Molecular Microbiology (1993) 8(3), 535-542

Regulation of the synthesis of unsaturated fatty acidsby growth temperature in Bacillus subtilis

Roberto Grau and Diego de Mendoza*Departamento de Microbiologia. Facultad de CienciasBioquimicas y Farmaceuticas. Universidad Nacional deRosario. Suipacha531. 2000 Rosario, RepublicaArgentina.

Summary

Bacillus subtilis synthesizes, almost exclusively, sat-urated fatty acids, when grown at 37'C. When cul-tures were transferred from 37 C to 20 C, a chloram-phenicol- and rifampicin-sensitive synthesis of a C-16unsaturated fatty acid was observed. Synthesis ofthis compound reached a plateau after 5h at 20 C,reaching levels of 20% of the total fatty acid content.['^C]-tabelled fatty acids attached as thioesters toacyi-carriers compounds, such as coenzyme A (CoA)or acyi-carrier protein (ACP) synthesized de novo byglycerol-requiring auxotrophs deprived of glycerol toarrest phospholipid synthesis, could not be desatu-rated at 20 C. Desaturation of these fatty acids wasreadily observed when glycerol was restored to thecultures allowing resumption of transfer of acyt-moi-eties from acyl-thioesters to phospholipid. It was alsoobserved that depletion of the pools of CoA and ACPby starvation of pantothenate auxotrophs had noeffect on the observed synthesis of unsaturated fattyacid at 20 C. The overall results indicate that synthe-sis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obli-gate intermediates In this fatty acid desaturationpathway.

Introduction

Bacteria, in common with most (if not all) olher organ-isms, synthesize phospholipids with a greater proportionof unsaturated fatty acids (UFAs) when grown at low,rather than high, temperatures (de Mendoza and Cronan.1983; de Mendoza and Farias; 1988; Russell, 1984). Thisregulatory mechanism system, called thermal control oftatty acid synthesis, is thought to be designed to ameli-orate the effects of temperature change on the physical

Received 10 August 1992; revised 9 November 1992: accepted 17 Jan-uary. 1993 'For correspondence. Tel. (41) 300309; Fax (41) 240445.

State of the membrane phospholipids (de Mendoza andCronan, 1983; de Mendoza and Farias. 1988; Russell.1984). It is well documented that the proportion of fluid(disordered) lipid to no fluid (ordered) lipid in cell mem-branes plays a major role in membrane function (Cronanand Rock, 1987; de Mendoza and Cronan. 1983; de Men-doza and Farias, 1988). Increased incorporation of UFAsdecreases the melting temperature of the membranephospholipids. whereas increased incorporation of satu-rated fatty acid has the opposite effect (Cronan and Rock,1987; de Mendoza and Cronan, 1983). The thermal regu-latory system can thus adapt the membrane lipid for opti-mal functioning at new growth temperatures.

The mechanism used to alter the unsaturatlon of bacte-rial membrane lipids in response to growth temperaturedepends on the mechanism of UFA synthesis. In bacteria,both anaerobic and aerobic mechanisms are responsiblefor the synthesis of UFAs (for reviews see de Mendozaand Farias. 1988; de Mendoza et ai, 1992). The anaero-bic pathway, elucidated in detail using E. coli, producesc/s-UFA by a specific 2.3-dehydrase acting at the C-10level (for a review see Bloch, 1970). In certain bacteria(usually Gram-positive), and in eukaryotes, the introduc-tion of double bonds into the fatty acids employs a com-mon general mechanism. The reaction is catalysed by anoxygen-dependent desaturation system requiring the par-ticipation of a specific electron transport chain (de Men-doza and Farias, 1988; de Mendoza et al., 1992).

The molecular mechanism of thermal control of UFAbiosynthesis has been extensively studied in E. coli (fora review see de Mendoza and Cronan. 1983), In thisorganism, the UFA synthesized in greater quantity at lowtemperature is c/s-vaccenic acid (de Mendoza et ai,1983). This regulatory response does net require proteinsynthesis (Cronan and Rock. 1987; de Mendoza and Cro-nan, 1983) and functions by thermal modulation of theactivity of the soluble enzyme 3-ketoacyl-acyl-carrier-pro-tein-synthase II that converts palmitoleic acid to c/s-vaccenic acid (de Mendoza etat.. 1983).

A different regulatory mechanism of UFA synthesishas been reported in a large number of Bacillus speciesthat synthesize UFAs by the oxygen-dependent desatura-tion mechanism (Fulco, 1983). In these bacteria a desatu-rating system is induced at low growth temperatures (forreviews see Fulco, 1983; de Mendoza etal., 1992). Thus,the regulatory system of bacilli seems to involve an "on or

536 R. Grau and 0. de Mendoza

^ SFAof UFA synthesis involves direct desaturation of the fatty-acyi groups of membrane phospholipids

18:116:1

ABCDE

Rg. 1, AutoradlographicpaHernof synthesis of fatty acids in S. subtilis a\different tempefatures. One-milliiJtre aiiquots from cultures of strain 168grown lo exponential phase at 37' C were shifted to 2O'"C (lanes B, C andD) for 1 mm and then exposed to 10 iiCi of ['•'C]-acetate. After 4 h of incu-bation at 20"C, lipids were extraclGd, fatty-add moieties converted tomethyl esters and chromatographed in the argentation system describedin tfie Experimental procedures. The radioactive compounds were locatedby autoradiography and then quantified by scintillation counting. About20 000 c.p,m. of radioactivity was loaded in all lanes.A. Cbltufe grown at 37"C and labelled wilh 10 jiCi of [^'C]-acetate at thistemperature. The radioactivity detected in the fraction containing satu-rated tatty acids (SFAs) was 15 000 c.p.m.B. Culture shifted trom 37-C to 20"C. The radioactivity detected in thefraction of SFAs and UFAs was 13 000 and 2500 c.p.m., respectively,C. Cuiture shifted from 37' C to 20"C with chloramphenicol (100ng mr ' )added 5 mm t>efore the temperature shift.D. Culture shifted from 37'C to 20-0 with rifampicin (100 Mg ml"' addedas in C).E. Radioaciive fatty acids synthesized by E. coli C600 at 30X, The tJFAssynthesized by tfiis strain are: palmitoleic acid (c)S-.i^-hexadecenoic) andcvs-vaccenicadd {cis-iV-octadecenoJc) (de Mendoza etaL. 1982), Thefinal positions of UFAs and SFAs are indicated in the right margm.

off' transcriptional regulatory system rather than modula-tion of the activity of a lipid biosynthetic enzyme. Tofurther define the molecular mechanism{s) of cold-induc-tion of UFA biosynthesis and the mechanism(s) for sens-ing temperature changes in bacilli it is essential to charac-terize the pathway{s) of desaturation of fatty acids Inthese bacteria. In this context, Guffanti etai. (1987) haverecently shown that mutants of Baciiius subtilis selectedfor resistance to the protonophore carbonyl cyanide m-chlorophenyihydrazone (CCCP) have decreased con-tents of mono-unsaturated fatty acids (c, 50% of the nor-mai levels). Dunkley et ai (1991) found that thesemutants lack an NADPH-dependent palmitoyl-CoA desat-urase that requires both membrane and cytosolic compo-nents. Although the CCCP-resistant mutants completelylack palmitoyl-CoA desaturase activity, the UFA contentIs decreased by only one-half (Dunkley e^ ai, 1991). Itwas concluded from these data that 6. subtilis must con-tain an alternate synthetic pathway accounting for theremaining UFA content (Dunkley ef ai. 1991). in thisreport, we have used 6, subtilis auxotrophs that ailowarrest either at phospholipid or fatty acid synthesis toexamine the cold-induced synthesis of UFAs. Our resultsprovide strong evidence to show that a 6. subtiiis pathway

ResultsIn vivo characterization of the synthesis of UFAs inB. subtilis.

Since the characteristics of the fatty acid desaturationsystem in B. subtilis have not been reported, we havestudied some of its properties. We first analysed the syn-thesis of radioactive fatty acids by growing B. subtilis(strain 168) in the presence of ['"CJ-acetate at 37"C. Asshown in Fig. 1 (lane A), only saturated fatty acids arefound to be synthesized under these conditions. How-ever, when a culture growing at 37'C was transferred to20X and pulsed with [^"^Cl-acetate, synthesis of an UFAwhich closely comigrated with palmitoleic acid {lane B)was observed. The desaturation of fatty acids at 20"Crequired de novo synthesis of RNA and proteins since itwas abolished by rifampicin or chloramphenicol addedbefore the temperature shift (Fig. 1, lanes C and D). Addi-tion of chloramphenicol 30 min after the temperature shiftdid not affect the observed desaturation (data not shown).These resuits indicate that the dramatic increase in thesynthesis of UFAs in cells shifted from 3 7 X to 2O''C is acold-inducible process.

The proportion of UFAs synthesized by S. subtilis aftertransferring the cultures from 37'-"'C to 20"C increased forabout 5 h before reaching a plateau (Fig. 2). It is interest-ing to note that the exponential growth of S. subtiiis at2O'C {after shift from 37 C) was not preceded by a lagphase and that the maximum level of UFA synthesis wasreached about 1 h before a growth division cycle wascompleted (Fig. 2). The proportion of UFAs initiaily foundin cultures transferred from 37"C to 20°C far exceededthe levels of synthesis found in cultures growing for sev-eral generations at 2O'C (Fig.3). In addition, the extent ofinduction of the synthesis of UFAs was lower after a shiftfrom 37"C to 30"C than from 37^C to 20'̂ 'C (Fig. 3).

Cold-induced desaturation of phospholipids synthesized

To determine if ['''*C]-labeiled phospholipids synthesizedby B. subtilis at 37'C are desaturated after a temperatureshift to 20"C, cultures of strain 168 were labelled with[^•'CJ-acetate at 37''C. The labelled cells were washed,resuspended in media without labelled acetate, grown at20°C for 3 h and UFA contents were then determined. Wefound that about 20% of the labelled fatty acids wereUFAs (Table 1). These results suggest that membranephosphoiipids may act as substrates for the desaturasesince the pools of labelled fatty-acyl thioester intermedi-ates should be small. The possibility that an acyl-thioester

Regulation of UFA biosynthesis in Bacillus subtilis 537

0 1 2 3 4 5 6

l l o m s l i f te r t rans fe r lu -iO"r.

Fig. 2. Time course of UFA synthesis by S. subtilis after shift from 37°C to20"C. Cells were grown to exponential phase at 37 'C and Itien 1 ml sam-ples were shifted lo 2O''C and exposed to 1O (iCi ol ["Cl-acetate duringthe times indicated in the Figure. Afler these incubatron periods, the llpidswere extracted and the fatty-acid methyl esters prepared as described inthe legend of Fig. 1 The resulting methyl esters (atxjut 20 000 c.p.m. persample) were separated into unsaturated and saturated tractions andQuantified as described in !he Experimental procedures. Results areexpressed as Ihe percentage ol Ihe total methyl esters recovered. Dataare means of ttiree experiments with the average error in percentage val-ues of UFAs being less than S%.

could also be a substrate, however, could not be ruledout.

UFA biosynthesis in the absence of phosphoiipidsynthesis

To test directly if a fatty-acyl thioester(s) can be desatu-rated in vivo by the B. subtilis desaturase, we uncoupledfatty acid synthesis from phospholipid synthesis by a spe-cific inhibition of the latter process. It has been shown pre-viously that glycerol starvation of glycerol-auxotrophs(glyc) of B. subtilis results in a profound inhibition of phos-pholipid synthesis, although the synthesis of fatty acidscontinues at a normal rate (Mindich, 1972). Because glyc-erol starvation of glyc mutants blocks the synthesis of theacyl acceptor, fatty acids cannot be transferred to phos-pholipids and. consequently, accumulate as free species(Mindich. 1972). This manipulation allows specificlabelling of the pools of thioester intermediates. To deter-mine if fatty-acyl moieties not bound to phospholipidscould be desaturated at 20 C. cultures of strain B42 weregrown at 37°C to exponential phase, starved for glycerol,and shifted to 2O''C to induce desaturation. At this timethe cultures were exposed to [^''C]-acetate, and incuba-tion was continued for 3h at 20°C before analysis of thelipid fraction. As shown in Fig. 4 (lane A), about 97% ofthe radioactivity found in these conditions was in the freefatty acid (FFA) fraction. The analysis of this fraction bysilver-nitrate-impregnated plates showed that the FFAs

accumulating at 2O''C did not contain UFAs (Fig. 4. laneC). In order to distinguish whether the acyl-thioesters syn-thesized in the absence of glycerol are not substrates ofthe desaturase or constitute a pool that is not metaboli-cally active, we starved B42 for glycerol at 37'C in thepresence of [''*C]-acetate until labelled FFAs accumu-lated. We then restored glycerol to the culture and simul-taneously removed the ['''C]-acetate. Following glycerotrestoration at 37°C, the label in the FFA fractiondecreased with an equivalent increase in the label foundin glycerolipids (Fig. 4, lane B). These labelled cultureswere then shifted to 2O'C for 2h. the phospholipidsextracted, purified by thin-layer chromatography, con-verted to methyl esters and analysed by argentation chro-matography. As shown in Fig. 4 (lane D). the acyl chainssynthesized in the absence of glycerol were incorporatedinto phosphoiipids and readily desaturated at 2O'"C.These results demonstrate that the FFAs accumulating inthe absence of glycero! are metabotically active, and fol-lowing re-acylation into phospholipids become substratesof the desaturase. Accordingly, these findings suggestthat the desaturase cannot desaturate fatty acids notbound to phospholipids. The possibility that an inhibitionof phospholipid synthesis may be responsible for theabsence of desaturation of acyl-thioesters synthesized at20"C in glycerol-deprived cells (e.g. blocking induction ofthe desaturation system) was ruled out as follows. Lipidsof glycerol-supplemented cultures of strain B42 werelabelled with ['^C]-acetate at STX. These cells were sub-sequently starved for glycerol, chased with cold acetate.

A B CD E

Tempera tu re sh i f t

Fig. 3. UFA synthesis in ft suW/Zis growing at a constant temperature andafter temperatiire shifts. In all cases the lipids of strain 168 growing expo-nentially were labelled with lO pCi ol I'^Cj-acetate lor a lime equivalent toone generation at the indicated temperature. Ttie percentage of UFAswas determined as described in the legend to Fig 2 Data are means otthree determinations with thB average error in percentage values ot UFAsbeing less than 5%. The temperature shifts from 37'C lo SO'C (B) or 30' C(D), respectively, were performed as described in Fig, 1. (A), (C) and (E)show results obtained with cultures growing at 20, 30 and 37 C. respec-tively.

538 R. Grau and D. de Mendoza

Table 1. Desaturation of pre-labelled phosplio-lipids by strains 168", 642". andURT".

Strain

168B42B42UR1UR1

Glycerol inculture

_

AbsentPresent-

-

Pantoate inculture

_--

AbsentPresent

Radioactivityin SFA"

93154106275974644581

Radioactivityin UFA"

21B5919593653293

Tolairadioactivity^

115005025335281174874

a. A 1 ml aliquot from a culture of strain 168 grown lo exponential phaseat 37'C was incubated with10 tiCi ot p''C]-acstate for 1 h. Ttie culture was then washed and transferred to growth medium con-taining 0.03 M ol non-labelled acetaie and shifted to 20"C for 3 h. The phospholipids were extractedand the fatty-acid moieties converted to meihyl eslers and chromatographed in the argeniation sys-tem. The radioactive compounds were located by auioradiography and quantified by scintillationcounting.b. Strain B42 was grown at 37"C to exponential phase in glycerol-supplemented medium. Two sam-ples {1 ml each) were incubated with tOijCi of ['"'Cl-acetatetor 1 h at 37"C. The cultures were thentransferred to a medium lacking glycerol but containing non-labelled acelate. One sample was sup-plemented with glycerol, and the other remained unsupplemented. Both cultures were then shifted to2O''C and incubated lor 3 ti. The chromatography of labelled methyl esters was periormed asdescribed above.c. Two samples (1 ml each) of a culture of strain UR1 grown to exponential phase at 37^0 were Incu-bated with lOjiCi ofl'^CJ-acetate (or 1 h. The cultures were deprived ol pantoate for 30 mm andshifted to 2O'C exactly as described for strain B42. The chromatography of labelled meihyl esterswas also performed as described atx>ve.d. The radioactivity is expressed in counts min"'. Data are means of three determinations wilh theaverage error in percentage values of UFAs being less than 5%.

and shifted to 20^0. The result of this experiment(Table 1) indicates that desaturation of prelabelted fattyacids from glycerol-starved or glycerol-supplementedcells is almost identical (in both cultures about 20% ofUFAs were synthesized). This demonstrates that thedesaturation system is induced and is active in theabsence of phospholipid synthesis.

UFA biosynthesis in cells depleted of CoA

The experiments performed with glycerol auxotrophsstrongly suggest that fatty acids must be incorporated intophospholipids in order to be desaturated at 20^0. Accord-ingly, these results are compatible with lipid-linked desat-uration in which the fatty acid remains esterified to themembrane lipid while being desaturated. Nevertheless,we could not firmly exclude the presence in 6, subtilis of apathway which involves deacylation of the membranelipid, esterlfication of the fatty acid to a coenzyme. desatu-ration of the fatty-acyl thioester, and reacylation of thelipid with the resultant UFAs. In order to determine if thispathway is operative in B. subtHis, we used the panEstrain URI, a strain with an inactive ketopantolc acidreductase (Baigori et ai, 1991). URI cannot synthesizepantoate. the precursor of pantothenate. Pantothenate isutilized primarily for the biosynthesis of CoA and ACP(Rock and Jackowski, 1982). Strain URI is phenotypicallyrecognized as a pantoate or pantothenate auxotroph andoptimal growth is achieved at about 1 mM pantothenate

(Baigori et ai. 1991). The generation times at 37°C ofURI growing in media supplemented with pantothenateor pantoate are about 30 min and 90 min. respectively (R.Grau and D. de Mendoza. unpublished). When strainURI is deprived of pantoate growth stops almost immedi-ately, presumably because of depletion of the CoA pool(see below and Jackowski and Rock. 1986). As shown inFig. 5. pantoate-deprlvation of URI results in a completeinhibition of the incorporation of p-atanine into CoA andACP. This result indicates that pantoate starvation veryefficiently blocks the synthesis of both acyl-carrier com-pounds. Moreover, the intracellular content of CoA andACP dropped precipitously (about 96%). when comparedwith pantoata-supplemented cultures, in the first 15 minfollowing the removal of pantoate (data not shown). Asexpected, pantoate-deprivation of strain UR1 results in97% inhibition of the incorporation of ['""CJ-acetate intophospholipids (data not shown). To determine if UFA syn-thesis takes place in the absence of acyl-carriers and fattyacid synthesis, cultures of URI were labelled with ['"C]-acetate at 37'C in the presence of pantoic acid. Thelabelled cells were then washed, chased with coldacetate, starved for pantoic acid and shifted to 2O''C.Table 1 shows that the desaturation levels of pre-labelledphospholipids from pantoate-starved or pantoate-supple-mented cells are almost identical. The data show that thecellular levels of acyl carriers can be reduced to <4% ofthe levels found in pantoate-supplemented cells without asignificant decrease in desaturation. Thus, these results

Regulation of UFA biosynthesis in Bacillus subtilis 539

FFA

DG

SFA

Origin . . I I Origin

B D

Fig. 4. Autoradiographic pattern of lipid synthesized by strain B42 growingIn the presence or absence of glycerol. Strain B42 growing exponentiallyat 37''C in glycerol-supplemented medium was deprived of this substrateas descntjed in ihe Experimental procedures. A i mt sample was trans-ferred to 2 0 ^ and exposed to 10 ̂ Ci of ['*C]-acetate for 3 fi at 2O'C (laneA). A second sample from Ihe same culture was maintained at 37^C in Ihepresence of {'''C)-acetate for 30 mm to allow synthesis of radioactiveFFAs. This culture was then washed free of [' *C]-acetate and resus-pended in a medium containing glycerol and non-radioactive acetate (0.03M) and incubated tor another 30 mm at 37°C (lane B). After these incuba-tion periods the radioactivity present in FFAs and glycerolipids of both cul-tures (lanes A ar>d B) were quantified as descrit}ed m the Experimentalprocedures. The sample in lane A contained 10 000 and 500 c.p.m. ofradioactivity In the FFA and phospholipid fractions, respectively. The sam-ple in lane B contained 5000, 4000 and 700 cp.m, of radioactivity in phos-pholipids, diacylglycerol (DG) and FFA, respectively. Similar results wereobserved in several independent experiments. The FFA Fraction shown inlane A was converted lo methyl esters with diazomethane and chro-matographed in the argentation system (lane C). A 2 ml sample of a cul-ture treated as described in lane B was transferred to 2O''C for 2 h. Afterincubation, the lipids were extracted and chromatographed as in lane B.The fraction corresponding to phospholipids was eluled from the silica geland the fatty acids converted to methyl esters with sodium methoxide andresolved into SFAs and UFAs (lane D). In lane C 9000 c.p.m. wasdetected in tha SFA fraction, whereas the sample of lane D contained9000 and 1500 c.p.m, in the SFA and UFA fractions, respectively. Essen-tially the same results were observed in several independent experiments.

Strongly suggest that the fatty-acyl moieties of the phos-pholipids can be desaturated without previously beingacylated to a coenzyme such CoA or ACP.

Discussion

Our understanding of the moiecuiar basis of the tempera-ture-induced controi by which iJFAs are synthesized bybacilli only at low growth temperatures is severely limited.Initial efforts to understand the moiecuiar mechanism ofinduction of the desaturation system were hampered bythe inability to use Bacillus species suitable tor geneticmanipulation. In this article we report experiments show-ing that B. subtilis, a bacterium widely used for geneticanalysis, contains a fatty acid desaturation system that is

induced by low temperatures (Fig. 1). We also found thatthe proportion of UFAs synthesized during the first growthdivision cycle at 20 '̂C was much higher than those of cul-tures growing for several generations at 20"C (Fig. 3).These results agree with those of Fulco and coworkers(Fujii and Fulco. 1977; Fulco, 1983). They reported thatthe levels of desaturation of exogenously suppliedpalmitic acid in cultures of Baciiius megaterium trans-ferred from SS^C to 2O''C far exceeded the Ieveis of desat-uration of cultures growing at 2O'C. To explain the dra-matic change in lipid composition of bacilli shifted from37X to 20X, Fujii and Fulco (1977) proposed that tran-scription of the fatty acid desaturase gene occurs only atlow temperatures. To account for the unexpectedly large

1200 -

900 -

600 -

300 -

1200 -

900

600

300 -

:to5 10 15 :̂ ()

fr«ction number

Rg. 5. Blocking of the synthesis of p-alanine-derived Intracellular metabo-lites in strain URt after starvation for pantoate. Strain URl (parjE) wasgrown on 4 ml of media supplemented with 0,8 mM pantoate to a densilyof 1 •< 10° cells m l ' . At this pointthe cells were depnved of pantoale asdescritied in the Expenmenlalprocedures The cells were itien resus-pended m 4 ml of the same media without pantoate. Half of this culturewas supplemented with 0 8 mM pantoate, while the other half remainedunsupplemented. Both cultures were then labelled with 1 jiCi of [i-[3-''Hl-atanlne at 37''C for 3 h. At the end ot the experiment, cells were har-vested, washed and lysed. Samples were chromatographed on silica gelH layers as described in the Experimental procsdures.

540 R. Grau and D. de Mendoza

initial degree of unsaturation seen immediately after adownward temperature shift. Fulco etai (Fujii and Fulco.1977; Fulco, 1983) postulated the existence of a modula-tor protein whose synthesis also proceeds at lower tem-peratures, but only following a brief delay. Thus, the rapiddesaturation taking place in freshly down-shifted cellswould soon be moderated to a rate yielding the steady-state level of fatty acid unsaturation characteristic of thattemperature. However, a molecular understanding of thiscold-induced hyperinduction of UFA clearly requiresmuch further investigation.

In Bacillus, it is a matter of dispute whether acyl-CoA orphospholipids constitute substrates for the desaturatingenzyme. Fulco et ai (Lombardi and Fulco, 1980; Fulco.1983) suggested that the substrates for desaturation arethe acyl groups present in intact membrane phospho-lipids, whereas Dunkley et ai (1991) recently reportedthat extracts of S. subtilis desaturate paimitoyl-CoA butphosphatidylethanolamine is not a substrate in vitro. In anattempt to elucidate the nature of the in vivo substrate(s)of the fi. subtilis desaturase, we have used auxotrophsthat allow arresting of phospholipid synthesis. The glycauxctrophs are blocked at the level of the acyl transfercaused by the lack of acyl acceptor, whereas panEmutants cannot synthesize fatty acids by depletion of acylcarriers. For direct testing of whether a fatty-acylthioester(s) such as acyl-ACP or acyl-CoA produced inthe synthetic fatty acid pathway can be desaturated invivo, we have used a system that allows us to uncouplefatty acid synthesis from pfiospholipid synthesis. Glycerolstarvation of B. subtilis glycerol auxotrophs inhibits phos-phoiipid synthesis, resulting in a significant accumulationof FFAs (see Fig. 4. lane A, and Mindich. 1972). Synthe-sis of UFAs from these de nova-produced fatty acidswould be expected in these cells after a temperature shiftif fatty-acyl thioesters are used as desaturation sub-strates. Moreover, these UFAs should be found in theFFA fraction, since it is accepted that free acyl moietiesaccumulated during glycerol starvation arise by hydroly-sis of fatty-acyl thioesters by thioesterase(s) (Rock andJackowski. 1982). Our resuits (Fig. 4, lane C) show thatfree UFAs are not formed after glycerol deprivation. Theinability of glycerol-starved cells to desaturate fatty-acylmoieties not bound to phospholipids cannot be attributedto the production of a non-metaboiizable pool of fattyacids. After addition of glycerol. these fatty acids arereadily incorporated into the phospholipids and subse-quently desaturated at 20"C (Fig. 4. lane D). These datasupport the conclusion that an acyl-ACP derived fromfatty acid biosynthesis or an acyl-CoA, that may beformed from acyl-ACP or from FFAs, are not desaturatedin vivo by B. subtilis. Therefore, our data strongly suggestthat, in vivo, membrane phosphotipids are obligate inter-mediates in the synthesis of UFAs during cold-adaptation.

The results obtained with starved glyc auxotrophs arecompatible with lipid-linked desaturation in which the fattyacid remains esterified to the membrane lipid white beingdesaturated. This latter desaturation mechanism hasbeen proposed for the cyanobacterium Anabaena varl-abilis (Sato et al.. 1986) and for the psychrophilic bac-terium Micrococcus cryophilus (foo\ etai, 1983). Never-theless, we needed to firmly exclude the presence inS. subtilis of a pathway proposed for Tetrahymena(Yasunaga etai, 1984) which involves deacylation of themembrane lipid. esterification of the fatty acid to a coen-zyme, desaturation of the fatty-acyl thioester, and reacy-lation of the lipid with the resultant UFAs. In this case, thesubstrate for desaturation couid be acyl-CoA since Dunk-ley et a/.(1991) have recently reported that, like someother bacteria that synthesize UFAs by the oxygen-dependent pathway, extracts of B. subtilis can desaturatepalmitoyi CoA in vitro. However, the data shown inTable 1 indicate that labelied phospholipids from pan-toate-starved panE auxotrophs are readily desaturated.We determined that pantoate starvation of panE mutantsresults in inhibition of the synthesis of CoA (Fig. 5) anddepletion of the CoA pool. This result indicates that thefatty-acid moieties of the phospholipids are desaturatedwithout any necessity for this coenzyme. Nevertheless,with the experiments reported here, we cannot excludethe possibility that a palmitoyt-CoA desaturase becomesinhibited when phospholipid synthesis is arrested by star-vation of giyc or pan auxotrophs. If this is the case, fi.subtilis would contain two different pathways for thebiosynthesis of UFAs: a paimitoyl-CoA desaturasecoupled to phospholipid synthesis and a desaturationmechanism, reported here, that is independent of phos-pholipid synthesis, which uses as substrates the fatty-acylgroups of intact phospholipids. Finally, that membranephospholipids may be directly desaturated could explainthe observation that fi. subtilis mutants lacking palmitoyi-CoA desaturase can still synthesize UFAs at a significantrate (Dunkley ©fa/., 1991).

Experimental procedures

Bacteriai strains and growth conditions

The following B. subtilis strains were used: strain 168 {trpC2),strain B42 (derived from strain 168), which is his. trpC2, glyc,and gIpD (Mindich. 1970). and strain UR1 {trpC2. thr5, panE)(Baigori etal.. 1991). In all experiments, the strains were grownin Spizizen minimal salts medium (Spizizen. 1958) supple-mented with glucose (0.5%). vitamin-free casein hydrolysate(0.1 %) and tryptophan (40 pg mp'). In addition, glycerol(25^igmr^) or pantoic acid (0.02%) were included to satisfygrowth requirements of strain B42 (glyc) or UR1 (panE).respectively.

Regulation of UFA biosynthesis in Baciiius subtilis 541

Glycerol or pantoic acid starvation

At the mid-exponential phase of growth, the growing culturewas subjected Io three cycles of centrifugation in media withoutglycerol or pantoic acid. Growth of strain B42 continued forabout one generation following removal of glycerol (Mindich,1970), while the growth of strain URl ceased after pantoic acidvcas removed (see the Resuits).

Analysis of radioactive lipids

For measurements of fatty acid synthesis, 8. subtiiis strainswere grown in the culture medium described above with aera-tion to exponential phase, Aiiquots of these cells (1 ml) wereexposed to lOpCi of sodium [1-'"*C]-acetate (59.0 mCi mmol"')for different limes at the temperatures described in the figurelegends. After these incubation periods, lipids were extractedfrom whole celis as previously described (de Mendoza ef ai,1982).

FFAs and glycerolipid fractions were separated by thin-layerchromatography on silica gel plates developed in petroleumether/ether/acetic acid (70:30:2). In this system the FFAsmigrate near the solvent front, whereas phospholipids remainin the origin of the thin-layer piate (de Mendoza et ai, 1982;Mindich, 1972). The FFA fraction was converted to methylesthers with diazomethane (Cronan et ai, 1975). Radioactivefatty acid melhyl esters were then separated into unsaturatedand saturated fractions by chromatography on 20% silver-nitrate-impregnated silica gel thin-layer plates (de Mendoza etai, 1982). The plates were developed in toluene at -17°C,autoradiographed, and the approplate areas of silica gel werescraped into vials containing scintillation solution (de Mendozaetai. 1982) to determine their radioactivity content.

Fatty acids from glycerolipids were converted to their methylesters with sodium methoxide (de Mendoza et ai. 1982). Theesters were then extracted in petroleum ether/ether (1:1), con-centrated under a nitrogen atmosphere and chromatographedon silver-nitrate-impregnated plates as described above. Theradioactive compounds were located by autoradiography andquantified by scintillation counting.

Chromatography of ^-alanine-derived metabolites

The B. subtilis strain URl was labeiied with [)-[3-^H]-alanine asdescribed in fhe figure legends. Extraction of p-alanine-derivedmetabolites was accomplished as described by Jackowski andRock (1981). Samples were analysed by thin-layer chromatog-raphy on silica gel H layers developed with ethanol/28%ammonium hydroxide, 4:1, (v/v) to 14 cm from the origin. In thissoivent system coenzyme A (CoA). acyl carrier protein (ACP)and p-alanine-derived phosphorylated metabolites remain inthe origin of the thin-layer plate, while p-alanine and pantothen-ate migrate with a defined R^ (Jackowski and Rock. 1981;Baigori et ai., 1991). The distribution of radioactivity on the thin-layer plate was determined by scraping 0.5 cm sections of thesilica gel layer into 3 ml of scintillation solution and ascertainingthe radioactive content.

Acknowtedgements

This work was supported by grants from Consejo Nactonal de

Investigaciones Cientificas y T6cnicas (CONICET) and Fun-dacion Antorchas (Argentina). R.G. is a fellow from CONiCETand D.deM. is a Career investigator from the same Institution.We \hank J. E. Cronan Jr for usefui discussions and numerouscomments on the manuscript. We also thank A, Viale and K,Magnuson for careful reading and ior correcting themanuscript, and M. Arevalo for technical assistance andpatience. We are grateful to L. Mindich for generously provid-ing strain B42, and to M. Gonzalez Sierra for helpful experi-mental advice.

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