JOURAL OF BACTERIOLOGY, June 1975, p. 1136-1143 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 3 Printed in U.SA.
Translational Control of Protein Synthesis in Staphylococcus aureus' SCOTT E. MARTIN2 AND JOHN J. IANDOLO* Division of Biology, Kansas State University, Manhattan, Kansas 66506 Received for publication 27 January 1975
The calculated in vivo polypeptide chain growth rate for Staphylococcus aureus MF-31 grown in nutritionally rich medium assuming all the ribosomes were functional was found to be approximately 16 amino acids/s/ribosome, but decreased to 10.2 amino acids/s/ribosome for cells grown in poor medium. An in vitro analysis revealed that 70S ribosomes isolated from rich medium cells were more active than similar 70S ribosomes derived from cells grown in poor medium. The 30S subunit was found responsible for the increased activity of the rich monosomes, whereas the 50S subunit appeared to be capable of either high or low activity. The composition of the growth medium has been shown to affect the rate of growth and the macromolecular composition of numerous procaryotic organisms. However, such studies have been limited to those genera possessing sufficient metabolic diversity to grow over a wide nutritional range. For example, when grown in nutritionally poor medium, Salmonella typhimurium (6, 14) and Bacillus lichiniformis (20) exhibited slower generation times and were found to contain less deoxyribonucleic acid, ribonucleic acid (RNA), and protein than when grown in nutritionally rich medium. In spite of these changes, the rate of amino acid polymerization (step time-time required to add an amino acid residue to a growing polypeptide chain) was found to be constant (approximately 66 ms) regardless of growth rate when between 0.5 and 1.9 doublings per h. On the other hand, when Escherichia coli 15 is grown at less than 1 doubling per h (7) or when S. typhimurium is starved for amino acids (6), a significant decrease in the average step time was observed. These observations suggest that ribosomes may be capable of more than an "all or none" function, and that they may be instrumental in controlling the rate of protein synthesis (i.e., translational control) under adverse environmental conditions. The possibility of distinct "classes" of ribosomes, in concert with changes in the distribution of ribosomal proteins that occur at vastly different growth rates, also suggests that the rates of polypeptide biosynthesis might be al-
tered as the growth rate changes. The heterogeneity of 30S ribosomal proteins in E. coli (3, 25) may indicate that different classes of 30S subunits exist with respect to functional abilities (11, 12). Furthermore, one or more of these classes might be enhanced as growth conditions are altered. In fact, two classes of 30S particles have been demonstrated, one able to complex with the synthetic messenger polyuridylic acid [poly(U) 1, and the other unable to interact with poly(U), presumably being dependent on the presence of the fractional protein 30S-1 (23). These data, however, are tentative, since S1 may be lost during purification (21). Nevertheless, an "initiating ribosome" has been found to be distinguishable from a "propagating ribosome," due to the presence, or absence, of protein 30S-21 (22). The number of either class may depend upon the rate of growth, since different growth media can influence the distribution of ribosomal proteins (4). Duesser (4) has found that two- to threefold greater amounts of proteins 30S-6, 30S-21, and 50L-12 were present in ribosomal populations isolated from cells grown in rich medium as compared to those obtained from cells grown in minimal medium. Because of the growth restrictions imposed by limiting media, nutritionally fastidious bacteria have not been studied in the manner described above. Such studies, however, are necessary to afford a better understanding of the regulation of protein synthesis in these organisms. Since fastidious organisms do not possess the metabolic alternatives available to more diverse microorganisms, the regulatory constraints on ' Contribution no. 1241, Kansas Agricultural Experiment biosynthesis are also likely to be less compliStation, Manhattan, Kan. 66506. 2Present address: Department of Medical Microbiology, cated. The elucidation of the mechanisms involved can provide important comparisons for University of Califomia at Irvine, Irvine, Calif. 92664. 1136
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Ribosomes were removed from the S-30 by centrifustudies in higher organisms. To achieve these goals, our approach utilized Staphylococcus gation for 2 h at 152,000 x g in a Beckman 5OTi rotor aureus grown in dissimilar nutritional condi- (48,000 rpm). The top 2/3 of the resulting supernatant (S-100) was aspirated and pooled. The S-100 was tions. dialyzed overnight against 500 volumes of 10 mM
MATERIALS AND METHODS Growth of bacteria. S. aureus MF-31, which was coagulase positive and B-hemolytic, was maintained on Trypticase soy agar slants at 4 C. Experiments were performed on cells grown in rich medium-trypticase soy broth (TSB), or a poorer defined medium (SDM). TSB contained the following (per liter): 17 g of Trypticase (GIBCO), 3 g of phytone (GIBCO), 5 g of NaCl, 2.5 g of K,HPO4, and 2.5 g of glucose. SDM was composed of 3 g of vitamin-free Casamino Acids (Difco) and 0.75 ml of minimal essential medium vitamins (GIBCO) per liter. For in vitro protein synthesis experiments, shake cultures of exponential-phase cells (300 ml) in the appropriate medium were inoculated into 37 liters of fresh medium at 37 C and grown under forced aeration. When turbidity was equal to 30 Klett units (4.4 x 107 cells/ml) in rich medium or 16 Klett units (4.2 x 107 cells/ml) in poor medium aeration was halted, and the culture was cooled in an ice bath. The cells were collected at 4 C in a DeLaval Cream Separator and resuspended in cold tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.8) (10 mM Tris, 50 mM NH4Cl, 10 mM magnesium acetate, 0.1 mM dithiothreitol [DTTJ-designated 10 mM Mg2+ buffer). The resuspended cells were pelleted by centrifugation for 10 min at 10,400 x g, washed two times in 10 mM Mg2+ buffer, and frozen in a dry ice-ethanol bath. The frozen cell pellets were stored at -70 C. For the quantitative analysis of cellular components and for the determination of growth curves, cells were grown under aeration at 37 C in 300 ml of medium. Inocula were taken from exponentially growing cultures in the appropriate medium. Preparation of cell fractions. Washed cells were thawed, and a 50% (wt/vol) suspension was made by the addition of 10 mM Mg2+ buffer. The suspension was warmed to 37 C, and 300 ;&g of lysostaphin (Schwarz/Mann) and 14 sg of deoxyribonuclease I (Sigma) were added per g of wet cells. After vigorous shaking for 10 min at 37 C, the lysate was cooled in an ice bath, and the cellular debris was removed by two centrifugations for 10 min each at 27,000 x g. The supernatant (S-30) was then mixed with an equal volume of preincubation mix and gently shaken. The final concentration of the reagents in the diluted preincubation mix was: 160 mM Tris-hydrochloride (pH 7.5), 10 mM magnesium acetate, 50 mM NH4Cl, 0.1 mM DTT, 5 mM adenosine triphosphate (potassium salt-ATP), 18 Mg of pyruvate kinase per ml, 1.1 mg of phosphoenol pyruvate (potassium salt-PEP) per ml, and 0.15 mM each of the following amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, hydroxyproline, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. After 30 min, the S-30 was cooled and centrifuged for 10 min at 27,000 x g.
Mg2+ buffer, divided into 1-ml aliquots, frozen in a dry ice-ethanol bath, and stored at -70 C. The remaining S-100 was removed and discarded. Ribosomes were resuspended in 10 mM Mg2+ buffer and clarified by centrifugation at 27,000 x g for 10 min. After pelleting, the ribosomes were washed twice in 10 mM Mg2+ buffer and finally resuspended in a minimal volume of 10 mM Mg2+ buffer. Any aggregates were removed by centrifugation at 27,000 x g for 10 min, and the resulting supematant fluids were treated in one of two modes. If 70S ribosomes were desired, the suspension was dialyzed overnight against 1,000 volumes of 10 mM Mg2+ buffer. Dialyzed 70S ribosomes were divided into aliquots, frozen in a dry ice-ethanol bath, and stored at -70 C. The concentration of the ribosomes was determined by assuming an extinction of 19 absorbance units per mg at 260 nm (A21*) (15). When subunits were desired, the ribosomal suspension was dialyzed for 24 h against 2,000 volumes of Tris-hydrochloride buffer (pH 7.8) (10 mM Tris, 50 mM NH4Cl, 0.1 mM magnesium acetate, 0.1 mM DTT-designated 0.1 mM Mg2+ buffer). The dialyzed ribosomal subunits were then separated at 25,000 rpm for 12 h on 36-ml gradients (SW-27 rotor) of 10 to 30% sucrose in 0.1 mM Mg2+ buffer. Fractions containing the appropriate subunits were pooled and concentrated by centrifugation at 152,000 x g for 18 h (5OTi rotor). The ribosomal subunit pellets were suspended in 10 mM Mg2+ buffer, frozen in a dry ice-ethanol bath, and finally stored at 70 C. Purity of the subunit preparations was determined by analysis on 5-ml, 10 to 30% sucrose gradients in a Beckman SW50.1 rotor for 3.5 h at 35,000 rpm. Salt-washed ribosomal subunits were prepared by the addition of high salt Tris-hydrochloride buffer (10 mM Tris, 10 mM magnesium acetate, 0.5 or 1.0 M NH4Cl, and 0.1 mM DTT-designated high salt buffer) to purified subunit suspensions. After 1 h at 4 C, the salt-washed subunits were collected by centrifugation in a Beckman SW50.1 rotor for 6 h at 45,000 rpm. The top 2/3 of the supernatant was removed and saved; the lower 1/3 was discarded. The pellet was suspended a second time in 5 ml of high salt buffer and allowed to stand ovemight at 0 C. The washed subunits were then pelleted, and the top 2/3 of the supernatant was removed and combined with the first upper supernatant. After discarding the lower 1/3, the washed particles were suspended in 10 mM Mg2+ buffer and frozen. The combined supernatants were concentrated with dry G-15 Sephadex, dialyzed against 10 mM Mg2+ buffer, and stored at -20 C. Preparation of tRNA. The procedures followed were slight modifications of those of von Ehrenstein and Lipmann (24) and Jacobson and Hedgcoth (10). The ether-washed transfer RNA (tRNA) was dissolved in water, made 2% potassium acetate, and precipitated for 2 h with 3 volumes of 95% ethanol. Residual ethanol was then removed by lyophilization. The tRNA precipitate was dissolved in water, divided into aliquots, and stored at -20 C.
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In vitro protein synthesis. The procedures followed were modifications of those by Mao (15) and Nirenberg and Matthaei (16). Solution I contained 100 mM Tris, 220 mM magnesium acetate, 500 mM NH4Cl, 10 mM ATP, and 0.45 mM each of guanosine triphosphate, cytidine triphosphate, and uridine triphosphate (pH 7.6). Solution II was prepared fresh daily and contained 0.5 ml of solution I, 0.022 ml of 1 mM DTT, 0.5 ml of 75 mM PEP, 125 ug of pyruvate kinase, 0.5 ml of a 0.6 mM amino acid solution (preincubation mixture, minus phenylalanine), 0.02 ml of [3 H]phenylalanine (Schwarz/Mann, 6.0 Ci/ mmol, side chain 2, 3-3H) or L-["C]phenylalanine (Schwarz/Mann, 460 mCi/mmol, uniformly labeled), and approximately 2.6 mg of tRNA. The standard reaction mixture (0.325 ml) was prepared by adding the following components in sequence: 0.15 ml of solution II, 0.01 ml (150 Mg) of poly(U) (Miles Laboratories), ribosomes at concentrations of 420 ,g (8 A2,0 units) of 70S ribosomes, or 250 Mg (4.75 A2*0 units) of 30S subunits followed by 125 Ag (2.38 A,*o units) of 50S subunits, and S-100 fraction from poor medium cells (0.1 ml, approximately 500 ug of soluble protein); the volume was then adjusted to 0.325 ml with 10 mM Mg2+ buffer. The final concentrations in the reaction mixture were 12.8 mM Tris, 22 mM magnesium acetate, 64 mM NH4Cl, 0.78 mM ATP, 0.04 mM each of guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, 0.054 mM DTT, 5.86 mM PEP, 6.3 zg of pyruvate kinase, 0.046 mM each of 20 amino acids, 1 MCi of ['HJphenylalanine, 150 ;g of tRNA, and 150 ug of poly(U). After addition of the S-100, the reaction tubes were removed from the ice bath and gently agitated at 37 C for 30 min. The reaction was halted by the addition of 2 ml of cold 10% trichloroacetic acid. The tubes were then heated for 20 min at 90 to 95 C and subsequently placed in ice for 30 min. The labeled precipitate was collected on membrane filters (HA; 25 mm; Millipore Corp.), washed with 25 ml of cold 5% trichloroacetic acid, and dried at 80 C. Radioactivity was measured in a Beckman LS150 Liquid Scintillation Spectrometer. Rate studies were performed in the manner described above, except that uniformly labeled ["C ]phenylalanine was used. At appropriate times, 0.025-ml samples were removed and mixed with 2 ml of cold 10% trichloroacetic acid and treated as described. Determination of the macromolecular components. The techniques used for quantitation of the macromolecular components were modifications of those described by Shatkin (18) and van Dijk-Salkinoja and Planta (20). Cells were grown in each medium with shaking at 37 C. When growth reached the appropriate level (cell number was determined by pour plating on Trypticase soy agar), the cells were collected by filtration through 0.45-Mm membrane filters (Millipore Corp.). The filters were washed, and the cells were rinsed from the filters. An equal volume of cold 10% trichloroacetic acid was added, and the mixture was allowed to stand in ice for 1 h. The precipitates were centrifuged for 20 min at 2,000 x g,
J. BACTERIOL.
and the supernatant solution was saved for analysis. The precipitates were suspended in 5% trichloroacetic acid and heated for 45 min at 95 C. The heated solutions were cooled and centrifuged for 20 min at 2,000 x g. The percentage of RNA found in the ribosomal fraction (orcinol test) was determined on lysostaphin lysed cultures. After removal of cellular debris by low-speed centrifugation, the ribosomes were collected by centrifugation at 152,000 x g for 18 h (SW50.1 rotor). RNA was then determined in the pellet and supernatant fractions. The concentration of deoxyribonucleic acid in the supernatants was determined by the diphenylamine reaction. The remaining precipitate was hydrolyzed by heating overnight at 45 C in 1 ml of 1 N NaOH. The protein concentration in the hydrolyzed residue, as well as that of the supernatant, was determined by the Lowry procedure, utilizing the Folin-Ciocalteau phenol reagent. Lysozyme (Sigma, grade I) was used as the standard.
RESULTS Determination of the in vivo rate of protein synthesis. S. aureus MF-31 was grown at 37 C in rich medium (TSB) and poor medium (SDM). A typical growth curve and the point at which cells were studied are shown in Fig. 1. During the exponential phase of growth, the rich medium culture had a generation time of 20 4 2 min, whereas in poor medium the generation time was about three times greater (60 : 6
min). To estimate the in vivo step times (20), a determination of the amounts of RNA and protein per cell was performed (Table 1). Cells grown in rich medium contained more abundant amounts of all macromolecules. From these data, and the observation that, respectively, 76% and 62% of the total RNA was present as ribosomal RNA in rich and poor medium-derived cells, an estimation of the number of ribosomes per cell was possible (Table 1). Cells grown in rich medium contained approximately 83,200 ribosomes/cell, whereas poor medium-derived cells had only 25,600 ribosomes/cell. Evaluation of the number of ribosomes per cell allowed an estimation of the in vivo protein synthesizing efficiency of cells grown in both media (Table 2). Assuming all the ribosomes were functional, cells grown in rich medium polymerized amino acids at a rate of 16 amino acids/s/ribosome in comparison to poor medium-derived cells whose polymerization rate was 10.2 amino acids/s/ribosome. These data showed that rich medium cells synthesized protein about 1.6 times faster than poor medium cells and suggested that translational modulation of protein synthesis may occur.
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subunits isolated from cells grown in the other medium) showed that 30S ribosomal particles were responsible for the increased activity (Table 3, experiment C). When rich 30S subunits (subunits derived from cells grown in rich medium) were incubated with poor 50S subunits (subunits derived from cells grown in poor medium), approximately 1.60 ± 0.34 times as much isotope was incorporated than in the control poor homologous system. The reciprocal mixture consisting of poor 30S subunits and rich 50S particles directed approximately the same it amount of incorporation as found in the control poor homologous system. Thus the 30S subunits :q, from cells grown in poor medium were responsic ble for the lower level of poly(U)-directed [8H Iphenylalanine incorporated. Rate studies over a time period of 100 min were performed on both homologous and heterologous systems (Fig. 2). During the first 30 min in the presence of rich 30S subunits, the rate of incorporation for both systems remained constant and was approximately 3.3 x 10-14 mol of phenylalanine/min. The average rate of incorporation in the two poor 30S subunit systems 0 /00 200 300 400 also remained constant, but for 60 min, and was Miniutes approximately 2 x 10-14 mol of phenylalanine/ FIG. 1. Growth of Staphylococcus aureus MF-31 min. In the initial period of constant rate, the in nutritionally rich or poor medium. Growth was rich 30S particle directed incorporation at 1.6 monitored with a Klett Summerson Colorimeter. times the rate of the poor 30S particle. However, Symbols: 0, growth in rich medium; 0, growth in after 100 min of incubation, the amount of poor medium; Ft, point of harvest for in vivo and in phenylalanine polymerized was approximately vitro studies. the same for all preparations, although poor 30S Determination of in vitro protein synthe- subunit systems never reached the levels sizing capacity. To confirm the in vivo results, achieved by the rich 30S subunit systems. As ribosomes isolated from cells grown in either TABLE 1. Macromolecular composition of medium were assayed for their ability to incorexponential-phase Staphylococcus aureus cells porate [3HJphenylalanine into polypeptide as Growth mediuma directed by the synthetic messenger RNA Experimental poly(U). Incubation at 37 C for 30 min revealed Rich Poor that 70S ribosomes derived from rich medium cells were 1.33 0.10 times as active as were 70S ribosomes derived from poor medium cells (Table 3, experiment A). These data as well as the following regarding in vitro protein synthesis represent averages of at least six individual experiments, each originating with different batches of cells. To determine whether this effect was subunit mediated, the activity of the system was tested using purified ribosomal subunits. Homologous systems composed of ribosomal subunits from rich medium cells directed incorporation of approximately 1.88 0.63 times as much phenylalanine as did poor homologous systems (Table 3, experiment B). Heterologous systems (systems composed of 30S subunits isolated from cells grown in one medium and 50S
2.96 4 0.29 + 4.78 + 3.26 4
Growth rateb DNA/cellc Protein/cellc RNA/cellc % rRNAd
rRNA/cellc Calculated no. of ribs/celle a
Mean
76 2.48 83,200
E 4
i
0.34 0.05 0.73 0.22
1.00 4 0.20 s 2.81 ± 1.21 ±
2 63 0.22 0.76 7,400 25,600
+
0.10 0.02 0.35 0.15 1 0.15 5,000
standard error of five experiments, each
analyzed in triplicate. ° Generations per hour. cGrams per cell x 10-3. DNA, Deoxyribonucleic acid. d Percentage of ribosomal RNA (rRNA) calculated as described in Materials and Methods. e Calculated by dividing the amount of ribosomal RNA by the weight of the ribosomal RNA in a 70S ribosome (2.98 x 10- Is g).
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TABLE 2. Protein synthesizing efficiency and step times of ribosomes from exponential-phase Staphylococcus aureus cells Medium
rich poor
Generation (min) time
20 60
±
+
2 6
Cell protein
4.78 2.81
+ +
0.73 0.35
Ribs/cell
83,200 + 7,400 25,600 ± 5,000
Efficiency ribosomesaof
19.9
±
12.7
+
3.8 2.9
acids/ Amino ribosome/s 16 10.2
a Calculated from the equation k = 0.692 (PIRt) where k is the efficiency, P is the amount of protein/cell, R is the number of ribosomes/cell, and t is the generation time. Results are expressed as grams of protein per minute per ribosome 10-20.
TABLE 3. Polyuridylic acid-dependent phenylalanine incorporation by 70S ribosomes and ribosomal subunits derived from cells grown in rich and poor mediuma Ribosomal components
Phenylalanine incorporated (counts/min)b
A
70S rich 70S poor
8,273 4 536 6,233 i 687
B
30S rich + 50S rich 30S poor + 50S poor
4,436 ± 185 2,509 66
C
30S rich + 50S poor 30S poor + 50S rich
4,221 61 2,656 ± 95
Expt
±
a The incorporation of [3H]phenylalanine (6 Ci/ mmol) was performed as described using 8 A,60 units of 70S ribosomes or 4.75 A..0 units of 30S subunits and 2.38 A,.0 units of 50S subunits. Incubation was at 37 C for 30 min. IAverage of at least six individual experiments, each originating with different batches of cells.
50 75 MINUTES
FIG. 2. Rate of polyuridylic acid-dependent phenwas the case in the heterologous systems, the ylalanine incorporation utilizing homologous and hetsource of the 50S subunits seemed to have little erologous subunits derived from cells grown in rich or poor medium. The poly(U)-dependent incorporation effect. To determine if the observed difference in of ['4Clphenylalanine (460 mCi/mmol) was performed using 0.5 1sCi of ["4CJphenylalanine, 4.75 activity of the two sources of 30S subunits was as described units 30S ribosomes, and 2.38 A,.0 units of 50S of A,,, due to a smaller proportion of poor 30S particles ribosomes. Incubation at 37 C. At appropriate being able to successfully interact with poly(U) time intervals, 0.025-mIwas samples were removed and and 50S subunits, 70S ribosomes from cells added to 2 ml of ice-cold 10% trichloroacetic acid and grown in both media were mixed just prior to subsequently assayed as previously described. Symincubation, or, alternatively, whole cells were bols: 0, 30S rich + 50S rich; *, 30S poor + 50S poor; mixed before lysis (Table 4). If only the propor- A, 30S rich + 50S poor; A, 30S poor + 50S rich. tion of "active" 30S subunits differed, then in the post-lysis mixture one would predict an factors (8), and therefore possibly fortuitiously activity halfway between that found for rich bound cytoplasmic proteins. One method to and poor medium-derived controls, and in the remove such proteins is to wash ribosomes in pre-lysis mixture a number slightly greater than buffers containing NH4Cl (8). If the difference midway. Such a situation was not found. observed between the ribosomes derived from Rather, these data suggest that the polymeriza- cells grown in either medium was due to nontion rate was controlled by the activity of the specific binding of a nonribosomal inhibitory poor 30S containing monosome, as the rate protein(s), then this should be removed by would be dependent upon the slowest compo- washing in NH4C1. When S. aureus ribosomes were washed in either 0.5 or 1.0 M NH4Cl, they nent of the system. Ribosomal subunits prepared as described lost their poly(U)-directed polyphenylalanine here have been found to carry soluble initiation synthesizing capacity. Experiments showed
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that this was due to the removal of several ribosomal proteins in the high salt wash. However, when those proteins removed by the high salt wash of the poor 30S subunit were added to functional systems, no inhibitory effect on either rich or poor 30S subunit systems was noted.
DISCUSSION The observation that S. aureus MF-31 grew with differing growth rates in rich and poor medium offered a system to study the regulation of protein synthesis in a fastidious, grampositive organism. The step time for S. aureus grown in rich medium, having a doubling time of 20 min, (62 ms), was very close to the step times calculated for Salmonella typhimurium (67 ms) and for Bacillus licheniformis (52 ms) when these organisms were grown with doubling times between 30 and 120 min (6, 14, 20). The similar value obtained increases the reliability of the result and suggested that the step time for S. aureus grown in poor medium with a doubling time of 60 min (100 ms) was also valid. These estimates are minimal values, however, as they do not include measurements of the number of active ribosomes (11). Estimation of the number of active ribosomes is often determined by polysome analysis in sucrose gradients and assumes that all the ribosomes in a polysome are actives (7). The conditions necessary to lyse S. aureus (incubation at 37 C for 10 min) unfortunately TABLE 4. Polyuridylic acid-dependent phenylalanine incorporation by post-lysis and pre-lysis mixturesa Expt
Ribosomal components
Phenylalanine incorporated (counts/min)
A
70S rich 70S poor 1/2 70S rich + /2 70S poor
7,759 6,512 6,538
B
30S rich + 50S rich 30S poor + 5OS poor Pre-lysis mixture
6,038 4,234 4,296
aThe incorporation of [3H]phenylalanine (6 Ci/ mmol) was performed as described. Incubation was at 37 C for 30 min. In experiment A, the mixture contained 4 A2*0 units of both 70S rich and 70S poor ribosomes, whereas the control systems contained 8 A2.0 units of ribosomes derived from cells grown in either medium. In experiment B, equal quantities (wet wt) of rich and poor medium-grown cells were mixed before lysis and simltaneously carried through the isolation and purification procedures with the control systems.
1141
preclude such estimation, although these measurements are subject to some question. Both the ionic environment of bacterial lysis (17) and the speed of centrifugation (9) also have been shown to influence the distribution of polysomes, monosomes, and ribosomal subunits within a sucrose gradient. In spite of the possible errors inherent in these calculations, the in vivo results with S. aureus suggested that ribosomes in rich medium-grown cells were more efficient than those in poor medium cells. This observation was in contrast to those of Engbaek et al. (6) and van Dijk-Salkinoja and Planta (20), who found that the growth medium, and hence the rate of growth up to approximately 120-min doubling times, had no effect on the polypeptide chain growth rate. Variations of the quantities of macromolecular components with the generation time were also comparable to those reported for S. typhimurium (14) and B. licheniformis (20): cells grown in poor medium contained less deoxyribonucleic acid, RNA, and protein than cells grown in rich medium. In conjunction with the lowered amount of RNA, cells grown in poor medium also had a smaller percentage of their RNA as ribosomal RNA. Both the test organism and the composition of poor medium could have influenced these differences. S. aureus was the most fastidious of the three organisms compared, and therefore its growth requirements, or their limitations, could have had more pronounced effects on the entire organism. If the cells were in a starved, or near starved, condition, the step times might have been adversely influenced. For example, Brunschede and Bremer (1) found that the in vivo step time of methionine and proline auxotrophs of E. coli was increased from 60 to 1000 ms during starvation for required amino acids. In this study, however, starvation was not evident in the poor medium since the organism was harvested at the midpoint of exponential growth and had the potential to double for two generations after the time of cell harvest. The results found in vitro, utilizing 70S ribosomes, are consistent with the in vivo observations. That is, 70S ribosomes from rich medium cells were more active in the synthesis of polyphenylalanine than were 70S ribosomes isolated from cells grown in poor medium. It seems unlikely that factors involved during initiation were influencing these results since the poly(U) message circumvents the requirement for soluble protein initiation factors (8), and the high magnesium concentration (22 mM) eliminated the need for formylmethionyl tRNA (2, 10). In addition, both rich and poor
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70S ribosomes were incubated with the poor S-100 fraction; similar differences were also evident when the rich S-100 fraction was utililized. The soluble elongation and termination factors were present in the same amounts in both systems, and therefore also not directly involved in the differences observed between the two 70S ribosomes. Moreover, the same standard reaction mixture was used with both systems, indicating that the energy-generating components, the ionic conditions, and all other related in vitro environmental components were identical. It is possible therefore to conclude that the reduced step time of the 70S poor ribosomes was due to the 70S ribosomes themselves and not to other variables. The subsequent observation that homologous subunit systems (30S and 50S particles isolated from cells grown in the same medium) possessed similar activities as found with 70S intact ribosomes also supported the hypothesis that rich ribosomes functioned more efficiently than poor ribosomes. Experiments involving heterologous subunit systems implied that the 30S particle was responsible for the altered activity of the entire systm. In all cases, the presence of the 30S subunit, isolated from cells grown in rich medium, stimulated the system to incorporate more phenylalanine than did systems containing poor 30S subunits. Further support that 30S subunits were important in determining activity was found in the kinetic experiments involving homologous and heterologous subunit systems. The source of the 30S particle dictated the rate of incorporation of phenylalanine, whereas the source of the 50S subunit seemed unimportant. Systems containing rich 30S particles incorporated isotope at a faster rate than did poor 30S subunit systems. Whereas the initial rates differed, both systems reached approximately the same levels after 100 min of incubation, indicating that, given enough time, the poor 30S subunits were capable of the same maximal amount of incorporation as were rich 30S particles. This implied that the differences found were due to the activity levels imposed by the 30S subunit, and not to substrate limitation, the presence of a unique nuclease or protease, or any other factor peculiar to one system. These findings are consistent with the theory of Kurland (11), who suggested that 30S subunits of E. coli were heterogeneous with respect to their ribosomal (r)-proteins, and hence possessed possible differences in functionality. They similarly corroborate the finding that E. coli 50S subunits are not heterogeneous in their protein composition and therefore probably not in their functional capabilities (11).
One explanation for the reduced activity of the poor 30S particles might be the presence of a new r-protein or the alteration of one or more existing r-proteins. Nevertheless, sucrose gradient co-cenrifugation of rich and poor 30S particles revealed that the two subunits were similar with respect to their molecular weights, as they sedimented in the same fraction (unpublished data). Neither particle contained gross amounts of different or contaminating proteins, although it is unlikely that this method would be sensitive to small protein differences. However, more detailed analysis by polyacrylamide gel electrophoresis (data not presented) resulted in only 16 bands in gels containing protein samples derived from either subunit. This number was identical to that reported by Traut et al. (19) for E. coli 30S r-proteins analyzed under similar conditions. In addition, no different bands were evident when gels containing rich 30S and poor 30S r-proteins were compared, suggesting no new or altered r-proteins were present. Analysis of 50S r-proteins by polyacrylamide gel electrophoresis revealed approximately 29 proteins, as compared to 22 found in E. coli 50S particles (19). No differences were seen in the protein patterns of rich or poor r-proteins. Although the one-dimensional analysis used does not afford the resolution obtained by two-dimensional techniques, it is apparent that there are no gross differences in the two types of 30S subunits with respect to protein composition. Many potentially interesting problems have been uncovered by this work. It is obvious that the regulation of protein synthesis in gram-positive organisms does not follow the guidelines established for E. coli. But, even in E. coli systems, the evidence for translational control is increasing. The specificity shown here with regard to 30S control may prove to be even more interesting, especially in relation to the rate of translation or possibly through a direct change in the efficiency of the 30S subunit. ACKNOWLEDGMENTS We thank P. S. Sypherd for his critical review of this manuscript. This work was supported by Public Health Service grants FR-7036 from the Division of Research Facilities and Resources and FD-00585 from the Food and Drug Administration.
LITERATURE CITED 1. Brunschede, H., and H. Bremer. 1971. Synthesis and
breakdown of proteins during amino-acid starvation. J. Mol. Biol. 57:35-57. 2. Clark, B. F. C., and K. A. Marcker. 1966. N-formyl-methionyl-s ribonucleic acid and chain initiation in protein biosynthesis: polypeptide synthesis directed by a bac-
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teriophage ribonucleic acid in a cell-free system. Nature (London) 211:378-380. 3. Craven, G. R., P. Voynow, S. J. S. Hardy, and C. G. * Kurland. 1969. Ribosomal proteins of E. coli. B. Chemical and physical characterization of 30S proteins. Biochemistry 8:2906-2915. 4. Deusser, E. 1972. Heterogeneity of ribosomal populations in E. coli cells grown in different media. Mol. Gen. Genet. 119:249-258. 5. Eisenstadt, J., and P. Lengyel. 1966. FormylmethionyltRNA dependent amino acid incorporation in extracts of trimethoprim-treated Escherichia coli. Science 154:524-527. 6. Engbaek, F., N. 0. Kjeldgaard, and 0. Maal0e. 1973. Chain growth rate of ,B-galactosidase during exponential growth and amino acid starvation. J. Mol. Biol. 75:109-118. 7. Forchhammer, J., and L. Lindahl. 1971. Growth rate of polypeptide chains as a function of the cell growth rate in a mutant of Escherichia coli 15. J. Mol. Biol. 56:563-568. 8. Goldman, E., and H. F. Lodish. 1972. Specificity of protein synthesis by bacterial ribosomes and initiation factors: absence of change after T4 infection. J. Mol. Biol. 67:35-47. 9. Infante, A. A., and R. Baierlein. 1971. Pressure-induced dissociation of sedimenting ribosomes: effect on sedimentation patterns. Proc. Natl. Acad. Sci. U.S.A. 68:1780-1785. 10. Jacobson, M., and C. Hedgcoth. 1970. Levels of 5,6-dihydrouridine in relaxed and chloramphenicol transfer ribonucleic acid. Biochemistry 9:2513-2519. 11. Kurland, C. G. 1972. Structure and function of the bacterial ribosome. Annu. Rev. Biochem. 197T: 377408. 12. Kurland, C. G., P. Voynow, S. J. S. Hardy, L. Randall, and L. Lutter. 1969. Physical and functional heterogeneity of E. coli ribosomes. Cold Spring Harbor Symp. Quant. Biol. 34:17-24. 13. Lucas-Lenard, J., and F. Lipmann. 1966. Separation of three microbial amino acid polymerization factors. Proc. Natl. Acad. Sci. U.S.A. 56:1562-1566. 14. Maaloe, O., and N. Kjeldgaard. 1966. Steady states of
1143
growth, p. 70-96. In 0. Maaloe and N. Kjeldgaard (ed.), Control of macromolecular biosynthesis. Benjamin, New York. 15. Mao, J. 1967. Protein synthesis in a cell-free extract from Staphylococcus aureus. J. Bacteriol. 94:80-86. 16. Nirenberg, M. W., and J. H. Matthaei. 1961. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polynucleotides. Proc. Natl. Acad. Sci. U.S.A. 47:1588-1602. 17. Phillips, L. A., B. Hotham-Iglewski, and R. M. Franklin. 1969. I. Effects of monovalent cations on the distribution of polysomes, ribosomes and ribosomal subunits. J. Mol. Biol. 40:279-288. 18. Shatkin, A. J. 1969. Colorimetric reactions for DNA, RNA, and protein determinations, p. 231-237. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press, New York. 19. Traut, R., H. Delius, C. Ahmad-Zadeh, T. A. Bickle, P. Pearson, and A. Tissieres. 1969. Ribosomal proteins of E. coli: stoichiometry and implication for ribosome structure. Cold Spring Harbor Symp. Quant. Biol. 34:25-38. 20. van Dijk-Salkinoja, M. S., and R. J. Planta. 1971. Rate of ribosome production in Bacillus licheniformis. J. Bacteriol. 105:20-27. 21. van Duin, J., and P. H. van Knippenberg. 1974. Functional heterogeneity of the 30S ribosomal subunit of Escherichia coli. HI. Requirement of protein S1 for translocation. J. Mol. Biol. 84:185-195. 22. van Duin, J., P. H. van Knippenberg, and M. Dieben. 1972. Functional heterogeneity of the 30S ribosomal subunit of Escherichia coli. II. Effect of S21 on initiation. Mol. Gen. Genet. 116:181-191. 23. van Duin, J., and C. G. Kurland. 1970. Functional heterogeneity of the 30S ribosomal subunit of E. coli. Mol. Gen. Genet. 109:169-176. 24. von Ehrenstein, G., and F. Lipmann. 1961. Experiments on hemoglobin biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 47:941-950. 25. Wittman, H. G., and G. Stoffler. 1972. Structure and function of bacterial ribosomal proteins, p. 285-351. In L. Bosch (ed.), The mechanism of protein synthesis and its regulation. American Elsevier, New York.
Vol. 122, No. 3 Printed in U.SA.
Translational Control of Protein Synthesis in Staphylococcus aureus' SCOTT E. MARTIN2 AND JOHN J. IANDOLO* Division of Biology, Kansas State University, Manhattan, Kansas 66506 Received for publication 27 January 1975
The calculated in vivo polypeptide chain growth rate for Staphylococcus aureus MF-31 grown in nutritionally rich medium assuming all the ribosomes were functional was found to be approximately 16 amino acids/s/ribosome, but decreased to 10.2 amino acids/s/ribosome for cells grown in poor medium. An in vitro analysis revealed that 70S ribosomes isolated from rich medium cells were more active than similar 70S ribosomes derived from cells grown in poor medium. The 30S subunit was found responsible for the increased activity of the rich monosomes, whereas the 50S subunit appeared to be capable of either high or low activity. The composition of the growth medium has been shown to affect the rate of growth and the macromolecular composition of numerous procaryotic organisms. However, such studies have been limited to those genera possessing sufficient metabolic diversity to grow over a wide nutritional range. For example, when grown in nutritionally poor medium, Salmonella typhimurium (6, 14) and Bacillus lichiniformis (20) exhibited slower generation times and were found to contain less deoxyribonucleic acid, ribonucleic acid (RNA), and protein than when grown in nutritionally rich medium. In spite of these changes, the rate of amino acid polymerization (step time-time required to add an amino acid residue to a growing polypeptide chain) was found to be constant (approximately 66 ms) regardless of growth rate when between 0.5 and 1.9 doublings per h. On the other hand, when Escherichia coli 15 is grown at less than 1 doubling per h (7) or when S. typhimurium is starved for amino acids (6), a significant decrease in the average step time was observed. These observations suggest that ribosomes may be capable of more than an "all or none" function, and that they may be instrumental in controlling the rate of protein synthesis (i.e., translational control) under adverse environmental conditions. The possibility of distinct "classes" of ribosomes, in concert with changes in the distribution of ribosomal proteins that occur at vastly different growth rates, also suggests that the rates of polypeptide biosynthesis might be al-
tered as the growth rate changes. The heterogeneity of 30S ribosomal proteins in E. coli (3, 25) may indicate that different classes of 30S subunits exist with respect to functional abilities (11, 12). Furthermore, one or more of these classes might be enhanced as growth conditions are altered. In fact, two classes of 30S particles have been demonstrated, one able to complex with the synthetic messenger polyuridylic acid [poly(U) 1, and the other unable to interact with poly(U), presumably being dependent on the presence of the fractional protein 30S-1 (23). These data, however, are tentative, since S1 may be lost during purification (21). Nevertheless, an "initiating ribosome" has been found to be distinguishable from a "propagating ribosome," due to the presence, or absence, of protein 30S-21 (22). The number of either class may depend upon the rate of growth, since different growth media can influence the distribution of ribosomal proteins (4). Duesser (4) has found that two- to threefold greater amounts of proteins 30S-6, 30S-21, and 50L-12 were present in ribosomal populations isolated from cells grown in rich medium as compared to those obtained from cells grown in minimal medium. Because of the growth restrictions imposed by limiting media, nutritionally fastidious bacteria have not been studied in the manner described above. Such studies, however, are necessary to afford a better understanding of the regulation of protein synthesis in these organisms. Since fastidious organisms do not possess the metabolic alternatives available to more diverse microorganisms, the regulatory constraints on ' Contribution no. 1241, Kansas Agricultural Experiment biosynthesis are also likely to be less compliStation, Manhattan, Kan. 66506. 2Present address: Department of Medical Microbiology, cated. The elucidation of the mechanisms involved can provide important comparisons for University of Califomia at Irvine, Irvine, Calif. 92664. 1136
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PROTEIN SYNTHESIS IN S. AUREUS
1137
Ribosomes were removed from the S-30 by centrifustudies in higher organisms. To achieve these goals, our approach utilized Staphylococcus gation for 2 h at 152,000 x g in a Beckman 5OTi rotor aureus grown in dissimilar nutritional condi- (48,000 rpm). The top 2/3 of the resulting supernatant (S-100) was aspirated and pooled. The S-100 was tions. dialyzed overnight against 500 volumes of 10 mM
MATERIALS AND METHODS Growth of bacteria. S. aureus MF-31, which was coagulase positive and B-hemolytic, was maintained on Trypticase soy agar slants at 4 C. Experiments were performed on cells grown in rich medium-trypticase soy broth (TSB), or a poorer defined medium (SDM). TSB contained the following (per liter): 17 g of Trypticase (GIBCO), 3 g of phytone (GIBCO), 5 g of NaCl, 2.5 g of K,HPO4, and 2.5 g of glucose. SDM was composed of 3 g of vitamin-free Casamino Acids (Difco) and 0.75 ml of minimal essential medium vitamins (GIBCO) per liter. For in vitro protein synthesis experiments, shake cultures of exponential-phase cells (300 ml) in the appropriate medium were inoculated into 37 liters of fresh medium at 37 C and grown under forced aeration. When turbidity was equal to 30 Klett units (4.4 x 107 cells/ml) in rich medium or 16 Klett units (4.2 x 107 cells/ml) in poor medium aeration was halted, and the culture was cooled in an ice bath. The cells were collected at 4 C in a DeLaval Cream Separator and resuspended in cold tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.8) (10 mM Tris, 50 mM NH4Cl, 10 mM magnesium acetate, 0.1 mM dithiothreitol [DTTJ-designated 10 mM Mg2+ buffer). The resuspended cells were pelleted by centrifugation for 10 min at 10,400 x g, washed two times in 10 mM Mg2+ buffer, and frozen in a dry ice-ethanol bath. The frozen cell pellets were stored at -70 C. For the quantitative analysis of cellular components and for the determination of growth curves, cells were grown under aeration at 37 C in 300 ml of medium. Inocula were taken from exponentially growing cultures in the appropriate medium. Preparation of cell fractions. Washed cells were thawed, and a 50% (wt/vol) suspension was made by the addition of 10 mM Mg2+ buffer. The suspension was warmed to 37 C, and 300 ;&g of lysostaphin (Schwarz/Mann) and 14 sg of deoxyribonuclease I (Sigma) were added per g of wet cells. After vigorous shaking for 10 min at 37 C, the lysate was cooled in an ice bath, and the cellular debris was removed by two centrifugations for 10 min each at 27,000 x g. The supernatant (S-30) was then mixed with an equal volume of preincubation mix and gently shaken. The final concentration of the reagents in the diluted preincubation mix was: 160 mM Tris-hydrochloride (pH 7.5), 10 mM magnesium acetate, 50 mM NH4Cl, 0.1 mM DTT, 5 mM adenosine triphosphate (potassium salt-ATP), 18 Mg of pyruvate kinase per ml, 1.1 mg of phosphoenol pyruvate (potassium salt-PEP) per ml, and 0.15 mM each of the following amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, hydroxyproline, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. After 30 min, the S-30 was cooled and centrifuged for 10 min at 27,000 x g.
Mg2+ buffer, divided into 1-ml aliquots, frozen in a dry ice-ethanol bath, and stored at -70 C. The remaining S-100 was removed and discarded. Ribosomes were resuspended in 10 mM Mg2+ buffer and clarified by centrifugation at 27,000 x g for 10 min. After pelleting, the ribosomes were washed twice in 10 mM Mg2+ buffer and finally resuspended in a minimal volume of 10 mM Mg2+ buffer. Any aggregates were removed by centrifugation at 27,000 x g for 10 min, and the resulting supematant fluids were treated in one of two modes. If 70S ribosomes were desired, the suspension was dialyzed overnight against 1,000 volumes of 10 mM Mg2+ buffer. Dialyzed 70S ribosomes were divided into aliquots, frozen in a dry ice-ethanol bath, and stored at -70 C. The concentration of the ribosomes was determined by assuming an extinction of 19 absorbance units per mg at 260 nm (A21*) (15). When subunits were desired, the ribosomal suspension was dialyzed for 24 h against 2,000 volumes of Tris-hydrochloride buffer (pH 7.8) (10 mM Tris, 50 mM NH4Cl, 0.1 mM magnesium acetate, 0.1 mM DTT-designated 0.1 mM Mg2+ buffer). The dialyzed ribosomal subunits were then separated at 25,000 rpm for 12 h on 36-ml gradients (SW-27 rotor) of 10 to 30% sucrose in 0.1 mM Mg2+ buffer. Fractions containing the appropriate subunits were pooled and concentrated by centrifugation at 152,000 x g for 18 h (5OTi rotor). The ribosomal subunit pellets were suspended in 10 mM Mg2+ buffer, frozen in a dry ice-ethanol bath, and finally stored at 70 C. Purity of the subunit preparations was determined by analysis on 5-ml, 10 to 30% sucrose gradients in a Beckman SW50.1 rotor for 3.5 h at 35,000 rpm. Salt-washed ribosomal subunits were prepared by the addition of high salt Tris-hydrochloride buffer (10 mM Tris, 10 mM magnesium acetate, 0.5 or 1.0 M NH4Cl, and 0.1 mM DTT-designated high salt buffer) to purified subunit suspensions. After 1 h at 4 C, the salt-washed subunits were collected by centrifugation in a Beckman SW50.1 rotor for 6 h at 45,000 rpm. The top 2/3 of the supernatant was removed and saved; the lower 1/3 was discarded. The pellet was suspended a second time in 5 ml of high salt buffer and allowed to stand ovemight at 0 C. The washed subunits were then pelleted, and the top 2/3 of the supernatant was removed and combined with the first upper supernatant. After discarding the lower 1/3, the washed particles were suspended in 10 mM Mg2+ buffer and frozen. The combined supernatants were concentrated with dry G-15 Sephadex, dialyzed against 10 mM Mg2+ buffer, and stored at -20 C. Preparation of tRNA. The procedures followed were slight modifications of those of von Ehrenstein and Lipmann (24) and Jacobson and Hedgcoth (10). The ether-washed transfer RNA (tRNA) was dissolved in water, made 2% potassium acetate, and precipitated for 2 h with 3 volumes of 95% ethanol. Residual ethanol was then removed by lyophilization. The tRNA precipitate was dissolved in water, divided into aliquots, and stored at -20 C.
1138
MARTIN AND IANDOLO
In vitro protein synthesis. The procedures followed were modifications of those by Mao (15) and Nirenberg and Matthaei (16). Solution I contained 100 mM Tris, 220 mM magnesium acetate, 500 mM NH4Cl, 10 mM ATP, and 0.45 mM each of guanosine triphosphate, cytidine triphosphate, and uridine triphosphate (pH 7.6). Solution II was prepared fresh daily and contained 0.5 ml of solution I, 0.022 ml of 1 mM DTT, 0.5 ml of 75 mM PEP, 125 ug of pyruvate kinase, 0.5 ml of a 0.6 mM amino acid solution (preincubation mixture, minus phenylalanine), 0.02 ml of [3 H]phenylalanine (Schwarz/Mann, 6.0 Ci/ mmol, side chain 2, 3-3H) or L-["C]phenylalanine (Schwarz/Mann, 460 mCi/mmol, uniformly labeled), and approximately 2.6 mg of tRNA. The standard reaction mixture (0.325 ml) was prepared by adding the following components in sequence: 0.15 ml of solution II, 0.01 ml (150 Mg) of poly(U) (Miles Laboratories), ribosomes at concentrations of 420 ,g (8 A2,0 units) of 70S ribosomes, or 250 Mg (4.75 A2*0 units) of 30S subunits followed by 125 Ag (2.38 A,*o units) of 50S subunits, and S-100 fraction from poor medium cells (0.1 ml, approximately 500 ug of soluble protein); the volume was then adjusted to 0.325 ml with 10 mM Mg2+ buffer. The final concentrations in the reaction mixture were 12.8 mM Tris, 22 mM magnesium acetate, 64 mM NH4Cl, 0.78 mM ATP, 0.04 mM each of guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, 0.054 mM DTT, 5.86 mM PEP, 6.3 zg of pyruvate kinase, 0.046 mM each of 20 amino acids, 1 MCi of ['HJphenylalanine, 150 ;g of tRNA, and 150 ug of poly(U). After addition of the S-100, the reaction tubes were removed from the ice bath and gently agitated at 37 C for 30 min. The reaction was halted by the addition of 2 ml of cold 10% trichloroacetic acid. The tubes were then heated for 20 min at 90 to 95 C and subsequently placed in ice for 30 min. The labeled precipitate was collected on membrane filters (HA; 25 mm; Millipore Corp.), washed with 25 ml of cold 5% trichloroacetic acid, and dried at 80 C. Radioactivity was measured in a Beckman LS150 Liquid Scintillation Spectrometer. Rate studies were performed in the manner described above, except that uniformly labeled ["C ]phenylalanine was used. At appropriate times, 0.025-ml samples were removed and mixed with 2 ml of cold 10% trichloroacetic acid and treated as described. Determination of the macromolecular components. The techniques used for quantitation of the macromolecular components were modifications of those described by Shatkin (18) and van Dijk-Salkinoja and Planta (20). Cells were grown in each medium with shaking at 37 C. When growth reached the appropriate level (cell number was determined by pour plating on Trypticase soy agar), the cells were collected by filtration through 0.45-Mm membrane filters (Millipore Corp.). The filters were washed, and the cells were rinsed from the filters. An equal volume of cold 10% trichloroacetic acid was added, and the mixture was allowed to stand in ice for 1 h. The precipitates were centrifuged for 20 min at 2,000 x g,
J. BACTERIOL.
and the supernatant solution was saved for analysis. The precipitates were suspended in 5% trichloroacetic acid and heated for 45 min at 95 C. The heated solutions were cooled and centrifuged for 20 min at 2,000 x g. The percentage of RNA found in the ribosomal fraction (orcinol test) was determined on lysostaphin lysed cultures. After removal of cellular debris by low-speed centrifugation, the ribosomes were collected by centrifugation at 152,000 x g for 18 h (SW50.1 rotor). RNA was then determined in the pellet and supernatant fractions. The concentration of deoxyribonucleic acid in the supernatants was determined by the diphenylamine reaction. The remaining precipitate was hydrolyzed by heating overnight at 45 C in 1 ml of 1 N NaOH. The protein concentration in the hydrolyzed residue, as well as that of the supernatant, was determined by the Lowry procedure, utilizing the Folin-Ciocalteau phenol reagent. Lysozyme (Sigma, grade I) was used as the standard.
RESULTS Determination of the in vivo rate of protein synthesis. S. aureus MF-31 was grown at 37 C in rich medium (TSB) and poor medium (SDM). A typical growth curve and the point at which cells were studied are shown in Fig. 1. During the exponential phase of growth, the rich medium culture had a generation time of 20 4 2 min, whereas in poor medium the generation time was about three times greater (60 : 6
min). To estimate the in vivo step times (20), a determination of the amounts of RNA and protein per cell was performed (Table 1). Cells grown in rich medium contained more abundant amounts of all macromolecules. From these data, and the observation that, respectively, 76% and 62% of the total RNA was present as ribosomal RNA in rich and poor medium-derived cells, an estimation of the number of ribosomes per cell was possible (Table 1). Cells grown in rich medium contained approximately 83,200 ribosomes/cell, whereas poor medium-derived cells had only 25,600 ribosomes/cell. Evaluation of the number of ribosomes per cell allowed an estimation of the in vivo protein synthesizing efficiency of cells grown in both media (Table 2). Assuming all the ribosomes were functional, cells grown in rich medium polymerized amino acids at a rate of 16 amino acids/s/ribosome in comparison to poor medium-derived cells whose polymerization rate was 10.2 amino acids/s/ribosome. These data showed that rich medium cells synthesized protein about 1.6 times faster than poor medium cells and suggested that translational modulation of protein synthesis may occur.
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PROTEIN SYNTHESIS IN S. AUREUS
subunits isolated from cells grown in the other medium) showed that 30S ribosomal particles were responsible for the increased activity (Table 3, experiment C). When rich 30S subunits (subunits derived from cells grown in rich medium) were incubated with poor 50S subunits (subunits derived from cells grown in poor medium), approximately 1.60 ± 0.34 times as much isotope was incorporated than in the control poor homologous system. The reciprocal mixture consisting of poor 30S subunits and rich 50S particles directed approximately the same it amount of incorporation as found in the control poor homologous system. Thus the 30S subunits :q, from cells grown in poor medium were responsic ble for the lower level of poly(U)-directed [8H Iphenylalanine incorporated. Rate studies over a time period of 100 min were performed on both homologous and heterologous systems (Fig. 2). During the first 30 min in the presence of rich 30S subunits, the rate of incorporation for both systems remained constant and was approximately 3.3 x 10-14 mol of phenylalanine/min. The average rate of incorporation in the two poor 30S subunit systems 0 /00 200 300 400 also remained constant, but for 60 min, and was Miniutes approximately 2 x 10-14 mol of phenylalanine/ FIG. 1. Growth of Staphylococcus aureus MF-31 min. In the initial period of constant rate, the in nutritionally rich or poor medium. Growth was rich 30S particle directed incorporation at 1.6 monitored with a Klett Summerson Colorimeter. times the rate of the poor 30S particle. However, Symbols: 0, growth in rich medium; 0, growth in after 100 min of incubation, the amount of poor medium; Ft, point of harvest for in vivo and in phenylalanine polymerized was approximately vitro studies. the same for all preparations, although poor 30S Determination of in vitro protein synthe- subunit systems never reached the levels sizing capacity. To confirm the in vivo results, achieved by the rich 30S subunit systems. As ribosomes isolated from cells grown in either TABLE 1. Macromolecular composition of medium were assayed for their ability to incorexponential-phase Staphylococcus aureus cells porate [3HJphenylalanine into polypeptide as Growth mediuma directed by the synthetic messenger RNA Experimental poly(U). Incubation at 37 C for 30 min revealed Rich Poor that 70S ribosomes derived from rich medium cells were 1.33 0.10 times as active as were 70S ribosomes derived from poor medium cells (Table 3, experiment A). These data as well as the following regarding in vitro protein synthesis represent averages of at least six individual experiments, each originating with different batches of cells. To determine whether this effect was subunit mediated, the activity of the system was tested using purified ribosomal subunits. Homologous systems composed of ribosomal subunits from rich medium cells directed incorporation of approximately 1.88 0.63 times as much phenylalanine as did poor homologous systems (Table 3, experiment B). Heterologous systems (systems composed of 30S subunits isolated from cells grown in one medium and 50S
2.96 4 0.29 + 4.78 + 3.26 4
Growth rateb DNA/cellc Protein/cellc RNA/cellc % rRNAd
rRNA/cellc Calculated no. of ribs/celle a
Mean
76 2.48 83,200
E 4
i
0.34 0.05 0.73 0.22
1.00 4 0.20 s 2.81 ± 1.21 ±
2 63 0.22 0.76 7,400 25,600
+
0.10 0.02 0.35 0.15 1 0.15 5,000
standard error of five experiments, each
analyzed in triplicate. ° Generations per hour. cGrams per cell x 10-3. DNA, Deoxyribonucleic acid. d Percentage of ribosomal RNA (rRNA) calculated as described in Materials and Methods. e Calculated by dividing the amount of ribosomal RNA by the weight of the ribosomal RNA in a 70S ribosome (2.98 x 10- Is g).
J. BACTERIOL.
MARTIN AND IANDOLO
1140
TABLE 2. Protein synthesizing efficiency and step times of ribosomes from exponential-phase Staphylococcus aureus cells Medium
rich poor
Generation (min) time
20 60
±
+
2 6
Cell protein
4.78 2.81
+ +
0.73 0.35
Ribs/cell
83,200 + 7,400 25,600 ± 5,000
Efficiency ribosomesaof
19.9
±
12.7
+
3.8 2.9
acids/ Amino ribosome/s 16 10.2
a Calculated from the equation k = 0.692 (PIRt) where k is the efficiency, P is the amount of protein/cell, R is the number of ribosomes/cell, and t is the generation time. Results are expressed as grams of protein per minute per ribosome 10-20.
TABLE 3. Polyuridylic acid-dependent phenylalanine incorporation by 70S ribosomes and ribosomal subunits derived from cells grown in rich and poor mediuma Ribosomal components
Phenylalanine incorporated (counts/min)b
A
70S rich 70S poor
8,273 4 536 6,233 i 687
B
30S rich + 50S rich 30S poor + 50S poor
4,436 ± 185 2,509 66
C
30S rich + 50S poor 30S poor + 50S rich
4,221 61 2,656 ± 95
Expt
±
a The incorporation of [3H]phenylalanine (6 Ci/ mmol) was performed as described using 8 A,60 units of 70S ribosomes or 4.75 A..0 units of 30S subunits and 2.38 A,.0 units of 50S subunits. Incubation was at 37 C for 30 min. IAverage of at least six individual experiments, each originating with different batches of cells.
50 75 MINUTES
FIG. 2. Rate of polyuridylic acid-dependent phenwas the case in the heterologous systems, the ylalanine incorporation utilizing homologous and hetsource of the 50S subunits seemed to have little erologous subunits derived from cells grown in rich or poor medium. The poly(U)-dependent incorporation effect. To determine if the observed difference in of ['4Clphenylalanine (460 mCi/mmol) was performed using 0.5 1sCi of ["4CJphenylalanine, 4.75 activity of the two sources of 30S subunits was as described units 30S ribosomes, and 2.38 A,.0 units of 50S of A,,, due to a smaller proportion of poor 30S particles ribosomes. Incubation at 37 C. At appropriate being able to successfully interact with poly(U) time intervals, 0.025-mIwas samples were removed and and 50S subunits, 70S ribosomes from cells added to 2 ml of ice-cold 10% trichloroacetic acid and grown in both media were mixed just prior to subsequently assayed as previously described. Symincubation, or, alternatively, whole cells were bols: 0, 30S rich + 50S rich; *, 30S poor + 50S poor; mixed before lysis (Table 4). If only the propor- A, 30S rich + 50S poor; A, 30S poor + 50S rich. tion of "active" 30S subunits differed, then in the post-lysis mixture one would predict an factors (8), and therefore possibly fortuitiously activity halfway between that found for rich bound cytoplasmic proteins. One method to and poor medium-derived controls, and in the remove such proteins is to wash ribosomes in pre-lysis mixture a number slightly greater than buffers containing NH4Cl (8). If the difference midway. Such a situation was not found. observed between the ribosomes derived from Rather, these data suggest that the polymeriza- cells grown in either medium was due to nontion rate was controlled by the activity of the specific binding of a nonribosomal inhibitory poor 30S containing monosome, as the rate protein(s), then this should be removed by would be dependent upon the slowest compo- washing in NH4C1. When S. aureus ribosomes were washed in either 0.5 or 1.0 M NH4Cl, they nent of the system. Ribosomal subunits prepared as described lost their poly(U)-directed polyphenylalanine here have been found to carry soluble initiation synthesizing capacity. Experiments showed
PROTEIN SYNTHESIS IN S. AUREUS
VOL. 122, 1975
that this was due to the removal of several ribosomal proteins in the high salt wash. However, when those proteins removed by the high salt wash of the poor 30S subunit were added to functional systems, no inhibitory effect on either rich or poor 30S subunit systems was noted.
DISCUSSION The observation that S. aureus MF-31 grew with differing growth rates in rich and poor medium offered a system to study the regulation of protein synthesis in a fastidious, grampositive organism. The step time for S. aureus grown in rich medium, having a doubling time of 20 min, (62 ms), was very close to the step times calculated for Salmonella typhimurium (67 ms) and for Bacillus licheniformis (52 ms) when these organisms were grown with doubling times between 30 and 120 min (6, 14, 20). The similar value obtained increases the reliability of the result and suggested that the step time for S. aureus grown in poor medium with a doubling time of 60 min (100 ms) was also valid. These estimates are minimal values, however, as they do not include measurements of the number of active ribosomes (11). Estimation of the number of active ribosomes is often determined by polysome analysis in sucrose gradients and assumes that all the ribosomes in a polysome are actives (7). The conditions necessary to lyse S. aureus (incubation at 37 C for 10 min) unfortunately TABLE 4. Polyuridylic acid-dependent phenylalanine incorporation by post-lysis and pre-lysis mixturesa Expt
Ribosomal components
Phenylalanine incorporated (counts/min)
A
70S rich 70S poor 1/2 70S rich + /2 70S poor
7,759 6,512 6,538
B
30S rich + 50S rich 30S poor + 5OS poor Pre-lysis mixture
6,038 4,234 4,296
aThe incorporation of [3H]phenylalanine (6 Ci/ mmol) was performed as described. Incubation was at 37 C for 30 min. In experiment A, the mixture contained 4 A2*0 units of both 70S rich and 70S poor ribosomes, whereas the control systems contained 8 A2.0 units of ribosomes derived from cells grown in either medium. In experiment B, equal quantities (wet wt) of rich and poor medium-grown cells were mixed before lysis and simltaneously carried through the isolation and purification procedures with the control systems.
1141
preclude such estimation, although these measurements are subject to some question. Both the ionic environment of bacterial lysis (17) and the speed of centrifugation (9) also have been shown to influence the distribution of polysomes, monosomes, and ribosomal subunits within a sucrose gradient. In spite of the possible errors inherent in these calculations, the in vivo results with S. aureus suggested that ribosomes in rich medium-grown cells were more efficient than those in poor medium cells. This observation was in contrast to those of Engbaek et al. (6) and van Dijk-Salkinoja and Planta (20), who found that the growth medium, and hence the rate of growth up to approximately 120-min doubling times, had no effect on the polypeptide chain growth rate. Variations of the quantities of macromolecular components with the generation time were also comparable to those reported for S. typhimurium (14) and B. licheniformis (20): cells grown in poor medium contained less deoxyribonucleic acid, RNA, and protein than cells grown in rich medium. In conjunction with the lowered amount of RNA, cells grown in poor medium also had a smaller percentage of their RNA as ribosomal RNA. Both the test organism and the composition of poor medium could have influenced these differences. S. aureus was the most fastidious of the three organisms compared, and therefore its growth requirements, or their limitations, could have had more pronounced effects on the entire organism. If the cells were in a starved, or near starved, condition, the step times might have been adversely influenced. For example, Brunschede and Bremer (1) found that the in vivo step time of methionine and proline auxotrophs of E. coli was increased from 60 to 1000 ms during starvation for required amino acids. In this study, however, starvation was not evident in the poor medium since the organism was harvested at the midpoint of exponential growth and had the potential to double for two generations after the time of cell harvest. The results found in vitro, utilizing 70S ribosomes, are consistent with the in vivo observations. That is, 70S ribosomes from rich medium cells were more active in the synthesis of polyphenylalanine than were 70S ribosomes isolated from cells grown in poor medium. It seems unlikely that factors involved during initiation were influencing these results since the poly(U) message circumvents the requirement for soluble protein initiation factors (8), and the high magnesium concentration (22 mM) eliminated the need for formylmethionyl tRNA (2, 10). In addition, both rich and poor
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70S ribosomes were incubated with the poor S-100 fraction; similar differences were also evident when the rich S-100 fraction was utililized. The soluble elongation and termination factors were present in the same amounts in both systems, and therefore also not directly involved in the differences observed between the two 70S ribosomes. Moreover, the same standard reaction mixture was used with both systems, indicating that the energy-generating components, the ionic conditions, and all other related in vitro environmental components were identical. It is possible therefore to conclude that the reduced step time of the 70S poor ribosomes was due to the 70S ribosomes themselves and not to other variables. The subsequent observation that homologous subunit systems (30S and 50S particles isolated from cells grown in the same medium) possessed similar activities as found with 70S intact ribosomes also supported the hypothesis that rich ribosomes functioned more efficiently than poor ribosomes. Experiments involving heterologous subunit systems implied that the 30S particle was responsible for the altered activity of the entire systm. In all cases, the presence of the 30S subunit, isolated from cells grown in rich medium, stimulated the system to incorporate more phenylalanine than did systems containing poor 30S subunits. Further support that 30S subunits were important in determining activity was found in the kinetic experiments involving homologous and heterologous subunit systems. The source of the 30S particle dictated the rate of incorporation of phenylalanine, whereas the source of the 50S subunit seemed unimportant. Systems containing rich 30S particles incorporated isotope at a faster rate than did poor 30S subunit systems. Whereas the initial rates differed, both systems reached approximately the same levels after 100 min of incubation, indicating that, given enough time, the poor 30S subunits were capable of the same maximal amount of incorporation as were rich 30S particles. This implied that the differences found were due to the activity levels imposed by the 30S subunit, and not to substrate limitation, the presence of a unique nuclease or protease, or any other factor peculiar to one system. These findings are consistent with the theory of Kurland (11), who suggested that 30S subunits of E. coli were heterogeneous with respect to their ribosomal (r)-proteins, and hence possessed possible differences in functionality. They similarly corroborate the finding that E. coli 50S subunits are not heterogeneous in their protein composition and therefore probably not in their functional capabilities (11).
One explanation for the reduced activity of the poor 30S particles might be the presence of a new r-protein or the alteration of one or more existing r-proteins. Nevertheless, sucrose gradient co-cenrifugation of rich and poor 30S particles revealed that the two subunits were similar with respect to their molecular weights, as they sedimented in the same fraction (unpublished data). Neither particle contained gross amounts of different or contaminating proteins, although it is unlikely that this method would be sensitive to small protein differences. However, more detailed analysis by polyacrylamide gel electrophoresis (data not presented) resulted in only 16 bands in gels containing protein samples derived from either subunit. This number was identical to that reported by Traut et al. (19) for E. coli 30S r-proteins analyzed under similar conditions. In addition, no different bands were evident when gels containing rich 30S and poor 30S r-proteins were compared, suggesting no new or altered r-proteins were present. Analysis of 50S r-proteins by polyacrylamide gel electrophoresis revealed approximately 29 proteins, as compared to 22 found in E. coli 50S particles (19). No differences were seen in the protein patterns of rich or poor r-proteins. Although the one-dimensional analysis used does not afford the resolution obtained by two-dimensional techniques, it is apparent that there are no gross differences in the two types of 30S subunits with respect to protein composition. Many potentially interesting problems have been uncovered by this work. It is obvious that the regulation of protein synthesis in gram-positive organisms does not follow the guidelines established for E. coli. But, even in E. coli systems, the evidence for translational control is increasing. The specificity shown here with regard to 30S control may prove to be even more interesting, especially in relation to the rate of translation or possibly through a direct change in the efficiency of the 30S subunit. ACKNOWLEDGMENTS We thank P. S. Sypherd for his critical review of this manuscript. This work was supported by Public Health Service grants FR-7036 from the Division of Research Facilities and Resources and FD-00585 from the Food and Drug Administration.
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