Bacillus licheniformis

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1 JOURNAL OF BACTERIOLOGY, January 1971, p Vol. 105, No. I Copyright 1971 American Society for Microbiology Printed in U.S.A. Rate of Ribosome Production in Bacillus licheniformis M. S. VAN DIJK-SALKINOJAt AND R. J. PLANTA Biochemisch Laboratorium, Vrije Universiteit, de Boelelaan 1085, Amsterdam, The Netherlands Received for publication 30 September 1970 The ribosome content of exponential-phase cells of Bacillus licheniformis was measured at three different growth rates. The average number of ribosomes per cell was about 92,000, 34,400, or 12,500 70S equivalents in balanced cultures growing at 37 C with generation times of 35, 60, and 120 min, respectively. Since the ribosomal particles were shown to be metabolically stable in exponentially growing cells, these figures implicate large differences in the quantity of ribosomes synthesized per unit of time in an individual cell grown under the various conditions. Nevertheless, the time required for the biosynthesis of a single 50S subunit was constant (about 10 min) and independent of the specific growth rate of the cell (within the limits studied). These results show that ribosome production is not regulated by control of the rate of assemblage of individual ribosomes, but rather by control of the number of the ribosomes in manufacture at a time. The ribosome content of bacterial cells is a function of growth rate (7, 16). During a shift to a higher growth rate, a cell increases its content of ribosomes (7). The mechanism of the regulation of ribosome formation is, however, still unknown. It has been proposed (12) that at higher growth rates the assembly of single 305 and 50S subunits will take place more rapidly. Nevertheless, no direct measurements of the kinetics of formation of single subunits as a function of growth rate have been made. In this paper such measurements are presented, together with measurements of the cellular ribosome contents, carried out for Bacillus licheniformis in balanced growth at three different growth rates. It is shown that the time required for the biosynthesis of a single subunit is independent of the growth rate of the cell. The results indicate that ribosome production is regulated by controlling the number of ribosomes in manufacture at a time. MATERIALS AND METHODS Chemicals. '4C-uracil (specific activity 55.4 mci/mmole) and 3H-uridine (specific activity 2.73 Ci/mmole) were purchased from the Radiochemical Centre (Amersham, England). Bovine pancreatic deoxyribonuclease (DN-C, type 1; EC ) and lysozyme (egg white, grade I; EC ) were obtained from Sigma Chemical Co. (St. Louis, Mo.); pancreatic lipase (EC ) (80,000 units/g) from E. Merck AG I Present address: Laboratorium von Chemische Physiologie, Vrije Universiteit, Amsterdam, The Netherlands. 20 (Darmstadt, Germany); and Pronase B grade from Calbiochem AG (Lucerne, Switzerland). The composition of the standard buffer was as follows: 10 mm tris(hydroxymethyl)aminomethane (Tris), 60 mm KCI, 10 mm magnesium acetate, 0.5 mm spermine, 0.75 mm spermidine, 1 mm dithiothreitol, and 100 1gg of chloramphenicol per ml. The ph was adjusted with HCI to 7.6 at 0 C. Organism and growth conditions. B. licheniformis S244 (wild type) was grown at 37 C in synthetic medium containing, per liter: 10 g of KH2PO4-3H20, 9 g of Tris, 2 g of NH4C1, I g of sodium citrate * 3H20, 0.2 g of MgSO4,7H,O, 40 mg of FeCl86H2O, and 1.6 mg of MnSO4*4H2O. Variation in growth rate was achieved by varying the carbon source. L-Glutamic acid (120 mg/liter or less) results in a doubling time of 120 min or more; glucose (5 g/liter) in addition to L-glutamic acid results in a doubling time of min, and a further supplementation with Casamino Acids (10 g/liter) in a doubling time of 35 i 2 min. The cultures were grown under vigorous aeration (2 liters of air per min per liter of culture) in a 7-liter laboratory fermentor (New Brunswick Scientific Co., Inc., New Brunswick, N.J.) except for the radioactive cultures. Growth of the cultures was followed by measuring the optical density in a Zeiss PMQ II spectrophotometer at 550 nm (pathlength I cm). In addition, viable cell counts were carried out at regular intervals. The cultures were diluted by 0.5 volume of fresh medium each time the culture reached a density of 2.0 x 108 cells/ml (in case of the 35-min culture, 1.2 x 108 cells/ml). In this way, balanced growth was maintained, as was judged from the fact that the dry weight and the macromolecular composition of the bacterial cell remained constant in each culture.

2 VOL. 105, RATE OF RIBOSOME PRODUCTION 21 The cultures were tapped off at the indicated cell densities (see Table 1) on frozen standard buffer under simultaneous addition of liquid N,. Preparation of labeled cells. The bacteria were grown at 37 C in a 6-liter flask under forced aeration with warm, moist air. The other conditions were identical to those employed for the culturing of unlabeled cells. The cells were labeled with '4C-uracil for at least 4 hr and then pulse-labeled with 'H-uridine for 75 sec. In some experiments no uniform labeling was done but only pulse-labeling for 5 min (see Fig. 2). The concentrations of the radioactive precursors are listed in legends to Fig. I and 2. Further incorporation was stopped by the addition of 3 volumes of warm medium and a large excess of unlabeled uridine (I g/liter). At appropriate times after the chase, samples of 50 to 250 ml were collected on frozen standard buffer under simultaneous addition of liquid N,. Lysis of cells and preparation of ribosomes. The bacteria were collected from the samples by centrifugation (10 min at 3,000 x g), washed twice with cold standard buffer, and stored in liquid N, until use. The cells were lysed with Brij-58 (0.5%, w/v) at 0 C after a 10-min exposure to lysozyme (0.5 mg/ml) at 0 C. The "membrane" fraction (separated from the "soluble" fraction by centrifuging the lytic mixture for 10 min at 7,000 x g) was solubilized by a combined digestion of lipase (65 isg/ml) with deoxyribonuclease (2 pg/ml) in the presence of Brij-58 (0.5%, w/v). Full details were given in a previous publication (17). Sucrose gradient sedimentation analysis. The "soluble fraction" (25-ml samples) and the "solubilized membranes" (10-mI samples) were analyzed by zonal centrifugation through a 10 to 40% exponential sucrose gradient (with a 75-ml cushion of 45% sucrose) in a Spinco B XIV titanium zonal rotor at 48,000 rev/min for 80 min. Details were given previously (17). Purified ribosome preparations were used in the radioactive experiments. These were obtained by centrifuging 1.5-ml samples of "soluble" and "solubilized membrane" fractions through 3 ml of 20% (w/v) sucrose (made in standard buffer with 0.5% Brij-58) for 3 hr at 64,000 rev/min in a Spinco SW65 titanium rotor. The transparent, nearly colorless ribosome pellet was suspended in standard buffer and then analyzed on linear 5 to 30% sucrose gradients in a Spinco SW25: I rotor. All sucrose gradients were made in standard buffer with the omission of dithiothreitol. Assay of radioactivity. Samples (0.8 ml) of purified ribosomes or 10-drop fractions from sucrose gradients were assayed for their contents of 'H and "IC in a Nuclear-Chicago scintillation counter (Mark I) with 15 ml of a dioxan scintillation fluid with 0.7% 2,5-diphenyloxazole, 0.005% 1,4-bis-2-(5-phenyloxazolyl)-benzene, and 9% naphthalene. Before measuring radioactivity, the ribosomes were digested with ribonuclease (20 Ag/ml, 3 hr at 37 C) and Pronase (15 sg/ml, 24 hr at 37 C). In this way the counting efficiency was enhanced and selfabsorption was eliminated. The counting efficiency was determined for each sample separately, making use of the channels ratio technique which relates counting efficiency to the ratio of net count rates produced by an external standard (1"Ba) in two separate analyzer channels. This method provides automatic quench correction data, enabling a calculation of the true number of disintegrations per minute for each sample. (For further details, see the instruction manual of the Mark I Nuclear- Chicago Scintillation Computer.) Other analytical procedures. Protein was determined by the biuret method (6) with pure lysozyme as a standard. Deoxyribonucleic acid (DNA) was assayed by the diphenylamine method (13) with highly polymerized salmon sperm DNA (Calbiochem) as a standard. The precipitation step was omitted in this procedure because the DNA had been digested during the solubilization of the membranes. Ribonucleic acid (RNA) was determined by the orcinol method (13) with purified ribosomes from B. licheniformis as a reference. The RNA content of the latter (53%, w/v) was first determined by a phosphorus assay by the method of Chen (2). Standardization of the orcinol method with a heterologous RNA preparation (such as commercial yeast RNA) is not sufficient for accurate measurements since the color development is known to be somewhat dependent on the base composition of the RNA assayed (9). The RNA content of pooled ribosomal fractions from sucrose gradients was estimated by assuming that an optical density of 25 at 260 nm corresponds to an RNA content of 1.0 mg/ml. This relation was found to be valid (in the optical density region of 0.05 to 1.5) for such preparations, as checked by phosphorus assay (2). The dry mass of 50-ml samples from balanced, exponentially growing cultures was determined by the ultrafiltering method (4). Viable cells were counted in the same samples by the plating method after an adequate dilution with cold sterile medium. The bacterial densities of the radioactive cultures were calculated from the optical density; a balanced culture, growing exponentially with a generation time of 35, 60, or 120 min, contains 1.3 x 10', 2.9 x 10', or 5.3 x 10' cells/ml, respectively, per one optical density unit (measured at 550 nm with 1-cm pathlength). RESULTS Relation of cellular amount of ribosomes to growth rate. To calculate the number of ribosomes per cell, information is required on (i) the average weight of an individual cell, (ii) the RNA contents of the bacterial mass, and (iii) the percentage of this RNA that is present in ribosomal particles. Tables 1 and 2 show such information for B. licheniformis growing at three different growth rates. Table I shows that the average weight of a cell is quite constant during balanced growth at a given growth rate. But if the growth rate is varied, the average mass of the bacteria varies accordingly and this approximately as a linear function of the growth rate. The macromolecular constitution-especially the contents of RNA-of the cells appears to correlate to the growth rate as well. To estimate the proportion of RNA present in

3 22 DIJK-SALKINOJA AND PLANTA J. BACTERIOL. TABLE 1. Dry weight and macromolecular composition of B. licheniformis cells grown exponentially with different growth rates Bacterial density Avg dry wt Macromolecular composition of the celila Growth ratel at harvest" of I bacteriumc Protein' DNA RNA 1.70 X ± X i X l ain generations per hour. Values indicate cells per milliliter x c Mean standard error of at least three separate cultures, each analyzed in triplicate. Values indicate grams x d Determined from lysates, per gram of dry weight. e Mean + standard error of at least three separate cultures, each analyzed in triplicate. TABLE 2. Analysis ofcellfractions oflysed B. licheniformis cells grown exponentially with different growth rates Total amt recovered Growth from lysates in' rate' rae Aaaay ~~~~~~Soluble Solubilized Recovery' fraction membranes 1.70 * 0.15 Protein 321 * DNA 4.8 i * RNA Total Ribosomall 32 * 2% % Protein * DNA * RNA Total *3 97 Ribosomall 20 * 2% 73 2% 0.50 i 0.02 Protein * DNA RNA Total 29 * 2 67 * 2 98 Ribosomall 9 * 1% 68 * 1% 'In generations per hour. Mean * standard error of at least three separate cultures, each determined in triplicate. Results expressed in milligrams per gram (dry weight). ' Results expressed at per cent of total amount present in the lysate. For the composition of the lysate, see Table 1. I Percentage of RNA sedimenting in the ribosomal region in zonal sedimentation analysis. ribosomes, exponentially growing cells from balanced cultures were lysed and fractionated as described above. The "soluble fraction" and "solubilized membranes" were assayed on nucleic acids and protein (Table 2) and then subjected to sedimentation analysis in a B XIV Spinco rotor for 80 min at 48,000 rev/min. A good separation of ribosomes from the slowly sedimenting RNA was obtained by the zonal centrifugation. We used a cushion of concentrated sucrose at the heavy end of the gradient to gather the polyribosomes and to prevent precipitation of the ribosomal particles on the rotor edge. After the run, the gradient was fractionated and the ribosomes were pooled for an assay of RNA. Owing to the fact that the amount of RNA in the complete samples also was known, the proportion of the RNA sedimenting in the ribosomal region of the two subcellular fractions could readily be calculated (Table 2). The "soluble fraction" and "solubilized membranes" together contain practically all of the nucleic acid and protein of the original lysate (last column, Table 2). And, indeed, when the residual pellet from the lysis (not shown in Table 2) was dissolved in I N NaOH and assayed, no protein or nucleic acid was found. Therefore, it probably consists of cell wall constituents and needs no further consideration here. We used the results given in Tables I and 2 to calculate the average quantity of ribosome-associated RNA per bacterial cell (see Table 3). The cellular quantity of ribosome-bound RNA can be expressed in 70S equivalents, if we assume that the ribosomes have a constant RNA content, corresponding to a molecular weight of 1.69 x 106 of the three ribosomal RNA species together (11), plus an additional contribution of 0.09 x 106 daltons for transfer RNA and messenger RNA present on the polysomal ribosomes. In this way, numerical ribosome contents of 92,000, 34,400, or 12,500 ribosomes per cell were calculated for the three different growth rates. Kinetics of the incorporation of labeled RNA

4 VOL. 105, 1971 RATE OF RIBOSOME PRODUCTION 23 TABLE 3. Calculation of the quantity ofrna and ribosomes in exponential-phase cells of B. licheniformis grown with different growth rates RNA sedimenting in nbosomal region Calculated no. Growth rate' RNA content/avg cell' of ribosomes/ Per cent RNA" Avg/cell' avg cell ± ± ,000 ± 8, ± ± ,400 ± 3, i ± ± ,500 X 1,000 a In generations per hour. Figures shown to be multiplied x Results expressed as grams per cell. Per cent of cellular RNA, calculated for the whole cells from the percentages given for both subcellular fractions in Table 2. A correction was made for incomplete recovery. precursor in ribosomal subunits at different growth rates. Balanced cultures, growing with generation times of 35, 60, and 120 min, were steady-state labeled with 14C-uracil and pulse-labeled with 3H-uridine for 75 sec. Further incorporation was stopped by a chase, and samples were collected at intervals of I or 2 min for a period of 25 min. The rate of 3H incorporation in ribosomal particles was studied by sedimentation analysis. Figure I shows a part of the long-spin (15 hr) gradients. Polyribosomes, and partly also the 70S particles, were precipitated on the bottom of the tube to obtain a good separation of the subunits. The ribosome preparations consisted of about 70% of polyribosomes and < 10% of subunits. During the first minutes after the chase, the 3H profiles do not coincide with the 14C profiles. (Since the 'IC profiles did not essentially differ from each other in the various runs, they are given only once for each generation time.) The 3H-labeled particles that sediment on both sides of 30S are probably ribosomal precursors. The smallest precursors (<<30S) were removed in our experiments during purification of the ribosomal preparations and are therefore not present in the sedimentation profiles given in Fig. 1. At 9 min after the chase, the 3H curve coincides with the 'IC curve for the first time and remains symmetrical to the 'IC curve (not shown in Fig. 1). The time needed for complete coincidence of the two profiles in the 50S region seems to be constant, irrespective of the growth rate (Fig. 1). Such coincidence of the ah and 14C curves in the 50S region can be regarded as an indication that the pulse-labeled 9H-RNA has become fully incorporated into mature subribosomal particles. This criterion is obviously not valid for the 30S region, because some precursors of 50S ribosomes are known to sediment at about 30S (8). The appearance of RNA label in the subunit region (comprising the ribosomal subunits plus their precursors) appears to cease rather abruptly after 8 to 9 min, for, beyond this point of time, the 8H activity in these particles (fractions 8 to 26 in Fig. 1) falls quite rapidly (insets, Fig. 1). From that time, the labeled subunits which have left the pool of free subunits because they are incorporated into polysomal complexes (12) are apparently no longer replaced by newly formed labeled subunits. This finding indicates that the pool of labeled nucleoside has been chased out very rapidly (within 1 or 2 min) in all three cultures. We therefore propose that the time necessary for the de novo formation of ribosomal subunits from labeled RNA precursors in B. licheniformis is constant (about 10 min) and hence does not depend at all on the growth rate of the bacteria. DISCUSSION The ribosome content of B. licheniformis was measured in these experiments for balanced, exponentially growing bacteria with three different growth rates. For this purpose, the bacterial cells were lysed gently and analyzed for their ribosome content by zonal centrifugation. We preferred this method, because it is not encumbered by the restriction of an incomplete recovery as is the case for all methods for extraction of RNA from the cells. Nuclease activity was very low in our preparations in which a high percentage of the ribosomes was present as polyribosomes (about 70%). The quantity of RNA per cell of B. licheniformis differs greatly in bacteria grown at different growth rates (Table 3). The bulk of the RNA (50 to 65%, varying a little with the growth rate) sediments in the ribosomal region. Calculation, on basis of the currently accepted molecular weights of the ribosomal RNA species (11), produced the number of ribosomes approximately 92,000, 34,400, or 12,500 70S equivalents per cell for bacteria grown with generation times of 35, 60, or 120 min, respectively. Put into terms of an average net production of ribosomes per cell per unit of time, the respective cultures should produce 2,600, 570, or 100 ribosomes per cell per minute.

5 24 DIJK-SALKINOJA AND PLANTA J. BACTERIOL. U0 I p lb 0 FRACTIONS FRACTIONS FRACTIONS FIG. 1. Balanced cultures of B. licheniformis, growing exponentially with doubling times, respectively, of (A), 60 i 4 (B), and (C) min, were steady-state labeled with '4C-uracil (0.2 lsci/ml, 3 hr). 3H-uridine was then added (1.2 MCi/ml in A, 2.2 g Ci/ml in B, and 3 ugci/ml in C); 75 sec later, further incorporation was stopped by adding 3 volumes offresh medium and unlabeled uridine (I g/liter). Samples (50 to 100 ml) were collected at 0, 3, 6.5, 8, 9, 11, 13, 15, 17, and 20 min after the chase. A 100-mg amount of unlabeled carrier cells was added in each sample; the cells were washed lysed, and ribosomes were isolated as described. Ribosomal preparations, containing in all cases over 60% ofpolyribosomes, were analyzed on sucrose gradients (5 to 30%, w/v) run for 15 hr at 22,500 rev/min. *, Solid line, 14C activity (disintegrations per minute per fraction). The 3H activities (disintegrations per minute per fraction) are given at 0 min (O), 6.5 min (A), 8 min (A), dashed line, and 9 min (0) after the chase. The insets give the sum of the 3H labeling of the fractions 8 to 26 at 3, 6.5, 8, 9, and 10 min after the chase (A). All 3H curves correspond to the same 14C curve; therefore the 3H activities can be compared directly. The regulation leading to such great differences in the production of ribosomes might operate in three basically different ways: (i) The time course of the biogenesis of a single ribosome can be varied. The cells then would increase the rate of synthesis of each individual ribosome when the growth rate increases. (ii) Not the rate of ribosome synthesis but, instead, their breakdown is regulated. A considerable turnover of ribosomes should then occur, especially at low growth rates. (iii) The number of the ribosomal particles synthesized simultaneously in one cell can be varied. The present results show that in B. licheniformis the kinetics of the biogenesis of the 50S subunit is not influenced by the growth rate. The labeled RNA precursor accumulated in the free 50S particles within 10 min after its addition to the cultures (9 min after the chase) in each of the three growth rates. In other experiments (not shown here), we used labeled amino acids instead of uridine to measure the formation time of the 50S ribosome. We found that the pulse-labeled protein accumulated in the mature subunits about 10 min after the chase (van Dijk-Salkinoja and Planta, Arch. Biochem. Biophys., in press). Sells and Davis studied the kinetics of incorporation of labeled amino acids into 50S ribosomes in Escherichia coli (3, 14). They found that the assemblage of labeled ribosomal protein into mature 50S particles takes as long as 20 to 30 min at the low growth rate of 0.5 generations per hr (14). However, they obtained this result for cultures growing at 28 C instead of 37 C. A decrease in growth temperature will undoubtedly result in a slow-down of the biosynthetc processes. The results of Davis and Sells (3), obtained at 37 C, are consistent with this opinion, for they showed that the pulse-labeled proteins then accumulate in the mature 50S ribosomes in about 10 min. We therefore feel that our conclusion that, independent of the growth rate, the 50S ribosomes are formed in about 10 min at 37 C is justified. It is true that this result is different from that obtained by Mangiarotti et al. (8) for E. coli at a growth rate 0.5 generations per hr at 37 C. However, the experimental conditions they used were very different from ours. For instance, in their ionic conditions (no K+ or polyamines, a relatively low concentration of Mg2+) the ribosomes were largely present as subunits (about 50%). In our preparations, 10% or less subunits were present. Therefore it is difficult to compare their results to ours. We prefer the use of 60 mm K+, 10 mm Mg2+, and 1.25 mm polyamines because this medium mimics the ionic environment of the intracellular fluid of bacteria (17). Hypothesis (ii) is not very likely, because it has been established that ribosomes are metabolically stable during exponential growth in E. coli (12). However, we felt a necessity to check this for our organism under our conditions. In these experiments (Fig. 2), the cells were pulse-labeled with 3H-uridine and then chased. We followed the chase of radioactivity from the ribosomes for some hours. This was done at two extreme

6 VOL. 105, 1971 growth rates. Figure 2 shows that the half-time of the dilution of the radioactivity in the ribosomes was 36 min, and it was 146 min in the two different cultures, corresponding exactly to the doubling time of the cultures. Therefore, in these cultures no measurable turnover of ribosomes takes place. The only possibility left is hypothesis iii, by which the quantity of the ribosomes synthesized simultaneously is regulated. For instance, the number of ribosome genes that are available for transcription might be regulated. Bacillus species are known to possess 9 to 10 ribosomal RNA genes per genome (15). Not all genes might be active at all growth rates. It is also known that bacteria contain more DNA per cell during rapid growth than at low growth rates (7). This is also true for B. licheniformis. At the rate of 1.7 generations per hr, the cells contain twice as much DNA (0.047 x g per cell, calculated from the data in Table 1) than at the rate of 0.5 generations per hr. In addition, rapidly growing cells have more than one DNA replication point per chromosome (1, 18) and are therefore relatively richer in early replicating markers than are slowly growing cultures, in which each chromosome has only one replication point. The ribosomal RNA genes belong to those early replicating markers in B. subtilis (10, 15). An excess of ribosomal DNA occurring in rapidly growing cells, differing from slowly growing cells-in which the ribosomal RNA genes may partly be repressed-could indeed lead to the RATE OF RIBOSOME PRODUCTION large differences observed in the rate of the ribosome production. Many ribosomal protein markers of B. subtilis are known to be clustered with the major group of the ribosomal RNA genes (5). Therefore, the production of messenger RNA for ribosomal proteins might be regulated in the same way as that of ribosomal RNA, leading to a coordinated synthesis of the ribosomal components. As the growth rate increases, the ribosomal protein becomes a larger fraction of the cellular protein: 8, 16, and 26% of the B. licheniformis protein is ribosomal at the growth rates of the 0.5, 1.0, and 1.7 generations per hr. [This was calculated from the data given in Tables I and 3 by making use of the known ratio of protein to RNA (47:53) of the B. licheniformis ribosomes.] To provide for the increasing demand of ribosomal proteins, rapidly growing cells might employ a greater proportion of their ribosomes to synthesize them than do the slowly growing cells. Or, if this is not true, the ribosomal protein-synthesizing ribosomes are bound to synthesize protein much more efficiently in rapidly growing bacteria than in the slowly growing cells. If we assume that the efficiency of the ribosomes that synthesize (stable) nonribosomal proteins is either constant or increasable with the growth rate, it will be manifest from the subjoined calculations that the proportion of the ribosome-synthesizing ribosomes will be higher in rapidly growing cells than in slowly growing cells. Our results support the idea that the protein-synthesizing efficiency 25 ID 0 1 hours after the chase FIG. 2. 3H uridine (0.12,Ci/ml) was added to balanced, exponentially growing cultures of B. licheniformis with doubling times of 35 i 2 min (o) or 144 i 5 min (@), respectively. After 5 min, further incorporation was stopped by adding 3 volumes offresh medium and unlabeled uridine (I glliter). Samples (50 to 250 ml) were collected at indicated points of time and ribosomes were isolated as described. The radioactivity of the ribosomal preparations is given as disintegrations per minute per unit ofabsorbance at 260 nm.

7 26 DIJK-SALKINOJA AND PLANTA J. BACTERIOL. of the ribosomal protein-synthesizing ribosomes is unchangeable and equal to the calculated overall efficiency, which is 19 amino acid residues incorporated in protein per second per 70S equivalent (see Appendix). APPENDIX Calculation of the protein-synthesizing efficiency of the ribosomes in B. licheniformis at three different growth rates. Theory. Let p be the total amount of protein in one cell at the time t after the latest cell division; r be the number of ribosomes in the cell at the time t; T be the generation time; P be the average amount of protein per cell and R the average of the number of the ribosomes per cell during the cell cycle; and k be the efficiency of the ribosomes in protein synthesis: dp/dt = k r (1) We choose p = P0 and r = R0 when t = 0. At the time t = T, p = 2 PO, and r =2 R0 (2) Let us postulate that the cell increases its number of ribosomes exponentially in time: r = Ro 21/T (3) The R can be calculated by integration from (3): Integration of (1) gives (5): TABLE 4. R = - rdt= (4) T In 2 pt p= Po+k r di (5) (5) can be solved by substituting (3) for r: p Po + (2hIT - 1) In R P0 can be calculated from (6) by setting p = 2 P0 at t = T (2): KRo T Po = In 2 and P can be calculated from (6) by integration and substituting P0 with (7): l jt kkrot krot krot P=. Pd Po + (8) T (in 2)2 In 2 (In 2)2 When also Ro is substituted in (8) by (4), we get: p = k, or k t 0.693P In 2 R T from which the efficiency (k) of the ribosomes can be calculated if the corresponding P, R, and T are experimentally measured. If, however, the cell increases its number of ribosomes linearly, rather than exponentially in time, then (3) and (4) should be replaced by (3a) and (4a): leading finally to (9a): r = R0 (1 + 1/7) R = 1.5 Ro P =-k R T, or k ;:z RT Calculation of the protein synthesizing efficiency of ribosomes in B. licheniformis at different growth rates Protein/cell No. of nbosomes/cell (R) Efficiency of nbosomes in synthesis of T (min) Total' Ribosomal1 d Total Ribosomal Nonribosomal (P) (rp) Total R i protein e protein 're protein ne (6) (7) (9) (3a) (4a) (9a) ,000 f3.92, f 1 -fs ,400 f2. 34, f3 15 f ,500 fl l2, fi 17 f Calculated from the data given in Table 1. Figures shown (in grams) to be multiplied x 'Calculated from the data given in Tables I and 3. The weight ratio of protein to RNA in the ribosomes is 47: 53. Figures shown (in grams) to be multiplied x From Table 3. d 'R, the number of ribosomes per cell that synthesize ribosomal protein. e Calculated from equation 9: k, = R i- rki = LT; nki = Pi where i = 1, 2, or 3. Re TfeRpTe' o)r (f rt s Figures shown to be multiplied x l0-1. Results expressed at grams of protein per minute per ribosome.

8 VOL. lo5, RATE OF RIBOSOME PRODUCTION 27 Application. Let the fraction of the ribosomes that synthesize ribosomal proteins (rp) in B. licheniformis be fa, f,, and f, at the growth rates of 0.5, 1.0, and 1.7 generations/hr, respectively. We call the proteinsynthesizing efficiency of these ribosomes rk,, tk5, and rk,, and that of the ribosomes synthesizing nonribosomal proteins nk5, "k5, and nk,, respectively. We assume that, when the growth rate increases, the efficiency of the ribosomes will not decrease. Then nk3 > "k2 > 5ki, or, after substituting the calculated values of "k from equation 9 (see Table 4): 14.6/(1 - f3) > 15.7/(1 - f2) > 17.3/(1 - f5). This can only be true if f5 > A > f1, that is, if the fraction of ribosomes that synthesize ribosomal protein is larger at higher growth rates. If we assume further that, at one of the growth rates, nk = 'k, we can calculate the numerical values of fl, f,, and f,, for instance, let "k, = rkc, by using equation 9 (see also Table 4), this equals 3.0/f2 15.7/ = - (1 f2), from which f2 = If we substitute this in the calculated values of k in nk, > nk2 "k, and, 'k k2 Ik, (see Table 4), we get 0.22 f, 0.27 and 0.08 > fi > (Similar values can be obtained if another "k is put equal to the corresponding rk.) The fraction of the cellular ribosomes that produce ribosomal protein (fi = 8%, f2 16%, and f, from 22 = to 27%) seems thus to be proportional to the amount of ribosomal protein produced (8, 16, or 26% of the total protein). The efficiency of the ribosomal proteinsynthesizing ribosomes is then equal to the overall efficiency of the ribosomes, which is 19 to g of protein synthesized per min (Table 4) (or about 19 amino acid residues incorporated per sec) per 70S equivalent. ACKNOWLEDGMENTS This investigation was supported by the Netherlands Foundation for Chemical Research (S.O.N.) and The Netherlands Organization for the Advancement of Pure Research (Z.W.O.). We are grateful to T. J. Stoof for his collaboration in some of the experiments and to E. M. van de Plassche, H. Mullink, W. H. Mager, and H. van Keuien for skilful technical assistance. LITERATURE CITED 1. Caro, L. G Chromosome replication in E. coli Segregation of chromosomal strands in multiforked replication. J. Mol. Biol. 48: Chen, P. S., T. Y. Toribara, and H. Warner Microdetermination of phosphorus. Anal. Chem. 28: Davis, F. C., and B. H. Sells Synthesis and assembly of ribosomal protein into 50 S subunits during recovery from chloramphenicol treatment. J. Mol. Biol. 39: De Vries, W., and A. H. Stouthamer Fermentation of glucose, lactose, galactose, mannitol and xylose by bifidobacteria. J. Bacteriol. 96: Goldthwaite, C., D. Dubnau, and 1. Smith Genetic mapping of antibiotic resistance markers in Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A. 65: Layne, E Spectrofotometric and turbidimetric methods for measuring proteins, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 7. Maaloe, O., and N. 0. Kjeldgaard Control of macromolecular synthesis, p W. A. Benjamin Inc., New York. 8. Mangiarotti, G., D. Apirion, D. Schlessinger, and L. Silengo. 1%8. Biosynthetic precursors of 30S and 50S particles in E. coli. Biochemistry 7: Munro, H. N., and A. Fleck Determination of nucleic acids, p In D. Glick (ed.), Methods of biochemical analysis, vol. 14. Interscience Publishers, Inc., New York. 10. Oishi, M., and N. Sueoka Location of genetic loci of ribosomal RNA on Bacillus subtilis chromosome. Proc. Nat. Acad. Sci. U.S.A. 54: Osawa, S Ribosome formation and structure. Annu. Rev. Biochem. 37: Schlessinger, D., and D. Apirion Escherichia coli ribosomes: recent developments. Annu. Rev. Microbiol. 23: Schneider, W. C Determination of nucleic acids in tissues by pentose analysis, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 14. Sells, B. H., and F. C. Davis Biogenesis of 50S particles in exponentially growing Escherichia coli. J. Mol. Biol. 47: Smith, 1., D. Dubnau, P. Morell, and J. Marmur Chromosomal location of DNA base sequences complementary to trna and to 5S, 16S and 23S rrna in Bacillus subtilis. J. Mol. Biol. 33: Sykes, J., and T. W. Young Studies on the ribosomes and ribonucleic acids of Aerobacter aerogenes grown at different rates in carbon-limited continuous culture. Biochim. Biophys. Acta 169: Van Dijk-Salkinoja, M. S., T. J. Stoof, and R. J. Planta The distribution of polysomes, ribosomes and ribosomal subunits in exponential-phase cells of Bacillus licheniformis. Eur. J. Biochem. 12: Yoshikawa, H., A. O'Sullivan, and N. Sueoka Sequential replication of the Bacillus subtilis chromosome. IIl. Regulation of initiation. Proc. Nat. Acad. Sci. U.S.A. 52: