JouRNAL OF BACTERIOLOGY, Feb. 1980, p. 626-6340021-9193/80/02-0626/09$02.00/0

Vol. 141, No. 2

A Rapidly Metabolizing Pool of Phosphatidylglycerol as aPrecursor for Phosphatidylethanolamiine and Diglyceride in

Bacillus megateriumFRANK J. LOMBARDI, SYBIL L. CHEN, AND ARMAND J. FULCO*

Department ofBiological Chemistry, University of California at Los Angeles Medical School, andLaboratory of Nuclear Medicine and Radiation Biology, Los Angeles, California 90024

Pulse-chase experiments in Bacillus megaterium ATCC 14581 with [U-14C]-palnitate, L-[U-_4C]serine, and [U-'4C]glycerol showed that a large pool of phos-phatidylglycerol (PG) which exhibited rapid turnover in the phosphate moiety(PGJ) underwent very rapid interconversion with the large diglyceride (DG) pool.Kinetics of DG labeling indicated that the fatty acyl and diacylated glycerolmoieties of PGt were also utilized as precursors for net DG formation. The [U-'4C]glycerol pulse-chase results also confirmed the presence of a second, meta-bolically stable pool of PG (PG.), which was deduced from [32P]phosphate studies.The other major phospholipid, phosphatidylethanolamine (PE), exhibited pro-nounced lags relative to PG and DG in "C-fatty acid, [14C]glycerol, and [P]-phosphate incorporation, but not for incorporation of L-[U-'4C]serine into theethanolamine group of PE or into the serine moiety of the small phosphatidyl-serine (PS) pool. Furthermore, initial rates of L-[U-'4C]serine incorporation intothe serine and ethanolamine moieties ofPS and PE were unaffected by cerulenin.The results provided compelling in vivo evidence that de novo PGt, PS, and PEsyntheses in this organism proceed for the most part sequentially in the orderPGt -+ PS -+ PE rather than via branching pathways from a common intermediateand that the phosphatidyl moiety in PS and PE is derived largely from thecorresponding moiety in PGt, whereas the DG pool indirectly provides an addi-tional source for this conversion by way of the facile PGt ± DG interconversion.

[32P]phosphate incorporation and chase ex-periments have shown that the predominantmembrane lipid of Bacillus megaterium ATCC14581, phosphatidylglycerol (PG), which com-prises 70% of the total phospholipids, is presentin two distinct pools(18). One pool (PG8; 27% oftotal phospholipids) is metabolically stable,whereas the second pool (PGt; 43% of total phos-pholipids) undergoes a rapid turnover in thephosphate moiety. The phosphate group in theother major phospholipid, phosphatidylethanol-amine (PE; 26 to 27% of total phospholipids) isstable to turnover. Analyses of [2-3H]glycerol-and [ U-14C]glycerol-labeled cultures demon-strated a large pool of diglycerides (DG), whichis comparable in size to the PE pool and whichcontains the 1,2- and 1,3-DG isomers in a ratioof about 1:2 (18). In addition, [32P]phosphate-equilibrated cultures revealed low levels (1 to2%) of phosphatidylserine (PS), traces (0.2 to0.4%) oflysophosphatidylglycerol (lyso-PG), andless than 0.5% cardiolipin. It was shown in thesestudies that [3P]phosphate incorporation intoPE lagged well behind incorporation into PG.Furthermore, incorporation of 3P into PE con-tinued for a limited period after the addition of

cerulenin, an inhibitor of fatty acid synthesis,reflecting continued PE accumulation at the ex-pense of the DG and PG pools. However, ceru-lenin had no significant effect on turnover of thephosphate moiety in the PGt pool and did notinhibit initial rates of [32P]phosphate incorpo-ration into PG (18).

In earlier work with this organisim it was foundthat when ['4C]palmitate was added to culturesat 200C after cerulenin addition, rapid and tran-sient accumulation of 1-[`4C]acyl-lyso-PG wasobserved (4, 5), and this in turn was followed byits gradual conversion to 1-["4C]acyl-PG viaacylation with endogenous acyl groups. If[I4C]palmitate was added before cerulenin, how-ever, no [I4C]lyso-PG was observed, and allphospholipid radioactivity was recovered as 1-[I4C]acyl-PG at the earliest times tested (5).Furthermore, basal levels of [32P]lyso-PG in[3P]phosphate-labeled cultures have beenshown to expand rapidly after cerulenin additionat 200C (18).

In this report we present results on pulse-chase experiments carried out with [U-14C]pal-mitate, L-[U-'4C]serine, and [U- 4C]glycerol; thiscomplements the [32P]phosphate data in the ac-

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GLYCEROLIPID BIOSYNTHESIS IN B. MEGATERIUM 627

companying paper (18), and the results in totoare considered from the standpoint of patternsof in vivo glycerolipid metabolism in B. mega-terium.

MATERIALS AND METHODSMaterials. [ U-'4C]palmitic acid (900,uCi/,umol) was

purchased from Amersham/Searle Corp. L-[U-'4C]ser-ine (150 ILCi/umol), [U-'4C]glycerol (100 ,uCi/,umol),and [2-3H]glycerol (250,tCi/,umol) were obtained fromNew England Nuclear Corp. Other materials wereobtained as noted previously (18).

Incubations and lipid separation. Bacteria weregrown and incubated in either medium S-M56-LP orGCN medium as described previously (18). Lipids wereextracted and separated into PS, PG, PE, and solventfront Lipids by silica gel thin-layer chromatography inCHCl3-CH3OH-H20 (65:25:4, vol/vol). The solventfront region was scraped, and the solvent front Lipidswere extracted and rechromatographed in benzene-ether-ethyl acetate-acetic acid (80:10:10:0.2, vol/vol)to obtain the purified DG and free fatty acid fractions(18). Lyso-PG was extracted and then purified by silicagel thin-layer chromatography in CHC13-CH30H-CH3COOH-H20 (65:25:8:4, vol/vol), as described pre-viously (18).

Fatty acid hydrolysis and analysis. For deter-mination of radioactivity in the fatty acyl moieties ofthe various lipids, the separated PG, PE, PS, and DGwere extracted from silica gel with CHC13-CH3OH-H20 (10:5:1, vol/vol), a portion from each sample wascounted, and a second portion was evaporated to dry-ness under N2. A 1-ml amount of 10% KOH in CH30H-H20 (1:1, vol/vol) was added, and the samples wereincubated at 700C overnight. After acidification with0.5 ml of 6 N HCl, the fatty acids were removed bytwo extractions with 4 ml of n-pentane, which wasfollowed by two extractions with 3 ml of CHC13. Theextracts were combined and evaporated under N2, andthe radioactivity of the residue fatty acids was meas-ured. Radioactivity in the water-soluble hydrolysisproducts was determined by counting the extractedaqueous phase in Aquasol-2 (New England NuclearCorp.)

Acetolysis ofphospholipids. The method ofRen-konen (23) was modified for quantitation ofradioactivelipid moieties. Duplicate samples of purified PG andPE were measured, and the disintegrations per minutepresent in the fatty acid moieties and in the glycerolmoieties (PG) or glycerol plus ethanolamine moieties(PE) were determined in one sample of each pair bythe KOH hydrolysis procedure described above. Non-radioactive carrier PG or PE was then added to thesecond sample of each pair. In the case of [U-'4C]-glycerol- or [2-3H]glycerol-labeled lipids, in which thefatty acids were devoid of radioactivity, the secondsample was also supplemented with a measuredamount of ['4C]PG or ['4C]PE which had been isolatedfrom a culture ofB. megaterium grown in the presenceof [U-'4C]palmitic acid and in which all radioactivitywas present in the fatty acid moieties, as shown by theKOH hydrolysis procedure. The samples were evapo-rated to dryness under N2, 20 1I of triethylamine wasadded to each of the PE-containing samples, and 0.4

ml of acetic anhydride was then added to all samples.After preliminary incubation at 700C for 30 min, 0.6ml of glacial acetic acid was added, and the sampleswere incubated for 4 h at 1450C. After cooling, 1 ml ofwater was added to each sample, and the crude di-fatty acyl-monoacetylglycerols were removed by threeextractions with 4-ml volumes of n-pentane. The com-bined extracts were evaporated, nonradioactive lipidstandards were added, and the lipid samples wereapplied in small volumes to silica gel thin-layer plates.The plates were developed for 70 min in benzene-ether-ethyl acetate-acetic acid (80:10:10:0.2, vol/vol).After radioscanning, the purified di-fatty acyl-mon-oacetylglycerols (migrating between 1,3-dipalmitinand triplamitin) were extracted with CHCl3-CH30H(2:1, vol/vol), and the disintegrations per minute pres-ent in the fatty acid moieties and in the glycerolmoiety were determined by the KOH hydrolysis pro-cedure described above. The results were converted todisintegrations per minute present in the fatty acidand acylated glycerol moieties of PG and PE by cor-recting for the somewhat variable yield of diglycerideacetate via the observed percent recovery in fatty aciddisintegrations per minute. Radioactivities in the non-acylated glycerol moiety of PG and in the ethanol-amine moiety of PE were calculated by subtractingthe combined fatty acid and acylated glycerol radioac-tivities from the total disintegrations per minute foreach sample.

RESULTSIncorporation of [U-1'Cjpalmitate. When

a culture ofB. megaterium ATCC 14581 growingat 20°C was pulsed with [ U-_4C]palmitate, radio-activity was rapidly incorporated into both PGand DG within 5 to 10 min and then graduallydeclined in both components (Fig. 1A). As shownpreviously (4, 5), radioactivity incorporated intoPG under these conditions is present exclusivelyin the 1-acyl position. In the presence of cerule-nin (Fig. 1B), initial rates of incorporation intoPG and DG were greatly reduced, and accumu-lation of radioactivity in these lipids was pre-ceded by transient accumulation of 1-[ U_'4C]pal-mitoyl-lyso-PG. This was followed by conversionof the latter to 1-[14C]acyl-PG (4) (via acylationwith endogenous acyl groups) and to [14C]DG.At early times in the cerulenin-treated culture,rapidly disappearing "4C-free fatty acid derivedfrom the [U-_4C]palmitate pulse was also ob-served (Fig. 1B). This was in accord with earlierwork (11) which showed that a fraction of addedtracer pahlnitate was rapidly incorporated intocomplex lipids of B. megaterium and was rela-tively stable thereafter, whereas the remainderwas broken down by fl-oxidation within 5 min.Moreover, the radioactivity present at latertimes in the lipids of ['4C]palmitate-pulsed cul-tures was nearly quantitatively recovered, afterhydrolysis, in palmitate itself or, at 200C, in itsdesaturation product cis-5-hexadecenoate (12).

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628 LOMBARDI, CHEN, AND FULCO

so-1070-

60

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. Wa .. ,l,v,s,o.,-.n,PE\FFA LYSO-PON~~~~~~S 90 120 1~~~~~~~~~~~~~..... ..0

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FIG. 1. Time course of [U-'4C]palmitate incorpo-ration into lipids of B. megaterium ATCC 14581 inthe presence and absence of cerulenin at 20'C. Aculture growing in medium S-M56-LP at a cell den-sity of200 Klett units was divided into two portions;cerulenin (20 pg/ml) was added to one portion (B),and [U-'4CJpalmitate (0025 nmol/ml; 900,Ci/mol)was then added to both portions (zero time). Incuba-tion was continued at 20"C, and 5-ml samples were

withdrawn at intervals for "4C-lipid analysis. (A)Control culture. Symbols: 0, PG; A, PE; 0l DG. (B)Cerulenin-treated culture. Symbols: 0, PG; A, PE;O, DG; V, lyso-PG; *, free fatty acid (FFA).

The incorporation of [U-_4C]palmitate intoDG observed in Fig. 1 lagged slightly but signif-icantly behind the incorporation into PG in bothcontrol and cerulenin-treated cultures. In con-

trast, incorporation into PE exhibited a pro-nounced lag (Fig. 1) and then continued to in-crease for over 2 h, concomitant with a gradualdecline in [14C]PG and [14C]DG. However, thedecrease in radioactivity in PG plus DG pluslyso-PG was roughly two- to threefold greaterthan the increase in [14C]PE and thus repre-sented a slow loss of total lipid radioactivity,presumably via gradual turnover of fatty acidsin PG and DG. Finally, the extent of 14C incor-poration into PG and DG, but not into PE, wasinhibited approximately 50% by cerulenin (Fig.1).Incorporation studies with L_U-m4Clser-

ine. Figure 2A shows that L-serine was rapidlyand specifically incorporated into the serine andethanolamine moieties of PS and PE, respec-

TIME (MIN.)FIG. 2. Incorporation of radioactivity from L -[U-

"4C]serine into water-soluble KOH hydrolysis prod-ucts (A) and fatty acids (B) of B. megaterium lipidsat 35°C. A 120-mi culture growing at a ceU density of50 Klett units in GCN medium was divided into twoportions; cerulenin (20 pg/ml) was added to one por-tion, and L -[U-14C]serine (0.33 pmol; 150 jXCi/umoi)was then added to both portions (zero time). Incuba-tion was continued at 35°C. and 5-mi samples wereremoved at intervals for "C-lipid separation andKOH hydrolysis. (A) Symbols: glycerol moieties ofPGin the presence (0) and absence (0) of cerulenin;glycerol plus ethanolamine moieties of PE in thepresence (A) and absence (A) of cerulenin; glycerolplus serine moieties of PS in the presence (U) andabsence (0) of cerulenin; glycerol moiety of DG ineither the presence or absence (*) of cerulenin. (B)Symbols: radioactivity in the fatty acid moieties ofPG (0), PE (A), and DG (O) in the absence ofcerulenin.

tively. Cultures were pulsed with L-[ U-4C]serlineat 350C (the tracer was exhausted after about 20min [Fig. 2]); samples were taken at intervals,and the lipids were purified and hydrolyzed withKOH (10). The radioactivity in the water-solu-ble hydrolysis products is shown in Fig. 2A, andthat in the fatty acid moieties is shown in Fig.2B. Label appeared with little or no delay in thenon-fatty acid portions of PS and PE, whereasonly low levels of radioactivity slowly accumu-lated in the glycerol moieties of PG and none

accumulated in the glycerol of DG (Fig. 2A).Acetolysis of the labeled PS and PE demon-strated that more than 90% of the radioactivityin the water-soluble hydrolysis products fromthese lipids resided in the serine and ethanol-amine portions, respectively. As Fig. 2A shows,the level of radioactivity in PS reached a maxi-mum before that of PE did and thereafter de-clined, whereas PE radioactivity remained high.These results are consistent with the notion that

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GLYCEROLIPID BIOSYNTHESIS IN B. MEGATERIUM 629

PS is a precursor ofPE in B. megaterium. More-over, blockage of de novo fatty acid synthesis bycerulenin had no effect on L-[14C]serine incor-poration into the serine and ethanolamine por-tions of these lipids.As Fig. 2B shows, radioactivity from L-[U-

"4C]serine was incorporated into the fatty acidsof PE, PG, and DG, presumably by way ofintermediate conversion to ['4C]acetyl coenzymeA. This incorporation exhibited properties sim-ilar to those of exogenous fatty acid incorpora-tion (Fig. 1). Thus, there was a rapid and exten-sive incorporation into PG, followed almost im-mediately by a rapid appearance of '4C in DG.This in turn was followed by a delayed, gradualappearance of label in PE, accompanied at latertimes by a slow decline in PG radioactivity (Fig.2B).

Pulse-chase studies with [U-14C]glycerol.A culture growing at 35°C was pulsed with [U-'4C]glycerol for 30 min and then chased withunlabeled glycerol (Fig. 3, curves 1, 3, 5, 7, and9). An identical culture was treated with ceru-lenin at 3 min before initiation of the pulse-chaseexperiment (Fig. 3, curves 2, 4, 6, and 8). Radio-activity was determined in the nonacylated glyc-erol moiety of PG (curves 1 and 2), the diac-ylated glycerol moiety of PG (curves 3 and 4),and the glycerol moieties ofDG (curves 5 and 6)and PE (curves 7 and 8) by a combination ofKOH hydrolysis and acetolysis. All lipid radio-activity isolated from cells under these condi-tions was recovered in the glycerol moieties ofthe purified lipids, and none was detected infatty acid or ethanolamine moieties, suggestingthat exogenous glycerol, unlike L-serine (Fig. 2),

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FymG.l3. Pulse-caseiit laeingth kintcslte ofyceB. moegteiumo lPid(using [Uand4C]lceo inthures presnce6)andP

moeyoG(curves 1and 2);radioactivity in the diacylated glycerolmoietyeofPGplsDpusP(curves39n ). (B)n

symbols represent control cultures, and closed symbols represent cerulenin-treated cultures.

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630 LOMBARDI, CHEN, AND FULCO

may not be oxidized appreciably under theseconditions. Moreover, although radioactivityfrom [U-14C]glycerol was incorporated into thelipid-free cell residue (data not shown) to anextent comparable to that in the total lipids,levels of 14C in the nonlipid fraction were stableduring a [j2C]glycerol chase, thus precluding thisfraction as a potential source of [UC]glycerolresidues for subsequent incorporation into lipid.

[U-'4C]glycerol incorporation into the nonac-ylated glycerol moiety of PG was approximatelyfour times more rapid than incorporation intothe diacylated glycerol of PG in the controlculture during the initial 10 to 15 min (Fig. 3A)and was about twice as fast as phosphate incor-poration into PG under comparable conditions(18). Moreover, like phosphate incorporation,initial rates of [U- 4C]glycerol incorporation intothe nonacylated glycerol ofPG were not affectedby cerulenin, although the level of '4C accumu-lation in this moiety eventually fell below thatin the control culture due to continued growthand pool expansion in the latter. Finally, like[32P]phosphate-labeled PG (18), the major por-tion of 14C in the nonacylated glycerol of PG wasrapidly lost during the chase, but radioactivityin this moiety soon stabilized at a plateau levelin both control and cerulenin-treated cultures(Fig. 3A), thus indicating that the nonacylatedglycerol moiety in PG., like the phosphate group,is metabolically stable.The two glycerol moieties of PG showed in-

teresting differences in their kinetics of [U-"C]glycerol labeling in the control culture (Fig.3A). Thus, the diacylated glycerol moiety ex-hibited a slight lag in "C uptake during theinitial 5 min (Fig. 3A, curve 3), whereas thenonacylated glycerol showed no lag (curve 1).This difference was more apparent in the chasephase of the experiment, where ''C in the non-acylated glycerol dropped precipitously duringthe first 5 min of the chase (curve 1), whereasthat in the diacylated glycerol continued to in-crease for the first 5 min (curve 3) before begin-ning its downward course. Similar 5-min lagshave been observed with PG in [tP]phosphateincorporation and chase experiments (18).

Figure 3B suggests that PE is derived largelyfrom PG plus DG pools in growing cultures ofB. megaterium. As noted above, there was arapid incorporation of radioactivity into the di-acylated glycerol moiety of PG during thepulse period (Fig. 3A, curve 3), which was fol-lowed by a decline in label in this group duringthe chase. Moreover, incorporation into DG oc-curred with a similar time course but at some-what less than one-half the rate (curve 5) andalso exhibited a pronounced decline during the

chase. In contrast, incorporation of label into PE(curve 7) again showed a marked lag during thepulse phase but continued to occur throughoutthe chase period. Indeed, the bulk of the [U-''C]glycerol incorporation into PE occurred dur-ing the chase phase and paralleled the decline inthe diacylated glycerol ofPG and DG beyond 35to 40 min. As observed also with [3P]phosphateincorporation (18), [''C]glycerol incorporationinto PE was concave upward throughout thepulse period, and the [''C]PE levels measuredduring the initial 10 min of incorporation wereless than 5% of those in the diacylated glycerolmoiety of PG.

It is of interest that the time course of DGlabeling with [U-"4C]glycerol (curve 5) laggedslightly behind that of the diacylated glycerolmoiety in PG (curve 3), as was also observedwith the fatty acid moieties labeled either ex-ogenously (Fig. 1) or endogenously (Fig. 2B).As shown previously (4, 18), cerulenin pro-

duces rapid and virtually complete inhibition ofnet lipid synthesis in B. megaterium under theconditions of the experiment shown in Fig. 3.Strikingly, [U-14C]glycerol incorporation ratesinto the diacylated glycerol moieties of PG, DG,and PE (Fig. 3, curves 4, 6, and 8) proceeded inthe cerulenin-treated culture at about one-fourth their rates in the control. That this incor-poration represented turnover of the diacylatedglycerol moiety in the lipids was confirmed bythe observation that total 14C in the diacylatedglycerol moieties of PG plus DG plus PE in thecontrol culture (curve 9) declined during thechase with unlabeled glycerol, thus demonstrat-ing turnover of the diacylated glycerol moiety inthe absence of cerulenin as well. Calculationsindicated that the rate of this turnover could bestimulated about twofold in the presence of ce-rulenin. Finally, during the chase phase in thecerulenin-treated culture, 14C was lost slowlyfrom the diacylated glycerol of PG (curve 4),whereas it gradually increased in PE (curve 8).

Pulse-chase experiments carried out with [2-3H]glycerol gave results similar to those in Fig.3. Moreover, all lipid radioactivity isolated from[2-3H]glycerol-pulsed cells was recoverable inthe glycerol moieties of the purified lipids, andnone was found in the fatty acids or other moie-ties.

DISCUSSIONThe results reported in this and the accom-

panying paper demonstrate a novel metabolicpattern of in vivo glycerolipid biosynthesis andmetabolism in B. megaterium. For purposes ofcomparison, the present results may be consid-ered from the viewpoint of known reactions of

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GLVCEROLIPID BIOSYNTHESIS IN B. MEGATERIUM 631

bacterial lipid metabolism, which have been es-tablished primarily in Escherichia coli (Fig. 4A).As Fig. 4 shows, the major lipids in E. coli, PEand PG, are formed in branching biosyntheticpathways from a common internediate, CDP-diglyceride (Fig. 4A, reactions 3 through 6). Do-nation of glycerophosphate units from PG toacceptors leads to DG formation either directly(reaction 10) or possibly via intermediate for-mation of cardiolipin (reaction 8) and subse-quent transfer (step 11). The DG produced isthen coverted in E. coli to phosphatidic acid bydiglyceride kinase (reaction 7) (21, 22). Further-more, phosphatidic acid is converted to CDP-diglyceride (reaction 2) (16) and then back toPG, thus completing the cycle. Fig. 4A alsoincludes the possibility of a phospholipase D-like hydrolysis of PG (reaction 9), which is con-sidered in the analysis below.

In Fig. 4B, a working hypothesis for glycero-lipid biosynthesis and metabolism in B. mega-terium is presented, which rationalizes all of thein vivo results obtained to date with this orga-nism (4, 5, 18). In this scheme, PG. and PGt

'%LYCERO-3- FATTY ACIDPHOSPHATE SYNTHESIS

(CERULENIN)

PHOSPHATIDATE

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GLYCEROL;: 3P

DG . - L PG-PHOSPHATEG-P-R PS

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2:PG PE

represent the stable and rapidly turning overpools of PG, respectively, and the numbers be-neath PG., PGt, PE, PS, lyso-PG, and DG denotetheir mole fractions (18). The dashed arrows inFig. 4B represent possible multistep pathwayshaving indeterminate numbers of intermediates.The scheme shown in Fig. 4B not only accountsfor the in vivo properties of glycerolipid metab-olism in B. megaterium, but also possesses sev-eral novel features, as indicated below.

(i) Distinct pools of PG. and PGt. The["4C]glycerol pulse-chase experiment involvingthe nonacylated glycerol moiety of PG (Fig. 3A)confirmed the presence of distinct PG. and PGtpools, which was deduced independently from[32P]phosphate studies (18). In addition, our re-sults show that the metabolic stability of PG.includes the nonacylated glycerol moiety as wellas the phosphate group. In contrast, a compari-son of the ['4C]glycerol results in Fig. 3A with[32P]PG incorporation and chase data (18) indi-cates that the nonacylated glycerol moiety inPGt turns over at approximately twice the rateof the phosphate moiety under the conditions

FA-CoA. I CH2OH 0L20or

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FIG. 4. Pathways ofglycerolipid synthesis and metabolism established in E. coli (A) and postulated for B.megaterium ATCC 14581 (B). In (A) solid arrows represent known reactions in E. coli catalyzed by thefollowing enzymes: 1, fatty acyl-sn-glycerol-3-phosphate acyltransferase (6); 2, CTP:phosphatidic acid cyti-dylyltransferase (16); 3, phosphatidylglycerophosphate synthetase (13); 4, phosphatidylglycerophosphate phos-phatase (3); 5, phosphatidylserine synthetase (17, 20); 6, phosphatidylserine decarboxylase (7); 7, diglyceridekinase (21, 22); 8, cardiolipin synthetase (14, 24). Dashed arrows in (A) denote interconversions whosemechanism or significance for E. coli are uncertain or hypothetical. In (B) a working hypothesis based on thepresent studies is presented for the in vivo pattern ofglycerolipid metabolism in B. megaterium ATCC 14581,in which PG. and PGt represent the stable and rapidly turning over pools of PG and the numbers givenbeneath PG., PGt, PE, PS, DG, and Iyso-PG denote their mole fractions (18). Dashed arrows in (B) representpossible multistep pathways involving an unknown number of intermediates; X and Y denote hypotheticalintermediate carriers of the glycerophosphoryl andphosphatidyl moieties ofPGt, respectively. In both (A) and(B) R represents an acceptor ofglycerophosphoryl moieties. FA-CoA, Fatty acid coenzyme A; FA-ACP, fattyacid acyl carrier protein.

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632 LOMBARDI, CHEN, AND FULCO

described in the legend to Fig. 3. Thus, a portionof the turnover and/or exchange in the nonac-ylated glycerol of PGt involves a process distinctfrom that associated with turnover of the phos-phate moiety and might reflect reversible trans-fer of the phosphatidyl moiety of PGt to a hy-pothetical intermediate carrier (represented byY in Fig. 4B), which is concomitant with arelease of free glycerol. A similar exchange withmedium glycerol is observed in E. coli; the non-acylated glycerol moiety in some of the PGmolecules of this organism has been shown toundergo rapid exchange with free glycerol (1). InB. megaterium, these observations may also berelated to the finding that the chase of thediacylated glycerol moiety in PG exhibited a 5-min lag, whereas that ofthe nonacylated glycerolshowed no discernible lag (Fig. 3A).

(ii) Facile PGt + DG interconversion. Fig-ures 1 through 3 indicate that the PGt poolundergoes very rapid interconversion with thelarge DG pool present in this organism (Fig. 4B).That the PGt and DG pools are in near-equilib-rium at both 20 and 350C is evidenced by thefinding that curves for PG and DG in pulse-chase experiments, except for a difference inscale, exhibited nearly identical time coursesboth in the diacylated glycerol moiety (Fig. 3B)and in the fatty acid moieties derived exoge-nously (Fig. 1) or via endogenous de novo syn-thesis (Fig. 2B). The scale difference favoringincorporation into PG over DG by approxi-mately 2:1 for the diacylated glycerol moiety(Fig. 3B) and endogenously synthesized fattyacyl groups (Fig. 2B) apparently reflects therelative sizes of the PGt and DG pools in thisstrain (18). On the other hand, exogenous['4C]palmitate was incorporated to approxi-mately the same extent in PG and DG in theexperiment shown in Fig. 1, suggesting that ex-ogenous and endogenous fatty acids may not beequivalent with regard to incorporation intocomplex lipids.The curves obtained for PG and DG in pulse-

chase experiments involving exogenously de-rived fatty acyl groups (Fig. 1), endogenouslyderived fatty acyl groups (Fig. 2B), and thediacylated glycerol moiety (Fig. 3B) show thatthe DG label lagged slightly but consistentlybehind [14C]PG in both pulse and chase phases.This suggests that the PGt pool is a precursorfor net DG formation (Fig. 4B).On the basis of the short lags observed for DG

relative to PG as compared with the long lagsnoted for PE labeling (Fig. 1 through 3) (18) andin view of the relatively large size of the PGtpool, it is apparent that the rate of the PGt-DGinterconversion must be many times faster than

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the rate of phosphate turnover in the PGt pooland is even considerably more rapid than therate of turnover and/or exchange in the nonac-ylated glycerol moiety of PGt (Fig. 3A). On theother hand, the PGt-DG interconversion is pre-sumably related to turnover of the phosphatemoiety in the PGt pool. These observations maybe reconciled if it is assumed that the glycero-phosphoryl moiety of PGt undergoes extremelyrapid reversible transfer to a hypothetical inter-mediate carrier (represented by X in Fig. 4B)with a release of DG, whereas exchange of thephosphate group ofPGt with medium phosphaterequires some additional slower step(s). The gly-cerophosphoryl moiety derived from PGt mightthen be transferred to acceptors (represented byR in Fig. 4B), such as nascent teichoic andlipoteichoic acids, as has been demonstrated inStreptococcus sanguis (8, 9). A similar transferof glycerophosphate units from PG to mem-brane-derived oligosaccharides in E. coli (26)has been shown to lead to the production of 1,2-DG (22).

(iii) PGt -.- PS -- PE. The pulse-chase datain Fig. 3B demonstrate that the bulk of the PEformed in growing cultures of B. megaterium isderived from the combined pools ofPG plus DG.Moreover, the combined pulse-chase results inFig. 1 through 3 together with [3P]phosphateincorporation data (18) provide compelling evi-dence that de novo PGt and PE syntheses pro-ceed sequentially in the order PGt -- PS -* PE,rather than via branching pathways from a com-mon intermediate (Fig. 4A), and that the phos-phatidyl moiety of PE is derived largely fromthe corresponding moiety in PGt, with the DGpool providing an indirect source of additionalDG moieties by way of the PGt-DG interconver-sion (Fig. 4B). The evidence for this conclusionis as follows.

(a) The long lags observed for incorporationof [32P]phosphate (18), exogenous 14C-fatty acid(Fig. 1), endogenous 14C-fatty acid (Fig. 2B), andacylated [U14C]glycerol moieties into PE ascompared with PG or DG are explained by di-lution of precursors in the large PGt plus DGpools. Indeed, the lag observed in [ P]phos-phate incorporation into PE at 35°C coincidedwith an initial rapid phase of incorporation intoPG and was followed by accelerated incorpora-tion into PE, concomitant with a somewhat low-ered rate into PG (18). Furthermore, the smallPS pool cannot contribute appreciably to thelong lags observed in the above studies, as shownby the insignificant lag in L-[U- 40]serine incor-poration into the ethanolamine moiety of PE(Fig. 2A).

(b) Rates of [3P]phosphate incorporation into

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GLYCEROLIPID BIOSYNTHESIS IN B. MEGATERIUM 633

PE (18) and rates of L-[U-'4C]serine incorpora-tion into the serine and ethanolamine moietiesof PS and PE (Fig. 2A) were unchanged for aperiod of 20 to 30 min after the addition ofcerulenin at a concentration sufficient to blockfatty acid (4) and PG syntheses (18) rapidly andcompletely. Since PE once formed is metabol-ically stable in all moieties (Fig. 1 through 3)(18), the continued incorporation into PE ob-served in the presence of cerulenin representednet PE accumulation at the expense of PGt andDG pools, as shown previously (18).

(c) Initial rates of [3P]phosphate and [U-"4C]glycerol incorporation into PE were less than3% (18) and less than 5% (Fig. 3B), respectively,of the corresponding rates of incorporation intoPG. These results are in accord with Fig. 4B, butare clearly incompatible with the scheme in Fig.4A, even assuming rapid PG turnover via DGand/or hypothetical phospholipase D-like hy-drolysis (reaction 9).

(d) The PG. pool is excluded as possible pre-cursor for PE formation due to its metabolicstability.

(e) The DG pool cannot serve as a directsource for PE fornation since this would pre-clude a lag in [3P]phosphate incorporation intoPE.

(f) Pulse-chase kinetics of PS labeling withL-[U-_4C]serine were consistent with the as-sumed role of PS as immediate precursor to PE(Fig. 4B).

(iv) PGt - - PG.. Figure 3A suggests thatPG., like PE, is derived from the combined PGtplus DG pools (Fig. 4B). As Fig. 3 shows, incor-poration of [U-14C]glycerol into the nonacylatedglycerol of PG. continued in the cerulenin-treated culture. Since PG., like PE, is stableunder these conditions (18), the continued in-corporation into PG. represented net accumu-lation at the expense of PGt plus DG pools.According to this conclusion, radioactive lipidprecursors which are incorporated initially intoPGt and DG pools during a brief pulse shouldeventually be displaced nearly completely intothe stable PE and PG. pools after a prolongedchase. This is of interest in view of earlier obser-vations in Bacillus lichenifornis, which indi-cated that, although [14C]palmitate was initiallyincorporated approximately equally intoDG andphospholipid, continued incubation of the cul-ture at 35°C led to a gradual decline in DGradioactivity and a corresponding increase inphospholipid radioactivity. Eventually, almostall of the radioactivity accumulated in phospho-lipid and was about evenly distributed betweenPG and PE (2, 19).

(v) [32P]phosphate + DG --- [2PJPGt.

According to the scheme in Fig. 4A, ceruleninwould be expected to inhibit the initial rate of[3P]phosphate incorporation into PG by at least50%. As shown previously, however, ceruleninhad no effect on initial rates of [32P]phosphateincorporation (18). This suggests that, insofar asthe phosphate moiety is concerned, PGt is de-rived from a lipid precursor lacking phosphorus(i.e., DG) (Fig. 4B).

(vi) [2P]PGt ---+ [32P]lyso-PG. [32P]phos-phate incorporation into lyso-PG lags behindincorporation into PG in both the presence andabsence of cerulenin (18). Thus, with regard tophosphate incorporation, the precursor-productsequence involving DG, PGt, and lyso-PG maybe in the order DG -- PGt -- lyso-PG (Fig. 4B).It is of interest that this sequence is the reverseof that observed for incorporation of exogenous['4C]palmitate into the lipids of cerulenin-treated cultures (i.e., free fatty acid, lyso-PG,PG, DG, and finally PE [Fig. 1]). Recent resultsindicate that this same pattern can be observedin control cultures containing no cerulenin at 20or 350C (Fulco, unpublished data). As pointedout previously (5), the [14C]palmitate results canbe rationalized if incorporation of exogenousfatty acids is assumed to require activation tothe coenzyme A derivative before acylation (15)and if the second acylation step in PG biosyn-thesis is assumed to require the fatty acyl carrierprotein derivative (Fig. 4B).A final complexity in the lipid metabolism of

B. megaterium was revealed in the finding thatthe diacylated glycerol moiety of the PG plusDG pools undergoes slow turnover at a ratecalculated to be 10 to 12% of the rate of netglycerolipid synthesis (Fig. 3B). Furthermore,this turnover and/or exchange was apparentlystimulated about twofold by cerulenin at 350C.This phenomenon was responsible for the [U-14C]glycerol incorporation into the diacylatedglycerol moieties of PG, DG, and PE observedin the presence of cerulenin (Fig. 3B).

Confirmation of the conclusions derived fromthese studies will require in vitro demonstrationof the postulated lipid interconversions andeventual genetic studies. Current work on denovo lyso-PG and PG syntheses in B. megate-rium includes in vivo and in vitro studies withlysophosphatidic acid, monoglyceride, and CDP-monoglyceride (25) analogs.

ACKNOWLEDGMENTSWe thank Edmund Yang and Sue Hill for excellent tech-

nical assistance in this work and Ellin James for preparationof the figures.

These studies were supported in part by contract EY-76-C-03-0012 between the Department ofEnergy and the Universityof California and by Public Health Service research grant Al-

VOL. 141, 1980

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634 LOMBARDI, CHEN, AND FULCO

09829 from the National Institute of Allergy and InfectiousDiseases.

LITERATURE CITED

1. Ballesta, J. P. G, C.L de Garcia, and M. Schaechter.1973. Tumover of phosphatidylglycerol in Escherichiacoli. J. Bacteriol. 116:210-214.

2. Chang, N. C., and A. J. Fulco. 1973. The effects oftemperature and fatty acid structure on Lipid metabo-lism in Bacillu licheniformis 9259. Biochim. Biophys.Acta 296:287-299.

3. Chang, Y.-Y., and E. P. Kennedy. 1967. Phosphatidylglycerophosphate phosphatase. J. Lipid Res. 8:456-462.

4. Chen, S. L., and A. J. Fulco. 1978. The cerulenin-in-duced formation of 1-acyl-lysophosphatidylglycerol inBacillw megaterium. Biochem. Biophys. Res. Com-mun. 80:126-132.

5. Chen, S. L, F. J. Lombardi, andA. J. Fulco. 1978. Thetemperature-mediated metabolism of I-acyl-lysophos-phatidylglycerol in cerulenin-treated cultures of Bacil-lus megaterium. Biochem. Biophys. Res. Commun. 80:133-139.

6. Cronan, J. E., Jr., and R. II Bell. 1974. Mutants ofEscherichia coli defective in membrane phospholipidsynthesis: mapping of 8n-glycerol-3-phosphate acyl-transferase K, mutants. J. Bacteriol. 120:227-233.

7. Dowhan, W., W. T. Wickner, and E. P. Kennedy.1974. Purification and properties of phosphatidylserinedecarboxylase from Ewcherichia coli. J. Biol. Chem.249:3079-84.

8. Emdur, L I., and T. H. Chiu. 1974. Tumover of phos-phatidyleycerol in Streptooaccus sanguis. Biochem.Biophys. Res. Commun. 59:1137-1143.

9. Emdur, L, and T. HE Chiu. 1975. The role of phospha-tidylglycerol in the in vitro biosynthesis of teichoic acidand lipoteichoic acid. FEBS Lett. 55:216-219.

10. Fischer, W., and H. R. Landgraf. 1975. Glycerophos-phoryl phosphatidyl kojibiosyl diacylglycerol, a novelphosphoglucolipid from Streptococcus faecalis.Biochim. Biophys. Acta 380:227-244.

11. Fulco, A. J. 1972. The biosynthesis of unsaturated fattyacids by bacilli. m. Uptake and utilization of exogenouspalmitate. J. Biol. Chem. 247:3503-3510.

12. Fulco, A. J., R. Levy, and K. Bloch. 1964. The biosyn-thesis of A"- and AW-monounaaturated fatty acids bybacteria. J. Biol. Chem. 239:998-1003.

13. Hirabayashi, T., T. J. Larson, and W. Dowhan. 1976.Membrane-asociated phosphatidylglycerophosphatesynthetase from Escherichia coli: purification by sub-strate affinity chromatography on-cytidine 5'-diphos-pho-1,2-diacyl-sn-glycerol Sepharose. Biochemistry 15:

J. BACTERIOL.

5205-5211.14. Hirschberg, C. B., and E. P. Kennedy. 1972. Mecha-

niam of the enzymatic synthesis of cardiolipin in Esch-erichia coli. Proc. Natl. Acad. Sci. U.S.A. 69:648-651.

15. Klein, K., R. Steinberg, B. Fiethen, and P. Overath.1971. Fatty acid degradation in Escherichia coli. Eur.J. Biochem. 19:442-450.

16. Langley, K. E., and E. P. Kennedy. 1978. Partial puri-fication and properties of CTP:phosphatidic acid cyti-dylyltransferaae from membranes of Escherichia coli.J. Bacteriol. 136:85-95.

17. Larson, T. J., and W. Dowhan. 1976. Ribosomal-asso-ciated phosphatidylserine synthetase from Escherichiacoli: purification by substrate-specific elution fromphosphocellulose using cytidine 5'-diphospho-1,2-dia-cyl-sn-glycerol. Biochemistry 15:5212-5218.

18. Lombardi, F. J., and A. J. Fulco. 1979. Two distinctpools of membrane phosphatidylglycerol in BaciUlusmegaterium. J. Bacteriol. 141:618-625.

19. Quint, J. F., and A. J. Fulco. 1973. The biosynthesis ofunsaturated fatty acids by bacilli V. In vio substratespecificities of fatty acid desaturases. J. Biol. Chem.248:6885-6895.

20. Raetz, C. R. H., and E. P. Kennedy. 1972. The associ-ation of phosphatidylserine synthetase with ribosomesin extracts of Escherichia coli. J. Biol. Chem. 247:2008-2014.

21. Raetz, C. R. H., and K. F. Newman. 1978. Neutral lipidaccumulation in the membranes of Escherichia colimutants lacking diglyceride kinawe. J. Biol. Chem. 253:3882-3887.

22. Raetz, C. R. H., and K. F. NewmanL 1979. Diglyceridekinase mutants of Escherichia coli: inner membraneassociation of 1,2-diglyceride and its relation to synthe-sis of membrane-derived oligosaccharides. J. Bacteriol.137:860-868.

23. Renkonen, 0. 1966. Individual molecular species of dif-ferent phospholipid classes. H. A method of analysis. J.Am. Oil Chem. Soc. 42:298-304.

24. Short, S. A., and D. C. White. 1972. Biosynthesis ofcardiolipin from phosphatidylglycerol in Staphylococ-cus aureus. J. Bacteriol. 109:820-826.

25. Thompson, W., and G. MacDonald. 1979. Isolation ofa specific arachidonyl coenzyme A: cytidine diphos-phate monoacylglycerol acyltransferase. J. Biol. Chem.254:3311-3314.

26. van Golde, L M. G., G. H. Schulman, and E. P.Kennedy. 1973. Metabolism of membrane phospholip-ids and its relation to a novel class of oligosaccharidesin Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 70:1368-1372.

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