the light-harvestingcomplex enriched in cells grown at low illumina-tion. In the present study, a 2.4-fold enrichmentin light-harvesting protein was observed in chro-matophores over a 100-fold decrease in lightintensity which is consistent with other results(39).The phospholipid composition of aerobic cells

grown in this study in a chemostat with a genera-tion time equivalent to that of cells grown at alow illumination level suggests that energy limi-tation is not the basis for the changes observedin the ratios of the phospholipid species duringphototrophic growth. Instead, the overall me-tabolism of phospholipids during phototrophicgrowth may be related by some other means tothe flux rate of quanta absorbed by the Bchl andcarotenoids of the light-harvesting apparatus.This is known to strongly influence the rates ofboth growth and membrane synthesis (17). Themechanism by which such changes in radiantenergy flux might regulate the levels of the polarphospholipid head groups within the membraneis unknown and requires further investigation.Speculations include changes in the oxidation-reduction state of the crucial regulatory elementas proposed for Bchl synthesis (27) and changesin membrane structure affecting enzyme activity(7, 15). However, the latter is not consistentwith a recent report on R. sphaeroides in which

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838 ONISHI AND NIEDERMAN

an in vivo intermembrane transfer of phospho-lipids to the chromatophore membrane was pro-

posed (6). Although this suggested that phospho-lipids are synthesized at a site distinct from theintracytoplasmic membrane, direct assays ofenzymes of phospholipid biosynthesis have re-

cently demonstrated substantial activity in puri-fied chromatophores (C. W. Radcliffe, G. M.Carman, and R. A. Niederman, unpublisheddata). Further studies on this question are in

progress here.

ACKNOWLEDGMENTS

This work was supported by National Science Foundationgrant PCM76-24142, Public Health Service grant GM 26248from the National Institute of General Medical Sciences, andthe Charles and Johanna Busch Memorial Fund. J.C.O. was

supported by the Merck Doctorate Program, and R.A.N. was

the recipient of Public Health Service Research Career Devel-opment Award GM 00093 from the National Institute ofGeneral Medical Sciences.We thank Ann C. St. John and Ronald C. Poretz for useful

suggestions, Lynn Paege for use of the gas chromatograph,and Cynthia W. Radcliffe for independent determinations ofphospholipid composition.

ADDENDUM

While this manuscript was in preparation, the pres-

ence of this apparent phospholipid species was alsoreported in R. sphaeroides by Cain et al. (6).

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