Proc. Nat. Acad. Sci. USAVol. 71, No. 9, pp. 3758-3762, September 1974

Regulation of the Fatty Acid Composition of the Membrane Phospholipidsof Escherichia coli

(gene dosage/enzyme levels)

JOHN E. CRONAN, JR.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510

Communicated by Konrad Bloch, June 13, 1974

ABSTRACT An increase in the dosage of the fabAgene, which codes for the enzyme 0-hydroxydecanoyl thio-esterase, causes an increase in the unsaturated fatty acylcontent of the phospholipids. This increase is small in cellsgrowing at a constant temperature. However, if cellsgrown at 37 or 420 are shifted to 150, the rate of unsaturatedfatty acyl moiety synthesis approaches twice the rate of anormal strain. These results indicate that the ratio ofunsaturated to saturated fatty acyl groups in phospho-lipids is partially determined by the levels of at least thisfatty acid biosynthetic enzyme.

Phospholipids play an important role in the structure andfunction of all biological membranes (1, 2). In virtually allnatural phospholipids, unsaturated fatty acids are prefer-entially esterified at position 2 and saturated fatty acids atposition 1 of the glycerol molecule (3). This asymmetric fattyacid distribution is thought to be of major importance in thefunctional and structural roles of phospholipids in membraneprocesses. Perturbations in the fatty acid content of mem-brane phospholipids can produce drastic disturbances in cellu-lar physiology (4-9). Thus, the enzymatic mechanisms con-ferring such positional specificity of fatty acid residues are ofcentral importance to the cell.Recent investigations (10-12) indicate that this specificity

can be attributed to the enzymes acylating sn-glycerol 3-phosphate to form phosphatidic acid, a key intermediate inthe phospholipid biosynthesis. Therefore, a mechanism toaccount for this asymmetric distribution which requires theselection by the acylating enzymes of the "proper" fatty acidfrom a pool of saturated and unsaturated fatty acid precursorsseems attractive. However, two lines of reasoning make such asimple mechanism unlikely. First, because of the close couplingof fatty acid to phospholipid synthesis in Escherichia coli(1, 13), the pools of phospholipid acyl moiety precursors areextremely small (8) [even when fatty acids are provided in theculture medium (8, 14) and when degradation is blocked (14)1.Therefore, a pool from which selection of fatty acyl chains bythe acyltransferase could occur, is not obvious. Second, theunsaturated fatty acid content of the phospholipids of E. coliincreases as the growth temperature is decreased (15). Thespecificity of the acylating enzymes changes with temperaturein a manner consistent with this in vivo situation (12), but theincreased amounts of unsaturated fatty acid utilized muststill be provided by the synthetic pathway. For these reasons,it seems apparent that the ratio of palmitate to unsaturatedfatty acid is controlled at (at least) two levels. One level isacylation into phospholipid; the other is at the level of supplyof fatty acid to the acylating enzyme systems.A test of this hypothesis would be to specifically increase the

synthesis of unsaturated acyl groups by the cell and to ex-

amine the effect of this alteration on the phospholipid fattyacid composition. An increase in the synthesis of phospholipidunsaturated acyl groups would indicate that control of thesaturated to unsaturated fatty acid resides at the level ofunsaturated fatty acid synthesis. The lack of an increase wouldbe consistent with the acyltransferase selection hypothesis.The most likely enzyme in the fatty acid synthetic scheme

of E. coli, at which the ratio of saturated to unsaturated fattyacyl moieties could be controlled, is fl-hydroxydecanoyl thio-ester dehydrase. This enzyme introduces the double bond ofthe unsaturated fatty acids (16). We have previously shownthat a decrease in the activity of this dehydrase produces anequivalent decrease in phospholipid unsaturated fatty acylmoieties (9). This result suggested that little excess of de-hydrase activity is present in E. coli and that the level of thisenzyme might be closely related to the level of unsaturatedfatty acyl moieties found in phospholipid. With this rationale,I increased the intracellular level of this dehydrase by geneticmanipulation and tested the effect of this alteration on theratio of saturated to unsaturated fatty acids found in theenvelope phospholipid.

MATERIALS AND METHODS

Strain Construction. Strains KL181 KLFG/KL181 have thegenotype: pyrDS4, thi-1, his-68, trp-4.5, recAl, F-. StrainKLF6/KL181 carries F106. Strain CY50 [formerly calledYAA1 (20)] is a pyrD+ fabA2 transductant of KL185 (a rec+derivative of KL181). Strains CY57 and CY59 are fadE62,recAl derivatives of CY50 and KL185, respectively. ThefadE62 and recAl lesions were introduced by mating as pre-viously described (17, 18). Strains CY97 and CY99 are KLF6-carrying derivatives of CY57 and CY59. Strains CY103 andCY104 are CYFl-carrying derivatives of strains CY57 andCY59, respectively. The CYF1 episome was derived fromKLF6 by homogenization (19) in a CY50 background. Allmerodiploid strains were grown under conditions which pre-clude the loss of the episome (without uracil for pyrD strainsand at 420 for fabA2 strains). The recAl lesion in all strainsstabilized merodiploid strains by preventing recombinationof the required episomal gene into the chromosome. Othergenetic methods (19-23) and the EC (8) and ECG (24) mediawere described previously.

Enzyme Assays. Extracts for measurement of ,3-hydroxy-decanoyl thioester dehydrase were prepared and assayed forisomerase or dehydrase activity exactly as previously de-scribed (9). A unit of activity is defined as the formation of1 nmol of trans-2-decenoyl-N-acetylcysteamine per min of in-cubation. An internal standard to correct for loss of enzyme

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during nucleic acid removal was added after cell disruption.This standard consisted of a sample of pure dehydrase com-

pletely inactivated with 4C-labeled 3-decynoyl-N-acetyl-cysteamine, a specific inhibitor which forms a covalent bond inthe active site of the enzyme (16). This standard was preparedby J. Stein of Harvard University and was the generous gift ofJ. Stein and K. Bloch. Recoveries of enzyme (monitored byscintillation counting) were >85% and close to 100%o in mostcases. al-Oxidation by intact cells was assayed as described byKlein et al. (14).

Lipid Analyses. Most analyses were done in lipids radio-actively labeled with [14C]acetate. Phospholipids were ex-

tracted from either cell pellets or cultures (25). Total fattyacids were obtained by saponification or acid hydrolysis ofcultures followed by extraction into ether. Phospholipids werepurified by thin-layer chromatography (25). Methyl esterswere prepared and analyzed by thin-layer or gas chromatog-raphy as previously described (24). Internal standards of 3H-labeled palmitic acid (for thin-layer chromatographic analy-sis) or of pentadecanoic acid (for gas chromatographic analy-sis) were used to monitor recoveries of fatty acids.

RESULTS

Strains of E. coli with two copies of the fabA gene were used toincrease the level of P-hydroxydecanoyl thioester dehydrase invivo. The fabA locus is the structural gene coding for the de-hydrase (9). This gene maps very close to the pyrD locus on

the E. coli genetic map (20), and hence is included with pyrDon the portion of the chromosome carried by the episome KL-F6 (Fig. 1). A strain carrying functionalfabA genes on both thechromosome and the KLF6 episome would thus contain twocopies of the fabA gene, and was expected to contain twice thenormal level of dehydrase activity. If such a merodiploidstrain did not contain twice the normal level of dehydraseactivity, this would suggest regulation of this enzyme at a

transcriptional or translational level. However, if twice thenormal level of dehydrase activity was found in the mero-

diploid strain, then examination of the lipid composition of thestrains should reveal any existing control of phospholipidfatty acid composition by the dehydrase level. Therefore, we

examined a pair of strains, KL181 and KLF6/KL181 (a giftfrom Dr. K. B. Low).

Strain KL181 is a normal strain carrying a single copy of thefabA gene. Strain KLF6/KL181 carries two functional fabAgenes, one on the chromosome and the other on the KLF6 epi-some. We first examined the dehydrase levels and fatty acidcomposition of cultures of these strains grown at 370 (Table1). Strain KLF6/KL181 (+/+) * does have twice the level ofdehydrase activity found in strain KL181 (+). This is truewhen either the isomerase activity or the dehydrase activity of-the enzyme was assayed. This result indicates that only genedosage controls the level of this enzyme as assayed in extracts.The unsaturated fatty acid content of the phospholipids of

strain KLF6/KL181 (+/+) is greater than that of strainKL181 in cultures grown at 37°. A similar enrichment for the

* The functionality of a given fabA gene is denoted by a + signor a - sign. Merodiploid strains have two such signs separatedby a slash (/). The sign to the left of the slash denotes the fabAgene on the episome, whereas the right hand sign denotes thechromosomal fabA gene. Monoploid strains have only a singlesign.

Regulation of Escherichia coli Lipid Composition 3759

KLF6

pdxC pyrD fobA pyrC purB trp

20 23 27

FIG. 1. The location of the fabA gene and the KLF6 chromo-somal region on the E. coli genetic map. The data are from refs.20 and 26. The KLF6 episome is systematically called F106 inref. 26.

+/+ strain is seen for cultures grown at other-temperatures(Table 1). However, these differences are considerably smallerthan might be expected on the basis of the dehydrase levels.Although this result might seem disappointing, a 2-fold in-crease in unsaturated acyl content was not expected. Such anincrease would result in cellular phospholipids containing nosaturated fatty acid, a circumstance known to be lethal to thecell (27). Also, it can be argued from both in vivo (28) andin vitro (10-12) data, that the specificity of the acyltrans-ferase enzymes may not allow position 1 of the phospholipid tobe completely occupied by unsaturated fatty acid. Therefore,I sought conditions under which an increased rate of unsatu-rated acyl moiety synthesis would not be detrimental to thecell and which also might alleviate any restrictions imposed byacyltransferase specificity.A sudden decrease in temperature of growing cultures of the

+ and +/+ strains provided the conditions sought. Therationale of this experiment follows from the work of Marr andIngraham (15) and of Sinensky (12). Marr and Ingraham (15)demonstrated that, following temperature decrease, E. coliincreases its rate of unsaturated acyl group synthesis. Thisphenomenon appears of minor importance to the cell (24),suggesting that a further increase in this rate would notdamage the cells. Sinensky (12) found that the acyltransferaseactivities of isolated cell envelopes of E. coli incorporated moreunsaturated fatty acid into phosphatidic acid as the incuba-tion temperature was decreased. He also showed that the

TABLE 1. Properties of strains KL181 and KLF6/KL181

KLF6/KL181 KL181(+) (+/+)

ft-hydroxydecanoylthioester dehydrase

activity*(U/mg of protein)

Isomerase assay 4.30 10.34Dehydrase assay 0.60 1.58

PhospholipidUFA/SFA ratios

Cultures grown at 150 1.27 1.56Cultures grown at 370 0.95 1.29Cultures grown at 42° 0.81 0.97

DPM/108 cellsincorporated at 15°t

Cultures shifted from 370 to 150Palmitoleic acid 13,450 16,400cis-Vaccenic acid 18,150 23,500Saturated fatty acid 4,200 2,600

UFA, unsaturated fatty acid; SFA, saturated fatty acid.* Cultures for enzyme assay were grown at 37°.t Cultures labeled with 1 ,uCi/ml of ['4C]acetate for 2.5 hr

after temperature shift.

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TABLE 2. Properties of bacterial strains

Growth at Ability to transferfabA Dehydrase activity UFA/SFA

Strain* genotypef 300 400 420 pyrD + fabA+ U/mg proteint 420

CY57 - + 0 0 0 0 <0. 01CY59 + + + + 0 0 5.51 0.80CY97 +/- + + + + + 5.21 0.78CY99 +/+ + + + + + 10.49 0.97CY103 -/- + + 0 + 0 <0.01CY104 -/+ + + + + 0 4.44 0.80

* All strains are his, trp, fadE62, recAl. CY59 also requires uracil for growth. Upon curing with acridine orange (22), strains CY99 andCY104 give rise to uracil requiring female strains indistinguishable from strain CY59. Curing of strains CY97 and CY103 gives rise touracil independent, temperature-sensitive female strains indistinguishable from strain CY57.

t The genotypes are symbolized as given in footnote 1.$ All strains were grown at 420. The cultures of CY57 and CY103 were supplemented with oleic acid. The values given are the average

of at least four determinations which varied <10%. The isomerase assay was used. UFA, unsaturated fatty acid; SFA, saturated fatty acid.

incubation temperature during assay (rather than the growthtemperature of the cells) determined the acyltransferasespecificity. Thus, a sudden drop in temperature should tem-porarily alleviate any acyltransferase-imposed restriction onthe incorporation of unsaturated fatty acids into phospho-lipid. Therefore, after a decrease in temperature, the rate ofsynthesis of phospholipid unsaturated acyl moieties should bedependent only on unsaturated fatty acid synthesis per se,and thus a larger difference between the + and +/+ strainsmight be observed.With this rationale, cultures of strains KLF6/KL181

(+/+) and KL181 (+) were grown at 370, then shifted to150 in the presence of [14C]acetate. As shown in Table 1, theaccumulation of [14C]acetate in either of the E. coli unsatu-rated fatty acids was greater in the +/+ strain than in themonoploid (+1) strain, whereas incorporation into saturatedfatty acyl groups was much greater in the + strain. Thecombination of these differences resulted in the ratios of un-saturated to saturated fatty acyl moieties (those accumulatedduring the first 2.5 hr after temperature shift) to be 7.35 forstrain KL181 and 14.0 for strain KLF6/KL181. Therefore, theKLF6 episome has an almost 2-fold effect on unsaturated acylgroup synthesis during an interval after temperature de-creaset. However, this time interval must be brief since cul-tures grown at 150 have only a slightly increased unsaturatedacyl content (Table 1).

Evidence for involvement of thefabA gene

The presence of the KLF6 episome does increase the rate ofsynthesis of phospholipid unsaturated acyl moieties (Table1). However, these results did not show that the fabA genecarried by the episome was responsible for this change in lipidsynthesis. Although the KLF6 episome carries only about 1%of the E. coli genome (Fig. 1), it was possible that the aboveresults were due to the presence on the episome of a gene (orgenes) other than fabA. Hence, the results of Table 1 could bedue to increasing the dosage of this gene rather than or in

t Both strains (as well as those listed in Table 2) commencegrowth at the 150 rate immediately upon shift to 15° from 370 or

420. The doubling time at 150 is 13.5 hr, about 10-fold greaterthan at 370 (or 420). The rates of acetate incorporation alsodiffer by 10-fold between 150 and 370 (or 420). Therefore, acetateincorporation at 150 measures de novo lipid synthesis rather thanincorporation via an exchange or turnover process.

addition to an increase in p-hydroxydecanoyl thioester de-hydrase activity.The strains described in Table 2 were constructed to test

this possibility. All possible combinations of + and - fabAalleles were constructed. As expected from Table 1, the +/+strain has twice the enzyme activity of those strains havingonly a single functional fabA gene. Those strains with no func-tional fabA genes have no activity. A comparison of the -strain, CY57, with strain CY103 (-/-) suggests the dehy-drase activities measured in vitro are a valid indication of thein vivo situation. Strain CY103 (-/-) grows at 400, whereasstrain CY57 (-) cannot, suggesting the former strain has anincreased level of dehydrase activity in vivo. A more quantita-tive experiment involved shift of cultures of these strains from300 to 42° (Fig. 2). Upon shift in temperature, both strainsquickly lose unsaturated fatty acid biosynthetic ability.However, the rate of this loss is greater in strain CY57 (-)than in strain CY103 (-/-), and the residual rate of syn-thesis in strain CY103 (-/-) is twice the residual rate instrain CY57 (-). This result is that expected if strain CY103produced twice as much thermolabile dehydrase as strainCY57 and thus argues that dehydrase activities assayed invitro accurately reflect the intracellular situation.With this characterization we then examined the involve-

ment of the fabA gene in the KLF6 episome effect. The mostimportant strains in this test are CY97 (+/-) and CY104(-/+). Each of these strains possesses all the genetic in-formation on the KLF6 episome, but contains only a singlefunctional fabA gene. Strain CY97 (+/-) has a functionalfabA gene on the episome, whereas, thefabA locus on the chro-mosome is defective. Strain CY104 (-/+) has the oppositearrangement of fabA alleles. If an episomal gene (or genes)other than fabA is the cause of the increased unsaturated acylgroup synthesis, then both strain CY97 (+/-) and strainCY104 (-/+) should show this increase. If, however, therate of unsaturated acyl group synthesis in the -/+ and+/- strains was found to be identical to the rate shown bythe + strain, then this would be proof that the additionalfabA gene in the +/+ strain caused increased acyl groupsynthesis. Therefore, strains CY97 (+/-) and CY104 (-/+)were compared with strains CY59 and CY99 (the closely re-lated + and +/+ strains).As shown in Table 2, when grown at 420, the ratio of un-

saturated fatty acids to saturated fatty acids for strain CY99(+/+) was higher than the ratios for strains CY59, CY97,

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Regulation of Eecherichia coli Lipid Composition 3761

Zco

0)

C:

LL

00)

cr

.c

>0

0 (

00.0ro-

Hours after shift

FIG. 2. Rates of unsaturated fatty acid synthesis in variousstrains following temperature increase. Cultures of strainsCY59 (+), CY57 (-), and CY103 (-/-) were grown at 300 inEC medium. When the cultures reached 108 cells/ml, they wereshifted to a 420 water bath shaker. At various time intervals, 1-mlsamples were removed to a parallel flask containing 10 MCi of[4C]acetate. After a 15-min exposure to the label, 6 ml of CHC1-CH30H (1/2, v/v) was added and the lipids were extracted. Theextracted lipids were assayed for phospholipid acyl moiety syn-thesis as given in Methods.

0-

. 11__

o )

CO

>00.-0- ess

a: ,-

B

2.5

2.0

1.5 -.--CY99(+)

. CY97 (+/-) -- -- a I1.0

0.5

I01 2 3 4 5Hrs after shift to 150

and CY104, the strains carrying only a single functional fabAgene. However, this experiment is rather insensitive. There-fore, a much more sensitive experiment involving pulse label-ing of cultures after temperature decrease was done4.The four strains were grown at 420 (in order to irreversibly

inactivate the temperature-sensitive fabA2 defect included inthe +/- and -/+ strains), then shifted to 15°. As shown inFig. 3A, the rate of synthesis of phospholipid cis-vaccenylmoieties shows a complex pattern after temperature shift.However, similar patterns were seen in all four strains exceptthat the rate of synthesis in strain CY99 (+/+) is 1.3-1.9times greater than the rate shown by the other strains. Thisdifference is more easily appreciated (Fig. 3B) when the dataat each time point are normalized to the datum at that timepoint of the + strain, CY59 (set at 1.0). During the first fourhours after temperature shift, the rate of unsaturated fattyacid synthesis in the +/+ strain is 1.67 0.21 times the rateshown by the +, +1-, and -/+ strains (mean of 15 deter-minations).

Since strains carrying +- and -/+ genotypes behave ina manner very similar to the + monoploid strain, I concludethat an increase in the dose of the fabA gene per se is respon-

sible for the increased synthesis of phospholipid unsaturatedacyl moieties observed in the +/+ strains.The increased rate of synthesis of unsaturated moieties seen

in the +/+ strain is transient (Fig. 3). This transient period

t Although the fabA - lesion used in these experiments is a tem-perature-sensitive defect (fabA + at temperatures <370), theenzyme coded by the fabA2 allele is irreversibly inactivated byheating at 420 either in vivo (Gelmann and Cronan, unpublished)or in vitro (9). Since the enzyme does not spontaneously renature,only after several generations of growth at 150 do + /- or -/+strains approximate a +/+ strain. However, since the effectsseen in the temperature decrease experiments are essentiallycomplete after 0.3 generation, the nature of thefabA lesion used isnot a complication.

FIG. 3. Rate of cis-vaccenic acid (CVC) synthesis in variousstrains after temperature decrease. Cultures of the above strainswere grown in parallel in EC medium at 420 for several genera-tions. When the culture density reached 108/ml, the cultures wereshifted to a 150 water-bath shaker. At various time intervals,40-ed samples were removed to 250-ml flasks containing 10 1ACi of["4C] acetate in the same water bath. After a 15-min exposure tothe label, the cells were quickly harvested and the lipids extractedand analyzed as described in Methods. For clarity, only the dataon cis-vaccenate synthesis are shown (the data for palmitoleatesynthesis are very similar). These data are given in panel A.In panel B, the datum for each strain at that time point hasbeen divided by the datum for strain CY59 (+) and thus eachtime point has been normalized to strain CY59 (+) which is setat 1.0. The 70-hr sample in panel B is from a parallel experimentin which cultures of CY59 and CY99 were diluted such as to main-tain the culture in log phase for 70 hr at 150.

ends when the strain attains the overall fatty acid compositioncharacteristic of a culture grown several generations at 150(Fig. 3).Coupling between unsaturated fatty acid andphospholipid synthesisIt was shown in Table 1, that during growth at a constanttemperature, the presence of an increased dehydrase level hadonly a small effect on the content of unsaturated fatty acylgroups. It was thought that perhaps an increased amount ofunsaturated fatty acids had been synthesized by these strainsbut was not observed since these acids were not phospholipidcomponents. Therefore, the possibility of synthesis of non-

phospholipid unsaturated fatty acid was examined. Thechloroform-methanol extraction used to obtain the phos-pholipid fraction would also extract free fatty acid (25).The amount of free fatty acid in all cultures examined -was<1% of the phospholipid fatty acid even after temperaturedecrease [a result contrary to that of Okuyama (29)]. How-ever, free fatty acid might have been absent due to its deg-

Ado.,, CYI04(-/+)

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radation via fl-oxidation. Another possibility is that the fattyacid was covalently bound to a hydrophilic molecule and thuswas not extracted by chloroform-methanol. The possibility offatty acid degradation was eliminated by the presence of alesion in the fadE gene (14) of the fl-oxidation pathway in thestrains given in Table 2. This lesion-reduces a-oxidation inthese strains to <1% of the normal activity. The possibility ofcovalent bonding of nonphospholipid fatty acid is unlikely,since neither acid nor basic hydrolysis of entire cultures (cellsplus medium) yields any unsaturated fatty acid not attrib-utable to the phospholipids. It should also be pointed outthat growth rate and the kinetics of cyclopropane fatty acidsynthesis in all these strains were identical under all conditionsof growth.

DISCUSSION

The data in this paper indicate that the level of g3-hydroxy-decanoyl thioester dehydrase limits the unsaturated fatty acidcontent of the phospholipids. Therefore, it is apparent that theacyltransferase specificity is not the sole determinate of theratio of saturated to unsaturated fatty acid in the membranephospholipids, but that the level of at least this fatty acid bio-synthetic enzyme is closely tuned to the demands of phos-pholipid synthesis.Unsaturated acyl moieties can be synthesized at almost

twice the normal rate (for a brief period) in strains possessingtwice the normal dehydrase activity, whereas under steadystate conditions the presence of the additional dehydrase ac-tivity has only a small effect on fatty acid composition. Theseresults indicate the presence of regulatory step (or steps)beyond the fatty acid biosynthetic pathway which controlsthe amounts of unsaturated acid found in phospholipid. Thisstep is temperature-controlled (Table 1) and becomes ap-parent after a temperature shift-down, at about the time thephospholipids reach the fatty acid composition characteristicof the growth temperature and the strain (Fig. 3). The tem-perature behavior of this regulatory site is consistent with thatattributed to the acyltransferases by Sinensky (12). Thesecond property may be a mechanism to maintain the physicalproperties of phospholipids within narrow limits such as thatobserved by Esfahani and co-workers (5, 6) for incorporationof exogenous fatty acids. Results similar to those of Esfahaniet al. (5, 6) were previously obtained by Silbert and coworkers(4, 28, 30) and an interpretation based on acyltransferase speci-ficity was suggested. Therefore, it seems likely that the regu-latory step operative after fatty acid biosynthesis is at theacyltransferase level. However, it should be noted that someof the data on which acyltransferase specificity is based havebeen recently challenged (31). These results must be recon-ciled with the previous data before a conclusion on the role ofthe acyltransferases in the regulation of fatty acid compositioncan be made.My results also suggest that unsaturated fatty acids are not

synthesized unless they can be incorporated into phospho-lipid. Similar conclusions have been made using mutants de-fective in phosphatidic acid synthesis (1, 13, 32). Therefore, it

appears that fatty acid and phospholipid synthesis are tightlycoupled to one another.

I thank Edward P. Gelmann for performing some of the earlyexperiments in this study and Dr. William Nunn for his valuableadvice on the work and the manuscript. This work was supportedby research grants from the National Institutes of Health (AI10186) and the National Science Foundation (GB 32063) and aResearch Career Development Award (I F02-AI 55,327) fromthe National Institutes of Health.1. Cronan, J. E., Jr. & Vagelos, P. R. (1972) Biochim. Bio-

phys. Acta 265, 25-60.2. Singer, S. J. & Nicolson, G. L. (1972) Science 175, 720-726.3. Van Deenen, L. L. M. (1965) in Progress in the Chemistry of

Fats and Other Lipids, ed. Holman, R. T. (Pergamon Press,New York), Vol. VII, Part I, pp. 1-115.

4. Silbert, D. F., Ruch, F. & Vagelos, P. R. (1966) J. Bacteriol.95, 1658-1685.

5. Esfahani, M., Ioneda, T. & Wakil, S. J. (1971) J. Biol.Chem. 246, 50-56.

6. Esfahani, M., Barnes, E. M., Jr. & Wakil, S. J. (1969)Proc. Nat. Acad. Sci. USA 64, 1057-1064.

7. Haest, C. W. M., DeGier, J., Van Es, G. A., Verkleij, A. J.& Van Deenan, L. L. M. (1972) Biochem. Biophys. Acta288, 43-53.

8. Nunn, W. D. & Cronan, J. E., Jr. (1974) J. Biol. Chem.249, 724-731.

9. Cronan, J. E. Jr. & Gelmann, E. P. (1973) J. Biol. Chem.248, 1188-1195.

10. Van den Bosch, H. & Vagelos, P. R. (1970) Biochim. Bio-phys. Acta 218, 233-248.

11. Ray, T. K., Cronan, J. E., Jr., Mavis, R. D. & Vagelos, P. R.(1970) J. Biol. Chem. 245, 6442-6448.

12. Sinensky, M. (1971) J. Bacteriol. 106, 449-455.13. Mindich, L. (1972) J. Bacteriol. 110, 96-102.14. Klein, K., Steinberg, R., Fiethen, B. & Overath, P. (1971)

Eur. J. Biochem. 19, 442-450.15. Marr, A. G. & Ingraham, J. L. (1962) J. Bacteriol. 84,

1260-1267.16. Bloch, K. (1971) in The Enzymes, ed. Boyer, P. D. (Aca-

demic Press, Inc., New York), Vol. 5, 3rd ed., pp.441-464.17. Cronan, J. E., Jr., Nunn, W. D. & Batchelor, J. G., (1974)

Biochim. Biophys. Acta 348, 63-75.18. Cronan, J. E., Jr. & Bell, R. M. (1974) J. Bacteriol., in

press.19. Cronan, J. E., Jr. & Godson, G. N. (1972) Mol. Gen. Genet.

116, 199-210.20. Cronan, J. E., Jr., Silbert, D. F. & Wulff, D. L. (1972) J.

Bacteriol. 112, 206-211.21. Cronan, J. E., Jr. & Bell, R. M. (1974) J. Bacteriol. 118,

598-05.22. Fan, P. (1969) Genetics 61, 351-359.23. Low, K. B. (1973) J. Bacteriol. 113, 798-812.24. Gelmann, E. P. & Cronan, J. E., Jr. (1972) J. Bacteriol. 112,

381-387.25. Cronan, J. E., Jr. & Wulff, D. L. (1969) Virology 38,

241-246.26. Low, K. B. (1972) Bacteriol. Rev. 36, 587-607.27. Harder, M. E., Beacham, I. R., Cronan, J. E., Jr., Beacham,

K., Honegger, J. L. & Silbert, D. F. (1972) Proc. Nat. Acad.Sci. USA 69, 3105-3109.

28. Silbert, D. F. (1970) Biochemistry 9, 3631-3640.29. Okuyama, H. (1969) Biochim. Biophys. Acta 176, 125-134.30. Silbert, D. F., Cohen, M. & Harder, M. E. (1972) J. BRio.

Chem. 247, 1699-1707.31. Okuyama, H. & Wakil, S. J. (1973) J. Biol. Chem. 248,

5197-5205.32. Bell, R. M. (1973) J. Bacteriol. 117, 1065-1076.

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