JOURNAL OF BACTERIOLOGY, Mar. 1974, p. 1280-1288Copyright © 1974 American Society for Microbiology

Vol. 117, No. 3Printed in U.S.A.

Growth of Ribonucleic Acid Bacteriophage f2 in a ConditionalPutrescine Auxotroph of Escherichia coli: Evidence for a

Polyamine Role in TranslationDELANO V. YOUNG AND P. R. SRINIVASAN

Columbia University, College of Physicians and Surgeons, Department of Biochemistry,New York, New York 10032

Received for publication 10 December 1973

The ribonucleic acid (RNA) bacteriophage, f2, grows poorly in a conditionalputrescine auxotroph during polyamine starvation. The addition of putrescinesimultaneously with f2 enhances phage growth, shortens the latent period, andincreases the burst size. The stimulation of f2 growth is reflected in higher ratesof phage RNA and protein syntheses as measured by radioactive labeling ofinfected cells in the presence of rifampin. Putrescine does not affect f2 adsorptionor the penetration of its RNA. Rather, in vitro assays demonstrate that inputrescine-supplemented cells more molecules of f2 replicase are made perincoming parental RNA than in polyamine-starved cultures. The ability ofpolyamines to stimulate the translation of a preformed messenger suggests aphysiological role for these organic cations in normal protein synthesis.

The polyamines putrescine and spermidine,found as natural products in most organisms,have been shown to be necessary for rapidgrowth in Escherichia coli through the use of amutant conditionally incapable of synthesizingthe initial precursor, putrescine (7, 11, 16a, 24).Earlier (24), we demonstrated that the additionof putrescine (or spermidine) to cells partiallydepleted of their polyamines initiated a definitesequence of macromolecular events, the firstdetectable effect being the stimulation of pro-tein synthesis (24). After approximately 60 min,increases in stable ribonucleic acid (RNA) anddeoxyribonucleic acid syntheses and an acceler-ation in the rate of cell division occurred. Thosepast experiments were not designed to measurechanges in messenger RNA (mRNA) synthesis.The immediate stimulation of protein synthe-

sis suggests that polyamines are needed in anormal cell for mRNA synthesis and/or for theefficient translation of pre-existing messengers.To test the second possibility, it was decided toutilize the special properties of the male-specific RNA bacteriophage f2. This RNAphage (12, 20, 26) carries its own preformedmessenger. After infection, the parental "plus"strand of 106 molecular weight, bearing infor-mation for only three genes, is partially trans-lated into the phage-specific subunit of the f2replicase (2, 4, 9, 14). Synthesis of double-stranded RNA and progeny plus strand RNA,and the translation of the other two genes for

coat and maturation proteins, follow in a defi-nite order (12, 20, 26). Since f2 inserts apreformed message whose immediate transla-tion product, the f2 replicase, can be assayed (2,3, 9, 14), it was felt that this system provided ameans of uncoupling translation from host tran-scription and offered an opportunity to observe,in vivo, any direct effect of polyamines ontranslation.

MATERIALS AND METHODSBacterial strains. The male-specific, RNA bacte-

riophage, f2, was kindly provided by Peter Model. E.coli K-12 strain C600 (K19) (5, 23), also provided byP. Model, was used to prepare large phage stocks andserved as the indicator strain for f2. The conditionalputrescine auxotroph, E. coli K-12 strain MA-135,(F-, argE, his, pro, trp, thi, speB, canR) whosemicrobiological properties have been described exten-sively elsewhere (11, 24), was converted into a suitablemale host for f2 by bacterial mating with the maledonor strain KLF 15/JC 1553 (F' his+/his, leu, met,arg,' lacY, str, recA) provided by Werner Maas.Transfer of the male genotype was indicated by loss ofthe histidine requirement in the female recipient andby the ability to grow f2. The conditional putrescinerequirement was tested as previously described (11,24) on arginine-containing plates. A suitable male,conditional putrescine auxotroph, designated strainED-5 (F', his+/his, argE, pro, trp, thi, canR, speB),was used in the following experiments.

Biochemicals. The polyamines, putrescine dihy-drochloride and spermidine trihydrochloride, were

1280

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

POLYAMINES AND TRANSLATION OF f2 RNA

purchased from Calbiochem. Rifampin was suppliedby the Dow Chemical Co. Ribonuclease-free deox-yribonuclease I was obtained from Worthington Bio-chemical Corp., whereas pancreatic ribonuclease waspurchased from both Worthington and Sigma Chemi-cal Co. The sodium salts of adenosine 5'-triphosphate(ATP), uridine 5'-triphosphate (UTP), cytidine 5'-tri-phosphate (CTP), and tetralithium guanosine 5-tri-phosphate (GTP) were all purchased from Schwarz/Mann, neutralized to pH 7.0, and stored frozen at - 20C before use. The tricyclohexylammonium salt ofphosphoenolpyruvate (converted to the potassiumsalt, neutralized to pH 7.0, and stored frozen at - 20 Cbefore use) and phosphoenolpyruvate kinase were theproducts of Boehringer Mannheim Corp. [5-3Hjuracil(500 sCi/ml, 7,800 AtCi/Lmol), L_ [14C Ileucine uni-formly labeled (50 MCi/ml, 312 gCi/Umol), L-["4C]his-tidine uniformly labeled (50 ACi/ml, 312 MCi/Mmol),and ['H JGTP (1,000 MCi/ml, 1,380 MCi/Mmol) were allpurchased from Schwarz/Mann. The f2 antiserumused in the adsorption experiments was the generousgift of Peter Model.

Bacterial media. Strain ED-5 was grown in eitherminimal medium or enriched, synthetic medium. Theminimal medium (24) contained the Davis-Mingiolisalts medium (6) plus 0.25% glucose, proline (100Ag/ml), tryptophan (100 ;ig/ml), and thiamine (2gg/ml). When either ornithine (100 ug/ml), arginine(200 jg/ml), or arginine plus putrescine (100 Ag/ml)were added, the medium was designated MMO,MMA, or MMAP, respectively. The enriched, syn-thetic medium, AF', was a modification of the previ-ously described AF medium (11). It contained the lowphosphate, tris(hydloxymethyl)aminomethane(Tris)-maleate buffered salts mixture (17), plus 0.5%glucose, all the common amino acids (except forhistidine, leucine, lysine, arginine, and ofithine),adenosine, and vitamins (including thiamine). Sincef2 penetration requires Ca2" (19), a low-phosphatemedium was necessary to prevent precipitation ofcalcium phosphate. The precise concentrations of allof the above components can be found in earlierreferences (11, 17). For the radioactive labeling exper-iments, uracil, leucine, and histidine were added ascalled for below. In general, when they were not usedas labels, these compounds were added as supple-ments. As with the minimal media, when ornithine,arginine, or arginine plus putrescine were added, theenriched, synthetic medium was designated AF',AF'A, or AF'AP, respectively. For details concerningthe high-phosphate media AFO and AFA a previousreport should be consulted (11).

Tryptone broth (15) was used for the growth ofstrain K19.

Polyamine starvation procedure. Polyamine-starved cells were prepared by the following procedure(24). Strain ED-5 was grown overnight at 37 C withaeration in MMO medium. The culture was thenrefrigerated for a minimum of 4 h at 4 C. A 1:20inoculum was added to MMA medium and allowed togrow with aeration at 37 C overnight. The followingmorning the cells were centrifuged and resuspended(5- to 10-fold diluted) in AF'A medium or anothersuitable medium.

Radioactive labeling of phage-specific RNA and

protein. A polyamine-starved culture of strain ED-5,prepared as outlined above, was centrifuged andresuspended (5- to 10-fold diluted) in AF'A medium.After growth at 37 C with aeration for 3 h, [5-3H ]uracil(0.25 MCi per 10 Ag per ml) and L-["C]leucine (0.30MCi per 10 Ag per ml) were added to the culture. Onehour later the culture was divided into three portions;all portions received f2 (multiplicity of infection[MOI] -30) and CaCl2 (2.5 mM). One minute laterputrescine (100 Ag/ml) was added to one culture(early putrescine addition). At 10 to 12 min afterinfection, rifampin (25 ug/ml) was added to allcultures. At 16 min postinfection, putrescine wasadded to a second culture (late putrescine addition).Samples of 0.5 ml were removed from all cultures atthe indicated intervals and added to 0.5 ml of coldtrichloroacetic acid (10%). The precipitates were col-lected on GF/C filters, washed with 5% trichloroaceticacid, dried, and counted in 2,5-diphenyloxazole-1,4-bis-2-(5-phenyloxazolyl)benzene toluene with a Nu-clear-Chicago Mark I liquid scintillation counter.

Preparation of f2 replicase containing infectedcell extracts and the f2 replicase assay. The follow-ing procedure for the preparation of infected cellextracts and the f2 replicase assay were derived withminor modifications from previously publishedmethods (2, 3, 9, 14).

Polyamine-starved or putrescine-supplementedcultures of strain ED-5 cells (10' to 2 x 10'/ml), atvarious times after infection with f2 (MOI-5), werepoured over frozen, crushed saline and collected bycentrifugation at 4 C. Each cell pellet (stored at -20C) was ground with twice its weight of alumina at 4 Cfor a few min. A volume of starting buffer (50 mMTris-chloride buffer, pH 7.5, 5 mM magnesium ace-tate, 1 mM ethylenediaminetetraacetic acid, 0.1 mMdithiothreitol, and 25% [vol/vol] glycerol) equal toeight times the cell weight was added to each extractand the mixture was centrifuged twice at 30,000 x gfor 10 min at 4 C. The supernatant, containing f2replicase activity, was stored at -70 C.The f2 replicase assay was carried out in a total

volume of 0.2 ml and contained 0.125 M potassiumphosphate buffer, pH 7.0, 0.5 mM dithiothreitol, 10mM magnesium acetate, 0.25 mM each of ATP, CTP,and UTP, ribonuclease-free deoxyribonuclease I (25Mg/ml), rifampin (5 Mg/ml), 10% (vol/vol) glycerol, 5mM phosphoenolpyruvate, potassium salt, phospho-enolpyruvate kinase (2 Mg/ml), 0.1 mM [3H]GTP (2MCi/assay, 100 MCi/Mtmol, or 44 counts per min perpmol of GMP incorporated), and infected cell extractin varying amounts. The reaction mixture was incu-bated at 25 C for 10 min and terminated by theaddition of 3 ml of cold trichloroacetic acid (5%)containing 0.02 M sodium pyrophosphate. The pre-cipitates were collected on GF/C filters, washedrepeatedly with 5% trichloroacetic acid which con-tained 0.02 M sodium pyrophosphate, dried, andcounted as above. As a suitable background thetrichloroacetic acid-precipitable activity found inuninfected cell extracts was subtracted from infectedcell activity- at each protein concentration. The pro-tein concentrations in infected and uninfected ex-tracts were determined by the method of Lowry et al.(16).

VOL. 117, 1974 1281

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

YOUNG AND SRINIVASAN

RESULTSOne-step growth curves. For the purpose of

our experiments, the conditional putrescineauxotroph, MA-135, was converted by conjuga-tion to a male strain, ED-5. Strain ED-5, whichlike strain MA-135 (11, 24) cannot synthesizeputrescine when arginine is present in theexternal medium and ornithine is absent. Itgrows with a generation time of 45 min intryptone broth and 90 min in the enrichedsynthetic medium, AFO. After overnight star-vation for polyamines, a generation time of 200to 300 min is obtained in the starvation me-dium, AFA. The procedure used here to starvethe cells of their endogenous pools of polya-mines causes the intracellular putrescine to fallto below detection levels, whereas spermidine ispresent at one-third to one-half of the valueobserved in putrescine-supplemented cultures(11).When f2 is grown in strain ED-5 under the

different culture conditions cited above, thelatent period of phage development and thefinal burst size reflect the cellular metabolicrate at the time of infection. The latent periodin tryptone broth is 30 min, in AF'O medium 50to 60 min, and for polyamine-starved cells inAF'A medium it is 80 to 90 min. Similarly, theburst size is highest in tryptone broth and fornonstarved cells in AF'O medium and lowest forpolyamine-starved cells in AF'A medium.To investigate the effects of polyamine star-

vation and supplementation on f2 growth, poly-amine-starved cells were grown in AF'A me-dium and infected with phage. Either shortlybefore or simultaneously with phage infection,putrescine was added to the cultures (Fig. 1). Itcan be seen (Fig. 1) that putrescine increasesthe initial burst size (at 180 min) and shortensthe latent period. Addition of putrescine 20 minprior to infection raises the burst size to a levelnear that of nonstarved, infected cells grown inan AF'O medium (unpublished data). At timesvery late after infection (300 min), the phageyield of the starved culture begins to approachthat of the culture supplemented with putres-cine at the time of infection.Phage-specific RNA and protein

syntheses. Rifampin is a well-known inhibitorof E. coli RNA polymerase (13, 22). Whenadded to an exponentially growing culture ofstrain ED-5, rifampin (25 ,ug/ml) immediatelystops RNA synthesis, as measured by radioac-tive incorporation, and within 5 min proteinsynthesis also ceases (Fig. 2). Since f2 does notshut down host RNA and protein syntheses, it isnecessary to add rifampin to infected cultures in

100,000

50P00

10,000

5,000

U-

a.

1,000

500

100

50

,,%\

0 2 3 4 5TIME (HOURS)

FIG. 1. Effect of polyamine starvation on the one-step growth of f2 in strain ED-5. Polyamine-starvedcells of strain ED-5 (see Materials and Methods) were

resuspended in AF'A medium (supplemented withhistidine and leucine) and grown at 37 C for -3 h to acell density of 0.6 x 10'/ml. At -20 min the culturewas divided into three portions and one portionreceived putrescine. At -5 min putrescine was addedto a second portion and all three then received NaCN(2 mM), CaCI2 (2.5 mM), and f2 (MOI 0.12). Attime zero the cultures were diluted 10-' and titratedby standard procedures (1, 15) without chloroform forextracellular phage released at the indicated times.The data presented as total PFU/ml includes bothadsorbed and unadsorbed phage. In this experimentabout one-third of the initial PFU represents ad-sorbed phage as determined by chloroform treatment.Strain K19 served as the indicator bacterial strain.Incubation of all tryptone broth-agar plates (15) wasat 37 C overnight. Symbols: A, no putrescine; 0,

putrescine added with f2; 0, putrescine added at -20min.

order to measure radioactively labeled phageRNA and protein (10).

Figures 3 and 4 summarize the results of atypical double-label experiment in which phageprotein was labeled with L"C Jleucine andphage RNA was labeled with [3Hluracil. In thisexperiment a polyamine-starved culture grow-ing in an AF'A medium was divided into threeparts. At zero time all cultures received f2 andCaCl2. The first culture also received putrescine

*0~~

1282 J. BACTERIOL.

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

POLYAMINES AND TRANSLATION OF f2 RNA

syntheses which is absent in the polyamine-I starved culture during the course of the experi-

ment.20* ct 6 Z Effect of putrescine on f2 adsorption and20 / / X penetration. Although the results obtained

x thus far suggest that polyamines can exert atRif 4 2 least some of their stimulatory effects on f2a.azo/ . ~ growth independently of host RNA transcrip-

0. wu 15 . z tion, the exact cause of these effects is stillx A U unclear. They might either act at some step in10 ~~~~~~~Do 52 the intracellular development of the phage, or_J / / <, simply assist in f2 adsorption and penetration.10// Table 1 shows that putrescine does not affect

v O <,Ri0f the adsorption of f2 to bacterial cells. In this

5 0

5400.~~~~~~~~~~~~~

/E( < 1 t>~~~~u300 /

x+Put

0 IhI 2 3 4 Z -ut

TIME (HOURS) J PXtFIG. 2. Inhibition of host RNA synthesis by rifam- Rit 5 A A

pin in exponentially growing cells. An overnight !culture of strain ED-5 in MMO medium was cen- 10 Co +f24trifuged and resuspended (10-fold diluted) at timezero in AF'O medium containing L-[14C]leucine (0.05 o 2 3MCi per 5 Ag per ml) and [5-3H]uracil (0.25 MCi per 10 TIME (HOURS)Mg per ml). At the indicated times, 0.5-ml samples FIG. 3. Stimulation of phage protein synthesis bywere removed and mixed with 0.5 ml of cold 10% polyamines. The exact experimental protocol is giventrichloroacetic acid. At 2 h and 40 min, the culture in detail in Materials and Methods. Rifampin (25was divided into four parts and rifampin was added to Mig/ml) was added at 11 min postinfection. Symbols:three of them at the concentrations of 10 jg/ml (0), A, no putrescine added; 0, putrescine added 1 min25 Mglml (A), and 50 jg/ml (A). The fourth portion after phage; 0, putrescine added 16 min after phage.(0, 0) remained rifampin free. The samples weretreated and counted, and the 3H and 'IC counts were 36distinguished as described in Materials and Methods. £

32 A to Put A

1 min after infection (early putrescine). At 11 A

min postinfection, rifampin (25 ug/ml) was 28 Puta. Puadministered to all the cultures. Finally, at 16 LImin, putrescine was added to the second culture O 24

(late putrescine). The third culture did not ,-Jreceive any putrescine. Both figures clearly i 20 P tshow that restoration of the cellular polyamine ' 0level causes significant increases in the rates of I 16 PutIphage RNA and protein syntheses. The time of Rifputrescine addition either before or after rifam- 12 4pin addition does not seem to affect the labelingcurves (the difference seen in the RNA curve

0 2 3was not confirmed by other experiments). The .S TIME (HOURS)FIG. 4. Stimulation of phage RNA synthesis bystimulatory effects of polyamines must, there- polyamines. These results were computed from thefore, occur independently of the host transcrip- double-label experiment described in Fig. 3. Symbols:tional system. Also, it should be noted that in 0, no putrescine added; A, putrescine added 1 minthe putrescine-supplemented culture there is a after phage (Early Put); A, putrescine added 16 mindistinct shut-off of both RNA and protein after phage (Late Put).

1283VOL. 117, 1974

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

YOUNG AND SRINIVASAN

TABLE 1. Effect of putrescine and spermidine on f2adsorption"

Infection media

Determination AF'A AFAP(polyamine- (polyamine-starved) supple-

mented)Bacterial cell count b/ml 6.8 x 106 6.8 x 106f2 Input (PFU/ml) 3.3 x 107 3.3 x 107Total PFU/ml 2.62 x 107 2.33 x 107Infectious centers/ml 1.74 x 106 1.85 x 106Chloroform-treated 1.7 x 10' 2.7 x 10'

supernatant (PFU/ml)Corrected infectious 1.57 x 106 1.58 x 106

centers/mlBacteria infected (%) 23:1 23.25Input f2 producing 4.76 4.79

infectious centers (%)

a A culture of polyamine-starved strain ED-5 cells,after growth in AF'A medium at 37 C to a density of0.5 x 10" to 1.0 x 108 ml, was divided, and putrescine(100 jAg/ml) was added to one portion. Immediately,CaCl2 (2.5 mM) and f2 (MOI - 3 to 5) were added,and phage adsorption was permitted for 10 min a't37 C. The infected cultures were then diluted 1:10with the Tris-maleate buffered salts medium (17)containing 2.5 mM CaCl2, and the total plaque-form-ing units (PFU) of adsorbed and unadsorbed phage("total PFU") of the diluted cultures was measured.The diluted cultures then received f2-specific an-tiserum and were kept at room temperature for 15 minto remove unadsorbed phage. The cultures were thentitrated to determine the amount of adsorbed f2("infectious centers"). Part of the remaining dilutedcultures were treated with CHCl3 at 37 C for 30 minand centrifuged, and the supernatant solutions weretitrated to measure any unadsorbed phage whichescaped antiserum neutralization ("chloroform-treated supernatant"). This number was subtractedfrom the value obtained for "infectious centers" toyield a better estimate of the adsorbed phage ("cor-rected infectious centers"). The "bacteria infected(%)" and the "input f2 producing infectious centers

were computed by dividing the values for the"corrected infectious centers" by the "bacterial cellcount" and the "f2 input."

h The "bacterial cell count" was determined for the1: 10 diluted cultures.

experiment a polyamine-starved culture wasinfected with f2 (MOI-5) in the presence andabsence of putrescine. Ten minutes later thecultures were diluted 10-fold and titrated for f2plaque-forming units, and sufficient f2 an-tiserum was added to remove all unadsorbedphage. After an additional 15 min the cultureswere again titrated and treated with chloro-form, and the chloroform-treated supernatantwas re-titrated. The corrected values for the

number of infectious centers were equal for bothcultures. Putrescine does not increase the frac-tion of bacteria susceptible to f2 adsorption.

Similarly, the addition of putrescine at thetime of infection does not facilitate the penetra-tion of f2 RNA into the host cell. It is knownthat ribonuclease and rifampin added prior to f2infection inhibit f2 plaque formation almostcompletely (8, 10, 19, 25; unpublished data).Addition of rifampin after infection causes lessinhibition of plaque formation, the degree ofinhibition being dependent on the time ofaddition of the drug after infection (8; unpub-lished results).The effect of ribonuclease, added prior to f2,

on phage-specific RNA labeling is illustrated inFig. 5. In polyamine-starved cells, ribonucleasegreatly reduces phage RNA synthesis for nearly2 h, although RNA is rapidly made later.Putrescine supplementation causes an increasein the labeling rate and this is also true in thescultures devoid of exogenous ribonuclease (Fig.4) and probably reflects a stimulation of intra-cellular events. On the other hand, if ribonu-clease is added 12 min after f2 infection there isno effect on phage RNA labeling either in theabsence or presence of putrescine (Fig. 6).Putrescine added before (at 1 min) or afterribonuclease (at 16 min) elicits no change in thelabeling pattern. In polyamine-starved cultures,

60

a. 50u +RNase Put

.i 40 / ^0,>, +Noase-Put

_4 0.cr~~~~~~-Nose -Pu

Rif~,30 1e

0 2 3TIME (HOURS)

FIG. 5. Inhibition of phage RNA synthesis by ribo-nuclease added prior to infection. The experimentalprotocol followed closely the procedure described inMaterials and Methods for radioactive labeling ofphage RNA with the major exception that ribonu-clease (20 1ig/ml) was added to all four cultures 10 minbefore infection with f2 (time zero). Putrescine andrifampin were added at I and 12 min after infection,respectively. Symbols: 0, no putrescine added, noribonuclease added; *, no putrescine, but with ribo-nuclease addition; A, putrescine added, but no ribo-nuclease; A, putrescine added, ribonuclease added.

1284 J. BACTERIOL.

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

POLYAMINES AND TRANSLATION OF f2 RNA

iS' Erly Put

a.

0 -Put

-24--j

RNose

2 3TIME (HOURS)

FIG. 6. Effect of ribonuclease added after infectionon phage RNA synthesis. The experimental procedureis identical to the experiment of Fig. 3 and 4 with theexception that ribonuclease (20,ug/ml) was added toall three cultures 12 min after f2 infection. Sym bols:O, no putrescine; A, putrescine added I min afterphage (Early Put); A, putrescine added 16 min afterphage (Late Put).

f2 adsorption and penetration are completed by12 min after infection. Therefore, in the experi-ments depicted in Fig. 3 and 4, putrescineaddition at 16 min could not have increased thealabeling rates by enhancing the efficiency of f2penetration. Since early and late additions yieldnearly identical labeling patterns, it is alsounlikely that early addition of putrescine affectsf2 penetration. The same conclusions can bemade from similar experiments in which rifam-pin, instead of ribonuclease, was added beforeinfection.

Stimulation of f2 replicase synthesis byputrescine. The first known event following thebinding of the incoming parental f2 RNA to hostribosomes is the translation of the replicasegene into the phage-specified subunit of the f2replicase (2, 4, 9, 14). All of the replicasemolecules produced in infected cells come fromthis initial, host transcription-independenttranslation of the parental RNA (14, 18). Fur-thermore, it has been shown that the f2 repli-case assay, in which contributions from hostRNA synthesizing systems are effectively pre-vented, gives a good approximation of theactual number of replicase molecules (10, 12,21). By direct in vitro assay of the f2 replicasesynthesized after infection, it is possible todetermine whether polyamines directly affectedthis initial, translational event.The addition of putrescine does augment the

synthesis of f2 replicase in infected cells (Fig. 7).

The first observable activity appears 15 minafter infection and exhibits a slow, linear in-crease in polyamine-starved cells. Putrescinesupplementation at the time of infection doesnot appreciably alter the time of initial appear-ance of enzyme, but does increase its rate ofsynthesis: the maximum difference occursshortly after 20 min. Eventually, this rapidreplicase synthesis in supplemented cells istermin'ated and replaced by a slower accumula-tion of enzyme. The replicase accumulationcurves for both starved and supplemented cul-tures bear a close resemblance to the labelingcurves of Fig. 3, 4, and 6, with the exceptionthat shut-off occurs at an earlier time in Fig. 7.The higher specific activity of extracts from

putrescine-supplemented cultures can be dem-onstrated over a wide range of protein concen-trations (Fig. 8), and is presumably due to anactual increase in the number of enzyme mole-cules. The maximum difference between sup-plemented and starved cultures is observed 20min after infection. The kinetics of the polymer-ization reaction using 20-min extracts (Fig. 9)again reveals the considerably higher specificactivity of the supplemented culture. Sincepolyamines added to an in vitro assay do notstimulate f2 replicase activity (Table 2), thepossibility that polyamines simply enhance theactivity of the enzyme without an actual in-

120-

IV 80

E

2 40-

C0 5 10 15 20 25 30 35 40

TIME (MINUTES)FIG. 7. Effect of putrescine supplementation on

the rate of appearance of f2 replicase activity in polya-mine-starved, infected cells. The details for thegrowth and harvesting of infected cells, the prepara-tion of infected cell extracts containing f2 replicaseactivity, and the f2 replicase assay are described inMaterials and Methods. Putrescine was added to oneculture at the time of infection (time zero). Rifampinwas not added to the cells prior to nor duringinfection. Symbols: 0, replicase activity in the polya-mine-starved culture; *, replicase activity in theputrescine-supplemented culture.

VOL. 117, 1974 1285

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

YOUNG AND SRINIVASAN

E

0. 40-20

20 - ,t.2

+ PutI1

O 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8[PROTEIN] (mg)

FIG. 8. Effect of increasing protein concentrationon f2 replicase activity from polyamine-starved andputrescine-supplemented infected cells. The detailsfor infected cell extract preparation and the replicaseassay appear in Materials and Methods. The abscissaindicates the amount of protein added per assay. Thefinal assay volume was 0.20 ml in each case. Infected,polyamine-starved and putrescine-supplemented (attime of infection) cells were harvested at 15, 20, and30 min after infection. The 20-min data show theresults of two, separate, infected cell preparations forboth starved and supplemented extracts. Symbols: 0,

no putrescine, harvested at 15 min; 0, putrescineadded, harvested at 15 min; A, 0, no putrescine,harvested at 20 min; A, *, putrescine added, har-vested at 20 min; 0, no putrescine, harvested at 30min, U, putrescine added, harvested at 30 min.

crease in the number of replicase molecules isruled out.

DISCUSSIONWe have previously suggested that polya-

mines in E. coli are involved in some aspect ofprotein synthesis (24). Because of the closeassociation between transcription and transla-tion in bacteria, it was not known whetherpolyamines influence transcription of mRNA,its translation, or both events. The RNA bacte-riophage f2 was, therefore, used in the aboveexperiments to dissociate host transcriptionfrom translation and to determine the effect, ifany, of polyamines on the latter event.The presence of polyamines enhances the

growth of f2 (Fig. 1). This is seen in a shortenedlatent period and in an increase in the size of theinitial burst (at 180 min). The labeling experi-ments confirm this conclusion; polyaminesstimulate phage-specific protein and RNA la-

beling (Fig. 3 and 4), and the labeling curvesobtained strongly resemble the one-step growthcurves of Fig. 1. The high backgrounds of RNAand protein syntheses found in polyamine-starved cells may be due to residual levels ofspermidine still present in these cells. Signifi-cantly, this stimulation by polyamines canoccur in the absence of host RNA transcription,demonstrating the growth-stimulating ability ofpolyamines in a system in which transcriptionand translation have been uncoupled.

Also seen in these experiments is the influ-ence of polyamines on the cessation of phagegrowth. Phage RNA and protein syntheses areclearly terminated by 150 to 180 min in theputrescine-supplemented cultures, whereas thepolyamine-starved cells continue to synthesize

120

8l

80

E

40-

40 5 10 15 20

TIME (MINUTES)FIG. 9. Time course of the f2 replicase-catalyzed

reaction from polyamine-starved and putrescine-sup-plemented infected cell extracts. The preparation ofinfected cell extracts and the replicase assay werecarried out as before. Putrescine was added to oneculture at the time of infection. In both cases theinfected cells were harvested 20 min after infection.The actual amounts of protein used in each assay(0.20 ml) were 551 ug and 501 gg for starved andsupplemented extracts, respectively. The results onthe ordinate have been normalized to I mg of proteinin each case. Individual assays in a series wereterminated at the indicated times and processed forcounting as described earlier (Materials andMethods). Symbols: 0, activity from polyamine-starved, infected cells; 0, activity from putrescine-supplemented, infected cells.

1286 J. BACTERIOL.

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

POLYAMINES AND TRANSLATION OF f2 RNA

phage products at a slow rate. This regulatoryphenomenon, also seen in normal infection (12,18), has been attributed to the gradual accumu-

lation of coat protein molecules which inhibit f2translation. The failure to observe this regula-tion in polyamine-starved cells during thecourse of the experiment suggests that coatprotein molecules may not have accumulated insufficient amounts.

Since Ca2" or other divalent metal ions are

required for f2 penetration (19), it was necessary

to explore the possibility that putrescine as-

sisted inS either adsorption or penetration. Thispossibility was clearly ruled out by experimentsin which rifampin and ribonuclease were addedprior to infection. Added before penetration,these agents inhibit phage RNA synthesis (Fig.5 and reference 8); when added 12 min after f2addition, however, they have no apparent effecton phage RNA labeling (Fig. 4 and 6). Theprocess of penetration must be complete by 12min in polyamine-starved cells, yet putrescineadded at 16 min exerts effects on phage RNAand protein labeling identical to those obtainedby early addition (Fig. 3 and 4).The mechanisms by which ribonuclease and

rifampin inhibit f2 growth are only incompletelyunderstood. Ribonuclease presumably cleavesf2 RNA after exposure of this molecule prior topenetration (19, 25). Once nicked, the single-stranded parental strand cannot produce viablephage progeny. However, penetration andtranslation of this message does occur, sincephage protein labeling is not affected by ribonu-clease treatment prior to infection (unpublishedresults). Although nothing is known about thequality of these proteins made from nickedmessengers, the sudden increase of phage RNAsynthesis in ribonuclease-treated, polyamine-starved cells (Fig. 5) is perhaps due to a slowaccumulation of active replicase molecules.The results of the f2 replicase in vitro assays

support the contention that polyamines can

directly stimulate the translation of a pre-formed message. The kinetics of replicase ap-

pearance (Fig. 7) after infection of putrescine-supplemented cells resembles, in general, previ-ously published reports (2, 14). It has beenshown (10, 12, 21) by polyacrylamide gel elec-trophoresis of phage proteins from rifampin or

actinomycin-treated infected cells that this in-crease in replicase activity after infection corre-

sponds to an increase in the formation ofreplicase molecules. Presumably, more repli-case molecules are made per parental RNA inthe presence of putrescine than in its absence.Whether this means an increase in the number

TABLE 2. Effect of putrescine and spermidine addedin vitro on f2 replicase activitya

Incorporation of GMP (pmol)

Additios.Extract from Extract frompolyamine- polyamine-starved supplemented

infected cells infected cells

None 22.8 49.20.05 mM spermidine 18.35 45.40.5 mM spermidine 16.95 425 mM spermidine 14.38 41.1None 23.95 49.20.2 mM putrescine 23.95 47.72 mM putrescine 28.65 46.22GmM putrescine 16.58 43.6

a Infected cell extracts were prepared and f2 repli-case activity was assayed as described in Materialsand Methods. Incubation was for 10 min at 25 C. Thepolyamine-starved extract assays contained 404 Ag ofprotein per assay; the putrescine-supplemented ex-tract assays had 453 Ag of protein per assay. Both wereharvested 20 min after phage infection. One picomoleof GMP incorporated equals 44 counts/min.

of translational initiation events or more com-plete translations of the entire gene is notknown. However, the alternative interpretationthat polyamines enhance f2 replicase activity,without increasing the number of enzyme mole-cules, is unlikely for the above-mentioned rea-sons. It is also unlikely because of the inabilityof polyamines to stimulate replicase activitywhen added to the in vitro assay (Table 2).Consequently, it may be tentatively concludedfrom these experiments that polyamines partic-ipate directly in some step of mRNA transla-tion.

ACKNOWLEDGMENTSThis investigation was supported by a Public Health

Service grant CA-12235 from the National Cancer Institute.Delano V. Young was supported by Public Health Servicetraining grant GM-255 from the National Institute of GeneralMedical Sciences.We are extremely grateful to Peter Model for his advice

and also for providing the strains and materials used in thisinvestigation. We thank Elazar Rabbani for his help in thepreparation of strain ED-5.

LITERATURE CITED1. Adams, M. H. 1959. Bacteriophages. Interscience Pub-

lishers, Inc., New York.2. August, J. T., S. Cooper, L. Shapiro, and N. D. Zinder.

1963. RNA phage induced RNA polymerase. ColdSpring Harbor Symp. Quant. Biol. 28:95-97.

3. August, J. T., L. Shapiro, and L. Eoyang. 1965. Replica-tion of RNA viruses. I. Characterization of a viralRNA-dependent RNA polymerase. J. Mol. Biol.11:257-271.

4. Blumenthal, T., T. A. Landers, and K. Weber. 1972.Bacteriophage QB replicase contains the protein bio-

VOL. 117, 1974 1287

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from

YOUNG AND SRINIVASAN

synthesis elongation factors EF Tu and EF Ts. Proc.Nat. Acad. Sci. U.S.A. 69:1313-1317.

5. Campbell, A. 1961. Sensitive mutants of bacteriophage A.

Virology 14:22-32.6. Davis, B. D., and E. S. Mingioli. 1950. Mutants of

Escherichia coli requiring methionine or vitamin B,2. J.Bacteriol. 60:17-28.

7. Dion, A. S., and S. S. Cohen. 1972. Polyamine stimula-tion of nucleic acid synthesis in an uninfected andphage-infected polyamine auxotroph of Escherichiacoli K12. Proc. Nat. Acad. Sci. U.S.A. 69:213-217.

8. Engelberg, H. 1972. Inhibition of RNA bacteriophagereplication by rifampicin. J. Mol. Biol. 68:541-546.

9. Fedoroff, N., and N. D. Zinder. 1971. Structure of thepoly (G) polymerase component of the bacteriophage f2replicase. Proc. Nat. Acad. Sci. U.S.A. 68:1838-1843.

10. Fromagoet, H. P. M., and N. D. Zinder. 1968. Growth ofbacteriophage f2 in E. coli treated with rifampicin.Proc. Nat. Acad. Sci. U.S.A. 61:184-191.

11. Hirshfield, I. N., H. J. Rosenfeld, Z. Leifer, and W. K.Maas. 1970. Isolation and characterization of a mutantof Escherichia coli blocked in the synthesis of putres-cine. J. Bacteriol. 101:725-730.

12. Kozak, M., and D. Nathans. 1972. Translation of thegenome of a ribonucleic acid bacteriophage. Bacteriol.Rev. 36:109-134.

13. Lancini, G. C., and G. Sartori. 1968. Rifamycins LXI: invivo inhibition of RNA synthesis by rifamycins. Ex-perientia 24:1105-1106.

14. Lodish, H., S. Cooper, and N. D. Zinder. 1964. Host-dependent mutants of the phage f2 IV. On the biosyn-thesis of a viral RNA polymerase. Virology 24:60-70.

15. Loeb, T., and N. D. Zinder. 1961. A bacteriophagecontaining RNA. Proc. Nat. Acad. Sci. U.S.A.47:282-289.

16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.

Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

16a. Morris, D. R. 1973. RNA and protein synthesis in a

polyamine requiring mutant of Escherichia coli, p.

111-122. In D. H. Russell (ed.), Polyamines in normaland neoplastic growth. Raven Press, New York.

17. Moyer, R. W., and J. M. Buchanan. 1970. Effect ofcalcium ions on synthesis of T5-specific proteins. J.Biol. Chem. 245:5897-5903.

18. Nathans, D., M. P. Oeschger, S. K. Polmar, and K.Eggen. 1969. Regulation of protein synthesis directedby coliphage MS2 RNA. I. Phage protein and RNAsynthesis in cells infected with suppressible mutants. J.Mol. Biol. 39:279-292.

19. Paranchych, W. 1966. Stages in phage R17 infection: therole of divalent cations. Virology 28:90-99.

20. Stavis, R. L., and J. T. August. 1970. The biochemistry ofRNA bacteriophage replication. Annu. Rev. Biochem.39:527-560.

21. Sugiyama, T. 1969. Translational control of MS2 RNAcistrons. Cold Spring Harbor Symp. Quant. Biol.34:687-694.

22. Umezawa, H., S. Mizuno, H. Yamazaki, and K. Nitta.1968. Inhibition of DNA-dependent RNA synthesis byrifamycins. J. Antibiot. 21:234-236.

23. Webster, R. E., D. L. Engelhardt, N. D. Zinder, and W.Konigsberg. 1967. Amber mutants and chain termina-tion in vitro. J. Mol. Biol. 29:27-43.

24. Young, D. V., and P. R. Srinivasan. 1972. Regulation ofmacromolecular synthesis by putrescine in a condi-tional Escherichia coli putrescine auxotroph. J. Bacte-riol. 112:30-39.

25. Zinder, N. D. 1963. Properties of a bacteriophage contain-ing RNA. Perspect. Virol. 3:58-67.

26. Zinder, N. D. 1965. RNA phages. Annu. Rev. Microbiol.19:455-472.

1288 J. BACTERIOL.

on June 5, 2018 by guesthttp://jb.asm

.org/D

ownloaded from