Temperature-Induced Synthesis of Specific Proteins in Escherichia coli

Vol. 142, No. 3

JOURNAL OF BACTERIOLOGY, June 1980, p. 843-851 0021-9193/80/06-0843/09$02.00/0

Temperature-Induced Synthesis of Specific Proteins in Escherichia coli: Evidence for Transcriptional Control TETSUO YAMAMORI AND TAKASHI YURA Institute for Virus Research, Kyoto University, Kyoto 606, Japan

Synthesis of several protein chains of Escherichia coli is markedly, though transiently, induced upon shift-up of a log-phase culture to or above the critical temperature (about 340C). Such induction occurs coordinately for at least three protein chains (76K, 73K, and 64K) examined. Studies of initial kinetics of induction using a specific inhibitor of transcription (rifampin) revealed that induction occurs at the level of transcription with very little lag, though actual synthesis of messenger ribonucleic acids and proteins requires about 1 min when temperature is shifted up from 30 to 420C. Evidence suggests that E. coli cells somehow "recognize" the temperature change and activate transcription of several distinct operons, one of which contains the mop (morphogenesis of phages; groE) gene.

Bacterial cells growing in steady state at various temperatures do not differ appreciably in the relative content of macromolecules, despite the fact that the growth rate varies widely with varying temperatures (10). The differential synthesis rates for most individual proteins during balanced growth also seem to be rather constant when cells are grown within "nornal" temperature range (23 to 3700) (3). However, when cells are transferred from one temperature to another, they enter a transient stage in which the synthesis rates of certain proteins are altered dramatically (9, 19). This is particularly striking in the case of temperature shift-up; we have previously shown that synthesis of at least several protein chains is markedly stimulated when cells that have been grown at 300C are suddenly exposed to 420C (19). Apparently there is a time period of cellular perturbation upon shift-up of temperature, in which synthesis of certain specific proteins is actively and transiently regulated. To further assess the physiological significance of such a phenomenon, we have investigated the mechanisms by which synthesis of specific proteins is induced upon temperature shift-up. As the first step, initial kinetics of induction were studied using rifampin, a specific inhibitor of transcription initiation in bacteria. The results obtained clearly indicate that induction occurs at the level of transcription with very little lag after temperature shift-up. MATERIALS AND METHODS Bacterial strains and media. A permeable mutant of Escherichia coli B (AS19; reference 14) and E. coli K-12 (W3350) were used in all experiments. Cells were grown in synthetic medium E (17) supplemented with 0.5% glucose and thiamine (2 ,ig/ml).

Chemicals. L-[4,5-3H]leucine (58 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, England, and acrylamide and N,N'-methylene-bisacrylamide were obtained from Wako Chemical Co., Osaka. Sodium dodecyl sulfate was the product of Nakarai Chemical Co., Kyoto. Rifampin was obtained from Sigma Chemical Co. Temperature shift-up. Temperature shift-up was carried out by one of two methods. (i) A log-phase bacterial culture (10 to 20 ml unless otherwise indicated) grown at 30°C in a water-bath shaker was divided into 1-nl portions and was transferred to another water-bath shaker at 420C. (ii) In a flask kept at 420C, a culture grown at 30°C was mixed with an equal volume of prewarmed (550C) culture filtrate that had been obtained from a culture of the same strain (3 x 108 cells per ml). Method (i) was used, unless otherwise stated. Pulse-labeling of cells. Cells were grown to the mid-log phase (2 x 108 to 4 x 108 cells per ml) and were shifted to a higher temperature as indicated for each experiment. Portions of 1 ml of the culture were removed at appropriate intervals, pulse-labeled with 10 ,uCi of L-[3H]leucine, and chased with 200,ug of Lleucine and 50 ug of L-isoleucine per ml. The time periods for pulse-labeling and chase are indicated for each experiment. Polyacrylamide gel electrophoresis of proteins. Dodecyl sulfate-polyacrylamide gel electrophoresis with a discontinuous buffer system described by Laemmli (7) was used. Slab gels of 10% acrylamide (1 mm thick) were used as the separation gel, and 5% acrylamide was used as the stacking gel. Pulse-labeled cells were chilled in ice and mixed with an equal volume of 10% trichloroacetic acid. The precipitates were collected by centrifugation and washed twice with cold 5% trichloroacetic acid and once with acetone. The precipitates were dissolved in sample buffer (0.0625 M Tris [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol, 10 mM dithiothreitol, 0.001% bromophenol blue) and heated at 100°C for 3 min. Electrophoresis was run for 3 h at 30 mA (constant current) or 843

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for 16 h at 75 V (constant voltage). Protein bands are generally sharper in the former condition, whereas proteins of higher molecular weight (>60K) are better separated in the latter. Molecular weights of proteins were determined using several reference proteins as described by Ito et al. (6). For detection of 3H-labeled proteins, fluorographs were taken by exposing Fuji Xray films to the scintillator-treated gel slabs. The films were preexposed to a brief flash of light (8). Densitometer tracing and quantitative estimation of proteins. Content of a given protein among total proteins synthesized was estimated from densitometer tracings (Joyce-Loebel densitometer MKIII) of fluorograms. Each peak cut out from tracing paper was weighed, and the weight was normalized to that of total proteins. Quantitative estimation of radioactivity incorporated into a protein band was carried out within the range in which a linear relationship exists between time of exposure and weight of paper corresponding to the protein.

RESULTS Effect of varying temperature on temperature-induced protein synthesis. We have shown previously that the patterns of total proteins synthesized in cells of E. coli K-12 (W3350) growing in steady state at low (300C) and high (420C) temperatures are similar to each other (19). This has been confirmed and extended to a permeable strain (AS19) of E. coli B (Fig. 1A). The gel electrophoretic patterns of proteins synthesized in cells grown at 30, 33, 36, 39, and 42°C revealed no major differences among them, despite the fact that the growth rate varied about 1.5-fold in this temperature range (see reference 3 for alterations of individual proteins). An apparent exception is that a protein chain of molecular weight 80K seems to be synthesized at the narrow temperature range between 36 and 420C. When a culture of the same strain is transferred from low to high temperature, protein bands of 87K, 76K, 73K, and 64K that are hardly detectable under steady-state growth become evident within several minutes, in agreement with the previous finding (19). In the present experiments, a culture was shifted up by 30C starting from various temperatures between 30 and 420C (Fig. 1B). Clearly, the extent of induction is greater with the increasing preshift temperature. Also, a more striking induction is observed with a greater change in temperature. Among the protein chains affected, the 87K protein band is not well separated from other protein(s) synthesized at 30°C under these conditions. A shift-up from 30 to 330C failed to give any appreciable induction, whereas that from 30 to 34°C gave slight but significant induction as judged by the present gel system (a shift-up from 23 to 34°C gave a similar extent of induc-

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FIG. 1. Dodecyl sulfate-polyacrylamide gel patterns of proteins synthesized during steady-state growth or upon temperature shift-up. Log-phase cells of E. coli AS19 were grown in medium E at a given temperature for at least two generations. A 1-ml portion of cells was taken and pulse-labeled with L[3H]leucine for 3 min, then chased with L-leucine and L-isoleucine for2min. Cells were treated with trichloroacetic acid, and the whole-cell proteins were analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis at 30 mA for 3 h. (A) Cells in steady-state growth were labeled at the following temperatures. (1) 300C, (2) 330C, (3) 360C, (4) 390C, (5) 42°C. Protein samples, each containing 100,000 cpm, were used for each lane, and the film was exposed for 4 days. (B) Cells were shifted from low to high temperature and labeled after 5 min. (1) Control cells labeled at 30°C. (2) to (9) Cells were transferred as follows: (2) from 30 to 330C, (3) from 33 to 360C, (4) from 36 to 390C, (5) from 39 to 420C, (6) from 42 to 450C, (7) from 30 to 420C, (8) from 30 to 340C, (9) from 36 to 420C. Each lane received 200,000 cpm of radioactive protein, and the film was exposed for 2 days.

tion; data not shown). It appears therefore that induced sTynthesis of these proteins occurs when a culture is shifted to or beyond a certain critical temperature (about 34°C), and that the extent of induction depends both on the preshift temperature and on the size of temperature shift. Essentially the same results have been obtained with E. coli K-12, strain W3350 (data not shown). Initial kinetics of induction of protein synthesis. Induced synthesis of the proteins upon temperature shift-up is a rapid process and reaches the maximum level after about 5 min when the temperature is shifted from 30 to 420C (19). The following experiments were carried out to determine whether any time lag exists in such induction.

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When 1 ml of culture grown at 30°C is transferred to 420C in a water-bath shaker, it requires approximately 0.5 min to reach 37°C, 1 min to reach 400C, and 1.5 min to reach 420C. Thus the culture reaches the critical temperature (340C or above) in about 30 s. During the period of temperature transition, the rate of bulk protein synthesis fluctuates to some extent (1.2 to 1.8 times the preshift level). Under these conditions, induction occurred within 2 min after temperature shift (Fig. 2A). In a similar experiment with E. coli K-12 (W3350), significant induction could be detected after 2 min but not after 1 min (data not shown). Thus a lag of 1 to 2 min seems to be needed for induction to take place. To determine the induction lag more precisely, we have adopted a procedure which permits an almost instantaneous shift-up of temperature (see Materials and Methods). Whereas about 90 s is required to raise the temperature from 30 to 420C by the ordinary procedure (1ml culture), only several seconds are required by

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this rapid shift procedure, the rate of temperature change being at least 20-fold faster. A shorter pulse-labeling (20 s) was used to obtain more accurate kinetics of induction. As seen in Fig. 2B, induction of 87K, 76K, 73K, and 64K proteins was observed just as in the previous experiments. The extent of induction does not seem to depend on the rate of temperature shiftup, at least within the range. Significant induction occurs only when cells are labeled after incubation for 60 s or longer at 420C (Fig. 2B). The densitometer tracing of the data permitted us to estimate the lag to be about 50 s for 76K protein and 60 s for 64K protein (Fig. 3). The bands for 73K protein were too faint to be traced accurately; however, induction seemed to follow the kinetics similar to those for 76K and 64K proteins. Since it is difficult to estimate the background (steady-state) level of these proteins from the present data, the lag period obtained may be somewhat overestimated. However, a short lag of less than 1 min seems to intervene

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FIG. 2. Time course of temperature-induced protein synthesis. (A) Cells of AS19 were shifted-up from 30 to 42°C, pulse-labeled at various times for 45 s with L-[3H]leucine, chased for 165 s with unlabeled L-leucine and L-isoleucine, and analyzed by dodecyl sulfate-gel electrophoresis. (1) Pulse-labeled at 30°C. (2) to (7) Pulselabeled at (2) 0 time, (3) 2 min, (4) 3 min, (5) 4 min, (6) 5 min, or (7) 8 min. Each lane received 100,000 cpm of labeled protein, and the film was exposed for 4 days. (B) Cells of AS19 were shifted up instantaneously to 42°C by mixing a 30°C culture with an equal volume of 55°C medium (see the text), pulse-labeled for 20 s with L-[3H]leucine, and chased for 3 min with unlabeled L-leucine and L-isoleucine. The labeled proteins were analyzed by gel electrophoresis at 75 V for 16 h. (1) Pulse-labeled at 30°C. (2) to (7) Pulse-labeled at (2) time 0, (3) 30 s, (4) 60 s, (5) 90 s, (6) 120 s, or (7) 150 s. Each lane received 100,000 cpm ofproteins, and the film was exposed for 4 days.

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have determined whether induction occurs at the transcriptional or translational level. A culture of E. coli AS19 was grown at 300C and transferred to 420C, and the rate of [3H]leucine incorporation into specific proteins was measured in the presence of rifampin added at various times after temperature shift. Rifampin interacts with bacterial RNA polymerase and specifically inhibits initiation of transcription (15, 18) with a lag of several seconds (12). As shown in Fig. 5, [3H]leucine incorporation into bulk protein decreased logarithmically with a half-life of 1.3 min after a lag of a few minutes upon rifampin addition. Similar kinetics were obtained whether the drug was added at the time of temperature shift (time zero) or 1.5 or 5 min thereafter. The lag observed presumably reflects the time for elongation of mRNA chains that had been initiated before drug addition. These results suggest that the induced synthesis of proteins may also continue for a few minutes after drug addition once induction has taken place. No appreciable induction was observed when rifampin was added at time zero (Fig. 6A). If induction were to occur at the posttranscriptional level, it would have been detected within

120

Time (sec) FIG. 3. Initial kinetics of synthesis of two proteins upon temperature shift-up. Relative rates of synthesis of 76K and 64K proteins were estimated from the densitometer tracings of a fluorogram (exposure, 1 day) taken from the gel used in Fig. 2B (see the text). The values are plotted at the midpoint of the time of each pulse-labeling. (0) 76Kprotein; (a) 64Kprotein.

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in the temperature-induced synthesis of these proteins under the conditions of present experiments.

Figure 4 summarizes the kinetics of temperature-induced synthesis of the three protein chains examined above (76K, 73K, and 64K). Induction was well coordinated for all these proteins; the differential synthesis rates reached the maximum level 4 to 5 min after temperature shift-up and gradually decreased thereafter. Such coordination suggests the possible involvement of a common mechanism for temperature induction of all these proteins in E. coli. It can also be estimated that the cellular content of 64K protein reaches the level 1.8 times that of 300C within 10 min after shift-up to 420C, thus rapidly attaining the new steady-state synthesis rate, as reported by Lemaux et al. (9). Induction at the transcriptional level. To obtain further insight into the mechanism of temperature induction of protein synthesis, we

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Time (min) FIG. 4. Coordinate synthesis of temperature-induced proteins. Cells of AS19 were shifted up from 30 to 42'C (as in Fig. 2B) and pulse-labeled for 45 s and chased for 165 s at the times indicated. Other conditions and procedures were as in Fig. 2B, except that the films for fluorography were exposed for 1 day. The differential synthesis rates of the three proteins (76K, 73K, and 64K) were estimated from densitometer tracing offluorographs and plotted as percentage of total proteins synthesized as a function of the time after temperature shift. Symbols: (0) 76K, (A) 73K, and (-) 64K.

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FIG. 5. Effect of rifampin on bulk protein synthesis temperature shift-up. Cells of AS19 were grown in 1-ml portions at 30°C and transferred to 42°C (time 0), and rifampin was added to 50 pg/ml at the times indicated by arrows. At appropriate intervals, each culture was pulse-labeled with L-[3H]leucine for 45 s and chased with unlabeled L-leucine and Lisoleucine for 165 s. Cells were harvested by centrifugation and washed with 5% trichloroacetic acid twice. The resulting acid precipitates were dissolved in 0.2 ml of sample buffer and adjusted to pH 6.8 by adding 1 M Trizma base. Radioactivities of each sample (5,ul) were determined in a liquid scintillation counter and normalized to the value at the time of drug addition. Rifampin was added at (a) time 0, (0) 1.5 min, or (A) 5 min. (5) Control without rifupon

ampin. a few minutes after drug addition, since mRNA's should remain active for a few minutes, under these conditions (Fig. 5; cf. Fig. 9). In contrast, induction clearly occurred when the drug was added 1.5 or 5 min after temperature shift (Fig. 6B and C). Similar experiments with a rifampinresistant mutant of AS19 showed that temperature-induced proteins were made even when rifampin was added at time zero (data not shown). These results suggest that de novo synthesis of RNA upon temperature shift-up is required for induction to occur; i.e., induction occurs at the transcriptional level. The data also indicate that at least mRNA's for 76K, 73K, and 64K proteins start to be synthesized within 1.5 min after temperature shift. Kinetics of mRNA synthesis for temperature-induced proteins. The amount of mRNA's for temperature-induced proteins was

847

then determined quantitatively by measuring the cellular capacity to synthesize those proteins under conditions where transcription initiation is inhibited. Cells were continuously labeled with [3H]leucine starting from the time of temperature shift-up, and rifampin was added at various times during subsequent incubation at 420C. Incubation for an additional 15 min in the presence of rifampin was enough to permit the cells to synthesize proteins in amounts proportional to those of mRNA's that had been synthesized or initiated at the time of drug addition. Also there was no appreciable degradation of temperature-induced proteins under these conditions. The results indicate that the fraction of radioactivity associated with 76K, 73K, and 64K proteins increases and then gradually decreases with increasing time of incubation before drug addition (Fig. 7A). The amounts of 76K and 64K proteins synthesized have been estimated by multiplying the observed fraction of each protein among total labeled protein by the 3H1 radioactivity incorporated into total protein (Fig. 7B). Assuming that rifampin inhibits only initiation of transcription, and that the amount of protein synthesized is proportional to that of mRNA, the amounts of mRNA reach their maximum levels about 3.5 min after temperature shift-up (Fig. 70). The apparent absence of mRNA for 76K protein after 8 to 10 min is doubtful and was probably caused by the decreased sensitivity in measurement of this protein under these conditions. The initial kinetics of mRNA synthesis were then followed more closely for the first few minutes after temperature shift (Fig. 8). It was found that very little lag (less than 15 s) exists in induction of mRNA initiation for 64K protein. Therefore the lag of 50 to 60 s for induction of 76K and 64K proteins observed above (Fig. 3) appears to be required largely for the actual synthesis of mRNA's and of proteins under the conditions of present experiments. Functional stability of mRNA's synthesized after temperature induction. Stability of mRNA's was determined by measuring the decay rate of the cellular capacity to synthesize the specific proteins in the presence of rifampin. The capacity to produce 76K, 73K, and 64K proteins decreased with a half-life of 1.3, 1.0, and 2.0 min, respectively (Fig. 9). About 50% of mRNA's for 64K protein seem to remain active for 5 min after addition of rifampin, whereas only 10% of mRNA's for 76K and 73K proteins are active. Such a differential mRNA stability might suggest the involvement of distinct species of RNA for synthesis of 76K (or 73K) and 64K proteins.

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DISCUSSION Synthesis of at least several protein chains is transiently induced upon temperature shift-up of E. coli cells, provided that the temperature is shifted to or above 340C. The actual extent of induction observed depends both on the preshift temperature and on the size of shift-up. The kinetic studies of induction under the standard conditions (shift from 30 to 42°C) revealed a lag of about 1 min in induction of such proteins. The lag seems to represent largely the time for actual synthesis of mRNA's coding for those proteins and of proteins themselves, rather than the time before mRNA initiation. The present evidence strongly suggests that a control mechanism is operative at the level of transcription in bringing about induced synthesis of the several specific proteins upon temperature shift-up. Induction of the three protein chains examined (76K, 73K, and 64K protein) occurs almost simultaneously, reaching maxi-

mum levels in about 5 min, and gradually leading to the new steady-state levels (Fig. 4). Moreover, synthesis of mRNA's for the three proteins also seems to be well coordinated, the maximum rate of synthesis being attained a few minutes before that for the synthesis of proteins (3.5 min after temperature shift; Fig. 7C). However, the three protein chains do not seem to be coded by a single mRNA molecule. On the contrary, the simultaneous induction observed tends to preclude such a possibility, since sequential synthesis of these proteins from one mRNA molecule would result in significantly different time lags for different proteins. Also, the functional halflife of mRNA's for 64K protein appears to differ from that for the two other proteins. It seems most likely that the 76K, 73K, and 64K proteins are coded by separate mRNA molecules and that synthesis of these RNAs is induced coordinately by a certain common mechanism. In support of such a possibility, we have recently found an amber mutant of E. coli K-12 defective in

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of addition of rifampicin Time (min) (min) FIG. 7. Kinetics of synthesis of mRNA for temperature-induced proteins. (A) A log-phase culture of AS19 grown at 30°C was mixed with an equal volume of 55°C medium containing L-[3HJleucine (5.3 Ci/mmol; 40 ,Ci/ml), and the mixture was shaken at 42°C. Portions of ml of the culture were removed at appropriate intervals and added to a tube containing 50 pg of rifampin. After incubation for 15 min at 42°C, the labeling was stopped by adding trichloroacetic acid, and the whole-cell proteins were subjected to gel electrophoresis at 75 V for 16 h. Incorporation of radioactivity into cells proceeded linearly during the experiment. After addition of rifampin, the labeling of 76K and 64K proteins reached the plateau at 5 and 7.5 min, respectively. Protein samples each containing 50,000 cpm were subjected to gel electrophoresis. The film was exposed for 2 days. Rifampin was added at (1) 0 min, (2) 1 min, (3) 2 min, (4) 3 min, (5) 4 min, (6) 5 min, (7) 6 min, (8) 7 min, (9) 8 min, or (10) 10 min. (B) The amounts of [3H]leucine incorporated into 76K (0) and 64K (U) proteins in the presence of rifampin were plotted as a function of the time of rifampin addition after temperature shift-up. Each value has been calculated by multiplying the fraction of each protein by the radioactivity incorporated into total protein in (A). (C) The amount of mRNA synthesized for 76K (0) and 64K (U) proteins has been estimated from the curve obtained in (B) by taking a differential for each time interval indicated. Time

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lime of addition of rifampicin (sec) FIG. 8. Initial kinetics of synthesis of mRNA for 64K protein. Experimental conditions and procedures were the same as in Fig. 7, except that the cells were labeled with L-[3H]leucine (10.5 Ci/mmol; 20 yCi/ml). The lag has been estimated by extrapolating the initial portion of the curve, which was drawn by the method of least squares, to the basal level of the protein synthesized at 30°C.

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FIG. 9. Kinetics of decay of the capacity to synthesize temperature-induced proteins. Samples from the experiment of Fig. 6C were used to estimate the capacities for synthesizing 76K, 73K, and 64K proteins. The fraction of radioactivity incorporated into each of these proteins among total proteins synthesized in the presence of rifampin was estimated by densitometer tracing of the fluorograph similar to that shown in Fig. 6C. Each value thus obtained was multiplied by the radioactivity incorporated into total protein, and the resulting product was normalized to that for the sample labeled 30 s after rifampin addition. (0) 76K protein; (A) 73K protein; (-) 64K protein.

J. BACTERIOL.

induction of several of these proteins upon temperature shift-up (Yamamori and Yura, manuscript in preparation). It thus appears that E. coli cells somehow "recognize" shift-up of temperature to or beyond a certain critical point and activate transcription of several distinct operons. In contrast to the temperature-induced proteins studied here, synthesis of proteins such as RNA polymerase subunits and ribosomal proteins is temporarily reduced upon temperature shift-up (9). The regulatory mechanism involved in the latter case appears to be quite distinct from that involved in the former; transient decrease in the synthesis of RNA polymerase subunits (a, ,B, ,B', but not a) occurred in a reLA + but not relA strain, whereas synthesis of 76K, 73K, and 64K proteins was induced in a relA as well as a relA + strain (Yamamori, unpublished data). The temperature-induced 64K protein has previously been identified as a monomer of ATPase, which is often copurified with RNA polymerase (5, 19). The 61K protein previously thought to represent another subunit of the ATPase was found to be an in vitro artifact; it may be identical to the 64K protein, which migrates faster when dodecyl sulfate-gel electrophoresis is run in the absence of a sufficient amount of dithiothreitol. Interestingly, the 64K protein has recently been identified as a product of the mop (=groE) gene, which is involved in morphogenesis of several coliphages (2, 4); this gene also seems to be essential for growth of E. coli cells (16). When E. coli proteins pulse-labeled at 5 min after temperature shift-up are compared with those synthesized at 30 or 42°C in two-dimensional electropherograms (11), several proteins whose synthesis is greatly induced by temperature shift-up can be detected (Yamamori and Yura, manuscript in preparation). Specific protein spots have been identifie-d for 87K, 76K, and 64K proteins, whereas two adjacent spots were identified for 73K protein. Besides, a couple of proteins with apparent molecular weights of 18K and 16K were found to be induced upon temperature shift-up (12% gel used) (13). Thus altogether seven polypeptides have been shown to be induced, the 64K protein being most abundant, although it remains unclear whether they all represent distinct polypeptides coded by the separate genes. Finally, the results reported here in E. coli seem to resemble basically those observed for "heat shock polypeptides" of Drosophila, which have been studied extensively (1). In both systems, rapid induction at the transcriptional level of a specific set of proteins occurs. Taken together with the occurrence of analogous phenomena in a variety of other organisms, it apa

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E. COLI TEMPERATURE-INDUCED PROTEIN SYNTHESIS

pears possible that temperature-induced protein synthesis is related to a general adaptive response of both eucaryotic and procaryotic cells to sudden temperature change in natural environments. ACKNOWLEDGMENTS We are grateful to the members of our Institute for discussions and in particular to A. Ishihama and S. Hiraga for helpful suggestions. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan.

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