Vol. 169, No. 8

DNA Replication Initiation, Doubling of Rate of PhospholipidSynthesis, and Cell Division in Escherichia coli

DANIJLE JOSELEAU-PETIT,* FRANCOIS KEIPES,t LYNE PEUTAT, RICHARD D'ARI, AND ADAM KEPESt

Institut Jacques Monod, Centre National de la Recherche Scientifique and Universite Paris 7, 75251 ParisCedex 05, France

Received 8 January 1987/Accepted 22 May 1987

In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about byglycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but preventedthe initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal,which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as littleas 10 min before the expected initiation. We conclude that the initiation ofDNA replication is not triggered bythe preceding DROPS but requires active phospholipid synthesis. Conversely, when DNA replication initiationwas specifically blocked in a synchronized culture of a dnaC(Ts) mutant, two additional DROPS were observed,after which phospholipid synthesis continued at a constant rate for at least 60 min. Similarly, when DNAelongation was blocked by thymine starvation of a synchronized culture, one additional DROPS was observed,followed by linear phospholipid accumulation. Control experiments showed that specific inhibition of celldivision by ampicillin, heat shock, or induction of the SOS response did not affect phospholipid synthesis,suggesting that the arrest of DROPS observed was due to the DNA replication block. The data are compatiblewith models in which the DROPS is triggered by an event associated with replication termination or

chromosome segregation.

It has been speculated that in bacteria the cell membranemay play a role in chromosome segregation (6, 7, 19).Subsequent work with Escherichia coli has provided evi-dence for attachment of the membrane to DNA or to oriC,the replication origin (5, 13, 15, 20), and a membrane proteinwith a specific binding site in oriC has been described (8, 9).However, little is known about possible regulatory intercon-nections between membrane synthesis and DNA replication(4, 18, 21).

In the bacterial cell cycle a small number of events havebeen characterized as occurring only at a precise cell age. Allof these events involve either the chromosome or the cellenvelope. On the one hand there are initiation and termina-tion of chromosome replication and nucleoid segregation,and on the other hand there are cell septation, the doublingof the rate of protein insertion into the outer membrane (2),and the doubling in the rate of phospholipid synthesis(DROPS; 10, 16, 17). The latter event involves both phos-phatidylethanolamine and phosphatidylglycerol, whose ratesof synthesis are constant with an abrupt doubling at a

particular moment of the cell cycle (10).We previously looked for a temporal relation between the

DROPS and replication initiation or cell division in severalE. coli strains growing in different media. The timing of theseevents was found to vary with the growth conditions (10).The work was done with cultures synchronized by repeatedphosphate starvation (11, 12), giving more than three cyclesof synchronous growth for all strains and media used.

In the present study, again using synchronized cultures,we analyzed (i) the effect of a specific block in phospholipidsynthesis on DNA initiation and chain elongation and (ii) theeffect of a specific block in either replication initiation or

* Corresponding author.t Present address: Department of Biochemistry, University of

California, Berkeley, CA 94720.t Deceased 18 April 1984.

chain elongation on the DROPS. The results suggest thatphospholipid synthesis is required for the initiation of DNAreplication but not for chain elongation, and they are com-

patible with models in which the DROPS is triggered bysome event linked to replication termination or nucleoidsegregation. Furthermore, each DROPS seems to provide a

stable phospholipid-synthesizing capacity which can con-

tinue to function in the absence of additional DROPS.

MATERIALS AND METHODS

Bacteria and growth conditions. The K-12 strains usedwere BB2636 (glpD3 glpR2 glpK14 plsB26 plsX50 relAlphoA8 pit-1O fhuA22) (1, 18), provided by Robert Bell; PC2[dnaC2(Ts) thy deo leu rpsL] (3), provided by Jean-PierreBouche; and AT18, a dnaC+ transductant of PC2. To ensurethat AT18 was not a revertant of PC2, a deo+ clone was

chosen from the thermoresistant colonies; AT18 is a spon-taneous deo mutant of this transductant, selected on 2.5 ,ugof thymine per ml.

Bacteria were grown in mineral salts medium bufferedwith Tris hydrochloride (5 x 10-2 M) or MOPS (morpholine-propanesulfonic acid)-KOH (5 x 10-2 M) at pH 7.3 at 37 or

30°C. These two media contained NaCl (4.6 g/liter), KCI (1.5g/liter), (NH4)2SO4 (2 g/liter), MgSO4 (0.2 g/liter), FeSO4 (0.5mg/liter), and thiamine (1 mg/ml). Glucose was the carbonsource. For strain BB2636, glycerol (0.5 g/liter) was added,and for strains PC2 and AT18, thymine (12 mg/liter) and an

amino acid mixture (methionine, histidine, arginine, leucine,threonine, and proline at 0.1 g of each per liter) were added.The phosphate concentration was 0.4 x 10-3 to 1 x 10-3 Min batch culture. In experiments, bacterial densities werebelow 100 ,ug/ml (dry weight).

Radiochemicals. [6-3H]thymidine (30 Ci/mmol), [2-14C]thy-mine (57 mCi/mmol), and [32P]phosphate (carrier free, 10mCi/ml) were purchased from the Commissariat a l'EnergieAtomique, Saclay, France.

3701

JOURNAL OF BACTERIOLOGY, Aug. 1987, p. 3701-37060021-9193/87/083701-06$02.00/0Copyright © 1987, American Society for Microbiology

3702 JOSELEAU-PETIT ET AL.

(DROPS) DROPS

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FIG. 1. DNA replication in the absence of phospholipid synthesis. Strain BB2636 was synchronized at 37°C in MOPS medium (Materialsand Methods). After 16 cycles of 75 min, the culture was diluted in phosphate-nonlimiting medium (time zero). At the times indicated, samplesA, B, C, and D were filtered on 0.45-p.m-pore-size Millipore filters, rapidly washed twice, and suspended in glycerol-free medium (A, B, andC) or in glucose plus glycerol medium (D) (the operation took less than 3 min). Mass increase was estimated by optical density at 600 nm inthe control (A) and in subcultures A (A) and D (A). Cell number was measured with a Coulter counter in the control (0) and in subculturesA (0), B (C), C (O), and D (0). The rate of DNA synthesis was measured by 2-min pulses of [6-3H]thymidine in the control (U) and insubcultures A (l), B (E), C (Oi), and D (El). The rate of phosphatidylethanolamine synthesis was measured by 5-min pulses of [(4C]acetatein the control culture (V). The ordinate scale is in arbitary units. The symbols Div, I, and DROPS below the figure indicate the time of themid-rise point in the curve for cell number, rate of DNA synthesis, and rate of phosphatidylethanolamine synthesis, respectively; the firstDROPS, shown in parentheses, was determined by assuming a plateau twofold below the first observed.

Synchronization procedure. Bacteria were synchronizedby 12 to 15 successive cycles of doubling followed by a shortphosphate starvation. At the end of each cycle, the culturewas automatically diluted twofold with limiting phosphatemedium (1 x 10-4 to 2 x 10-' M), which permitted one massdoubling and one cell doubling before the next starvation(11). Synchronized cells were harvested at the phase ofphosphate starvation and diluted for batch culture innonlimiting phosphate medium (0.4 to 1 mM).

Cell counting. A 5- to 80-p.l volume of culture was dilutedin 20 ml of "counting solution" (7.5 g of NaCl and 5 ml ofFormol per liter). Cells were counted in a Coulter Counter(model ZB; Coultronics S.A., Margency, France).Measurements of DNA synthesis. DNA synthesis was mea-

sured by pulse labeling with [3H]thymidine or by incorpora-tion of [14C]thymine in thymine auxotrophs.For pulse-labeling, 0.4 ml of culture was incubated with

[6-3H]thymidine (2 x 10-6 M; 4 Ci/mmol) and deoxyuridine(5 x 10-5 M) for 2 min. The reaction was stopped byprecipitation with cold 5% trichloroacetic acid containing

thymidine (10-4 M). Insoluble material was collected onMillipore filters (0.45-,um pore size), which were dried andcounted.For [14C]thymine incorporation, the culture was incubated

with [2-14C]thymine (1.7 x 10-4 M; 20 mCi/mmol). Samplesof 100 ,ul were removed at close intervals and precipitatedwith cold trichloroacetic acid containing thymine (10-3 M).Measurement of phospholipid synthesis. Since patterns of

synthesis of phosphatidylethanolamine and phosphatidyl-glycerol were similar during the cell cycle (10), phospholipidsynthesis was measured by [IPlphosphate incorporationinto phosphatidylethanolamine. A portion of the culture wasincubated with [32Plphosphate (0.6 x 10-3 to 1 x 10-3 M; 50to 200 p.Ci/,pmol). At indicated times, 30-p.l samples weredeposited directly on silicated paper (SG81; Whatman) anddried. Chromatograms were developed with solvent systemA (chloroform-methanol-water, 100:36:5, vol/vol). Phospho-lipid spots were located by autoradiography; phosphatidyl-ethanolamine spots were cut out, and the radioactivity wascounted in liquid scintillation mixture.

J. BACTERIOL.

DNA INITIATION AND PHOSPHOLIPID SYNTHESIS IN E. COLI

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FIG. 2. Phospholipid synthesis in the absence of initiation of

DNA replication. Strain PC2 was synchronized at 30°C in Trismedium (Materials and Methods). After 11 cycles of 107 min, theculture was diluted in phosphate-nonlimiting medium (time zero),and ['4C]thymine or [32P]phosphate was added 80 min later. At thetime indicated (A), a sample was shifted to 41°C. Mass increase wasestimated by optical density at 600 nm in the control (A) and insubculture A (A). Cell number was measured with a Coulter counterin the control (0) and in A (0). DNA synthesis was measured bycontinuous incorporation of [2-14C]thymine in the control (A) and insubculture A (l). Phosphatidylethanolamine synthesis was mea-sured by continuous incorporation of [32P]phosphate in the control(V) and in A (V). Below the figure are indicated the times at whichdivision (Div), replication initiation (I), and DROPS took place,calculated as explained in the legend of Fig. 1.

For pulse-labeling, SO-,ul samples of the culture wereincubated for 5 min with [14C]acetate (2 x 10-' M; 5

mCi/mmol). Samples of 30 ,ul were treated as describedabove; after migration in solvent system A, the chromato-grams were developed with solvent system B (petroleumether-ethyl ether; 70:30, vol/vol) and then with solventsystem C (water-acetic acid, 100:2, vol/vol).

RESULTSDNA replication in the absence of phospholipid synthesis.

To block phospholipid synthesis specifically, we used the E.

coli mutant BB2636 (1). This strain is unable to reducedihydroxyacetone phosphate to glycerol 3-phosphate, theprecursor of all phospholipids. It therefore requires exoge-nous glycerol for growth but uses this glycerol solely forphospholipid synthesis, other metabolites and energy beingderived from an exogenous sugar, generally glucose. Thus inthis strain starvation for glycerol causes an immediate andcomplete arrest of phospholipid synthesis, without interfer-ing with other metabolic pathways; at present this is the onlymeans of blocking phospholipid synthesis specifically andquickly in E. coli.

In the experiment shown in Fig. 1, strain BB2636 wassynchronized (see Materials and Methods); at time zero, theculture was diluted into phosphate-nonlimiting medium con-taining glucose plus glycerol (doubling time, 60 min). At theindicated times, portions of the culture (A, B, C, and D)were filtered. The bacteria were washed and suspended inglycerol-free medium for A, B, and C (Fig. la) and in glucoseplus glycerol medium for D (Fig. lb). Cell recovery (gener-ally >70%) was evaluated by cell counts, and the experi-mental curves were adjusted accordingly. In all three glyc-erol-starved subcultures, mass increased about twofold andcell number increased less than 10% (Fig. la), consistentwith earlier reports on exponential cultures (1). Theunstarved control (Fig. lb) shows that the filtration intro-duced a 15-min delay but did not otherwise affect the rate ofmass or number increase.The rate of DNA synthesis was measured by [3H]thymi-

dine pulse-labeling. The filtration-resuspension processcaused a drop in the rate of DNA synthesis for 15 to 20 min,after which the rate returned to 90% of that of the unfilteredculture, with initiations slightly delayed (Fig. lb). In subcul-ture B, in which glycerol was removed in mid-cycle duringthe rise in the rate of phospholipid synthesis and during theplateau in DNA synthesis, the rate of DNA synthesis re-mained constant for 40 to 50 min and then decreased.Subculture A was starved for glycerol at the time of repli-cation initiation, i.e., during the rise in the rate of DNAsynthesis and just before the extrapolated DROPS; it exhib-ited an increase in rate of about 20%, followed 50 min laterby a sharp decrease. Subculture C, in which glycerol wasremoved just before the next initiation and at the end of theDROPS, showed a 10% increase in the rate of DNA synthe-sis, followed 50 min later by a sudden decrease.These results show that the rate of DNA elongation is not

perturbed by a block in phospholipid synthesis and suggestthat little or no initiation takes place under these conditions.If phospholipid synthesis is blocked before or during the risein DNA synthesis rate, the increase does not exceed 20%,suggesting that initiation is blocked rapidly after glycerolremoval. The initiation block was observed whether phos-pholipid synthesis was arrested before (Fig. 1, A), during(B), or after (C) the DROPS.

Phospholipid synthesis in the absence of DNA replicationinitiation. A specific block in the initiation of cycles of DNAreplication is provided in E. coli PC2, in which initiation istemperature sensitive due to the dnaC2 mutation (3). Thisstrain was synchronized at 30°C, and at time zero the culturewas diluted into phosphate-nonlimiting medium containingglucose plus the growth requirements (doubling time, 80 minat 30°C). A portion of the culture was shifted to the nonper-missive temperature at the time indicated (Fig. 2). The rateof mass increase accelerated immediately to a doubling timeof 55 min and remained exponential for 120 min at 41°C.DNA accumulation at 30°C, monitored by continuous['4C]thymine labeling, was linear with a rapid doubling in

VOL. 169, 1987 3703

3704 JOSELEAU-PETIT ET AL.

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FIG. 3. Effect of a temperature shift on phospholipid synthesis. Exponential-phase culture of strains (a) AT18 (dnaCC) and (b) PC2 (dnaC),growing in MOPS medium at 30°C, were divided into three parts 1.5 generation times before time zero; one part was labeled with[32P]phosphate, and another was labeled with ['4C]thymine. At time zero, a portion of each unlabeled culture was shifted to 41°C. Symbolsrepresent optical density at 600 nm at 30°C (A) and 41°C (A), [2-14C]thymine incorporation at 30°C (U) and 41°C (Li), and [32P]phosphateincorporation in phosphatidylethanolamine at 30°C (V) and 41°C (V).

slope at the time of initiation. In the 41°C subculture noreinitiation was observed, and DNA accumulation essen-tially stopped. Phospholipid synthesis, on the other hand,continued unperturbed at 41°C for 180 min. The incorpora-tion of [32P]phosphate into phosphatidylethanolamine under-went two successive DROPS in the 41°C subculture and thencontinued linearly at the same rate for at least 60 min longer.A control experiment was carried out with a dnaC+

transductant of strain PC2 (see Materials and Methods) toevaluate the effect of a temperature shift alone on phospho-lipid synthesis. DNA synthesis and phosphatidylethanol-amine synthesis were measured in this strain and in strainPC2 during exponential growth at 30°C and after a shift to41°C. In the dnaC+ strain, the temperature shift caused arapid acceleration in DNA synthesis and phosphatidyletha-nolamine synthesis, which remained exponential and inconstant proportion for the 4 h of the experiment (Fig. 3a).Assuming that phospholipid synthesis proceeds by succes-sive DROPS in exponential culture, this result shows that atemperature shift does not affect the DROPS. The resultswith the dnaC mutant were compatible with those obtainedin synchronized growth: at 41°C DNA synthesis stoppedafter 60 min, and phosphatidylethanolamine synthesis was asat 30°C for 110 min and then continued linearly for over 90min longer (Fig. 3b). This is the incorporation patternexpected if there are only two DROPS after the temperatureshift.

The above results taken together indicate that a block inthe initiation of new rounds of DNA replication specificallyleads to two further DROPS, followed by an extended periodof linear phospholipid accumulation.

Phospholipid synthesis in the absence of DNA elongation.We next investigated the effect on phospholipid synthesis ofan arrest of DNA chain elongation. Elongation was blockedby thymine starvation. To maintain the same genetic back-ground as in the initiation experiments, we used strain PC2,which is thyA; the experiment was carried out at 30'C, apermissive temperature for dnaC2. Bacteria were synchro-nized at 30'C, as described above, and at different times,portions of the culture (Fig. 4, A, B, and C) were filtered.Bacteria were washed and suspended at 30°C in thymine-freemedium for A and B and in thymine-containing medium forC. In the latter, control subculture it can be seen that thefiltration and resuspension caused a lag of about 20 min, afterwhich the curves of cell number and DNA and phospholipidsyntheses were parallel to those of the unshifted culture (Fig.4b). In subcultures A and B, cell division stopped rapidly,although mass continued to increase, indicating filamenta-tion. Phosphatidylethanolamine synthesis, after a short lag,rose parallel to that in the control culture for 80 min, duringwhich time one DROPS occurred; synthesis then continuedlinearly at the same rate until the end of the experiment, withno further DROPS (Fig. 4a).We conclude that when DNA chain elongation is blocked,

J. BACTERIOL.

DNA INITIATION AND PHOSPHOLIPID SYNTHESIS IN E. COLI

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FIG. 4. Phospholipid synthesis in the absence ofDNA elongation. Strain PC2 was synchronized at 30°C as described in the legend of Fig.2. At the times indicated, samples A, B, and C were filtered on 0.45-,um-pore-size Millipore filters. The bacteria were washed twice,suspended in thymine-free medium (A and B) or in thymine-containing medium (C), and incubated at 30°C. Mass increase was estimated bythe optical density at 600 nm in the control (A) and in subcultures A (A), B (A), and C (A). Cell number was measured with a Coulter counterin the control (0), in A (0), in B (C), and in C (0). DNA synthesis was measured by continuous incorporation of [2-14C]thymine in the control(i) and in C (E). Phosphatidylethanolamine synthesis was measured by continuous incorporation of [32P]phosphate in the control (V), in A(V), in B (V ), and in C (vW ). The symbols Div, I, and DROPS below the figure indicate the time of the mid-rise point in the curve for cellnumber, rate of DNA synthesis, and rate of phosphatidylethanolamine synthesis, respectively.

only a single DROPS can take place, followed by an ex-

tended period of linear phospholipid synthesis.Phospholipid synthesis in the absence of cell division. Dur-

ing thymine starvation and, to a lesser extent, during expres-

sion of the dnaC mutation, cell division was blocked (Fig. 2and 4). To see whether the arrest of the DROPS observed inthese conditions was a consequence of the cell divisionblock, we measured phospholipid synthesis under conditionsin which cell division was specifically blocked but DNAsynthesis continued. Ampicillin was added to a portion of anexponentially growing culture of strain PC2 at 30°C. Celldivision stopped, whereas mass continued to increase expo-

nentially, in parallel to that in the control. Phosphatidyleth-anolamine synthesis also remained exponential for over twomass doublings. Similar results were obtained with a syn-

chronized culture of E. coli ML30, in which two DROPSwere clearly seen in the presence of ampicillin (data notshown).These observations suggest that the extended period of

linear phospholipid synthesis without DROPS observed atlate times after DNA initiation or elongation blocks is not a

result of the absence of cell division.

DISCUSSION

In E. coli, both chromosomal DNA synthesis and enve-

lope phospholipid synthesis undergo sudden increases atspecific points in the cell cycle, viz., replication initiationand the DROPS, respectively. The experiments reportedhere were designed to reveal possible causal relationshipsbetween DNA synthesis and phospholipid synthesis by usingsynchronized cultures. The results answer several ques-tions.The first question is: how does a block in phospholipid

synthesis affect DNA synthesis? For these studies we usedstrain B132636, which requires exogenous glycerol for phos-pholipid synthesis but cannot use glycerol for other meta-bolic needs. Our results showed that phospholipid synthesisis not needed for DNA chain elongation but is required forthe initiation of a new replication cycle. The initiation blockwas observed rapidly after the removal of glycerol, wellbefore the membrane structure changed. Our results are

consistent with those reported by Bell (1), who isolatedstrain BB2636, and by Pierucci and Rickert, who more

recently reached a similar conclusion after studying the same

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3705VOL. 169, 1987

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3706 JOSELEAU-PETIT ET AL.

strain in exponential growth (18). Our experiments withsynchronous cultures further showed that initiation wasblocked independently of the cell age at which phospholipidsynthesis was arrested. In particular, it was possible to stopphospholipid synthesis after the DROPS, but before initia-tion, and still block initiation. We conclude that the DROPSis not a sufficient trigger for the next replication initiation.However, we cannot rule out other roles of the DROPS ininitiation; investigation of this possibility would require ameans of specifically stopping the DROPS without blockingphospholipid synthesis.The second question that our results address is: how does

a block in DNA replication affect the DROPS? When repli-cation was arrested specifically at the initiation step, using adnaC(Ts) mutant, two further DROPS were observed at thenonpermissive temperature, after which phospholipid syn-thesis continued at a constant rate for at least one moregeneration. Thus replication initiation cannot be the triggerfor the subsequent DROPS. When DNA chain elongationwas blocked by thymine starvation, only one DROPS wasobserved before phospholipid synthesis became linear. Thedifference between the initiation block (two DROPS) and theelongation block (one DROPS) cannot be attributed to theheat shock in the former experiment, as shown in a dnaC'control, to induction of the SOS response in the latter case(14), as shown in a recA (Tif) dnaC' control (unpublisheddata), or to differences in the extent of cell division inhibitionin the two cases.

It is noteworthy that whenever the DROPS was abolished,phospholipid synthesis continued at the same rate for severalgeneration times. Each DROPS thus seems to provide astable phospholipid-synthesizing capacity which can con-tinue to function in the absence of additional DROPS.Our results suggest that the failure of the cells to carry out

more than one or two DROPS in the absence of DNAsynthesis may be a direct result of the DNA block. Since twoDROPS were observed when initiation was blocked butongoing rounds of replication were allowed to terminate,whereas only one DROPS was observed when DNA chainelongation was blocked, one is tempted to speculate that theDROPS may be triggered by replication termination ornucleoid segregation.

ACKNOWLEDGMENTS

We thank Robert Bell and Jean-Pierre Bouche for providingbacterial strains.

This work was financed in part by the Centre National de laRecherche Scientifique (ATP "Microbiologie' 960113).

LITERATURE CITED1. Bell, R. M. 1974. Mutants of Escherichia coli defective in

membrane phospholipid synthesis: macromolecular synthesis inan sn-glycerol 3-phosphate acyltransferase K,,, mutant. J. Bac-teriol. 117:1065-1076.

2. Boyd, A., and I. B. Holland. 1979. Regulation of the synthesis ofsurface protein in the cell cycle of E. coli B/r. Cell 18:287-296.

3. Carl, P. L. 1970. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol. Gen. Genet. 109:107-122.

4. Clark, D. J. 1968. The regulation of deoxyribonucleic acidreplication and cell division in Eshchrichia (oli B/r. J. Bacteriol.96:1214-1224.

5. Craine, B. L., and C. S. Rupert. 1978. Identification of abiochemically unique DNA-membrane interaction involving theEscherichia (c/li origin of replication. J. Bacteriol. 134:193-199.

6. Helmstetter, C. E. 1974. Initiation of chromosome replication inEscherichia co/i. J. Mol. Biol. 84:21-36.

7. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation ofDNA replication in bacteria. Cold Spring Harbor Symp. Quant.Biol. 28:329-347.

8. Jacq, A., and M. Kohiyama. 1980. A DNA-binding proteinspecific for the early replication region of the chromosomeobtained from Eslheirichia coli membrane functions. Eur. J.Biochem. 105:25-31.

9. Jacq, A., M. Kohiyama, H. Lother, and W. Messer. 1983.Recognition sites for a membrane-derived DNA binding proteinpreparation in the E. coli replication origin. Mol. Gen. Genet.191:460-465.

10. Joseleau-Petit, D., F. Kepes, and A. Kepes. 1984. Cyclic changesof the rate of phospholipid synthesis during synchronous growthof Escherichia co/i. Eur. J. Biochem. 139:605-611.

11. Kepes, F., and A. Kepes. 1980. Synchronisation automatique dela croissance de E. coli. Ann. Microbiol. (Paris) 131:3-16.

12. Kepes, F., and A. Kepes. 1981. Long-lasting synchrony of thedivision of enteric bacteria. Biochem. Biophys. Res. Commun.99:761-767.

13. Kusano, T., D. Steinmetz, W. G. Hendrickson, J. Murchie, M.King, A. Benson, and M. Schaechter. 1984. Direct evidence forspecific binding of the replicative origin of the Escherichia colichromosome to the membrane. J. Bacteriol. 158:313-316.

14. Little, J. W., and D. W. Mount. 1982. The SOS regulatorysystem of E. coli. Cell 29:11-22.

15. Nicolaidis, A. A., and I. B. Holland. 1978. Evidence for thespecific association of the chromosomal origin with outer mem-brane fractions isolated from Escherichiia coli. J. Bacteriol.135:178-189.

16. Pierucci, 0. 1979. Phospholipid synthesis during the cell divi-sion cycle of Escheric/hia (0li. J. Bacteriol. 138:453-460.

17. Pierucci, O., M. Melzer, C. Querini, M. Rickert, and C. Kra-jewski. 1981. Comparison among patterns of macromolecularsynthesis in Escherichia (cli B/r at growth rates of less and morethan one doubling per hour at 37°C. J. Bacteriol. 148:684-696.

18. Pierucci, O., and M. Rickert. 1985. Duplication of Escherichiacoli during inhibition of net phospholipid synthesis. J. Bacteriol.162:374-382.

19. Sandler, N., and A. Keynan. 1981. Cell wall synthesis andinitiation of deoxyribonucleic acid replication in Bacillus sub-tilis. J. Bacteriol. 148:443-449.

20. Wolf-Watz, H., and M. Masters. 1979. Deoxyribonucleic acidand outer membrane: strains diploid for the oriC region showelevated levels of deoxyribonucleic acid-binding protein andevidence for specific binding of the oriC region to outer mem-brane. J. Bacteriol. 140:50-58.

21. Zaritsky, A., and C. L. Woldringh. 1978. Chromosome replica-tion rate and cell shape in Escherichia coli: lack of coupling. J.Bacteriol. 135:581-587.

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