uridine transport and incorporation into nucleic acids in escherichia coli

BIOCHIMICA ET BIOPHYSICA ACTA :21

BBA 96022

U R I D I N E TRANSPORT AND INCORPORATION INTO NUCLEIC ACIDS IN

E S C H E R I C H I A COLI

HANS B R E M E R AND DOROTHY YUAN Southwest Center ]or Advanced Studies, P.O. Box 30365, Dallas, Texas 7523 ° (U.S.A.) (Received May 2oth, 1968) (Revised manuscr ipt received Ju ly i6th, 1968)

SUMMARY

Growing Escherichia coli bacteria take up radioactive uridine without lag at a rate depending on its concentration (vmax. ~ 7 re#moles/rain per lO 9 cells, K,~ ~ 1.3 /,M). Within 15 sec all label taken up is in pyrimidine nucleotides. At uridine concentrations below I/zM, the uptake rate is lower than the rate of cellular pyrimidine consumption for RNA synthesis; at higher uridine concentrations, when the initial uptake rate is higher than the rate of pyrimidine consumption, the uptake rate is reduced in 2 steps, occurring during the first minute and after about IO rain.

The incorporation of medium-derived pyrimidines into nucleic acids proceeds at an initially increasing rate and becomes constant after about 4 rain. In a well aerated culture, this final rate is equal to the final rate of uridine uptake. In RNA, UMP becomes labelled before CMP; the kinetics of the changes in the relative UMP or CMP incorporation are independent of the rate of uridine uptake.

From the kinetics of nucleic acid labelling and of uridine uptake the size of the bacterial pool of pyrimidine nucleotides is estimated to increase from 7 to IO m/~- moles per lO 9 cells after feeding bacteria with a high concentration of uridine. Fur- ther, it is inferred from the kinetics of UMP labelling in RNA, that cellular pyrimidine synthesis is repressed to its final extent within the first minute after uridine feeding.

INTRODUCTION

The labelling of bacterial RNA with radioactive RNA precursors such as uridine or uracil, added to the growth medium, is widely used for determination of the rate of RNA synthesis in bacteria, especially in the s tudy of messenger RNA, which, as a minor fraction of the total cellular RNA, can hardly be detected without radioactive labelling. The RNA labelling rate, however, is not always a measure of the rate of RNA synthesis, since it depends also on the rate of t ransport of the labelled compounds from the medium into the cells and on the extent to which the radioactive RNA precursors are diluted by non-radioactive precursors, which are pre-existent or continuously synthesized in the cell. In the experiments to be reported here we have studied the kinetics of uptake of radioactive uridine into both bacteria and bacterial nucleic acids (mainly RNA) at various uridine and cell concentrations. The results have mainly practical value for labelling experiments with radioactive uridine.

Biochim. Biophys. Acta, 169 (1968) 21-34

22 H. BREMER AND D. YUAN

R e c e n t l y , PETERSON, BONIFACE AND KOCH 1 have studied the uridine transport in Escherichia coll. Their results, however, are difficult to compare with the results to be reported here, since they used uridine-adapted cells, growing on uridine as the only carbon source. The cells were then harvested, " t reated with chloramphenicol (IO-5O #g/ml) and chilled until used". Presumably, after harvesting, the cells were resuspended in a medium free of uridine and other carbon sources. These cells convert uridine to uracil (measured at 28 °) at a ten-fold increased rate, and, while the sugar-residue of the uridine enters the cell metabolism, the uracil (or most of it) is metabolically released into the medium. They then use the amount of this uracil in the medium as a measure for the uridine uptake into the cell. In our studies, where formation of uracil occurs to a much lesser extent, we measure only uridine which remains in the ce*l under normal growth conditions (at 37°), presumably as a whole nucleoside. I t is likely tha t the two kinds of uridine uptake observed under these two growth conditions involve different enzymes.

MATERIALS AND METHODS

Growth o/ bacteria E. cull B were grown at 37 ° in a shaking apparatus in the glucose-salts medium M 9

(11. 3 g/1 Na2HPO 4. 7H2 0, 3.0 g/1 KH~PO 4 I.O g/1 NHaC1, IO -3 M MgSO4, IO -4 MCaC12, IO -6 M FeC12, 5 g/1 glucose). Growth was followed by measuring the absorbaney at 55 ° m/~ (A550 ma) in a Gilford Spectrophotometer (I cm light path,) I A550 m~ unit was found to correspond to a liter of 4" lOS cells/ml. Doubling time was 46:~2 rain.

Radioactive labelling A sample ( i - 5 ml) of the culture was taken at the desired A~5 om/, and added

to 2o-5o#1 of an aqueous solution of E5-3H]uridine (Schwarz BioResearch, Inc., Orangeburg, N.Y., 20 C/mmole) and non-radioactive uridine, in an aeration tube in a 37 ° water bath. Samples (o.I ml) were taken with an automatic pipette. During prolonged incubation some evaporation (0.2 ml/h) may take place. This evaporation may cause a hazard, since at the end of a labelling experiment the medium con- tains tr i t iated water (see RESULTS). In some experiments (Figs. I -3) , the sample of the culture was taken at an A~50 m/, - - 0.2, filtered through a Millipore filter (0.45 # pore size), and the filter containing the bacteria was shaken for I rain with a known volume of fresh, prewarmed medium in order to resuspend the bacteria to the desired concentration. After removal of the filter the A550 m~ was checked (resuspension was always found to be more than 9 ° °/o complete) and then a sample of this suspen- sion was used for labelling. The labelling kinetics obtained in this way were similar to those obtained from bacteria left in their original medium, but at high uridine concentrations some differences were found depending on the length of t ime (1-4 rain) the bacteria were kept after resuspension before the addition of uridine.

Lysis o/ bacteria The radioactive samples were added either directly to o.5 ml of lysis mixture

(o.I iV[ NaC1, o.oi M Tris-C1 (pH 7.5), 0.o2 M EDTA, 0.5 ~o sodium dodecyl sulfate

Biochim. Biophys. Acta, I69 (1968) 21-34

URIDINE TRANSPORT IN E. coli 23

kept at IOO ° in a boiling-water bath, or, in case of a "chase" experiment (see RESULTS, Section I) they were first incubated for 45 rain with 50 #1 of a 4-1o mM solution of non-radioactive uridine, then 0.5 ml of IOO ° lysis mixture was added. After addition of lysis mixture samples were held for 3 rain at IOO ° and then cooled to room temper- ature. Lysis of the samples was found to be necessary in order to determine repro- ducibly the 8H radioactivity in cellular nucleic acids.

Determination o/radioactivity For determination of radioactivity incorporated into nucleic acids, the lysed

samples were chilled in an ice ba th together with I ml of a 50 ~g/ml solution of yeast RNA as carrier, and the nucleic acids were precipitated with 0.5 ml 3 lVf trichloroacetic acid. The precipitates were collected and washed (4× I ml o.oi Mtrichloro- acetic acid) on Millipore filters (0.45 # pore size). The dried filters were placed into vials containing 5 ml of to luene-PPO-POPOP (Packard Co.) scintillation fluid and counted in a Beckman LS-Ioo scintillation spectrometer. The counting efficiency was 3 1 % (3H) under these conditions.

Radioact ivi ty in a fluid sample was determined by adding 4 ° ¢,1 to IO ml of the toluene scintillation fluid (see above) with 0.2 ml of a 50 ~o methanol solution of hydroxide of Hyamine (Packard). The counting efficiency for 8H in this mixture was also 3 1 % .

For calculation of the specific radioactivity of the ~aH]uridine it has been assumed that only 75 0/o of the ~H radioactivity can be incorporated into nucleic acids, the rest is lost as tri t iated water, see RESULTS, Section 2.

Background o/ radioactivity For determination of the initial rate of uridine uptake by the "chase" method

(experiment in Fig. 4) a background of radioactivity of o.I % of the total radioact ivi ty present was used. This background has been determined by "labelling" for IO sec a culture which had been pre-incubated for 40 rain with IOO/~g/ml of chloramphenicol, then incubating for an additional 45 rain with an excess of non-radioactive uridine. The value observed after processing this sample as normally corre- sponded to o. I °/o of the total radioactivity in the sample and is the same as found if bacteria were omitted. I t is concluded, therefore, that no measurable amount of uridine binds to the bacteria unspecifically in a way that renders it acid precipitable.

Electrophoresis o/RNA hydrolysates The nucleic acids of a cell lysate were precipitated with I mg of carrier RNA

in 6 ml I M trichloroacetic acid. The precipitates were collected by centrifugation (IO rain, io ooo×g) , redissolved (2 ml o.I M phosphate buffer, pH 7.5), reprecipi- rated and centrifuged as before, then washed with 2 ml 7 ° % ethanol and dissolved for hydrolysis in 0.2 ml I M KOH. After 16 h incubation at 37 °, the hydrolysates were neutralized with Dowex-5o W, X 2 (mesh 5O-lOO) -pyridine and applied to Whatman paper 3 MM for electrophoresis. The electrophoresis was carried out in 0.05 g sodium citrate buffer (pH 3.9) for 7 ° rain at 50 V/cm. After drying and mark-


2 4 H. BREMER AND D. YUAN

ing the positions of the optical markers in ultraviolet light, the paper was cut into I-cm-wide strips, and each strip (I cm ×4-3 cm), after cutting it in halves, placed into a scintillation vial containing 1.5 ml of water. The radioactive compounds were elated from the paper by heating the vials for I h at 60 ° under repeated shaking and counted after addition of 5 ml of 1. 4 dioxane (Baker, reagent grade), o.5 g naphtha- lene and 0.o03 g PPO (Packard). The recovery of radioactivity was 80-90 % for nucleosides and nucleoside monophosphates. In the electrophoretic analysis of the pyrimidine nucleotide pool (see table) nucleoside di- and triphosphates gave somewhat lower recoveries (7o-8o %) and their radioactivity in the vials decreased in time, presumably due to readsorption of the compounds to the paper.

RESULTS

(z) Determination o/uridine uptake into the cellular nucteotide pool For reasons of technical simplicity, and to avoid measurement of uridine bound

only temporarily to the cells, an indirect assay for the uridine uptake was used which is demonstrated in the experiment of Fig. I.

TIME AFTER ADDtTION OF L=~Urd (MIN)

I0 20 30 4 0 50 ~_- I [ I - - i I

~ 2 5 b

E

~

~ e

- - D 0 ~0 20 30 4 0 50 60

TiME AFTER ADDITION OF NON-RAD, Urd(MIN)

Fig. i . E x h a u s t i o n of r ad ioac t ive nucle ic acid p recu r so r s in t h e bac te r ia l nuc leo t ide pool a f te r d i lu t ion of t h e specif ic r a d i o a c t i v i t y of [~HJuridine fed to t h e bac ter ia . (a) [aHJuridine (Io m # - moles /ml , 52 ooo coun t s / r a i n per re#mole) was a d d e d to 7.2. lO 7 cel ls /ml and t h e k ine t ics of nucle ic acid label l ing were fol lowed (C)). A t 2, 5 and 5.5 ra in ( ~ ) a l iquo t s (I ml) of t h e cu l tu re were added to non - r ad ioac t ive u r id ine (o.5-1 ml) to r e su l t in a 13o- (2 a n d 5.5 rain) or 5oo-fold (5 rain) di lu- t i on of t h e specif ic r a d i o a c t i v i t y of t he ur id ine ; t h e k ine t ics of nucle ic acid label l ing were also fol lowed in t he se t u b e s ([~, &), k ine t ics s t a r t i n g a t 5 m i n is n o t shown, see (b). (b) Rep lo t of t h e d a t a f r om Fig. Ia, i nc lud ing t h e k ine t ics s t a r t i n g a t 5 rain (&) . On t h e abscissa , t i le t i m e of add i t i on of non- rad ioac t ive u r id ine is se t equa l to zero; t he e x p a n d i n g t ime scale correc ts for t h e cu l tu re g r o w t h du r ing t h e label l ing (expans ion factor , 2t/D - i ; D = doub l ing t ime) . On t h e or- d ina te , t h e r ad ioac t i v i t y in nucleic ac ids a t t h e m o m e n t of add i t i on of non- rad ioac t ive u r id ine is se t equa l to zero. The f inal p a r t of t i le k ine t ics e x t r a p o l a t e d to t = o (- - -) ind ica tes t he rad io- a c t i v i t y in t he nuc leo t ide pool a t t = o. T he decreas ing d i s t ance be tween t h e da shed and t h e ob- s e rved k ine t ics ind ica te s t h e e x h a u s t i o n of r ad ioac t ive nuc leo t ides in t h e pool.

B i o c h i m . B i o p h y s . A c t a , 169 (1968) 21-34


Radioactive uridine was fed to bacteria, and the kinetics of labelling of nucleic acids were followed (Fig. Ia, O). 2 and 5.5 rain later aliquots from the culture were removed and added to a small volume of non-radioactive uridine (dilution of the specific radioactivity of uridine in the medium -- 13o fold); these aliquots were further incubated, and also here the kinetics of incorporation of radioactivity into nucleic acids were followed (Fig. Ia, [~, zx). In a third aliquot, taken at 5.0 min, the dilution factor of the specific radioactivity was 500, the kinetics obtained from this aliquot is shown in Fig. Ib, • .

The conclusions of this experiment, which become clearer in the replot of the data shown in Fig. Ib, are the following: (I) the initial rate of incorporation of radioactivity into nucleic acids observed after dilution of the specific radioactivity of uridine is independent of the dilution factor (I3o or 500 fold) if the dilutions are done nearly simultaneously (at 5.0 or 5.5 rain), indicating that the radioactive nucleotides in the cell are no longer in rapid equilibrium with the uridine in the medium, or that once "inside" the bacteria, they do not leak out rapidly. (2) The final slope of the three kinetics is, corresponding to the dilution factors, 13o or 500 fold, respectively, smaller than the slope in the kinetics observed without dilution of the specific radioactivity of uridine (Fig. Ia, O). Accordingly, each kinetics in Fig. Ib shows actually two su- perimposed kinetics, the continuing incorporation of radioactive uridine from the medium, and the exhaustion of radioactive nucleotides from the pool. The incorporation from the pool approaches a maximum which corresponds to the amount of radioactive nucleotides present in the pool at the moment of addition of the non-radioactive uridine; this maximum is determined as the intercept of the straight line given by the final part of the observed kinetics with the ordinate. (3) At 5 rain after addition of radioactive uridine to the culture there are more radioactive pyrimidine nucleotides in the pools than at 2 rain, indicating that between 2 and 5 rain the radioactive labelling of the nucleotide pool is still approaching the equilibrium at which the flows of radioactive pyrimidines into and from the pool become equal. (4) I t takes about 15 rain until 90 % of the nucleotides present in the pool at the beginning of the "chase" are incorporated into nucleic acids.

In the experiments to follo ~ the dilution factor of the specific radioactivity of uridine was IOOO-iO ooo, and after dilution, o.I-ml samples were further incubated for 45 min. Thus, according to Fig. Ib ( • ) , the incorporation of uridine from the medium occurring after dilution was less than I °/o of the incorporation from the pool and was therefore neglected. With this small sample volume there was no need for aeration after the addition of non-radioactive uridine.

(2) Utilization o/f5-SH]uridine Since E5-3H~uridine was used, some radioactive hydrogen can be expected to

be removed from the uracil residue during the methylat ion in the synthesis of the thymine residue. The release of 3H from E5-aH]dUMP into water in the thymidylate synthetase reaction has been shown to occur in vitro 2. The following experiment (Fig. 2) shows that this reaction occurs also in vivo to a considerable extent, more than one would expect from the amount of thymine needed for DNA synthesis. In addition, the experiment shows that most radioactive uridine taken up is transferred to acid-precipitable compounds in presence of excess non-radioactive uridine, a fact

Biochim. Biophys. Acta, 169 (1968) 2I-34


~00

~o

60

f_ ~_ 4 o

~¢ 2 0

.o . . . .

&

IOC

oo I

601

40 i

201

C

'4 C

c

5~

V-

O

°o ,'o 2'0 3'0 20 5'o 6o ' 2o ~o , o 5o ~o TIME (MINUTES) TIME (MINUTES)

Fig. 2. Ut i l i za t ion of r ad ioac t ive [5-3H]- and [2-14C]uridine (0.32 m/ ,mole /ml ) added to a cu l tu re of 2. 5. l • T cells/ml. R a d i o a c t i v i t y inco rpora t ed in to nucle ic ac ids ( O ) ; r ad ioac t iv i t y in nucleic ac ids plus bacter ia l nucle ic acid precursors , d e t e r m i n e d as in t h e e x p e r i m e n t of Fig. I (O) ; t o t a l r a d i o a c t i v i t y in cells, d e t e r m i n e d b y col lect ing a n d w a s h i n g wi th 4.5 ml wa t e r o.5 m l of t h e bacter ia l cu l tu re on a Millipore fil ter, fol lowed by r e suspens ion and lysis of t he bac t e r i a in 2 ml lys- ing m e d i u m (see METHODS) (2X); r e m a i n i n g r ad ioac t iv i t y in t h e m e d i u m d e t e r m i n e d in t h e Milli- pore f i l t ra te of t he cu l tu re ( A ) ; s u m of r ad ioac t iv i t i e s in t he cells plus m e d i u m ([2]).

on which the method employed here for measuring the uridine uptake is based. A mixture of both [2-14C~ - and E5-3Hluridine was added to a growing bacterial culture. After various times the following samples were taken.

(a) One sample was lysed and assayed for acid-preeipitable radioactivity (Fig. 2, e ) . The incorporation values obtained in this way reach a plateau, corresponding to 65 % of aH or 75 % of 14C radioactivity present in the culture, showing that an appreciable fraction of the radioactive compounds fed to the bacteria does not end up in precipitable nucleic acids.

(b) A second sample was incubated with an excess of non-radioactive uridine and only then lysed and assayed for acid-precipitable radioactivity as described in the preceding section. The values obtained (Fig. 2, ©) increase faster than those from the first sample, reflecting the uptake of radioactive uridine into both cellular nucleotide pool and nucleic acids. These values also reach a plateau, the level of which is about as high as (or perhaps slightly higher than) the level reached by immediate precipitation of samples removed at the same time (Fig. 2, • ) . This was to be expected, since, after exhaustion of radioactive nucleic acid precursors in the medium, those radioactive nucleotides still present in the cellular nucleotide pool should finally become "chased" into nucleic acids by non-radioactive nucleotides synthesized in the cell.

(c) The third sample was filtered through a Millipore filter and the filtrate, including wash fluid, was assayed for radioactivity. The values obtained correspond to the radioactivity in the medium (= " total radioactivity present in the culture minus radioactivity taken up by the cells plus radioactivity leaked out of the cells"). The kinetics (Fig. 2, A) show an initial decrease, reflecting the metabolic iemoval of radioactive uridine from the medium, and a leveling out when the medium con-



tains 25 % of the *H and 12 % of the 14C radioactivity present in the culture. The filtrate of the sample taken at 60 rain was distilled (not to dryness) and the distillate (presumably water) assayed for radioactivity. The distillate was found to contain 9 ° °/o of the 3H, but less than 0.5 % of the 14C radioactivity present in the medium before distillation, suggesting that, as had been suspected, tritiated water has been formed in the bacteria. (Radioactive medium before addition of bacteria was shown to contain no volatile radioactivity.) The unincorporated 14C radioactivity has not been further analyzed and may consist of impurities in the [~4C~uridine preparation or phosphorylated compounds which have leaked out of the bacteria.

(d) The fourth sample was a radioactivity assay of a lysate of the washed cells remaining on the filter after filtration. The kinetics obtained show the net uptake of radioactivity into bacteria. The uptake of both 3H and 14C determined in this way (Fig. 2, A) is about the same as the uptake determined by the "chase" experiment (Fig. 2, C)), which indicates that most of the radioactive pyrimidine nucleotides in the ceJ1 are not released again into the medium, but become incorporated into ~cid- precipitable compounds, presumably nucleic acids. At any time, the sum of the ra dio- activity in the medium plus radioactivity in the cells must be equal to the total radioactivity present in the culture, which, in fact, is observed (Fig. 2, Z]). How- ever, some unexplained deviations from this expectancy occur which appear to be systematic.

For the quantitative evaluation of the experiments to follow it is assumed that the fraction of 3H radioactivity transferred to water is independent of the rate of uptake of uridine from the medium. This was shown by the finding that the aH/~4C ratio in the nucleic acids labelled with a given proportion of [5-3Hluridine to C2-1*C~- uridine does not vary with different concentrations of total uridine in the medium (checked in the range of o.16-1o m/~moles/ml of uridine), although the uridine concentration does determine the rate of uptake of uridine, as will be shown in the following.

(3) Kinetics o/ uridine uptake into bacteria Immediately after bacteria are exposed to uridine, the rate of uridine uptake

into the bacteria is highest; later the rate drops in one or two relatively sudden steps (Fig. 3). This initially high uptake suggests that an enzyme catalyzing the uridine transport into the cell has not to be induced, but rather must be already present when the bacteria are grown in absence of nridine. This conclusion is supported by an experiment in which the kinetics of uridine uptake was followed in presence of chloramphenicol (IOO/,g/ml, given 5 rain before uridine, at a concentration of 5 m#- moles/ml, was added). With chloramphenicoI, the rate of uridine uptake (measured by the "chase" method which presupposes nucleic acid synthesis during the 45-rain chase) was for the first 3 rain slightly higher than the uptake rate measured in absence of chloramphenicol. Later the uptake rate dropped. Similarly, I{URLAND AND MAALOE 3 observed an initially increased, later decreased incorporation of radioactive uracil into RNA in presence of chloramphenicol.

Furthermore, the decrease in the rate of uridine uptake and its assimilation to the rate of cellular pyrimidine consumption suggests that the uptake is redaced as a consequence of the accumulation of some pyrimidine nucleotides which are gener- ated faster than they are consumed.

Biochim. Biophys. Ac!a, 169 (1968) 21-34


6

5

4

z 70

~: so

8 _z 50

40

3c

20

Io

0

o 5.0 z 5 kO

- - o c -

_ 0.05 m/~ moles/ml 0 0.55

• • . o . 2 s

/ / 0.10

- - b TIME (MINI

7.5 m~ motes/ml

- - 0 •

4 8 12 16 20 24 TIME {MINUTES)

Fig. 3. Kinetics of uridine uptake into bacteria and nucleic acids. (a) o.o5, (b) 7.5 m#moles]ml of uridine; uptake into bacteria (O) and nucleic acids (Q). (c) Initial kinetics of uptake at various uridine concentrations, o.o5- 5 m/~moles/ml. Cell concentration was 2.2. Ioe/ml for (a) and 2.5" lOT/ ml for (b) and (c). All experiments contained 480 ooo counts/min per ml.

At low uridine concentrations (Fig. 3a) the uptake immediately attains a constant rate which, after xo min, drops to the final rate.

At higher uridine concentrations ( > o.25 m#mole/ml, Fig. 3 b) the kinetics of uridine uptake show three phases (the initial phases are seen clearer in Figs. 3c and 4) : (z) An initial phase, during which the rate of uptake drops from its maximum value (at t -+ o) to a minimum value (at t = z mini and during which at least 2 m#moles of uridine are taken up per zo 9 cells ( = zo 6 molecules/cell). Electrophoretic analysis of the cellular nucleotide pool after x5 sec of labelling with [3H]uridine (see Table I) shows all radioactivity taken up in electro-negative compounds, among them the mono-, di- and triphosphates of uridine and cytidine, indicating that the initial rapid uptake does not reflect an unspecific binding of uridine to the ceils but that the uridine is rapidly phosphorylated. (2) During the next zo-15 min the rate of uridine uptake increases for about x min and then remains constant (secondary rate), but still slightly faster than the labelling rate of nucleic acids. (3) After this t ime the uptake rate drops 2o %, to its final rate.

For determination of the "ra te per cell" from graphs with expanded time scale, as in Fig. 3, the original nnexpanded time scale must be used, which agrees with the time scale shown only at t ~ o.

Biochim, Biophys. Acta, I69 (I968) 21-34

URIDINE TRANSPORT IN E. coli 2 9

TABLE I

L A B E L L I N G OF CELLULAR NUCLEIC ACID PRECURSOR W I T H [ a H ] u R I D I N E

I ml of cell cul ture (8. Io ~ cells/ml) was labelled wi th 0.5 m/~mole/ml [3Hjuridine (700 ooo counts / min per ml). o.2 ml of labelled cul ture was lysed a t 15 sec (see METHODS) . I O o ] ~ l of this lysate was precipi ta ted for direct measurement of the a m o u n t of label in nucleic acids. Another 50/zl of lysate was analysed by electrophoresis (see METHODS). Radioact iv i ty remain ing a t the origin of the electrophoresis d is t r ibut ion was taken to be f rom nucleic acids, radioact iv i ty at uridine posit ion taken to be unincorpora ted label, radioact iv i ty in electro-negative compounds taken to be cellular nucleotides. (The original [SH]uridine did not contain detectable a m o u n t s of electro- negat ive compounds . ) Total recovery f rom electrophoresis was 8 1 % (566 000:700 ooo) of radio- ac t iv i ty spotted. Three fu r ther o.2-ml samples of labelled cul ture were diluted wi th an excess of non-radioact ive uridine at 20, 4 ° and 6o sec, incubated for an addit ional 50 min, then lysed and precipi ta ted for the direct measu remen t of the nucleotide pool ( = value ext rapola ted from this kinetics to 15 sec).

2~lethods o/ determination

Direct Electrophoresis

Counts/min per mI % Counts/rain per ml %

Nucleic acids 17 500 2. 5 17 ooo 3 Nucleotide pool 187 500 27 192 ooo 34 Unincorpora ted -- -- 351 ooo 63

Total 700 ooo ioo 566 ooo IOO

(4) Dependence o~ the rate ol uridine uptake on the uridine concentration If the initial rate of uridine uptake is plotted in reciprocal co-ordinates as a

function of the uridine concentration, a straight line is obtained (Fig. 4 a) suggesting

kb,/ 3

2:

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I I I I Y 2o~ _A

g j eO.O

0 o Io 20 30 TIME (SECONDS}

310 - - 0 0 5 I0 15 2 0

I/URIDINE CONCENTRATION (m~. moles /ml )-=

Fig. 4. (a) Ini t ial ra te of uridine up take as a funct ion of uridine concentra t ion in reciprocal co- ordinates. Ini t ial ra te ~ average ra te between o and IO sec, except for concentra t ions below o.i m/zmole/ml, where the ra te observed after io sec was used, to exclude an addit ional background, perhaps due to some non-specific binding of uridine to the cell membrane . (b) Ini t ial kinetics on which the plot in (a) is based. 2.5"1o T cells/ml were labelled wi th 0.05 m/~mole/ml of [SH]uridine (480 ooo coun t s /min per ml) and var ious a m o u n t s of non-radioact ive uridine as indicated on each kinetics (in to ta l mt, moles/ml ). The kinetics were followed for 3 min; the last pa r t of the kinetics not shown here was used for Fig. 5 c.

Biochim. Biophys. Aeta, 169 (1968) 21-34

3 ° H. BREMER AND D. YUAN

that the initial rate of uptake is determined by one enzyme, perhaps a kinase, which phosphorylates the uridine in the cell membrane. From the intercepts of this line with the co-ordinates the maximum rate of uridine transport (at infinite uridine concentration) can be seen to be 7 re#moles/rain per lO 9 cells (3 times faster than the rate of pyrimidine consumption for nucleic acid synthesis), and the half maximum rate is seen to occur at a uridine concentration of 1.3 m#mole/mi.

At uridine concentrations above 0.25 m/~mole/ml the rate of uridine uptake becomes limited less than a minute after addition of uridine to the culture by secondary and perhaps tertiary reactions in the cell which reduce the net rate of uridine transport into the cell, as was shown in the preceding. If the initial and secondary rates from the experiments of Fig. 4 and some others done at different cell concentrations, (Figs. 5a, 5 b) are plotted as a function of uridine concentration (Fig. 5c) it is seen that the secondary rate does not exceed 3 re#moles/rain per lO 9 cells and

b

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0.37 me. mole/ml Q

: j 4107

IO me. mo le /m l b

8,10 7

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TIME (MINUTES) 8.0

~ 6.0

[ 5.0

~ 4.0

m 3.0

~- I.O

I 1 I I 0 2 4 6 8 I0

URIDINE CONCENTRATION (m e. m o t e s / m l )

Fig. 5. Uridine up take a t different cell- and uridine concentrat ions. (a, b) Kinetics of up take a t 0.37 and I.O m/zmole/ml of uridine at 2. 5- lO T, 4" IO7 and 8. Io 7 cells/ml. Each culture contained 480 ooo coun t s /min per ml of [3H]uridine. (c) Ini t ial (open symbols) and secondary (closed symbols) ra te of uridine up take (from the kinetics in a, b and others no t shown) as a funct ion of uridine concent ra t ion at 2.5' IO 7 (71, I ) , 4" I°~ ((2), 0 ) and 8. io ~ (A, A) cells/ml.

Biochim. Biophys. Acta, 169 (I968) 21-34


that the initial and secondary rates of uridine uptake are independent of cell concentration. The final rate of uridine uptake, measured in other experiments, was always found to be 15-25 ~/o lower than the secondary rate.

(5) Kinetics o/nucleic acid labelling At the low uridine concentration of o.o5 m/zmole/ml the labelling rate of nu-

cleic acids (Fig. 3a, lower curve) becomes constant after about I rain, equal to o.25 m/zmole/min per IO 9 cells. At the higher uridine concentration (7.5 m/zmoles/ml (Fig. 3b, lower curve)) the final labelling rate of nucleic acids is reached after about 4 rain and is seen to correspond to 2.0 re#moles/rain per IO 9 ceils.

0_ 0.5 0.5

a 0 0.4 ~ 0.6 ~ .,

o.3 • o.z g

0.2 08 _~

o.o5 . . . . . . . ,m, ,.", ,.", [ ~: OI I I I [ I m --~o~ 3__ 4 8 12 I 16 0"9 0

~0 0t01 ~ TIME (MINUTES) / TotoI

3 0 " " i

Z ~. To~.I - ~ Unstable _~ UMP -o > Unstable

&2o ~ o / o }~ /

CMP . , ' ,o / ~ . ~ t o b ,

o_ ~. I o ° O / I' )p _ ~ . . 7 - u.e,ab,,

z_ t / }M

0 4 8 12 16 O0 4 8 12 16 0 T I M E " - - ~

TIME (MINUTES) TIME (MINUTES)

Fig. 6. Labelling of UMP and CMP residaes in RNA. (a) Relative labelling of UMP and CMP, obtained from electrophoretic analysis of RNA hydrolysates, as a function of labelling time at 0.05 (O), 0.25 (Lx), i.o ( 0 ) and 5.0 (A) m/,moles/ml of uridine. Left ordinate fraction of radio- activities CMP[ (CMP + UMP), right ordinate UMP/(CMP + UMP). Curve shown = assumed average. (b) and (c), kinetics of UMP and CMP labelling in IRNA at two different uridine concentrations, obtained by multiplication of the values of the totM pyrimidine incorporation into nucleic acids, (corrected for incorporation into DNA, see text) from Figs. 3 a and b with the fractions (assumed average) of UMP and CMP labelling in Fig. 6a.

Fig. 7. Theoretical labelling kinetics of unstable (mRNA), stable (rRNA, tRNA) and total RNA (= s table+unstable) with radioactive precursors under the assumption tha t (a) no cellular precursor poolis present and mRNA breakdown products do not flow back into a precursor pool; (b) no precursor pool is present but mRNA breakdown products are reused as precursors; (c) precursor pool is present, mRNA breakdown products flow back into pool. I t is assumed tha t the actual situation is represented by (c). The negative ordinate values correspond to non-radioactive RNA precursors present in the cell before labelling in either mRNA (M) or pool (P) under tile presupposition tha t the flow rate of label into the cell is constant in time (in case of uridine labelling, this presupposition is given only when the bacteria are pre-fed with unlabelled uridine for at least 15 mill). In all 3 graphs it is assumed tha t the synthesis rate of unstable RNA is equal to tha t of stable RNA.

l?iochim. Biophys. Mcta, 169 (1968) 21-34

32 It. BREMER AND D. YUAN

The experiment in Fig. 6a shows that the radioactive CMP incorporation into RNA, relative to the total incorporation (CMP+UMP), increases with labelling time until it reaches the final value corresponding to the mole fraction of cytosine in E. coli RNA pyrimidines (about 45 %). Similar observations were reported by McCAR- THY AND BRITTEN 4, who have labelled E. coli RNA with ~14C]uracil. The kinetics of the relative CMP incorporation are seen to be independent of the uridine concentrat ion in the medium.

Using the kinetics in Fig. 6a, the kinetics of total aH incorporation into nucleic acids (Figs. 3a, 3b, lower curves) can be split into two separate kinetics of [3H]UMP and ~3H]CMP incorporation into RNA (Figs. 6b, 6c). (In order to correct for the incorporation into DNA it has been assumed that 5 % of the radioactivity in nucleic acids is in DNA, taking into account that only dCMP, but not dTMP, can be labelled from [5-3H]uridine.)

At the high concentration of uridine (Fig. 6c), the initial rate of UMP labelling is only slightly lower than the final rate of UMP labelling, suggesting that cellular synthesis of UTP, and thus also of CTP, is repressed to its final extent within the first minute after addition of uridine to the culture. Furthermore, it may be concluded from this short lag, in contrast to the long lag in CMP labelling, that the formation of UTP from CTP in the reaction equilibrium UTP ~- CTP occurs at a very small rate.

(6) In[luence o[ aeration on nucleic acid labelling In a bacterial culture of 3" Iov cells/ml and at low uridine concentration (o.05

m/,mole/ml) absence of aeration (no bubbling of air through the culture, but still diffusion of air from the surface, culture filled 15 m m high in the tube) does not reduce uridine uptake and incorporation into nucleic acids. At high uridine concentration (5 m, umoles/ml), however, the rate of uptake was found to be 2o °/o, and the rate of nucleic acid labelling 80 °/o reduced in an unaerated culture, whereas the doubling time of the bacteria was normal. These observations suggest that, at this cell concentration, synthesis of UTP from medium-derived uridine is limited by aeration, but not normal cell metabolism, including UTP synthesis from cellular pyrimidine precursors. Possibly the phosphorylations of pyrimidine-nucleotides which occur after feeding bacteria with aridine are linked to some energy-supplying oxidative reactions.

DISCUSSION

Uptake and release o[ uridine In the experiments reported here, the net rate of uracil uptake was measured

and assumed to be equal to the rate of uridine uptake, which implies a negligible rate of uracil efflux (in contrast to the observations by PETERSON et al., see INTRO- DUCTION). That under our conditions, the rate of uracil efflux is, in fact, negligible, is suggested by the experiments in Figs. I and 2 which show that most, if not all, radioactive nucleotides in the cell, measured after collecting cells on a Millipore filter, become incorporated into nucleic acids when uptake of radioactive uridine is stopped by addition of excess non-radioactive uridine to the culture.



Comparison o/uridine uptake and RNA synthesis Previously reported experiments indicated that the rate of RNA synthesis in

E. coli is independent of the presence of uridine in the medium 5. Here we observed that the net rate of uridine uptake and incorporation into nucleic acids vary with the uridine concentration in the medium, if this concentration is below 2/~M (Fig. 5c). Furthermore, it was found that the labelling rate, but not the synthesis rate, of nucleic acids may vary with the extent of aeration of the culture. Thus, only when cul- tures are grown under identical conditions and when the concentration of uridine is above 2 ~lVf, the incorporation of medium-derived pyrimidines into RNA corresponds to the total pyrimidine incorporation into RNA and labelling rates of RNA may be taken as a measure of RNA synthesis rates.

Influence o/messenger RNA turnover on the kinetics o/nucleic acid labelling When bacteria begin to take up radioactive uridine, breakdown products of

non-radioactive mRXA dilute the radioactive RNA p:ecursors in the nucleotide pools, such that stable and unstable nucleic acids are labelled at a reduced rate. Later, when mRNA and its breakdown products become labelled, the specific radioactivity in the nucleotide pool increases such that nucleic acids become labelled at a higher rate. This increase in the labelling rate of total nucleic acids is compensated, however, by a decreasing (net) labelling rate of mRNA. Since for every initially non-radioactive mRNA nucleotide "chased" into stable nucleic acids a radioactive nucleotide is incorporated into mRNA, the obtained kinetics of total nucleic acid labelling are the same as if mRNA were a stable fraction of the total nucleic acids (Fig. 7). Only at the beginning of labelling slight differences from this expectation may occur due to a delayed equilibration of mRNA breakdown products with the flow of radioactive nucleotides from the medium.

Size o/the pyrimidine nucleotide pool When the pyrimidine nucleotides are homogeneously labelled their amount in

the pools can be determined as the quotient "radioactivi ty in the pool" over "specific radioactivity of pool nucleotides".

Assuming that the synthesis of pyrimidines is completely inhibited at a uridine concentration of 7.5 m~moles/ml, the size of the bacterial pyrimidine pool after it has become constant can be read directly from Fig. 3b as the vertical distance after 18 min between the two kinetics shown ( = difference " tota l incorporation into bacteria" minus "incorporation into nucleic acids"), which is seen to correspond to 12 m~moles/ io 9 cells. The value obtained varies somewhat in different experiments and may be as low as 8 m~moles/ io 9 cells. Also by prefeeding bacteria for 15 min with a high concentration of non-radioactive uridine, the pool size may be determined, according to the principle illustrated in Fig. 7, from the final labelling kinetics linearly extrapolated to the time of addition of label. Such experiments (not included in RESULTS) gave values of 8-IO m/zmoles/Io 9 cells when the concentration of non-radioactive uridine during the prefeeding period was IO re#moles/m1.

At the very low uridine concentration of 0.05 ml~mole/ml (Fig. 3a) the final amount of radioactive nucleotides in the pool corresponds to 0. 9 m/~mole/Io 9 cells



and the final specific radioactivity of pool nucleotides corresponds to o.125 of the specific radioactivity of uridine in the medium (since the rate of incorporation into nucleic acids of labelled nucleotides corresponds to o.25 re#mole/rain per lO 9 cells, which is o.125 of the maximum incorporation rate in the experiment of Fig. 3b); thus, the final size of the pyrimidine nucleotide pool corresponds to o.9/o.125 = 7.2 m#- moles/Io 9 cells. This value is somewhat lower than the amount of pyrimidine nucleotides in cells grown at 7.5 m/zmoles/ml of uridine, and may be close to the value of the pool size of cells grown in absence of uridine.

An expansion of the pyrimidine nucleotide pool after addition of uridine to the cells is plausible in connection with the idea that cellular pyrimidine synthesis is inhibited by cytidine nucleotides 6. The dependence of the final rate of nucleic acid labelling on the uridine concentration suggests that the degree of inhibition of cellular pyrimidine synthesis and thus also the concentration of cytidine nucleotides in the cell varies with the uridine concentration in the medium. Above 3 re#moles per ml of uridine, when the final rates of uridine uptake and of nucleic acid labelling have reached a maximum, the size of the pyrimidine nucleotide pool may also have reached its maximum; this would be consistent with the findings of SALSER, TANIN AND LEVINTHAL 7, who reported that the guanine nucleotide pool in B. subtilis does not expand any further if the guanine concentration in the medium is raised from 0.3 /zg/ml ( ~ 2 m/,moles/ml) to higher values. These authors further conclude from the absence of an initial lag in the nucleic acid labelling observed when radioactive guanine is given to cells grown in the absence of guanine, that, under those conditions, the guanine nucleotide pool rapidly expands. This conclusion, however, does not take into account the possible partial compartmentalization of the nucleotide pool, which has been shown to exist for the pyrimidine pool s. On the basis of a similar experiment it has also been concluded a,9 that the pyrimidine nucleotide pool does not expand after addition of uracil to the bacteria. Although this conclusion is consistent with direct measurements of the pyrimidine nucleotide pool in E. coli cells grown in presence of (5o m#moles/ml) uracil s, which gave a value of 7.2 m#moles/Io 9 cells, it ap- pears likely that also uracil causes some pool expansion which then causes the inhibition of cellular pyrimidine synthesis.

ACKNOWLEDGEMENTS

We wish to thank J. 1V[cCoRQUODALE for his advice during the preparation of this manuscript.

The research has been supported by the National Institutes of Health, Grants No. I ROI, G1V[ 15142-Ol and No. POI GM I3234-olA2.

R E F E R E N C E S

i R. N. PETERSON, J. BONIFACE AND A. L. KOCH, Biochim. Biophys. Acta, 135 (i967) 771. 2 M. I. SMITH LOMAX AND G. R. GREENBERG, J. Biol. Chem., 242 (1966) lO9. 3 C. G. KURLAND AND O. MAALOE, J. Mol. Biol., 4 (1962) 193. 4 B. J. MCCARTHY AND R. J. BRITTEN, Biophys. J . , 2 (i962) 35- 5 n . BREMER AND D. YUAN, J. Mol. Biol., 34 (1968) 527. 6 J. C. GERHARD AND A. B. PARDEE, Cold Spring Harbour Syrup. Quant. Biol., 28 (1963) 491. 7 W. SALSER, J. TANIN AND C. LEVINTHAL, J. Mol. Biol., 31 (1968) 237. 8 K. MULLER AND H. BREMER, J. Mol. Biol., in the press. 9 D. P. NIERLICH AND W. VIELMETTER, J. Mol. Biol., 32 (1968) 135.

Biochim. Biophys. Hcta, 169 (1968) 21-34

uridine transport and incorporation into nucleic acids in escherichia coli

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