JOURNAL OF BACTERIOLOGY, Jan. 1981, p. 472-4780021-9193/81/010472-07$02.00/0

Vol. 145, No. 1

Lipid Synthesis During the Escherichia coli Cell CycleC. E. CARTY AND L. 0. INGRAM*

Department ofMicrobiology and Cell Science and Department ofImmunology and Medical Microbiology,University ofFlorida, Gainesville, Florida 32611

Lipid synthesis was examined in Escherichia coli cells at different stages of celldivision. Exponentially growing cells were pulse-labeled with appropriate isotopesfor 0.1 generation time, inactivated, and separated by size on a sucrose gradient.An abrupt increase in the rate of lipid synthesis occurred which was coincidentwith the initiation of cross walls. In contrast, the rate of protein synthesis duringthis same interval remained constant, resulting in an increased lipid/protein ratioin dividing cells. No changes in the composition of phospholipid head groups,fatty acids, or phospholipid molecular species were observed in cells at differentstages of division. The observed increase in the rate of lipid synthesis may reflecta means by which the activities of membrane-associated enzymes are modulatedduring cross wall formation.

Bacterial cell division requires the complexcoordination of diverse cellular processes. Agreat deal is known about the temporal couplingof DNA synthesis to the division process (9, 15,21-23). The completion of a round of genomereplication is essential for the initiation of a newcross wall. However, relatively little is knownabout the conditions that actually initiate crosswall formation (5). The rate of peptidoglycansynthesis increases during cross wall formation(32, 33) as do the activities of murein hydrolases(3, 20). The synthesis ofa new cross wall dependsupon the coordination of these processes withother membrane-associated processes such aslipopolysaccharide synthesis, membrane proteinsynthesis, and lipid synthesis.Membrane lipids could be involved in the

coordination of biosynthetic activities whichproduce cross walls. Lipids are essential for theactivities of many membrane-bound enzymes(10). Both the types and quantities of lipids canalter enzyme activity (10, 17, 18, 24, 41). Mem-brane lipids are highly regulated in bacterialsystems (11, 36). Extreme alterations in mem-brane lipid composition have been shown toblock the processing and proper insertion ofsome membrane proteins (13,14). The inhibitionof lipid synthesis prevents the expression butnot the synthesis of aminopeptidase N (29).Changes in the total activity of specific mureinhydrolases during the cell cycle (3, 20) may bethe result of differential expression of hydrolaseenzyme activity rather than a differential rate ofsynthesis of the hydrolase enzyme per se (31).Such changes in the expression of enzyme activ-ity could be the result of regulation involvingthe membrane lipids.

Ballesta and Schaechter (1), Starka and Mo-

ravova (40), and Johnson and Grula (28) havedemonstrated differences in the phospholipidcompositions of dividing and division-inhibitedcells. Daniels (12) reported a change in the rateof synthesis of phospholipids during the cellcycle of Bacillus megaterium. Recent studieshave detailed cyclic changes in lipid synthesisand the lipid composition of intracytoplasmicmembranes of Rhodopseudomonas sphae-roides during the cell cycle (16, 30).

Studies describing phospholipid synthesisduring the division cycle in Escherichia coli areboth ambiguous and contradictory. Churchwardand Holland (8) and Ohki (34) have reportedthat phospholipid synthesis increases exponen-tially during the cell cycle. Daniels (12), Hack-enbeck and Messer (19), and Pierucci (35) havereported a stepwise increase in the rate of lipidsynthesis late in the cell cycle (1). Ohki (34)noted a stepwise turnover of phosphatidylglyc-erol during the division process. However, Zu-chowski and Pierucci (42) reported that nochanges in the rate of phosphatidyl glycerolsynthesis occurred during the cell cycle. Shul-man and Kennedy (37) have demonstrated thatthe rapid turnover of membrane lipids is notessential for normal cell division.The contradictory nature of these observa-

tions may be due, in part, to differences in thestrains of organisms studied and, in part, to thesynchronization methods used by the inivestiga-tors. Most synchronization techniques used in-volve some type of stress on the populationunder study such as temperature shock, centrif-ugation, filter binding, or starvation. These ex-ternal stresses are known to induce changes inprotein synthesis during studies of the cell cycle(31) and may also affect lipid metabolism. In an

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LIPID SYNTHESIS IN E. COLI 473

attempt to resolve the conflicting reports, wehave examined the lipid composition of both theK-21 and B/r strains of E. coli by using undis-turbed cell populations.

(Part of this research was presented at theAnnual Meeting of the American Society forMicrobiology, May 1980, Miami Beach, Fla.).

MATERIALS AND METHODSBacterial strain and growth conditions. E. coli

strain TB4 is a fadE derivative of E. coli K-12 strainCSH-2 (7). Strain B/rA was a gift from 0. Pierucciand C. Helmstetter, Bacillus subtilis strain BR151was kindly provided by N. Mendelson. Cells weregrown at 37°C in a shaking water bath. The minimalmedium employed, designated medium G, contained(in grams per liter of distilled water): NaCl, 1.5; KCl,10; NH4Cl, 1.5; MgSO4 7H20, 0.2; citric acid, 2.0; andmorpholinepropanesulfonic acid (MOPS), 10. The pHwas adjusted to 7.4. Potassium phosphate was addedto a final concentration of 1 mM. After being auto-claved, the basal medium was supplemented with glu-cose (10 g/liter), thiamine (5 mg/liter), and L-trypto-phan (40 mg/liter).Growth was monitored by measuring optical density

at 550 nm on a Bausch & Lomb Spectronic 70 spectro-photometer. The generation times ofthe E. coli strainswere 57 min for strain TB4 and 59 min for strain B/r.B. subtilis had a generation time of 62 min.

Size (age) separation of cells. Cultures (40 ml/250-ml Erlenmeyer flask) were inactivated during ex-

ponential growth by the addition of either formalde-hyde (1.8%, final concentration) or chloramphenicol(200 ,ug/ml, final concentration) plus sodium azide (5mM, final concentration). Similar results were ob-tained with both methods. The inactivated cells wereharvested by centrifugation (5,000 x g, 6 min, 25°C)and gently suspended in fresh basal medium G (nosupplements). This suspension was layered onto a 5 to30% (wt/vol) sucrose gradient (50 ml) and centrifugedfor 12 min at 1,300 x g in a swinging bucket centrifugeat 22 to 25°C. Gradients were harvested from the topby using a Searle Densiflow II and a Gilson fractioncollector. The cells were contained in 15 to 16 tubes(2 ml each). The first two cell-containing tubes were

pooled and referred to as fraction 1; these representedthe smallest cells. The next nine tubes were analyzedindividually and referred to as fractions 2 through 10,respectively. The next two tubes were pooled anddesignated as fraction 11. The final two or three tubeswere pooled and formed fraction 12. Fractions 11 and12 represented the largest and oldest cells. The fre-quency of cells containing cross walls was determinedby direct examination by using phase-contrast micros-copy.

Inactivation of biosynthesis by formaldehydeor chloramphenicol plus azide. The inhibition ofbiosynthesis by formaldehyde or chloramphenicol plusazide was determined as follows. Exponentially grow-

ing cultures were pulse-labeled with [2-'4C]thymidine(0.1 ,uCi/ml; 20 Ig/ml), L-[4,5-3H]leucine (1 ,tCi/ml; 15Ig/ml), or nPO42- (0.16 ,uCi/ml) and sampled at 1-minintervals. After 6 min, either formaldehyde (1.8%, finalconcentration) or chloramphenicol (200 ILg/ml, final

concentration) plus azide (0.1 mM, final concentra-tion) was added, and sampling was continued. Wheneither thymidine or leucine was used as the label,sampling consisted of placing 0.1-ml portions on disksand processing the disks as described below. To ex-amine lipid synthesis, 2-ml aliquots of 32PO42--labeledsamples were mixed with 5 ml of carrier cells; lipidswere extracted with chloroform and methanol andquantitated by liquid scintillation counting (26, 27).Measurement of DNA and protein synthesis.

The relative rates of protein synthesis by various sizesof cells were determined radiochemically. Cells wereuniformly labeled for five generations with L-[6-3H]-tryptophan (1 luCi/ml; 15 ug/ml) before inactivation ofthe culture and subsequent fractionation. Replicatesamples (0.1 ml) were spotted on filter paper diskswhich were washed three times with 5% trichloroaceticacid, followed by three washes in 70% aqueous ethanol.Disks were dried, placed in scintillation vials, andcounted in a Beckman liquid scintillation counter.DNA synthesis was measured relative to cell protein

in the various size classes. Exponential cell cultureswere uniformly labeled for five generations with L-[4,5-3H]leucine (1 ,uCi/ml; 15 jg/ml) and pulse-labeledwith [2-14C]thymidine (0.1 ,uCi/ml; 20 ,g/ml) for a 0.1generation time before inactivation and size separa-tion. Replicate samples (0.1 ml) were spotted on filterpaper disks and processed as described above. Resultsare expressed as the ratio of 14C incorporated intoDNA versus that of 3H incorporated into protein.Measurement of lipid synthesis. Lipid synthesis

was measured relative to cell protein in cells of varioussizes. Log-phase cells were continuously labeled forfive generations with L-[4,5-3H]leucine (1 ,uCi/ml; 15,ug/ml) and then pulse-labeled for a 0.1 generationwith either 'PO42- (0.16 ,Ci/ml) or [1-_4C]acetate (0.1yCi/ml; 10 ,g/ml) before inactivation and separationinto size classes. Replicate samples (0.1 ml) were spot-ted on filter paper disks and processed as describedabove to determine the incorporation of label intoproteins. The remainder of each fraction was com-bined with trichloroacetic acid-inactivated carriercells. Lipids were extracted as previously described(26). Samples of lipid extracts were dried in scintilla-tion vials and counted. Results are expressed as theratio of the counts per minute incorporated into lipidsrelative to that incorporated into protein. In experi-ments involving the continuous labeling of both lipidsand protein, results are expressed as the ratio of mi-cromoles of lipid per micromoles of protein.

lipid extraction and analysis. Cell fractionswere mixed with 5 ml of trichloroacetic acid-inacti-vated carrier cells and concentrated by centrifugation.Total lipids were extracted overnight. Lipid synthesiswas estimated by counting a sample of the washedlipid extract. The remainder of the sample was usedfor the analysis of composition by using thin-layerchromatography. Phospholipid composition was de-termined as described previously (27) by using cellslabeled with PO42-. Fatty acid composition was de-termined by using cells labeled with "C-acetate.Methyl esters were prepared by the method of Silbertet al. (39) and separated by argentation chromatogra-phy as described previously by Buttke et al. (6). Cellswere labeled with 14C-acetate for the analysis of phos-

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474 CARTY AND INGRAM

pholipid molecular species. Monoacyl diglycerideswere prepared by the method of Silbert (38) andseparated by the method of Berger et al. (4).

Chemicals. Radiochemicals were purchased fromAmersham Corp., Arlington Heights, Ill. Chromatog-raphy solvents were obtained from Mallinckrodt, St.Louis, Mo. Chloramphenicol, MOPS buffer, thiamine,and L-tryptophan were obtained from Sigma ChemicalCompany, St. Louis, Mo. Authentic lipid standardswere obtained from Supelco, Inc., Bellefonte, Pa., andApplied Science Laboratories, Inc., State College, Pa.Precoated analytical Silica Gel G plates (Rediplates)were purchased from the Fisher Scientific Company,Pittsburgh, Pa.

RESULTSEffect of formaldehyde and chloram-

phenicol-azide on cells. Figure 1 shows theeffects of formaldehyde and chloramphenicol-azide on the incorporation of 32PO42- into lipid(A) and [3H]leucine into protein (B) by E. coliB/r. Both treatments caused a nearly instanta-neous cessation of incorporation of all labeledcompounds, with formaldehyde being a slightlymore rapid agent (15 versus 30 s for chloram-phenicol-azide). The incorporation of thymidinewas also rapidly inhibited by these treatments(data not shown). Similar results were obtainedwhen the TB4 strain of E. coli was used.To ensure that these treatments did not affect

our fractionation of cellular lipids, both cell cul-tures and authentic lipid standards were chro-matographed after having been treated withchloramphenicol plus azide or formaldehyde.

Neither of these agents affected the mobility ofthe lipids in various thin-layer chromatographysystems employed.Size (age)-dependent separation of cells.

Separation of the cells into discrete fractionscontaining bacteria at various stages of the di-vision cycle was accomplished by sucrose-gra-dient centrifugation of chemically inactivatedcultures. Phase microscopic examination of thefractions indicated that small newly formed cellsremained near the top of the gradient and con-tained few cross walls (Fig. 2). Subsequent frac-tions contained larger cells with a higher fre-quency of cross walls (Fig. 2).

Figure 3 shows that the relative rate of DNAsynthesis for both B/r (A) and TB4 (B) variedamong the fractions in the gradient. DNA syn-thesis increased in the first third of the sizefractions, with maximal synthesis occurringaround fraction 5. The relative rate of synthesisof DNA decreased in the subsequent fractionsexcept for a minor peak around fraction 9 or 10.The significance of this minor peak is unknown.During the latter third of this cycle, the rate ofsynthesis ofDNA by the celLs declined.Lipid and protein syntheses during the

cell cycle. The rate of protein synthesis wasexamined during the cell cycle. Cells were uni-formly labeled with 3H]tryptophan and pulsedwith [14Clleucine before inactivation and sepa-ration. The relative rate of protein synthesis perunit of cell protein (continuous label) remainedconstant during the cell cycle as was observed

4400O

4000 -

3600 -

3200-J7 2800 -

7 2400-a.O 2000 -

1600-

1200 -

800 -

400-

A

x..

XI

1 I I I I I I I I I I

0 2 4 6 8 10 12 0 2 4 6 8 10 12TIME (MINUTES) TIME(MINUTES)

FIG. 1. Effects offormaldehyde and chloramphenicol plus azide on the incorporation of32pO42- into lipids(A) and L-[4,5-3H]leucine into protein (B) by E. coli Blr. Symbols: x, control cells; 0, cells to whichchloramphenicolplus azide was added; 0, cells to which formaldehyde was added. Arrows indicate the timesat which the inhibitory solutions were added.

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!40t 0g4ot lf

20 200 ~~~~0

O 4 8 12 12FRACTION NO. FRACTION NO.

FIG. 2. Percentage of cells having visible crosswalls as a function ofposition in thegradient. (A) E.coli Blr; (B) E. coli TB4. Bars indicate standarddeviations firom three experiments.

20 20

AA

12 I._

0~~~~~~

201 l , 10-

0 4 a 12 0 4 12FRACTION NO. FRACTION NO.

FIG. 3. DNA synthesis as a function of ceosize(age). Cells were unifostolabeled with[gHtleucine,pulsed for 0.1 generation with [l4C]thymidine, andinactivated with formalaehyde before separationinto size firactions. Results are expressed as countsper minute of "4C (DNA) per counts per minute of3H(protein). Bars indicate standard deviations fromthree experiments. (A) Strain Blr; (B) strain TB4.

by other workers (18, 30).The relative rate of lipid synthesis was mea-

sured in each size fraction by pulse-labeling with32PO42- (Fig. 4). Six26larresuts were obtainedwith both strain B/r and strain TB4. The rela-tive rate of lipid synthesis was lowest in themiddle size fractions and highest in the largecells containing abundant cross walls. The rela-tive rate of lipid synthesis declined in fractions1 to 3 and remained low in the middle 3 or 4

LIPID SYNTHESIS IN E. COLI 475

fractions. The relative rate of lipid synthesisbegan to increase around fraction 6 or 7, coinci-dent with cross wall formation. In both cases,the relative rate of lipid synthesis peaked infraction 11. Similar results were obtained when"4C-acetate was used to measure lipid synthesisindicating that the change in the relative rate oflipid synthesis occurred in both the phospholipidhead groups and the acyl chains.The lipid/protein ratio in the various size

fractions was determined with cells of strainTB4 which were uniformly labeled with both'PO42 (lipid) and [3H]leucine (protein). Theoldest cells, those in the process of completingcross walls, contained approximately twice thelevel of lipid found in the younger cells (Table1).Lipid composition. The compositions of the

lipids synthesized in the various size classes weredetermined. The results with strain TB4 are

0.24-

0.22

0.20-

a 0.16-

0.16

z 0.14-

0.12-0

I 0.10-

9 0.06-0.

-J 0.06

0.04-

n eno-

A

0.02 V~~~~~~~.0C02 4 68 10 1214 0i2 4 6 01214

FRACTION NUMBER FRACTION NUMBER

FIG. 4. Lipid synthesis as a function of cell size(age). (A) E. coli B/r; (B) E. coli TB4. Cells were

uniformly labeled with [3Hlleucine, pulsed for 0.1generation with 32PO42, and inactivated by the ad-dition offormaldehyde or chloramphenicol-azide be-fore separation into size fractions. Results are ex-

pressed as countsper minute of32P incorporated intolipids per counts per minute of 3H (protein). Resultsrepresent the average of duplicate experiments.

TABLE 1. Lipid/protein ratios of cells at variousstages of the cell cycle

Lipid/protein ratioa

Baba 8llsb MidphaseOrganimBaby cells Mpceb Large cells

E. coli TB4 0.168 ± 0.02 0.153 ± 0.01 0.23 ± 0.054B. subtilis 0.143 ± 0.05 0.113 ± 0.04 0.278 ± 0.06BR151a Ratios are expressed as micromoles of phospho-

lipid per micromole of protein ± the standard error.b Derived from fractions 1 and 2.'Derived from fractions 6, 7, and 8.d Derived from fractions 11 and 12.

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476 CARTY AND INGRAM

summarized in Fig. 5. The compositions of thephospholipid head groups (27), fatty acids (26),and phospholipid molecular species (4) agreedwell with those previously reported. The pro-portions of lipid components synthesized duringa pulse of 0.1 generation remained constant inall size classes. Similar results were obtainedwith strain B/r.Lipid synthesis in B. subtilis. By using the

methods employed in the E. coli experiments,both the relative rate of phospholipid synthesisand the lipid/protein ratios of B. subtilis strainBR151 were determined. In B. subtilis, an in-crease in the rate of phospholipid synthesis wasobserved late in the division process. The timeof this increase was coincident with the time ofcross wall formation (Fig. 6). Changes in thelipid/protein ratios of continuously labeled cellswere also observed. Phospholipids were moreabundant in the older, dividing cells than eitherthe newly formed daughter cells or the mid-phase-growing cells (Table 1).

DISCUSSIONOur results with cells which were labeled dur-

ing an undisturbed division cycle were in excel-lent agreement with the results of Pierucci (35),Daniels (12), and Hackenbeck and Messer (19).The rate of lipid synthesis increased in a step-wise fashion during the cell cycle at a time nearthe initiation of a new cross wall. These resultsare in conflict with those of Churchward andHolland (8), Ohki (34), and Bauza et al. (2), whofound that lipid synthesis increased exponen-tially, like cell mass, during the division cycle.These workers all used glycerol incorporation asa measure of lipid synthesis. Although it is un-clear why this should not accurately reflect lipidsynthesis, our data with 32P02, our studies and

those of Hackenbeck and Messer (19) with ra-dioactive acetate, and those of Pierucci with["4C]serine as a measure of phosphatidyl etha-nolamine synthesis (35) provide compelling evi-dence that a change in the rate of lipid synthesisdoes occur during the cell cycle.The composition of the phospholipid head

groups synthesized at different stages in the cellcycle remained constant in our studies, consist-ent with the results of Ohki (34) and Pierucci(35). The composition of the acyl chains synthe-sized also remained constant, as did the compo-sition of the phospholipid molecular species.Lipid composition is extensively regulated in E.coli in response to growth temperature. Acylchain composition and polar head groups canalter the activities of membrane-bound enzymes(18, 24, 41). Our results indicate that no changes

0 2 4 6 a 10 12 14FRACTION NUMBER

FIG. 6. Lipid synthesis and cross wall abundancein various size fractions of B. subtilis strain BR151.Symbols: 0, percentage of cells containing crosswalls, *, counts per minute of 32p042- incorporatedinto lipid per counts per minute of L -[4,5-3H]leucineincorporated into protein.

A

IP

=-4 PG

C.LCL

1001 B

go-

a0-

60-

1 40-

' 20-

40.

35.

30.

3_++F 4 i l e ' .~~~~~~~~~lSATUPATED 20.

f { 4 § I Ii -i 4 I 116:I i

at 5-

16:0/16:1

i $ 16:0/16:1

0isa1wa1ts'el:/Is:,

0 2 4 6 a 10 12 0 2 4 6 a lo 12 0 2 4 6 6 10 12FRACTION NO. FRACTION NO. FRACTION NO.

FIG. 5. Compositions of the lipids synthesized in the various size fractions. Strain TB4 was pulsed for 0.1generation with either 32PO42 (A) or 14C-acetate (B, and C) and inactivated by the addition offormaldehydeor chloramphenicol-azide before separation into size classes. Lipids were extracted, fractionated by thin-layerchromatography, scraped, and counted. Results are expressed as a percentage of the total. Bars representstandard deviations from five determinations. (A) Phospholipid composition: 0, phosphatidyl ethanolamine(PE); A, phosphatidyl glycerol (PG); 0, cardiolipin (CL). (B) Fatty acid composition: 0, saturated fatty acids(16:0 + 14:0, primarily); A, vaccenic acid; 0, palmitoleic acid. (C) Phospholipid molecular species: A, 16:0/16:1; 0, 16:0/18:1; A, 18:1/18:1, 0, saturated fatty acid/saturated fatty acid; U, 18:1/16:1.

100-

ol o-

l 60-

40-

20-

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VOL. 145, 1981

in the synthesis of these molecules occurredduring the cell cycle, eliminating this as a pos-sible mechanism for regulation.

Cyclic changes in the density and compositionof the intracellular membranes of R. sphae-roides have been elegantly established (16, 30).Our studies with E. coli and B. megateriumindicate that the lipid/protein ratio of cellschanges during the division process, with thehighest proportion of lipids being in cells nearingthe completion of the division process. Althougha direct comparison of these two cell types iscomplicated by differences in the surface area ofintact cells, results indicate that cell cycle-de-pendent changes occur.The rate of peptidoglycan synthesis increases

during cross wall formation (32, 33) as do theactivities of several murein hydrolases (3, 20,25). It appears likely that these changes in totalenzyme activity result from changes in cell cycle-dependent enzyme expression rather than in-creased transcription of hydrolase enzymes perse (31). Membrane lipids could be involved inthis regulation of enzyme activity. At least twoof the enzymes of peptidoglycan synthesis areknown to be sensitive to changes in their lipidenvironment: the C-55 isoprenoid phosphoki-nase (17) and the muramyl-peptide translocase(41). Lipid content has been shown to controlthe expression of another cytoplasmic enzyme,aminopeptidase N (29). This enzyme continuesto be synthesized in the absence of lipid synthe-sis but in an inactive form, which is activated bythe restoration of lipid synthesis. Other enzymesinvolved in cross wall formation may also beaffected by lipid content.Rhodopseudomonas, Escherichia, and Bacil-

lus species exhibit changes in the rate of lipidsynthesis during the cell cycles. The common-ality of this change suggests that it may repre-sent an important feature of the bacterial cellcycle.

ACKNOWLEDGMENTSWe thank M. G. Pate for her technical assistance and L. C.

Eaton for his critical reading of this manuscript.This investigation was supported by Public Health Service

grant 1 ROI GM 24059-19 from the National Institutes ofHealth, by grant 1 ROI AA 03816-01 from the National Insti-tute of Alcohol Abuse and Alcoholism, and by the FloridaAgricultural Experiment Station (publication no. 2511). L.O.I.is the recipient of a Career Development Award from theNational Institute of Alcohol Abuse and Alcoholism (1 K0200036-01).

LITERATURE CITED1. Ballesta, J. P. G., and M. Schaechter. 1971. Effect of

shift-down and growth inhibition on phospholipid me-tabolism of Escherichia coli. J. Bacteriol. 107:251-258.

2. Bauza, M. T., J. R. De Loach, J. J. Aguanno, and A.R. Larrabee. 1976. Acyl carrier protein prosthetic

LIPID SYNTHESIS IN E. COLI 477

group exchange and phospholipid synthesis in synchro-nized cultures of a pantothenate auxotroph of Esche-richia coli. Arch. Biochem. Biophys. 174:344-349.

3. Beck, B. D., and J. T. Park. 1976. Activity of threemurein hydrolases during the cell division of Esche-richia coli K-12 as measured in toluene-treated cells. J.Bacteriol. 126:1250-1260.

4. Berger, B., C. E. Carty, and L 0. Ingram 1980. Alco-hol-induced changes in the phospholipid molecular spe-cies of Escherichia coli. J. Bacteriol. 142:1040-1044.

5. Burdett, I. D. J., and R. G. E. Murray. 1974. Septumformation in Escherichia coli: characterization ofseptalstructure and effects of antibiotics on cell division. J.Bacteriol. 119:303-324.

6. Buttke, T. M., and L 0. Ingram. 1978. Inhibition ofunsaturated fatty acid synthesis in Escherichia coli bythe antibiotic cerulenin. Biochemistry 17:5285-5286.

7. Buttke, T. M., and L. 0. Ingram. 1978. Mechanism ofethanol-induced changes in lipid composition of Esch-erichia coli: inhibition of saturated fatty acid synthesisin vivo. Biochemistry 17:637-644.

8. Churchward, G. G., and J. B. Holland. 1976. Envelopesynthesis during the cell cycle in Escherichia coli B/r.J. Mol. Biol. 105:245-261.

9. Clark, D. J. 1968. The regulation of DNA replication andcell division in Escherichia coli B/r. Cold Spring Har-bor Symp. Quant. Biol. 33:823-38.

10. Coleman, R. 1973. Membrane-bound enzymes and mem-brane ultrastructure. Biochem. Biophys. Acta 300:1-30.

11. Cronan, J. E., Jr. 1978. Molecular biology of bacterialmembrane lipids. Annu. Rev. Biochem. 47:163-189.

12. Daniels, M. J. 1969. Lipid synthesis in relation to the cellcycle ofBacillus megateriumKM and Escherichia coli.Biochem. J. 115:697-701.

13. DiRienzo, J. M., and M. Inouye. 1979. Lipid fluidity-dependent biosynthesis and assembly ofthe outer mem-brane proteins of E. coli. Cell 17:155-161.

14. DiRienzo, J. M., K. Nakamura, and M. Inouye. 1978.The outer membrane proteins of gram-negative bacte-ria: biosynthesis, assembly, and function. Annu. Rev.Biochem. 47:481-582.

15. Donachie, W. D. 1979. The cell cycle of Escherichia coli,p. 11-37. In J. H. Parish (ed.), The developmentalbiology of prokaryotes. Blackwell Scientific Publica-tions, Oxford.

16. Fraley, R. T., D. R. Lueking, and S. Kaplan. 1979. Therelationship of intracytoplasmic membrane assembly tothe cell division cycle in Rhodopseudomonas sphae-roides. J. Biol. Chem. 254:1980-1986.

17. Gennis, R. B., and J. L. Strominger. 1976. Activationof C5-isoprenoid alcohol phosphokinase from Staphy-lococcus aureus. I. Activation by phospholipids andfatty acids. J. Biol. Chem. 251:1264-1269.

18. Grisham, C. M., and R. E. Barnett. 1973. The role oflipid-phase transitions in the regulation of the sodium+ potassium adenosine triphosphatase. Biochemistry12:2635-2637.

19. Hackenbeck, R., and W. Messer. 1977. Oscillations inthe synthesis of cell wall components in synchronizedcultures of Escherichia coli. J. Bacteriol. 129:1234-1238.

20. Hackenbeck, R., and W. Messer. 1977. Activity of mu-rein hydrolases in synchronized cultures of Escherichiacoli. J. Bacteriol. 129:1239-1244.

21. Helmstetter, C. E. 1967. Rate of DNA synthesis duringthe division cycle of Escherichia coli B/r. J. Mol. Biol.24:417-427.

22. Helmstetter, C. E., and 0. Pierucci. 1976. DNA syn-thesis during the division cycle of three substrains ofEscherichia coli B/r. J. Mol. Biol. 102:477-486.

23. Helmstetter, C. E., 0. Pierucci, M. Weinberger, M.Holmes, and M.-S. Lang. 1979. Control of cell division

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in Escherichia coli, p. 517-579. In L. N. Omston and J.R. Sokatch (ed.), The bacteria, vol. VII. AcademicPress, Inc., New York.

24. Hidalgo, C., N. Ikemoto, and J. Gergely. 1976. Role ofphospholipids in the calcium-dependent ATPase of thesarcoplasmic reticulum. Enzymatic and ESR studieswith phospholipid-replaced membranes. J. Biol. Chem.251:4224-4232.

25. Hoffmann, B., W. Messer, and U. Schwarz. 1972.Regulation of polar cap formation in the life cycle ofEscherichia coli. J. Supramol. Struct. 1:29-32.

26. Ingram, L. 0. 1976. Adaptation of membrane lipids toalcohols. J. Bacteriol. 125:670-678.

27. Ingram, L 0. 1977. Preferential inhibition of phosphati-dyl ethanolamine synthesis in E. coli by alcohols. Can.J. Microbiol. 23:779-789.

28. Johnson, J. H., and E. A. Grula. 1980. Cell membranephospholipids and their constituent fatty acids in divid-ing and non-dividing cells ofMicrococcus lysodeikticus.Can. J. Microbiol. 26:658-665.

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