J. Mol. Biol.

(1971) 55, 549-562

In viva Assay of Protein Synthesizing Capacity of Escherichia coli from Slowly Growing Chemostat Cultures
ARTHURL. KOCH
Indiana
AND

CAROL S. DEPPE-~

Department of Microbiology
University

Bloomington, Indiana 47401, U.S.A. (Received 2 February 1970, and in revised form 17 August 1970)

The incorporation of short pulses of [14C]guanine and [14C]tryptophan by chemostat cells grown with different nutrient limitations throughout a shift-up into enriched medium containing glucose, sulfate, vitamins and amino acids, but not purines and very little or no tryptophan wss followed. We found that the increase in the rate of protein synthesis following the shift-up of a sulfate-limited chemostat paralleled the cumulative increase in raet RNA. On the other hand, the rate of protein synthesis in glucose-limited or in alanine-limited cells increased abruptly 4*4- to 6*6-fold immediately after the shift, and after the initial spurt also increased at a rate that paralleled cumulative net RNA synthesis. We interpret these results to mean that in the caee of carbon-limitation, but not in the case of sulfate-limitation, there is a change in the efficiency of protein synthesis per unit amount of RNA immediately after the shift. It is also concluded that virtually all the RNA in carbon-limited cells can become functional but only a small fraction of the RNA of sulfate-limited cells becomes functional.

1. Introduction
When enteric organisms are shifted from a nutritionally poor environment to a nutritionally rich environment they are capable of faster growth. Fast-growing cells need (and have) a higher content of ribosomes per genome to synthesize the needed amount of protein in the shorter doubling time. Thus, one of the first effects of a nutritional enrichment is an increased rate of RNA synthesis (Kjeldgaard, Maalse, & Schaechter, 1958; Maalee & Kjeldgaard, 1966). Several years ago Koch (1965) showed that this increase in the rate of RNA synthesis was in some circumstances very rapid indeed. In his experiments, this rate was measured, at five-second intervals after the shift, by 20-second pulses of [14C]guanine entering into the trichloroacetic acid-insoluble fraction. It was found that when cells growing with a five-hour doubling time in nL-alanine as a carbon source are shifted to medium supporting a 29-minute doubling time they linearly increase their rate of RNA synthesis over a 40-fold range, and achieve the maximum rate within 100 seconds. When the shift was from the starvation for a needed amino acid or for uracil, the increase in the rate of RNA synthesis
t Present address: Department of Biology, Harvard 549 University, Cambridge, Mass. 02318, U.S.A.

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was a bit slower. Even in these cases, this same maximum rate of RNA synthesis (the rate characteristic of cells grown indefinitely on the rich complete medium) was achieved within three minutes. The rapid shift in the rate of RNA synthesis indicates that cells grown in a nutritionally poor environment, or starved of some needed factor, have a reserve supply of the enzymes for purine and pyrimidine biosynthesis and of RNA polymerase which were not being used to capacity. Furthermore, it demonstrates that if there were any limitations in the availability of metabolic energy or metabolic intermediates involved in RNA biosynthesis, this lack also is evidently remedied within a very short time following the shift. This observation suggested that the complementary study of the rate of protein synthesis during such shifts could yield information about the content of functional ribosomal RNA. It would be expected that any energy limitation for protein synthesis would also be quickly relieved. Also, any limitation for amino acids would no longer be present since all the protein amino acids except tryptophan are given in large excess in the enrichment medium. (Tryptophan is also not limiting since its addition does not further increase the growth rate.) We can therefore argue that in times equal to or less than the 100 seconds or the three minutes quoted above, these cells should become capable of protein synthesis to the knit of the new nutritional environnaent, but with the previously synthesized translation machinery, i.e. the available levels of transfer-RNA, ribosomes and activating enzymes. These times are sufficiently short compared with the new doubling time so that the cells cannot appreciably augment the translation machinery by new synthesis. Actually, we found it useful to extrapolate back to the time of the shift from data collected over about 30 minutes in order to correct for new synthesis. We can thus measure the protein synthesizing capacity and take this as a measure of the amount of translation machinery in cells which had grown under a variety of growth conditions, but were tested under the same optimal conditions. If the extrapolation leads to the same rate of protein synthesis as the cells were carrying out in the previous environment, then the translation machinery was working to full efficiency in the previous environment. If it leads to a higher value, then ribosomal RNA (free, in particles, or as intact ribosomes) and other elements of the translation machinery was present which was either not functioning at all or not functioning at maximum efliciency in protein synthesis before the shift. In the theoretical section of this paper, expressions are derived which relate the change in net rate of RNA and protein synthesis per unit amount of cellular protein or dry weight to time after a nutritional shift. This kinetic analysis then serves as a basis for analyzing data obtained from experiments in which cells were growing under steady-state chemostat conditions with various growth limitations and were suddenly nutritionally enriched by the addition of a mixture of glucose, sulfate, amino acids and vitamins. We conclude that under circumstances of carbon limitation the cells have formed translation machinery during the slow growth which is not used to maximum etIiciency, but is rapidly converted to maximum efticiency when the environment is enriched. Under other circumstances, such as sulfate limitation, the kinetic analysis indicates that new protein synthesizing capacity parallels new synthesis of stable RNA. The bulk of the RNA present in the cell at the time of the shift does not function in protein synthesis and does not appear to be subsequently used for protein synthesis.

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2. Materials and Methods

coli strain B requiring uracil was grown in continuous culture at 37C. The design and construction of the chemostats is described elsewhere (Norris, 1970; Koch, 1971). The carbon source limitation was either 0.02% glucose, or O*04h DL-alanine in 1946). For sulfate limitation, the MgSO, of the the basic salt medium MQ (Anderson, medium wss replaced with MgClz and 10 mM-SOiwas added to achieve a satisfactory concentration of cells. In this case 0.2% glucose was the carbon source. Uracil at 10 pg/ml. was present in all media. The nutritional enrichment consisted of raising the final concentration to 0.2% in glucose, vitamins to a final concentration of 1 y0 of that supplied in the concentrate MEM N.Y.) and a mixture of vitamin solution (Grand Island Biological Co., Grand Island, amino acids. In most of the experiments the amino-acid enrichment consisted of the addition of C&amino acids to a &al concentration of 0.2%. In the acid hydrolysis used to make this product from casein the tryptophan is destroyed almost completely. A small amount remains, varying from batch to batch. The amount remaining is enough to dilute the radioactive tryptophan significantly. This dilution decreases the specific activity l-47- to 2.5-fold in different experiments with different batches of Casamino acids. Correction for this dilution is only important when we need to calculate an absolute rateor compare the shift-up conditions to the preshift conditions. The dilutions of specific activity due to the contaminating tryptophan were calculated through appropriate isotope dilution experiments. For the sulfate-limited chemostat of Fig. 5 a synthetic mixture of 19 amino acids at a final concentration of 10 pg/ml. each was used. The washed trichloroacetic acid precipitates on Millipore filters were dissolved in dioxane-based scintillation fluid and counted in a Beckman scintillation counter in the the case of the guanine samples. In the case of the tryptophan samples the filters were glued to planchets and counted in a Nuclear Chicago gas-flow mylar window counter at an efficiency of 33%. At least 3000 counts were recorded for each sample. A background correction for the physical background and the radioactivity adsorbing onto the bacteria and the filter was subtracted from all data. Under any of the conditions of balanced growth employed here the rate of incorporation of [2-14C]tryptophan into trichloroacetic acid-insoluble material is linear with time (or dry weight concentration if growth is signiiicant) and exhibits no significant lag. The concentration of cellular material was determined turbidimetrically from the apparent absorbance of cultures at 420 nm in a Cary 16 spectrophotometer. A calibration curve valid for bacterial cells growing at a variety of growth rates was used. The concentration of cellular material in mg dry wt/ml. is given; by 0.1361 A + 0.0362 A2 where A is the absorbance. The expression is applicable up to A = 1.1. The amount of RNA was determined by the Fleck & Begg (1965) method. The method has been successfully modified in this laboratory by Ehrenfeld, Koch, Norris, Deppe, Bernstein & Alton so that it is directly applicable to E. coli in MQ growth medium. 5 ml. ice-cold 1.2 N-perchloric acid is added to 20 ml. of chilled culture at approximately 0.05 mg dry weight/ml. After 20 min on ice, the samples are centrifuged, decanted, washed once with 5 ml. of 0.2 N-perchloric acid, and drained well. The pellet is taken up in 4.0 ml. of 0.3 N-NaGH and hydrolyzed at 37C for 45 to 60 min. Then 2.0 ml. ice-cold 1.2 r?-perchloric acid is added and the samples allowed to precipitate for 20 mm in an ice bath. The samples are centrifuged and measured at 260 nm and 231 nm against an appropriate blank. If the ratio A,,,/A,,1 is 2.8 to 3.3, then the RNA sample is spectrally pure and the RNA content in pg/ml. is computed as 8.90 A,,,.

Escherichia

3. Theoretical Section
In this section, equations are developed for the kinetics of protein formation in bacteria subsequent to a shift in environmental conditions. The treatment is more general than that previously presented (Koch, 1970) in that the shift in RNA synthesis need not be abruptly discontinuous, but may only gradually change from the

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value characteristic of balanced cells growing in the pre- to the post-shift medium. We assume that the ribosomal RNA present functions at constant eflloienoy in protein synthesis very shortly after the shift. We can then define k, the rate constant for protein synthesis, as follows :

d(p) = k(r) dt

(1)

where (p) is the concentration of protein per ml. of culture, (T) is the concentration of ribosomal RNA per ml. of culture, t is the time, and k is the rate constant of protein synthesis per unit amount of ribosomal RNA. The rate of synthesis of ribosomes depends on the number of genetic copies of the regions coding for rRNA in the cell and on the cellular control mechanisms which determine the rate at which a gene will be read. As above we write d(r)/dt = k,(d), where k, is the rate constant for the synthesis of ribosomes per unit amount of DNA, and (d) is the DNA concentration per ml. of culture. Since it is empirically found that the concentration of DNA and the concentration of protein are nearly proportional to each other under any growing conditions, then the ratio C = (d)/(p) under the steady-state conditions of balanced growth is essentially constant (Maalee & Kjeldgaard, 1966; Koch, 1970). On this basis the rate of ribosomal RNA synthesis can be rewritten as follows : d(r) = k,(d) = k,C(p). dt (2)

Under conditions of balanced growth, k, would be constant. Under conditions of a shift, k, changes from the values that existed under the pre-shift conditions until a new steady state is re-established. Even though the values of k, and k may change as a result of a change in the environment, if they both quickly change and they remain constant thereafter, equations (1) and (2) can be solved to give (T) and (p) as a function of time. In the case of the sulfate-limited chemostat bacteria the shift in k, is gradual, but if we can write a suitable equation approximating the behavior of k, as a function of time after the shift, the equations can still be solved. A suitable function defining k, is the following expression : k, = k,, (1 -fx eeBt) *

(P)

In this expression (p) is the concentration of protein at the time of the shift, /3 is a transient rate constant, krf is the final rate constant after the new steady-state is established, and f is a constant adjusting for the fold increase in the rate of ribosomal RNA synthesis. The rate at t = 0, is k, = krf (1 - f). From experimental measurements of the rate of nucleic acid synthesis all three constants /3, k,, and f can be determined, on the assumption that the synthesis of ribosomal RNA is proportional to the synthesis of net nucleic acid measured by the [14C]guanine incorporation. This is reasonable because ribosomal RNA is the major species of net nucleic acid at all growth rates. Furthermore, since dry weight and protein content are proportional

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under all the experimental conditions employed here, we assume that the guanine pulse incorporation data per unit dry weight are experimental measurements of

as a function of time after the shift. On substituting equation (3) into equation (2), differentiating equation (l), and then eliminating d(r)/dt between the two equations, a single differential equation of the second degree results : d(p) = MC,, c (1 - f3 6) dt2 (PI (p) (4)

It can be shown (Koch, 1970) that the growth rate constant under balanced growth conditions, A, is equal to kk,,C. Substituting this expression into equation (4) and solution by standard methods yields (p) = Cl2 + c&-At + C,e-pt. (5)

It can be shown that this expression for (p) can be made to satisfy equation (4) and the initial boundary conditions if the constants are given by

(7)

In these expressions g is given by :

g is also equal to h/h if the translation machinery functions at the same efficiency before as after the shift. Equation (5) with these values of C,, C, and C, expresses the concentration of protein at any time after a shift. This equation will be used below to calculate the lag in the growth curves, but first we would like to relate it to the experimental observations on the specific net rate of protein synthesis measured by the [lC]tryptophan incorporated in a pulse per unit dry weight of culture. Because protein is a quite constant fraction of cell dry weight no matter how fast the cells are growing, the specific net rate of protein synthesis is proportional to and measured by d(p)/(p)dt. To obtain an expression in this form, equation (5) for (p) is differentiated and the resulting equation divided by equation (5) to yield

d(p) -=
(p)dt

AC,Zt - hCZ*-At - pC3e-Bt

C,eAt + c/t + Cp-@

(10)

The importance of equations (5) and (10) is that they contain no adjustable parameters not essentially fixed from the specific rate of nucleic acid synthesis.

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Growth

time after shift-up

(units of one new doubing time)

FIG. 1. Theoretical curves for the specific synthesis rates of RNA and protein. Calculation based on equation (10) for various values of the RNA transient rate constant j. The value of the g is 40 for all the calculations shown. For clarity the RNA curves for p > 1.5 have been omitted.

Based on this equation, theoretical curves are presented in Figure 1 for the rate of RNA and protein synthesis per unit protein normalized to the final steady-state values. In this graph, time is measured in units of one new doubling time, the arbitrary parameters are the severity of the shift, g; and the rate constant, p, for the speed of the RNA transient. Theoretical curves are also shown in other Figures with parameters chosen to fit the experimental data. A form of equation (10) applicable where the shift of RNA synthesis from the old rate to the new rate is sufficiently fast that it can be treated as being instantaneous is useful. It can be derived from equation (lo), or independently developed (Koch, 1970) : 1-9-l 0) -= Wt h l+g-l
g+l

e-2At s+l
0-21t

(11)

Equation (5) predicts a lag in growth after a shift-up before the new steady-state of exponential growth is established. The magnitude of this lag is readily calculated and useful since it can be related to simple growth experiments based on turbidity measurement on the culture (see below). When the new steady state is established :

(24 = (y - &)
If there had been no lag then (24 = (p)e+At

(z4e+At.

(12)

(13)

Therefore, because of the existence of the lag, the culture after a long time contains

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fewer cells. The lag time, y, is the length of time required to grow from the quantity in parentheses in equation (12) to 1 at the new growth rate. This is (14) This can be rewritten using equation (9) as

2g (B+al y=lln --. h s+l j3g+h

(15)

In summary of this theoretical section, measurements of the specific rate of net nucleic acid synthesis can be fitted to equation (3) on the assumption that ribosomal RNA synthesis is proportional to net nucleic acid synthesis. If the constant efficiency hypothesis holds, then a knowledge of h and X and the kinetics of the net rate of RNA synthesis are enough to specify the constants in equations (10) or (11) for protein synthesis. If the constant efficiency hypothesis does not hold, there will be an immediate jump in the specific rate of protein synthesis after the shift-up. The subsequent increase in the specific rate of protein synthesis will follow equations (10) or (11)) but, where the constants must be recomputed with some value of g less than the value of g = h/h which applies in the constant efficiency case.

4. Results
It was previously shown (Koch, 1965) that the net rate of synthesis of RNA changes abruptly upon enrichment of a culture growing in a poorly utilized carbon source. In these studies, cultures were used of cells growing with a five-hour doubling time in D,L-alanine, a very poor carbon source for E. coli strain B. We now extend this observation to cells taken from very slowly growing chemostat cultures. In Figure 2 we see a plot of the time-course of increase in rate of RNA synthesis after

Time (min) --+

FIO. 2. Specific RNA synthesis ra& after a shift-up at 0 min of a 24&r glucose-limited Radioactive guanine incorporation for IO-sea pulses before and after an enrichment 30-min doubling time (T$).

ahemostat. that gave a

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enrichment of a glucose-limited chemostat with a 24-hour doubling time. The initial rate of net RNA synthesis is overestimated in the Figure for two reasons. First, there is a partial turn-on of the rate of RNA synthesis during the 20-second pulse measurements of guanine uptake measured in the enriched medium. Second, at slow growth rates the net RNA synthesis is so small that exchange across the cell membrane and incorporation into messenger lead to significant incorporation beyond net RNA synthesis (Koch, unpublished observation). Even so, the rate of RNA synthesis that we measure increases very abruptly. The rate of net synthesis achieved after two minutes is the definitive rate as measured at much later times in this experiment. This same fmal rate is found in many other completely independent experiments which measure the rate of RNA synthesis of cells growing under balanced conditions in the enriched medium. The implication is that even in a 24-hour chemostat, all (or at least, very nearly all) the cells in the population are capable of immediate RNA synthesis after enrichment. The specific rate of protein synthesis in such chemostat cultures (Fig. 3) changes much more slowly than does the specific rate of RNA synthesis (Fig. 2) in reaching the new steady-state value. This is necessarily so if the increase in the rate of protein synthesis is dependent upon the production of new translation machinery. However, it can be seen that the specific rate of protein synthesis measured in these twominute pulses shifts abruptly by a factor of nearly sevenfold within two minutes of the enrichment. Further increase in rate is considerably more gradual, taking an hour to approach the eventual steady-state level. The ratio in specific rate of protein synthesis in the new steady-state to that of the old steady-state is substantially the same as the ratio of the growth constants. This must be so if the tryptophan pulse experiments measure set protein synthesis. The abrupt rise we take as indicative of the existence, or rapid assembly in the cells, of translation machinery. This available translation machinery has a total capacity for much more protein synthesis than actually takes place in the cells growing in the chemostat under glucose limitation. This result is strong evidence that the net rate of protein synthesis in the slow-growing, glucose-limited cells is not limited by the amount of any macromolecular component of the translation machinery, i.e. mRNA, tRNA, rRNA, ribosomal protein, enzymes, or other components that require significant periods of time to augment their amounts. The solid curve fitted through the data follows equation (ll), the equation applicable when there is a rapid change in specific rate of RNA synthesis. The factor g that fits the data best is 2.5. (That is, the rate of protein synthesis changes after the shift in the way that one would predict if the ratio of initial to final growth rates, h/X was 2.5.) From the growth rate data, however, h/h = 16.5. Therefore there is 16.512.5 = 6.6 times more protein-synthesizing capacity present in these slowgrowing cells than is being used. If the bulk of the RNA of the cell can become functional when cells are shifted-up, then the ratio of total RNA per unit dry weight in cells growing with a 30-minute doubling time to those growing with an 11-hour doubling time should be 25, the value of g that fits the data of Figure 3. From the data of Norris quoted in Koch (1970) the amount of RNA in this ratio is 2.7 and thus almost all the RNA is capable of being used at maximal efficiency after the shift. Almost identical results were obtained with a D,L-alanine limited chemostat. The RNA data are not shown but the shift is just as abrupt in this case. The specific rate of protein synthesis data are shown in Figure 4. In this case g = 3 fits the data points, while X/,\ is 13.3. Tbrrcforc~, tbercx appears to bc 4.4 times more protein-

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tll
-2 BE K/

v) t: 0 z l0 Pre +Enrlchment 0 I IO I 20 I 30 I 40 I 50 t 60 /_1

CD

Time (min)

Fm. 3. Specific protein synthesis rate after a shift-up of an 11-hr glucose-limited ohemostat. Radioactive tryptophan uptake for 2-min pulses before and after an enriohment that gave a IO-mm doubling time (Ta). Also shown are experimental results on a oontrol culture growing with a 30-min doubling time in the enriched medium. The curve has been fitted to equation (13) with g = 2.5.

synthesizing capacity in (or quickly assembled in) these cells than is actually functioning. Typical results with a sulfate-limited chemostat (Fig. 5) are in marked contrast to the results with glucose or n,L-alanine-limited chemostats. Although the reason why the rate of RNA synthesis does not change abruptly under these circumstances is not yet clear, by using the equations derived in the theoretical section we can Cnd out if new RNA synthesis is needed to achieve new protein synthesizing capacity.

,,,--IO-hr

DL-alanine-limited chemostot

rc Enrichment
I IO I 1 I I 50 I 60 OJ

20

30

40
Time

(min)

Fro. 4. Speoiiic protein-synthesis rate after a shift-up of a lo-hr n,L-alanine-limited The doubling time after the shift was 45 min. The curve has been fitted with g = 3.0.

chemostat.

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G Time (min)

ILU

co

FIG. 5. The speciiic synthesis rates for both RNA and protein after a shift-up of a sulfatelimited chemostat. The RNA data (the thin horizontal lines) were fitted to theorv as described in the text to yield the thin solid line; the theory also predicted the specific rate of protein syn. thesis shown by the thicker solid curve. If new RNA synthesis parallels new protein synthetic capacity then the thick solid line, should fit the experimental values (the thick horizontal lines) and the growth rate data (triangles).

First, the specific rate of synthesis of RNA was fitted approximately from the initial and final specific rates and from the time to make the rate half of the final specific rate. A computer program for a Wang 370 was used to compute the specific rates of RNA and protein synthesis at various times after the shift. A slightly better fit to the RNA data was obtained by varying the choice of the constants slightly until the fit to the RNA data shown as a thin line in Figure 5 was obtained. The predicted line for specific rate of protein synthesis approaches the experimental data reasonably well at later times as do the apparent growth rates (shown as triangles). These were calculated from the tangents to the curve which relates the log of dry weight concentration to time after the shift. The discrepancy between the theory and the experiment is larger at earlier times, i.e. initially, the specific rate of RNA synthesis and the specificrateof proteinsynthesis are higher than predicted. The change in the latter is in the right direction to suggest that a more complicated and closer fitting of the RNA data would lead to a closer agreement of theory and observation for the protein rate data. Thus, indeed, it seems that the increase in specific rate of protein synthesis runs parallel with the accumulation of RNA resulting from net RNA synthesis as measured by the [14C]guanine assay. Therefore, in this case, in contrast to the carbon-limited situation, there is reason to believe that some element of the translation machinery is limiting the rate at which protein is synthesized in the chemostat. Moreover, the translation machinery which does function is used with the same efficiency before and after the shift. Further new synthesis proportional to net RNA synthesis is needed for new net production of protein-synthesizing capacity. Both statements follow,

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because the growth rate-constant changes 31-fold in this experiment and the specific rate of protein synthesis also changes 31-fold, i.e. the best-fitting value of g is equal to h/h. The ratio of total RNA to dry weight for the cells of the chemostat culture shown in Figure 5 was 120 pg/mg. This value is within the range of those expected from a glucose-limited chemostat with a 20-hour doubling time (T. Norris, personal communication). Therefore, in this case, more than 8076 of the RNA in the cells is very inefficiently utilized for protein synthesis, even after such long times as 30 to 60 minutes after the cells are brought into a rich environment. Sulfate limitation might be equivalent to a specific amino-acid limitation, although we cannot reject the possibility that some sulfur-containing cofactor or certain tRNAs are limiting. At least, the results of an experiment shown in Table 1 indicate that. an TABLE 1
Inactivation

of protein sy&esizing

capacity by growth in chloramphenicol

Pulse

incorporation of [14C]tryptoph8n (corr. cts/min/mg dry wt/2-min pulse)

Cam? in assay With Cam in &ssay 2450 f 200

Without Pm-chlommphenicol Effect Immediately filtering after 1 min 2 min 3 min Effect Immediately after filtering Pulse started after : 1 min 2 min 5 min 20 rnin 25 min 30 min t Abbreviation

44,000 f 2000 of 3-min exposure 24,000 24,000 28,000 54,000 of 2%min exposure 3300 5500 5200 5000 21,800 24,400 32,900 used : Cam, ohlorampheniool. to 60 pg Cam/ml. to 50 pg Cam/ml.

6300

9u

inhibition of protein synthesis itself suffices to cause some of the characteristics we find in sulfate-limited cells. A culture growing rapidly in the enrichment medium was exposed to chloramphenicol at 50 pg/ml. for three minutes or 28 minutes. Then the culture was filtered, washed, and resuspended in enrichment medium lacking chloramphenicol. At times thereafter samples of culture were exposed to two-minute pulses of [14C]tryptophan. The top part of the Table shows that under these conditions chloramphenicol acts immediately. If cells that have been exposed to the drug for three minutes are filtered and washed, the protein-synthesizing ability is recovered fully in three minutes. After 28 minutes in chloramphenicol, the protein-synthesizing capacity of the cells is also almost completely inhibited. However, after these cells are washed, the protein-synthesizing capacity recovers only very slowly. Even after 20 minutes it is only back to one-half the original rate.
37

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Because the three-minute incubation data and the eventual recovery of cells after 28 min incubation clearly indicates that the washing procedure does eliminate the effect of chloramphenicol, we conclude that some part of the translation machinery itself is rendered less functional by the longer incubation. It should be remembered that RNA synthesis continues during chloramphenicol inhibition, One possibility that suggests itself is that ribosomal protein can redistribute itself to all of the rRNA in cells. In cases where the amount of ribosomal protein is insufficient to make complete ribosomes of all the rRNA present (which must be the case where ribosomal protein synthesis, but not rRNA synthesis, has been inhibited by chloramphenicol, and which might be the case in sulfate-limited cells if sulfate limitation is limiting upon protein synthesis) few or none of the ribosomes in the cell would operate at maximum efficiency. At any rate, the addition of chloramphenicol is known to inhibit protein synthesis and when the restriction on protein synthesis is removed, these cells, like shifted-up sulfate-limited cells, demonstrate only a very gradual increase in their protein synthesizing capacity. Finally in Table 2 we present the growth lag data taken from the experiments of Figures 3 to 5, but consistent with other experiments with chemostats varying in
TABLE 2

Changes in growth rate during shifts conditions Chemostat

doubling time Doubling time observed after shift-up Lag time observed Lag time calaulated

Cultural

Gluoose-limited chemostat r&-ah+nine-limited chemostat Sulfate-limited chemostat

11 hr 11 hr 20 hr

42

min

16 min 15 min 67 min

22 min 24 min 71 mill

40 min 30 min

growth rate from 10 to 24 hours. In all cases the lag is short after shift from carbon limitation to the rich medium. This is consistent with the original observation of Kjeldgaard et al. (1958) in the shift of Salmonella typhinmrium from glucose minimal medium to broth, although the shifts used in present experiments span a much larger range of growth rates. If we assume that rRNA operates at a constant efficiency and if the rate of RNA synthesis changes discontinuously at the shift, equation (15) shows that the lag time should equal the new doubling time. It is in fact much shorter. It can be seen that this can almost be accounted for quantitatively by rejecting the constant efficiency hypothesis and using the fitted values of g found above. For sulfate-limited chemostats the lag is longer than the new doubling time, accounted for by the gradual shift in the rate of RNA synthesis. The value of the constant /3 best iltting the data for Figure 5 was (ln 2)/60 minutes. Substituting this and a value of g = 31 into equation (16) yields a calculated lag that is little different from that experimentally observed.

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5. Discussion
In an effort to understand the control mechanisms whereby the common colon bacillus adjusts its macromolecular synthesis to altered nutritional circumstances, pulse tracer methods have been developed to follow the changes in the specific rates of protein and RNA synthesis following a nutritional enrichment in order to measure the capability of such cells for protein synthesis under the conditions of previous growth. The results show that the rate of protein synthesis per unit RNA changes when carbon-limited chemostat cultures are enriched, and that this change in the efficiency with which the RNA is utilized occurs before significant new macromolecular synthesis can take place. Whether this change in efficiency involves all or only part of the rRNA is not known. That is, our change in efficiency of total RNA could result from a change in efficiency of functioning ribosomes or from a change in the fraction of total RNA that occurs in the form of functioning ribosomes. The level of RNA known to be present in such slowly growing chemostat cells is eight times greater than is expected from the growth rate if one assumes that the majority of the available RNA in such cells is present as ribosomal and transfer RKL4 functioning as efficiently as they do in cells growing at higher growth rates (Norris, 1970; Koch, 1970). Experiments to determine whether the extra RNA is in the form of ribosomes, ribosomal particles of precursors, or free RNA are currently in progress. One other observation, however, is relevant to this question. Experiments (Coffman, Norris & Koch, manuscript in preparation) show that the delay time between addition of inducer and the completion of translation of ,B-galactosidase is only slightly longer in cells from slow chemostat cultures compared with batch cultures; this observation suggests that at least some of the ribosomes from slowly growing cells operate at maximum efficiency once polypeptide synthesis has begun. This does not exclude the possibility that these ribosomes might be less efficient with respect to binding mRNA or initiating polypeptide synthesis. Now there are two hypotheses that could explain the existence of the extra RNA without rejecting the hypothesis that the RNA in E. coli cells operates at a constant eficiency independent of h. These possibilities have been considered in greater detail elsewhere (Koch, 1970) but are mentioned here since they are excluded as the major raison detre of extra RNA. The first possibility is that in slowly growing chemostats a fraction of the cells at any given time are in a state approaching a stationary or lag state. We found that although all the cells are alive and will respond rapidly to an enrichment, one-third of the cells in a population with a doubling time of 24 hours are temporarily not synthesizing ,!l-galactosidase (Koch & Coffman, 1970). If these cells are not synthesizing any protein, then they do not contribute toward any measurement of total protein synthesis, but do contribute to measurements of RNS content or RNA synthesis of the culture. This phenomenon can account for only a small percentage of the extra RNA. The second hypothesis is that the extra RNA is functioning in the slowly growing cells, but much of the protein is then degraded, and therefore net protein synthesis is smaller. In fact, a small portion of the proteins of slowly growing cells are synthesized and then degraded (Nath & Koch, 1970). Moreover, a larger proportion of the translation machinery of the cell is utilized for the synthesis of these unstable proteins at low growth rates than at high growth rates. This second hypothesis can, however, only account for a few percent of the extra RNA. The two hypotheses together account

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for a 70 or 80% increase above expectation for cells with a doubling time of ten hours. Since actually there appears to be seven to eight times as muoh translation machinery as is being used, these hypotheses fail quantitatively, to account for it. When the experiments described above were proposed, yet another possible outcome was envisaged. If it took a considerable time for a ribosome to mature, then there would be a lag before the specific rate of protein synthesis increased after a shift-up. Actually, no lag was detected in the shift-up from carbon-limited growth, where there was an essentially discontinuous shift in RNA synthesis. This means that the time required for maturation of ribosomes must be only a few minutes or less. Thus our results are consistent with the rapid synthesis of ribosomal subunits observed by Kelley & Schaechter (1970) but are much faster than the findings from Schlessingers laboratory (Mangiarotti, Apirion, Schlessinger & Silengo, 1968) with fragile bacterial strains. In both types of chemostat cultures the specific rate of RNA synthesis after enrichment parallels functional protein synthesizing capacity. Shortly after the shift they are both low with sulfate-limited chemostat culture while both k, and k becomes quickly maximal in cells taken from a carbon-limited chemostat. The same parallelism between protein-synthesizing capacity and level of RNA synthesis occurred in the chloramphenicol experiment reported here. These reuslts are consistent with the hypothesis of Stent (1966) that transcription may be pulled by the translation process. Further support for the thesis that excess translational machinery is available in cells growing slowly under certain circumstances comes from the results of Mateles, Ryu & Yasadu (1965) with nitrogen-limited chemostat cultures and Harvey (1970) with glycerol-minimal cultures. In both laboratories, it was clearly found that the synthesis of protein increased rapidly after shifts which doubled the growth rate, although the detailed kinetics were not established.
We wish to thank Dr Tom Norris and Mr Tom Alton for RNA determinations. Work in our laboratory is supported by the National Science Foundation grants GB7022 and GB7846 and by the U.S. Public Health Service grant A19337. REFERENCES Anderson, E. H. (1946). Proc. Nat. Acd Sci., Wash. 32, 120. Fleck, A. & Begg, D. (1965). Biochim. biophys. Acta, 108, 333. Harvey, R. J. (1970). J. Bact. 101, 574. Kelley, W. S. & Schaechter, M. (1970). J. Mol. Biol. 50, 170. Kjeldgaard, N. O., Maalee, 0. BE Schaechter, E. (1958). J. Gen. Microbial. 19, 607. Koch, A. L. (1966). Nature, 205, 801. Koch, A. L. (1970). J. Zheoret. Biol. 28, 203. Koch, A. L. (1971). Advanc. Microbial. Physiol. in the press. Koch, A. L. & Coffman, R. (1970). Biotech. Bioeng. 12, 651. Maalee, 0. & Kjeldgaerd, N. 0. (1966). CodroE of Mucromlecular Synthesis. New York: W. A. Benjamin, Inc. Mangiarotti, G., Apirion, D., Schlessinger, D. & Silengo, L. (1968). Biochemietry, 7, 466. Mateles, R. I., Ryn, D. Y. & Yaauda, I. (1965). Nature, 208, 263. N&h, K. & Koch, A. L. (1970). J. Biol. Chem. 246, 2889. Norris, T. E. (1970). Ph.D. Dissertation, Indiana University. Stent, G. (1966). Proc. Roy. Sot. B,164, 181.