Folia Microbiol. 19, 2 6 4 - - 2 7 1 (1974)

Growth and Cell Division of Escherichia coli 15 TAU after Transfer to Deficient Media with Different Sources of Carbon and Energy M. OPEKAROVi* and V. VONDREJS Department o] Biophysics, Faculty of Science, Charles University, 120 O0 Prague 2 Received August 20, 1973

ABSTRACT. W e s t u d i e d g r o w t h a n d ceU division of Escherichia cell 15 T A U after t r a n s f e r to t h y m i n e - f r e e m e d i u m w i t h different sources of c a r b o n a n d e n e r g y or to t h e s a m e m e d i u m in w h i c h n o t o n l y t h y m i n e b u t also arginino a n d / o r uracil wore o m i t t e d . A f t e r t r a n s f e r to t h y m i n e - f r e e m e d i u m o n l y a f r a c t i o n o f cells divides once. T h e size of t h e dividing f r a c t i o n is p r e d e t e r m i n e d p a r t i c u l a r l y b y c o n d i t i o n s o f b a l a n c e d g r o w t h before i n h i b i t i o n of D N A s y n t h e s i s a n d only slightly a f f e c t e d b y c o n d i t i o n s a f t e r t r a n s f e r , while t h e g r o w t h r a t e a f t e r s h i f t to m e d i u m w i t h different source of c a r b o n a n d e n e r g y c h a n g e s a b r u p t l y . Following t r a n s f e r to arginine-dofioiont m e d i a cell division proceeds m u c h m o r e slowly t h a n in o t h e r eases t e s t e d . T h e f r a c t i o n of cells w h i c h causes a d e v i a t i o n of rate m a i n t e n a n c e a f t e r s h i f t - u p a n d s h i f t - d o w n (Cooper, 1969) s e e m s to be t h e s a m e as t h e cell fraction d i v i d i n g a f t e r t r a n s f e r to t h y m i n e - f r ~ o m e d i u m

Janderovs and Vondrejs (1970) observed t h a t during inhibition of DNA synthesis by thymine starvation a fraction of cells of Escherichia cell 15 TAU still divides once. Similar results were obtained when DNA synthesis was inhibited with nalidixie or oxolinic acid (~ulcovs etal., 1974). The fraction of dividing cells is larger when the carbon and energy source in the medium is richer and the culture is, therefore, growing faster (Janderov~ and Vondrejs, 1970). In this paper we focus our attention on the following question connected with cell division of Escherichia cell 15 TAU during inhibition of DNA synthesis: "To what extent is the fraction of dividing cells determined by growth conditions before and during inhibition?" Two aspects of growth condition were studied: (1) the role of carbon and energy source and (2) extensive synthesis of RNA and proteins. We believe t h a t the answer connected with aspect (1) is obtained on the basis of two sets of experiments in which growth and cell division after transfer to thymine-free medium with carbon and energy source different from t h a t of the original medium were studied and compared with cell division after transfer to the same medium where only t h y m i n e was omitted (Janderovs and Vondrejs, 1970). Aspect (2) is at least partially clarified by the results of experiments in which cell division during inhibition of DNA synthesis was studied under conditions in which synthesis of RNA and proteins was also inhibited in uracil- and arginine-free medium. MATERIALS AND METHODS

Escherichia cell 15 TAU requiring thymine (T) arginine (A) and uracil (U) was kindly supplied by Dr. Speyer (Cold Spring Harbor Laboratory of Quantitative Biology, N.Y., USA) several years ago. * Present address: I n s t i t u t e of Microbiology, Czechoslovak A c a d e m y of Sciences, 142 20, P r a g u e 4.

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All cultivation media were modifications of the medium M9 (Kellenberger, Lark and Bolle, 1962) supplemented with 0.02 g thymine, 0.02 g uracil and/or 0.34 g arginine per 1000 ml (glucose T A U medium; if thymine, arginine and/or uracil were omitted, this is indicated b y a minus sign next to the corresponding letter of the medium abbreviation). The M9 medium was in some cases supplemented with 1.5 g Bactocasamino acids (Difco) per 1000 ml (further glucose-casamino acids TAU medium) or glucose was substituted b y 3.68 g of glycerol per 1000 ml (further glycerol T A U medium). When bacteria were diluted or washed, buffer p H 7.2 containing 10 g Na~H1)O4, 4.3 g K t t 2 P 0 4 , 0.7 g NaC1 and 1.1 g NHaC1 per 1000 ml was used. All chemical compounds, if not otherwise indicated, were supplied b y Laehema Company, Brno. Inorganic components were of purity 1).A. Bacteria were cultivated in thin-walled test tubes diameter 17 mm, with intensive aeration in selected medium at 37~ The media were exchanged using filtration through a Synpor 6 (Synthesia, Czechoslovakia) membrane filter, diameter 5 cm and porosity 0.4 ~m (Janderovs et al., 1971). Turbidity of cultures was measured on a Hilger-Spekker colorimeter with a C3 orange filter. Cultivation tubes served also as colorimeter cuvettes. Determination of cell number. One ml of culture was added to 9 ml of 1 ~ formaldehyde solution in dilution buffer. The cells were counted 20 min after the sample had been placed in a Neubauer counter. The total number of cells was always determined by counting 250 to 800 cells using a phase contrast microscope (Carl Zeiss, Jena). RESULTS

Transfer to a new medium containing richer source of carbon and energy

Two media differing in source of carbon and energy were selected for this set of experiments. Doubling time of Escherichia coli 15 T A U growing exponentially in glycerol TAU medium was about 56 rain and in richer glucose-casamino acids T A U medium about 36 min (Fig. 1). When an exponentially growing culture of Escherichia coli 15 TAU was transferred from glycerol TAU medium to glucose-casamino acids T - A U medium the turbidity of culture increased faster than at pre-shift conditions (Fig. 1). The growth continued until about 60 rain, when the turbidity of the culture reached 3 times the original value. The same increase in growth rate was observed when the culture was shifted up to glucose-casamino acids T A U medium. In Fig. 2 the relative increase of cell number after transfer from glycerol T A U medium to glucose-casamino acids T - A U medium is compared with the time relationship of the cell number after shift-up to glucose-casamino acids T A U medium. Cell division continues after shift in both cases for about 30 min and then stops in thymine-free medium, slows down in medium with thymine. The first relative increase of cell number is about the same in both media independently of the presence of thymine. The second rapid increase in the cell number after transfer to the new medium was observed only in the presence of thymine. Transfer to poorer medium When a culture of Escherichia coli 15 T A U growing exponentially in glucose-casamino acids T A U medium was transferred to glycerol T - A U or glycerol T A U

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FIG. 1. G r o w t h of Escherichia coli 15 T A U a f t e r t r a n s f e r f r o m glycerol T A U m e d i u m to glucoseeasamino acids T A U or T - A U m e d i u m . A bacterial culture growing exponentially in glycerol T A U m e d i u m was t r a n s f e r r e d at time 0 to glucosecasamino acids T A U -- C) --, glucosa-casamino acids T - A U - - D - - , or glycerol T A U m e d i u m - - & - - . F o r ease of viewing t h e curve s h o w i n g the g r o w t h of a culture of Escherichia coli 15 T A U following the t r a n s f e r f r o m m e d i u m glucose-casamino acids T A U to t h e s a m e fresh m e d i u m (from Fig. 3) is s u p e r i m p o s e d on this figure as a d o t t e d line. T is t u r b i d i t y of culture; rain, time a f t e r r e s u s p e n s i o n in a now m e d i u m .

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Fzo. 2. Cell division of Escherichia cell 15 T A U after t r a n s f e r f r o m glycerol T A U m e d i u m to glucosoe a s a m i n o acids T A U or T - A U m e d i u m . A bacterial c u l t u r e growing e x p o n e n t i a l l y in glycerol T A U m e d i u m was t r a n s f e r r e d at t i m e 0 to glucose-easamino acids T A U O or T - A U -- g - - medium. Curve s h o w i n g coil division after t r a n s f e r f r o m glycerol T A U m e d i u m (unbroken line) or glucoso-easamino acids m e d i u m (dotted line) t o the s a m e m e d i u m b u t w i t h o u t t h y m i n e is s u p e r i m posed on this figure f r o m the r e p o r t of J a n d o r o v ~ and Vondrojs (1970). Nt is the total n u m b e r of cells at t i m e t rain f r o m rosusponsion of coils in a n e w m e d i u m . No is the t o t a l cell n u m b e r at t i m e 0.

medium an immediate stop of growth was observed in both cases (Fig. 3). Some time after the shift, turbidity of the culture began to increase with the rate of growth similar to t h a t observed during the balanced growth of Escherichia cell 15 T A U in glycerol T A U medium. In the absence of thymine, however, the growth continued only for some time. The increase of relative turbidity after shift to thyminefree medium with poorer source of carbon and energy is smaller than that after transfer to richer thymine-free medium (Fig. 1). The first increase of cell number observed after shift to glycerol T - A U or T A U medium (Fig. 4) is larger than after shift from glycerol T A U to glucose-casamino acids T - A U or T A U medium (Fig. 2). Similarly as in the shift-up experiment, the second increase in cell number was observed only in medium with thymine, while in thymine-free medium only a fraction of cells divided once. More than 60~o of cells divided during the first 20 min interval after transfer from glucose-casamino acids T A U medium to glycerol T - A U or T A U medium. This is approximately the same as the fraction of cells which divided after transfer from glucose-easamino acids T A U medium to the thymine-free medium with unchanged source of carbon a n d

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Fzo. 3. G r o w t h of Escherichia cell 15 T A U after t r a n s f e r f r o m glucose-easamino acids TAU med i u m to glycerol T A U or T - A U medium. A culture growing exp o n e n t i a l l y in g l u c o s e - e a s a m i n o acids T A U m e d i u m w a s t r a n s f e r rod a t t i m e 0 to glycerol T A U -O--, T-AU@-- or glucose- c a s a m m o acids T A U m e d i u m -- 9 C u r v e s h o w i n g cell g r o w t h in glycerol T A U m e d i u m is superimposed on t h i s figure f r o m Fig. 1 as a d o t t e d line. T is t u r b i d i t y o f culture; rain, time m e a s u r e d f r o m r o s u s p e n s i o n in a now m e d i u m .

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F i e . 4. Cell division o f Escherichia cell 15 T A U a f t e r t r a n s f e r f r o m g l u e o s o - c a s a m i n o acids T A U m e d i u m to glycerol T A U or T - A U m e d i u m . A cult u r e g r o w i n g e x p o n e n t i a l l y in g l u e o s e - c a s a m i n o a c i d s T A U m e d i u m w a s t r a n s f e r r e d a t t i m e 0 to glycerol T A U - - O - - or T - A U - - @ - - m e d i u m . C u r v e s h o w i n g cell d i v i s i o n following a t r a n s f e r f r o m glycerol T A U m e d i u m ( u n b r o k e n line) or g l u c o s o - c a s a m i n o acids T A U m e d i u m (dotted line) to t h e same medium but without thymine is s u p e r i m p o s e d o n t h i s figure f r o m t h e r e p o r t o f J a n d o r o v ~ a n d V o n d r e j s {1970). N t is t h e t o t a l n u m b e r of cells a t t i m e t rain f r o m r e s u s p e n s i o n in a now m e d i u m . No is t h e total cell n u m b e r a t t i m e 0.

energy (Janderovs and Vondrejs, 1970). The cell fraction dividing after the transfer from glycerol T A U medium to glucose-casamino acids T - A U medium is, on the other hand, the same or only slightly larger than the fraction of cells dividing after transfer of Escherichia cell 15 TAU from glycero~ T A U medium to the thymine-free medium with unchanged source of carbon and energy. The results of experiments described up to now have the following common features: Cell division of Escherichia coli 15 T A U after transfer to thymine-free medium is limited. Only a cell fraction, which seems to be determined particularly b y the pre-shift conditions of the exponentially growing culture, divides. The kinetics of division in thymine-free medium does not seem to be remarkably affected b y the change in the source of carbon and energy. This result is of particular interest because the doubling time of exponential growth and division in poorer glycerol T A U medium is about 1.6 times longer than in glucose-casamino acids TAU medium. I n addition, after shift-up the culture of Escherichia cell 15 T A U immediately grows faster than at pre-shift conditions while after shift-down the growth stops.

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I F i e . 5. Ceil division o f Escherichia coli 15 T A U after t r a n s f e r f r o m glucose T A U m e d i u m to glucose T - A U or T - A U - m e d i u m . A culture g r o w i n g exponentially in glucose T A U m e d i u m w a s transferred a t t i m e 0 to gluooee T - A U -- O -- or T - A U --0-m e d i u m . Nt is the t o t a l cell n u m b e r a t time t rain m e a s u r e d f r o m rosuspension in a n e w m e d i u m . No is the t o t a l cell n u m b e r a t t i m e 0.

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Fro. 6. Cell division of Escherichia cell 15 T A U after t r a n s f e r f r o m glucose m e d i u m to glucose T - A - U ' T - A - U - or glucose-free T - A - U - m e d i u m . A culture g r o w i n g exponentially in glucose T A U m e d i u m w a s t r a n s f e r r e d at t i m e 0 to glucose T-A-U --O--, T - A - U - -- 9 or glucose-free T - A - U - - - A - - m e d i u m . Nt is t h e total cell n u m ber a t time t m i n m e a s u r e d f r o m rosuspension in a now m e d i u m . N0 is the t o t a l cell n u m b e r at time 0.

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Transfer to thymine-free media deficient in arginine, uracil and~or source of carbon and energy In order to determine whether the fraction of dividing cells and kinetics of division after the transfer to thymine-free media is quite independent of the new conditions, R N A synthesis and/or protein synthesis was inhibited in addition to D N A synthesis and in one experiment even a source of carbon and energy was omitted. Total cell number after transfer from glucose T A U medium to selected medium was determined in dependence on time of cultivation measured from the moment of resuspension in thymine-free medium. The results of this set of experiments are summarized in Figs. 5 and 6. In comparison with the relative increase of cell number in glucose T - A U medium (Fig. 5) the fraction of dividing cells in other deficient thymine-free media is slightly smaller. No remarkable difference was observed between the rate of division after the transfer of the culture to glucose T - A U and glucose T - A U - media (Fig. 5). The rate of increase of cell number in thymine-free media without arginine, uracil and also glucose was similar to that observed in glucose T - A - U - medium. In the former case, however, an even smaller fraction of cells divided (Fig. 6). A culture of Escherichia cell 15 T A U grows in complete glucose T A U medium with a generation time of approximately 45 min. Following transfer to a deficient glucose T-AU, T - A - U , T - A U - or T - A - U - medium or to glucose-free T - A - U medium, the t u r b i d i t y of the culture does not remarkably increase (Fig. 7). DISCUSSION

The experimental results summarized in this paper are in good agreement with results of J a n d c r o v s and Vondrejs (1970) showing t h a t there is a fraction of cells

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to glucose T - A U --O--, T - A - U --O--, T - A U -- A --, T - A - U - -- 9 T A - U - -- [ ] - or glucosefree T - A - U 9 medium. G r o w t h in glucose TAU medium --• is shown for comparison. T is turbidity of the m]lture; rain, time from re. suspension in a n e w medium.

in an exponentially growing culture of Escherichia coli 15 T A U which have reached the state when their division cannot be prevented by transfer to thymine-free medium. As the pool of thymine-containing precursors is very small in thymine-auxotrophic derivatives of Es~herichia coli 15, DNA synthesis is interrupted almost instanteneously (Lark and Lark, 1969). For this reason we assume that the division of Escherichia coli 15 T A U observed in thymine-free media occurs under conditions when D N A synthesis is not taking place. Because the fraction of dividing cells and kinetics of division is not remarkably affected b y the carbon and energy sources in a new thymine-free medium and depends particularly on conditions before inhibition of D N A synthesis, it seems hkely t h a t those cells have reached the state when "decision is made". Such cells are independent not only of D N A synthesis b u t also of other changes following shift to the medium with different source of carbon and energy: i.e. change in growth rate and rate of I~NA synthesis (Maaloe and Kjeldgaard, 1966). Our method of determination of cell number, however, does not allow us to exclude the possibility t h a t new conditions have a small effect. The main conclusions from our nutritional shift-up and shift-down experiments to medium with thymine are in good agreement with the results obtained originally with Salmonella typhimurium (Maalae and Kjeldgaard, 1966) and with shift-up experiments using Escherichia coli B/r (Cooper, 1969). After shift-up to a richer medium an immediate increase of growth rate appeared. Shift-down was followed b y immediate stop of growth. In both cases a definitive post-shift rate of division was reached after some period of time. Kjeldgaard et al. (1958) noted that after shift to a richer medium the rate of cell division continued at the pro-shift value before an abrupt increase to the new rate. A similar rate maintenance was described after shift-down (Maaloe and Kjeldgaard, 1966). Results summarized in this paper show regular deviation in "rate maintenance" after shift-up and also after shift-down. A stepwise increase of cell number appea-

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red, before it began to increase at definitive post-shift rate instead of continuing at the pre-shift rate. It does not seem likely that this deviation is induced by the washing procedure because a similar observation was made by Cooper (1969) who studied cell division after shift-up of Escherichia coli B/r from glucose to glucosecasamino acids medium. I n that experiment the conditions were changed only by adding casamino acids solution to the original medium. The same phenomenon is hinted at by the published experimental values of Kjeldgaard et al. (1958) and Maaloe and Kjeldgaard (1966). Slight deviations from rate maintenance can be explained, according to Cooper (1969) by variations in time schedule of cell cycle. A comparison of cell division after shift-up or shift-crown with cell division after transfer to thymine-free medium containing the same or different source of carbon and energy indicates that the cells dividing during the period of first increase of cell n u m b e r after shift are those which already before shift reached the state when "decision was m a d e " ; they can divide even in the absence of DNA synthesis. The results of experiments in which cell division after transfer to media deficient not only in t h y m i n e was studied show t h a t the process of division in the absence of DNA synthesis is not quite independent of other conditions. I n the stringent strain Escherichia coli 15 TAU (Maaloe and Kjeldgaard, 1966) dependent on thymine, arginine and uracil, the synthesis of RNA and proteins is inhibited when uracil and/or arginine is omitted from the medium. In all deficient media tested the total fraction of cells dividing after transfer decreases, but only in the absence of arginine does cell division also slow down. The fraction of cells dividing under these conditions is still large enough even when thymine, arginine, uracil and glucose as the only source of carbon and energy are omitted. These results enable us to conclude t h a t cell division in absence of t h y m i n e is at least in a subfraction of cells dependent on synthesis of proteins or RNA or both and on synthetic activity of cells, but we cannot say on the basis of these experiments anything more about the mechanism of this effect. A similar suggestion was made by Smith and Pardee (1970) who observed accumulation of a protein required for division during the cell cycle of Escherichia coli. Experiments of Clark (1968) and particularly of Helmsteter and Pierucci (1968) led to the interesting suggestion t h a t all cells in an exponentially growing culture of Escherichia coli B/r t h a t have reached the point of termination of replication will divide after replication is inhibited. The expected cell number increments were observed also with Escherichia coli 15TAU (Janderovs and Vondrejs, 1970) on the basis of the assumption that this strain exhibits the same time interval between termination of replication cycle and division as Escherichia coli B/r. Results summarized in this paper and the communication of Janderovs and Vondrejs (1970) support the idea of critical event at which "decision about division is made". This e v e n t is in time coincidence with, and m a y be in causal connection with or equiv a l e n t to termination of replication cycle. In this sense our results support the hypothesis of Clark (1968) and Helmstetter and Pierucci (1968) mentioned above. BEFEHENCES CLARK D. J.: Regulation of dooxyribonuoleio aoid replioation and o9ll division in Eseheriohla eoli B/r J. Baoteriol. 96, 1214 (1968). CooPEg S.: Cell division and DNA replication following a shift to riohor medium. J. MoL B4ol. 48, 1 (1969). HELMSTETTER 0. E., PI~.RtrCer O.: Coil division during inhibition of dooxyribonueleio acid synthesis in Escherichia coll. d. Bacteriol. 95, 1627 (1968). J~'~D~.aovJ, B., VO~DREZS V.: Division of cells of Escherichia coli 15 TAU during thymine starvation. Falia Microbiol. 15, 500 (1970).

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JANDEROV~. B., •IRSCH I., VONDREJS V.: Phases of thymineloss d e a t h in Escherichia r 15 TAU. z~olia Mi~robioL | 6 , 303 (1971). KELLENB~.RGER E., L~LRK K. G., BELLE A.: Amino acid dependent control of D N A synthesis in bacteria and vegetative phage. Prec. Nat. Acad. ~ci. 48, 1860 (1962). KJELDG~.F,D 1~'. O., MAAr~a~.., SCHAEC~T~R M.: The transition between different physiological states during balanced growth of ~almo~ella typhimuriurn. J. (~en. Bacteriol. 19, 607 (1958). L ~ . K C., Li~t~r K. G.: Evidence for two distinct aspects of t h e mechanism regulating ehromosmo replication in Escherichia coli. J. MoL Biol. 1O, 120 (1969). MAALOE O., KJELI)G~-RD N. O.: Control of macromoleoular synthesis. W. A. Benjamin, Inc., New York (1966). SMITH H. S., P~aDEE A. B.: Acumulation of a protein required for division during cell cycle of E. ceil J. Bacteriol. 191, 901 (1970). ~uLcov~_ V., HA~OV/~-MORAVOV/LJ., ~.r A., VONI)~JS V.: Effect of derivatives of T-quinolinecarboxylic acid of DNA synthesis, growth and division in E. coli 15 TAU. Folia Microbiol. 19, 281 (1974).