Folia Microbiol. 18, 81--89 (1973)

DNA Synthesis in a Synchronized Culture of Escherichia coli 15 TAU bar after Continuous and Interrupted Thymine Starvation M. OPrx_~novi and V. VO~CDRSJS Department of Biophysics, Faculty of Sciences, Charles University, Prague 2 Received July 25, 1972

ABSTRACT. After transfer to a thymine-containing medium the DI~A synthesis did not increase with increasing intervals of t h y m i n e starvation. On the other hand, the starvation interrupted a t regular intervals by 5 rain thymine pulses resulted in an increased DlqA synthesis. Induction of a bacteriophage which is prevented b y the pulses is discussed as a possible reason for the observed difference in kinetics of the DNA synthesis following continuous and i n t e r r u p t e d thymine starvation. Turbidity of the culture increased roughly three times, both during the continuous and the interrupted thymine starvation. The increase of the turbidity after prolonged interrupted starvation was lower t h a n t h a t which would correspond to the observed increase of the DXA synthesis according to the hypothesis of a critical mass o f t h e cell resulting in the initiation (Donachie, 1968).

The increase in the rate of DNA synthesis in Escherichia coli after thymine starvation was observed by Barner and Cohen (1956) and Nakada ~1960). This increase could be caused either by an accelerated replication or by an increase in the number of replication points (Hanawalt et al., 1961). Pritchard and Lark (1964) found that after thymine starvation the DNA synthesis continues at replication points in which it was interrupted due to a shortage of the precursor at the beginning of the starvation and, in addition, the replication starts from the terminal end of the chromosome. A similar phenomenon was observed after removal of the inhibition of the DNA synthesis by nalidixic acid (Boyle et al., 1967; Ward et al., 1970), by ultraviolet radiation (Hewit and Bfllen, 1964, 1965; Kalenbach and Ma, 1968) or, in mutants with thermosensitive DNA synthesis, after an interval of cultivation at non-permissive temperature (Stein and Hanawalt, 1969; Worcel, 1970). I t was found in experiments with related strains of Escherichia coli 15 TAU or 15 T A U bar that after thymine starvation the rate of DNA synthesis is not increased as much as in other strains or that it may be even decreased. As both strains carry a defective prophage, which can be induced by thymine starvation, the hypothesis was postulated that the induction of prophages is responsible for the observed abnormal behavior of the above-mentioned strains. It was found in Escherichia coli 15 TAU (Dobiw163 Hirsch and Vondrejs, 1972) that a suitable schedule of short thymine pulses interrupting the thymine starvation may prevent the bacteriophage 15 from entering the lyric cycle. Therefore, the DNA synthesis was studied in both strains in more detail, both after continuous and interrupted thymine starvation. The results of the experiments obtained with Escherichia coli 15 TAU bar which arc in agreement with the postulated hypothesis are reported in the present communication.

82

M. O P E K A R O V A AND V. V O N D R E J S

Vol. 18

MATERIALS AND METHODS

Escherichia coli 15 T A U bar requiring thymine (T), arginine (A), uracil (U) and other amino acids -- methionine, t r y p t o p h a n and proline -- was kindly supplied b y Dr. Kanawalt (Biophysics Laboratory, Stanford University, USA). Bacteria were grown at 37~ in an aerated medium M 9 (Kellenberger, Lark and Bolle, 1962) supplemented with 2 ~g thymine, 340 ~g arginine, 20 ~g uracil, 30 ~g methionine, 14 ~g t r y p t o p h a n and 20 ~g proline per one ml of the medium; the above medium is designated as the T a a U medium in the present communication. The T a a - U - medium containing no amino acids and no uracil was used for synchronization of the replication (Maaloe and Hanawalt, 1961). Synchronization was performed under the same conditions as those used for the cultivation and always lasted 120 rain. Thymine starvation was performed in the T - a a U medium which did not contain t h y m i n e and thus differed from the cultivation medium. Short pulses of thymine were started b y adding a concentrated thymine solution to a culture starving in the T - a a U medium. Final concentration of thymine was 2 ~g per ml of the medium. The pulse, always lasting 5 min, was terminated b y filtration and washing of the culture which was subsequently resuspended in a fresh T - a a U medium preheated to 37~ Designation T* instead of T is used for the medium containing, in addition to 2 ~g non-labelled thymine, thymine tritiated in its methyl group (Institute for Research, Production and Uses of Radioactive Isotopes, Prague). Labelled thymine of a specific radioactivity of 19.96 Ci/mMol was added in such quantity as to make its final activity equal to 4 ~Ci per ml of the medium. Media were changed b y means of filtration through membrane filters Synpor 6 (Synthesia, Czechoslovakia) (Janderovs I-Iirsch and Vondreis, 1971). D N A synthesis was followed b y incorporation of tritiated thymine. Bacteria were grown for 200 min in the T*aaU medium and then in the synchronization medium T * a a - U - . After different intervals of continuous or interrupted starvation in the T - a a U medium the culture was transferred again to the T * a a U medium or to the T * a a - U - medium. Samples were taken at regular intervals and the radioactivity incorporated into D N A assayed. Assay of radioactivity incorporated into DNA. 0.6 ml of the labelled bacterial suspension was mixed with the same volume of 10% TCA. After at least 12 h standing at 4~ one ml of a well-mixed sample was taken and filtered through a membrane filter Synpor 6 (diameter 13 mm, pore size 0.45 ~). The filtrate was washed with 10 ml of boiling water and with one ml of ethanol. Prior to use the filters were shortly boiled in distilled water. Filters with the washed samples were dried for 2 h in an oven at 80~ E a c h filter was immersed in 3 ml of a scintillation liquid SLT 41 (Spolana, Neratovice, Czechoslovakia). Radioactivity was assayed in a coincidence system NZB (Tesla, P~emy~leni, Czechoslovakia). Relative radioactivity A was calculated from the measured values using the following equation: A=

Pn - - P0

P1 -- P0 where Pn is radioactivity of the n-th sample in the sampling series, P1 is radioactivity of the first sample in the sampling series and P0 is background activity determined b y measuring radioactivity of the scintillation liquid with a filter not containing the sample. Growth of the culture was followed b y measuring t u r b i d i t y in a colorimeter tIilger-Spekker using a blue filter. Samples were measured in cultivation cylindrical

AQI 1973

DNA SYNTHESIS

I N E. C O L I

8~

0.6

FIG. 1. G r o w t h o f a s y n c h r o n i z e d c u l t u r e o f E ~ cherichia coli 15 TALr bar i n m e d i u m T a a l J and[ T-aaIJ. Exponentially growing culture was transf e r r e d f r o m m e d i u m TaaLr to m e d i u m T a a - L r - and[ s y n c h r o n i z e d for 120 rain. A p o r t i o n o f t h e c u l t u r e w a s t h e n t r a n s f e r r e d to t h e T a a U m e d i u m ( O ) a n d a n o t h e r p o r t i o n to t h e T - a a U m e d i u m ( O ) A b s o r b a n c e (A) o f t h e c u l t u r e w i t h a blue f i l t e r w a s m e a s u r e d f r o m t h i s m o m e n t (t in m i n ) .

0.4 0.3

02

0.1

o.o~ 0

50

Ir

I 1,50

I00

FIG. 2. G r o w t h o f a s y n c h r o n i z e d c u l t u r e of E~cherichia coli 15 T A U bar d u r i n g i n t e r r u p t e d t h y m i n e s t a r v a t i o n . E x p o n e n t i a i l y g r o w i n g culture. w a s t r a n s f e r r e d f r o m t h e T a a U m e d i u m to t h e T a a - U - m e d i u m a n d s y n c h r o n i z e d for 120 m i r a T h e c u l t u r e w a s t h e n d i v i d e d into six p a r t s a n d s u b j e c t e d either to c o n t i n u o u s s t a r v a t i o n in t h e ~ T - a a U m e d i u m ( O ) , or to s t a r v a t i o n interruptec[ a f t e r 50 rain (~/), a f t e r 50 a n d 70 m i n ( ~ ) , a f t e r 50, 70 a n d 90 m i n ((~), or a f t e r 50, 70, 90 a n d l l 0 m i n ( O ) , b y 5-rain p u l s e s o f t h y m i n e . T i m e (rain} t = ton ~- tn, w h e r e ton is t h e s u m of all int e r v a l s o f s t a r v a t i o n u p to t h e last i n t e r r u p t i o n b y t h e p u l s e a n d d u r a t i o n o f all p u l s e s a n d tn is t h e t i m e e l a p s e d since t h e l a s t pulse. V a l u e s o f a b s o r b a n c e (A) in t h e c o n t r o l r e p r e s e n t t h e m e a s u r e d v a l u e s , in o t h e r s a m p l e s t h e y were c a l c u l a t e d f r o m

A 1.0 08 0.6

04 03 02

0.1 I 0

i 50

t

i I00

m 150

A ~ At-

*~kkt On

w h e r e At is t h e v a l u e m e a s u r e d in

A to n

a g i v e n s a m p l e at t i m e t, At0n is t h e v a l u e m e a s u r e d in t h e s a m p l e i m m e d i a t e l y a f t e r w a s h i n g a n d r e s u s p e n s i o n in t h e T - a a U m e d i u m a f t e r t h e l a s t p u l s e , a n d Akron is t h e v a l u e m e a s u r e d in t h e cont r o l s t a r v i n g in t h e T - a a U m e d i u m for a corres p o n d i n g t i m e ton rain. T h e t i m e s c h e d u l e o f t h e e x p e r i m e n t is s u m m a r i z e d i n T a b l e I.

c u v e t t e s , 1.7 cm in d i a m e t e r . T h e c u l t i v a t i o n m e d i u m f r o m w h i c h all c a r b o n cont a i n i n g c o m p o n e n t s were o m i t t e d s e r v e d as a blank. A b s o r b a n c e v a l u e o f 0.1 corr e s p o n d e d in t h e e x p o n e n t i a l phase to 5 • l 0 T eells/ml. T~BL~ I. T i m e s c h e d u l e o f c u l t i v a t i o n , ton is t h e t i m e i n c l u d i n g s t a r v a t i o n b e t w e e n p u l s e s a n d t o t a l t i m e o f pulses. Sample number

S a m p l e c o n s e c u t i v e l y c u l t i v a t e d in m e d i u m (rain)

rain

9 AUi TAU J T AUl TAU I T ACI TAU J T AUI TAU f T AUI TAr

Control

tk

1

50

5

2

50

3

50

4

50

5

tl 20

5

t~

5

20

5

20

5

t3

5

20

5

20

5

20

5

t4

0 55 80' 105 130

84

M. OPEKAROV.~ AND V. V O N D R E J S

Vol. 18

RESULTS

Growth of th~ syrt~hro~gu~ c~dture of E~eherichia cell 15 T A U bar in complete medium and durin I co~'~in~t)~t~ t h y ~ s~arv~ion The culture of Escherichia cell in the exponential phase of growth was transferred for 120 rain to the synchronization medium T a n - U - . The medium was again substituted b y the complete TaaU ,medium. Growth of the synchronized culture was followed turbidimetrically (Fig. 1). No increase could be o b s e r v e d after about 15 min following the transfer of the synchronized culture to the T a a U medium. The phase of exponential growth then followed, during which the time required for doubling of turbidity of the culture was roughly 38 min. The increase of absorbance slowed down at 0.4 and terminated at 0.48. Growth of the synchronized culture transferred to the T - a a U medium was slightly different (Fig. 2). After a lag of about5 20 min the t u r b i d i t y increased much more slowlythan in the culture not starving for thymine. Growth terminated about 100 min. During this interval the turbidity of the culture increased roughly three times. In the following series of experiments the growth of the synchronized culture subjected to interrupted thymine starvation was followed (Fig. 2). The time schedule of the interrupted starvation of individual samples is summarized in Table I. The first sample served as a control. The synchronized culture was starved continuously as in the former case. The course of t u r b i d i t y in the Continuously starved culture is illustrated b y the basic curve in Fig. 2. As it was of p r i m a r y interest to establish whether a short pulse or short pulses of thymine would stimulate an increase of biomass after the pulse, in further cases, the culture of Escherichia cell 15 T A U bar, cultivated according to the schedule summarized in Table I, was transferred after t h e last pulse of thymine to the T - a a U medium and the turbidity of the culture was measured beginning with this time interval. As each pulse was terminated b y filtration, washing and resuspension in a fresh T - a a U medium, the loss of a portion of cells could not be avoided. Thus, as during the resuspension of the culture, a portion of the cells remained adsorbed to the filter, the t u r b i d i t y of the culture naturally decreased. I n order to find out whether and to what e x t e n t the pulses stimulate the growth of the culture in the T - a a U medium, it was necessary to normalize curves expressing the relationship between t u r b i d i t y and the t i m e of starvation obtained after different numbers of pulses in the following way: (1) Individual points of the curve describing the time dependence were plotted in such a w a y that time corresponds to a respective turbidity t ~ ton A- tn, where ton is a sum of time intervals for which the synchronized culture (sample n) was cultivated in media T - a a U and T a a U and tn is the interval between the last transfer from the T a a U medium to the T - a a U medium and the time when the t u r b i d i t y of the culture was measured. (2) Values of t u r b i d i t y plotted were calculated from v a l u e s measured in the following way: the measured value was multiplied b y a fraction of turbidity reached b y the continuously starved culture ton min after the transfer to the T - a a U and the turbidity of the corresponding previously i n t e r m i t t e n t l y starved culture immediately after the resuspension in the T - a a U medium following the last pulse. The course of all normalized curves of turbidity after different time intervals of starvation interrupted b y pulses did not differ substantially from the curve illustrating the relationship between t u r b i d i t y of the continuously starved culture of Escherichia cell 15 T A U bar and time of starvation (Fig. 2). I t thus follows t h a t short thymine pulses did not substantially stimulate a g r o w t h of the culture in the T - a a U medium.

1973

D N A S Y N T H E S I S IN E . COL1

Q

lO

0

50

I00

150

200

250

300

t

4 0

3

l

I

I

1

0

50

I00

150

"

8,~

FIG. 3. I)NA synthesis in Escherichia cell 15 T A I / bar m t h e T * a a U m e d i u m after continuous t h y m i n e s t a r v a t i o n . A culture growing exponentially in t h e T * a a U m e d i u m was t r a n s f e r r e d to t h e T ' a s - U m e d i u m . After 120 rain s y n c h r o n i z a t i o n the culture was divided into four parts. Portions of the c u l t u r e were s t a r v e d for 50 rain ( O ) , 70 rain (O), 110 rain ( O ) , a n d 150 rain ( ~ ) in t h e T-aaTJ m e d i u m . After s t a r v a t i o n t h e cultures were transferred t o t h e T * a a U m e d i u m . Values of the relative a c t i v i t y incorporated into t h e cells (a) are related always to t h e values of a c t i v i t y i m m e d i a t e l y after resuspension in the T * a a U m e d i u m after t h y m i n e starvation; t is the time in rain, including t h e interval of s t a r v a t i o n a n d the t i m e of cultivation in t h e T * a a U m e d i u m after starvation. FIQ. 4. I ) N A synthesis i n ~,scherichia coli 15 TAD" bar in t h e T * a a - U - m e d i u m after continuous t h y m i n e starvation. A culture growing exponentially in the T * a a U m e d i u m was transferred t o t h e T + a a - U - m e d i u m . After 120 m l n synchroniZation t h e culture was divided into four p a r t s . Portions of t h e culture were s t a r v e d ibr 50 rnin ( ~ ) , 70 rain (O), 110 rain (O), a n d 150 rain ((~), in t h e T - a a U m e d i u m . A f t e r t h y m i n e s t a r v a t i o n all portions were transferred to the T ' a s - U - m e d i u m . Values of relative a c t i v i t y incorporated i n t o t h e cells (a) are related t o t h e value of a c t i v i t y incorporated after t h y m i n e s t a r v a t i o n at the beg i n n i n g of cultivation in t h e T * a a - I ~ - m e d i u m ; t is t h e time in m i n of cultivation in t h e T * a a - U medium.

D N A synthesis after continuous thymine starvation The culture of Escherichia coli 15 T A U bar was cultivated in the medium TaaU: synchronized in the T * a a - U - medium and thymine starved for 50, 70, 110 and 150 min. At the end of each interval of starvation samples were taken from the basic culture and transferred either to the T * a a U or to the T * a a - U - medium. Samples were taken at regular time intervals and the activity of the incorporated labelled thymine was assayed. Fig. 3 summarizes the results of the experiments in which the incorporation was studied of labelled t h y m i n e into the ceils growing after various time intervals o f continuous thymine starvation in the complete T*aaU~ medium. In order to make the fi~ure more illustrative the time is determined in all cases in such a w a y t h a t the time interval of t h y m i n e starvation is also included. I t was shown that in all cases the relative a m o u n t of incorporated labelled thymine increases exponentially at the beginning and growth decelerates after a certain interval. The initial rate of I ) N A synthesis was found to decrease with a decreasing time of thymine starvation. Fig. 4 summarizes the results of the experiments following the incorporation o f labelled thymine into the cells growing after different intervals of continuous t h y m i n e starvation in the T ' a s - U - medium, i.e. under conditions of limited D N A synthesis. I t could be shown t h a t after 50 min t h y m i n e starvation under conditions of limited growth the activity in the ceils roughly doubled as compared~with t h e situation prior to starvation. After 70 rain starvation a small increase of the ~total incorporated activity with respect to the 50 rain s t a r v a t i o n could be detected. After prolonged

86

M. O P E K . A R O V A A N D V. V O N D R E J S

:30 2O

I0 8 6

J

I 50

I I00

l 150 t

I 200

I 250

I 300

3

0

50

I'

I00

.150

Vol. 18 F I e . 5. D N A s y n t h e s i s in Escherichia cell 15 T a u bar in t h e T*aaLY m e d i u m a f t e r i n t e r r u p t e d t h y m i n e s t a r v a t i o n . A c u l t u r e g r o w i n g e x p o n e n t i a l l y in t h e T * a a U m e d i u m w a s t r a n s f e r r e d to t h e T ' a n - U m e d i u m . A f t e r 120 m i n s y n c h r o n i z a t i o n t h e c u l t u r e w a s d i v i d e d into f o u r parts. T h e first p o r t i o n w a s s t a r v e d for 50 rain in t h e T - a a U m e d i u m ( O ) , t h e r e m a i n i n g p o r t i o n s w e r e s t a r v e d i n t e r m i t t e n t l y for 70 rain ((~), 110 rain ( Q ) , a n d 150 rain ( ~ ) . T h e t i m e s c h e d u l e of i n t e r r u p t e d s t a r v a t i o n is s u m m a r i z e d in T a b l e I I . A f t e r t h e g i v e n i n t e r v a l o f s t a r v a t i o n t h e c u l t u r e s were t r a n s f e r r e d to t h e T * a a U m e d i u m . T h e relative a c t i v i t y i n c o r p o r a t e d into cells (a) is r e l a t e d to t h e a c t i v i t y i n c o r p o r a t e d a t t h e b e g i n n i n g o f c u l t i v a t i o n in t h e T * a a U m e d i u m a f t e r s t a r v a t i o n ; t is t i m e in min, i n c l u d i n g s t a r v a t i o n or t o t a l t i m e of i n t e r r u p t e d s t a r v a t i o n , a n d t h e t i m e of c u l t i v a t i o n in t h e T * a a U m e d i u m after starvation. FrG. 6. ]:)NA s y n t h e s i s in Escherichia coli 15 TALr bar in t h e T*aa-LT- m e d i u m a f t e r i n t e r r u p t e d t h y m i n e s t a r v a t i o n . A culture g r o w i n g e x p o n e n t i a l l y in t h e T*aaLr m e d i u m w a s t r a n s f e r r e d t o t h e T * a a - L ? - m e d i u m . A f t e r 120 m i n s y n c h r o n i z a t i o n t h e c u l t u r e w a s d i v i d e d into six p a r t s : t h e first p o r t i o n w a s s t a r v e d c o n t i n u o u s l y for 50 rain in t h e T - a a U m e d i u m ((~), t h e r e m a i n i n g p o r t i o n s were s t a r v e d i n t e r m i t t e n t l y for 70 m i n ( ~ ) , 90 rain ( O ) , 110 m i n ( = ) , 130 m i n ( O ) , or 150 m i n ({D)A f t e r s t a r v a t i o n t h e c u l t u r e s were t r a n s f e r r e d t o t h e T * a a - U - m e d i u m . T h e relative a c t i v i t y inc o r p o r a t e d into t h e cells (a) is r e l a t e d to t h e act i v i t y i n c o r p o r a t e d a t t ~ 0; t is t h e t i m e o f cult i v a t i o n in t h e T ' a n - U - m e d i u m a f t e r s t a r v a t i o n .

starvation the total increase of incorporated radioactivity under conditions of limited D N A synthesis was again twofold, similar to the cultivation in the T ' a n - U medium after 50 min cultivation w i t h o u t thymine. I t follows from the previous results (Figs. 3, 4) that in Escherichia coli 15 T A U bar s u b j e c t e d to continuous thymine starvation, no increase of the initial D N A synthesis occurred after the transfer to the T * a a U medium; the total amount of D N A synthesized after the transfer to the T ' a n - U - medium did not increase significantly either.

D N A synthesis after interrupted thymine starvation The culture of Escherich~a coli 15 T A U bar was cultivated in the T ' a n - U - medium a n d then thymine-starved in such a w a y t h a t starvation was interrupted b y 5 min pulses according to the schedule presented in Table II. After 50 min of continuous starvation the culture was transferred to the T*aaU medium. Samples were t a k e n a t regular time intervals and the total amount of activity incorporated into the ceils was determined (Fig. 5). I t was found that starvation, interrupted b y shor~ pulses, results in a regular increase of the rate of D N A synthesis after the final addition of thymine, as compared with the continuous starvation. The rate of t h e thymine incorporation decreased in all cases, again to a value corresponding roughly to the time of doubling of D N A of a b o u t 80 min.

1973

D N A S Y N T H E S I S I N E. C O L I

T~rE

87

I I . Time schedule of i n t e r m i t t e n t s t a r v a t i o n , t is the t o t a l time of i n t e r m i t t e n t s t a r v a t i o n .

t rain 50 70 90 110 130 150

Sample consecutively c u l t i v a t e d in m e d i u m (min)

T-A I 50 50 50 50 50 50

I T-a : I TA , I T-A--I 5 5 5 5 5

20 20 20 20 20

5 5 5 5

I T-Art

20 20 20 20

5 5 5

20 20 20

i T-a l TA. I T-A

5 5

20 20

5

20

When the culture was starved continuously for 50 min and intermittently for 70 90, 110, 130 or 150 rain, according to Table II, and transferred after the last interval of starvation to the T * a a - U - medium, an increase in the total a m o u n t of synthesized DNA, related to the total time of starvation, could be observed (Fig. 6). In other words, also in this case a pronounced difference between continuous and interrupted starvation was demonstrated. Table I I I summarizes and compares relative increases TABLE I I I . Comparison o f t h e increase of b i o m a s s d u r i n g i n t e r m i t t e n t t h y m i n e s t a r v a t i o n w i t h the increase of D N A d u r i n g c u l t i v a t i o n in the T * a a - U m e d i u m after s t a r v a t i o n . Values t a k e n f r o m Fig. 2 a n d 6. Time schedule of i n t e r m i t t e ~ t s t a r v a t i o n is s u m m a r i z e d in Table I I . At is t h e absorbance of t h e culture at the end of s t a r v a t i o n . A0 is the absorbance of the culture at t h e beginning of starv a t i o n ; a0 is the a c t i v i t y i n c o r p o r a t e d after t r a n s f e r to the T * a a - U - m e d i u m ; am is the value of a c t i v i t y i n c o r p o r a t e d into cells d u r i n g cultivation in t h e T * a a - U - medium after thymine starvation. Total time of s t a r v a t i o n rain

100. At/Ao

aM/aO

50 70 90 110 130 150

170 230 280 300 310 320

180 230 290 360 450 520

%

100.

%

of the total amount of incorporated t h y m i n e with increases of turbidity after the transfer of the intermittently starved culture to the T a a - U - medium. The above d a t a show that increases of incorporated thymine, under conditions of long-term interrupted starvation, are higher than the increase of turbidity. DISCUSSION

I t was demonstrated that Escherichia coli 15 T A U bar, similarly to most strains derived from Escherichia coli 15, contains at least one defective phage (Frampton and Brinkley, 1965). Whereas induction of defective prophages in most related strains

88

M. OPEXA.ROV2~ AND V. V O N D R E J S

Vol. 18

results in lysis of the cells (Menningmann, 1964; Sandoval et al., 1965; Endo et al., 1965; Frampton and Brinkley, 1965; Vondrejs and Hirseh, 1969; Medoff and Schwartz, 1969; Yudelewieh and Gold, 1969), Escherichia coli 15 TAU bar does not lyse, but incomplete phage particles were observed after the induction in cells broken with lysozyme (Frampton and Brinkley, unpublished data, according to Frampton and Brinkley, 1965). The induction of the prophage could explain the fact that with increased time of thymine starvation, the increase in the rate of DNA synthesis and the total amount of DNA synthesized due to premature initiation of replication (Pritchard and Lark, 1964), analogous to those observed in the related strain Escherichia coli JG 151 freed of the prophage (Donachie, Hobbs and Masters, 1968), could not be demonstrated (Donachie and Hobbs, 1967). The above hypothesis is also supported by other results: Short pulses of thymine interrupting the thymine starvation of Escherichia coli 15 TAU prevent lysis of this strain after the addition of thymine to the medium (Dobi~ovs Hirsch and Vondrejs, 1972) and probably prevent induction or inhibit the lyric cycle already prior to the replication of the phage DNA (Hirsch, personal communication). When the starvation of Escherichia coli 15 TAU bar is interrupted by short pulses, the expected increase in the rate of DNA synthesis after transfer to the T*aaU medium and the increase of the total amount of DNA after transfer to the T*aa-U- do occur with extended time of starvation. As with prolonged continuous starvation, no substantial increase in the total amount of DNA occurs after transfer to the T*aa-U- medium. It may be expected that a new initiation of DNA replication does not take place under conditions leading to phage induction. A mechanism preventing the initiation can hardly be explained on the basis of the present results. It is possible that a factor determined by DNA of the prophage modifies RNA polymerase of the bacterium, RNA synthesis of the bacterium required for initiation thus being inhibited (Lark, 1972). In addition to the difference in behaviour of the culture after continuous and interrupted starvation, it is of interest that short thymine pulses interrupting the thymine starvation induce a considerable increase of DNA synthesis after the final addition of thymine but do not induce a significant increase of absorbance as compared with the continuously starved culture, in which the increased synthesis was not demonstrated. Thus, after a prolonged interrupted starvation DNA synthesis increases more than the biomass measured as absorbance. This finding is not in agreement with the hypothesis of Donachie (1968) that the initiation of replication takes place only when the cell reaches a certain critical mass, which explains the behavior of the cells during balanced growth. The above-mentioned findings indicate that the initiation depends on another parameter rather than the size or mass of the egll. This parameter would change proportionally with the size or mass of the cell only under conditions of balanced growth. REFERENCES BARlqER H. D., COH~Slr S. S.: Synchronization of division of a thymineless m u t a n t of Escherichia coU. J. Bacteriol. 72, 115 (1956). BOYLE J. ~7., GRoss W. A., Cook T. M.: Induction of excessive deoxyribonucleic acid synthesis in Escherich~a coli by nalidixic acid. J. Bactcriol. 94, 1664 (1967). DOBIw ~r tttRSCH I., VOI~DREJS V.: Death and lysis of Escherichia coli 15 TAIY cells after pulse-interrupted thymine starvation. Folia Microbiol. 17, 39 (1972). DOtr W. D.: Relationship between cell size and time of initiation of DI~TA replication. Nature 219, 1077 (1968).

1973

DNA S Y N T H E S I S I N E. COLI

89

DO~ACKIE W. D., HOBBS D. G.: Recovery from "thymineless d e a t h " in ~scherichia cell 15 T - . Bioahem. Biophys. Res. Commun. 29, 172 (1967). DO~CAC]tIE W. D., HONES D. G., MASTERS M.: Chromosome replication a n d cell division in .Escherich~ cell 15 T after growth in t h e absence of DNA synthesis. Nature 219, 1079 (1968). E~cDO H. K., AYABE K., AMA~rO K., TAXEYA K.: Inducible phage of Escherichia coli 15. Virology 25, 468 (1965). F~AMPTO~r E. F., B R I ~ T . ~ y E. R.: Evidence of lysogeny in derivatives of Escherichia coli. J. Bactsriol. 90, 446 (1965). HANAWALT P. C., MAXLe~ 0., C U ~ I ~ G S D. J., SCHA~C~T~R M.: The normal D N A replication cycle I I . J . Mol. Biol. 3, 156 (1961). H ~ w I ~ A., B ~ E ~ D.: Alteration in the sequence of deoxyribonucleic acid synthesis by exposure t o ultraviolet light. Biochem. Biophys. Res. Cpmraun. 15, 588 (1964). H E w t ~ r A., BIBLES D.: Reorientation of chromosome replication after exposure to ultraviolet light o f Escherichia coli. J. Mol. Biol. 13, 40 (1965). J~rCDEI~OVX B., ~ S C H L , VO~D~EJS V.: Phases of thymineless d e a t h in Escherichia coli 15 TAU. Folia Microbiol. 16, 303 (1971). K~T.~rzAcrr N. R., MA R.: Initiation of deoxyribonucleic acid synthesis after t h y m i n e starvation o f Bacillus subtilis. J. Baeteriol. 95, 304 (1968). K~LI,ENBEROER E., LARK K. G., BELLE A.: Aminoacid dependent control of D N A synthesis in bacteria a n d vegetative phage. Prec. nat. Acad. Scl. 48, 1860 (1962). :LA~K K. G.: Evidence of the direct involvement of R N A in the initiation of D N A replication in Escherichix, cell 15 T . J. MoL Biol. 64, 47 (1972). MAALOE O., HA~AW.~LT P. C.: Thymine deficience a n d the normal DI~rA replication cycle I. J. Mol. Biol~ 3, 144 (1961). MEDOlrF Go, S(3]tW~LRTZM. ]~.-" I n d u c t i o n of a defective phage DNA methylation in Escherichia cell 15 T - . J. Gen. Virol. 4, 15 {1969). Mms~r~GMAN~ H. D.: I n d u c t i o n in Escherichla cell 15 of the colicinogenic factor b y thymlne-less d e a t h . Biochem. Biophys. Res. Commun. 16, 373 (1964). N~DA D.: I n v o l v e m e n t of newly-formed protein in the synthesis of deoxyribonucleic acid. Biochem. Biophys. Acta 44, 241 (1960). P~ITCHARD R. H., LA~K K. G.: Induction of replication b y t h y m i n e starvation a t t h e chromosome origin in Esch~richia coli. J . ~ o l . Bigl. 9, 288 (1964). SANDOVAX~H. K., REALLY H. C., TANDLEI~ ]~.." Co~iein 15: possibly a defective bacteriophage. Nature ~{]~, 522 (1965). S r ~ r r G., HANAWA~ P. C.: Initiation of DNA replication cycles in Escheriehia coli following DNA syn* thesis inhibition. J. Mol. Biol. 46, 135, 1969. VONDREJS V., ~c[IRSCH I.: Thymineless death. (In Czech) Biologickg listy 34, 38 (1969). WARD G. B., HAN~. M. W., GL.~SER D. A.: Synchronous reinitiation of chromosome replication in Esche* richia coli B/r after nalidixic acid t r e a t m e n t . Prec. Nat. Acad. ~ci. 66, 365 (1970). ~rO~CEL A.: Induction of chromosome re-initiations in a thermosensitive DXNTAm u t a n t of ~scherichia coli. J. Mol. Biol. 52, 371 (1970). Y~D~L~W~CH A., GOLD M.: A specific DNA methylase induced b y bacteriophage 15. J. Mot. Biol. 40, 77 (1969).