The effects of polyamines on the replication of T4rII mutants in Escherichia coli K12 (λ)

VIROLOGY26, 221-227 (1965)

The Effects of Polyamines on the Replication of T4rll Mutants in Escherichia coli K12 (X) ~'2 M. L O U I S E B R O C K ~

Department of Bacteriology, Indiana University, Bloomington, Indiana Accepted February 9, I965 While rlI mutants of bacteriophage T4 can replicate in Escherichia coli K12S, replication is blocked when this host is lysogenized by X phage. Polyamines, Mg++, and streptomycin increase the yield of rII deletion mutants in K12(X) to varying degrees below the normal yield obtMned with wild-type phage. Of a homologous series of diamines, 1,7-diaminoheptane is about 7 times more effective than 1,4-diaaminobutane (putrescine) or Mg++. The critical portion of the latent period for dinmine stimulation is between 10 and 20 minutes after infection. In the absence of dinmine, UV induction of X replication permits subsequent rII replication; further stimulation by 1,7-diaminoheptane still occurs under these conditions. Diamlnes and Mg++, unlike UV irradiation, do not bring about induction of X phage. Uninfected ceils of K12(X) and K12S were found to contain nearly identical amounts of putrescine. Severalindependent methods were used to examine permeability differences between the two types of infected cells; in MI cases, no significant differences in permeability to large or small molecules were found. Therefore, the inability of rH mutants to replicate in K12(X) cannot be attributed to generalized leakage resulting from phage infection. Several alternatives are suggested for the mode of action of these substances. INTRODUCTION A class of m u t a n t s of coliphage T4 designated as r l I m u t a n t s causes rapid lysis of Escherichia coli strain B and forms typical "r" plaques when plated on this host. T h e y are distinguished from ocher types of r mutants, however, by their inability to replicate in E. coli strain K12 lysogenie for X unless, as Garen (1961) discovered, the medium is supplemented with magnesium or calcium ions. Since polyamines are normal constituents of bacterial cells, act similarly to magnesium ions in m a n y systems (Tabor et al., 1961), and occur along with magnesium i Aided by National Institutes of Health Grant CA-02772. A preliminary report of these data was presented at the 64th Annual Meeting of the American Society for Microbiology, May 1964, Washington, D.C. Present address: 509 S. Highland St., Bloomingtort, Indiana.

ions in bacteriophage particles (Ames et al., 1958; Ames and Dubin, 1960), the effects of polyamines on the replication of T 4 r H mutants in E. coli K12(X) are presented in this paper. MATEt~IALS AND METHODS

Bacterial strains. E. coil Hershey and K12(X) were from our own collection. Strain K12S was kindly provided by Dr. Seymour Benzer. Phage strains. T4 was from our laboratory; the following mutants w e r e obtained from Dr. Benzer: r1272, a deletion m u t a n t of the entire r l I region; r184, a deletion m u t a n t of the A cistron; r638, a deletion m u t a n t of the B cistron. Media. P Y medium: 0.7 % proteose peptone, 0.3% yeast extract, 0.1% glucose, 0.3% NaC1, 0.002% Na2HPO4.2H20, prepared with deionized distilled water, p H adjusted to 7.0. Agar media: 1% tryptone,

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0.8% NaC1, 0.1% glucose, 1.5 × 10-3 M rained between the number of infective NaOH, 1.0 × 10-4 M CaC12, pH adjusted centers predicted by Poisson distribution and to 7.0; bottom layer 1% agar, top layer the reduction in viable count after phage 0.6 % agar. Adsorption buffer: 0.003 % gela- infection. The phage techniques in general tin, 2.05 X 10-~ M Na2HP04, 1.1 X 10-2 M were the standard methods described by KH2P04, 6.9 X 10-2 M NaC1, 2.9 N 10-2 M Adams (1959). K~S04, pH adjusted to 7.0; chloramphenicol Assay of putrescine by gas chromatography. and tryptophan were added to give final The bacteria from one liter of cells at 2 X concentrations of 25 #g/ml and 20 #g/ml, 10a/ml were harvested according to the procedure of Dubin and Rosenthal (1960). After respectively. Diamines. Stock solutions were prepared the removal of TCA, the aqueous extract in deionized distilled water at a final con- was placed on ice and saturated with solid centration of 1.0 M, final pH of 7. NaOH by the slow addition of 4 pellets Phage stocks. High titer stocks were pre- (about 400 mg) per milliliter of extract. pared from confluent lysis plates using E. Quantitative removal of putrescine from coli Hershey as described by Adams (1959). the alkaline extract was achieved in reconExperimental methods. Stationary-phase struction experiments by extracting it with host cells were grown in P ¥ medium to six successive equal volumes of ether, poolwhich MgS04 and CaCI: had been added to ing the ether extracts. Samples of the ether give final concentrations of i X 10-3 M and extract were then assayed using a gas chro1 X 10-4 M, respectively. These cells were matograph (ChromAlyser-100, Dynatronics diluted 1/50 into PY medium, and after Instrument Corporation) equipped with a aeration at 37 ° to a cell density of 2 >( thermal conductivity detector, operated at 10S/ml the cells were centrifuged and con- 250 ma, minimum attenuation. The Carbocentrated fivefold in adsorption buffer. wax-coated alkaline Celite packing described by Smith and Radford (1961) was used in a After 5 minutes' aeration at 37 °, phage were added and i0 minutes was allowed for ad- column 8' x }~6" at 160 ° with H2 as the carrier. The putrescine content of a sample sorption; after the addition of antiserum, was determined by comparing the resulting another 5 minutes was allowed for inactivation of unadsorbed phage. The infected cells peak size with that from a known amount were then diluted twofold with cold buffer of putrescine free base in ether. This method of assaying the diamine lacking chloramphenico] and tryptophan, content of biological materials is advancentrifuged, washed with i0 ml cold PY metageous because of its specificity in contrast dium, resuspended to 2 X 108/mi in 37 ° PY to other procedures (Tabor et al., 1961), but medium, and aerated. The time of resuspension in 37 ° growth medium is considered 0 its accuracy is dependent upon the sensitivity of the detector. Less than 0.05 #mole minutes. Except where indicated otherwise, samples were transferred at 4 minutes to of putrescine could not be measured under aeration tubes containing various cations. the conditions described above. Samples for phage assay were chloroformed RESULTS and plated by the agar layer method with E. coli Hershey which was grown in PY Stimulation of Replication medium with added Ca ++ and Mg ++. The A typical growth curve of r1272 in K12(h) rise of intracellular phage begins at 15 minutes and the maximal titer is obtained at 45 is shown in Fig. 1, in which it can be seen minutes under these conditions. All attempts that little increase in phage titer occurs in PY medium not supplemented with Mg ++. to assay directly the number of r[[-KI2(X) infective centers failed. The number of The optimal concentration of Mg ++ for infected cells and the multiplicity of infec- giving the largest yield was found to be 0.02 M, higher and lower concentrations tion were determined indirectly from colony counts on samples taken before and after reducing the yield below 2-3 phage per ininfection, and from assays of initial and un- fected cell. The polyamine spermidine, at an optimal adsorbed phage; excellent agreement was ob-

EFFECTS OF POLYAMINES

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FIG. 1. I~eplication of r1272 in K12(X). Average multiplicity of infection: control and 1,7-diaminoheptane, 2.5; Mg++, 6.6. concentration of 0.014 M, also caused an increase of r1272 above the control level, but the maximum titer obtained was only an average of 1 phage per 2 infected cells. When putrescine (1,4-diaminobutane) was tested for its ability to increase the yield of r I I phage in K12(X), the optimal concentration and the maximum yield were the same as those found with Mg ++. Since both Mg ++ and putrescine had identical effects in permitting the replication of the three r I [ mutant.s tested, r184, r638 and r1272, mutant r1272 lacking the enth~e r I I region was used for all subsequent studies. Of a homologous series of polymethylene diamines, ranging from 1,3-diaminopropane to 1,10-diaminodecane, tested at 0.02 M, 1,7-diaminoheptane was found to be the most active in stimulating ~'II replication in K12(X) (Fig. 1). The results of these experiments are shown in Table 1, which includes comparable data on the effects of these

diamines on the replication of r + in K12(X) and r I I in K12S. It should be noted that although 1,7-diaminoheptane at the optimal concentration of 0.02 M strongly stimulates r I I replication in K12(X), the average yield is only 15 phage per infected cell, as compared with yields of 46 r I I phage per K12S cell and 136 r + phage per K12(X) cell under identical conditions. Streptomycin (SM) promotes the replication of r1272 in a streptomycin-resistant host K12(X)SM R. The maximal yield in the presence of 1 mg/ml SM was only 1/~0th that obtained with 1,7-diaminoheptane. Time of Stimulation

Putrescine or SM added at 10 minutes stimulated phage replication as well as when they were added earlier; when the time of addition was delayed to 20 or 30 minutes, the phage yields were significantly lower. Delaying the time of addition of putrescine

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BROCK TABLE 1 EFFECT OF POLYMETHYLENE DIAMINES ON PHAGE ~EPLICATION

Diamine

Relative increase due to diamlnea r1272 in K12 (X)

r + in K12 (~,)

r1272 in K12S

1,3-Diaminopropane 1,4-Diaminobutane

135 650

0.015 0.041

0.39 0.81

1,5-Diaminopentane 1,6-Diaminohexane 1,7-Diaminoheptane 1,8-Diaminoctane 1,9-Diaminononane 1,10-Diaminodecane Control

590 770 2925 6.2 X 10-a 5.3 X 10-8 -(1)b

1.4 1.3 1.0 1.3 X 10-G 2.6 X 10-6 2.6 X 10-° (1)~

1.2 1.3 0.91 7.1 X 10-6 5.8 X 10-7 3.0 X 10-° (1)d

a Phage yield at 45 minutes in presence of diamine, divided by control yield at 45 minutes. b Actual yield 4.9 X 10-8 phage per infected cell; average multiplicity 2.5. c Actual yield 136 phage per infected cell; average mutliplicity 5.8. d Actual yield 51 phage per infected cell; average multiplicity 1.1. did not cause a corresponding delay in the time at which the maximal titer of phage occurred. Further work was done with 1,7-diaminoheptane which was added to the infected

cells at i minute and removed at I0 minutes (by centrifuging and washing) ; there was no subsequent increase in phage titer over that of the parallel control. When 1,7-diaminoheptane was added or removed at intervals during the latent period, it could be shown that it need not be present until i0 minutes to be effective, and that the critical interval during which it relieved the block was between I0 and 20 minutes of the latent period. Garen (1961) has shown that Mg ++ is not required for the first 10 minutes of the latent period, but it is required thereafter for phage maturation to, occur.

rII Replication in UV-Induced KI2(X ) Mutant phage yield in K12()~) was increased 600-fold b y ultraviolet light induetion of the host 45 minutes prior to rII infection. Even in the UV-induced host 1,7-diaminoheptane is still stimulatory, permitting a further 45-fold increase. Mg ++ or 1,7-diaminoheptane also increases the yield in a strain not inducible b y UV, K12(X)ind(obtained from Mr. G. R. L. Worthington). Numerous attempts failed to demonstrate induction of X phage replication in K12(X) or K12()~)ind- by Mg ++, putreseine, or 1,7diaminoheptane; neither do these cations

have a significant effect on the latent period, rate of increase, or final yield of X phage produced b y UV-induced K12(),)J I t is concluded from these experiments that the cations do not relieve the r[I block b y inducing X replication as UV induction of the host does.

Other Observations I have not been able to obtain yields of r N phage in K12(X) equivalent to that from the wild-type T4 in the presence of added M g ++ or diamine. However, a combination of these gave higher yields than either one alone. The low yields cannot be attributed to inhibition by K + ions, as discussed b y Garen (1961), since identical results were obtained when N a + was substituted for K + in the adsorption buffer. Neither was the yield greater when chloramphenieol was omitted from the adsorption buffer, or when 0.2% tryptone was used instead of P Y medium. Substances used to stabilize protoplasts, such as sucrose and Carbowax (Spizizen, 1962), when added to P Y medium, did not promote replication. Phage titers obtained after lysozymeVersene lysis of the infected cells were similar to those obtained b y chloroform lysis. The yield of r1272 in K12(X)/6 (kindly 4 The technical assistance of Mrs. Doris Harris in these experiments is greatly appreciated.

EFFECTS OF POLYAMINES provided by Dr. Garen) was the same as that obtained in K12(X) under the mass lysis conditions described, with or without added Mg++ putrescine, or 1,7-diaminoheptane.

Putrescine Content In view of the fact that polyamines synthesized by the bacterial host are incorporated into phage DNA (Ames et al., 1958; Ames and Dubin, 1960), the possibility was considered that the stimulation of r I I phage replication in K12(X) by these compounds could result from the inability of the host to synthesize them. A comparison of uninfected K12(X) with K12S showed, however, that they contained nearly identical amounts of putrescine, 4.8 and 4.2 mnoles per 2 X 10u cells, respectively, as determined by gas chromatography. Permeability Since it has been suggested by Garen (1961) that a possible function of cations in relieving the r l I block could be to somehow effect repah" of the damage to the cell surface resulting from phage infection, it seemed important to examine this hypothesis in order to define the role of cations as reversing agents. Several independent criteria were used in an attempt to demonstrate differences in permeability between r + and r//-infeeted K12(X) cells in the absence of reversing agents. The multiplicity of infection in the following experiments was 2-3 phage per cell, and no cations were added to promote r l I replication. Under these conditions, lysis does not occur in the r//-infeeted cell, as also shown by Garen (1961). Gross permeability. Strain W1317 of K12(X) (obtained from Dr. H. V. Riekenberg) constitutively synthesizes ~-galaetosidase and the galactoside permease. This strain was used for experiments in which the amounts of fl-galaetosidase and 260 raftabsorbing material in the culture supernatant fluids of both r +- and r/I-infected cells were found to increase very little for the first 20 minutes of the latent period, after which there were rapid increases for the r+-infeeted culture, attributable to lysis, but little further increases for the r//-infected

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culture. This experiment was done using a synthetic medium lacking added Ca ++ and Mg++; replication of the wild-type phage was normal, while that of the mutant was inhibited. ~-Galactosidase was assayed as described by Brock and Brock (1961). Results similar to these were found for the appearance of p32 in membrane filtrates of infected cells in PY medium, tile host K12(X) having incorporated p~2 prior to phage infection. These measurements indicate that no gross changes in permeability leading to the loss of large molecules results from infection of K12(X) with the r / / mutant, and that the changes which occurred during the first 20 minutes of the latent period were no different from those resulting from infection with T4. Ability to concentrate a nonmetabolizable co~npound. Another method used to evaluate changes in the infected cells during the latent period was to compare the ability of infected cells of strain W1317 to concentrate methyl - C ~4 - ¢~ - D - thiogalactopyranoside (TMG) (a gift of Dr. H. V. Rickenberg). One-milliliter samples of cells at 2 X 108/ml were transferred to tubes containing 0.1 ml of C~4-TMG (1 X 10-a M, 6.8 X 105 epm/ml final), shaken in a water bath at 37 ° for 2 minutes, filtered on a membrane filter, and washed with 1 ml of cold buffet-. The filters were glued to planehets, and radioactivity was determined with a Nuclear Chicago gas flow counter. Uninfected cells concentrate 11-13 times more C~4-TMG than the amount which would be expected to equilibrate nonspeeifitally, while pretreatment for 15 minutes with sodium azide (0.02 M) reduces the amount to threefold. Both types of phageinfected cells rapidly lose the ability to concentrate C~4-TMG. This observation could indicate either a decrease per se in the serum amount which was incorporated, or an increase in permeability which would prevent the infected cells from retaining C 14TMG. The significant fact for the present discussion, however, is that no significant difference is observed between r +- and rII-infeeted K12(X) cultures. Streptomycin binding. Streptomycin binds

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rapidly to E. coli cell walls and also binds to intraeeliular components under conditions of increased permeability (Anand et al., 1960). The binding of monohydromonotritiostreptomycin (MHTS) (a gift of Dr. T. D. Brock) by K12(X) was measured by incubating 2 X l0 s cells for 1 minute at 37 ° with 3.5 )4 104 cpm (0.025 t~g) of MHTS, filtering on a membrane filter and washing the cells with 1 ml of distilled water. Radioactivity of the dried filters was determined with a Nuclear Chicago scintillation counter. During phage adsorption the amount of MHTS bound by both r +- and rlI-infected cells increases, and this is followed during the latent period by a rapid decline in the amount of MHTS bound by both types of infected cells, reaching a minimum by about 7 minutes. Similarly treated uninfected cells show an increase rather than a decrease in the amount of MILTS bound during the equivalent of the latent period. These changes could very well reflect the increase in permeability and subsequent repair of cell surface damage resulting from phage infection described by Puck and Lee (1955), and if so, indicate that repair is complete by 10 minutes during the latent period. DISCUSSION Numerous characteristics of E. coli K12S are not altered when this strain is lysogenized by the phage X. The r I I mutants of phage T4 fail to replicate in these lysogenized cells, however, while T4 replieates normally. An obvious explanation of these observations is that X, in attaching itself to the host genome, does alter some process of little consequence to the cell under ordinary laboratory growth conditions but, necessary to the r l I mutants because they lack the genetic information for a substitute process. It is known that the DATA of the r I I phages enters and that replication proceeds to the point of manufacture of phage proteins and of some 10-15 phage units of DNA (Garen, 1961). The addition of divalent cations, as shown by Garen, or, in the present work, of certain polyamines or streptomycin, relieves the block encountered by deletion mutants, to varying degrees. The effect of these low molecular weight substances appears to be on the host process blocked by X.

Why large amounts of polyamines or magnesium must be supplied externally to relieve the block is not immediately obvious. It is possible that the normal amount present in K12(X) is insufficient for some unknown reason to support replication in the absence of the r I I function. It is also possible that the specific leakage of polyamine from rIIinfected K12(X) (FerroLuzzi-Ames and Ames, 1965), unassociated with generalized cell leakage, reduces the intracellular polyamine concentration below a critical level. An attractive mechanism whereby streptomycin, Mg ++, and even polyamines could act indirectly is that of affecting the host ribosomes, permitting the successful function of a K12(X) host product which can substitute for the r l I product. The mode of action of these cations has been reviewed by Brock (1964). The high efficiency of reversal by some of the diamines and Mg ++ by this process would seem to militate against this type of mechanism, although it is a reasonable explanation for the action of streptomycin. An interesting parallel is the similar low efficiency of reversal by streptomycin in a different phage-host system (Zinder and Valentine, 1964). Polyamines, Mg ++, and streptomycin could also act by functioning as cofactors or stabilizers of a host product, again part tially converting KI2(X) to a permissive host as far as rl[ replication is concerned. A high efficiency of reversal such as that obtained with Mg ++ and certain diamines would be expected for this type of function. While none of the experimental evidence rules out the possibility that the r/I-infected cells are unable to retahl a specific substance necessary for replication, they do indicate that the general permeability changes which have been measured occur to the same extent in X12@) infected with the wild-type phage. Therefore the hypothesis that the r I I block could be due simply to general leakage caused by the lack of repair of cell surface damage after phage infection no longer appears tenable. Further knowledge of the nature of lysogeny, as well as studies on virulent phageinfected cells, will hopefully lend greater insight to the intriguing problem of the r I I block.

EFFECTS OF POLYAMINES ACKNOWLEDGMENTS The author is extremely grateful for the advice and encouragement of Dr. Dean Fraser, in whose laboratory this work was carried out. She is Mso indebted to Drs. G. FerroLuzzi-Ames and B. N. Ames for providing information on their unpublished work and for their helpful suggestions. REFERENCES AD_~MS, M. H. (1959). "Bacteriophages." Wiley (Interscience), New York. AM~S, B. N., and DUmN, D. T. (1960). The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J. Biol. Chem. 235,769-775. AMES, B. N., DUmN, D. T., and ROSn~XTm~L,S. ~[. (1958). Presence of polyamines in certain bacterial viruses. Science 127,814-816. ANAND, N., DAVIS, B. D., and AnMm'_aGE,A. K. (1960). Uptake of streptomyci)~ by Escheriehia coli. Nature 185, 23-24. B~mCK, T. D. (1964). The mode of action of streptomycin and related antibiotics. Federation Proc. 23,965-975. BROCK, T. D., and Bnoc~, M. L. (1961). Reversal of azaserine by phenylManine. J. Baeteriol. 81, 212-217. DUmN, D. T., and R o s ~ L , S. M. (1960). The

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acetylation of polyamines in E.vcherichia coli. J. Biol. Chem. 235, 776-782. FEnnoLuzz>AMEs, G., and AMEs, B. N. (1965). The multiplication of T4rII phage in E. coli K12(X) in the presence of polyamines. Biochem. Biophys. Res. Cornmun. 18, 639-647. G,~n~N, A. (1961). Physiological effects of rII mutations in bacteriophage T4. Virology 14, 151-163. Pvc~:, T. T., and LEn, H. H. (1955). Mechanism of cell wall penetration by viruses. II. Demonstration of cyclic permeability change accompanying virus infection of Escherichia coli B cells. J. Exptl. Med. 101,151-175. S~IT~, E. D., and R~DFORD, R. D. (1961). Modification of gas chromatographic substrates for the separation of aliphatic diamines. Anal. Chem. 33, 1160-1162. SHZIZE~X, J. (1962). Preparation and use of protoplasts. In "Methods in Enzymology" (S. P. Colowick and N. O. Kaplan, eds.), Vol. V, pp. 122-134. Academic Press, New York. TaBon, H., T.¢BOR, C. W., and ROS~NTmkL, S. M. (1961). The biochemistry of the polyamines: spermidine and spermine. Ann. Rev. Biochem. 30, 579-604. ZIxJ)En, R. C., and V.}~LENTINI~,N. D. (1964). Phenotypic repair of RNA-bacteriophage mutants by streptomycim Science 144, 14581459.