Vol. 122, No. 1 Printed in U.SA.

JOURAL o0 BAcrERIWoY, Apr. 1975, p. 89-92 Copyright 0 1975 American Society for Microbiology

Variation of Ribosomal Proteins with Bacterial Growth Rate ALEXANDER N. MILNE,I W. WAI-NAM MAK, MD J. TZE-FEI WONG* Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Received for publication 7 January 1975

The composition of ribosomal proteins has been examined as a function of the growth rate of Escherichia coli cells. Seven sets of cultural conditions, utilizing different combinations of carbon and nitrogen sources, were employed to provide a 36-fold spread in growth rate. The cellular content of most of the ribosomal proteins in ribosomes decreased to a similar extent in the very slow-growing cultures. Major exceptions were proteins S6 and L12, which exhibited a much more pronounced decrease, and S21, which exhibited an increase. None of the proteins remained invariant with growth rate. of 20 mM, centrifuged, suspended in 10 mM tris(hydroxymethyl)aminomethane acetate with 10 mM magnesium acetate (pH 7.4; TM buffer), and lysed in a French pressure cell. Ribosomal particles were sedimented at 220,000 x g for 1 h. After one wash in TM buffer, followed by two washes in TM buffer containing 0.5 M ammonium chloride and 10 mM mercaptoethanol (7), the ribosomal proteins were extracted with a 66% acetic acid-0.03 M magnesium acetate solution (6). After lyophilization, they were separated by two-dimensional polyacrylamide-gel electrophoresis, by the method of Kaltschmidt and Wittmann (8), and identified on the basis of the strain B pattern established by these workers. Analyss of II/14C ratios. Radioactive labeling of the ribosomes were performed by adding 2 mCi of ['Hleucine (29.8 Ci/mmol; New England Nuclear, Boston, Mass.) to a 200-ml sample of each of the seven cultures that had been grown at a steady-state for at least five generations. The cells were harvested 0.5 generation later. Samples of culture 1 were also labeled with 0.2 mCi of ['4C]leucine (254 mCi/mmol; New England Nuclear) and harvested similarly. Each 'H-labeled sample was combined with a "4C-labeled MATERIALS AND MEIHODS sample, and the different ribosomal proteins from the Escherichia coli ATCC 12632, a pyrimidine auxo- combined sample were extracted from the two-dimentroph with no other nutrient requirement, was grown sional polyacrylamide-gel after electrophoresis by the in a mineral medium containing (per liter): KH,P04, method of Zaitlin and Hariharasubramanian (15). 13.6 g; MgSO4-7H,O, 0.3 g; and FeCl,.6H,O, 0.5 mg. The "H/ 4C ratio of each protein was determined by The pH was adjusted to pH 7.2, and the medium was the channel ratio method with an external standard supplemented with 30 mg of uridine per liter. The and a Nuclear-Chicago Mark II liquid scintillation carbon and nitrogen sources were varied to bring spectrometer. An F value was calculated for each of about seven different steady-state growth rates (Table cultures 2 through 7, which expressed the ['HIleucine 1). The amino acid supplements added to culture 2 labeling of the various ribosomal proteins in these contained 50 mg each of glycine, alanine, serine, cultures relative to culture 1, as follows: threonine, methionine, valine, isoleucine, aspartic Fvalue for culture i = 'H/"IC ratio for culture i acid, glutamic acid, asparagine, glutamine, lysine, IH/14C ratio for culture 1 arginine, cysteine, phenylalanine, and tryptophan per liter. To extract ribosomes from a culture, cells were Calculated on this basis, the F value provides a harvested by being poured over crushed frozen me- measure of the fraction of the ['H Jleucine pulse that dium containing sodium azide at a final concentration enters into any ribosomal protein on the ribosomes in I Present address: Connaught Laboratories Limited, Wil- cells of culture i relative to the fraction that enters into the same protein in cells of culture 1. lowdale, Ontario, Canada M2N 5T8.

Ribosomes have a dynamic structure, and the exact stoichiometry of their constituent proteins is subject to variation with the functional state of the subunit particles (1, 4, 13, 14). In relation to cell growth, Deusser (3) made the important observation that the composition of ribosomal proteins differed for two different rates of steady-state growth. When cells were grown in an enriched medium, proteins S6, S21, and L12 were found to occur in two or three times the quantities occurring in cells grown in a minimal medium. However, with a study of only two steady-state growth rates, it is difficult to assess the limits by which the synthesis of ribosomal proteins could be modulated by growth rate. Accordingly, the present study was directed towards an examination of the variations in ribosomal proteins over as wide a range of steady-state growth rates as possible to define further this regulatory process.





RESULTS The E. coli cells exhibited different steadystate growth rates when grown on different carbon and nitrogen sources. The seven sets of cultural conditions shown in Table 1 were chosen to provide a 36-fold spread in the growth rate. (Extensive searches failed to uncover any cultural condition yielding a slower steady-state

growth rate than the gycerol-histidine combination of culture 7.) The fraction of a small pulse of [3 H]leucine incorporated into any ribosomal protein in each of cultures 2 through 7, relative to the incorporation in culture 1, was estimated in terms of the F value. Tables 2 and 3 show the F values for various ribosomal proteins in the small and the large subunits.

TABLE 1. Variation of growth rate with carbon and nitrogen sources

DISCUSSION The F values shown in Tables 2 and 3 indicate Carbon and nitrogen sources Gratwth a number of aspects regarding the regulation of Culture ribosomal proteins by bacterial growth rate. First, no single ribosomal protein gave an F 1 0.25% Glucose, 20 mM NH4Cl 0.86 value that remained invariant with growth rate. 2 0.25% Glucose, 20 mM NH4Cl, The general decrease of F values with growth amino acids 1.73 rate was in accord with the known enrichment 3 0.2% Sodium succinate, 20 mM 0.78 NH4Cl of ribosomal particles in fast-growing cells and 4 0.2% Alanine, 0.2% histidine 0.29 depletion in slow-growing cells (5). However, 0.2% Aspartate 5 0.17 the decrease was by no means linear, and the 6 0.2% Aspartate, 0.2% histidine 0.11 anomalously high F values for culture 5 are 7 0.25% Glycerol, 0.2% histidine 0.047 particularly striking in this regard. This deviaa Growth rate was determined as the reciprocal of tion from linearity suggests that growth rate is the mass doubling time of each of the cultures in an important, but not the sole, control of ribosome biosynthesis. Second, the F values for hours. TABLE 2. Variation of F values of small-subunit proteins with growth ratea Growth rate (doublings/h) Protein

1.73 (culture 2)

0.78 (culture 3)

0.29 (culture 4)

0.17 (culture 5)

0.11 (culture 6)

0.047 (culture 7)

Culture 7 after shift-up

Si S2 S3 S4 S5 S6 S7 S8 S9 S1o S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

1.13 1.28 1.24 1.14 1.18 1.71 1.15 1.17 1.13 1.16 1.16 1.00 1.23 1.16 1.16 1.14 1.23 1.14 1.17 1.19 1.42

0.74 0.74 0.72 0.71 0.69 0.61 0.71 0.71 0.73 0.70 0.70 0.67 0.73 0.74 0.72 0.64 0.80 0.68 0.77 0.70 0.79

1.29 1.04 0.74 0.84 0.77 0.36 0.85 0.77 0.78 0.93 0.87 1.11 0.82 0.80 0.81 0.86 0.91 0.99

1.79 1.31 0.96 1.03 1.01 0.50 1.00 1.01 0.96 1.18 1.12 1.09 1.04 1.39 1.01 1.09 1.14 1.16 1.04 0.98 1.17

0.66 0.69 0.54 0.50 0.53 0.23 0.55 0.56 0.52 0.65 0.72 0.60 0.57 0.68 0.55 0.57 0.73 0.54 0.42 0.43 0.68

0.36 0.38 0.31 0.31 0.26 0.08 0.31 0.26 0.28 0.34 0.39 0.41 0.33 0.38 0.30 0.36 0.48 0.36 0.35 0.36 0.68

0.40 0.39 0.29 0.33 0.34 0.15 0.33 0.33 0.33 0.36 0.35 0.33 0.38 0.37 0.31 0.31 0.31 0.33 0.30 0.30 0.41

0.84 0.85 1.34

a The F values for any protein express the incorporation of l3H Ileucine into the protein in cultures 2 through 7 relative to its incorporation in culture 1 (growth rate = 0.86 doublings per h). After labeling with [3H]leucine for 0.5 generation, a sample of culture 7 was also shifted-up nutritionally by the addition of 0.25% glucose and 20 mM NH4Cl, and 30 min after shift-up the culture was harvested; the F values of its ribosomal proteins are indicated in the last column of this table and Table 3. All F values were the average from three replicate experiments, and the range of F values for the replicates was mostly within 10%.

VOL. 122, 1975



TABLE 3. Variation of F values of large-subunit proteins with growth ratea



1.73 (culture 2)

0.78 (culture 3)

Li L2 L3 L4 L5 L6 L7 L8 L9 L10 Lll L12 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L26 L27 L28 L29 L30

1.24 1.54 1.25 1.19 1.22 1.24 1.11 1.14 1.22 1.30 1.21 1.61 1.24 1.25 1.29 1.20 1.25 1.24 1.23 1.05 1.18 1.28 1.23 1.18 1.25 1.19 1.15 1.26 1.28 1.39

0.71 0.71 0.72 0.79 0.73 0.72 0.79 0.77 0.74 0.70 0.69 0.48 0.70 0.69 0.74 0.73 0.71 0.71 0.69 0.64 0.79 0.70 0.71 0.71 0.73 0.70 0.66 0.69 0.74 0.81

Growth rate (doublings/hr) 0.11 0.17 0.29 (culture 6) (culture 5) (culture 4)

0.72 0.65 0.71 0.70 0.76 0.79 0.94 0.86 0.85 0.77 0.72 0.42 0.74 0.71 0.75 0.68 0.69 0.68 0.77 0.89 1.51 0.72 0.81 0.78 0.88 0.85 0.74 0.74 0.82 0.74

0.87 0.88 0.87 0.95 0.98 0.96 1.19 1.12 1.05 1.02 0.95 0.68 0.93 0.94 0.96 0.89 0.92 0.90 1.00 1.11 1.76 0.95 1.02 1.02 1.06 0.98 0.97 1.00 0.94 0.94

0.50 0.50 0.48 0.52 0.55 0.52 0.64 0.60 0.64 0.53 0.54 0.31 0.49 0.51 0.55 0.53 0.47 0.49 0.57 0.48 1.06 0.49 0.53 0.49 0.58 0.43 0.52 0.50 0.53 0.53

0.047 (culture 7)

Culture 7 after shift-up

0.21 0.24 0.24 0.28 0.28 0.27 0.20 0.21 0.29 0.23 0.26 0.08 0.24 0.27 0.26 0.30 0.23 0.24 0.28 0.39 0.48 0.24 0.27 0.28 0.31 0.36 0.45 0.52 0.24 0.40

0.34 0.34 0.33 0.33 0.38 0.37 0.31 0.28 0.32 0.28 0.34 0.16 0.34 0.38 0.37 0.34 0.31 0.36 0.31 0.33 0.43 0.30 0.31 0.39 0.39 0.30 0.37 0.38 0.30 0.35

See footnote a, Table 2.

most of the proteins varied only slightly from the average trend over the entire 36-fold range of growth rate between cultures 2 and 7. This underlines the extensive coordination, already indicated by the studies of Deusser (3) and Dennis (2) on a more restricted range, of the biosynthesis of most of the ribosomal proteins. Third, Deusser (3) had observed that proteins S6, S21, and L12 varied more strongly with growth rate than the other proteins. Our results (Tables 2 and 3) confirmed that the amounts of S6 and L12 were particularly growth rate sensitive: their F values decreased to well below most of the other proteins at very slow growth rates. However, the behavior of S21 was distinct from that of S6 and L12. Although all three proteins were increased more than most others in the fast growth of culture 2 in agreement with Deusser's finding, S6 and L12 decreased in the very slow growths of cultures 6 and 7, whereas S21 remained high, higher in fact than all other proteins. Protein L12 represents the nonacyl-

ated form of L7 (12), and the L12/L7 ratio was found by Ramagopal and Subramanian (11) to rise during the early log phase of a growing culture but to fall as the stationary phase was approached. Accordingly, the low F values of L12 in cultures 6 and 7 were most likely due to the regulation of the acetylation reaction by growth conditions. Kung et al. (9) found that the replacement of L7 and L12, or vice versa, did not affect significantly any of the measurable ribosomal functions. Finally, in contrast to L12, the low F values of S6 in cultures 6 and 7 cannot be explained as yet by the regulation of acetylation or other posttranslational modifications. Accordingly, the simple explanations are: (i) a decreased synthesis of S6 relative to the other ribosomal proteins; (ii) a decreased assembly of S6 into the 30S particle; and (iii) an increased metabolic turnover of S6. To test explanation ii, culture 7 cells were labeled with [3H Ileucine and then subjected to a nutritional shift-up,




with the addition of glucose and ammonium ACKNOWLEDGMENTS chloride, before harvesting. On the one hand, if We wish to thank Lloyd Porter for his valuable assistance. there existed a large fraction of synthesized but This study was supported by the Medical Research Council of unassembled S6 in the cells, much of the Canada. unassembled S6 would be expected to move into LITERATURE CITED particulate form after a shift-up to culture 1 1. Bickle, T. A., G. A. Howard, and R. R. Traut. 1973. conditions and its F value would be expected to Ribosome heterogeneity. J. Biol. Chem. 248:4862-4864. rise to the same range as those of the other 2. Dtennis, P. 0. 1974. In vivo stability, maturation and relative differential synthesis rates of individual riboribosomal proteins. On the other hand, if there somal proteins in Escherichia coli B/r. J. Mol. Biol. was no large unassembled fraction of S6 in the 88:25-41. cells, its F value would remain lower than the 3. Deusser, E. 1972. Heterogeneity of ribosomal populations other F values even after the nutritional shiftin Escherichia coli cells grown in different media. Mol. up. Our data (Table 2) show that the F value for Gen. Genet. 119:249-258. S6 after the shift-up was 0.15, which was still 4. Deusser, E., H. J. Weber, and A. R. Subramanian. 1974. Variations in stoichiometry of ribosomal proteins in only half the F values of the other proteins. Escherichia coli. J. Mol. Biol. 84: 249-256. Consequently, explanation ii appears to be 5. Ecker, R. E., and M. Schaechter. 1963. Bacterial growth under conditions of limited nutrition. Ann. N.Y. Acad. entirely inadequate. It is concluded that either Sci. 102:549-563. the synthesis of $6 was specifically retarded in S. G. S., C. G. Kurland, P. Voynow, and G. Mora. the slow-growing cultures, relative to the other 6. Hardy, 1969. The ribosomal proteins of Escherichia coli. I. proteins, or S6 was metabolically less stable Purification of the 30S ribosomal proteins. Biochemistry 8:2897-2905. than the other proteins, or both. Recently, Dennis (2) observed an accelerated metabolism 7. Hindennach, I., G. Stoffler, and H. G. Wittmann. 1971. Ribosomal proteins. Isolation of the proteins from 30S of S6 and S21 in E. coli cells. In culture 7, the F ribosomal subunits of Escherichia coli. Eur. J. Biovalue of S6 was lower than those of the other chem. 23:7-11. proteins, but the F value of S21 was actually 8. Kaltschmidt, E., and H. G. Wittmann. 1970. Ribosomal proteins. VII. Two-dimensional polyacrylamide gel higher. This suggests that explanation iii might electrophoresis for finger printing of ribosomal probe of relatively minor significance; thus a reteins. Anal. Biochem. 36:401-412. duced synthesis of S6 in the slow-growing cul- 9. Kung, H. F., J. E. Fox, C. Spears, N. Brot, and H. Weissbach. 1973. Studies on the role of ribosomal ture 7 is indicated. Although the exact mechaproteins L7 and L12 in the in vitro synthesis of nism responsible for this reduction is unclear, it ,8-galactosidase. J. Biol. Chem. 248:5012-5015. would require S6 to be regulated by a growth 10. Maaloe, O., and N. 0. Kjeldgaard. 1966. Control of rate-sensitive operon that did not code for other macromolecular synthesis. W. A. Benjamin, Inc., New York. ribosomal proteins. Indeed, although the synthesis of ribosomal ribonucleic acid is known to 11. Ramagopal, S., and A. R. Subramanian. 1974. Alteration in the acetylation level of ribosomal protein L12 during be highly growth rate sensitive (10), the rethe growth cycle of Escherichia coli. Proc. Nat. Acad. duced content of S6 per ribosome implies that Sci. U.S.A. 71:2136-2140. the synthesis of S6 was even more growth rate 12. Terhorst, C. P., B. Wittman-Liebold, and W. Moller. 1972. 50S ribosomal proteins. Peptide studies on two sensitive than that of ribosomal ribonucleic acidic proteins, A, and A,, isolated from 50S ribosomes acid. Furthermore, ribosomes obtainable from of Escherichia coli. Eur. J. Biochem. 25:13-19. culture 7, since they contained only about 13. Voynow, P., and C. G. Kurland. 1971. Stoichiometry of one-fourth the level of S6 compared to ribothe 30S proteins of Escherichia coli. Biochemistry 10:517-524. somes obtainable from culture 1, might be H. J. 1972. Stoichiometric measurements of 30S useful for investigating the functions of S6. It 14. Weber, and 50S ribosomal proteins from Escherichia coli. Mol. would be important to determine whether the Gen. Genet. 119:233-248. culture 7 ribosomes exhibit any functional defi- 15. Zaitlin, M., and V. Hariharasubramanian. 1970. An improvement in the procedures for counting tritium ciencies and whether such deficiencies can be and carbon-14 in polyacrylamide gels. Anal. Biochem. overcome by the addition of purified S6 to the 35:296-297. ribosomes.

Variation of ribosomal proteins with bacterial growth rate.

The composition of ribosomal proteins has been examined as a function of the growth rate of Escherichia coli cells. Seven sets of cultural conditions,...
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