JouRNAL OrBAcTaoLoGY, Apr. 1972, p. 96-102Copyright @ 1972 American Society for Microbiology

Vol. 110, No. 1Printed in USA.

Control of Fatty Acid Synthesis in BacteriaLEONARD MINDICH

Department of Microbiology, The Public Health Research Institute of the City ofNew York, Inc., New York,New York 10016

Received for publication 26 November 1971

When glycerol-requiring auxotrophs of Bacillus subtilis are deprived of glyc-erol, the synthesis of fatty acids continues at an apparent rate of 20 to 50% thatof supplemented cultures. The newly synthesized fatty acids are not incorpo-rated into phospholipid and accumulate as free fatty acids. These moleculesundergo a much more rapid turnover than phospholipid fatty acids, and therate of turnover is sufficient to indicate that the rate of fatty acid synthesis inglycerol-deprived cultures is similar to that in supplemented ones. The average

chain length of the free fatty acids is greater than that of the phospholipid fattyacids. Cells deprived of required amino acids also show a diminution in theapparent rate of fatty acid synthesis; however, in this case, the fatty acids ac-cumulate in phospholipid, and no increase of the free fatty acid fraction is ob-served. It is argued on the basis of these fmdings that the control of lipid syn-thesis does not operate at the level of transacylation but must act on one ormore of the reactions of the fatty acid synthetase.

The synthesis of fatty acids has been studiedin both eukaryotic and prokaryotic organisms.Although there has been excellent progress inthe elucidation of the individual steps of fattyacid synthesis, there has been rather smalladvancement made in determining the mannerin which the process is regulated (8) both interms of the extent of synthesis and the finalcomposition of fatty acids. Acetyl coenzyme A(CoA) carboxylase is the rate-limiting enzymein fatty acid synthesis in vitro, and many ofthe schemes for regulating fatty acid synthesisinvolve the regulation of the activity of thisenzyme. Regulation of the rate of fatty acidsynthesis can involve variations in the amountof enzyme per cell, feedback inhibition byproducts of fatty acid synthesis such as fattyacids or their derivatives, or modulation of ac-tivity by the allosteric interaction of othermolecules with the components of the fattyacid synthetase.Evidence is presented in this paper that the

rate of fatty acid synthesis in at least two spe-cies of bacteria, Bacillus subtilis and Staphylo-coccus aureus, is not regulated by feedbackinhibition mechanisms involving free fattyacids, nor is the control of phospholipid syn-thesis at the transacylation step. We have in-vestigated the synthesis of fatty acids in bac-terial mutants that are defective in glycerolbiosynthesis (10, 11). These organisms cease

net phospholipid synthesis when deprived ofglycerol. However, they continue to incorpo-rate radioactivity from glucose or acetate intolipid, albeit at a diminished rate (10, 11, 16).We have found that fatty acids are made atnormal rates in the absence of complex lipidformation and that the apparent diminution ofthe synthetic rate is due to the concurrent deg-radation of these fatty acids.

MATERIALS AND METHODSBacterial strains and media. Two strains of B.

subtilis were used: strain 168-2, obtained from DavidDubnau; and strain B42, a mutant of strain SB1 thatis auxotrophic for histidine (his), tryptophan (try),and glycerol (glyc) and lacks glycerol phosphatedehydrogenase (glpD) (10). Escherichia coli strainGR-1, auxotrophic for glycerol, was obtained from C.F. Fox (5). Cells were cultured in a defined mediumconsisting of salts, phosphate buffer, and aminoacids as described previously (10). Media werechanged by filtering and washing cells on membranefilters and resuspending the cells in prewarmed me-dium.

Isolation and analysis of lipids. Lipids were ex-tracted by the method of Bligh and Dyer (1) andsubjected to thin-layer chromatography (TLC) onBrinkman silica gel plates in petroleum ether-di-ethyl ether-acetic acid (70:30:2), followed by autora-diography and counting of material scraped off theplates in the areas corresponding to the radioactivespots (14). Standard mixtures containing mono-, di-,and triglycerides and palmitic acid (Applied Science

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CONTROL OF FATTY ACID SYNTHESIS

Laboratories, TLC-8) or ,8-hydroxy, ,8-keto- and a-a-unsaturated fatty acids (gift of M. Pullman) wererun along with radioactive samples on silica gelplates. Samples were also chromatographed onWhatman SG-81 silica gel-loaded paper in solvent 1of Wuthier (17). Radioactive spots were cut out afterautoradiography. Radioactivity from TLC plates wasdetermined in a Beckman LS2B scintillation counterin a mixture of dioxane, napthalene, and 2,5 diphen-yloxasole; radioactivity from the paper was deter-mined in a toluene-based mixture. Fatty acids wereisolated from cells by saponification as describedpreviously (10). Methyl esters of the phospholipidfatty acids were prepared by transmethylation inmethanol-benzene-sulfuric acid (87.5: 12.5:2) at 60 Cfor 3 hr or by reaction with boron trifluoride-meth-anol reagent [Applied Science Laboratories, Inc. (9)]after saponification. Methyl esters of free fatty acidswere prepared with the boron trifluoride-methanolreagent. Fatty acids were decarboxylated by the pro-cedure of Brady, Bradley, and Trams (2) and Gold-fine and Bloch (4). Methyl esters of fatty acids weresubjected to gas chromatography, using a column ofethylene glycol-succinate (15% Hi Eff 2BP by weighton Gas Chrom P; Applied Science Laboratories)with a 6-min period at 120 C and a programmedtemperature rise of 7.5 degrees per min until 170 Cwas reached. Chain length was determined by com-parison with standard mixtures and with the knowncomposition of B. subtilis fatty acids (6). Samples ofthe methyl esters were collected for determination ofradioactivity by placing. pasteur pipettes lightlyplugged with cotton on the outlet of the sample

A

column before the detector (unpublished procedureof M. Pullman). Sample recoveries were 83%.

RESULTSAccumulation of free fatty acids during

glycerol deprivation. When a culture of B.subtilis, auxotrophic for glycerol, is deprived ofglycerol, net phospholipid synthesis ceases(10). The incorporation of radioactivity from"4C-glucose into lipid is immediately depressedto a rate that is approximately 20 to 50% of thatin glycerol-supplemented cultures (10). Todetermine the nature of the lipid in which theradioactivity was accumulating, we chromato-graphed samples of lipid from cells grown with"4C-acetate or "4C-glucose in the presence andabsence of glycerol. The distribution of ra-dioactivity in various lipids is shown in Fig. 1.It is clear from Fig. 1B that phospholipid andneutral lipids are labeled in the presence ofglycerol and that only neutral lipid is labeledin the absence of glycerol. The chromatogramshown in Fig. 1A, which was developed in aless polar solvent than that used in 1B, re-solves the neutral lipids and clearly indicatesthat the neutral lipid that is labeled in theabsence of glycerol has the same RF as pal-mitic acid. The material in this spot was fur-ther identified as free fatty acid on the basis ofthe partitioning of the radioactivity between

,

.

.. ...i.

NL

PL4'. * FA

.;DG

Pe.. + .................................o. .

N t|

FIG. 1. Autoradiographic patterns of B. subtilis lipids after chromatography. An exponentially growingculture of strain B42 was filtered, washed, and suspended in medium with 0.01 M sodium acetate-l- "C (51sCi/ml). One-half was incubated with glycerol for 45 min (G) and one-half without glycerol (N). The cellswere then collected by centrifugation, and the lipids were extracted by the method of Bligh and Dyer (1) andchromatographed (A) on silica gel plates in petroleum ether-diethyl ether-acetic acid (70:30:2) along with"IC-labeled palmitic acid (P); or (B) on Whatman SG-81 silica gel-loaded paper in the solvent of Wuthier(17). NL, neutral lipid; PL, phospholipid; FA, free fatty acid; DG, diglyceride.

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pentane and aqueous solvents at high and lowpH and the behavior of methylated derivativesduring gas-liquid chromatography. The ra-dioactivity from deprived cells did not migrateon silica gel plates in a manner similar to un-saturated fatty acids, ,B-keto-fatty acids, fi-

hydroxy-fatty acids, monoglycerides, diglycer-ides, or triglycerides. The accumulation of ra-

dioactivity into various lipid classes in cellsincubated in the presence or absence of glyc-erol was shown for S. aureus and B. subtilis intwo papers (11, 16).

Lipids from the spot for the presumed freefatty acids were methylated with the boron tri-fluoride-methanol reagent, and the productswere examined by gas-liquid chromatography.Figure 2 shows the distribution of methyl es-ters of the free fatty acids of a deprived cultureand those from the phospholipids of a supple-mented culture. The latter sample was methyl-ated after saponification. Although the peaksin the sample from deprived cells are in thepositions characteristic of methyl esters offatty acids found in B. subtilis, the relativeamounts are changed to a preponderance oflonger-chain fatty acids. Whereas the normallipids contain mostly Cl0 and Cl7 fatty acids,the free fatty acid fraction from the deprivedcells is predominantly C17 and C,., with a sig-nificant amount of fatty acid that might beC i. The radioactivity of the fatty acids followsthe pattern of mass rather closely in both thefree fatty acids and the phospholipid fattyacids. Since the longer chains could be due to

Cl7 cig

10 20

minutes

FIG. 2. Gas-liquid chromatogram, on an ethyleneglycol-succirate column, of the methyl esters of thefatty acids isolated from B. subtilis B42 phospho-lipids (b) and of the free fatty acids isolated from asimilar culture deprived of glycerol for 45 min (a).

a lengthening of preformed chains, we deter-mined the ratio of radioactivity in the terminalcarboxyl groups of the fatty acids relative tothat of the total molecules. Table 1 shows thatthe fraction of radioactivity in the terminalcarboxyl group was approximately the samefor lipids from deprived as well as supple-mented cells. In fact, the ratio of the terminalto total radioactivity is close to what would bepredicted on the basis of the average chainlength of the free fatty acids in the deprivedcells (18 carbon atoms) and of the phospho-lipid-associated fatty acids of the supple-mented cells (16 carbon atoms), if one assumesthat the bulk of these molecules are synthe-sized from a five-carbon precursor that islengthened by the addition of two-carbon units(7). If the fatty acids were labeled by the addi-tion of radioactive acetate to preformed (phos-pholipid-derived) fatty acids, the ratio of totalradioactivity to carboxyl radioactivity wouldhave approached 1.Turnover of the free fatty acids. We found

that the level of free fatty acid in the cell wasthe resultant of synthesis and breakdown ofthese molecules. A culture grown in the ab-sence of glycerol for 45 min with '4C-labeledacetate accumulated radioactivity in the freefatty acid fraction. If the culture was then fil-tered, and nonradioactive acetate was added tothe medium, the amount of radioactivity inthe free fatty acids declined, as shown in Fig.3. The rate constant for this decay is 0.026 permin. When the culture was supplemented with

TABLE 1. Decarboxylation of fatty acids isolatedfrom cells of B. subtilis incubated with and without

glycerola

Radio- Radio-Glycerol in activity activity Total/CO2Glyceol in in total in CO,culture sample relemsdFon Exetd(counts/ (counts/ Found Expected'

min) min)

Absent 8,270 1,200 6.89 6.5Present (20 16,365 3,029 5.40 5.5

4g/ml)a Strain B42 was incubated for 1 hr with acetate-l-

14C in the presence or absence of glycerol. The totalfatty acids were collected by saponification and de-carboxylated as described in the text. Total radioac-tivity was compared with that released as carbondioxide.

'Expected on the basis of an average chain lengthof C1. for the deprived culture and C,, for the sup-plemented one. Since these fatty acids are synthe-sized from an initial five-carbon unit and elongatedby two-carbon additions, we expect 5.5 two carbonunits in the former case and 6.5 in the latter.

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a 0.8z

z

0.6

0.4

1-

h!

0 20 40

minutes

a

c-

z

zz

w

U1)1--z

z

0

41

U&.

0.8k

0.61_

0.4j

0 4020minutes

FIG. 3. Turnover of fatty acids labeled in the ab-sence of glycerol. A culture of B. subtilis B42 was

incubated for 45 min with '4C-acetate in the absenceof glycerol to label free fatty acids. The cells were

then washed and transferred to medium still lackingglycerol but containing 0.01 M acetate with no ra-

dioactivity. Samples were saponified, and radioac-tivity of the fatty acids was measured. Ordinate hasa logarithmic scale.

glycerol after filtration, the decline of radioac-tivity in the lipid fraction was less, and labelwas found in the phospholipid fraction of thecells. Radioactive acetate incorporated in thepresence of glycerol has a very low rate ofturnover during subsequent incubation in thepresence or absence of glycerol (Fig. 4).The rate of accumulation of radioactivity in

the free fatty acid fraction of the deprived cellswas 32 counts per min per min per ml. If therate constant for turnover in these cells is0.026/min and the amount of radioactivity infree fatty acids is 1,420 counts per min per mlat the time of the turnover measurement, thenthe rate of fatty acid turnover is 37 counts permin per min per ml. The rate of synthesis isthe sum of the rate of accumulation and therate of turnover, i.e., 69 counts per min per minper ml. The rate of accumulation is therefore46% of the rate of synthesis. This figure agreesvery well with the observed rates of fatty acidsynthesis in glycerol-deprived cells relative tosupplemented ones. We usually observe a rateof accumulation that is between 20 and 50%the rate of normal fatty acid synthesis.

This finding implies that the glycerol depri-vation is not affecting the rate of fatty acidsynthesis. The deprived cells are making fattyacids at their normal rate; however, these fattyacids are subject to breakdown and thereforeaccumulate at the observed low rate. We haveseen the same results in glycerol auxotrophs of

FIG. 4. Thrnover of fatty acids labeled in thepresence of glycerol. A culture of B. subtilis B42 was

incubated for 45 min with "C-acetate in the pres-

ence of glycerol. The cells were then washed andtransferred to medium containing 0.01 M acetatewith (0) or without (0) 20 ug of glycerol/ml. Sam-ples were saponified, and radioactivity of the fattyacids was measured. Ordinate has a logarithmicscale.

S. aureus (11). In a glycerol auxotroph of E.coli (5), we have found a much lower level ofincorporation into free fatty acids; however, we

found no such incorporation into glycerol-sup-plemented cells. Figure 5B shows the incorpo-ration of "C-acetate into the free fatty acidfraction of glycerol-deprived E. coli, and Fig.5A shows the incorporation into total phospho-lipids.

Effect of amino acid deprivation on fattyacid synthesis. It has been shown that aminoacid deprivation leads to a reduction in theapparent rate of fatty acid synthesis in E. coli(15). We find the same result with B. subtilis.The fatty acids that are synthesized in theabsence of required amino acids are incorpo-rated into phospholipid and do not remain as

free fatty acids; there is no increased appear-ance of free fatty acids during amino acid de-prival. The normal level was found to be 2% ofthe total fatty acids both in strain B42 and instrain 168-2, which is prototrophic for glycerol.Figure 6 shows the incorporation of "4C-acetateinto free fatty acids and phospholipids underconditions of amino acid deprivation, glyceroldeprivation, and chloramphenicol addition (70ug/ml). One can see that glycerol deprivalleads to free fatty acid accumulation, but thisdoes not occur when protein synthesis is inhib-ited either by amino acid deprival or chloram-phenicol. When cells are deprived of aminoacids and glycerol simultaneously (unpub-

0

* 0

o 0

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80 0 20minutes

FIG. 5. Incorporation of radioactive acetate into phospholipids and free fatty acids of E. coli strain GR-1(5). An exponentially growing culture was filtered and resuspended in medium with 0.01 M sodium acetate-l-'IC (5 uCi/ml) with 20 pg of glycerol/ml (0) or without glycerol (S). Samples were taken at indicated times,and lipids were extracted and isolated by thin-layer chromatography. Accumulation of radioactivity intophospholipids is shown in (a) and into free fatty acids in (b).

a b 12

~~~~05-10

0

4- ~~~~~~~~~~~~~~8~~~~~~~~~~0

3-z3

w

2-0 4

wA

~~~~~~~~~~~~~~~2

0~~~~~~~~~~~~~~~~~~~~~~~~~~~~120 40 60 80 20 40 60 80

m inut es

FIG. 6. Incorporation of radioactive acetate into phospholipids and free fatty acids of B. subtilis B42 underconditions of inhibition of protein synthesis or glycerol deprivation. An exponentially growing culture was fil-tered and resuspended in medium with 0.01 M sodium acetate-l-14C (5 uCi/mI) with amino acids and glycerol(20 pg/mI) (0), with amino acids but lacking glycerol (0), with glycerol but lacking tryptophan and histidine(0), or with glycerol, amino acids, and chloramphenicol (70 ug/ml) (A). Samples were taken at indicatedtimes, and lipids were extracted and isolated by thin-layer chromatography. Accumulation of radioactivityinto phospholipids is shown in (a) and into free fatty acids in (b).

lished data), the rate of fatty acid accumula- prived culture, there is a rapid resumption oftion does not decrease below that for glycerol the normal rate of fatty acid synthesis.deprival alone, and the fatty acids accumulatein their free form.The inhibition of fatty acid synthesis ob- DISCUSSION

served after amino acid deprivation is quicklyreversed by the readdition of amino acids. This The cell membrane is a complex and impor-is illustrated in Fig. 7, where it can be seen tant structure. The synthesis of the membranethat, upon readdition of amino acids to a de- components must be under some sort of regu-

EC.)0

0

a:z

w

wC..

.1uIt

0

3

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CONTROL OF FATTY ACID SYNTHESIS

6ECL

0.

to

04

4

z

wX0

0

FIG. 7. Incorporfatty acids of B.amino acid depriLture was filtered i

0.01M sodium a

mixture containinlacking these am

sample of the depwith tryptophanremoved at the inradioactivity of th

lation so as to k

tity of the memfor the econom'

previously (10, Ibrane proteinsi

of lipid synthesmembrane. Instrated that fatthe control oferol auxotrophsand therefore Elipids accumula

mately 20 to 5(thesis in supplrate of synthesthat in the sup

acids synthesizappear as free fhave only a ver3acids, about 2%proportion can r

Whereas the Iare stable duriniof free fatty acic

The free fatty acids are also longer in chainlength than the phospholipid-bound fattyacids. This supports the possibility that thedeterminant of chain length is the transacylaseand that, in the absence of an acceptor, thechains are allowed to elongate further beforebeing freed from the acyl carrier protein on

which they are synthesized (8). It is possiblethat the transacylase is normally the means by

o which fatty acid is split from the acyl carrierprotein but that the specificity is altered whenwater instead of glycerol phosphate is the ac-ceptor. It is also possible that a different en-

zyme system is responsible for the cleavageo* reaction. It seems clear as a result of these

t1

findings that, at least for B. subtilis and S.aureus, neither glycerol phosphate nor a feed-back mechanism (3) controls fatty acid syn-

I_____________________________________lthesis.The results with E. coli are of interest in

20 40 60 that we find much less accumulation of free

minutes fatty acids during glycerol deprivation in thisspecies than was found with the two gram-

ration of radioactive acetate into positive species. These results can be recon-

subtilis B42 under conditions of ciled by several possibilities. The first would

)al. An exponentially growing cul- be that there is rapid turnover of theresuspended in medium with

.u.i.icetate-l-"4C with an amino acidfattyacids in colithan in subtolisLgtryptophan and histidine (0) or

and S. However,

measurementof

mino acids (0). After 30 min, a turnover rate in E. coli shows that it is not suf-

)rived culture was resupplemented ficient to explain the lack of accumulation.and histidine (A). Samples were The accumulation of fatty acids would also bedicated times and saponified, and expected to be much greater in E. coli growne fatty acids was measured. on glucose because of the catabolic repression

of the fatty acid degradation system (13). Al-;eep the composition and quan- though more free fatty acids accumulate in E.

brane within acceptable limits coli grown with glucose as a carbon source asy of the cell. We have shown compared to cells grown on amino acids, the11) that the synthesis of mem- level of accumulation is still much lower thans not regulated by the progress that found for the gram-positive cultures. The3is or the lipid content of the alternative possibilities would be that (i) thethis paper we have demon- acyl carrier protein derivatives of the fattyty acid synthesis is not under acids are more stable in E. coli and the synthe-complex lipid synthesis. Glyc- sis of new fatty acids is limited by the availabil-that are deprived of glycerol ity of acyl carrier protein; (ii) the fatty acids are

are not synthesizing complex synthesized by being incorporated into non-ite free fatty acids at approxi- extractable compounds such as lipid A of theD% the rate of fatty acid syn- cell envelope (12); or (iii) a feedback controlemented cultures. The actual mechanism does operate on E. coli, and theis appears to be the same as low levels of free fatty acids observed are suf-iplemented cultures. The fatty ficient to prevent further synthesis.z

ed in the deprived cultures Another difference between B. subtilis andatty acids. Ordinarily the cells E. coli in the control of fatty acid synthesis is

y small proportion of free fatty the response to chloramphenicol. Sokawa et al.); in the deprived cultures this (15) demonstrated that stringent cultures of E.r

each as high as 20%. coli make fatty acids at a reduced rate whenphospholipid-bound fatty acids deprived of amino acids. They found thatg glycerol deprivation, the pool chloramphenicol partially reversed this effectis is turning over quite rapidly. in a manner somewhat similar to the control of

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MINDICH

ribosomal ribonucleic acid (RNA) synthesis.We found that amino acid deprivation reducesthe rate of fatty acid synthesis in B. subtilisB42, which is stringent for RNA synthesis, butthat chloramphenicol does not reverse the ef-fect of amino acid deprival on fatty acid syn-thesis although it does reverse the effects onRNA synthesis (unpublished data). Moreover,we found that chloramphenicol inhibits fattyacid synthesis even in the presence of aminoacids. Rifamycin and actinomycin D, anti-biotics which inhibit RNA synthesis and con-sequently protein synthesis, also inhibit fattyacid synthesis. Although it is tempting to im-plicate the same regulatory mechanism in bothRNA and fatty acid synthesis, it appears, atleast for B. subtilis, that although similaritiesexist there is not an identical control mecha-nism. It seems more likely that protein syn-thesis is necessary for normal rates of fattyacid synthesis. The observations (8) that acetylCoA carboxylase and fatty acid synthetaseshow changes in level in starved animals isperhaps a similar phenomenon.The low level of accumulation of labeled free

fatty acids during amino acid deprivation of B.subtilis B42 is probably not the result of con-trol at the level of the transacylase reaction,since it is clear that the free fatty acids doaccumulate in this organism when glycerol ismissing. The most likely means of controlwould therefore be a change in the levels of thefatty acid synthetase enzymes resulting fromreduced rate of synthesis along with turnoveror a modulation of the enzymatic activities byas yet unknown effector molecules.

ACKNOWLEDGMENTSThis investigation was supported by Public Health

Service grant AI-09861-04 from the National Institute ofAllergy and Infectious Diseases.

The experiments were performed with the excellent tech-nical assistance of Valerie Dubac. I greatly appreciate theadvice of M. Pullman and D. C. White on lipid chemistrytechniques, and that of L. Day on kinetics.

LITEATURE CITED1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of

total lipid extraction and purification. Can. J.Biochem. Physiol. 37:911-917.

2. Brady, R. O., R. M. Bradley, and E. G. Trams. 1960.Biosynthesis of fatty acids. I. Studies with enzymesobtained from liver. J. Biol. Chem. 235:3093-3098.

3. Esfahani, M., T. loneda, and S. J. Wakil. 1971. Studieson the control of fatty acid metabolism. III. Incorpo-ration of fatty acids into phospholipids and regulationof fatty acid synthetase of Escherichia coli. J. Biol.Chem. 246:50-56.

4. Goldfine, H., and K. Bloch. 1961. On the origin of un-saturated fatty acids in Clostridia. J. Biol. Chem. 236:2596-2601. &

5. Hsu, C. C., and C. F. Fox. 1970. Induction of the lactosetransport system in a lipid-synthesis-defective mutantof Escherichia coli. J. Bacteriol. 103:410-416.

6. Kaneda, T. 1963. Biosynthesis of branched chain fattyacids. I. Isola-tion and identification of fatty acidsfrom Bacillus subtilis (ATCC 7059). J. Biol. Chem.238:1222-1228.

7. Kaneda, T. 1963. Biosynthesis of branched chain fattyacids. II. Microbial synthesis of branched long chainfatty acids from certain short chain fatty acid sub-strates. J. Biol. Chem. 238:1229-1235.

8. Majerus, P. W., and P. R. Vagelos. 1967. Fatty acid bio-synthesis and the role of the acyl carrier protein.Advan. Lipid Res. 5:1-33.

9. Metcalfe, L. D., and A. A. Schmitz. 1961. The rapidpreparation of fatty acid esters for gas chromato-graphic analysis. Anal. Chem. 33:363-364.

10. Mindich, L. 1970. Membrane synthesis in Bacillus sub-tilis. I. Isolation and properties of strains bearingmutations in glycerol metabolism. J. Mol. Biol. 49:415-432.

11. Mindich, L. 1971. Induction of Staphylococcus aureuslactose permease in the absence of glycerolipid syn-thesis. Proc. Nat. Acad. Sci. U.S.A. 68:420-424.

12. Osborne, M. J. 1969. Structure and biosynthesis of thebacterial cell wall. Annu. Rev. Biochem. 38:501-538.

13. Overath, P., Pauli, G., and H. U. Schairer. 1969. Fattyacid degradation in Escherichia coli. An inducibleacyl-CoA synthetase, the mapping of old-mutationsand the isolation of regulatory mutants. Eur. J.Biochem. 7:559-574.

14. Skipsky, V. P., and B. Barclay. 1969. Thin-layer chro-matography of lipids, p. 530-598. In J. M. Lowenstein(ed.), Methods in enzymology, vol. 14. AcademicPress Inc., New York.

15. Sokawa, Y., E. Nakao, and Y. Kaziro. 1968. On the na-ture of the control by RC gene in E. coli: amino acid-dependent control of lipid synthesis. Biochem. Bio-phys. Res. Commun. 33:108-112.

16. Willecke, K., and L. Mindich. 1971. Induction of citratetransport in Bacillus subtilis during the absence ofphospholipid synthesis. J. Bacteriol. 106:514-518.

17. Wuthier, R. E. 1966. Two-dimensional chromatographyon silica gel-loaded paper for microanalysis of polarlipids. J. Lipid Res. 7:544-550.

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