Effect of a bacteriocin produced by Enterobacter cloacae on protein biosynthesis

122

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96871

E F F E C T OF A BACTERIOCIN PRODUCED BY E N T E R O B A C T E R ON P R O T E I N BIOSYNTHESIS

CLOACA E

F. K. D E G R A A F , R. J. P L A N T A AND A. H. S T O U T H A M E R

Biological Laboratory, Microbiology Department and Biochemical Laboratory, Free University, Amsterdam (The Netherlands) (Received J a n u a r y 26th, 1971)

SUMMARY

I. Bacteriocin DF13, produced by Enterobacter cloacae strain DF13, inhibits protein synthesis in Enterobacter cloacae strain 02, while RNA and DNA synthesis continue at a reduced rate. 2. Ribosomes from bacteriocin-treated cells are unable to support phage MS 2RNA directed incorporation of ~4C-labeled amino acids. The ribosomes retain the ability to bind mRNA, but appear not to bind fMet-tRNA fMet under direction of MS~-RNA. 3. Ribosomes from bacteriocin-treated cells are physically intact with respect to their sedimentation behaviour in sucrose gradients, the integrity of their RNA components, and the composition of ribosomal proteins. 4- Addition of bacteriocin to exponentially growing cells induces a leakage of K ÷ and an accumulation of 3o-S and 5o-S ribosomal subunits with a concomitant decrease in larger ribosomal particles.

INTRODUCTION

Bacteriocins are high molecular weight antibiotics, produced by certain strains of bacteria, which have a killing action on strains of the same or related species. The production of bacteriocins is determined by a plasmid (the bacteriocinogenic factor) which can be transferred by conjugation 1,~. Bacteriocins adsorb to specificreceptor sites on the surface of sensitive bacteria ~, and kill the cells according to single hit kinetics ~. Studies on the mode of action of various bacteriocins have revealed that they have quite different specific biochemical effects on sensitive bacteria. The colicins A (ref. 5), EI (ref. 6), and K (ref. 6), produced by Escherichia coli, and pyocin 7, produced by Pseudomonas aeruginosa, inhibit a whole series of energy-dependent reactions, including the synthesis of nucleic acids and proteins. Colicin E 2 (ref. 8), produced by Escherichia coli, megacin C (ref. 9), produced by Bacillus megaterium, and vibriocin (ref. IO), produced by Vibrio comma, cause degradation of the bacterial DNA. Colicin E 3 (ref. 8), produced by Escherichia coli inhibits solely protein synthesis. Enterobacter cloacae strain DF13 produces a bacteriocin which is active against strains of the genera Enterobacter and Klebsiella n. The bacteriocin has been purified, and consists of a simple protein molecule with a tool. wt. of 56 ooo (ref. 12). The Biochim. Biophys. Acta, 24o (1971) 122-136

BACTERIOCIN

DFls

AND PROTEIN BIOSYNTHESIS

12 3

bacteriocinogenic factor was shown to be a small closed circular DNA molecule with a contour length of 3 . o i o . 2 / z m (refs. 13, I4). As described previously 1~ bacteriocin DF~s, like colicin E 3, has a pronounced inhibitory effect on protein synthesis. The experiments presented in this paper were undertaken to elucidate further the biochemical effects of bacteriocin action. The results show that ribosomes isolated from bacteriocin-treated cells are unable to support in vitro protein synthesis in a phage MS2-RNA directed system. This block in protein synthesis is shown to be the result of an inhibition of the binding of formylmethionyl-tRNA to the MSz-RNA-ribosome complex, which is accompanied b y an accumulation of ribosomal subunits. Furthermore addition of bacteriocin induces a leakage of K + from sensitive cells. The data suggest that this leakage is not the prim a r y cause for the inhibition of protein synthesis.

MATERIALS AND METHODS

Bacterial strains Enterobacter cloacae strain o2, and Klebsiella edwardsii var. edwardsii strain S15, were used as bacteriocin-sensitive strains. Mutants of Enterobacter cloacae strain 02, requiring cysteine, uridine or thymidine were used for incorporation experiments with these substrates. The bacteriocin was prepared from Enterobacter cloacae strain DFls, as described previously 13. Bacteriocin concentrations are given in killing units per ml, designated as killing units/ml. One killing unit represents lOs killing particles 15. Media and huller solutions Bacteria were grown in nutrient broth containing 13 g Standard Bouillon (Oxoid), 5 g Bacteriological Peptone (Oxoid), and 5 g NaC1 per 1. Standard TrisHC1 buffer (pH 7.8) was o.oI M Tris chloride, o.oi M magnesium acetate, 0.06 M KC1 and 0.006 M fl-mercaptoethanol. Standard urea phosphate buffer (pH 6.5) was 6 M urea, 0.05 M NaH2PO 4, o.o12 M methylamine and 0.006 M fl-mercaptoethanol. I n vivo incorporation o/labeled substrates Incorporation of Ex4Clcysteine, E14C]uridine, and L14C]thymidine into acid-insoluble material was measured as described previously JS. Preparation o/cell-/ree extracts, ribosomes, and supernatant /ractions Cell-free extracts were prepared from exponentially growing cells b y grinding the cells for IO min with alumina (Alcoa). The extracts were suspended in standard Tris-HC1 buffer (pH 7.8), and preincubated to remove endogenous m R N A as described by VOORMA et al. le. For preparation of the ribosomes a preincubated extract was centrifuged for 5 h at 20o ooo ×g. The ribosomal pellet was suspended in standard Tris-HC1 buffer and used without further purification. The upper two thirds of the supernatant was first dialyzed against standard Tris-HC1 buffer and then used for amino acid incorporation. All procedures were carried out at 4 ° . I n vitro incorporation o[ 14C-labeled amino acids Incorporation of ~4C-labeled amino acids in cell-free systems under direction of phage MS~-RNA was carried out as described b y VOORMA et al. is. Biochim. Biophys. Acta, 240 (1971) I22-136

124

F . K . DE GRAAF et al.

Preparation o] RNA-labeled ribosomes and o] labeled r R N A Cells were grown for several generations in nutrient broth supplemented with E14C~uridine (2.5/~C/ml). An excess of bacteriocin (IOOO killing units/ml) was then added and incubation was continued for another 60 min. The cells were chilled, washed with standard Tris-HC1 buffer and mixed with a 3o-fold excess of non-radioactive untreated cells. Ribosomes were prepared from this cell mixture according to the method of GODSONAND SINSHEIMER 17, followed by dialysis against standard TrisHC1 buffer but containing lO -4 M magnesium acetate. EI4C]RNA was prepared from a crude cell extract as described by GILLESPIE AND SPIEGELMAN 18.

Preparation o[ labeled ribosomal protein Two cultures of Klebsiella edwardsii var. edwardsii strain $15 were grown under aeration at 37 ° in nutrient broth, one culture containing 14C-labeled amino acid mixture (0.75 #C/ml), the other 3H-labeled amino acid mixture (3 #C/ml). Tile radioactive amino acid mixtures had exactly the same composition and the same specific activity (250 mC/mmole). To the 14C-labeled culture excess of bacteriocin (iooo killing units/ml) was added as soon as the turbidity of the culture at 660 m/, became 0.7, and the incubation was continued for 30 rain. The cells of the two cultures were collected by centrifugation (30 min at 23 ooo ×g), washed twice with standard TrisHC1 buffer and resuspended in a small volume of the same buffer. The two suspensions were mixed and the bacterial cells were disrupted by sonication (6 sonication pulses of 30 sec with shaking and cooling in between, using the Model $75 Sonifier, Branson Instruments Inc., set at maximum power). Deoxyribonuclease was added to a final concentration of 5 #g/ml and the suspension was centrifuged for 30 rain at 3 ° o o o × g to remove cell debris. The ribosomal particles were collected from the supernatant by centrifugation for 5 h at 200 ooo ×g. The ribosomal pellet was resuspended in standard Tris-HC1 buffer but containing lO-4 M magnesium acetate, and the suspension was layered on the top of a linear lO-3O % sucrose gradient in the same low magnesium buffer and centrifuged for 16 h in a SW-25 I rotor at 22 500 rev./min. The separated ribosomal subunits were collected from the gradient fractions by centrifugation for 16 h in a SW-5o rotor at 5° ooo rev./min. The labeled particles were suspended in a small volume (about 2 ml) of o.oi M Tris-HC1 buffer (pH 7.4) containing 0.003 M succinic acid and o.oi M MgC12, and the ribosomal proteins were isolated by extraction with acetic acid in the presence of o.I M MgC12 as described by HARDY et al. 19. The acetic acid solutions of ribosomal proteins were lyophilized and the residues were dissolved in standard urea phosphate buffer (pH 6.5). At this stage unlabeled ribosomal proteins, isolated from corresponding unlabeled ribosomal subunits from untreated cells in the same manner as described above, were added to obtain a final protein concentration of 5-1o mg/ml. Prior to chromatographic fractionation the protein solutions were dialyzed overnight against standard urea phosphate buffer.

Analysis o/ ribosomal breakdown Cells were grown for several generations in nutrient broth supplemented with E14C]uridine (0.5 #C/ml). Then the cells were harvested by centrifugation, washed twice with standard Tris-HC1 buffer, and resuspended in non-radioactive nutrient broth. Incubation was continued for another 20 min to ensure that all E14Cluridine Biochim. Biophys. Acta, 240 (1971) 122-136

BACTERIOCIN DF~3 AND PROTEIN BIOSYNTHESIS

12 5

was incorporated into cellular RNA. Subsequently, an excess of bacteriocin (IOOO killing units/ml) was added to one half of the culture and incubation continued for another 60 rain. During this incubation o.5-ml samples were taken at Io-min intervals from both the bacteriocin-treated and untreated culture, and mixed with 0.5 ml of IO °/o trichloroacetic acid, heated for 15 min at 9 o°, cooled, and filtered on Whatman GF/C glass fibre filters. The radioactivity of the filtrate, containing acid-soluble material was assayed. The acid-insoluble material, collected on the filter was washed twice with 5 % trichloroacetic acid, dried on the filter and also assayed for radioactivity.

Binding assays The binding of MS2-E3H~RNA to ribosomes, and the binding of E35S~fMett R N A fMet to complexes of ribosomes and MS2-RNA was performed as described b y ALBRECHT et al. ~°. See further the legends to Figs. 6 and 7, and Table I I I . Radioactivity measurements All radioactive samples were counted in a liquid scintillation counter (Mark I, Nuclear Chicago). Glass fibre filters with radioactive material were dried at 7 °0 for 1.5 h and then placed in scintillation counting vials. After incubation with I ml of methanol for I h at room temperature, the filters were counted in 15 ml of scintillation liquid containing 4 g PPO and 5 ° mg P O P O P per 1 of toluene. Aqueous radioactive samples were mixed with distilled water to a final volume of 1.5 ml and then added to 15 ml of Bray's solution containing 60 g naphthalene, 4 g PPO, 5 ° g POPOP, ioo ml of methanol, and 20 ml of ethyleneglycol per 1 of dioxane. Materials MS2-RNA, MS2-E3HIRNA, and E~S]fMet-tRNA ~et were a gift from Dr. H. O. Voorma. Ll'CJCysteine, I14C~uridine, and E14C3thymidine were obtained from the Radiochemical Centre (Amersham, England). 14C- and 3H-labeled amino acid mixtures were purchased from NEN Chemicals G m b H (Frankfurt, Germany). E. coli B t R N A was purchased from General Biochemicals (Chagrin Falls, Ohio), phosphodiesterase, Type II, from Sigma (St. Louis, Mo.).

RESULTS

E//ect o/bacteriocin on growth and macromolecular synIhesis An excess of bacteriocin (IO00 killing units/ml), added to exponentially growing cells of Enterobacter cloacae strain 02, induces an almost abrupt growth inhibition after a lag time of about 20 rain (Fig. I). A similar result was obtained with Klebsiella edwardsii var. edwardsii strain $15, as described previously 15. With the latter strain it was found that the lag time before complete growth inhibition was dependent on the bacteriocin concentration 15. Although more t h a n 9 ° °/o of the cells irreversibly adsorb the bacteriocin within 2 min after addition, the observed lag time, in broth, was never shorter than 15 min, if an excess of bacteriocin (600 killing units/ ml) was used. Enterobacter cloacae strain 02 is somewhat less sensitive to the bacteriocin t h a n Klebsiella edwardsii var. edwardsii strain S15. Therefore, all experiments Biochim. Biophys. Acta, 240 (I97 I) 122-136

126

F . K . DE GRAAF et

al.

were performed with a bacteriocin concentration of iooo killing units/ml, corresponding to about 20o killing particles per cell, which for both strains represents an excess, as addition of more bacteriocin did not show any stronger effect.

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Fig. I. Effect of bacteriocin DFzs on g r o w t h of Enlerobacter cloacae s t r a i n o2 in n u t r i e n t b r o t h . A n excess of bacteriocin (iooo killing u n i t s / m l ) was a d d e d a t a t u r b i d i t y of 0. 5 a t 660 m # to one h a l f of t h e culture. S a m p l e s for r i b o s o m e analysis, as described in Fig. 6, were t a k e n a t t h e t i m e s indic a t e d in t h e figure. S a m p l e I f r o m u n t r e a t e d cells, S a m p l e s II, III, a n d IV f r o m bacteriocint r e a t e d cells. 0 - 0 , g r o w t h of t h e control culture; C)-C), g r o w t h of t h e b a c t e r i o c i n - t r e a t e d culture. Fig. 2. E f f e c t of bacteriocin DFIS on m a c r o m o l e c u l a r s y n t h e s i s in Enterobacter cloacae s t r a i n 02. C u l t u r e s were g r o w n in m i n i m a l m e d i u m s u p p l e m e n t e d w i t h t h e r e q u i r e d 1*C-labeled s u b s t r a t e (see MATERIALS AND METHODS). A n excess of bacteriocin (iooo killing u n i t s / m l ) w a s a d d e d a t t = o. (A) I n c o r p o r a t i o n of F14CJcysteine. (B) I n c o r p o r a t i o n of [14CJuridine. (C) I n c o r p o r a t i o n of E14CJ-thymidine. G - C ) , i n c o r p o r a t i o n in control cultures; S - - - 0 , i n c o r p o r a t i o n in bacteriocint r e a t e d cultures.

The effect of the bacteriocin on protein and nucleic acid synthesis was determined b y measuring the incorporation of E14Clcysteine, [x4CJuridine, and [14Clthymidine into acid-insoluble material, in m u t a n t strains which require these nutrients. The results presented in Fig. 2 show t h a t addition of bacteriocin causes a total cessation of protein synthesis after a characteristic lag time of 15-2o min, while RNA and DNA synthesis continue, although at a reduced rate.

Amino acid incorporation in cetl-/ree systems Cell-free extracts and ribosomes were prepared from untreated and bacteriocin-treated cells and compared for the ability to support in vitro amino acid incorporation. The results in Table I show that the preincubated cell-free extracts from bacteriocin-treated cells have only a slight capacity to incorporate z4C-labeled amino acids under the direction of phage MS2-RNA. Addition of bacteriocin to an extract of untreated cells has no effect on the incorporation activity, indicating that there is no direct interaction between components of the protein synthesizing system and the bacteriocin itself. The results in Table I I show that the effect of the bacteriocin on the amino acid incorporation must be localised on the ribosomes which lost almost all their incorporation activity. The supernatant fractions of bacteriocin-treated cells appear to support in vitro amino acid incorporation even better than a supernatant from untreated cells. Similar results as described for Enterobacter cloacae strain o2 (Tables I and II) were obtained with Klebsiella edwardsii var. edwardsii strain Sly Biochim. Biophys. Acta, 240 (1971) 122-136

BACTERIOCIN

DF13 AND

PROTEIN BIOSYNTHESIS

12 7

TABLE I INCORPORATION OF 14C-LABELED AMINO ACIDS IN A CELL-FREE SYSTEM WITH EXTRACTS DERIVED FROM BACTERIOCIN-TREATED AND UNTREATED CELLS* The i n c u b a t i o n m i x t u r e (total v o l u m e o.i ml) contained 0.03 ml of p r e i n c u b a t e d cell-free e x t r a c t a n d 2o # g M S , - R N A (see MATERIALS AND METHODS). After i n c u b a t i o n for 6o mid a t 37 ° the reaction was t e r m i n a t e d b y adding I ml of 5 % trichloroacetic acid. After centrifugation the precipit a t e w a s resuspended in 5 % trichloroacetic acid, heated for 15 miD at 9 o°, cooled, collected on W h a t m a n GF/C glass fibre filters, and w a s h e d twice w i t h 5 % trichloroacetic acid. Filters were dried a n d counted in a liquid scintillation counter.

Incubation mixture

Incorporation (counts/miD)

Control

Complete s y s t e m minus 1V~S~-RNA plus bacteriocin

Bacteriocin-treated

Complete s y s t e m minus M S f R N A

23 568 949 22 806 320 189

Cell-free e x t r a c t s were p r e p a r e d from cells of Enterobacter cloacae strain 02. Bacteriocin t r e a t m e n t was 60 miD w i t h iooo killing units/ml.

TABLE II INCORPORATION OF x4C-LABELED AMINO ACIDS IN A CELL-FREE SYSTEM WITH RIBOSOMES AND SUPERNATANT FRACTIONS DERIVED FROM BACTERIOCIN-TREATED AND UNTREATED CELLS* The incubation m i x t u r e (total v o l u m e / o . i ml) was the same as described in Table I, except t h a t instead of the p r e i n c u b a t e d cell-free e x t r a c t was added: iSo/~g unpurified ribosomes, 0.03 ml of s u p e r n a t a n t fraction, a n d IO/zg t R N A .

Incorporation (counts~miD)

Incubation mixture Ribosomes

Supernatant

Complete system

Minus MS2-RNA

Control

Control Bacteriocin-treated Control Bacteriocin-treated

4 896 6 586 320 229

6o9 8o9 240 188

Bacteriocin-treated

* Ribosomes and s u p e r n a t a n t fractions were p r e p a r e d from cells of Enterobacter cloacae strain o2. Bacteriocin t r e a t m e n t was 60 min w i t h iooo killing units/ml.

Integrity o/ribosomes and rRNA No changes could be observed in the sedimentation behaviour of 3o-S and 5o-S ribosomal particles isolated from bacteriocin-treated cells, compared with ribosomal particles from control cells (Fig. 3). Sucrose gradient sedimentation analysis of the rRNA, prepared by phenol extraction from control and bacteriocin-inactivated ribosomes, also shows no differences in sedimentation pattern except for a slight increase in the amount of radioactivity observed in the slowly sedimenting region (Fig. 4)The possibility of RNA degradation to acid-soluble material for the RNA derived from bacteriocin-inactivated ribosomes was explored by labeling the cellular RNA with [14C]uridine followed by bacteriocin treatment, and assaying the amount of radioactivity in acid-soluble and acid-insoluble material at appropriate intervals as described under MATERIALSAND METHODS. It was found that no degradation of cellular RNA occurred, at least not during the first hour after addition of the bacterioBiochim. Biophys. Acta, 24 ° (197 I) 122-136

F.K. DE GRAAF et al.

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Fig. 3. Sucrose gradient s e d i m e n t a t i o n analysis of ribosomes isolated from bacteriocin-treated and u n t r e a t e d cells. The ribosome m i x t u r e was p r e p a r e d as described u n d e r MATERIALS AND METHODS, and layered on the t o p of a 15-3 o % sucrose gradient in s t a n d a r d Tris-HC1 buffer b u t containing IO-* M m a g n e s i u m acetate. The gradient was centrifuged for 16 h in a MSE 3 × 23 ml So-rotor at 22 ooo rev./min. A capillary probe was slowly lowered to the b o t t o m of the tube, and t h e gradient was r e m o v e d b y siphoning. A2s 0 mu was m e a s u r e d c o n t i n u o u s l y with a Uvicord I I L K B 83oo UV A b s o r b t i o m e t e r equipped with a L K B 6520 A recorder. F r a c t i o n s of o. 5 ml were assayed for radioactivity as described in MATERIALS AND METHODS. - - - , A280 ln,u of ribosomes from unt r e a t e d cells; O - - - O , 14C radioactivity of ribosomes from bacteriocin-treated cells. Fig. 4. Sucrose gradient s e d i m e n t a t i o n analysis of r R N A extracted from control and bacteriocininactivated ribosomes. The r R N A was p r e p a r e d as described u n d e r MATERIALS AND METHODS. The R N A m i x t u r e was layered on t h e t o p of a 5-20 % sucrose gradient in s t a n d a r d Tris-HC1 buffer b u t containing lO -4 M m a g n e s i u m acetate. The gradient was centrifuged for 6 h in a MSE 3 × 23 ml SO-rotor at 39 ooo rev./min. A 260 mu a n d x4C-radiactivity were m e a s u r e d as described in the legend to Fig. 3. , A2eo mu of RblA from control ribosomes; O - - - O , xiC radioactivity of I~NA from bacteriocin-inactivated ribosomes.

cin. With the same method it has been shown that vibriocin induces DNA degradation 1° and that high "multiplicities" of colicin E 2 besides DNA degradation 21, induces degradation of rRNA after a lag period of about 30 rain (refs. 22, 23). All experiments described in this paragraph were performed with both indicator strains which gave the same result.

Size distribution o/ribosomal particles Exponentially growing cells of Enterobacter cloacae strain o2 or Klebsiella edwardsii var. edwardsii strain $15 were treated with an excess of bacteriocin (I000 killing units/ml) for about 2 h. Samples were taken at intervals as indicated in Fig. I. The ribosomes were isolated from the cells according to the method of GODSO~ AND SINSHEIMER 17, and analysed on sucrose gradients. The results presented in Fig. 5 show that the size distribution of the ribosomal particles of bacteriocin-treated cells, during the lag time, is similar to the distribution in untreated cells. However, as soon as growth was completely inhibited, a gradual accumulation of 3o-S and 5o-S ribosomal subunits concomitant with a decrease in the number of 7o-S and larger ribosomal particles was observed. About 2 h after addition of the bacteriocin most of the ribosomes are present as 3o-S and 5o-S subunits, which do not spontaneously associate at optimal concentrations of K + and Mg *+, present during isolation and centrifugation of the ribosomes. The data suggest that bacteriocin-inactivated ribosomes are defect in some binding function which prevents reassociation. It has been shown b y KAEMPFER et al. ~ that all ribosomal particles cycle periodically through a free pool of 3o-S and 5o-S ribosomal subunits. These subunits, in vivo, associate to 7o-S Biochim. Biophys. Acta, 240 (1971) 122-136

BACTERIOCINDFI3 AND PROTEIN BIOSYNTHESIS

12 9

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Fig. 5. Size distribution of ribosomes isolated from bacteriocin-treated cells. Ribosomes, isolated from various samples (see Fig. i), were layered on the top of a Io-25 % sucrose gradient in standard Tris-HC1 buffer. The gradients were centrifuged for 4.5 h in a MSE 3 × 20 ml SO-rotor at 29 ooo rev./min. A,60mu was measured continuously as described in the legend to Fig. 3. The numbers I-IV refer to the samples indicated in Fig. i. initiation particles in the presence of mRNA and formylmethionyl-tRNA. The experiments described below were undertaken to test the binding functions of bacteriocin-inactivated ribosomes. Binding o/ m R N A The binding function for m R N A was investigated using phage MS~-RNA. ALBRECHT et al. 2° have shown that MS=-RI~A binds primary to the 3o-S particles. This binding is fully dependent on ribosomal factors present in unpurified ribosomes. No mRNA-7o-S complexes are formed in the absence of fMEt-tRNA fua. Unpurified ribosomes, isolated from cells of Enterobacter cloacae strain 02 treated with an excess of bacteriocin (iooo killing units/ml) for 60 min, were suspended in standard Tris-HC1 buffer but with i0 -4 M magnesium acetate, and mixed with MS2-E3HJRNA. After incubation for IO min the reaction mixture was analyzed on a sucrose gradient (Fig. 6). It was found that the 3o-S ribosomal particles from bacteriocin-treated cells, although inactive in protein biosynthesis, are still capable to bind MS2-RNA, as a first step in polypeptide chain initiation. Binding o//Met-tRNA/Mato the m R N A - r i b o s o m e complex The second step in the initiation of polypeptide synthesis directed by phage RNA is the binding of fMet-tRNA f~tet to the mRNA-ribosome complex 2°,*~,ze. As bacteriocin-inactivated ribosomes bind MS2-RNA, it was necessary to test whether these ribosomes can promote the binding of fMet-tRNA fMa. The results presented in Table I I I show that bacteriocin-inactivated ribosomes are unable to bind E35SjfMet-tRNA fMa. The inability to promote the formation of the initiation complex containing MS2-[3HJRNA, ribosomes, and [35S]fMet-tRNAfMa was also demonstrated by sucrose gradient sedimentation analysis of the reaction mixture. If both the binding of m R N A and of formylmethionyl-tRNA to the 3o-S ribosomal subunit is still possible, addition of E35S~fMet-tRNAfMa to a mixture of MS2-~3HJRNA and unpulified ribosomes should result in a transfer of the tritiated MS2-RNA from the 3o-S peak (Fig. 6) to the 7o-S peak on the gradient. From Fig. 7, it can be seen that with conBiochim. Biophys. Acta, 24o (i971) z22-136

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Fig. 6. Binding of MS,-[3H]RNA to unpurified ribosomes from Enterobacter cloacae strain 02. A m i x t u r e of 2o # g MS=-[aH]RNA (specific activity 72o c o u n t s / m i n per Fg RNA), a n d 3o0/~g unpurified ribosomes w a s incubated at 37 ° for IO min as described u n d e r MATERIALS AND METHODS. Then, 3 o # g phosphodiesterase was added to degrade u n b o u n d m R N A , and i n c u b a t i o n was continued for a n o t h e r 45 min. Subsequently, t h e reaction m i x t u r e was cooled and layered on the t o p of a 15-35 % sucrose gradient w i t h the same ionic composition as the incubation m i x t u r e . The gradient was centrifuged for 2o h in a MSE 3 × 20 ml SO-rotor at 19 500 rev./min..4260 my and radioactivity were m e a s u r e d as described in the legend to Fig. 3. (A) Ribosomes f r o m u n t r e a t e d cells. (B) Ribosomes from bacteriocin-treated cells, - - , .4,e0 mu; O - - - O , aH radioactivity. TABLE III BINDING OF [35S]fl~ET-tR~q'AfMet TO COMPLEXES OF RIBOSOMES AND MS,-RNA* A m i x t u r e of 15o/,g unpurified ribosomes, 2o/zg MS=-RNA and 3 ° pmoles [a~s]fMet-tRNAfMet w a s incubated as described u n d e r MATERIALS AND METHODS. After 12 rain incubation at 37% t h e reaction m i x t u r e was s u p p l e m e n t e d w i t h 3 ml of buffer w i t h the same ionic composition as t h e i n c u b a t i o n m i x t u r e a n d filtered on Millipore H A W P 025 filters. The filters were w a s h e d twice, dried, and c o u n t e d in a liquid scintillation counter.

Incubation mixture

[35S]flViet-tRNAfMet (counts/min)

Control ribosomes

Complete s y s t e m minus MS~-RNA

1236 342

Bacteriocin-iuactivated ribosomes

Complete s y s t e m minus MS~-RNA

325 312

* Ribosomes were isolated from cells of Enterobacter cloacae strain o2. Bacteriocin t r e a t m e n t was 6o min w i t h iooo killing units/ml.

trol ribosomes both MS2-I3HIRNA and E85SlfMet-tRNA fM~t are detected in the 7o-S region indicating the formation of a 7o-S initiation complex. The conversion of MS~RNA to the 7o-S peak is never complete ~5. The tritium label found in the 3o-S region is also due to the presence of unbound, free MS2-RNA, which has a sedimentation constant of about 28 S. With bacteriocin-inactivated ribosomes, however, no radioactivity was found in association with the 7o-S ribosomes. The MS~-E3HIRNA was still detected in the 3o-S region, while EzsS]fMet-tRNA~M~t remains in the surface layer. As binding of formylmethionyl-tRNA is a prerequisite for the binding of other aminoacyl-tRNA's (refs. 25, 27), it m a y be concluded from these experiments t h a t bacteriocin-inactivated ribosomes are unable to support protein synthesis, because of a defect in polypeptide chain initiation. Biochim. Biophys. Acta, 24 ° (1971) 122-136

BACTERIOCIN D F I s AND PROTEIN BIOSYNTHESIS

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Fig. 7. B i n d i n g of MS=-[SH]RNA a n d [ssS]fNiet-tRNAfMet to u n p u r i f i e d r i b o s o m e s f r o m Enterobacter cloacae s t r a i n 02. T h e reaction m i x t u r e (see T a b l e h i ) w a s applied to a 15-35 % sucrose g r a d i e n t w i t h t h e s a m e ionic c o m p o s i t i o n as t h e i n c u b a t i o n m i x t u r e . T h e g r a d i e n t was c e n t r i f u g e d for 17 h in a M S E 3 × 23 m l SO-rotor a t 20 ooo r e v . / m i n . As60 m, a n d SH a n d ssS r a d i o a c t i v i t i e s were m e a s u r e d as described in t h e legend to Fig. 3. (A) R i b o s o m e s f r o m u n t r e a t e d cells. (B) R i b o s o m e s f r o m b a c t e r i o c i n - t r e a t e d ceils. - - , A~60 m/*; O - - - O , 35S r a d i o a c t i v i t y ; O . . . O , ~H r a d i o a c t i v i t y .

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Fig. 8. C o c h r o m a t o g r a p h y of 14C-labeled r i b o s o m a l p r o t e i n f r o m b a c t e r i o c i n - t r e a t e d cells, a n d *H-labeled r i b o s o m a l p r o t e i n f r o m u n t r e a t e d cells of Klebsiella edwardsii var. edwardsii s t r a i n Sis. T h e m i x t u r e of *4C- a n d SH-labeled r i b o s o m a l p r o t e i n (3 ° m g ) f r o m b o t h 3o-S a n d 5o-S r i b o s o m a l s u b u n i t s , p r e p a r e d as described u n d e r MATERIALS AND METHODS, WaS applied to a p h o s p h o c e l l u l o s e c o l u m n (30 c m × 2 cm) e q u i l i b r a t e d w i t h s t a n d a r d u r e a p h o s p h a t e buffer. T h e p r o t e i n s were e l u t e d f r o m t h e c o l u m n w i t h a linear g r a d i e n t of o - o . 6 M NaC1 in x2oo ml s t a n d a r d u r e a p h o s p h a t e b u f f e r (pH 6.5) a c c o r d i n g to HARDY et al.Zt T h e flow r a t e w a s 35 m l / h . To 6-ml fractions cold 4 ° ~o trichloroacetic acid w a s a d d e d to a final c o n c e n t r a t i o n of IO °/o. A f t e r s t a n d i n g for I h a t r o o m t e m p e r a t u r e , t h e p r o t e i n p r e c i p i t a t e s were collected on W h a t m a n G F / C glass fibre filters a n d a s s a y e d for r a d i o a c t i v i t y as described in MATERIALS AND METHODS. T h e SH c o u n t s (. . . . ) of t h e p r o t e i n s f r o m u n t r e a t e d cells are i n d i c a t e d b y t h e h i g h e r r a d i o a c t i v i t y scale, t h e 14C c o u n t s ( ) of t h e p r o t e i n s f r o m b a c t e r i o c i n - t r e a t e d cells b y t h e lower r a d i o a c t i v i t y scale. Biochim. Biophys. Acta, 240 (1971) I 2 2 - i 3 6

132

F.K.

D E G R A A F et

al.

Chromatographic comparison o/the ribosomal protein composition ol bacteriocin-treated and untreated cells The fact that ribosomes from bacteriocin-treated cells are unable to bind fMett R N A fMet under direction of MS2-RNA might be due to the absence of one or more functional ribosomal proteins (e.g. initiation factors). Therefore, the composition of 14C-labeled rib•soma1 protein isolated from bacteriocin-treated cells was compared with the composition of 3H-labeled ribosomal protein from control cells by cochromatography on phosphocellulose. As shown in Fig. 8 the chromatographic patterns of the two ribosomal protein preparations were virtually identical. Electrophoretic analysis of the various main peaks in 15 °/o polyacrylamide gels at p H 4.8 according to LEBOY el al. ~8 also revealed no differences between the band pattern obtained by staining with 0.5 % amid• black (representative for the added ribosomal protein from unlabeled control cells) and the band pattern obtained by autoradiography (representative for the t4C-labeled ribosomal proteins of bacteriocin-treated cells) following the procedure of FAIRBANKS et al. 29. So it can be concluded that the bacteriocin does not exert an effect on the protein composition of the ribosomes.

Leakage o/K+ Colicin E 1 and colicin K have been reported to inhibit active transport of K + (refs. 8, 30). Therefore, we investigated the possibility that bacteriocin DFlz too has an effect on the K+ transport of sensitive cells, moreover because K + are known to be necessary for protein synthesis 31. An excess of bacteriocin (IOOOkilling units/ml) was added to an exponentially growing culture and the K + content of the cells was measured at appropriate intervals. As shown in Fig. 9 A, baeteriocin-treated cells began to loose K + after a lag time of 15-2o rain, which leakage continued for about i h. The inhibition of growth, concomitant with the leakage of K +, very much resembles

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F i g . 9. L e a k a g e of K + f r o m b a c t e r i o c i n - t r e a t e d c e l l s of E n t e r o b a c t e r cloacae s t r a i n 02. A. Cells g r o w n i n n u t r i e n t b r o t h . B . Cells g r o w n i n n u t r i e n t b r o t h s u p p l e m e n t e d w i t h o.2 M K +. K + c o n t e n t w a s m e a s u r e d , ~s d e s c r i b e d b y TEMPEST et al. s=. A n e x c e s s of b a c t e r i o c i n ( i o o 0 k i l l i n g u n i t s / m l ) was added at the time indicated by the arrow. • 0 , g r o w t h of t h e c o n t r o l c u l t u r e ; • • g r o w t h of t h e b a c t e r i o c i n - t r e a t e d c u l t u r e ; C)- - -C), K + c o n t e n t c o n t r o l cells; A - - - ~ , K + c o n t e n t b a c t e r i o c i n - t r e a t e d cells. B i o c h i m . B i o p h y s . A c t a , 2 4 o (1971) 1 2 2 - 1 3 6

133

BACTERIOCIN DF13 AND PROTEIN BIOSYNTHESIS

the effect of potassium starvation on the growth of mutants of Escherichia colt a n d Bacillus subtilis defective in K + retention 83,~. If, as with these mutants, growth and protein synthesis are inhibited as a result of K ÷ shortage, addition of high concentrations of K + might protect the cells against bacteriocin action. However, as shown in Fig. 9 B, in the presence of high K + concentrations (0.2 M), the growth of

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Fig. i I. Effect of bacteriocin DF13 on g r o w t h of E n t e r o b a c t e r cloacae strain o2 in n u t r i e n t b r o t h s u p p l e m e n t e d w i t h a high concentration (o.2 M) of different cations. An excess of bacteriocin (IOOO killing units/ml) was added at the time indicated b y t h e arrow. Q - O , g r o w t h of the control culture; • - • , g r o w t h of the bacteriocin-treated culture w i t h o u t e x t r a cations; O - - - O , g r o w t h of the bacteriocin-treated culture in the presence of different cations. B i o c h i m . B i o p h y s . A c t a , 240 (1971) 122-136

134

F.K. DE GRAAF et al.

sensitive cells is still inhibited by the bacteriocin, but after a longer lag time. Fulthermore, under these conditions K + leakage still occurs although to a smaller extent. The delay in bacteriocin action in the presence of high K + concentrations appears to be due to a slower adsorption, as demonstrated b y the experiments illustrated in Fig. io. In nutrient broth, removal of the bacteriocin after IO rain of incubation has no influence on the lag time before growth inhibition (Fig. IoA). In nutrient broth supplemented with 0.2 M K +, however, this treatment causes a further increase in lag time (Fig. loB). Moreover, it was observed that besides K +, other mono- and divalent cations have the same effect (Fig. I I ) . Divalent cations are more effective than monovalent cations. The effect of high cation concentrations is quite similar to the effect of low bacteriocin concentrations described previously 15.

DISCUSSION

The results presented in this paper show that adsorption of bacteriocin DFI~ onto sensitive bacteria, induces a specific inhibition of protein biosynthesis. After a lag time of 15-2o min, the incorporation of [14C]cysteine into acid-insoluble material ceases completely and growth is arrested. Nucleic acid synthesis, however, continues at normaP 5 or at a reduced rate depending on the indicator strain used. I n vitro experiments with ribosomes isolated from bacteriocin-treated cells show that these ribosomes are unable to support protein synthesis in a cell-free system under direction of phage MS2-RNA. Binding function experiments with MS2-RNA and bacteriocin-inactivated ribosomes have shown that these inactive ribosomes can still bind mRNA. However, subsequent binding of formylmethionyl-tRNA, as the second step in polypeptide chain initiation, is inhibited. This inability to promote the formation of an initiation complex, in this system, also prevents the ribosomes from the binding of aminoacylt R N A other than fMet-tRNA fMet (refs. 25, 27). These experiments do not rule out, however, the possibility that, in vivo, synthesis of polypeptide chains which had already begun before initiation of protein synthesis was stopped b y the bacteriocin m a y be completed. KONISKY AND NOMURA~5 obtained similar results for colicin E 3. They found that treatment of sensitive cells with colicin E~ leads to a structural alteration in the 3o-S ribosomal subunit. The result of this alteration is an inhibition of the poly U-directed binding of phenylalanine-tRNA, while binding of the poly U itself on both 3o-S and 7o-S particles remains intact. Although their experiments do not distinguish non-specific random binding of the m R N A from the correct specific binding, the effect of colicin E 3 resembles that of bacteriocin DF13. Both bacteriocins have also m a n y similarities in amino acid composition 1~'36, but a completely different activity spectrum 11. Inactive ribosomes isolated from cells treated with bacteriocin DF13 seem to be structurally unaltered. The sedimentation coefficient of the inactive ribosomal subunits, and the integrity of their RNA constituents are indistinguishable from that of control particles. The possibility was considered that the inactive ribosomes missed one or more functional ribosomal proteins, for instance certain initiation factors, or a ribosomal protein of the type discovered by OZAKI et a l Y which determines the Biochim. Biophys. Acta, 240 (1971) 122-136

BACTERIOCIN DF13 AND PROTEIN BIOSYNTHESIS

135

sensitivity of the 30-S subunit to streptomycin. However, an extensive comparison of the ribosomal proteins of inactive ribosomal subunits and those of control particles do not reveal a n y difference in ribosomal protein composition. Although we cannot completely exclude the possibility that bacteriocin-inactivated ribosomes are missing one of the so-called fractional proteins 38, it seems more likely to suppose that the inactivity of these ribosomes must be ascribed to a subtle, perhaps localized, conformational change of the ribosomal subunits. A similar phenomenon of ribosome interconversion from the active to the inactive state, in vitro, has recently been described b y MISKIN et al. 39. They reported that removal of monovalent cations renders the ribosome inactive as a result of some structural rearrangement without any lack of essential components or change in sedimentation behaviour. The ribosomes could only be reverted to the fully active state if the reaction mixture was supplemented with a sufficient concentration of the appropriate cation and also heated to supply the required activation energy. These findings make it very likely that the leakage of K ÷ from bacteriocin-treated cells plays an important role in the conformational change which renders the ribosomes inactive. A number of previous reports have already shown that K+, as well as Mg~+, are essential cations for the protein synthesizing system 81,4°,41. Lu~IN AND ENNIS4° have shown that protein synthesis in cell-free extracts of K+-dependent mutants at low K + concentrations, is limited by the transfer of amino acid from aminoacyl-tRNA to the polypeptide. SPYRIDES41, and PESTKA AND NIRENBERG 42, showed that the binding of aminoacyl-tRNA to the mRNA-3o-S ribosome complex is stimulated b y K +. However, in contrast with bacteriocin-treated sensitive cells, addition of a sufficient amount of K + allows growth as well as protein synthesis in K+-dependent mutants. Furthermore, the in vitro activity of bacteriocin-inactivated ribosomes is not restored under conditions which support protein synthesis on ribosomes of K+-dependent mutants. These data show that ribosome inactivation as a result of bacteriocin action is not comparable with the inhibition of protein synthesis as a result of K + starvation. Apparently, the interaction of bacteriocin DFls with the cytoplasmic membrane of sensitive cells includes more than only an effect on K ÷ transport. As mentioned before the effects of colicin E3 and bacteriocin DF13 on protein synthesis show m a n y similarities although colicin E 3 does not induce K + leakage s. While this paper was in preparation, SENIOR et al. 4~ presented evidence that colicin E3, in vivo, causes a gradual loss of polysomes. In contrast with bacteriocin DFlz , this loss of polysomes is accompanied by approximately identical increases in the amounts of both 7o-S particles and ribosomal subunits. The majority of the polysomes, isolated during the period that protein synthesis was progressively inhibited, were unstable and rapidly dissociate, in vitro, at low Mg ~+ concentrations. These authors further showed that, in vivo, protein synthesis was blocked b y an inhibition of polypeptide chain elongation or termination and they supposed that this inhibition is the direct result of an alteration of a polysomal component. However, it must be emphasized that these authors did not properly investigate the effect of colicin Ea on the initiation of protein synthesis. Therefore, it m a y be possible that for both bacteriocins the primary effect on protein synthesis is a block in polypeptide chain initiation.

Biochim. Biophys. Acta, 24o (1971) i22-I36

136

E . K . DE GRAAF et al.

ACKNOWLEDGEMENTS

We wish to thank Dr. H. 0. Voorma for advice about amino acid incorporation and binding assays with phage MS~-RNA, and Mrs. J. M. van Gasteren-Vlaskamp for excellent technical assistance in the analysis of ribosomal proteins. This investigation was supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.0.). REFERENCES P. FREDERICQ, Compt. Rend. Soc. Biol., 148 (1954) 399. R. C. CLOWES, Nature, 19o (196o) 986. P. FREDERICQ, Syrup. Soc. Exptl. Biol., 12 (1958) lO 4. F. JAcoB, L. SIMINOVlTCH and E. WOLLMAN, Ann. Inst. Pasteur, 83 (I952) 28I. R. NAGEL DE ZWAIG, J. Bacteriol., 99 (1969) 913. K. L. FIELDS AND S. E. LURIA, J. Bacteriol., 97 (1969) 64. Y. KAZIRO AND M. TANAKA, J. Biochem., 57 (1965) 689. ]V[. NOMURA and A. MAEDA, Zentr. Bakteriol. Parasitenk..dbt. I Orig., 196 (1965) 216. I. ]3. HOLLAND, Biochem. Biophys. Res. Commun., 13 (1963) 246. A. J&YAWARDENE AND H. FARKAS-HIMSLEY, J. Bacteriol., lO2 (197o) 382. A. H. STOUTHAMER AND G. A. TIEZE, Antonie van Leeuwenhoek, 32 (1966) I 7 I . F. K. DE GRAAF, L. E. GOEDVOLK-DE GROOT AND A. H. STOUTHAMER, Biochim. Biophys. Acta, 22I (197 o) 566. 13 F. K. DE GRAAF, G. A. TIEZE, SJ. WENDELAAR ]~ONGA AND A. H. STOUTHAMER, J. Bacteriol., 95 (1968) 631. 14 G. A. TIEZE, A. H. STOUTHAMER, H. S. JANSZ, J. ZANDBERG AND E. F. J. VAN BRUGGEN, Mol. Gen. Genetics, lO6 (1969) 48. 15 F. I~. DE GRAAF, ELISABETH A. SPANJAERDT SPECKMANN AND A. H. STOUTHAMER, Antonie van Leeuwenhoeh, 35 (1969) 287. 16 H. O. VOORMA, P. W. GOUT, J. VAN DUIN, ]3. W. HOOGENDAM AND L. BOSCH, Biochim. Biophys. Acta, 95 (1965) 446. 17 G. N. GODSON AND R. L. SINSHEIMER, Biochim. Biophys. Acta, 149 (1967) 476. 18 D. GILLESPIE AND S. SPIEGELMAN, J. Mol. Biol., 12 (1965) 829. 19 S. J. S. HARDY, C. G-. KURLAND, P. VOYNOW AND G. MORA, Biochemislry, 8 (1969) 2897. 20 J. ALBRECHT, B. W. HOOGENDAM, W. ROZENBOOM, N. J. VERHOEF, H. O. VOORMA AND L. BOSCH, Biochim. Biophys. Acta, 19 ° (1969) 504 • 21 M. NOMURA, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 315 . 22 K. NOSE, D. MIZUNO AND H. OZEKI, Biochim. Biophys, Acta, 119 (I966) 636. 23 K. NOSE AND D. MIZONO, J. Biochem., 64 (1968) i. 24 R. O. R. KAEMPFER, IV[. MESELSON AND J. RASKAS, J. Mol. Biol., 31 (1968) 277. 25 H. O. VOORMA, R. BENI~E AND F. H. SCHOLTE TER HORST, J. Mol. Biol., 45 (1969) 423 • 26 M. NOMURA AND C. LOWRY, Proe. Natl. Aead. Sci. U.S., 58 (1967) 946. 27 R. VV. ERBE, M. M. NAU AND P. LEDER, J. Mol. Biol., 38 (1969) 441. 28 P. S. LEBOY, E. C. Cox AND J. G. FLAKS, Proe. Natl. Acad. Sci. U.S., 52 (1964) 1367. 29 G. FAIRBANKS, JR., C. LEVlNrHAL AND R. H. REEDER, Biochem. Biophys. Res. Commun, 20 (1965) 393. 3 ° S. E. LURIA, Ann. Inst. Pasteur, Suppl., 5 (1964) 67. 31 M. LUmN, Federation Proc., 23 (1964) 994. 32 D. \V. TEMPEST, J. W. DicKs AND J. R. HUNTER, J. Gen. Microbiol., 45 (1966) 135. 33 H. L. ENNIS AND M. LUBIN, Biochim. Biophys. Acta, 5 ° (1961) 399. 34 D. B. \VILLIS AND H. L. ENNIS, J. Bacteriol., 96 (1968) 2035. 35 J. KONISKY AND M. NOMURA, J. Mol. Biol., 26 (1967) 181. 36 H. R. HERSCHMAN AND D. R. HELINSKI, J. Biol. Chem., 242 (1967) 536o. 37 M. OZAKI, S. MIZUSHIMA AND M. NOMURA, Nature, 222 (1969) 333. 38 C. G. KURLAND, P. VOYNOW, S. J. s. HARDY, L. RANDALL AND L. LUTTER, Cold Spring Harbor Syrup. Quant. Biol., 34 (1969) 17. 39 R. MISKIN, A. ZAMIR AND D. ELSON, J. Mol. Biol., 54 (I97 °) 355. 4 ° M. LUBIN AND ]7{. L. ENNIS, Bochim. Biophys. Acta, 80 (1964) 614. 41 G. J. SPYRIDES, Proc. Natl. Acad. Sci. U.S., 51 (1964) 122o. 42 S. PESTKA AND ~V[. NIRENBERG, J. Mol. Biol., 21 (1966) 145. 43 ]3. \V. SENIOR, J. KWASNIAK AND I. B. HOLLAND, J. Mol. Biol., 53 (197 °) 205. I 2 3 4 5 6 7 8 9 io II 12

Biochim. Biophys. Acta, 240 (1971) i 2 2 - I 3 6