Vol. 121, No. 3 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Mar. 1975. p. 753-758 Copyright 0 1975 American Society for Microbiology
Stability of Escherichia coli Polysomes at High Hydrostatic Pressure DANIEL H. POPE,* NINA T. CONNORS, AND JOSEPH V. LANDAU Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181 Received for publication 12 December 1974
The stability of Escherichia coli polysomes at increased hydrostatic pressure was investigated in actively growing cells, in which the initiation of transcription was blocked by rifampin. In these cells, [3H juridine incorporation into messenger ribonucleic acid and the subsequent degradation of the message (and therefore of polysomes) by ribonuclease could be observed. Evidence is presented that the activity of the RNases is unaffected by a pressure of 680 atm, that protein synthesis is completely inhibited at 680 atm but immediately resumes at the 1 atm rate on release of pressure, and that no degradation of messenger ribonucleic acid in polysomes occurs at 680 atm. The effects of pressure; puromycin, and chloramphenicol on polysomal degradation are discussed. These results indicate that, contrary to some previous reports, polysomes are probably stabilized by high pressure. Therefore, we consider that polysomal instability is not a factor in the inhibition of protein synthesis by high pressures.
Several investigators (1, 11, 12, 19) have studied the rates of deoxyribonucleic acid, ribonucleic acid (RNA), and protein synthesis in Escherichia coli at increased hydrostatic pressures. There seems to be general agreement among these workers that, of these biosynthetic processes, protein synthesis is the most sensitive to high pressures and is probably the limiting phase of the growth processes of this organism at high pressure. Pope and Berger (20), in a brief review, have suggested that inhibition of protein synthesis by high pressure may, in fact, be the factor limiting the growth of most, if not all, prokaryotes at high pressures. Landau (13), using the data of hydrostatic pressure experiments on E. coli, has calculated that protein synthesis in this organism is accompanied by a volume change of activation (AV*) of about 100 cm3/mol. The AV* for RNA synthesis was found to be only about 55 cm3/ mol. Schwarz and Landau (21, 22), utilizing both whole cells and a cell-free protein-synthesizing system from E. coli, investigated the effect of increased hydrostatic pressures on several steps in the translation process. They found that a pressure of 670 atm at 37 C was sufficient to completely inhibit protein synthesis but has no effect on aminoacyl transfer RNA (AA-tRNA) formation, amino acid permeability, or polysomal integrity. The one isolated step found by these authors to be inhibited by pressure in a manner quantitively identical to the inhibition found both in whole cells and in a disrupted cell protein-synthesizing system was
the binding of AA-tRNA to the ribosome-messenger RNA (mRNA) complex. It would seem, therefore, that this step in the translational phase of protein synthesis is the rate-limiting reaction that has particular sensitivity to high pressures. Of special interest was the finding that polysomal integrity was unaffected at high pressure. This is consistent with reports (8-10, 16, 25) of experiments using extracts of E. coli, skeletal muscle, and sea urchins, which indicate that the pressures generated during ultracentrifugation can cause a dissociation of ribosome "dimers" into "monomeric" components (e.g., 70S into 50S and 30S) but do not seem to dissociate polysome complexes. These findings are in contrast to the report by Hermolin and Zimmerman (6), which demonstrated a marked reduction in the amount of polysomes recovered from pressurized cells of the eukaryotic protozoan, Tetrahymena pyriformis, as compared to the 1-atm samples. Other investigators (2, 7) have studied the effect of high pressure on various steps in the translation process, but have drawn different conclusions as to the importance of the pressure inhibition of these reactions to the overall inhibition of protein synthesis by pressure. Especially interesting is the thesis of Arnold and Albright (2) that dissociation of the polysomes occurs at high pressure and, further, that on release of pressure the polysomes immediately reassociate to give the form present prior to the application of pressure. As these authors point out, if this thesis were
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correct then studies in which a measure of polysomal stability, under pressure, is obtained only after the release of pressure might fail to demonstrate dissociation. Some support for this hypothesis was reported by Hildebrand and Pollard (7). Because of these differences of opinion regarding the question of polysomal stability at high pressure, their association on pressure release, and the obvious consequences in the determination of the mechanism and site of pressure effects, we considered it necessary to design an experiment in which polysomal stability could be measured in actively growing cells while still under pressure. For such experiments we have used the techniques recently reported by Pato et al. (17, 18). These techniques utilize growing E. coli cells and allow one to follow both the synthesis of mRNA (and thus the formation of polysomes) and the subsequent degradation by ribonuclease (RNase). These workers reported that the addition of chloramphenicol at the proper time largely prevented the degradation of the mRNA, presumably by inhibiting ribosome movement and thus stabilizing polysomal configuration (see 14, 15). On the other hand, addition of puromycin increased the rate of message degradation. It is believed that this antibiotic causes a dissociation of the ribosomes from the mRNA, thereby exposing more of the message to the action of the RNase. Our approach was, then, to apply a pressure that would totally inhibit protein synthesis, in lieu of the aforementioned antibiotics, and measure the rate of message degradation while under pressure. A result paralleling the effect of chloramphenicol could be interpreted as evidence that dissociation does not occur at high pressure. It was, however, necessary to first establish that the activity of the RNase was not decreased at high pressure. (This work is taken in part from a thesis to be submitted [N.T.C.] in partial fulfillment of the requirements tor an M.S. degree in the Department of Biology, Rensselaer Polytechnic Institute.) MATERIALS AND METHODS Bacterial strains. Strain AS-19, a derivative of E. coli B (23), highly permeable to many antibiotics including rifampin, was kindly provided by M. L. Pato, National Jewish Hospital and Research Center, Denver, Colo. Growth conditions. Stock cultures were maintained on nutrient agar slants and were checked for purity by streaking on nutrient agar plates prior to experiments. In preparation for the experiment, the cells were grown on a phosphate minimal medium (4)
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which was supplemented with 0.2% glucose as suggested by Pato et al. (17). One-liter cotton-stoppered flasks containing 400 ml of the liquid medium were inoculated from an overnight culture of the organism grown in the same medium. These were placed on a rotary shaker at 125 rpm and 37 C. Growth was followed spectrophotometrically at 560 nm with a Spectronic 20 spectrophotometer. The cultures were grown exponentially for two generations prior to use and were never used when the absorbancy at 560 nm was greater than approximately 0.25 (mid-log phase). Radioactive labeling of RNA and protein. Rifampin (20 gg/ml), nalidixic acid (20 g/ml), and [3H]uridine (2 gg/ml), as final concentrations in the reaction mixture, were added to 10 ml of exponentially growing cells contained in a vial in a 37 C water bath. Samples to be pressurized were removed from this mixture not more than 45 s prior to the application of pressure, and injected into the pressure cylinder also maintained at 37 C. The total time required to pressurize the sample to 680 atm was approximately 5 to 10 s. Upon release of pressure, the sample was removed, by means of the attached syringe, to a vial in the 37 C water bath. Samples (0.2 ml) were then removed at the times indicated for each experiment. Total time elapsed from pressure release until the first 0.2-ml sample was processed was approximately 15 s. Incorporation of [3H ]uridine into RNA was determined in the following manner. Samples (0.2 ml) were removed from the reaction mixture by means of a Biopette (Schwarz/Mann, Orangeburg, N.Y.) at the times indicated and lysed with sodium dodecyl sulfate (3). These were then precipitated with 3 M trichloroacetic acid, filtered onto membrane filters (0.45 Am, Millipore Corp.), and rinsed with two 10-ml samples of 0.1 M trichloroacetic acid. Samples for determining incorporation of "4C-labeled amino acids into protein (0.2 ml) were added to 2 ml of ice-cold 5% trichloroacetic acid, heated to 80 C for 30 min, and returned to the ice bath for 30 min. The samples were then filtered onto membrane filters (0.45 um, as above) and rinsed twice with 10-ml samples of ice-cold 5% trichloroacetic acid. All filters were dried at 80 C in scintillation vials. Ten milliliters of the scintillation fluid (Scintiverse, Fisher Scientific Co.) was added, and the radioactivity was determined in an Intertechnique SL-30 liquid scintillation counter at approximately 60% efficiency. Pressure cylinder and pressure apparatus. The pressure cylinder, permitting rapid sampling, was kindly loaned to us by L. R. Berger, University of Hawaii. This apparatus (24) permits rapid application of pressures to the sample and rapid sampling of the sample once pressure is released. Pressure was applied by means of an Enerpac model 11-400 hydraulic pump using water as the hydraulic fluid. Chemicals. [5- 3H luridine (28 gCi/mmol) and uniformly '4C-labeled amino acids were purchased from New England Nuclear, Boston, Mass. Rifampin and nalidixic acid were obtained from Calbiochem, La Jolla, Calif. Puromycin was obtained from Nutritional Biochemicals Corp., Cleveland, Ohio, and chloramphenicol was from Sigma Chemical Co., Saint Louis, Mo.
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RESULTS Inhibition of protein synthesis by pressure and its recovery after pressure release. It was first necessary to determine whether the effect of pressure on protein synthesis in E. coli AS-19 was similar to that observed for other strains of this organism, e.g., E. coli K-12 (11-13). For this measurement, "4C-labeled amino acids were added to a sample of actively growing E. coli AS-19. A portion of these cells was pressurized at 680 atm for 2 min. The pressure was released and incorporation of the labeled amino acids into protein was followed for another minute. The results (Fig. 1) demonstrate that no protein synthesis occurred while the sample was at 680 atm. The synthesis of proteins resumed immediately upon release of the pressure, at the same rate as that observed in the 1-atm control sample. These results are identi7x
5
-i/x I, u
x
v, 0
3 Minutes
5
FIG. 1. Effect of 680 atm on protein synthesis by E. coli AS-19 at 37 C. Uniformly "4C-labeled amino acids were added to an exponentially growing culture of E. coli AS-19. At 2 min, a 1-ml portion of the culture was pressurized to 680 atm. At 4 min, the sample was returned to 1 atm. At the times indicated, 0.2-mi samples were transferred to 2 ml of 5% trichloroacetic acid for determination of incorporation of '4C-labeled amino acids into protein. ( ) Periods during which atmospheric control and pressure samples were at 1 atm; (--) period during which samples were pressurized.
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cal, as to the extent of pressure inhibition and recovery, to those reported previously (11-13). Effect of rifampin and nalidixic acid on RNA and protein synthesis. In our experiments, the patterns of RNA synthesis and degradation and of protein synthesis by E. coli AS-19, in which initiation of transcription was blocked with rifampin, were identical to those of Pato et al. (17). Therefore, these data are not shown here. We agree with their interpretation of the results of such experiments (see Introduction). Effect of puromycin and chloramphenicol on the rate of mRNA degradation. The results of our experiments done to determine the effects of puromycin and chloramphenicol on the rate of mRNA degradation also confirm those of Pato et al. (17) and are not, therefore, presented here. Again, we believe that the interpretation of the data given by those authors to be correct (see Introduction). Stability of RNase activity at high pressures. Preliminary experiments with cell-free preparations (data not shown) had indicated that the ability of the E. coli K-12 RNase to degrade either polyuridylate or endogenous mRNA from E. coli K-12 was unimpaired at 680 atm. Experiments were done to confirm that the RNase activity was not decreased in actively growing E. coli AS-19 cells at 680 atm (Fig. 2). Puromycin was added to the reaction mixture at 1 min 30 s after the addition of rifampin and nalidixic acid. Thirty seconds later part of the sample was pressurized to 680 atm. The added puromycin should have resulted in the cells containing mRNA with portions devoid of ribosomes, allowing the RNase to act on large portions of mRNA. The results clearly show that the rate of mRNA degradation at 1 and 680 atm are almost identical. Therefore, we could assume that the activity of the RNase was not inhibited at 680 atm in whole cells of E. coli AS-19. Stability of polysomes at increased hydrostatic pressure. The results described thus far gave us a whole-cell system in which we could allow the cells to produce a limited amount of mRNA, and hence polysomes, after which it was possible to follow degradation of the polysomes (mRNA) at 680 atm, a pressure completely inhibiting protein synthesis (Fig. 1). Samples were treated with rifampin and nalidixic acid as for the previous experiment. Pressure (680 atm) was applied to a portion of the sample at 2 min (approximately the time of maximal uridine incorporation into RNA) and maintained at that pressure for periods of 2.5 or 5 min. The pressure was then released, and the
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these experiments that little or no polysomal dissociation or breakdown occurs in actively growing cells of E. coli at pressures to at least 680 atm, at least over relatively short time
periods. DISCUSSION The results presented here demonstrate that mRNA degradation by RNase in whole cells of E. coli AS-19 is unaffected by a pressure of 680 atm (Fig. 2). Therefore, if the polysomal dissociation hypothesis of Arnold and Albright (2) were correct, loss of mRNA (mRNA degradation), while under pressure, should have been observed at a rate approximating that seen when puromycin was added to a pressurized sample (Fig. 2). Instead, almost no degradation of mRNA was observed while the sample was
1%.X E
0
4 10 2
6
~
~
~
-
0
Minutes
FIG. 2. Effect of 680 atm on the rate of mRNA degradation by RNase at 37 C. Rifampin (20 ug/ml), nalidixic acid (20 zg/ml), and [3H]uridine (2 Ag/ml) were added to an exponentially growing culture of E. coli AS-19 at time zero. Puromycin (200 qg/ml) was added to the culture after 1.5 min. Pressure (680 atm) was applied to a portion of the culture (A) at 2 min and released at 4.5 min. A control sample was left at 1 atm (0). At the intervals indicated, 0.2-ml samples from control and pressurized samples were lysed with sodium dodecyl sulfate and then precipitated with 3 M trichloroacetic acid to determine the amount of [3HJuridine-labeled RNA present. (1t) Sample pressurized; (4,) sample to 1 atm.
10
$
III
2
sample was removed to the water bath and followed for another 5 min at 1 atm. Four determinations were made for each time period on 2 separate days with the same results. The results (Fig. 3) are representative of all the data obtained. It can be seen clearly (Fig. 3) that little or no degradation of mRNA occurred while the samples were at 680 atm and that, on release of pressure, mRNA degradation immediately resumed at the 1-atm rate. In some of the samples held at 680 atm for 5 min a small decrease in the amount of mRNA, approximately 15 to 20%, did occur. This might be attributed to the very slow movement of the ribosomes on the message during the period of pressurization or to some other unexplained phenomenon. A similar gradual loss in the amount of mRNA is seen in cultures with added chloramphenicol (17). We have concluded from
2
6
10
Minutes
FIG. 3. Effect of 680 atm on the rate of polysomal mRNA degradation at 37 C. At time zero, rifampin (20 jg/ml) and [3H]uridine (2 ag/ml) were added to exponentially growing culture of E. coli AS-19. Two experiments are shown, one in which a pressure of 680 atm was applied to a portion of the culture at 2 min and released at 4.5 min (x) and one in which pressure was applied at 2 min and released at 7 min (0). In both cases, the samples were followed for 5 min after the release of pressure. ( ) Samples at 1 atm; (- -) samples at 680 atm; (t) sample to 680 atm; (J) sample to I atm. At the intervals indicated, 0.2-ml samples were lysed with sodium dodecyl sulfate and then precipitated with 3 M trichloroacetic acid to determine the amount of [3H]uridine-labeled RNA present.
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under pressure (Fig. 3). In fact, the amount of mRNA lost while the sample was pressurized* was less than that observed when the polysomes were "stabilized" by the addition of chloramphenicol (17, 18), thus indicating that a pressure of 680 atm may be even more effective than chloramphenicol in preventing polysomal dissociation, possibly by preventing ribosome movement on the mRNA. We do not, however, wish to imply that the mechanisms whereby pressure and chloramphenicol act are identical. Rather, we believe that if Pato et al. (17, 18) are correct in their conclusions regarding the effects of chloramphenicol and puromycin on polysomal stability then our data are best interpreted as showing that the overall effect of pressure on polysomal stability is similar to that of chloramphenicol, i.e., the polysome is stabilized. There is also the possibility that polysomes are disaggregated by pressure in a manner unlike that caused by puromycin. We believe our data to be inconsistent with such a conclusion for the following reasons. (i) The observation that degradation of the message resumes, at the 1-atm rate, immediately upon the release of pressure (Fig. 3) is further indication that the polysomes may be stabilized or "frozen" while under pressure and that, over short periods of time under pressure, little or no change takes place in basic polysome configuration. (ii) The fact that protein synthesis immediately resumes at the 1-atm rate (Fig. 1) would seem to indicate that no degradation of the mRNA or disassociation of the polysome could have occurred while the sample was pressurized, since, if either has occurred under pressure, resumption of protein synthesis should have been at a lower rate than that originally observed at 1 atm, or there should have been a brief lag before the rate resumed at the original 1-atm rate. Our results, therefore, would seem to confirm those of Schwarz and Landau (21, 22) and be contradictory to those of Arnold and Albright (1) and those of Hildebrand and Pollard (7). Those authors (1, 7), however, used only specific portions of protein-synthesizing systems, a synthetic mRNA (polyuridylate), and incubation times of up to 80 min under pressure. These procedures raise some questions as to the validity of an extrapolation of their results to the events occurring in whole cells under pressure. The use of lengthy incubation periods under pressure has always been suspect, considering the interdependence of the multitude of reactions involved in the biosynthesis of proteins. Even if only one reaction in the protein-synthesizing machinery were affected by pressure per se and all the other reactions were initially unaffected, one should, over ex-
757
tended periods of time, observe changes in the rates of almost all the reactions involved as a consequence of the initial effect. Several investigators (1, 5, 7) have suggested that the effect of pressure on protein synthesis and presumably, therefore, on the growth of E. coli is attributable to no one reaction initially, but to the combined effects of pressure on several of the steps in the protein-synthesizing process, e.g., activity of the AA-tRNA synthetases, stability of the AA-tRNA complex, polysomal stability, and binding of the AA-tRNA to the ribosome-mRNA complex. We find it difficult to attribute a significant role in the inhibition of protein synthesis by high pressure to decreased activity of the AA-tRNA synthetases under these conditions, since the data of Hardon and Albright (5) indicate that the synthetase activity is only about 60 to 70% inhibited at 500 atm, a pressure at which there is almost complete inhibition of protein synthesis (5). In addition, Schwarz and Landau (21) have reported the absence of any such effect in whole-cell preparations of E. coli. It is also difficult to see how AA-tRNA instability could contribute significantly to a complete and immediate inhibition of protein synthesis, when the level of this molecule after 5 min at 600 atm (5) has been shown to decrease by only 3 to 7% from the amount originally present. In fact, the level of AA-tRNA reportedly drops by only about 30 to 45% after as long as 45 min at 600 atm. We do not consider that these effects of pressure can account for the immediate cessation of protein synthesis at high pressures, and the immediate resumption of synthesis at the 1-atm rate on the release of pressure would also seem to argue against their importance as anything but secondary events, of negligible importance in all but long-term experiments (Fig. 1; references 6, 16). The results presented here support the view that polysomal dissociation does not occur at high pressure and, therefore, cannot be responsible for the failure of protein synthesis to occur at high pressure. All investigators agree that the binding of AA-tRNA to the ribosome-mRNA complex is susceptible to inhibition by pressure, and Schwarz and Landau (21) have shown that this is quantitatively identical to that shown for protein synthesis in whole cells and cell-free extracts. It is our contention, therefore, that the rate-limiting step in protein synthesis that is particularly susceptible to pressure is the binding of AA-tRNA to the ribosome-mRNA complex. Furthermore, a forthcoming report (now in preparation) will indicate that the magnitude of the pressure effect is a characteristic inherent in the ribosome.
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This paper was supported by National Science Foundation grant no. GB38137.
LITERATURE CITED 1. Albright, L. J. 1969. Alternate pressurization-depressuri-
zation effects on growth and net protein RNA and DNA synthesis in Escherichia coli and Vibrio marinus. Can. J. Microbiol. 15:1237-1240. 2. Arnold, R. M., and L. J. Albright. 1971. Hydrostatic pressure effects on the translation stages of protein synthesis in a cell-free system from Escherichia coli. Biochim. Biophys. Acta 238:347-354. 3. Bremer, H., and D. Yuan. 1968. Uridine transport and incorporation into nucleic acids in Escherichia coli. Biochim. Biophys. Acta 169:21-34. 4. Clark, D. J., and 0. Maaloe. 1967. DNA replication and the division cycle in Escherichia coli. J. Mol. Biol. 23:99-112. 5. Hardon, M. J., and L. J. Albright. 1974. Hydrostatic pressure effects on several stages of protein synthesis in Escherichia coli. Can. J. Microbiol. 20:359-365. 6. Hermolin, J., and A. M. Zimmerman. 1969. The effect of pressure on synchronous cultures of Tetrahymena: a ribosomal study. Cytobios 1:247-256. 7. Hildebrand, C. E., and E. C. Pollard. 1972. Hydrostatic pressure effects on protein synthesis. Biophys. J. 12:1235-1250. 8. Infante, A. A., and R. Baierlein. 1971. Pressure-induced dissociation of sedimenting ribosomes: effect on sedimentation patterns. Proc. Nat. Acad. Sci. U.S.A. 68: 1780-1785. 9. Infante, A. A., and P. N. Graves. 1971. Stability of free ribosomes, derived ribosomes, and polysomes of the sea urchin. Biochim. Biophys. Acta 246:100-110. 10. Infante, A. A., and M. Krauss. 1971. Dissociation of ribosomes induced by centrifugation: evidence for doubting conformational changes in free ribosomes. Biochim. Biophys. Acta 246:81-99. 11. Landau, J. V. 1966. Protein and nucleic acid synthesis in Escherichia coli: pressure and temperature effects. Science 153:1273-1274. 12. Landau, J. V. 1967. Induction, transcription and translation in Escherichia coli: a hydrostatic pressure study.
Biochim. Biophys. Acta 149:506-512.
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Landau, J. V. 1970. Hydrostatic pressure on the biosynthesis of macromolecules, p. 45-70. In A. M. Zimmerman (ed.), High pressure effects on cellular processes. Academic Press Inc., New York. 14. Levinthal, C., D. F. Fan, A. Higa, and R. A. Zimmerman. 1963. The decay and protection of messenger RNA in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:183-190. 15. Mangiarotti, G., and D. Schlessinger. 1966. Polysome 13.
16.
17.
18.
19.
20. 21. 22.
23. 24.
25.
metabolism in Escherichia coli. J. Mol. Biol. 20:123-143. Martin, T. E., F. S. Rolleston, R. B. Low, and I. G. Wool. 1969. Dissociation and reassociation of skeletal muscle ribosomes. J. Mol. Biol. 43:135-149. Pato, M. L., P. M. Bennett, and K. Von Meyenburg. 1973. Messenger ribonucleic acid synthesis and degradation in Escherichia coli during inhibition of translation. J. Bacteriol. 116:710-718. Pato, M. L., and K. Von Meyenburg. 1970. Residual RNA synthesis in Escherichia coli after inhibition of initiation of transcription by rifampin. Cold Spring Harbor Symp. Quant. Biol. 35:497-504. Pollard, E. C., and P. K. Weller. 1966. The effect of hydrostatic pressure on the synthetic processes in bacteria. Biochim. Biophys. Acta 112:573-580. Pope, D. H., and L. R. Berger. 1973. Inhibition of metabolism by hydrostatic pressure: what limits microbial growth? Arch. Mikrobiol. 93:367-370. Schwarz, J. R., and J. V. Landau. 1972. Hydrostatic pressure effects on Escherichia coli: site of inhibition of protein synthesis. J. Bacteriol. 109:945-948. Schwarz, J. R., and J. V. Landau. 1972. Inhibition of cell-free protein synthesis by hydrostatic pressure. J. Bacteriol. 112:1222-1227. Sekiguchi, M., and S. Iida. 1967. Mutants of Escherichia coli permeable to actinomycin. Proc. Nat. Acad. Sci. U.S.A. 58:2315-2320. Shen, J., and L. R. Berger. 1974. Measurement of active transport by bacteria at increased hydrostatic pressure, p. 173-179. In R. Colwell and R. Morita (ed.), Effect of ocean environment on microbial activities. Proceedings of the Second U.S.-Japan Conference on Marine Microbiology. University of Maryland Press, College Park. Subamanian, A. R., and B. D. Davis. 1971. Rapid exchange of subunits between free ribosomes in extracts of Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 68:2453-2457.
JOURNAL OF BACTERIOLOGY, Mar. 1975. p. 753-758 Copyright 0 1975 American Society for Microbiology
Stability of Escherichia coli Polysomes at High Hydrostatic Pressure DANIEL H. POPE,* NINA T. CONNORS, AND JOSEPH V. LANDAU Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181 Received for publication 12 December 1974
The stability of Escherichia coli polysomes at increased hydrostatic pressure was investigated in actively growing cells, in which the initiation of transcription was blocked by rifampin. In these cells, [3H juridine incorporation into messenger ribonucleic acid and the subsequent degradation of the message (and therefore of polysomes) by ribonuclease could be observed. Evidence is presented that the activity of the RNases is unaffected by a pressure of 680 atm, that protein synthesis is completely inhibited at 680 atm but immediately resumes at the 1 atm rate on release of pressure, and that no degradation of messenger ribonucleic acid in polysomes occurs at 680 atm. The effects of pressure; puromycin, and chloramphenicol on polysomal degradation are discussed. These results indicate that, contrary to some previous reports, polysomes are probably stabilized by high pressure. Therefore, we consider that polysomal instability is not a factor in the inhibition of protein synthesis by high pressures.
Several investigators (1, 11, 12, 19) have studied the rates of deoxyribonucleic acid, ribonucleic acid (RNA), and protein synthesis in Escherichia coli at increased hydrostatic pressures. There seems to be general agreement among these workers that, of these biosynthetic processes, protein synthesis is the most sensitive to high pressures and is probably the limiting phase of the growth processes of this organism at high pressure. Pope and Berger (20), in a brief review, have suggested that inhibition of protein synthesis by high pressure may, in fact, be the factor limiting the growth of most, if not all, prokaryotes at high pressures. Landau (13), using the data of hydrostatic pressure experiments on E. coli, has calculated that protein synthesis in this organism is accompanied by a volume change of activation (AV*) of about 100 cm3/mol. The AV* for RNA synthesis was found to be only about 55 cm3/ mol. Schwarz and Landau (21, 22), utilizing both whole cells and a cell-free protein-synthesizing system from E. coli, investigated the effect of increased hydrostatic pressures on several steps in the translation process. They found that a pressure of 670 atm at 37 C was sufficient to completely inhibit protein synthesis but has no effect on aminoacyl transfer RNA (AA-tRNA) formation, amino acid permeability, or polysomal integrity. The one isolated step found by these authors to be inhibited by pressure in a manner quantitively identical to the inhibition found both in whole cells and in a disrupted cell protein-synthesizing system was
the binding of AA-tRNA to the ribosome-messenger RNA (mRNA) complex. It would seem, therefore, that this step in the translational phase of protein synthesis is the rate-limiting reaction that has particular sensitivity to high pressures. Of special interest was the finding that polysomal integrity was unaffected at high pressure. This is consistent with reports (8-10, 16, 25) of experiments using extracts of E. coli, skeletal muscle, and sea urchins, which indicate that the pressures generated during ultracentrifugation can cause a dissociation of ribosome "dimers" into "monomeric" components (e.g., 70S into 50S and 30S) but do not seem to dissociate polysome complexes. These findings are in contrast to the report by Hermolin and Zimmerman (6), which demonstrated a marked reduction in the amount of polysomes recovered from pressurized cells of the eukaryotic protozoan, Tetrahymena pyriformis, as compared to the 1-atm samples. Other investigators (2, 7) have studied the effect of high pressure on various steps in the translation process, but have drawn different conclusions as to the importance of the pressure inhibition of these reactions to the overall inhibition of protein synthesis by pressure. Especially interesting is the thesis of Arnold and Albright (2) that dissociation of the polysomes occurs at high pressure and, further, that on release of pressure the polysomes immediately reassociate to give the form present prior to the application of pressure. As these authors point out, if this thesis were
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correct then studies in which a measure of polysomal stability, under pressure, is obtained only after the release of pressure might fail to demonstrate dissociation. Some support for this hypothesis was reported by Hildebrand and Pollard (7). Because of these differences of opinion regarding the question of polysomal stability at high pressure, their association on pressure release, and the obvious consequences in the determination of the mechanism and site of pressure effects, we considered it necessary to design an experiment in which polysomal stability could be measured in actively growing cells while still under pressure. For such experiments we have used the techniques recently reported by Pato et al. (17, 18). These techniques utilize growing E. coli cells and allow one to follow both the synthesis of mRNA (and thus the formation of polysomes) and the subsequent degradation by ribonuclease (RNase). These workers reported that the addition of chloramphenicol at the proper time largely prevented the degradation of the mRNA, presumably by inhibiting ribosome movement and thus stabilizing polysomal configuration (see 14, 15). On the other hand, addition of puromycin increased the rate of message degradation. It is believed that this antibiotic causes a dissociation of the ribosomes from the mRNA, thereby exposing more of the message to the action of the RNase. Our approach was, then, to apply a pressure that would totally inhibit protein synthesis, in lieu of the aforementioned antibiotics, and measure the rate of message degradation while under pressure. A result paralleling the effect of chloramphenicol could be interpreted as evidence that dissociation does not occur at high pressure. It was, however, necessary to first establish that the activity of the RNase was not decreased at high pressure. (This work is taken in part from a thesis to be submitted [N.T.C.] in partial fulfillment of the requirements tor an M.S. degree in the Department of Biology, Rensselaer Polytechnic Institute.) MATERIALS AND METHODS Bacterial strains. Strain AS-19, a derivative of E. coli B (23), highly permeable to many antibiotics including rifampin, was kindly provided by M. L. Pato, National Jewish Hospital and Research Center, Denver, Colo. Growth conditions. Stock cultures were maintained on nutrient agar slants and were checked for purity by streaking on nutrient agar plates prior to experiments. In preparation for the experiment, the cells were grown on a phosphate minimal medium (4)
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which was supplemented with 0.2% glucose as suggested by Pato et al. (17). One-liter cotton-stoppered flasks containing 400 ml of the liquid medium were inoculated from an overnight culture of the organism grown in the same medium. These were placed on a rotary shaker at 125 rpm and 37 C. Growth was followed spectrophotometrically at 560 nm with a Spectronic 20 spectrophotometer. The cultures were grown exponentially for two generations prior to use and were never used when the absorbancy at 560 nm was greater than approximately 0.25 (mid-log phase). Radioactive labeling of RNA and protein. Rifampin (20 gg/ml), nalidixic acid (20 g/ml), and [3H]uridine (2 gg/ml), as final concentrations in the reaction mixture, were added to 10 ml of exponentially growing cells contained in a vial in a 37 C water bath. Samples to be pressurized were removed from this mixture not more than 45 s prior to the application of pressure, and injected into the pressure cylinder also maintained at 37 C. The total time required to pressurize the sample to 680 atm was approximately 5 to 10 s. Upon release of pressure, the sample was removed, by means of the attached syringe, to a vial in the 37 C water bath. Samples (0.2 ml) were then removed at the times indicated for each experiment. Total time elapsed from pressure release until the first 0.2-ml sample was processed was approximately 15 s. Incorporation of [3H ]uridine into RNA was determined in the following manner. Samples (0.2 ml) were removed from the reaction mixture by means of a Biopette (Schwarz/Mann, Orangeburg, N.Y.) at the times indicated and lysed with sodium dodecyl sulfate (3). These were then precipitated with 3 M trichloroacetic acid, filtered onto membrane filters (0.45 Am, Millipore Corp.), and rinsed with two 10-ml samples of 0.1 M trichloroacetic acid. Samples for determining incorporation of "4C-labeled amino acids into protein (0.2 ml) were added to 2 ml of ice-cold 5% trichloroacetic acid, heated to 80 C for 30 min, and returned to the ice bath for 30 min. The samples were then filtered onto membrane filters (0.45 um, as above) and rinsed twice with 10-ml samples of ice-cold 5% trichloroacetic acid. All filters were dried at 80 C in scintillation vials. Ten milliliters of the scintillation fluid (Scintiverse, Fisher Scientific Co.) was added, and the radioactivity was determined in an Intertechnique SL-30 liquid scintillation counter at approximately 60% efficiency. Pressure cylinder and pressure apparatus. The pressure cylinder, permitting rapid sampling, was kindly loaned to us by L. R. Berger, University of Hawaii. This apparatus (24) permits rapid application of pressures to the sample and rapid sampling of the sample once pressure is released. Pressure was applied by means of an Enerpac model 11-400 hydraulic pump using water as the hydraulic fluid. Chemicals. [5- 3H luridine (28 gCi/mmol) and uniformly '4C-labeled amino acids were purchased from New England Nuclear, Boston, Mass. Rifampin and nalidixic acid were obtained from Calbiochem, La Jolla, Calif. Puromycin was obtained from Nutritional Biochemicals Corp., Cleveland, Ohio, and chloramphenicol was from Sigma Chemical Co., Saint Louis, Mo.
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RESULTS Inhibition of protein synthesis by pressure and its recovery after pressure release. It was first necessary to determine whether the effect of pressure on protein synthesis in E. coli AS-19 was similar to that observed for other strains of this organism, e.g., E. coli K-12 (11-13). For this measurement, "4C-labeled amino acids were added to a sample of actively growing E. coli AS-19. A portion of these cells was pressurized at 680 atm for 2 min. The pressure was released and incorporation of the labeled amino acids into protein was followed for another minute. The results (Fig. 1) demonstrate that no protein synthesis occurred while the sample was at 680 atm. The synthesis of proteins resumed immediately upon release of the pressure, at the same rate as that observed in the 1-atm control sample. These results are identi7x
5
-i/x I, u
x
v, 0
3 Minutes
5
FIG. 1. Effect of 680 atm on protein synthesis by E. coli AS-19 at 37 C. Uniformly "4C-labeled amino acids were added to an exponentially growing culture of E. coli AS-19. At 2 min, a 1-ml portion of the culture was pressurized to 680 atm. At 4 min, the sample was returned to 1 atm. At the times indicated, 0.2-mi samples were transferred to 2 ml of 5% trichloroacetic acid for determination of incorporation of '4C-labeled amino acids into protein. ( ) Periods during which atmospheric control and pressure samples were at 1 atm; (--) period during which samples were pressurized.
755
cal, as to the extent of pressure inhibition and recovery, to those reported previously (11-13). Effect of rifampin and nalidixic acid on RNA and protein synthesis. In our experiments, the patterns of RNA synthesis and degradation and of protein synthesis by E. coli AS-19, in which initiation of transcription was blocked with rifampin, were identical to those of Pato et al. (17). Therefore, these data are not shown here. We agree with their interpretation of the results of such experiments (see Introduction). Effect of puromycin and chloramphenicol on the rate of mRNA degradation. The results of our experiments done to determine the effects of puromycin and chloramphenicol on the rate of mRNA degradation also confirm those of Pato et al. (17) and are not, therefore, presented here. Again, we believe that the interpretation of the data given by those authors to be correct (see Introduction). Stability of RNase activity at high pressures. Preliminary experiments with cell-free preparations (data not shown) had indicated that the ability of the E. coli K-12 RNase to degrade either polyuridylate or endogenous mRNA from E. coli K-12 was unimpaired at 680 atm. Experiments were done to confirm that the RNase activity was not decreased in actively growing E. coli AS-19 cells at 680 atm (Fig. 2). Puromycin was added to the reaction mixture at 1 min 30 s after the addition of rifampin and nalidixic acid. Thirty seconds later part of the sample was pressurized to 680 atm. The added puromycin should have resulted in the cells containing mRNA with portions devoid of ribosomes, allowing the RNase to act on large portions of mRNA. The results clearly show that the rate of mRNA degradation at 1 and 680 atm are almost identical. Therefore, we could assume that the activity of the RNase was not inhibited at 680 atm in whole cells of E. coli AS-19. Stability of polysomes at increased hydrostatic pressure. The results described thus far gave us a whole-cell system in which we could allow the cells to produce a limited amount of mRNA, and hence polysomes, after which it was possible to follow degradation of the polysomes (mRNA) at 680 atm, a pressure completely inhibiting protein synthesis (Fig. 1). Samples were treated with rifampin and nalidixic acid as for the previous experiment. Pressure (680 atm) was applied to a portion of the sample at 2 min (approximately the time of maximal uridine incorporation into RNA) and maintained at that pressure for periods of 2.5 or 5 min. The pressure was then released, and the
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POPE, CONNORS, AND LANDAU
J. BACTERIOL.
these experiments that little or no polysomal dissociation or breakdown occurs in actively growing cells of E. coli at pressures to at least 680 atm, at least over relatively short time
periods. DISCUSSION The results presented here demonstrate that mRNA degradation by RNase in whole cells of E. coli AS-19 is unaffected by a pressure of 680 atm (Fig. 2). Therefore, if the polysomal dissociation hypothesis of Arnold and Albright (2) were correct, loss of mRNA (mRNA degradation), while under pressure, should have been observed at a rate approximating that seen when puromycin was added to a pressurized sample (Fig. 2). Instead, almost no degradation of mRNA was observed while the sample was
1%.X E
0
4 10 2
6
~
~
~
-
0
Minutes
FIG. 2. Effect of 680 atm on the rate of mRNA degradation by RNase at 37 C. Rifampin (20 ug/ml), nalidixic acid (20 zg/ml), and [3H]uridine (2 Ag/ml) were added to an exponentially growing culture of E. coli AS-19 at time zero. Puromycin (200 qg/ml) was added to the culture after 1.5 min. Pressure (680 atm) was applied to a portion of the culture (A) at 2 min and released at 4.5 min. A control sample was left at 1 atm (0). At the intervals indicated, 0.2-ml samples from control and pressurized samples were lysed with sodium dodecyl sulfate and then precipitated with 3 M trichloroacetic acid to determine the amount of [3HJuridine-labeled RNA present. (1t) Sample pressurized; (4,) sample to 1 atm.
10
$
III
2
sample was removed to the water bath and followed for another 5 min at 1 atm. Four determinations were made for each time period on 2 separate days with the same results. The results (Fig. 3) are representative of all the data obtained. It can be seen clearly (Fig. 3) that little or no degradation of mRNA occurred while the samples were at 680 atm and that, on release of pressure, mRNA degradation immediately resumed at the 1-atm rate. In some of the samples held at 680 atm for 5 min a small decrease in the amount of mRNA, approximately 15 to 20%, did occur. This might be attributed to the very slow movement of the ribosomes on the message during the period of pressurization or to some other unexplained phenomenon. A similar gradual loss in the amount of mRNA is seen in cultures with added chloramphenicol (17). We have concluded from
2
6
10
Minutes
FIG. 3. Effect of 680 atm on the rate of polysomal mRNA degradation at 37 C. At time zero, rifampin (20 jg/ml) and [3H]uridine (2 ag/ml) were added to exponentially growing culture of E. coli AS-19. Two experiments are shown, one in which a pressure of 680 atm was applied to a portion of the culture at 2 min and released at 4.5 min (x) and one in which pressure was applied at 2 min and released at 7 min (0). In both cases, the samples were followed for 5 min after the release of pressure. ( ) Samples at 1 atm; (- -) samples at 680 atm; (t) sample to 680 atm; (J) sample to I atm. At the intervals indicated, 0.2-ml samples were lysed with sodium dodecyl sulfate and then precipitated with 3 M trichloroacetic acid to determine the amount of [3H]uridine-labeled RNA present.
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POLYSOMES AT HIGH HYDROSTATIC PRESSURE
under pressure (Fig. 3). In fact, the amount of mRNA lost while the sample was pressurized* was less than that observed when the polysomes were "stabilized" by the addition of chloramphenicol (17, 18), thus indicating that a pressure of 680 atm may be even more effective than chloramphenicol in preventing polysomal dissociation, possibly by preventing ribosome movement on the mRNA. We do not, however, wish to imply that the mechanisms whereby pressure and chloramphenicol act are identical. Rather, we believe that if Pato et al. (17, 18) are correct in their conclusions regarding the effects of chloramphenicol and puromycin on polysomal stability then our data are best interpreted as showing that the overall effect of pressure on polysomal stability is similar to that of chloramphenicol, i.e., the polysome is stabilized. There is also the possibility that polysomes are disaggregated by pressure in a manner unlike that caused by puromycin. We believe our data to be inconsistent with such a conclusion for the following reasons. (i) The observation that degradation of the message resumes, at the 1-atm rate, immediately upon the release of pressure (Fig. 3) is further indication that the polysomes may be stabilized or "frozen" while under pressure and that, over short periods of time under pressure, little or no change takes place in basic polysome configuration. (ii) The fact that protein synthesis immediately resumes at the 1-atm rate (Fig. 1) would seem to indicate that no degradation of the mRNA or disassociation of the polysome could have occurred while the sample was pressurized, since, if either has occurred under pressure, resumption of protein synthesis should have been at a lower rate than that originally observed at 1 atm, or there should have been a brief lag before the rate resumed at the original 1-atm rate. Our results, therefore, would seem to confirm those of Schwarz and Landau (21, 22) and be contradictory to those of Arnold and Albright (1) and those of Hildebrand and Pollard (7). Those authors (1, 7), however, used only specific portions of protein-synthesizing systems, a synthetic mRNA (polyuridylate), and incubation times of up to 80 min under pressure. These procedures raise some questions as to the validity of an extrapolation of their results to the events occurring in whole cells under pressure. The use of lengthy incubation periods under pressure has always been suspect, considering the interdependence of the multitude of reactions involved in the biosynthesis of proteins. Even if only one reaction in the protein-synthesizing machinery were affected by pressure per se and all the other reactions were initially unaffected, one should, over ex-
757
tended periods of time, observe changes in the rates of almost all the reactions involved as a consequence of the initial effect. Several investigators (1, 5, 7) have suggested that the effect of pressure on protein synthesis and presumably, therefore, on the growth of E. coli is attributable to no one reaction initially, but to the combined effects of pressure on several of the steps in the protein-synthesizing process, e.g., activity of the AA-tRNA synthetases, stability of the AA-tRNA complex, polysomal stability, and binding of the AA-tRNA to the ribosome-mRNA complex. We find it difficult to attribute a significant role in the inhibition of protein synthesis by high pressure to decreased activity of the AA-tRNA synthetases under these conditions, since the data of Hardon and Albright (5) indicate that the synthetase activity is only about 60 to 70% inhibited at 500 atm, a pressure at which there is almost complete inhibition of protein synthesis (5). In addition, Schwarz and Landau (21) have reported the absence of any such effect in whole-cell preparations of E. coli. It is also difficult to see how AA-tRNA instability could contribute significantly to a complete and immediate inhibition of protein synthesis, when the level of this molecule after 5 min at 600 atm (5) has been shown to decrease by only 3 to 7% from the amount originally present. In fact, the level of AA-tRNA reportedly drops by only about 30 to 45% after as long as 45 min at 600 atm. We do not consider that these effects of pressure can account for the immediate cessation of protein synthesis at high pressures, and the immediate resumption of synthesis at the 1-atm rate on the release of pressure would also seem to argue against their importance as anything but secondary events, of negligible importance in all but long-term experiments (Fig. 1; references 6, 16). The results presented here support the view that polysomal dissociation does not occur at high pressure and, therefore, cannot be responsible for the failure of protein synthesis to occur at high pressure. All investigators agree that the binding of AA-tRNA to the ribosome-mRNA complex is susceptible to inhibition by pressure, and Schwarz and Landau (21) have shown that this is quantitatively identical to that shown for protein synthesis in whole cells and cell-free extracts. It is our contention, therefore, that the rate-limiting step in protein synthesis that is particularly susceptible to pressure is the binding of AA-tRNA to the ribosome-mRNA complex. Furthermore, a forthcoming report (now in preparation) will indicate that the magnitude of the pressure effect is a characteristic inherent in the ribosome.
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POPE, CONNORS, AND LANDAU ACKNOWLEDGMENT
This paper was supported by National Science Foundation grant no. GB38137.
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