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Relationships between non-extractable DNA and the bacterial growth cycle
22
Biochimica et Biophysica Acta, 378 ( 1 9 7 5 ) 2 2 - - 3 4 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s BBA 98184
RELATIONSHIPS BETWEEN NON-EXTRACTABLE DNA AND THE BACTERIAL GROWTH CYCLE
J O S E P H I N E S. SALSER and M. EARL BALIS
Sloan-Kettering Institute for Cancer Research, New York, N.Y. 10021 (U.S.A.)
Summary
Escherichia coli DNA has been fractionated into extractable and nonextractable DNA after deproteinization of detergent-lysed cell preparations with chloroform--isoamyl alcohol. The former was extracted with dilute buffered saline whereas the latter remained in the interphase layer associated with residual cellular debris from which almost 40% could be released by incubating with pronase. About 20--25% more amino acid residues were bound to the pronase-released DNA than to the extractable DNA, but the relative distribution of the residues in the two DNA samples was virtually identical. The specific activities and the relative amounts of denser (1.709 g • cm -3) and lighter (on the surface of CsC1 gradients) DNA fractions from E. coli, grown in the presence of labeled thymidine, indicated that these two corresponded to extractable and non-extractable DNA, respectively. The relative amounts of the two fractions varied with the growth phase, primarily as a function of the growth rate. Age and metabolic state of cells i n the culture or those used as inocula could m o d i f y this relative distribution. When growth rate was maximal, the ratio of the two remained at about 1. During lag phase when no appreciable net synthesis of DNA could be detected, there was a rapid and preferential incorporation of labeled thymidine into non-extractable DNA. A disproportionate increase in the fraction of the total DNA, which was extractable, was also observed but only when stationary phase cultures were used as inocula. Complete equilibration of the label in the two DNA fractions was attained only after cultures had reached mid-log phase of growth. Similar results were obtained when prelabeled cells were used. These data have been interpreted as suggesting that the rate of cell growth and DNA synthesis are related to the number or size of sites of attachment of DNA to some cellular structure. Newly synthesized DNA would be attached to different sites on this structure and initiation of DNA replication in lag phase would require reorientation o f the two kinds of DNA. Small peptides which are firmly bound to the DNA and which vary quantitatively with the rate of DNA synthesis could perhaps be involved in the a t t a c h m e n t to the sites.
23 Introduction
When detergentAysed bacteria are extracted by conventional procedures, part of the DNA can not be isolated w i t h o u t considerable degradation and denaturation of the polynucleotide [1--3]. The possibility of association(s)of the non-extractable DNA fraction with some cellular structures has been suggested by diverse experimental observations; e.g. preferential partitioning of this fraction into the interphase during deproteinization and decreased b u o y a n t density in cesium chloride gradients [1,4--6], electron-microscopic demonstrations of linkages between the bacterial chromosome and cell membrane [7] and isolation of DNA--membrane complexes free from bulk cellular proteins [5,8--11]. The nature of these associations is still not clear although some of the non-extractable D N A can be released by treating with lysozyme [12], detergent [4,8,10], pronase [5,6,13] or pronase in conjunction with either ribonuclease [6] or detergent [14]. The metabolic role of such a non-extractable DNA fraction is also far from well defined. In exponentially growing cultures, a presumptive role in DNA replication has been based on a preferential accumulation of pulse label in non-extractable DNA and a rapid chase of this label into extractable DNA [1,4,15--17]. Studies from this laboratory [18,19] have shown that irrespective of cellular origin, DNA which is extractable with dilute buffered saline contains amino acid residues which can n o t be dissociated without extensive degradation of the polynucleotide. These residues in bacterial DNA varied in amount and composition with the growth cycle [19]. For any given growth phase, the amount of these amino acids associated with extractable DNA changed with the composition o f the medium. However, it was essentially unaffected by irradiation of the cultures [2] in spite of increases in the amounts of nonextractable D N A which have resulted as a consequence of the greater crosslinking o f DNA and proteins under these conditions [3]. There was a steady decrease in the a m o u n t o f amino acid residues as a culture progressed through the various growth phases. If there were any direct relationships between the amino acid residues in extractable DNA and the association of DNA with other cellular c o m p o n e n t s in non-extractable DNA, the relative amounts of the latter would also vary with the stages of the growth cycle. In this report, the amounts and the fate of DNA extractable and non-extractable with buffered saline from detergent-lysed bacteria at different growth phases and the amino acid residues associated with both DNA fractions have been examined. Methods Bacterial strains and growth conditions Escherichia coli strain B and the thymine-requiring strain 15T- were grown aerobically at 37°C in M9 medium [21] containing 0.4% glucose. This was supplemented with thymidine (10 ttg per ml) for 15T-. Growth was followed by absorbance readings at 540 nm (As 40 ). Unless otherwise specified, overnight cultures (16--17 h) consisting primarily of stationary phase cells were diluted over 100-fold into fresh medium to give an initial As 40 of 0.035--0.060 which was equivalent to 1.8 • 1 0 7 - 3 . 0 • 107 cells
24 per ml. Cell titers were determined using a Petroff--Hauser counter and viable cells counts by an agar plating m e t h o d [19]. Earlier studies on amino acid residues bound to DNA indicated that a larger fraction of the total cell DNA was consistently extracted with dilute buffered saline from lag, early-log and late-log phase cells. To reexamine these observations, large scale cultures were used so that relatively large volumes could be withdrawn during the early time periods when cell density was very low and all samples for the various phases of a growth cycle could be sequentially taken from the same set of cultures. Three 10-1 cultures were grown in a New Brunswick FS 314 Laboratory Fermentor. Parallel growth was maintained in these cultures by careful control of aeration and stirring rate. At various intervals, equal volumes were withdrawn from these cultures for DNA extraction; the pooled samples contained at least 150--200 As 4 o units.
Isotope incorporation studies [Me-3H]Thymidine (10 Ci/mmole) was obtained from International Chemical and Nuclear Corporation, California. T o follow DNA synthesis through the different phases of the growth cycle, 0.67 mCi of [Me -3H]thymidine was added with the required a m o u n t of unlabeled thymidine to each 10 1 of culture at the times indicated in the figures. Prelabeled 15T- cells were prepared either by growing overnight on a rotary shaker with 0.44 mCi of [Me-3 HI thymidine per 500 ml of medium or by continuing growth for 3 h after the addition of 0.67 mCi of labeled thymidine per 10 1 to a mid-log phase culture. Growth curves showed that at harvest, the former culture was in the stationary phase and the latter culture in the late-log phase of growth. The cultures were chilled and the cells, collected by centrifuging at 4°C, washed twice in cold glucose-free M9 medium and finally resuspended in prewarmed M9 containing the required a m o u n t of unlabeled thymidine. Dilution of the label in DNA was used as a measure of the changes in the extractable and non-extractable DNA during the growth cycle. Samples (1--2 ml) were taken at 10--15 min intervals to monitor the growth of the cultures. At the times indicated in the figures, larger samples were withdrawn for DNA extraction. These were rapidly chilled by pumping through a stainless steel coil immersed in an alcohol--ice bath and the cells collected by centrifugation. When sample volumes were greater than 500 ml, a Sorvall KSB Continuous Flow System was used. The packed cells were resuspended and washed twice with cold 0.15 M NaC1--0.1 M EDTA, pH 8, a medium which removes lysed cells present in the suspensions. About 85--90% of the cells were consistently recovered. In some samples, three-fourths of the resuspended cells were set aside for studies on amino acid residues bound to DNA. Washed packed cells were stored frozen at --20 ° C. Isolation o f DNA The procedure described by Marmur [22] was used with the following modifications. Cells were lysed with larger volumes of saline--EDTA--sodium dodecylsulfate (1%), i.e. 30 ml per g wet weight of packed cells. To insure quantitative recovery of the extractable DNA, the interphase layer resulting from deproteinization with chloroform--isoamyl alcohol was reextracted twice
25 with 0.015 M NaC1--0.0015 M trisodium citrate, pH 7.5 (dilute saline--citrate). The combined aqueous fractions were dialyzed against dilute saline--citrate (5 changes), concentrated 5-fold b y lyophilization and adjusted to a final concentration of 0.15 M NaC1--0.15 M trisodium citrate (standard saline--citrate). DNA was precipitated from this solution by the addition of t w o volumes of ethanol (95%), purified b y t w o reprecipitations and dried by a graded ethanol (70--100%) and ether wash. The interphase layer containing the non-extractable DNA was washed two more times with standard saline--citrate and dried by a similar alcohol--ether wash. Perchlorate extracts of the original suspensions and the isolated D N A fractions were used for DNA determination by the diphenylamine reaction [23]. The radioactivity incorporated into DNA was assayed in aliquots of the neutralized extracts. Some DNA samples were also fractionated by gradient centrifugation. Crude cell lysate--CsC1 mixtures (overall density 1.7 g per ml) were centrifuged f o r 72 h at 29 000 rev./min in the SW 39 rotor of a Beckman Model L2 preparative ultracentrifuge. The tubes (containing 5 ml of sample per tube) were punctured and three-drop fractions were collected in tubes containing 0.5 ml of dilute saline--citrate. Aliquots were removed and assayed for radioactivity. Combined peak fractions from repeated runs were desalted by gel filtration and concentrated to a minimal volume by lyophilization. DNA was recovered by the usual alcohol precipitation procedure. DNA was also extracted from the material floating on the top of the gradient; the latter was recovered b y repeated washing of the drained tubes. When amino acid residues associated with DNA were examined, the extractable DNA was isolated and purified as previously described [19]. A b o u t 40% of the non-extractable DNA could be recovered from the interphase layer after pronase treatment. The interphase material was washed with three volumes of ethanol (90%) followed b y two volumes of dilute saline--citrate (two times). After resuspending in standard saline--citrate, pronase (Calbiochem, 45 000 proteolytic units per g, preincubated for 3 h at 37°C) was added to give a final concentration of 0.5 mg per ml and the suspension incubated with gentle shaking for 16 h at 37°C. The suspension was centrifuged and DNA was precipitated with ethanol from the clear supernatant solution. This DNA was not readily soluble in dilute saline--citrate unless sodium dodecylsulfate was added to a final concentration of 0.25% and the solution allowed to stand overnight at r o o m temperature. The detergent was subsequently removed by dialyzing against standard saline--citrate. This DNA was further purified in the same manner as extractable DNA. Hydrolysis of DNA samples and amino acid determinations have previously been described [ 1 9 ] . Results
Preliminary studies carried o u t to establish optimal experimental conditions for large scale cultures showed that when stationary phase (overnight) cultures were diluted over 100-fold (instead of 15- to 20-fold) into 10 1 of prewarmed medium, lag phase could be prolonged from less than 25 min to about 100 min. Although there was little change in total DNA during this prolonged lag, increments of 30% or more were observed in cell density, i.e. cell
26 mass. When these cultures entered early-log phase, the rates of increase in cell density and total D N A paralleled each other, reaching and maintaining maximal values during mid-log phase. By late-log phase, however, there was a slowing down of both these rates of increase, that of total D N A was much greater than that of cell mass. In spite of the prolonged growth cycle, these findings were consistent with those observed in smaller cultures [ 2 4 ] . Total D N A determined by direct analysis of aliquots of the cultures was found to be virtually the same as the sum of the extractable and non-extractable DNA. Variations in replicate samples of either of these two D N A fractions were ~< 3%. Operationally, extractable D N A is defined as the D N A which was extracted with buffered saline when detergent-lysed cell preparations were deproteinized with chloroform--isoamyl alcohol and non-extractable as that which remained associated with the interphase material. To optimize utilization of thymidine [ 2 5 ] , only the thymine-requiring strain 15T- was used in these studies. Results from a typical experiment with 15T- are given in Fig. 1. [Me -3 H] Thymidine was added to cultures which had been equilibrated for 15 min in the absence of thymidine. Under these condi80
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F i g . 1. D i s t r i b u t i o n o f D N A d u r i n g t h e g r o w t h c y c l e o f E. coli s t r a i n 1 5 T - . O v e r n i g h t c u l t u r e s w e r e u s e d d i r e c t l y as i n o c u l u m . A f t e r a 1 5 r a i n e q u i l i b r a t i o n p e r i o d i n u n s u p p l e m e n t e d glucose--M9 medium, 100 m g o f t h y m i d i n e c o n t a i n i n g 0 . 6 7 m C i [ M e - 3 H ] t h y m i d i n e w e r e a d d e d t o e a c h 1 0 1. S a m p l e s w e r e r e m o v e d at t h e t i m e s i n d i c a t e d i n t h e f i g u r e a n d f r a c t i o n a t e d a n d a n a l y z e d as d e s c r i b e d u n d e r M e t h o d s . C e l l growth was monitored by A540 nm- The amount of DNA in the extractable and non-extractable fractions, determined by the diphenylamine reaction [23], was used to calculate the ratio of the two f r a c t i o n s a n d t h e t o t a l D N A p e r m l o f c u l t u r e , i.e. t h e s u m o f t h e t w o i n e a c h m l o f t h e o r i g i n a l s a m p l e . The specific activity has been calculated from the radioactivity detenmine in aliquots of each fraction. After complete equilibration of the label between the two DNA fractions, the specific activity was 177 + 0.5 (mean + S.E.) cpm per/~g DNA.
27
tions, the lag period before onset of thymineless death was 23 min. The viable cell c o u n t at 15 min was over 95% of that present in the original inoculum. Although there was no appreciable net synthesis of DNA during the first 120 min of incubation, there was a doubling of cell density, a pronounced incorporation of label into D N A as well as the distinct shift in the ratio of extractable to non-extractable DNA. A b o u t 30% and 96% of the final specific activity was found in extractable and non-extractable DNA, respectively. Since the ratio of the a m o u n t of DNA in the two fractions at this time was a b o u t 4 : 1, almost 45% of the total DNA was labeled. This initial period was followed by a rapid increase in total DNA which paralleled the logarithmic increase in cell density until late-log phase was reached. The increments observed in the specific activity of extractable DNA corresponded to the values which can be calculated if one assumes that all newly synthesized DNA was completely labeled and no extractable DNA is converted to non-extractable DNA. When these cultures approached mid-log phase, about 90% of the total DNA was labeled. The ratio of the a m o u n t of extractable to non-extractable D N A at this time was 1.2 : 1. When [Me -s H] thymidine was added to mid-log phase cultures, incorporation of the label was essentially instantaneous (Fig. 2). Although the increase in 8C ~- _~4 0 --//'1
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Fig. 2. D i s t r i b u t i o n o f D N A in m i d - l o g a n d late-log p h a s e E. coli s t r a i n 1 5 T - . A 10 1 c u l t u r e w a s g r o w n in glucose--M9 s u p p l e m e n t e d with unlabeled t h y m i d i n e until mid-log phase was approached, at which time ( 1 8 5 m i n as i n d i c a t e d b y t h e a r r o w ) 0 . 6 7 m C i o f [Me-3H]thymidine w a s a d d e d t o t h e m e d i u m . S a m p l e s w e r e t a k e n o u t f o r D N A f r a c t i o n a t i o n as d e s c r i b e d u n d e r M e t h o d s . Details f o r t h e v a r i o u s p a r a m e t e r s p r e s e n t e d are giving in Fig. 1. W h e n label w~s c o m p l e t e l y e q u H b r a t e d b e t w e e n e x t r a c t a b l e a n d n o n e x t r a c t a b l e D N A , t h e specific a c t i v i t y w a s 1 6 9 -+ 0 . 2 ( m e a n + S.E.) c p m p e r / ~ g D N A .
28 cell mass was only 20%, over 80% of the DNA was labeled within 15 min. The specific activities of extractable and non-extractable DNA were 70% and 90%, respectively, of the final values attained. The two fractions were equally labeled after one cell doubling. The ratio of the amounts of the two fractions remained at unity throughout mid-log phase. The patterns of changes in D N A of the non-thymine requiring strain B were rather similar to those of strain 15T- (Fig. 1). However, instead of the initial shift in the ratio of the amounts of extractable to non-extractable DNA; there was a steady decrease in the ratio during the prolonged lag phase. Trace amounts of label added to the culture were completely incorporated by the end of lag phase. Although the total radioactivity in DNA remained constant throughout the rest of the growth cycle, the continuing synthesis of unlabeled DNA by strain B resulted in a steady dilution of the specific activities of both DNA fractions. At 30 min, the specific activity of the non-extractable DNA was over three times that of the extractable but by mid-log phase, the two were not only identical b u t also less than one-fourth of the initial specific activity of the extractable DNA. Studies using prelabeled cells as inoculum confirmed the changes observed during lag phase. The more rapid dilution of label in the non-extractable DNA occurring early in the lag phase (Table I) paralleled that more rapid incorporation of label into this fraction in continuous label experiments (Fig. 1). There was a shift in the pattern of distribution of the two DNA fractions when stationary phase cultures were used as inoculum (cf. also Fig. 1). The increase in the ratio of extractable to non-extractable D N A reflects both an increase in the former and a decrease in the latter. Such an increase in the ratio of the two fractions did not occur when late-log phase cells were used as inoculum. This TABLE I C H A N G E S I N E X T R A C T A B L E A N D N O N - E X T R A C T A B L E D N A I N E. C O L I S T R A I N 1 5 T P r e l a b e l e d l a t e - l o g cells w e r e p r e p a r e d b y a d d i n g [ M e - 3 H ] t h y m i d i n e ( 0 . 6 7 m C i p e r 10 1 o f c u l t u r e ) t o m i d - l o g p h a s e cells (see Fig. 2) w h i l e s t a t i o n a r y p h a s e cells w e r e g r o w n o v e r n i g h t in t h e p r e s e n c e o f 0 . 4 4 m C i l a b e l e d t h y m i d i n e p e r 5 0 0 m l o f c u l t u r e . G r o w t h o f t h e w a s h e d and r e s u s p e n d e d cells in 3 H - f r e e m e d i u m w a s m o n i t o r e d b y A 5 4 0 n m - S a m p l e s w e r e r e m o v e d at t h e t i m e s i n d i c a t e d for fTactionation o f D N A as d e s c r i b e d u n d e r M e t h o d s . T h e D N A c o n t e n t o f t h e v a r i o u s f r a c t i o n s w a s d e t e r m i n e d b y t h e d i p h e n y l a m i n e r e a c t i o n [ 2 3 ] . T h e t o t a l a m o u n t and t h e t o t a l a c t i v i t y o f t h e D N A p e r m l o f original c u l t u r e h a v e b e e n c a l c u l a t e d as t h e s u m s o f t h e e x t r a c t a b l e ( E x t ) a n d n o n - e x t r a c t a b l e ( N o n - E x t ) D N A . Inoculum
Time (rain)
A540 n m
Total DNA ( btg/ml culture)
E x t / N o n - E x t Specific activity Ext (cpm//~g)
Non-Ext (cpm//~g)
Total activity (cpm/ml culture)
Late-log
0 68 128 200 255
0.036 0.054 0.086 0.178 0.362
0.34 0.37 0.44 0.85 1.76
2.70 2.40 2.15 1.35 1.15
168 143 95 78 41
166 123 60 29 15
49 53 50 49 50
Stationary
0 80 134 215 280
0.045 0.080 0.132 0.275 0.562
0.28 0.30 0.37 0.76 1.46
3.28 5.16 3.18 2.41 1.01
2208 1998 1771 940 595
2220 1224 537 273 160
619 561 544 537 548
29
difference between the two kinds of inocula was consistently observed in at least a dozen separate experiments. An analogous redistribution of the two fractions, however, was seen in mid-log strain 15T-A-U- (a derivative of 15Twhich requires thymine, arginine and uracil for growth) synchronized by sequential arginine and thymidine starvation [26,27]. This shift was observed during the period of amino acid starvation, shortly after the cessation of the small residual DNA synthesis in the inoculum. There was a steady decrease in the ratio of the two fractions during the subsequent thymidine starvation and the early phases of growth in fully supplemented medium. The pattern of changes in DNA following arginine starvation was very similar to that observed in the parent strain 15T-. In general, the amount of non-extractable DNA was proportional to cell density during mid-log phase whereas that of extractable DNA appeared to be proportional during most of the growth cycle with disparate increases occurring during late-log phase and continuing into stationary phase. Since there was a slowing down of the rate of increase in total DNA during these two phases, there was relatively little, if any, increase in the amounts of non-extractable DNA. Increases in the latter were initiated during early-log phase; a maximum level was reached and maintained during mid-log phase. The ratio of extractable
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Fig. 3. R e l a t i v e a m o u n t o f e x t r a c t a b l e a n d n o n - e x t r a c t a b l e D N A as a f u n c t i o n o f g r o w t h r a t e . E. coli s t r a i n B w a s g r o w n in u n s u p p l e m e n t e d g l u c o s e - - M 9 m e d i u m a n d s t r a i n 1 5 T - i n t h e s a m e m e d i u m supp l e m e n t e d w i t h t h y m i d i n e ( 1 0 ~ g p e r m l ) . G r o w t h c o n d i t i o n s , m o n i t o r i n g o f cell g r o w t h a t 5 4 0 n m a n d f r a c t i o n a t i o n o f D N A w e r e as d e s c r i b e d u n d e r M e t h o d s e x c e p t t h a t n o l a b e l e d t h y m i d i n e w a s u s e d . S a m p l e s were r e m o v e d at v a r i o u s phases of the g r o w t h cycle. T h e ratio of e x t r a c t a b l e to n o n - e x t r a c t a b l e D N A w a s c a l c u l a t e d f r o m t h e a m o u n t o f D N A in e a c h o f t h e s e f r a c t i o n s as d e t e r m i n e d b y t h e d i p h e n y l a m i n e r e a c t i o n [ 2 3 ] . T h e v a l u e s b e f o r e a n d a f t e r t h e t i m e o f m a x i m u m g r o w t h r a t e , i.e. m i d - l o g p h a s e , a r e d e s i g n a t e d , r e s p e c t i v e l y , as Q a n d • f o r s t r a i n 1 S T - a n d ® a n d X f o r s t r a i n B.
30
to non-extractable D N A varied with the growth cycle, apparently as a function of the growth rate which can be defined as (AA/A × time) X 100. The data from replicate experiments with strains B and 15T- are summarized in Fig. 3. The ratio of the two D N A fractions reached a value of unity at mid-log phase when growth rate was maximal, i.e. 1.0--1.5. Although early-log and late-log phase cultures have essentially the same growth rates, the ratios of the two fractions were not exactly the same. When aliquots of cell lysates were centrifuged in CsC1 gradients, the relative distribution and the specific activities of D N A with a buoyant density of 1.709 g • cm -3 and D N A associated with the lighter material floating on the surface of these gradients were virtually identical to those of extractable and non-extractable DNA, respectively, which had been fractionated from another aliquot of the sample (Table II). The specific activity of the pronase-released D N A appeared to be the same as that of the DNA which resisted this treatment. The total amino acid residues associated with the pronase-released D N A was 20--25% higher (variations of replicate isolations were ~< 5% than those bound to the corresponding extractable D N A (Table III). Reagent blanks and controls containing purified E. coli D N A reisolated after treatment with pronase indicated that there were no contributions of amino acid residues by either the reagents or the pronase preparations. There were increases in almost all the amino acids and the relative distribution of basics, acidics (aspartic, T A B L E II COMPARISON OF EXTRACTABLE STRAIN 15T-
A N D N O N - E X T R A C T A B L E D N A I S O L A T E D F R O M E. C O L I
F r a c t i o n a t i o n o f D N A b y e x t r a c t i o n w i t h b u f f e r e d saline o r b y CsCl c e n t r i f u g a t i o n and p r o n a s e t r e a t m e n t o f n o n - e x t r a c t a b l e D N A w e r e carried o u t as d e s c r i b e d u n d e r M e t h o d s . T h e lag phase s a m p l e ( 8 0 r a i n ) w a s f r o m t h e e x p e r i m e n t using p r e l a b e l e d s t a t i o n a r y cells as i n o c u l u m ( s e e T a b l e I); t h e o t h e r t h r e e s a m p l e s w e r e f r o m t h e e x p e r i m e n t s u m m a r i z e d in Fig. 1. Specific activity ( c p m / p g )
B u f f e r e d saline Extractable Non-extractable CsC1 c e n t r i f u g a t i o n * Denser (1.709 g . e m -3) Lighter Buffered saline--pronase Extractable. Non-extractable Pronase (+)* * Pronase ( - )
Lag (80 r a i n )
Early-log (120 min)
Mid-log (333 min)
Late-log (539 min)
1998 1224
55 172
154 180
169 177
2010 1218
60 166
2002
158
170
1208 1226
174 178
172 175
The d e n s i t y o f t h e w e l l - d e f i n e d d e n s e r D N A p e a k w a s d e t e r m i n e d b y refractive i n d e x m e a s u r e m e n t s . T h e lighter D N A w a s a s s o c i a t e d w i t h m a t e r i a l f l o a t i n g on the surface o f the gradients. The D N A f r a c t i o n w h i c h w a s e x t r a c t e d w i t h b u f f e r e d saline w a s f o u n d t o have a d e n s i t y o f 1 . 7 1 0 g" c m -3. The ratios o f d e n s e r t o lighter f r a c t i o n s in lag and early-log s a m p l e s w e r e 5.0 : 1 a n d 4.0 : 1, respectively. T h e ratios o f e x t r a c t a b l e t o n o n - e x t r a c t a b l e D N A in t h e s e s a m p l e s w e r e 5.2 : 1 a n d 4.1 : 1, respectively. Pronase (+) is u s e d t o d e s i g n a t e the p o r t i o n s o l u b i l l z e d b y p r o n a s e t r e a t m e n t and p r o n a s e ( - ) , that w h i c h r e m a i n e d w i t h the cellular debris.
31 T A B L E III AMINO ACIDS RESIDUES ASSOCIATED WITH EXTRACTABLE AND NON-EXTRACTABLE DNA E x t r a c t a b l e ( E x t ) D N A and pronase-released n o n - e x t r a c t a b l e D N A ( p r N o n - E x t ) w e r e prepared as described u n d e r M e t h o d s . Pttrification o f t h e isolated D N A and analyses o f the a m i n o acid residues associated w i t h t h e D N A have b e e n p r e v i o u s l y described [ 1 8 , 1 9 ] . Mid-log and late-log phase cells o f E. c o l i strain 1 5 T - w e r e f r o m t h e e x p e r i m e n t s u m m a r i z e d in Fig. 1. V a l u e s given for t o t a l a m i n o acid residues are averages o f replicate analyses. T h e variations in t h e s e a n a l y s e s w e r e ~ 1%. T h e relative d i s t r i b u t i o n o f the a m i n o acids is given in t e r m s o f the m o l e f r a c t i o n o f basics, acidics (aspartic acid, glutamic acid and their respective a m i d e s ) and neutrals (all e x c e p t t r y p t o p h a n and glycine). T y r o s i n e and p h e n y l a l a n i n e appear as a single u n r e s o l v e d p e a k in the D N A f r o m mid-log cells. Mid-log ( 3 3 3 m i n )
Late-log (539 m i n )
Ext
prNon-Ext
Ext
prNon-Ext
T o t a l ( # m o l e s / 1 0 0 mg D N A ) 15.72 Mole fraction Basics 0.18 Acidics 0.27 Neutrals 0.55 A r o m a t i c a m i n o acids ( p m o l e s / 1 0 0 mg D N A )
18.64
9.32
11.48
0.20 0.27 O. 53
0.12 0.28 0.60
0.13 0.29 0.58
} 2.99
} 3.28
0.69 0.44
0.72 0.45
Tyrosine Phenylalanine
glutamic acid and their respective amides) and neutrals (all except tryptophan and giycine, cf. ref. 19) was essentially the same. The unresolved aromatic amino acid peak characteristic of early-log and mid-log phase cells [19] was present in both DNA fractions isolated from mid-log phase 15T-, 333 min (cf. Fig. 1) but not in those from late-log phase 15T-, 539 min (cf. Fig. 1). Discussion
The results presented here indicate that the relative amounts of extractable and non-extractable DNA from E. coli vary with the growth phase, primarily as a function of the growth rate. When the latter was maximal, i.e. during mid-log phase, the ratio of the two fractions was approximately 1. Labeled thymidine added to cultures at this stage of the growth cycle was rapidly incorporated into DNA and the label was equilibrated between the two fractions in a single cell doubling. The greater part o f the DNA from cells in other growth phases was extractable with dilute buffered saline. Factors such as age and metabolic states of either the cultures themselves or those used as inoculum, however, could also modify the relative distribution of the two fractions. The ratios of extractable to non-extractable DNA in early-log phase cells were higher than those in late-log phase cells with similar growth rates, probably reflecting physiological differences in cells from these two growth phases. Moreover, although growth rates were minimal throughout the prolonged lag phase o f 15T-, increases in the fraction that was extractable occurred only during the early part of this phase (shortly after thymidine starvation) and only when stationary phase cultures were used as inocula. A similar increase in the fraction o f extractable DNA observed after DNA synthesis had ceased in arginine-starved mid-log phase 15T-A-U- is consistent with the postulate of a completion of the DNA replication cycle during amino acid starvation, fol-
32 lowed by liberation of the newly finished DNA [27,28]. The changes in the relative amounts of the t w o fractions could be part of a cellular alignment necessary for the initiation of the next round of DNA synthesis. When stationary phase (overnight) cultures of 15T- were diluted over 100-fold into new medium, the delay in the onset of DNA synthesis, in spite of an increase in cell mass, is qualitatively similar to that observed during transitions from poor to rich growth media [29]. Although there was no appreciable net synthesis of DNA during lag phase, there were considerable changes in the DNA, e.g. rapid incorporation of label into DNA, relatively rapid dilution of DNA from prelabeled cells used as inoculum and a transitory increase in the fraction of the total DNA that was extractable. The latter was observed only when stationary phase cultures were used as inoculum. It is rather unlikely that mere reutilization of DNA from lysed cells carried over with the inoculum could account for all these changes. Identical patterns were obtained when prelabeled cells washed free of spent medium were used as inoculum. Labeled thymidine was incorporated more rapidly into non-extractable than into extractable DNA. Complete equilibration of label between the two DNA fractions was a function of the number of cell doublings (generation times) which are necessary for a culture to reach mid-log phase of growth. A single complete cycle of cell replication was adequate only when growth rate was maximal, i.e. during mid-log phase. When label was added to cultures during the prolonged lag phase, considerably more cell doublings were required; e.g. 2.5 in synchronized 15T-A-U- (unpublished observations) and almost 4 in both B and 15T-. The generation times in these large scale cultures with the prolonged lag were generally 1.5 times as long as those reported for small cultures [ 3 0 ] . The prolonged lag apparently could delay and extend the basic processes involved in D N A replication without any other readily discernible changes in growth characteristics. Variations in the abilities of individual cells to reinitiate DNA replication could perhaps account for the large number of cell doublings which elapsed before maximal specific activity was attained in extractable DNA. Such variations in small synchronized populations have been observed to extend the replication of pulse-labeled DNA over half a generation time [ 3 1 ] . This rapid and preferential incorporation of label into non-extractable DNA, the resistance of this D N A to deproteinization as well as its association with the lighter density material found on the surface of CsCI density gradients are reminiscent of the nascent-bound DNA described in both bacterial [1,5,6,32] and mammalian [33--36] systems. There is no evidence in the data presented here for the binding of non-extractable DNA to any specific cellular structure. However, the release of an amino acid-rich D N A fraction from interphase DNA by treatment with pronase suggests a close association of the DNA with cellular proteins. Earlier studies have shown that the total amount of amino acid residues b o u n d to extractable DNA not only decreases as a culture progresses through the various growth phases [ 1 9 ] , but also changes within a few minutes after growth conditions are altered [37]. Although there were more amino acid residues in the pronase-released DNA, the relative distribution o f the residues b o u n d to it was virtually the same as that of the corresponding extractable DNA. The observations suggest that some of the amino acids may
33
be involved in linkages which are broken and lost during the redistribution of non-extractable DNA. Any definitive explanation of the relationship between amino acid residues and the attachment of DNA to cellular structures must take into account the constancy of the amounts of these residues after irradiation of bacteria under conditions which cause large increases in the amount of non-extractable DNA [20] and the relatively slow equilibration of label observed in the DNA fractions when the label was added during the early part of lag phase. The foregoing data as well as previous work by this [19,20] and other laboratories [1,2,4--6,32,37] could be explained by the assumption that the rate of cell growth, i.e. DNA synthesis, is affected or effected by the number of sites of attachment of DNA to a cellular structure, perhaps the cell membrane. All newly synthesized DNA is attached to this structure by linkages. Some of these could conceivably consist of small peptides most of which not only remain bound to the DNA after its release, but also vary with the rate of DNA synthesis. During lag phase, preparation for cell division requires reorientation involving some new polynucleotide synthesis, e.g. joining of partially formed fragments to existing macromolecular DNA, which must occur prior to reinitiation of DNA replication. The increased rate of DNA replication during mid-log phase requires either a larger number of attachment sites or much large sites per se.
Acknowledgements The authors wish to acknowledge the expert technical assistance of Mrs Sheila Clavey and Mr Stanley Zeichner. This investigation was supported in part by U.S.P.H.S. Grant CA-08648. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Goldstein, A. and Brown, B~J. (1961) Biochim. Biophys. Acta 53, 19--28 Hanawalt, P.C. and Ray, D.S. (1964) Proc. Natl. Acad. Sci. U.S. 5 2 , 1 2 5 - - 1 3 2 Smith, K.C. (1964) P h o t o c h e m . Photobiol. 3, 415--427 Ganesan, A.T. and Lederberg, J. (1965) B i o c h e m . Biophys. Res. C o m m u n . 1 8 , 8 2 4 - - 8 3 5 Smith, D.W, and Hanawalt, P.C. (1967) Biochim, Biophys. A c t a 1 4 9 , 5 1 9 - - 5 3 1 Porter, B. and Fraser, D ~ . (1968) J. Bacteriol. 96, 98--104 Ryter, A. (1968) Bacteriol. Rev. 32, 39--54 Tremblay, G.Y., Daniels, M.J. and Schaechter, M. (1969) J. Mol. Biol. 40, 65--76 Str//tling, W. and Knippers, R. (1971) Eur. J. Biochem. 20, 330--339 Fuchs, E. and Hanawalt, P. (1970) J. Mol. Biol. 52, 301- -322 Tessler, P.M., Loos, L.J. and salivar, W.O. (1972) Arch. Mikrobiol. 84, 161--190 Smith, K.C. (1962) Biochem. Biophys. Res. C o m m u n . 8 , 1 5 7 - - 1 6 3 Davern, C.I. (1966) Proc. Natl. Acad. Sci. U.S. 55, 792--797 Ivarie, R.D. and Pene, J~J. (1970) J. Bacteriol. 104, 839- -850 Lark, K.G. (1 966) Bacteriol. Rev. 30, 3--32 Sueoka, N. and Quinn, W.G. (1968) Cold Spring Harbor Syrup. Quant. Biol. 33, 695--705 Jacob, F., Ryter, A. and Cuzin, F. (1966) Proc. R. Soc. Lond., Set. B 164, 267--278 Salser, J.S. and Balis, M.E. (1967) Biochim. Biophys. A c t a 1490 220--227 Salser, J.S. and Balis, M.E. (1969) J. Biol. Chem. 244, 822--828 Balis, M.E., Salser, J.S. and Elder, A. (1964) Nature 203, 1170--1171 Davis, B.D. and Mingioli, E.S. (1950) J. Baeteriol. 60, 17--28 Marmur, J. (1961) J. Mol. Biol. 3, 208--218 Burton, K. (1956) Biochem. J. 62, 314--323
34 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Schaechter, M., Maal~be, O. and Kjeldgaard, N.O. (1958) J. Gen. Microbiol. 19, 592--606 Fangman, W.L. and Novick, A. (1966) J. Bacteriol. 91, 2390--2391 Lark, K.G., Repko, T. and Hoffman, E. (1963) Biochim. Biophys. A c t s 76, 9--24 Hanawalt, P.C. and Wax, R. (1964) Science 145, 1061-- 1063 Maal~be, O. and Hanawalt, P. (1961) J. Mol: Biol. 3 , 1 4 4 - - 1 5 5 Maal~e, O. and Kjeldgaaxd, N.O. (1966) in Control of Macromolecular Synthesis, pp. 97--117, W.A. Benjamin, New Yo rk Lark, K.G. (1966) in Cell Synchrony, pp. 54--80, Academic Press, New Y ork Lark, K.G. and Renger, H. (1969) J. Mol. Biol. 4 2 , 2 2 1 - - 2 2 5 Kidson, C. (1966) J. Mol. Biol. 17, 1--9 Ben-Porat, T., Stere, A. and Kaplan, A.S. (1962) Biochim. Biophys. Acta 6 1 , 1 5 0 - - 1 5 2 Levis, A.G., Krsmanovic, V., Miller-Faures, A. and Errera, M. (1967) Ettr. J. Biochem. 3, 57--60 Friedman, D.L. and Mueller, G.C. (1969) Biochim. Biophys. Acta 174, 253--263 O'Bxien, R.L., Sanyal, A.B. and Stanton, R.H. (1972) Exp. Cell Res. 70, 106--112 Webb, S.J. (1967) Can. J. Microbiol. 13, 57--68
Biochimica et Biophysica Acta, 378 ( 1 9 7 5 ) 2 2 - - 3 4 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s BBA 98184
RELATIONSHIPS BETWEEN NON-EXTRACTABLE DNA AND THE BACTERIAL GROWTH CYCLE
J O S E P H I N E S. SALSER and M. EARL BALIS
Sloan-Kettering Institute for Cancer Research, New York, N.Y. 10021 (U.S.A.)
Summary
Escherichia coli DNA has been fractionated into extractable and nonextractable DNA after deproteinization of detergent-lysed cell preparations with chloroform--isoamyl alcohol. The former was extracted with dilute buffered saline whereas the latter remained in the interphase layer associated with residual cellular debris from which almost 40% could be released by incubating with pronase. About 20--25% more amino acid residues were bound to the pronase-released DNA than to the extractable DNA, but the relative distribution of the residues in the two DNA samples was virtually identical. The specific activities and the relative amounts of denser (1.709 g • cm -3) and lighter (on the surface of CsC1 gradients) DNA fractions from E. coli, grown in the presence of labeled thymidine, indicated that these two corresponded to extractable and non-extractable DNA, respectively. The relative amounts of the two fractions varied with the growth phase, primarily as a function of the growth rate. Age and metabolic state of cells i n the culture or those used as inocula could m o d i f y this relative distribution. When growth rate was maximal, the ratio of the two remained at about 1. During lag phase when no appreciable net synthesis of DNA could be detected, there was a rapid and preferential incorporation of labeled thymidine into non-extractable DNA. A disproportionate increase in the fraction of the total DNA, which was extractable, was also observed but only when stationary phase cultures were used as inocula. Complete equilibration of the label in the two DNA fractions was attained only after cultures had reached mid-log phase of growth. Similar results were obtained when prelabeled cells were used. These data have been interpreted as suggesting that the rate of cell growth and DNA synthesis are related to the number or size of sites of attachment of DNA to some cellular structure. Newly synthesized DNA would be attached to different sites on this structure and initiation of DNA replication in lag phase would require reorientation o f the two kinds of DNA. Small peptides which are firmly bound to the DNA and which vary quantitatively with the rate of DNA synthesis could perhaps be involved in the a t t a c h m e n t to the sites.
23 Introduction
When detergentAysed bacteria are extracted by conventional procedures, part of the DNA can not be isolated w i t h o u t considerable degradation and denaturation of the polynucleotide [1--3]. The possibility of association(s)of the non-extractable DNA fraction with some cellular structures has been suggested by diverse experimental observations; e.g. preferential partitioning of this fraction into the interphase during deproteinization and decreased b u o y a n t density in cesium chloride gradients [1,4--6], electron-microscopic demonstrations of linkages between the bacterial chromosome and cell membrane [7] and isolation of DNA--membrane complexes free from bulk cellular proteins [5,8--11]. The nature of these associations is still not clear although some of the non-extractable D N A can be released by treating with lysozyme [12], detergent [4,8,10], pronase [5,6,13] or pronase in conjunction with either ribonuclease [6] or detergent [14]. The metabolic role of such a non-extractable DNA fraction is also far from well defined. In exponentially growing cultures, a presumptive role in DNA replication has been based on a preferential accumulation of pulse label in non-extractable DNA and a rapid chase of this label into extractable DNA [1,4,15--17]. Studies from this laboratory [18,19] have shown that irrespective of cellular origin, DNA which is extractable with dilute buffered saline contains amino acid residues which can n o t be dissociated without extensive degradation of the polynucleotide. These residues in bacterial DNA varied in amount and composition with the growth cycle [19]. For any given growth phase, the amount of these amino acids associated with extractable DNA changed with the composition o f the medium. However, it was essentially unaffected by irradiation of the cultures [2] in spite of increases in the amounts of nonextractable D N A which have resulted as a consequence of the greater crosslinking o f DNA and proteins under these conditions [3]. There was a steady decrease in the a m o u n t o f amino acid residues as a culture progressed through the various growth phases. If there were any direct relationships between the amino acid residues in extractable DNA and the association of DNA with other cellular c o m p o n e n t s in non-extractable DNA, the relative amounts of the latter would also vary with the stages of the growth cycle. In this report, the amounts and the fate of DNA extractable and non-extractable with buffered saline from detergent-lysed bacteria at different growth phases and the amino acid residues associated with both DNA fractions have been examined. Methods Bacterial strains and growth conditions Escherichia coli strain B and the thymine-requiring strain 15T- were grown aerobically at 37°C in M9 medium [21] containing 0.4% glucose. This was supplemented with thymidine (10 ttg per ml) for 15T-. Growth was followed by absorbance readings at 540 nm (As 40 ). Unless otherwise specified, overnight cultures (16--17 h) consisting primarily of stationary phase cells were diluted over 100-fold into fresh medium to give an initial As 40 of 0.035--0.060 which was equivalent to 1.8 • 1 0 7 - 3 . 0 • 107 cells
24 per ml. Cell titers were determined using a Petroff--Hauser counter and viable cells counts by an agar plating m e t h o d [19]. Earlier studies on amino acid residues bound to DNA indicated that a larger fraction of the total cell DNA was consistently extracted with dilute buffered saline from lag, early-log and late-log phase cells. To reexamine these observations, large scale cultures were used so that relatively large volumes could be withdrawn during the early time periods when cell density was very low and all samples for the various phases of a growth cycle could be sequentially taken from the same set of cultures. Three 10-1 cultures were grown in a New Brunswick FS 314 Laboratory Fermentor. Parallel growth was maintained in these cultures by careful control of aeration and stirring rate. At various intervals, equal volumes were withdrawn from these cultures for DNA extraction; the pooled samples contained at least 150--200 As 4 o units.
Isotope incorporation studies [Me-3H]Thymidine (10 Ci/mmole) was obtained from International Chemical and Nuclear Corporation, California. T o follow DNA synthesis through the different phases of the growth cycle, 0.67 mCi of [Me -3H]thymidine was added with the required a m o u n t of unlabeled thymidine to each 10 1 of culture at the times indicated in the figures. Prelabeled 15T- cells were prepared either by growing overnight on a rotary shaker with 0.44 mCi of [Me-3 HI thymidine per 500 ml of medium or by continuing growth for 3 h after the addition of 0.67 mCi of labeled thymidine per 10 1 to a mid-log phase culture. Growth curves showed that at harvest, the former culture was in the stationary phase and the latter culture in the late-log phase of growth. The cultures were chilled and the cells, collected by centrifuging at 4°C, washed twice in cold glucose-free M9 medium and finally resuspended in prewarmed M9 containing the required a m o u n t of unlabeled thymidine. Dilution of the label in DNA was used as a measure of the changes in the extractable and non-extractable DNA during the growth cycle. Samples (1--2 ml) were taken at 10--15 min intervals to monitor the growth of the cultures. At the times indicated in the figures, larger samples were withdrawn for DNA extraction. These were rapidly chilled by pumping through a stainless steel coil immersed in an alcohol--ice bath and the cells collected by centrifugation. When sample volumes were greater than 500 ml, a Sorvall KSB Continuous Flow System was used. The packed cells were resuspended and washed twice with cold 0.15 M NaC1--0.1 M EDTA, pH 8, a medium which removes lysed cells present in the suspensions. About 85--90% of the cells were consistently recovered. In some samples, three-fourths of the resuspended cells were set aside for studies on amino acid residues bound to DNA. Washed packed cells were stored frozen at --20 ° C. Isolation o f DNA The procedure described by Marmur [22] was used with the following modifications. Cells were lysed with larger volumes of saline--EDTA--sodium dodecylsulfate (1%), i.e. 30 ml per g wet weight of packed cells. To insure quantitative recovery of the extractable DNA, the interphase layer resulting from deproteinization with chloroform--isoamyl alcohol was reextracted twice
25 with 0.015 M NaC1--0.0015 M trisodium citrate, pH 7.5 (dilute saline--citrate). The combined aqueous fractions were dialyzed against dilute saline--citrate (5 changes), concentrated 5-fold b y lyophilization and adjusted to a final concentration of 0.15 M NaC1--0.15 M trisodium citrate (standard saline--citrate). DNA was precipitated from this solution by the addition of t w o volumes of ethanol (95%), purified b y t w o reprecipitations and dried by a graded ethanol (70--100%) and ether wash. The interphase layer containing the non-extractable DNA was washed two more times with standard saline--citrate and dried by a similar alcohol--ether wash. Perchlorate extracts of the original suspensions and the isolated D N A fractions were used for DNA determination by the diphenylamine reaction [23]. The radioactivity incorporated into DNA was assayed in aliquots of the neutralized extracts. Some DNA samples were also fractionated by gradient centrifugation. Crude cell lysate--CsC1 mixtures (overall density 1.7 g per ml) were centrifuged f o r 72 h at 29 000 rev./min in the SW 39 rotor of a Beckman Model L2 preparative ultracentrifuge. The tubes (containing 5 ml of sample per tube) were punctured and three-drop fractions were collected in tubes containing 0.5 ml of dilute saline--citrate. Aliquots were removed and assayed for radioactivity. Combined peak fractions from repeated runs were desalted by gel filtration and concentrated to a minimal volume by lyophilization. DNA was recovered by the usual alcohol precipitation procedure. DNA was also extracted from the material floating on the top of the gradient; the latter was recovered b y repeated washing of the drained tubes. When amino acid residues associated with DNA were examined, the extractable DNA was isolated and purified as previously described [19]. A b o u t 40% of the non-extractable DNA could be recovered from the interphase layer after pronase treatment. The interphase material was washed with three volumes of ethanol (90%) followed b y two volumes of dilute saline--citrate (two times). After resuspending in standard saline--citrate, pronase (Calbiochem, 45 000 proteolytic units per g, preincubated for 3 h at 37°C) was added to give a final concentration of 0.5 mg per ml and the suspension incubated with gentle shaking for 16 h at 37°C. The suspension was centrifuged and DNA was precipitated with ethanol from the clear supernatant solution. This DNA was not readily soluble in dilute saline--citrate unless sodium dodecylsulfate was added to a final concentration of 0.25% and the solution allowed to stand overnight at r o o m temperature. The detergent was subsequently removed by dialyzing against standard saline--citrate. This DNA was further purified in the same manner as extractable DNA. Hydrolysis of DNA samples and amino acid determinations have previously been described [ 1 9 ] . Results
Preliminary studies carried o u t to establish optimal experimental conditions for large scale cultures showed that when stationary phase (overnight) cultures were diluted over 100-fold (instead of 15- to 20-fold) into 10 1 of prewarmed medium, lag phase could be prolonged from less than 25 min to about 100 min. Although there was little change in total DNA during this prolonged lag, increments of 30% or more were observed in cell density, i.e. cell
26 mass. When these cultures entered early-log phase, the rates of increase in cell density and total D N A paralleled each other, reaching and maintaining maximal values during mid-log phase. By late-log phase, however, there was a slowing down of both these rates of increase, that of total D N A was much greater than that of cell mass. In spite of the prolonged growth cycle, these findings were consistent with those observed in smaller cultures [ 2 4 ] . Total D N A determined by direct analysis of aliquots of the cultures was found to be virtually the same as the sum of the extractable and non-extractable DNA. Variations in replicate samples of either of these two D N A fractions were ~< 3%. Operationally, extractable D N A is defined as the D N A which was extracted with buffered saline when detergent-lysed cell preparations were deproteinized with chloroform--isoamyl alcohol and non-extractable as that which remained associated with the interphase material. To optimize utilization of thymidine [ 2 5 ] , only the thymine-requiring strain 15T- was used in these studies. Results from a typical experiment with 15T- are given in Fig. 1. [Me -3 H] Thymidine was added to cultures which had been equilibrated for 15 min in the absence of thymidine. Under these condi80
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F i g . 1. D i s t r i b u t i o n o f D N A d u r i n g t h e g r o w t h c y c l e o f E. coli s t r a i n 1 5 T - . O v e r n i g h t c u l t u r e s w e r e u s e d d i r e c t l y as i n o c u l u m . A f t e r a 1 5 r a i n e q u i l i b r a t i o n p e r i o d i n u n s u p p l e m e n t e d glucose--M9 medium, 100 m g o f t h y m i d i n e c o n t a i n i n g 0 . 6 7 m C i [ M e - 3 H ] t h y m i d i n e w e r e a d d e d t o e a c h 1 0 1. S a m p l e s w e r e r e m o v e d at t h e t i m e s i n d i c a t e d i n t h e f i g u r e a n d f r a c t i o n a t e d a n d a n a l y z e d as d e s c r i b e d u n d e r M e t h o d s . C e l l growth was monitored by A540 nm- The amount of DNA in the extractable and non-extractable fractions, determined by the diphenylamine reaction [23], was used to calculate the ratio of the two f r a c t i o n s a n d t h e t o t a l D N A p e r m l o f c u l t u r e , i.e. t h e s u m o f t h e t w o i n e a c h m l o f t h e o r i g i n a l s a m p l e . The specific activity has been calculated from the radioactivity detenmine in aliquots of each fraction. After complete equilibration of the label between the two DNA fractions, the specific activity was 177 + 0.5 (mean + S.E.) cpm per/~g DNA.
27
tions, the lag period before onset of thymineless death was 23 min. The viable cell c o u n t at 15 min was over 95% of that present in the original inoculum. Although there was no appreciable net synthesis of DNA during the first 120 min of incubation, there was a doubling of cell density, a pronounced incorporation of label into D N A as well as the distinct shift in the ratio of extractable to non-extractable DNA. A b o u t 30% and 96% of the final specific activity was found in extractable and non-extractable DNA, respectively. Since the ratio of the a m o u n t of DNA in the two fractions at this time was a b o u t 4 : 1, almost 45% of the total DNA was labeled. This initial period was followed by a rapid increase in total DNA which paralleled the logarithmic increase in cell density until late-log phase was reached. The increments observed in the specific activity of extractable DNA corresponded to the values which can be calculated if one assumes that all newly synthesized DNA was completely labeled and no extractable DNA is converted to non-extractable DNA. When these cultures approached mid-log phase, about 90% of the total DNA was labeled. The ratio of the a m o u n t of extractable to non-extractable D N A at this time was 1.2 : 1. When [Me -s H] thymidine was added to mid-log phase cultures, incorporation of the label was essentially instantaneous (Fig. 2). Although the increase in 8C ~- _~4 0 --//'1
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I
360
Fig. 2. D i s t r i b u t i o n o f D N A in m i d - l o g a n d late-log p h a s e E. coli s t r a i n 1 5 T - . A 10 1 c u l t u r e w a s g r o w n in glucose--M9 s u p p l e m e n t e d with unlabeled t h y m i d i n e until mid-log phase was approached, at which time ( 1 8 5 m i n as i n d i c a t e d b y t h e a r r o w ) 0 . 6 7 m C i o f [Me-3H]thymidine w a s a d d e d t o t h e m e d i u m . S a m p l e s w e r e t a k e n o u t f o r D N A f r a c t i o n a t i o n as d e s c r i b e d u n d e r M e t h o d s . Details f o r t h e v a r i o u s p a r a m e t e r s p r e s e n t e d are giving in Fig. 1. W h e n label w~s c o m p l e t e l y e q u H b r a t e d b e t w e e n e x t r a c t a b l e a n d n o n e x t r a c t a b l e D N A , t h e specific a c t i v i t y w a s 1 6 9 -+ 0 . 2 ( m e a n + S.E.) c p m p e r / ~ g D N A .
28 cell mass was only 20%, over 80% of the DNA was labeled within 15 min. The specific activities of extractable and non-extractable DNA were 70% and 90%, respectively, of the final values attained. The two fractions were equally labeled after one cell doubling. The ratio of the amounts of the two fractions remained at unity throughout mid-log phase. The patterns of changes in D N A of the non-thymine requiring strain B were rather similar to those of strain 15T- (Fig. 1). However, instead of the initial shift in the ratio of the amounts of extractable to non-extractable DNA; there was a steady decrease in the ratio during the prolonged lag phase. Trace amounts of label added to the culture were completely incorporated by the end of lag phase. Although the total radioactivity in DNA remained constant throughout the rest of the growth cycle, the continuing synthesis of unlabeled DNA by strain B resulted in a steady dilution of the specific activities of both DNA fractions. At 30 min, the specific activity of the non-extractable DNA was over three times that of the extractable but by mid-log phase, the two were not only identical b u t also less than one-fourth of the initial specific activity of the extractable DNA. Studies using prelabeled cells as inoculum confirmed the changes observed during lag phase. The more rapid dilution of label in the non-extractable DNA occurring early in the lag phase (Table I) paralleled that more rapid incorporation of label into this fraction in continuous label experiments (Fig. 1). There was a shift in the pattern of distribution of the two DNA fractions when stationary phase cultures were used as inoculum (cf. also Fig. 1). The increase in the ratio of extractable to non-extractable D N A reflects both an increase in the former and a decrease in the latter. Such an increase in the ratio of the two fractions did not occur when late-log phase cells were used as inoculum. This TABLE I C H A N G E S I N E X T R A C T A B L E A N D N O N - E X T R A C T A B L E D N A I N E. C O L I S T R A I N 1 5 T P r e l a b e l e d l a t e - l o g cells w e r e p r e p a r e d b y a d d i n g [ M e - 3 H ] t h y m i d i n e ( 0 . 6 7 m C i p e r 10 1 o f c u l t u r e ) t o m i d - l o g p h a s e cells (see Fig. 2) w h i l e s t a t i o n a r y p h a s e cells w e r e g r o w n o v e r n i g h t in t h e p r e s e n c e o f 0 . 4 4 m C i l a b e l e d t h y m i d i n e p e r 5 0 0 m l o f c u l t u r e . G r o w t h o f t h e w a s h e d and r e s u s p e n d e d cells in 3 H - f r e e m e d i u m w a s m o n i t o r e d b y A 5 4 0 n m - S a m p l e s w e r e r e m o v e d at t h e t i m e s i n d i c a t e d for fTactionation o f D N A as d e s c r i b e d u n d e r M e t h o d s . T h e D N A c o n t e n t o f t h e v a r i o u s f r a c t i o n s w a s d e t e r m i n e d b y t h e d i p h e n y l a m i n e r e a c t i o n [ 2 3 ] . T h e t o t a l a m o u n t and t h e t o t a l a c t i v i t y o f t h e D N A p e r m l o f original c u l t u r e h a v e b e e n c a l c u l a t e d as t h e s u m s o f t h e e x t r a c t a b l e ( E x t ) a n d n o n - e x t r a c t a b l e ( N o n - E x t ) D N A . Inoculum
Time (rain)
A540 n m
Total DNA ( btg/ml culture)
E x t / N o n - E x t Specific activity Ext (cpm//~g)
Non-Ext (cpm//~g)
Total activity (cpm/ml culture)
Late-log
0 68 128 200 255
0.036 0.054 0.086 0.178 0.362
0.34 0.37 0.44 0.85 1.76
2.70 2.40 2.15 1.35 1.15
168 143 95 78 41
166 123 60 29 15
49 53 50 49 50
Stationary
0 80 134 215 280
0.045 0.080 0.132 0.275 0.562
0.28 0.30 0.37 0.76 1.46
3.28 5.16 3.18 2.41 1.01
2208 1998 1771 940 595
2220 1224 537 273 160
619 561 544 537 548
29
difference between the two kinds of inocula was consistently observed in at least a dozen separate experiments. An analogous redistribution of the two fractions, however, was seen in mid-log strain 15T-A-U- (a derivative of 15Twhich requires thymine, arginine and uracil for growth) synchronized by sequential arginine and thymidine starvation [26,27]. This shift was observed during the period of amino acid starvation, shortly after the cessation of the small residual DNA synthesis in the inoculum. There was a steady decrease in the ratio of the two fractions during the subsequent thymidine starvation and the early phases of growth in fully supplemented medium. The pattern of changes in DNA following arginine starvation was very similar to that observed in the parent strain 15T-. In general, the amount of non-extractable DNA was proportional to cell density during mid-log phase whereas that of extractable DNA appeared to be proportional during most of the growth cycle with disparate increases occurring during late-log phase and continuing into stationary phase. Since there was a slowing down of the rate of increase in total DNA during these two phases, there was relatively little, if any, increase in the amounts of non-extractable DNA. Increases in the latter were initiated during early-log phase; a maximum level was reached and maintained during mid-log phase. The ratio of extractable
0 0 ®
<3
g
® 0 ®
Oxo ° O® 0
I O. 50 Growth Rate
I 1. O0 A x time
I 1.50
x
Fig. 3. R e l a t i v e a m o u n t o f e x t r a c t a b l e a n d n o n - e x t r a c t a b l e D N A as a f u n c t i o n o f g r o w t h r a t e . E. coli s t r a i n B w a s g r o w n in u n s u p p l e m e n t e d g l u c o s e - - M 9 m e d i u m a n d s t r a i n 1 5 T - i n t h e s a m e m e d i u m supp l e m e n t e d w i t h t h y m i d i n e ( 1 0 ~ g p e r m l ) . G r o w t h c o n d i t i o n s , m o n i t o r i n g o f cell g r o w t h a t 5 4 0 n m a n d f r a c t i o n a t i o n o f D N A w e r e as d e s c r i b e d u n d e r M e t h o d s e x c e p t t h a t n o l a b e l e d t h y m i d i n e w a s u s e d . S a m p l e s were r e m o v e d at v a r i o u s phases of the g r o w t h cycle. T h e ratio of e x t r a c t a b l e to n o n - e x t r a c t a b l e D N A w a s c a l c u l a t e d f r o m t h e a m o u n t o f D N A in e a c h o f t h e s e f r a c t i o n s as d e t e r m i n e d b y t h e d i p h e n y l a m i n e r e a c t i o n [ 2 3 ] . T h e v a l u e s b e f o r e a n d a f t e r t h e t i m e o f m a x i m u m g r o w t h r a t e , i.e. m i d - l o g p h a s e , a r e d e s i g n a t e d , r e s p e c t i v e l y , as Q a n d • f o r s t r a i n 1 S T - a n d ® a n d X f o r s t r a i n B.
30
to non-extractable D N A varied with the growth cycle, apparently as a function of the growth rate which can be defined as (AA/A × time) X 100. The data from replicate experiments with strains B and 15T- are summarized in Fig. 3. The ratio of the two D N A fractions reached a value of unity at mid-log phase when growth rate was maximal, i.e. 1.0--1.5. Although early-log and late-log phase cultures have essentially the same growth rates, the ratios of the two fractions were not exactly the same. When aliquots of cell lysates were centrifuged in CsC1 gradients, the relative distribution and the specific activities of D N A with a buoyant density of 1.709 g • cm -3 and D N A associated with the lighter material floating on the surface of these gradients were virtually identical to those of extractable and non-extractable DNA, respectively, which had been fractionated from another aliquot of the sample (Table II). The specific activity of the pronase-released D N A appeared to be the same as that of the DNA which resisted this treatment. The total amino acid residues associated with the pronase-released D N A was 20--25% higher (variations of replicate isolations were ~< 5% than those bound to the corresponding extractable D N A (Table III). Reagent blanks and controls containing purified E. coli D N A reisolated after treatment with pronase indicated that there were no contributions of amino acid residues by either the reagents or the pronase preparations. There were increases in almost all the amino acids and the relative distribution of basics, acidics (aspartic, T A B L E II COMPARISON OF EXTRACTABLE STRAIN 15T-
A N D N O N - E X T R A C T A B L E D N A I S O L A T E D F R O M E. C O L I
F r a c t i o n a t i o n o f D N A b y e x t r a c t i o n w i t h b u f f e r e d saline o r b y CsCl c e n t r i f u g a t i o n and p r o n a s e t r e a t m e n t o f n o n - e x t r a c t a b l e D N A w e r e carried o u t as d e s c r i b e d u n d e r M e t h o d s . T h e lag phase s a m p l e ( 8 0 r a i n ) w a s f r o m t h e e x p e r i m e n t using p r e l a b e l e d s t a t i o n a r y cells as i n o c u l u m ( s e e T a b l e I); t h e o t h e r t h r e e s a m p l e s w e r e f r o m t h e e x p e r i m e n t s u m m a r i z e d in Fig. 1. Specific activity ( c p m / p g )
B u f f e r e d saline Extractable Non-extractable CsC1 c e n t r i f u g a t i o n * Denser (1.709 g . e m -3) Lighter Buffered saline--pronase Extractable. Non-extractable Pronase (+)* * Pronase ( - )
Lag (80 r a i n )
Early-log (120 min)
Mid-log (333 min)
Late-log (539 min)
1998 1224
55 172
154 180
169 177
2010 1218
60 166
2002
158
170
1208 1226
174 178
172 175
The d e n s i t y o f t h e w e l l - d e f i n e d d e n s e r D N A p e a k w a s d e t e r m i n e d b y refractive i n d e x m e a s u r e m e n t s . T h e lighter D N A w a s a s s o c i a t e d w i t h m a t e r i a l f l o a t i n g on the surface o f the gradients. The D N A f r a c t i o n w h i c h w a s e x t r a c t e d w i t h b u f f e r e d saline w a s f o u n d t o have a d e n s i t y o f 1 . 7 1 0 g" c m -3. The ratios o f d e n s e r t o lighter f r a c t i o n s in lag and early-log s a m p l e s w e r e 5.0 : 1 a n d 4.0 : 1, respectively. T h e ratios o f e x t r a c t a b l e t o n o n - e x t r a c t a b l e D N A in t h e s e s a m p l e s w e r e 5.2 : 1 a n d 4.1 : 1, respectively. Pronase (+) is u s e d t o d e s i g n a t e the p o r t i o n s o l u b i l l z e d b y p r o n a s e t r e a t m e n t and p r o n a s e ( - ) , that w h i c h r e m a i n e d w i t h the cellular debris.
31 T A B L E III AMINO ACIDS RESIDUES ASSOCIATED WITH EXTRACTABLE AND NON-EXTRACTABLE DNA E x t r a c t a b l e ( E x t ) D N A and pronase-released n o n - e x t r a c t a b l e D N A ( p r N o n - E x t ) w e r e prepared as described u n d e r M e t h o d s . Pttrification o f t h e isolated D N A and analyses o f the a m i n o acid residues associated w i t h t h e D N A have b e e n p r e v i o u s l y described [ 1 8 , 1 9 ] . Mid-log and late-log phase cells o f E. c o l i strain 1 5 T - w e r e f r o m t h e e x p e r i m e n t s u m m a r i z e d in Fig. 1. V a l u e s given for t o t a l a m i n o acid residues are averages o f replicate analyses. T h e variations in t h e s e a n a l y s e s w e r e ~ 1%. T h e relative d i s t r i b u t i o n o f the a m i n o acids is given in t e r m s o f the m o l e f r a c t i o n o f basics, acidics (aspartic acid, glutamic acid and their respective a m i d e s ) and neutrals (all e x c e p t t r y p t o p h a n and glycine). T y r o s i n e and p h e n y l a l a n i n e appear as a single u n r e s o l v e d p e a k in the D N A f r o m mid-log cells. Mid-log ( 3 3 3 m i n )
Late-log (539 m i n )
Ext
prNon-Ext
Ext
prNon-Ext
T o t a l ( # m o l e s / 1 0 0 mg D N A ) 15.72 Mole fraction Basics 0.18 Acidics 0.27 Neutrals 0.55 A r o m a t i c a m i n o acids ( p m o l e s / 1 0 0 mg D N A )
18.64
9.32
11.48
0.20 0.27 O. 53
0.12 0.28 0.60
0.13 0.29 0.58
} 2.99
} 3.28
0.69 0.44
0.72 0.45
Tyrosine Phenylalanine
glutamic acid and their respective amides) and neutrals (all except tryptophan and giycine, cf. ref. 19) was essentially the same. The unresolved aromatic amino acid peak characteristic of early-log and mid-log phase cells [19] was present in both DNA fractions isolated from mid-log phase 15T-, 333 min (cf. Fig. 1) but not in those from late-log phase 15T-, 539 min (cf. Fig. 1). Discussion
The results presented here indicate that the relative amounts of extractable and non-extractable DNA from E. coli vary with the growth phase, primarily as a function of the growth rate. When the latter was maximal, i.e. during mid-log phase, the ratio of the two fractions was approximately 1. Labeled thymidine added to cultures at this stage of the growth cycle was rapidly incorporated into DNA and the label was equilibrated between the two fractions in a single cell doubling. The greater part o f the DNA from cells in other growth phases was extractable with dilute buffered saline. Factors such as age and metabolic states of either the cultures themselves or those used as inoculum, however, could also modify the relative distribution of the two fractions. The ratios of extractable to non-extractable DNA in early-log phase cells were higher than those in late-log phase cells with similar growth rates, probably reflecting physiological differences in cells from these two growth phases. Moreover, although growth rates were minimal throughout the prolonged lag phase o f 15T-, increases in the fraction that was extractable occurred only during the early part of this phase (shortly after thymidine starvation) and only when stationary phase cultures were used as inocula. A similar increase in the fraction o f extractable DNA observed after DNA synthesis had ceased in arginine-starved mid-log phase 15T-A-U- is consistent with the postulate of a completion of the DNA replication cycle during amino acid starvation, fol-
32 lowed by liberation of the newly finished DNA [27,28]. The changes in the relative amounts of the t w o fractions could be part of a cellular alignment necessary for the initiation of the next round of DNA synthesis. When stationary phase (overnight) cultures of 15T- were diluted over 100-fold into new medium, the delay in the onset of DNA synthesis, in spite of an increase in cell mass, is qualitatively similar to that observed during transitions from poor to rich growth media [29]. Although there was no appreciable net synthesis of DNA during lag phase, there were considerable changes in the DNA, e.g. rapid incorporation of label into DNA, relatively rapid dilution of DNA from prelabeled cells used as inoculum and a transitory increase in the fraction of the total DNA that was extractable. The latter was observed only when stationary phase cultures were used as inoculum. It is rather unlikely that mere reutilization of DNA from lysed cells carried over with the inoculum could account for all these changes. Identical patterns were obtained when prelabeled cells washed free of spent medium were used as inoculum. Labeled thymidine was incorporated more rapidly into non-extractable than into extractable DNA. Complete equilibration of label between the two DNA fractions was a function of the number of cell doublings (generation times) which are necessary for a culture to reach mid-log phase of growth. A single complete cycle of cell replication was adequate only when growth rate was maximal, i.e. during mid-log phase. When label was added to cultures during the prolonged lag phase, considerably more cell doublings were required; e.g. 2.5 in synchronized 15T-A-U- (unpublished observations) and almost 4 in both B and 15T-. The generation times in these large scale cultures with the prolonged lag were generally 1.5 times as long as those reported for small cultures [ 3 0 ] . The prolonged lag apparently could delay and extend the basic processes involved in D N A replication without any other readily discernible changes in growth characteristics. Variations in the abilities of individual cells to reinitiate DNA replication could perhaps account for the large number of cell doublings which elapsed before maximal specific activity was attained in extractable DNA. Such variations in small synchronized populations have been observed to extend the replication of pulse-labeled DNA over half a generation time [ 3 1 ] . This rapid and preferential incorporation of label into non-extractable DNA, the resistance of this D N A to deproteinization as well as its association with the lighter density material found on the surface of CsCI density gradients are reminiscent of the nascent-bound DNA described in both bacterial [1,5,6,32] and mammalian [33--36] systems. There is no evidence in the data presented here for the binding of non-extractable DNA to any specific cellular structure. However, the release of an amino acid-rich D N A fraction from interphase DNA by treatment with pronase suggests a close association of the DNA with cellular proteins. Earlier studies have shown that the total amount of amino acid residues b o u n d to extractable DNA not only decreases as a culture progresses through the various growth phases [ 1 9 ] , but also changes within a few minutes after growth conditions are altered [37]. Although there were more amino acid residues in the pronase-released DNA, the relative distribution o f the residues b o u n d to it was virtually the same as that of the corresponding extractable DNA. The observations suggest that some of the amino acids may
33
be involved in linkages which are broken and lost during the redistribution of non-extractable DNA. Any definitive explanation of the relationship between amino acid residues and the attachment of DNA to cellular structures must take into account the constancy of the amounts of these residues after irradiation of bacteria under conditions which cause large increases in the amount of non-extractable DNA [20] and the relatively slow equilibration of label observed in the DNA fractions when the label was added during the early part of lag phase. The foregoing data as well as previous work by this [19,20] and other laboratories [1,2,4--6,32,37] could be explained by the assumption that the rate of cell growth, i.e. DNA synthesis, is affected or effected by the number of sites of attachment of DNA to a cellular structure, perhaps the cell membrane. All newly synthesized DNA is attached to this structure by linkages. Some of these could conceivably consist of small peptides most of which not only remain bound to the DNA after its release, but also vary with the rate of DNA synthesis. During lag phase, preparation for cell division requires reorientation involving some new polynucleotide synthesis, e.g. joining of partially formed fragments to existing macromolecular DNA, which must occur prior to reinitiation of DNA replication. The increased rate of DNA replication during mid-log phase requires either a larger number of attachment sites or much large sites per se.
Acknowledgements The authors wish to acknowledge the expert technical assistance of Mrs Sheila Clavey and Mr Stanley Zeichner. This investigation was supported in part by U.S.P.H.S. Grant CA-08648. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
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