Cycle of Escherichia coli (ribosome heterogeneity/regulation of acetylation/stationary phase adaptation)

Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp. 2136-2140, May 1974 Alteration in the Acetylation Level of Ribosomal Protein L12 During Growth Cycle of Escherichia coli (ribosome heterogeneity/regulation of acetylation/stationary phase adaptation) S. RAMAGOPAL AND ALAP R. SUBRAMANIAN* Biochemical Research Laboratory, Massachusetts General Hospital and Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02114 Communicated by Herman M. Kalckar, March 4, 1974 ABSTRACT The relative content in ribosomes of L7 and L12, the two forms of a protein in the 50S subunit specifically involved in GTP hydrolysis, is found to undergo a striking shift with the growth phase of E. coli. The content of L12 (nonacetylated form) increases during early logarithmic phase, becoming about 85% of the total before midlogarithmic phase. Thereafter, L7 (N-acetylated form) content begins to increase, eventually becoming 75-80% in stationary phase. The L7 + L12 content per ribosome, however, remained constant during this shift. Our evidence suggests that the shift did not occur through modification of preexisting ribosomes. The data further indicate that the E. coli celi may contain more than one structurally distinct (with regard to L7 or L12 content) 50S subunit population. Proteins L7 and L12 of Escherichia coli ribosome (nomenclature as given in ref. 1) form a unique pair among the 55 proteins in this particle since they possess the same aminoacid sequence and differ solely by the presence of an aminoterminal acetyl group on L7 (2, 3). Their stoichiometry, determined by a variety of methods, indicate that together they may be present in two copies or more (between two and four) per ribosome (4, 5). It is generally believed that a ribosome may contain at least one copy of each form (2, 6). In vitro studies have established that this pair of proteins is specifically involved in several steps in the ribosome cycle. They are required for the GTPase action of ribosomes at initiation (7, 8) and elongation (9-12) steps, and they appear involved also in the termination step (13). However, such studies have so far failed to reveal a significant functional attribute for the acetylated form, since ribosomes with L12 alone were as active in the several reactions tested (including DNA-dependent synthesis of an active enzyme) as ribosomes with a mixture of L12 and L7 or with L7 alone (11). Nevertheless, the possibility of distinct roles in vivo is suggested by the finding (14) that ribosomes from broth-grown cells contained two to three times as much L12 as ribosomes from cells grown in glucose-minimal medium, while L7 content showed only slight variation under the same conditions. It has also been found recently that polysomal ribosomes may contain a somewhat greater amount of both L7 and L12 than free ribosomes (15). In this paper we report the finding of a remarkable shift in the L12/L7 ratio of ribosomes in E. coli cells as a culture progresses from early logarithmic to stationary phase. The results indicate that L7 and L12 may have distinct roles in vivo during the growth cycle. * To whom correspondence may be addressed. 2136 MATERIALS AND METHODS Strains and Growth. Most studies were done with E. coli MRE600 (16); other strains used were CP78, CP79, and ML308. Cells were grown in Ibroth [1% bactotryptone, 0.2% yeast extract, 0.5% glucose, and 0.5% NaCl (w/v) I with vigorous aeration at 37. Growth was followed by optical density measurement at 540 nm in a Zeiss spectrophotometer (culture 10-fold diluted for optical densities above 1.5), and cells were harvested after rapid chilling as described (17). - The cell paste was washed once in buffer I (10 mm Tris HCl, ph 7.8-50 mm KCl-10 mm Mg acetate), frozen in solid COr-acetone, and stored at -90. Cell Breakage and Ribosomal Protein Extraction. Cells were ruptured by grinding with alumina and an S-30 fraction was prepared (17) in buffer II (buffer I with 1.0 mm dithiothreitol). This was centrifuged at 270,000 X g (Beckman 65 rotor) for 2 hr to pellet ribosomes. The ribosomal pellet was resuspended in buffer II and the proteins were extracted with acetic acid (18). The extract, after dialysis in the cold against 8 M urea with 7 mm 2-mercaptoethanol, was used for L7/L12 determination. Ribosome. concentration was determined from absorbance measurement at 260 nm (18). Determination of L7 and L12. A newly developed polyacrylamide gel electrophoretic procedure, capable of separating L7 and L12 from each other and from all other components of unwashed ribosomes, was used for this study (K. Li and A. R. Subramanian, manuscript in preparation). In this procedure L7 and L12, which are the most acidic proteins of E. coli ribosomes (2), migrate into gel as anions while the bulk of ribosomal proteins stay behind. For quantitation, gels (stained with Coomassie blue), were scanned at 550 nm in a Gilford spectrophotometer with attachments. The areas were determined by cutting out peaks on the recorder chart tracing and weighing them. Materials. Chloramphenicol and rifampicin were purchased from Sigma. The compounds used in the separation procedure have been described in connection with a 2-dimensional electrophoretic procedure for E. coli ribosomal proteins (18). RESULTS Alteration in the Content of L7 and L12 with Growth Phase. The separation of L7 and L12 (from the mixture of proteins in unwashed ribosomes) obtained with the new electrophoretic procedure is illustrated in Fig. 1. The two proteins travel well

Proc. Nat. Acad. Sci. USA 71 (1974) Alteration of L7/L12 Ratio During Growth Cycle 2137 31 1.00 b. Late Log c. Stationary I') 'C) 2 I E 0.75 "It 0.50 0.25 I1i t 2 4 6 8 MIGRA TION (cm) --* FIG. 1. Separation of L7 and L12 from unwashed ribosomes. Ribosomal proteins (60 Mg) from E. coli MRE600 were subjected to polyacrylamide gel electrophoresis for 3.7 hr at 2 ma per gel (Methods). After they were stained with Coomassie blue, the bands were scanned (1 cm/min) in a densitometer. Identification of the L7/L12 bands were made with pure proteins generously provided by Dr. N. Brot. ahead of the few other acidic proteins that also migrate into the gel. Since protein L7 is the more acidic, it forms the leading peak. Conclusive identification of the bands was made with authentic L7 and L12 samples. It was also shown that L7 and L12 bands were not contaminated with any other protein component by means of further separation in the second dimension (Li and Subramanian, manuscript in preparation). Fig. 1 shows a nearly equal distribution between L7 and L12. The ribosomes used in this analysis were isolated from cells in late logarithmic phase. When similar analyses were carried out with ribosomes from early logarithmic and stationary phase cells, a dramatic shift in the relative levels of these two proteins was observed (Fig. 2). About 85% of the total area was under the L12 peak in ribosomes of early logarithmic phase cells. The relative distribution shifted with the age of the culture toward L7, and in ribosomes from stationary phase culture 65% area was under L7 peak. The L7 content increased somewhat further as the cells were kept longer (with aeration at 370) in stationary phase; 24-hr-old cultures contained 75% as L7. This remarkable shift in the relative amounts of L7 and L12 between early logarithmic phase and stationary phase cells was not restricted to MRE600. Early logarithmic phase cells of CP79 (K strain) and ML308 also contained 70-75% of L12. Surprisingly, CP78 (the isogenic rel+ strain of CP79) consistently showed a somewhat lower level of L12. In stationary phase all the above strains contained 75-80% in L7 form. Continuity in the Shift of L12/L7 Ratio During Growth. When the L12/L7 ratio of ribosomes in a culture was analyzed at several points, from early logarithmic to stationary phase, the data fitted a smooth unimodal distribution (Fig. 3). The culture, started with a fresh overnight inoculun, started with an L12/L7 ratio of about 0.5. As the cell mass increased, the ratio began to climb up and it reached a maximum value MIGRATION -- L7/L12 content of ribosomes from early logarithmic, FIG. 2. late logarithmic, and stationary phase cells of E. coli MRE600. Ribosomal proteins were subjected to electrophoresis and staining as in the legend of Fig. 1. The bands were scanned at 0.5 cm/min. of about 6 just before midlogarithmic phase. Thereafter, the ratio smoothly declined until it fell below 1 as the culture attained stationary phase. The highest L12 content was found in cultures reaching an optical density of 1.0, which would correspond to midlogarithmic phase, since these cells appear to turn off exponential growth and begin to grow linearly near an optical density of 2.0 (Fig. 3). At this point the ribosomes contained 84-86% : cp,9 %C4 1 2 3 4 5 6 7 TIME (hr) 10.0 0 1.0 _. FIG. 3. Shift in L12/L7 ratio during growth of E. coli MRE- 600. Cells grown in ILbroth were harvested at different cell densities (1 OD64o unit = 4 X 108 cells per ml) throughout the growth phase, and L7 and L12 were separated and scanned as in the legend of Fig. 2. The ratio of the two areas was determined and plotted. The different sets of points are from completely independent experiments. 0.1 CO)

2138 Biochemistry: Ramagopal and SubramanianP Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 1. Constancy of L7 and L12 content per ribosome during growth L7 + L12 L12/L17 ratio Peak area (mg) Area (normalized) 3.1 856 0.89 5.3 1043 1.06 4.3 865 0.88 1.2 1000 1.02 0.82 1156 1.17 Average 984 1.00 IrzJ OL7 E. coli MRE600 was grown and L7/L12 ratio was determined as in the legend of Fig. 3. Peak areas represent the weight of densitometer chart tracing per 100 pg of ribosomal protein applied on gel. L12 and only 14-16% L7. Since cultures are generally started with an overnight inoculum which is rich in L7, a part of the L7 content in these midlogarithmic phase cells could be derived from the inoculum. In order to minimize this contribution we grew a culture with an inoculum of midlogarithmic phase cells and harvested it at optical density of 1.0. Ribosomes from such cells also contained 14-16% of L7. Therefore, L7 is made even in early logarithmic phase and it would appear that both forms are required in the cell. The results in Fig. 3 were obtained with L-broth. When E. coli was grown in other media [Nutrient broth, Zubay's Medium (19), glucose-minimal medium], the highest L12 content attained was found to be related to the growth rate. The higher the growth rate supported by the medium, the greater was the L12 content. However, the pattern of shift in the L12/L7 ratio during the growth cycle was quite similar in all the media tested. Evidence for Subpopulations of Ribosomes In Vivo. Although the relative content of L7 and L12 underwent such a dramatic shift, the sum of their content per ribosome was found to remain constant. As shown in Table 1, this sum remained constant (within experimental error) over a change in L12/L7 ratio from 5 to 1.0. Evidently the large shifts in relative amounts take place with no apparent change in the number of copies of L7 and L12 per ribosome. I'In 10 0.26 0.70 1.87 5.50 6.90 CELL DENS/TY (OD540nm) FIG. 4. Total content of L12 and L7 in a 100-mil culture of E. coli MRE600 at different growth phases. Ribosomes were prepared and L7 and L12 contents determined as in the legend of Fig. 3. The total contents of L7 and L12 were calculated from the content of each per mg of ribosome (expressed as the weight of chart paper under the peak) and the total mg of ribosomes obtained from each culture. Stoichiometric measurements have indicated that between two and four copies of these two proteins together are present in ribosome (5). Since the L12/L7 ratio reached a value of 6 (i.e., 86% L12 and 14% L7) at the peak in Fig. 3, it would appear that sufficient L7 to distribute one copy on each ribosome may not be present under these conditions. If the number of copies of L7 + L12 per ribosome were two, one copy of L7 on all ribosomes would require 50% L7 content; if there were four copies per ribosome, one copy of L7 would still require a content of 25%. The observed L7 content near midlogarithmic phases of only 14% would, therefore, suggest TABLE 2. Effect of chloramphenicol treatment on L7 and L1 content of ribosomes Content (100 ml of culture) Cell Cell density as peak area (g) at harvest Percent Growth phase Treatment (ODW) L7 L7 L12 A. Early logarithmic None (O hr) 0.54 64 0.68 0.39 Chloramphenicol (1.5 hr; 200 pg/ml) 0.65 67 0.65 0.31 None (1.S hr) 1.75 43 3.12 4.21 B. Late logarithmic None (O hr) 1.38 2a 1.43 4.59 Chloramphenicol (5 hr; 200 pg/mil) 1.62 34 1.37 2.69 None (S hr) 5.25 56 14.86 11.64 In experiment A, a stationary phase culture was diluted 10-fold with fresh L-broth at 370 and divided into three equal portions. At 0 time one portion was harvested. The second was treated with chloramphenicol, the third was left untreated; both were incubated with shaking for the indicated time. It should be noted that stationary phase cells yielded fewer ribosomes than growing cells. In experiment B, a culture in L-broth grown beyond midlogarithmic phase was divided into three portions and treated as in experiment A.

Proc. Nat. Acad. Sci. USA 71 (1974) the existence of at least two subpopulations of ribosomes: (a) those containing both L7 and L12, and (b) those containing solely L12. A further subpopulation containing only L7 may also exist in stationary phase, since cells in this phase contained only 20-25% of L12 in their ribosomes. The proportion between these subpopulations in cell would depend on the actual number (yet unknown) of copies of L7 + L12 per ribosome, as well as on the growth phase. We attempted a separation between these ribosome subpopulations based on the plausible assumption that L12 may occur solely in polysomes and not in free ribosomes since exponentially growing cells, which contain 70-80% of ribosomes in polysomes (20, 21), also contain the highest L12 content (Fig. 3). Therefore, we prepared a gentle lysate of cells in late logarithmic phase and fractionated the ribosomes into polysomes and free ribosomes by methods described (22). The two fractions, however, gave the same proportion of L7 and L12. Evidently, functioning ribosomes (i.e., polysomes) contain both forms of this protein (15). Mechanism of the L12/L7 Ratio Shift. The L12/L7 ratios shown in Fig. 3 represent the levels of these two forms in ribosomes at different points in the growth cycle. The observed shift could be accomplished with the conversion of L7 to L12 (or vice versa) in preexisting ribosomes. Such a conversion would result in net loss in the total content of L7 in early logarithmic phase and of L12 in late logarithmic phase cells. We tested for such loss by estimating the total amount of L7 or L12 in 100 ml of culture at different points in growth cycle. The estimation was made from the total amount of ribosomes recovered from such a culture; this yield was found to be proportional to cell density (20 ± 3 jig of ribosomes/od54o0nm per ml.). The results, presented as a histogram (Fig. 4), reveal a significant finding. There was no net loss in L7 during early logarithmic phase when L12/L7 ratio was increasing or, even more striking, there was no loss in L12 during late logarithmic phase when the above ratio was rapidly decreasing. It appears that the shift in L12/L7 ratio is accomplished by preferential synthesis of either L12 in early logarithmic phase or of L7 in late logarithmic phase, but without net conversion of these proteins in preexisting ribosomes. An enzyme activity that transfers an acetyl group from acetyl CoA to the NHrterminus of L12 has been described in the post-ribosomal supernatant of E. coli (6, 23). Although the substrate appeared to be specifically free L12 at first (6), appreciable acetylation of ribosome-bound L12 was also reported later (23). In addition, existence of a deacetylase that acts on ribosome-bound L7 has been proposed (6). Since the data in Fig. 4 did not support modifications in vivo in preexisting ribosomes, we carried out further experiments using antibiotics to inhibit protein synthesis in growing cells. The assembly of new ribosomes would be halted in such cells, but the activity of an already made enzyme (acetylase or deacetylase) that modifies preexisting ribosomes should remain unaffected. Results from these experiments using chloramphenicol are shown in Table 2. Experiment A, using cells with a high content of L7 (64%), was aimed at testing the putative deacetylase. There was no change in L7 content after 1.5 hr in the presence of chloramphenicol, while it dropped to 43% in the untreated control. In experiment B. cells with a low content (25%) of L7 were used in order to test the extent of possible Alteration of L7/L12 Ratio During Growth Cycle 2139 acetylation in preexisting ribosomes. After a long exposure to chloramphenicol these cells showed some increase in L7 content, but it was considerably less than the increase (56%) in the untreated control. Similar results were also obtained when rifampicin was used to inhibit protein synthesis. These two experiments suggest that acetylation or deacetylation of preexisting ribosomes does not occur in vivo to any significant extent. Unexpectedly, exposure of E. coli to chloramphenicol for 5 hr led to a 35% reduction in ribosome recovery even though no cell lysis was apparent (Table 2). A slight loss was also seen even after 1.5 hr of exposure. It is perhaps significant that this loss appeared selective and occurred almost entirely in L12 content. Such a selective loss of L12 may explain the limited relative increase in L7 noted in experiment B of Table 2. It also would suggest an adaptive value of L7 accumulation for stationary phase cells. DISCUSSION Since GTP hydrolysis provides the driving force for the ribosome cycle, the reactions that are coupled to this hydrolysis are considered pivotal in mrna translation (24). Therefore, the discovery that the ribosomal protein specifically involved in this reaction exists in two forms, as acetylated L7 and as nonacetylated L12, was of unusual interest (9, 10). In this paper we show that the relative levels of these two forms are subject to control in a growing culture of E. coli. During early logarithmic phase, cells build up increasing amounts of L12; by midlogarithmic phase over 85% is present in this form. Beyond midlogarithmic phase, increasing amounts of L7 are made so that cells in stationary phase contain 75% in this form. This shift in relative amounts apparently requires production of new ribosomes and does not depend on modification of preassembled ribosomes. Although such a dramatic shift takes place in the relative levels, the average content of L7 + L12 per ribosome remained constant throughout growth phase. It is generally believed that all ribosomes may contain at least one copy of both these forms (2, 6), but it should be pointed out that the available evidence (2-5, 14) does not exclude the alternative possibility of two distinct classes of ribosomes, each containing only a single form. Our results show that the relative proportion of L7:L12 in ribosomes shifts from 1:6 to 3:1 during growth cycle of E. coli. It is not clear whether this shift reflects a change in the relative number of copies of L7 and L12 in a single ribosome population or rather a shift in the levels of two subpopulations, each with only one form. It appears that both forms L7 and L12 are essential for cell growth since there was a basal level of either protein at all stages of growth. The well-ordered variation in L12/L7 ratio would suggest some specific cellular function that varies with growth phase, but no correlation was found between L7/L12 content and participation in polysomes. However, data from the chloramphenicol experiment (Table 2) indicate that ribosomes containing L7 may survive better in a nongrowing culture. Since L7 content characteristically rises at the close of exponential growth, the shift may indicate an adaptation toward stationary phase. There are other significant cellular alterations known to occur in E. coli during the transition from logarithmic phase to stationary phase. The relative level of the two isoaccepting

2140 Biochemistry: Ramagopal and Subramanian tyrosyl-trnas (trna1tyr and trna2tyr) in late logarithmic is reported to be different from that in early logarithmic phase (25). The lipid composition of E. coli cell envelope is known to undergo marked changes in late logarithmic phase, such as the modification of unsaturated fatty acids into cyclopropane derivatives, resulting in lowered membrane fluidity (26). An enzyme in the biosynthetic pathway of E. coli phospholipids has been found (in cell extracts) in tight association with ribosomes (27), but it remains unclear whether there may exist a common determinant for these multiple alterations observed in late logarithmic phase. We thank Dr. H. M. Kalckar for his interest and discussions and K. Li for assistance in the early phase of this problem. This work was supported in part by a grant from the American Cancer Society (Massachusetts Division) to A.R.S. 1. Kaltschmidt, E. & Wittmann, H. G. (1970) Proc. Nat. Acad. Sci. USA 67, 1276-1282. 2. M6ller, W., Groene, A., Terhorst, C. & Amos, R. (1972) Eur. J. Biochem. 25, 5-12. 3. Terhorst, C. P., Moller, W., Laursen, R. & Wittmann- Liebold, B. (1973) Eur. J. Biochem. 34, 138-152. 4. Weber, N. J. (1972) Mol. Gen. Genet. 119, 233-248. 5. Thammana, P., Kurland, C. G., Deusser, E., Weber, H. J., Maschler, R., Stoffler, G. & Wittmann, H. G. (1973) Nature New Biol. 242, 47-49. 6. Brot, N. & Weissbach, H. (1972) Biochem. Biophys. Res. Commun. 49, 673-679. 7. Kay, A., Sander, G. & Grunberg-Manago, M. (1973) Biochem. Biophys. Res. Commun. 51, 979-986. 8. Fakunding, J. L., Traut, R. R. & Hershey, J. W. (1973) J. Biol. Chem. 248, 8555-8559. Proc. Nat. Acad. Sci. USA 71 (1974) 9. Kischa, K., Moller, W. & Stoffler, G. (1971) Nature 233, 62-63. 10. Hamel, E. & Nakamoto, T. (1972) J. Biol. Chem. 247, 805-814. 11. Kung, H., Fox, J. E., Spears, C., Brot, N. & Weissbach, H. (1973) J. Biol. Chem. 248, 5012-5015. 12. Highland, J. H., Bodley, J. W., Gordon, J., Hasenbank, R. & St6ffler, G. (1973) Proc. Nat. Acad. Sci. USA 70, 147-150. 13. Brot, N., Tate, W. P., Caskey, C. T. & Weissbach, H. (1974) Proc. Nat. Acad. Sci. USA 71, 89-92. 14. Deusser, E. (1972) Mol. Gen. Genet. 119, 249-258. 15. Deusser, E., Weber, H. J. & Subramanian, A. R. (1974) J. Mol. Biol., in press. 16. Cammack, K. H. & Wade, H. E. (1965) Biochem. J. 96, 671-680. 17. Subramanian, A. R. (1972) Biochemistry 11, 2710-2714. 18. Subramanian, A. R. (1974) Eur. J. Biochem., in press. 19. Zubay, G., Chambers, D. A. & Cheong, L. C. (1970) in The Lactose Operon, eds. Beckwith, J. R. & Zipser, D. (Cold Spring Harbor Laboratory, Cold Spring Harbor), pp. 375-391. 20. Forcchammer, J. & Lindahl, L. (1971) J. Mol. Biol. 55, 563-568. 21. Davis, B. D. (1971) Nature 231, 153-157. 22. Subramanian, A. R. & Davis, B. D. (1973) J. Mol. Biol. 74, 45-56. 23. Brot, N., Marcel, R., Cupp, L. & Weissbach, H. (1973) Arch. Biochem. Biophys. 155, 475-477. 24. Lipmann, F. (1969) Science 164, 1024-1031. 25. Gross, H. J. & Raab, C. (1972) Biochem. Biophys. Res. Commun. 46, 2006-2011. 26. Cronan, J. E., Jr. & Vagelos, P. R. (1972) Biochim. Biophys. Acta 265, 25-60. 27. Raetz, C. R. & Kennedy, E. P. (1972) J. Biol. Chem. 247, 2008-2014.