JOURNAL OF VIROLOGY, JUlY 1975, p. 62-69 Copyright 0 1975 American Society for Microbiology
Vol. 16, No. 1 Printed in U.S.A.
Presence of Active Polyribosomes in Bacterial Cells Infected with T4 Bacteriophage Ghosts KEIICHI TAKEISHI AND AKIRA KAJI*
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Received for publication 24 January 1975
Host protein synthesis of Escherichia coli stops abruptly after T4 bacteriophage ghost infection. When infection was carried out in the presence of 10 mM Mg2+, infected cells still have active polyribosomes despite the complete stoppage of protein synthesis. On the other hand, when T4 ghost infection was carried out in the presence of 1 mM Mg2+, no polyribosomes were observed and most of the ribosomes were 30S and 50S subunit particles. Subunits obtained from extracts of ghost-infected cells at 1 mM Mg2+ concentration could not be converted to polyribosomes, even when Mg2+ concentration was adjusted to 10 mM after ghost infection. There was very little difference in amino acid incorporation activities between polyribosomes from ghost-infected and uninfected cells. In addition, the activity of 70S ribosomes isolated from uninfected cells was identical to that from cells infected with ghosts at 10 mM Mg2+.
Infection of growing Escherichia coli B with DNA-less T4 phage particles, or T4 ghosts, causes abrupt stoppage of host protein synthesis (2, 3, 5, 6). We previously reported that the cellular level of aminoacyl tRNA did not change significantly upon ghost infection, and that there was virtually no leakage or degradation of proteins and RNA into acid-soluble materials after infection (6). It has been suggested that ghost infection may result in a damage of ribosomal function for protein synthesis (13). Recent studies carried out by Goldman and Lodish (7) suggested that the mechanism of inhibitory effect of T4 ghosts on host protein synthesis appears to be different from the mechanism through which the phage infection influences host protein synthesis. In most of the studies mentioned above, infection of the cells with T4 ghosts has been carried out in the presence of 1 mM Mg2+. On the other hand, it is known that the leakage of cellular 35S into the medium induced by phage infection is markedly inhibited by 25 mM Mg2+ which does not influence adsorption of phage particles to host cells (14) and that the leakage of K+ from phage-infected cells can be prevented by the presence of 25 mM MgCl2 upon infection (16). In an attempt to obtain characteristics of translational machinery in the ghost-infected cells, the present studies were carried out on cells infected with ghosts in the presence of 10 mM Mg2+ to minimize the secondary effect of 62
permeability change due to the ghost infection. Contrary to the expectation that translationalmachinery of ghost-infected cells may be damaged, active polyribosomes were detected in ghost-infected cells. MATERIALS AND METHODS Bacteria and bacteriophage. E. coli B was used as the host bacteria sensitive to bacteriophage T4 D and its ghosts. Phage T4 D was used for the preparation of T4 D phage ghosts. E. coli B was grown at 37 C as described previously (6). Unless noted otherwise, cells were infected with T4 ghosts at a multiplicity of 10 in the presence of L-tryptophan (20 jg/ml). Infection was carried out at 37 C for 5 min. Under these conditions, more than 99.95% of the cells had lost their viability. Culture media used in this study were as follows. Tris medium contained 0.12 M Tris-hydrochloride, pH 7.5, 0.08 M NaCl, 0.02 M KCl, 0.02 M NH4Cl, 10-3 M MgSO4, 3.5 x 10-4 M CaCl2, 2 x 10-6 M FeCl3, 2.5 x 10- 3M Na2SO4, and 10- 4M K2HPO4 (9). TG medium was the Tris medium supplemented with glycerol to a final concentration of 0.5% by volume. M9S medium contained 0.041 M Na2HPO4, 0.022 M KH2PO4, 0.009 M NaCl, 0.019 M NH4CL, 10-3 M MgSO4, 10-6 M FeCl3, 0.4% glucose, 0.002% L-tryptophan, and 0.25% Casamino Acids (1). Bacteriophage T4 D stocks. The phage was prepared by infecting E. coli B at 37 C in M9S medium with phage T4 D at a multiplicity of 0.1. Infected cultures were lysed with chloroform 3 h after infection and then incubated with crude DNase (10 ,g/ml) at room temperature for 30 min. Lysates were cen-
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trifuged at 5,000 x g for 10 min to remove cell debris. Phage particles were pelleted by centrifugation at 25,000 rpm for 1 h in a Beckman Spinco rotor (no. 30) and resuspended overnight at 4 C in Tris medium. A large scale preparation of phage T4 D was performed by precipitation with polyethylene glycol 6000 (7). To the cell lysates from which cell debris was removed, NaCl (0.5 M) and polyethylene glycol 6000 (8% wt/vol) were added, and the mixture was allowed to stand 1 h at 4 C. The precipitate was collected by centrifugation at 9,000 x g for 10 min and suspended in the Tris medium. The final phage suspension was centrifuged at 5,000 x g for 10 min to remove insoluble materials. It was stored over chloroform at 4 C. Preparation and titration of T4 ghosts. The procedure for preparation of crude T4 ghosts was described previously (6). The purification of the ghosts was performed according to the method of Duckworth (3) with a slight modification as described below. Thus, the crude ghosts were layered on a 10 to 30% discontinuous sucrose gradient in Tris medium and centrifuged in a SW27 Beckman Spinco rotor at 17,000 rpm for 60 min. The discontinuous gradient was prepared in a centrifuge tube by placing 5 ml of 30% sucrose, 15 ml of 20% sucrose, and 15 ml of 10% sucrose in this order. Fractions (1.1 ml) were collected from the top of the tube. Fractions containing ghosts were detected by their bacteriocidal effect, pooled, and stored at 4 C in Tris medium. The ghost preparation was contaminated with less than 0.002% viable phage after this treatment. The ghosts thus purified were assayed by a modification of the method of Duckworth and Bessman which measures inhibitory effect of ghosts on the induction of ,B-galactosidase (4) as described previously (6). Preparation of cell extracts and mRNA. Unless otherwise stated the S.200 extract (soluble protein) was prepared as described by Nirenberg and Matthaei (12), except that the ribosomes were removed from the "incubated S30" by centrifugation at 200,000 x g for 3 h. A crude mixture of initiation factors from E. coli Q13 was prepared according to Iwasaki et al. (8). E. coli and T4 mRNA were prepared as described by Wilhelm and Haselkorn (19). RNA of MS2 phage was prepared as described by Strauss and Sinsheimer (18). Preparation of polyribosomes. Procedure I was a modification of the procedure as reported by Schwartz et al. (15). This procedure was used for the preparation of polysomes from T4 ghost-infected cells. E. coli B was grown in TG medium at 37 C to a density of 2.5 x 106 cells/ml. The culture (160 ml) was mixed with 1.6 ml of 1 M MgSO4 and 3.2 ml of L-tryptophan (1 mg/ml), infected with T4 ghosts as described in the preceding section and rapidly chilled to 0 C with a mixture of dry ice and acetone. The cells were pelleted by centrifugation at 10,000 rpm for 5 min in a Sorvall SS-34 rotor and resuspended in 1.6 ml of solution I containing 20% sucrose, 10 mM MgSO4, and 0.1 M Tris-hydrochloride, pH 8.1. The suspension was mixed with 0.4 ml of solution II containing 1.5 mg of
lysozyme/ml, 10 mM MgSO4, and 0.1 M Tris-hydrochloride, pH 8.1 and allowed to stand for 3 min at 0 C. To this mixture was added 0.4 ml of 0.05 M MgSO4 followed by 1.64 ml of the lytic mixture which consists of 0.2 ml of 10% Brij 58, 0.4 ml of 2% sodium deoxycholate, 0.04 ml of 5 mg of pure DNase/ml, and 1 ml of 4 mM Tris-hydrochloride, pH 8.1. After incubation at 0 C for 5 min, the lysate was centrifuged at 10,000 rpm for 10 min in a Sorvall SS-34 rotor. The supernatant fluid containing the polysomes was carefully aspirated. The pellets were resuspended in 1.6 ml of solution I and treated again with lysozyme and detergents in the same manner as described above. The supematant fluid (4 ml) was layered onto 4.9 ml of 20% sucrose in buffer A containing 10 mM Tris-hydrochloride, pH 7.8, 50 mM NH4Cl, 10 mM MgCl3, and 6 mM 2-mercaptoethanol. The tube was centrifuged in a Spinco 65 rotor for 3 h at 50,000 rpm. The pellets were rinsed twice with buffer A and resuspended in the same buffer. The suspension was clarified by centrifugation at 3,000 rpm for 10 min in a Sorvall SS-34 rotor. The yield of polyribosomes by this procedure was approximately 75% of total ribosomes. The amounts of polyribosomes were expressed by their protein content. Procedure II was a modification of procedure I and was used for the preparation of polysomes from uninfected cells. The modifications were: no addition of L-tryptophan and MgSO4 to the cell culture, omission of 10 mM MgSO4 from solution I, and substitution of 10 mM MgSO4 by 50 mM EDTA in solution II. After the addition of solution II, 0.4 ml of 0.12 M MgSO4 was added instead of 0.05 M MgSO4. Other ionic conditions during the lysis were identical to those of procedure I. The yield of polysomes in this procedure was approximately 60%. The repeated cell lysis with the lytic mixture was omitted. Procedure III utilized moderate sonic treatment for disruption of cells. Cultures (200 ml) of cells (3 x 108/ml) either uninfected or infected with T4 ghosts were rapidly chilled to 0 C. The cells were pelleted by centrifugation at 10,000 rpm for 5 min and resuspended in 5 ml of buffer A. Portions (2.5 ml) were sonically treated in a tube (17.5 by 100 mm) surrounded by ice for a total of 90 s with intervals between each 30 s of sonic treatment. The narrow tip (3-mm diameter) of MSE ultrasonic disintegrator was used. The sonic extracts were centrifuged at 6,500 rpm for 10 min in a Sorvall SS-34 rotor. The unbroken cells were subjected to an additional two sonic treatments to obtain approximately 90% breakage of cells. The sonic supernatant fluid thus obtained was further centrifuged in a Spinco 65 rotor at 50,000 rpm for 90 min. The top three-fourths of the supematant fluid was carefully aspirated to be used as the source of S.200 (see Fig. 6, 7, and 8). The pellets were gently rinsed twice with 1 ml of buffer A and resuspended in 3.5 ml of buffer A. The suspension was clarified by centrifugation at 6,500 rpm for 10 min in a Sorvall SS-34 rotor and stored in liquid N,. Preparation of monoribosomes from sonic extracts. The E. coli culture, (100 ml) 4 x 108 cells/ml of PG medium, was supplemented with
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MgSO4 (10 mM) and L-tryptophan (20 sg/ml) and was infected with T4 ghosts. For the control culture, Tris medium was added in place of ghost suspension. After being rapidly chilled to 0 C, the cells were collected by centrifugation in a Sorvall SS-34 rotor at 10,000 rpm for 10 min and suspended in 2 ml of buffer A. The cell suspension thus obtained was sonicated in an ice bath for a total of 2 min with intervals between each 15 s of sonic treatment. A sonicator rod of 9.5-mm diameter was used. Under these conditions, polysomes were broken down because of high sonic energy than in procedure III. The sonic extracts were centrifuged in a Beckman Spinco SW50.1 rotor at 18,000 rpm for 30 min. The supernatant fluid was then centrifuged at 50,000 rpm for 3 h in a Spinco 65 rotor. The precipitated ribosomes were carefully rinsed with buffer A and resuspended in 1.5 ml of the same buffer. The insoluble materials were removed by centrifuging at 6,500 rpm for 15 min in a Sorvall SS-34 rotor. The ribosome suspension was stored in liquid N2 until its
mM Mg2+ ghosts exert an inhibitory effect on the host protein synthesis in vivo, experiments shown in Fig. 2 were performed. This figure shows the time course of amino acid incorporation by exponentially growing cells. Immediately after the addition of T4 ghosts, almost immediate cessation of amino acid incorporation by the host cells occurred. It should be pointed out that in the experiment shown in Fig. 1 there were slight differences between the procedures used for the lysis of uninfected and ghost-infected cells. To compare polyribosomes of ghost-infected cells with those of the control cells under identical conditions, we developed a new procedure for isolation of polysomes. When cells were subjected to a moderate sonic treatment, polysomes were still present and could be isolated (Fig. 3). An important point in use. this experiment is the fact that the Mg2+ Materials. The following materials were pur- concentration at the time of infection with chased: E. coli tRNA and sucrose (RNase free), ghosts has to be 10 mM to be able to obtain Schwarz/Mann; GTP, P.L. Biochemicals; poly(U), Miles; puromycin, Nutritional Biochemicals; chlor- polysomes from the infected cells. Under these amphenicol, Sigma; fusidic acid, Leo Pharmaceuti- conditions an almost identical profile of polycals; sparsomycin, Upjohn; aurintricarboxylic acid, somes was observed with ghost-infected cells as Aldrich Chemicals; sodium deoxycholate, Difco Lab; Brij-58, Atlas Chemicals; phosphoenolpyruvate, pyruvate kinase, ATP, and tetracycline, Calbiochem; lysozyme (eggwhite, 2x crystallized), bovine pancreatic DNase, Worthington Biochemicals. Folinic acid obtained as Ca2+ salt from General Biochemicals was used as a formyl donor after being converted to the K+ salt by treatment with Dowex 50 W x 2 (K+ form). All the radioactive compounds and omnifluor were the products of New England Nuclear Corp. Other methods. Protein concentration was determined colorimetrically by the method of Lowry et al. (11). The radioactivity incorporated into protein was determined by counting in 5 ml of scintillation fluid (4 g of Omnifluor dissolved in 1 liter of toluene) in a Packard liquid scintillation counter. RESULTS
Presence of polyribosomes in T4 ghost-infected cells. In the experiment indicated in Fig. 1, cells were infected with ghosts in the presence of 10 mM Mg2+ or 1 mM Mg2+, lysed with lysozyme and detergents, and subjected to sucrose density gradient centrifugation. It is clear from this figure that just as many polysomes were present in cells infected with ghosts at 10 mM Mg2+ as were in uninfected cells. In fact, 56% of the total ribosomes were represented as polysomes in the infected cells, whereas 45% were polysomes in the uninfected cells. On the other hand, when cells were infected with ghosts in TG medium with 1 mM Mg2+, no appreciable polysomes were observed (Fig. lb). To ascertain that even in the presence of 10
(a) 70S 50S 3Os
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FIG. 1. Effect of Mg2+ in the growth media on the polysomal pattern of host cells infected with T4 ghosts; lysis with detergent. E. coli B was grown in TG medium to a density of 2.5 x 108 cells/ml. The cell lysate was prepared from 30 ml of the culture. The cell lysates (0.14 ml) were layered onto 5 ml of a linear 15 to 30%o (wt/vol) sucrose gradient in buffer B containing 10 mM Tris-hydrochloride, pH 7.4, 50 mM NH4CI, and 10 mM MgS04. Centrifugation was performed for 60 min at 45,000 rpm in a Spinco SW50.1 rotor. Gradients were analyzed using ISCO model D density gradient fractionator attached with model UA-2 ultraviolet analyzer. (a) The lysate was prepared from uninfected cells according to procedure II. (b) The lysate was prepared from ghost-infected cells according to procedure I except that the Infection was carried out in TG medium. (c) The lysate was prepared from cells infected with ghosts in TG medium supplemented with MgSO4 to 10 mM according to procedure I.
POLYRIBOSOMES IN BACTERIAL CELLS
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65
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Time (minutes) FIG. 2. Effect of T4 ghost infection in the presence of 10 mM Mg2+ on valine incorporation into proteins by E. coli B. The culture (4 ml) of E. coli B (5 x 108 cells/ml) in TG medium was mixed with 0.2 ml of solution containing 40 #mol of MgSO,, 80 pg of L-tryptophan, 6 pg of unlabeled valine, and 0.8 pCi of ["iC valine (210 mCi/mmot), divided into two parts (2 ml each), and incubated with shaking at 37 C for 8.5 min. At this point one part was infected with T4 ghosts at a multiplicity of 10. Portions (0.1 ml) were withdrawn and the radioactivity insoluble in hot trichloroacetic acid was determined. Symbols: 0, uninfected cells; *, T4 ghost-infected cells.
compared to uninfected cells (Fig. 3d and g). On the other hand, no significant polysomes were detected when T4 ghosts were given in the presence of 1 mM Mg2+ (Fig. 3a). Under similar conditions, some trace of polysomes remain in uninfected cells, but most of their ribosomes were 30S and 50S subunits (Fig. 3e). Raising magnesium ion concentration after sonic treatment did not increase polysomes appreciably in the extract of infected or control cells (Fig. 3b and f). Even when Mg2+ concentration was adjusted to 10 mM immediately after ghost infection and further incubated for 5 min at 37 C, no appreciable amount of polysomes was detected. However, 70S ribosomes were formed by this procedure from ribosomal subunits produced upon ghost infection (Fig. 3c). In vitro amino acid incorporation activity of polyribosomes isolated from ghost-infected cells. The surprising result that polysomes were present in the ghost-infected cells prompted us to examine the amino acid incorporating activity of these polysomes. In the experiment shown in Fig. 4, the amino acid incorporation by these polysomes isolated from cells treated with lysozyme and detergents was tested under conditions such that the incorporation was linearly dependent upon the amount of polysomes. The soluble factors used in these experiments were
Volume (ml)
BOTTOM
FIG. 3. Effect of Mg2+ on ribosomal sedimentation profile of infected and uninfected cells; disruption by sonication, E. coli B culture (3 x 10' cells/ml) in TG medium was supplemented with L-tryptophan (20 pg/ml) and infected with T4 ghosts at either 1 or 10 mM Mg'+. The cell suspension (2.5 ml) was rapidly cooled to 0 C and sonically treated briefly as described in Materials and Methods. The sonic extracts were centrifuged at 6,500 rpm for 10 min in a Sorvall SS-34 rotor. The supernatant fluid (0.5 ml) was layered on 4.8 ml of a linear 15 to 30% sucrose density gradient in buffer B (10 mM Mg'+), centrifuged and analyzed as in Fig. 1. (a and e) Ghost infection and sonic treatment were at 1 mM Mg'+. tb and t) Infection and sonic treatment at 1 mM Mg2+ but immediately after sonication Mg'+ was adjusted to 10 mM. (c) Infection was 1 mM Mg2+ but immediately after the infection the culture medium was adjusted to 10 mM Mg2+ and incubated at 37 C for 5 mmn before sonic treatment. (d and g) Infection and sonication were in 10 mMMg2+.
derived from uninfected cells. It is clear from this figure that the activity of the polysomes from the ghost-infected cells was only about 30% less than the activity of the control polysomes. In the experiment shown in Fig. 5, aurintricarboxylic acid (an inhibitor of the chain initiation step [17]) was added to the amino acid incorporation system. As shown in this figure, even in the presence of the initiation inhibitor, the difference between polysomes derived from infected and uninfected cells was only about 30%. It should be pointed out that ghost-infected cells from which these polysomes were derived had lost 100% of the protein synthesis activity. It is possible that 30% decrease in amino acid incorporating activity observed with polysomes from ghost-infected cells may be due to an increased susceptibility of the polysomes to detergents (deoxycholate and Brij 58) used in the isolation procedures. To avoid this complication, polysomes prepared by moderate sonic treatment were used in the next
J. VIROL.
TAKEISHI AND KAJI
66
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isolated from uninfected and ghost-infected Time (minutes) cells; lysis by detergents. Polysomes from ghostinfected and uninfected cells were isolated by proceFIG. 5. Kinetics of amino acid incorporation by dures I and II, respectively, as described in Materials and Methods. The reaction mixture (0.5 ml) for assay polysomes isolated from ghost-infected and uninfected cells; lysis by detergents. Polysomes (2.3 Mg of of amino acid incorporation contained 50 mM Tris- protein 0.1 ml) and the reaction mixture were the hydrochloride, pH 7.8, 50 mM NH4CI, 6 mM 2-mer- same asper Fig. 4 except that incubation was at 30 C. captoethanol, 10 mM MgCl2, 0.4 mM GTP, 1 mM Aliquots in(0.1 ml) were withdrawn at the indicated ATP, 10 mMphosphoenolpyruvate, 20 Mg of pyruvate the radioactivity incorporated into hot time and "4C-labeled kinase, 250 Mg of E. coli tRNA, 0.5 ACi of amino acids mixture (0.125 MCi each of alanine [156 trichloroacetic acid-insoluble materials was counted. mCi/mmolJ, leucine [311 mCi/mmol], phenylalanine (a) No aurintricarboxylic acid was added. Symbols: [412 mCi/mmol], and valine [210 mCi/mmol]), 0.5 0, polysomes from uninfected cells; 0, polysomes MM each of 16 unlabeled amino acids (minus alanine, from ghost-infected cells; A, no polysomes. (b) Aurinleucine, phenylalanine, and valine), 450 Mg of protein tricarboxylic acid (0.1 mM) was added. Symbols are from S.200 extracts, and polysomes as indicated. The the same as indicated in (a). final pH was adjusted to 7.8 with 0.2 N KOH if necessary. The mixture was incubated at 37 C for 60 0 1 - (a min and 2.5 ml of 10% trichloroacetic acid was added. - (b) - (c) Symbols: 0, radioactivity incorporated into proteins x 124 by polysomes from uninfected cells; 0, by polysomes E from ghost-infected cells. somes
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experiment (Fig. 6). In this experiment, soluble E 6factors were derived from either uninfected or 4 infected cell sonic extracts. In either case, no appreciable difference in the amino acid incor2 poration catalysed by polysomes from infected E. and uninfected cells was observed (Fig. 6a and 0 0 20 40 0 20 40 20 40 b). In Fig. 7, the time course of amino acid Polyribosomes ( ag protein) incorporation dependent on polysomes derived from uninfected and ghost-infected cells shows FIG. 6. Comparison of amino acid incorporation that there was no appreciable difference be- activity between polyribosomes isolated from unintween the initial rates of amino acid incorpora- fected and ghost-infected cell sonicates. Polyribotion of these polysomes. These observations somes and the soluble extract (S.200) were prepared clearly indicate that the protein synthesizing from cell sonicates by procedure III as described in activity tested in vitro of polysomes from unin- Materials and Methods. The reaction mixture (0.25 fected and ghost-infected cells was almost iden- ml) for amino acid incorporation and the incubationit were essentially the same as in Fig. 4 except that tical. contained either 109 Mg of protein of the soluble That the incorporation of amino acid ob- extract (S.200) of uninfected cells (a), 96Mug of protein served in the preceding experiment represented of the S.200 from ghost-infected cells (b), or no S.200 protein synthesis and not an abnormal incorpo- (c). The S.200 had been dialyzed against buffer A. ration such as N-terminal addition of amino Symbols: 0, polysomes from uninfected cells; 0, acid (10) was shown in the experiment described polysomes from ghost-infected cells. .'
0
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POLYRIBOSOMES IN BACTERIAL CELLS
in Table I. It is noted in this table that the incorporation by polysomes from sonic extracts of ghost-infected cells was sensitive to typical protein synthesis inhibitors such as fusidic acid, aurintricarboxylic acid, tetracycline, chloramphenicol, sparsomycin, and puromycin. The inhibition of the ghost-infected polysomes by these antibiotics was similar to that of control polyribosomes. Lack of effect of ghost infection on soluble protein factors and on ribosomes. The preceding experiment suggested that soluble fractions necessary for protein synthesis may not be influenced by ghost infection. To establish this point, various amounts of soluble fractions from cells infected with ghosts at 10 mM Mg2+ and uninfected cells were tested for their capacity to support amino acid incorporation by polysomes. There was no appreciable difference between uninfected and infected cells (Fig. 8). The observation was confirmed with the use of polysomes from either infected or uninfected cells (Fig. 8a and b). The preceding results obtained from the experiments on amino acid incorporation by polysomes indicated that the peptide chain elongation machinery was not damaged upon ghost infection in the presence of 10 mM Mg2+. This conclusion was further supported by the results shown in Fig. 9. In this experiment, initiation
TABLE 1. Effect of various antibiotics on amino acid incorporation by polysomesa
67
% Amino acid incorporation with
Additions
None ............ Fusidic acid ....... ATAb ............ Tetracycline ......
Chloramphenicol .. Sparsomycin ...... Puromycin ........
Concn. (MM)
0.5 0.1 0.1 0.3 0.1 0.5
polysomes Uninfected
Infected Ifce
100 68 37 39 27 16 0
100 77 37 34 24 19 0
a The polysomes were prepared from sonic extracts as described in Fig. 6. The reaction mixture for the incorporation of amino acids into protein was as described in Fig. 6. The mixture (0.1 ml) contained 89 gg of protein of S.200 extracts and either 10 ug of protein of polysomes from uninfected cells or 15 ug of protein of polysomes from ghostinfected cells. Incubation was carried out at 37 C for 60 min. The 100% incorporations by polysomes from uninfected and ghost-infected cells were 4,478 and 5,180 counts/min, respectively. Aurintricarboxylic acid.
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Time (minutes) FIG. 7. Time course of amino acid incorporation by polysomes isolated from ghost-infected and uninfected cell sonicates. Polysomes and the reaction mixture were the same as in Fig. 6, except that the reaction mixture (0.1 ml) contained 12 Mg of protein of the polysomes and 54 Mg of protein of the S.200 supematant from uninfected cell sonicates. Symbols:
0, polysomes from sonicates of uninfected cells; 0, polysomes from sonicates of ghost-infected cells; 0, no addition of polysomes.
(4g protein)
FiG. 8. Comparison of the amino acid incorporation activity of the soluble extracts from uninfected and ghost-infected cell sonicates. Polysomes, S.200, and the reaction mixture (125 MI) were the same as in Fig. 6. (a) Polysomes (16 Mtg of protein) from uninfected cells; (b) polysomes (23 Mg of protein) from ghost-infected cells; (c) no polysomes. Symbols: 0, S.200 from uninfected cell sonicates; 0, S.200 of ghost-infected cell sonicates.
factors from uninfected cells were added to the protein synthesizing system. The initial rate of amino acid incorporation programmed by either MS2RNA, T4 mRNA, or E. coli mRNA was identical for the ribosomes from control and ghost-infected cells. In a separate experiment, the final level of amino acid incorporation under these conditions was also identical. These experiments indicate that the protein synthesiz-
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(a) MS2 RNA 2 8
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(b)T4mRNA 6 (c)E.coli mRNA
programmed by
the natural mRNA. These observations strongly suggest that the initiation
and elongation steps of these ribosomes are not damaged by the ghost infection. It therefore 6_ 4 appears possible that mechanisms other than o Xcn c 1 o damage to protein synthesis machinery are re3_ / .a 1 4- D )( /4sponsible for the abrupt cessation of host protein ovc after ghost infection at 10 mM Mg2+. 2 synthesis (.)0 2 Although the Mg2+ concentration did not C0 influence the inhibitory effect of ghost infection ±~~~~~~ ~on host protein synthesis, it has another im4 8 0 4 8 0 4 8 0 portant influence on the effect of phage ghosts on the host. Thus, it has been shown that Ribosomes (/Lg protein) FIG. 9. Activity of monoribosomes from uninfected Mg2+ can suppress the lysis of cells by ghost and ghosit-infected cells for amino acid incorporation (2) and that leakage of cellular components programrned by natural mRNA's. Monoribosomes upon phage infection was prevented by 25 mM were prejvared from cell sonicates as in Materials and Mg2+ (14, 16). In the preceding studies it has Methods..The reaction mixture (0.05 ml) contained 50 been concluded that blockage of protein synmM Triss-hydrochloride, pH 7.8, 50 mM NH4CI, 6 thesis must occur somewhere after the formation mM 2-mEercaptoethanol, 9 mM MgCI2, 0.4 mM GTP, of aminoacyl tRNA (6). Our preliminary results 1 mM ALTP, 10 mM phosphoenolpyruvate, 5.9 Mg suggest that at least a part of the protein synof pyruv ate kinase, 25 Mg of E. coli tRNA, 0.05 thesizing activity of the ghost-infected cells MCi of [1 'Clvaline, 19 unlabeled amino acids (minus (at 10 MM Mg2+) can be restored by the valine), 4 M AM each, 60 Mg of folinic acid, 30 Mg of at mM G an be crude ini tiation factors, 27 Mg of protein from S.200 addition of of ATP, GTP, and their generathe generating extract, natural mRNA, and monoribosomes a system (unpublished observation). These conindicated1. Natural mRNAs added to the mixture siderations, together with the presence of (0.05 ml) were 29 Mg of MS2RNA (a), 34 Mg of active polysomes as described in this paper, T4 mRN[A (b), and 35 Mg of E. coli mRNA (c). may point to the possibility that cessation Incubatio)n was at 37 C. Initial rates of valine (counts! of protein synthesis after ghost infection may min x 1(9- /min) incorporation were determined and be caused by a sudden leakage of a key complotted a1gainst the amount of ribosomes. Symbols: 0, pound such as GTP. In fact, it is already monoribo somes from uninfected cells; *0 monoribo- known that ghost infection causes rapid deplesomes fro m ghost-infected cells. tion of the soluble pools of nucleotides (3, 20). are currently in progress to explore ing activ 'ity of oftcapact ribosomes, including the caclty Studies this possibility. L iniLtLate new protein synLnesls ana peptiae chain elongation, was almost identical in ghostLITERATURE CITED infected and uninfected cells. C
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-E
DISCUSSION It is surprising that active polyribosomes are present in ghost-infected cells which have completely stopped synthesizing protein. From the experiment performed in the presence of 1 mM Mg2+, it appeared that not only polyribosomes were disintegrated but also that the ribosome itself was damaged by ghost infection (13; unpublished observation). Despite the fact that Mg2+ concentration has a profound effect on the host polyribosomes during ghost infection, the overall inhibitory effect of ghost infection on the host protein synthesis was not influenced by Mg 2+ concentration. The polyribosomes isolated from ghost-infected cells at 10 mM Mg2+ were just as active as the control polyribosomes for amino acid incorporation. The ribosomes isolated from these polyribosomes were also active in the in vitro protein synthesis system
1. Champe, S. P., and S. Benzer. 1962. Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequences in messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 48:532-546. 2. Duckworth, D. H. 1970. Biological activity of bacteriophage ghosts and "take-over" of host functions by bacteriophage. Bacteriol. Rev. 34:344-363. 3. Duckworth, D. H. 1970. The metabolism of T4 phage ghost-infected cells. I. Macromolecular synthesis and transport of nucleic acid and protein precursors. Virology 40:673-684. 4. Duckworth, D. H., and M. J. Bessman. 1965. Assay for the killing properties of T2 bacteriophage and their "ghosts". J. Bacteriol. 90:724-728. 5. Fabricant, R., and D. Kennel. 1970. Inhibition of host protein synthesis during infection of E. coli by bacteriophage T4. J. Virol. 6:772-781. 6. Fukuma, I., and A. Kaji. 1972. Effect of bacteriophage ghost infection on protein synthesis in E. coli. J. Virol. 10:713-720. 7. Goldman, E., and H. F. Lodish. 1973. T4 phage and T4 ghosts inhibit f2 phage replication bv different mechanisms. J. Mol. Biol. 74:151-161. 8. Iwasaki, K., S. Sabol, A. Wahba, and S. Ochoa. 1968. Translation of the genetic message. VII. Role of initia-
VOL. 16, 1975
9. 10.
11.
2.-
13.
tion factors in formation of the chain initiation complex with E. coli ribosomes. Arch. Biochem. Biophys. 125:542-547. Kaempfer, R. 0. R., and B. Magasanik. 1967. Effect of infection with T-even phage on the inducible synthesis of ,-galactosidase in E. coli. J. Mol. Biol. 27:453-468. Kaji, A., H. Kaji, and G. D. Novelli. 1965. Soluble amino acid incorporating system. I. Preparation of the system and nature of the reaction. J. Biol. Chem. 240:1185-1191. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275. Nirenberg, M. W., and J. H. Matthaei. 1961. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U.S.A. 47:1588-1602. Nugent, K., and D. Kennell. 1972. Polypeptide synthesis by extracts from E. coli treated with T2 ghosts. J. Virol. 10:1199-1204.
14. Puck, T. T., and H. H. Lee. 1954. Mechanism of cell wall penetration by viruses. I. An increase in host cell
POLYRIBOSOMES IN BACTERIAL-CELLS
15.
16.
17. 18. 19. 20.
69
permeability induced by bacteriophage infection. J. Exp. Med. 99:481-494. Schwartz, T., E. Craig, and D. Kennell. 1970. Inactivation and degradation of messenger ribonucleic acid from the lactose operon of E. coli. J. Mol. Biol. 54:299-311. Silver, S., E. Levine, and P. M. Spielman. 1968. Cation fluxes and permeability changes accompanying bacteriophage infection of E. coli. J. Virol. 2:763-771. Stewart, M. L., A. P. Grollman, and M. T. Huang. 1971. Aurintricarboxylic acid: inhibitor of initiation of protein synthesis. Proc. Natl. Acad. Sci. U.S.A. 68:97-101. Straus, J. H., Jr., and R. L. Sinsheimer. 1963. Purification and properties of bacteriophage infection of E. coli. J. Mol. Biol. 7:43-54. Wilhelm, J., and R. Haselkorn. 1971. In vitro protein synthesis directed by RNA from T4-infected E. coli. Methods Enzymol. 20:531-536. Winkler, H. H., and D. H. Duckworth. 1971. Metabolism of T4 bacteriophage ghost-infected cells: effect of bacteriophage and ghosts on the uptake of carbohydrates in E. coli B. J. Bacteriol. 107:259-267.
Vol. 16, No. 1 Printed in U.S.A.
Presence of Active Polyribosomes in Bacterial Cells Infected with T4 Bacteriophage Ghosts KEIICHI TAKEISHI AND AKIRA KAJI*
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Received for publication 24 January 1975
Host protein synthesis of Escherichia coli stops abruptly after T4 bacteriophage ghost infection. When infection was carried out in the presence of 10 mM Mg2+, infected cells still have active polyribosomes despite the complete stoppage of protein synthesis. On the other hand, when T4 ghost infection was carried out in the presence of 1 mM Mg2+, no polyribosomes were observed and most of the ribosomes were 30S and 50S subunit particles. Subunits obtained from extracts of ghost-infected cells at 1 mM Mg2+ concentration could not be converted to polyribosomes, even when Mg2+ concentration was adjusted to 10 mM after ghost infection. There was very little difference in amino acid incorporation activities between polyribosomes from ghost-infected and uninfected cells. In addition, the activity of 70S ribosomes isolated from uninfected cells was identical to that from cells infected with ghosts at 10 mM Mg2+.
Infection of growing Escherichia coli B with DNA-less T4 phage particles, or T4 ghosts, causes abrupt stoppage of host protein synthesis (2, 3, 5, 6). We previously reported that the cellular level of aminoacyl tRNA did not change significantly upon ghost infection, and that there was virtually no leakage or degradation of proteins and RNA into acid-soluble materials after infection (6). It has been suggested that ghost infection may result in a damage of ribosomal function for protein synthesis (13). Recent studies carried out by Goldman and Lodish (7) suggested that the mechanism of inhibitory effect of T4 ghosts on host protein synthesis appears to be different from the mechanism through which the phage infection influences host protein synthesis. In most of the studies mentioned above, infection of the cells with T4 ghosts has been carried out in the presence of 1 mM Mg2+. On the other hand, it is known that the leakage of cellular 35S into the medium induced by phage infection is markedly inhibited by 25 mM Mg2+ which does not influence adsorption of phage particles to host cells (14) and that the leakage of K+ from phage-infected cells can be prevented by the presence of 25 mM MgCl2 upon infection (16). In an attempt to obtain characteristics of translational machinery in the ghost-infected cells, the present studies were carried out on cells infected with ghosts in the presence of 10 mM Mg2+ to minimize the secondary effect of 62
permeability change due to the ghost infection. Contrary to the expectation that translationalmachinery of ghost-infected cells may be damaged, active polyribosomes were detected in ghost-infected cells. MATERIALS AND METHODS Bacteria and bacteriophage. E. coli B was used as the host bacteria sensitive to bacteriophage T4 D and its ghosts. Phage T4 D was used for the preparation of T4 D phage ghosts. E. coli B was grown at 37 C as described previously (6). Unless noted otherwise, cells were infected with T4 ghosts at a multiplicity of 10 in the presence of L-tryptophan (20 jg/ml). Infection was carried out at 37 C for 5 min. Under these conditions, more than 99.95% of the cells had lost their viability. Culture media used in this study were as follows. Tris medium contained 0.12 M Tris-hydrochloride, pH 7.5, 0.08 M NaCl, 0.02 M KCl, 0.02 M NH4Cl, 10-3 M MgSO4, 3.5 x 10-4 M CaCl2, 2 x 10-6 M FeCl3, 2.5 x 10- 3M Na2SO4, and 10- 4M K2HPO4 (9). TG medium was the Tris medium supplemented with glycerol to a final concentration of 0.5% by volume. M9S medium contained 0.041 M Na2HPO4, 0.022 M KH2PO4, 0.009 M NaCl, 0.019 M NH4CL, 10-3 M MgSO4, 10-6 M FeCl3, 0.4% glucose, 0.002% L-tryptophan, and 0.25% Casamino Acids (1). Bacteriophage T4 D stocks. The phage was prepared by infecting E. coli B at 37 C in M9S medium with phage T4 D at a multiplicity of 0.1. Infected cultures were lysed with chloroform 3 h after infection and then incubated with crude DNase (10 ,g/ml) at room temperature for 30 min. Lysates were cen-
63
VOL. 16, 1975
POLYRIBOSOMES IN BACTERIAL CELLS
trifuged at 5,000 x g for 10 min to remove cell debris. Phage particles were pelleted by centrifugation at 25,000 rpm for 1 h in a Beckman Spinco rotor (no. 30) and resuspended overnight at 4 C in Tris medium. A large scale preparation of phage T4 D was performed by precipitation with polyethylene glycol 6000 (7). To the cell lysates from which cell debris was removed, NaCl (0.5 M) and polyethylene glycol 6000 (8% wt/vol) were added, and the mixture was allowed to stand 1 h at 4 C. The precipitate was collected by centrifugation at 9,000 x g for 10 min and suspended in the Tris medium. The final phage suspension was centrifuged at 5,000 x g for 10 min to remove insoluble materials. It was stored over chloroform at 4 C. Preparation and titration of T4 ghosts. The procedure for preparation of crude T4 ghosts was described previously (6). The purification of the ghosts was performed according to the method of Duckworth (3) with a slight modification as described below. Thus, the crude ghosts were layered on a 10 to 30% discontinuous sucrose gradient in Tris medium and centrifuged in a SW27 Beckman Spinco rotor at 17,000 rpm for 60 min. The discontinuous gradient was prepared in a centrifuge tube by placing 5 ml of 30% sucrose, 15 ml of 20% sucrose, and 15 ml of 10% sucrose in this order. Fractions (1.1 ml) were collected from the top of the tube. Fractions containing ghosts were detected by their bacteriocidal effect, pooled, and stored at 4 C in Tris medium. The ghost preparation was contaminated with less than 0.002% viable phage after this treatment. The ghosts thus purified were assayed by a modification of the method of Duckworth and Bessman which measures inhibitory effect of ghosts on the induction of ,B-galactosidase (4) as described previously (6). Preparation of cell extracts and mRNA. Unless otherwise stated the S.200 extract (soluble protein) was prepared as described by Nirenberg and Matthaei (12), except that the ribosomes were removed from the "incubated S30" by centrifugation at 200,000 x g for 3 h. A crude mixture of initiation factors from E. coli Q13 was prepared according to Iwasaki et al. (8). E. coli and T4 mRNA were prepared as described by Wilhelm and Haselkorn (19). RNA of MS2 phage was prepared as described by Strauss and Sinsheimer (18). Preparation of polyribosomes. Procedure I was a modification of the procedure as reported by Schwartz et al. (15). This procedure was used for the preparation of polysomes from T4 ghost-infected cells. E. coli B was grown in TG medium at 37 C to a density of 2.5 x 106 cells/ml. The culture (160 ml) was mixed with 1.6 ml of 1 M MgSO4 and 3.2 ml of L-tryptophan (1 mg/ml), infected with T4 ghosts as described in the preceding section and rapidly chilled to 0 C with a mixture of dry ice and acetone. The cells were pelleted by centrifugation at 10,000 rpm for 5 min in a Sorvall SS-34 rotor and resuspended in 1.6 ml of solution I containing 20% sucrose, 10 mM MgSO4, and 0.1 M Tris-hydrochloride, pH 8.1. The suspension was mixed with 0.4 ml of solution II containing 1.5 mg of
lysozyme/ml, 10 mM MgSO4, and 0.1 M Tris-hydrochloride, pH 8.1 and allowed to stand for 3 min at 0 C. To this mixture was added 0.4 ml of 0.05 M MgSO4 followed by 1.64 ml of the lytic mixture which consists of 0.2 ml of 10% Brij 58, 0.4 ml of 2% sodium deoxycholate, 0.04 ml of 5 mg of pure DNase/ml, and 1 ml of 4 mM Tris-hydrochloride, pH 8.1. After incubation at 0 C for 5 min, the lysate was centrifuged at 10,000 rpm for 10 min in a Sorvall SS-34 rotor. The supernatant fluid containing the polysomes was carefully aspirated. The pellets were resuspended in 1.6 ml of solution I and treated again with lysozyme and detergents in the same manner as described above. The supematant fluid (4 ml) was layered onto 4.9 ml of 20% sucrose in buffer A containing 10 mM Tris-hydrochloride, pH 7.8, 50 mM NH4Cl, 10 mM MgCl3, and 6 mM 2-mercaptoethanol. The tube was centrifuged in a Spinco 65 rotor for 3 h at 50,000 rpm. The pellets were rinsed twice with buffer A and resuspended in the same buffer. The suspension was clarified by centrifugation at 3,000 rpm for 10 min in a Sorvall SS-34 rotor. The yield of polyribosomes by this procedure was approximately 75% of total ribosomes. The amounts of polyribosomes were expressed by their protein content. Procedure II was a modification of procedure I and was used for the preparation of polysomes from uninfected cells. The modifications were: no addition of L-tryptophan and MgSO4 to the cell culture, omission of 10 mM MgSO4 from solution I, and substitution of 10 mM MgSO4 by 50 mM EDTA in solution II. After the addition of solution II, 0.4 ml of 0.12 M MgSO4 was added instead of 0.05 M MgSO4. Other ionic conditions during the lysis were identical to those of procedure I. The yield of polysomes in this procedure was approximately 60%. The repeated cell lysis with the lytic mixture was omitted. Procedure III utilized moderate sonic treatment for disruption of cells. Cultures (200 ml) of cells (3 x 108/ml) either uninfected or infected with T4 ghosts were rapidly chilled to 0 C. The cells were pelleted by centrifugation at 10,000 rpm for 5 min and resuspended in 5 ml of buffer A. Portions (2.5 ml) were sonically treated in a tube (17.5 by 100 mm) surrounded by ice for a total of 90 s with intervals between each 30 s of sonic treatment. The narrow tip (3-mm diameter) of MSE ultrasonic disintegrator was used. The sonic extracts were centrifuged at 6,500 rpm for 10 min in a Sorvall SS-34 rotor. The unbroken cells were subjected to an additional two sonic treatments to obtain approximately 90% breakage of cells. The sonic supernatant fluid thus obtained was further centrifuged in a Spinco 65 rotor at 50,000 rpm for 90 min. The top three-fourths of the supematant fluid was carefully aspirated to be used as the source of S.200 (see Fig. 6, 7, and 8). The pellets were gently rinsed twice with 1 ml of buffer A and resuspended in 3.5 ml of buffer A. The suspension was clarified by centrifugation at 6,500 rpm for 10 min in a Sorvall SS-34 rotor and stored in liquid N,. Preparation of monoribosomes from sonic extracts. The E. coli culture, (100 ml) 4 x 108 cells/ml of PG medium, was supplemented with
64
TAKEISHI AND KAJI
J. VIROL.
MgSO4 (10 mM) and L-tryptophan (20 sg/ml) and was infected with T4 ghosts. For the control culture, Tris medium was added in place of ghost suspension. After being rapidly chilled to 0 C, the cells were collected by centrifugation in a Sorvall SS-34 rotor at 10,000 rpm for 10 min and suspended in 2 ml of buffer A. The cell suspension thus obtained was sonicated in an ice bath for a total of 2 min with intervals between each 15 s of sonic treatment. A sonicator rod of 9.5-mm diameter was used. Under these conditions, polysomes were broken down because of high sonic energy than in procedure III. The sonic extracts were centrifuged in a Beckman Spinco SW50.1 rotor at 18,000 rpm for 30 min. The supernatant fluid was then centrifuged at 50,000 rpm for 3 h in a Spinco 65 rotor. The precipitated ribosomes were carefully rinsed with buffer A and resuspended in 1.5 ml of the same buffer. The insoluble materials were removed by centrifuging at 6,500 rpm for 15 min in a Sorvall SS-34 rotor. The ribosome suspension was stored in liquid N2 until its
mM Mg2+ ghosts exert an inhibitory effect on the host protein synthesis in vivo, experiments shown in Fig. 2 were performed. This figure shows the time course of amino acid incorporation by exponentially growing cells. Immediately after the addition of T4 ghosts, almost immediate cessation of amino acid incorporation by the host cells occurred. It should be pointed out that in the experiment shown in Fig. 1 there were slight differences between the procedures used for the lysis of uninfected and ghost-infected cells. To compare polyribosomes of ghost-infected cells with those of the control cells under identical conditions, we developed a new procedure for isolation of polysomes. When cells were subjected to a moderate sonic treatment, polysomes were still present and could be isolated (Fig. 3). An important point in use. this experiment is the fact that the Mg2+ Materials. The following materials were pur- concentration at the time of infection with chased: E. coli tRNA and sucrose (RNase free), ghosts has to be 10 mM to be able to obtain Schwarz/Mann; GTP, P.L. Biochemicals; poly(U), Miles; puromycin, Nutritional Biochemicals; chlor- polysomes from the infected cells. Under these amphenicol, Sigma; fusidic acid, Leo Pharmaceuti- conditions an almost identical profile of polycals; sparsomycin, Upjohn; aurintricarboxylic acid, somes was observed with ghost-infected cells as Aldrich Chemicals; sodium deoxycholate, Difco Lab; Brij-58, Atlas Chemicals; phosphoenolpyruvate, pyruvate kinase, ATP, and tetracycline, Calbiochem; lysozyme (eggwhite, 2x crystallized), bovine pancreatic DNase, Worthington Biochemicals. Folinic acid obtained as Ca2+ salt from General Biochemicals was used as a formyl donor after being converted to the K+ salt by treatment with Dowex 50 W x 2 (K+ form). All the radioactive compounds and omnifluor were the products of New England Nuclear Corp. Other methods. Protein concentration was determined colorimetrically by the method of Lowry et al. (11). The radioactivity incorporated into protein was determined by counting in 5 ml of scintillation fluid (4 g of Omnifluor dissolved in 1 liter of toluene) in a Packard liquid scintillation counter. RESULTS
Presence of polyribosomes in T4 ghost-infected cells. In the experiment indicated in Fig. 1, cells were infected with ghosts in the presence of 10 mM Mg2+ or 1 mM Mg2+, lysed with lysozyme and detergents, and subjected to sucrose density gradient centrifugation. It is clear from this figure that just as many polysomes were present in cells infected with ghosts at 10 mM Mg2+ as were in uninfected cells. In fact, 56% of the total ribosomes were represented as polysomes in the infected cells, whereas 45% were polysomes in the uninfected cells. On the other hand, when cells were infected with ghosts in TG medium with 1 mM Mg2+, no appreciable polysomes were observed (Fig. lb). To ascertain that even in the presence of 10
(a) 70S 50S 3Os
.0 0
).5
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c
.0
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0. 0.
2
-
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2
3
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4
r
5 BOTTOM
Volume (ml)
FIG. 1. Effect of Mg2+ in the growth media on the polysomal pattern of host cells infected with T4 ghosts; lysis with detergent. E. coli B was grown in TG medium to a density of 2.5 x 108 cells/ml. The cell lysate was prepared from 30 ml of the culture. The cell lysates (0.14 ml) were layered onto 5 ml of a linear 15 to 30%o (wt/vol) sucrose gradient in buffer B containing 10 mM Tris-hydrochloride, pH 7.4, 50 mM NH4CI, and 10 mM MgS04. Centrifugation was performed for 60 min at 45,000 rpm in a Spinco SW50.1 rotor. Gradients were analyzed using ISCO model D density gradient fractionator attached with model UA-2 ultraviolet analyzer. (a) The lysate was prepared from uninfected cells according to procedure II. (b) The lysate was prepared from ghost-infected cells according to procedure I except that the Infection was carried out in TG medium. (c) The lysate was prepared from cells infected with ghosts in TG medium supplemented with MgSO4 to 10 mM according to procedure I.
POLYRIBOSOMES IN BACTERIAL CELLS
VOL. 16, 1975
65
E cJ
IT
0
to
0 cL
C 0 0
0 D
:
.0 0CL C.)
0
10
20
Time (minutes) FIG. 2. Effect of T4 ghost infection in the presence of 10 mM Mg2+ on valine incorporation into proteins by E. coli B. The culture (4 ml) of E. coli B (5 x 108 cells/ml) in TG medium was mixed with 0.2 ml of solution containing 40 #mol of MgSO,, 80 pg of L-tryptophan, 6 pg of unlabeled valine, and 0.8 pCi of ["iC valine (210 mCi/mmot), divided into two parts (2 ml each), and incubated with shaking at 37 C for 8.5 min. At this point one part was infected with T4 ghosts at a multiplicity of 10. Portions (0.1 ml) were withdrawn and the radioactivity insoluble in hot trichloroacetic acid was determined. Symbols: 0, uninfected cells; *, T4 ghost-infected cells.
compared to uninfected cells (Fig. 3d and g). On the other hand, no significant polysomes were detected when T4 ghosts were given in the presence of 1 mM Mg2+ (Fig. 3a). Under similar conditions, some trace of polysomes remain in uninfected cells, but most of their ribosomes were 30S and 50S subunits (Fig. 3e). Raising magnesium ion concentration after sonic treatment did not increase polysomes appreciably in the extract of infected or control cells (Fig. 3b and f). Even when Mg2+ concentration was adjusted to 10 mM immediately after ghost infection and further incubated for 5 min at 37 C, no appreciable amount of polysomes was detected. However, 70S ribosomes were formed by this procedure from ribosomal subunits produced upon ghost infection (Fig. 3c). In vitro amino acid incorporation activity of polyribosomes isolated from ghost-infected cells. The surprising result that polysomes were present in the ghost-infected cells prompted us to examine the amino acid incorporating activity of these polysomes. In the experiment shown in Fig. 4, the amino acid incorporation by these polysomes isolated from cells treated with lysozyme and detergents was tested under conditions such that the incorporation was linearly dependent upon the amount of polysomes. The soluble factors used in these experiments were
Volume (ml)
BOTTOM
FIG. 3. Effect of Mg2+ on ribosomal sedimentation profile of infected and uninfected cells; disruption by sonication, E. coli B culture (3 x 10' cells/ml) in TG medium was supplemented with L-tryptophan (20 pg/ml) and infected with T4 ghosts at either 1 or 10 mM Mg'+. The cell suspension (2.5 ml) was rapidly cooled to 0 C and sonically treated briefly as described in Materials and Methods. The sonic extracts were centrifuged at 6,500 rpm for 10 min in a Sorvall SS-34 rotor. The supernatant fluid (0.5 ml) was layered on 4.8 ml of a linear 15 to 30% sucrose density gradient in buffer B (10 mM Mg'+), centrifuged and analyzed as in Fig. 1. (a and e) Ghost infection and sonic treatment were at 1 mM Mg'+. tb and t) Infection and sonic treatment at 1 mM Mg2+ but immediately after sonication Mg'+ was adjusted to 10 mM. (c) Infection was 1 mM Mg2+ but immediately after the infection the culture medium was adjusted to 10 mM Mg2+ and incubated at 37 C for 5 mmn before sonic treatment. (d and g) Infection and sonication were in 10 mMMg2+.
derived from uninfected cells. It is clear from this figure that the activity of the polysomes from the ghost-infected cells was only about 30% less than the activity of the control polysomes. In the experiment shown in Fig. 5, aurintricarboxylic acid (an inhibitor of the chain initiation step [17]) was added to the amino acid incorporation system. As shown in this figure, even in the presence of the initiation inhibitor, the difference between polysomes derived from infected and uninfected cells was only about 30%. It should be pointed out that ghost-infected cells from which these polysomes were derived had lost 100% of the protein synthesis activity. It is possible that 30% decrease in amino acid incorporating activity observed with polysomes from ghost-infected cells may be due to an increased susceptibility of the polysomes to detergents (deoxycholate and Brij 58) used in the isolation procedures. To avoid this complication, polysomes prepared by moderate sonic treatment were used in the next
J. VIROL.
TAKEISHI AND KAJI
66
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isolated from uninfected and ghost-infected Time (minutes) cells; lysis by detergents. Polysomes from ghostinfected and uninfected cells were isolated by proceFIG. 5. Kinetics of amino acid incorporation by dures I and II, respectively, as described in Materials and Methods. The reaction mixture (0.5 ml) for assay polysomes isolated from ghost-infected and uninfected cells; lysis by detergents. Polysomes (2.3 Mg of of amino acid incorporation contained 50 mM Tris- protein 0.1 ml) and the reaction mixture were the hydrochloride, pH 7.8, 50 mM NH4CI, 6 mM 2-mer- same asper Fig. 4 except that incubation was at 30 C. captoethanol, 10 mM MgCl2, 0.4 mM GTP, 1 mM Aliquots in(0.1 ml) were withdrawn at the indicated ATP, 10 mMphosphoenolpyruvate, 20 Mg of pyruvate the radioactivity incorporated into hot time and "4C-labeled kinase, 250 Mg of E. coli tRNA, 0.5 ACi of amino acids mixture (0.125 MCi each of alanine [156 trichloroacetic acid-insoluble materials was counted. mCi/mmolJ, leucine [311 mCi/mmol], phenylalanine (a) No aurintricarboxylic acid was added. Symbols: [412 mCi/mmol], and valine [210 mCi/mmol]), 0.5 0, polysomes from uninfected cells; 0, polysomes MM each of 16 unlabeled amino acids (minus alanine, from ghost-infected cells; A, no polysomes. (b) Aurinleucine, phenylalanine, and valine), 450 Mg of protein tricarboxylic acid (0.1 mM) was added. Symbols are from S.200 extracts, and polysomes as indicated. The the same as indicated in (a). final pH was adjusted to 7.8 with 0.2 N KOH if necessary. The mixture was incubated at 37 C for 60 0 1 - (a min and 2.5 ml of 10% trichloroacetic acid was added. - (b) - (c) Symbols: 0, radioactivity incorporated into proteins x 124 by polysomes from uninfected cells; 0, by polysomes E from ghost-infected cells. somes
1')
0
o
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experiment (Fig. 6). In this experiment, soluble E 6factors were derived from either uninfected or 4 infected cell sonic extracts. In either case, no appreciable difference in the amino acid incor2 poration catalysed by polysomes from infected E. and uninfected cells was observed (Fig. 6a and 0 0 20 40 0 20 40 20 40 b). In Fig. 7, the time course of amino acid Polyribosomes ( ag protein) incorporation dependent on polysomes derived from uninfected and ghost-infected cells shows FIG. 6. Comparison of amino acid incorporation that there was no appreciable difference be- activity between polyribosomes isolated from unintween the initial rates of amino acid incorpora- fected and ghost-infected cell sonicates. Polyribotion of these polysomes. These observations somes and the soluble extract (S.200) were prepared clearly indicate that the protein synthesizing from cell sonicates by procedure III as described in activity tested in vitro of polysomes from unin- Materials and Methods. The reaction mixture (0.25 fected and ghost-infected cells was almost iden- ml) for amino acid incorporation and the incubationit were essentially the same as in Fig. 4 except that tical. contained either 109 Mg of protein of the soluble That the incorporation of amino acid ob- extract (S.200) of uninfected cells (a), 96Mug of protein served in the preceding experiment represented of the S.200 from ghost-infected cells (b), or no S.200 protein synthesis and not an abnormal incorpo- (c). The S.200 had been dialyzed against buffer A. ration such as N-terminal addition of amino Symbols: 0, polysomes from uninfected cells; 0, acid (10) was shown in the experiment described polysomes from ghost-infected cells. .'
0
VOL. 16, 1975
POLYRIBOSOMES IN BACTERIAL CELLS
in Table I. It is noted in this table that the incorporation by polysomes from sonic extracts of ghost-infected cells was sensitive to typical protein synthesis inhibitors such as fusidic acid, aurintricarboxylic acid, tetracycline, chloramphenicol, sparsomycin, and puromycin. The inhibition of the ghost-infected polysomes by these antibiotics was similar to that of control polyribosomes. Lack of effect of ghost infection on soluble protein factors and on ribosomes. The preceding experiment suggested that soluble fractions necessary for protein synthesis may not be influenced by ghost infection. To establish this point, various amounts of soluble fractions from cells infected with ghosts at 10 mM Mg2+ and uninfected cells were tested for their capacity to support amino acid incorporation by polysomes. There was no appreciable difference between uninfected and infected cells (Fig. 8). The observation was confirmed with the use of polysomes from either infected or uninfected cells (Fig. 8a and b). The preceding results obtained from the experiments on amino acid incorporation by polysomes indicated that the peptide chain elongation machinery was not damaged upon ghost infection in the presence of 10 mM Mg2+. This conclusion was further supported by the results shown in Fig. 9. In this experiment, initiation
TABLE 1. Effect of various antibiotics on amino acid incorporation by polysomesa
67
% Amino acid incorporation with
Additions
None ............ Fusidic acid ....... ATAb ............ Tetracycline ......
Chloramphenicol .. Sparsomycin ...... Puromycin ........
Concn. (MM)
0.5 0.1 0.1 0.3 0.1 0.5
polysomes Uninfected
Infected Ifce
100 68 37 39 27 16 0
100 77 37 34 24 19 0
a The polysomes were prepared from sonic extracts as described in Fig. 6. The reaction mixture for the incorporation of amino acids into protein was as described in Fig. 6. The mixture (0.1 ml) contained 89 gg of protein of S.200 extracts and either 10 ug of protein of polysomes from uninfected cells or 15 ug of protein of polysomes from ghostinfected cells. Incubation was carried out at 37 C for 60 min. The 100% incorporations by polysomes from uninfected and ghost-infected cells were 4,478 and 5,180 counts/min, respectively. Aurintricarboxylic acid.
0 5x
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(b)
(c)
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2
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10
20
30
Time (minutes) FIG. 7. Time course of amino acid incorporation by polysomes isolated from ghost-infected and uninfected cell sonicates. Polysomes and the reaction mixture were the same as in Fig. 6, except that the reaction mixture (0.1 ml) contained 12 Mg of protein of the polysomes and 54 Mg of protein of the S.200 supematant from uninfected cell sonicates. Symbols:
0, polysomes from sonicates of uninfected cells; 0, polysomes from sonicates of ghost-infected cells; 0, no addition of polysomes.
(4g protein)
FiG. 8. Comparison of the amino acid incorporation activity of the soluble extracts from uninfected and ghost-infected cell sonicates. Polysomes, S.200, and the reaction mixture (125 MI) were the same as in Fig. 6. (a) Polysomes (16 Mtg of protein) from uninfected cells; (b) polysomes (23 Mg of protein) from ghost-infected cells; (c) no polysomes. Symbols: 0, S.200 from uninfected cell sonicates; 0, S.200 of ghost-infected cell sonicates.
factors from uninfected cells were added to the protein synthesizing system. The initial rate of amino acid incorporation programmed by either MS2RNA, T4 mRNA, or E. coli mRNA was identical for the ribosomes from control and ghost-infected cells. In a separate experiment, the final level of amino acid incorporation under these conditions was also identical. These experiments indicate that the protein synthesiz-
68
TAKEISHI AND KAJI 0 x
(a) MS2 RNA 2 8
J. VIROL
(b)T4mRNA 6 (c)E.coli mRNA
programmed by
the natural mRNA. These observations strongly suggest that the initiation
and elongation steps of these ribosomes are not damaged by the ghost infection. It therefore 6_ 4 appears possible that mechanisms other than o Xcn c 1 o damage to protein synthesis machinery are re3_ / .a 1 4- D )( /4sponsible for the abrupt cessation of host protein ovc after ghost infection at 10 mM Mg2+. 2 synthesis (.)0 2 Although the Mg2+ concentration did not C0 influence the inhibitory effect of ghost infection ±~~~~~~ ~on host protein synthesis, it has another im4 8 0 4 8 0 4 8 0 portant influence on the effect of phage ghosts on the host. Thus, it has been shown that Ribosomes (/Lg protein) FIG. 9. Activity of monoribosomes from uninfected Mg2+ can suppress the lysis of cells by ghost and ghosit-infected cells for amino acid incorporation (2) and that leakage of cellular components programrned by natural mRNA's. Monoribosomes upon phage infection was prevented by 25 mM were prejvared from cell sonicates as in Materials and Mg2+ (14, 16). In the preceding studies it has Methods..The reaction mixture (0.05 ml) contained 50 been concluded that blockage of protein synmM Triss-hydrochloride, pH 7.8, 50 mM NH4CI, 6 thesis must occur somewhere after the formation mM 2-mEercaptoethanol, 9 mM MgCI2, 0.4 mM GTP, of aminoacyl tRNA (6). Our preliminary results 1 mM ALTP, 10 mM phosphoenolpyruvate, 5.9 Mg suggest that at least a part of the protein synof pyruv ate kinase, 25 Mg of E. coli tRNA, 0.05 thesizing activity of the ghost-infected cells MCi of [1 'Clvaline, 19 unlabeled amino acids (minus (at 10 MM Mg2+) can be restored by the valine), 4 M AM each, 60 Mg of folinic acid, 30 Mg of at mM G an be crude ini tiation factors, 27 Mg of protein from S.200 addition of of ATP, GTP, and their generathe generating extract, natural mRNA, and monoribosomes a system (unpublished observation). These conindicated1. Natural mRNAs added to the mixture siderations, together with the presence of (0.05 ml) were 29 Mg of MS2RNA (a), 34 Mg of active polysomes as described in this paper, T4 mRN[A (b), and 35 Mg of E. coli mRNA (c). may point to the possibility that cessation Incubatio)n was at 37 C. Initial rates of valine (counts! of protein synthesis after ghost infection may min x 1(9- /min) incorporation were determined and be caused by a sudden leakage of a key complotted a1gainst the amount of ribosomes. Symbols: 0, pound such as GTP. In fact, it is already monoribo somes from uninfected cells; *0 monoribo- known that ghost infection causes rapid deplesomes fro m ghost-infected cells. tion of the soluble pools of nucleotides (3, 20). are currently in progress to explore ing activ 'ity of oftcapact ribosomes, including the caclty Studies this possibility. L iniLtLate new protein synLnesls ana peptiae chain elongation, was almost identical in ghostLITERATURE CITED infected and uninfected cells. C
5
-E
DISCUSSION It is surprising that active polyribosomes are present in ghost-infected cells which have completely stopped synthesizing protein. From the experiment performed in the presence of 1 mM Mg2+, it appeared that not only polyribosomes were disintegrated but also that the ribosome itself was damaged by ghost infection (13; unpublished observation). Despite the fact that Mg2+ concentration has a profound effect on the host polyribosomes during ghost infection, the overall inhibitory effect of ghost infection on the host protein synthesis was not influenced by Mg 2+ concentration. The polyribosomes isolated from ghost-infected cells at 10 mM Mg2+ were just as active as the control polyribosomes for amino acid incorporation. The ribosomes isolated from these polyribosomes were also active in the in vitro protein synthesis system
1. Champe, S. P., and S. Benzer. 1962. Reversal of mutant phenotypes by 5-fluorouracil: an approach to nucleotide sequences in messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 48:532-546. 2. Duckworth, D. H. 1970. Biological activity of bacteriophage ghosts and "take-over" of host functions by bacteriophage. Bacteriol. Rev. 34:344-363. 3. Duckworth, D. H. 1970. The metabolism of T4 phage ghost-infected cells. I. Macromolecular synthesis and transport of nucleic acid and protein precursors. Virology 40:673-684. 4. Duckworth, D. H., and M. J. Bessman. 1965. Assay for the killing properties of T2 bacteriophage and their "ghosts". J. Bacteriol. 90:724-728. 5. Fabricant, R., and D. Kennel. 1970. Inhibition of host protein synthesis during infection of E. coli by bacteriophage T4. J. Virol. 6:772-781. 6. Fukuma, I., and A. Kaji. 1972. Effect of bacteriophage ghost infection on protein synthesis in E. coli. J. Virol. 10:713-720. 7. Goldman, E., and H. F. Lodish. 1973. T4 phage and T4 ghosts inhibit f2 phage replication bv different mechanisms. J. Mol. Biol. 74:151-161. 8. Iwasaki, K., S. Sabol, A. Wahba, and S. Ochoa. 1968. Translation of the genetic message. VII. Role of initia-
VOL. 16, 1975
9. 10.
11.
2.-
13.
tion factors in formation of the chain initiation complex with E. coli ribosomes. Arch. Biochem. Biophys. 125:542-547. Kaempfer, R. 0. R., and B. Magasanik. 1967. Effect of infection with T-even phage on the inducible synthesis of ,-galactosidase in E. coli. J. Mol. Biol. 27:453-468. Kaji, A., H. Kaji, and G. D. Novelli. 1965. Soluble amino acid incorporating system. I. Preparation of the system and nature of the reaction. J. Biol. Chem. 240:1185-1191. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275. Nirenberg, M. W., and J. H. Matthaei. 1961. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U.S.A. 47:1588-1602. Nugent, K., and D. Kennell. 1972. Polypeptide synthesis by extracts from E. coli treated with T2 ghosts. J. Virol. 10:1199-1204.
14. Puck, T. T., and H. H. Lee. 1954. Mechanism of cell wall penetration by viruses. I. An increase in host cell
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17. 18. 19. 20.
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permeability induced by bacteriophage infection. J. Exp. Med. 99:481-494. Schwartz, T., E. Craig, and D. Kennell. 1970. Inactivation and degradation of messenger ribonucleic acid from the lactose operon of E. coli. J. Mol. Biol. 54:299-311. Silver, S., E. Levine, and P. M. Spielman. 1968. Cation fluxes and permeability changes accompanying bacteriophage infection of E. coli. J. Virol. 2:763-771. Stewart, M. L., A. P. Grollman, and M. T. Huang. 1971. Aurintricarboxylic acid: inhibitor of initiation of protein synthesis. Proc. Natl. Acad. Sci. U.S.A. 68:97-101. Straus, J. H., Jr., and R. L. Sinsheimer. 1963. Purification and properties of bacteriophage infection of E. coli. J. Mol. Biol. 7:43-54. Wilhelm, J., and R. Haselkorn. 1971. In vitro protein synthesis directed by RNA from T4-infected E. coli. Methods Enzymol. 20:531-536. Winkler, H. H., and D. H. Duckworth. 1971. Metabolism of T4 bacteriophage ghost-infected cells: effect of bacteriophage and ghosts on the uptake of carbohydrates in E. coli B. J. Bacteriol. 107:259-267.
