Vol. 15, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Feb. 1975, p. 232-237 Copyright © 1975 American Society for Microbiology
Canavanine-Mediated Depletion of Polyamine Pools in Escherichia coli: Effect on Head Morphogenesis and DNA Synthesis REX W. BOLIN* AND DONALD J. CUMMINGS Department of Microbiology, University of Colorado Medical Center, Denver, Colorado 80220 Received for publication 12 September 1974
We have found that L-canavanine inhibited the synthesis of polyamines in T4-infected Escherichia coli. These polyamines are known to be required for T4 DNA synthesis and may be involved in phage morphogenesis. The new data indicate that the inhibition of polyamine synthesis is not primarily responsible for the L-canavanine-mediated inhibition of DNA synthesis nor does it seem to be involved in the induction of lollipops. L-Canavanine does influence the relative amounts of putrescine and spermidine found in the phage particle, but it does not influence the amount of DNA phosphate neutralized by polyamines.
It has been previously shown that when a T-even bacteriophage-infected cell is exposed to canavanine followed by an arginine chase a viable monster phage particle termed a "lollipop" is produced. The lollipops usually represent about 3% of the phage particles and, due to their abnormally large size, up to 30% of the assembled proteins (8). The addition of canavanine to T4-infected cells markedly inhibits viral DNA synthesis and the virus yield (2, 7). Net RNA and protein synthesis remain unaffected. However, the cleavages of the head proteins P22, P23, P24, and IPIII are inhibited and two additional proteins, P20 (head protein) and P12 (tailplate protein), do not appear (3). Canavanine also induces the appearance of highermolecular-weight forms of P10 (tailplate protein) and P18 (sheath protein) in addition to the normal forms. Polyheads similar to those produced by mutants defective in gene 20 are produced in the presence of canavanine. Addition of arginine to canavanine-treated infected cells results in a stimulation of the viral DNA synthesis, a partial restoration of the phage yield, and the appearance of the monster particles. The induction of the lollipops requires a phage late function and, under the most favorable conditions, a 3-min exposure to canavanine (2). The length of the lollipop depends to some extent on the length of the exposure to canavanine. Our data indicate that canavanine induces the formation of lollipops by interfering with a step in the virus prohead assembly which is critical for the control of normal head length (3). The block allows the accumulation of a lollipop precursor which is converted into a lollipop upon the addition of arginine and the restoration of some vital function. Several possi232
ble events could be affected by canavanine. The termination of the elongation events during head assembly possibly involves a cleavage; therefore, canavanine may be incorporated into proteins such that either the cleaving enzymes are inactive or the substrate proteins are not susceptible to cleavage. The inhibition of DNA synthesis could be responsible, although there is evidence that the normal proheads are formed prior to the packaging of the DNA (12, 13, 17). The purpose of this paper is to investigate a third possibility. Canavanine is an analogue of arginine and, since arginine is a potential precursor to polyamines, it is possible that canavanine interferes with the viral processes by interfering with the polyamine accumulation in the infected cells. In Escherichia coli, polyamines are synthesized by two independent pathways (15, 16). In the absence of exogenous arginine, the principle pathway involves the decarboxylation of ornithine, a precursor to arginine, directly to putrescine (14), which can be converted to spermidine. In the presence of exogenous arginine, the majority of the polyamines arise from the conversion of arginine to agmatine, which is then converted to putrescine. Canavanine could interfere with the polyamine pathways by (i) acting like arginine to repress the ornithine to putrescine pathways or (ii) possibly by being converted to an agmatine analogue which is converted to the putrescine analogue which might impair normal polyamine function. Following T4 infection of E. coli, the amounts of both polyamines increase dramatically in the cell (5). The polyamines have been shown to neutralize approximately 50% of the DNA phosphates in the phage head, although the phage
VOL. 15, 1975
POLYAMINE POOLS AND HEAD MORPHOGENESIS
polyamine content is dependent on the polyamine content of the bacteria (1) which is a function of the growth conditions. T4 DNA synthesis is dependent on the presence of polyamines (10), but the accumulation of polyamines is not coupled to DNA synthesis (9). High concentrations of polyamines have been shown to increase the production of smallheaded phage in T4 infections and to result in aberrant tail attachment (6). In this report we have investigated the possibility that polyamines may be responsible for the canavanine-induced inhibition of T4 DNA synthesis as well as for the induction of lollipops. Our data indicate that canavanine does reduce the polyamine pools in the infected cell. However, the reduction in the polyamine content is probably not primarily responsible for the inhibition of DNA synthesis or for the induction of lollipops. MATERIALS AND METHODS Growth conditions. E. coli B was grown to 2 x 108 cells/ml in minimal medium supplemented with 5 Ag of an amino acid supplement per ml (6) and then infected with T4B at a multiplicity of infection of five phage per bacterium. Tryptophan at 10 gg/ml was added prior to infection to enhance adsorption. The bacteria were superinfected 7 to 9 min later with the same amount of virus. The infected cells were incubated at 37 C with aeration. The polyamines (Calbiochem), L-canavanine sulfate (Calbiochem), and L-arginine were added as noted in the text. Assays. For estimation of intracellular polyamines, 2.0-ml samples of cells were removed from cultures as noted in the text and filtered through membrane filters (0.45-Mum pore size, type HA; Millipore Corp.). The filters were washed with 6.0 ml of medium and then were placed in 1.0 ml of ice cold 0.2 N perchloric acid for 1 h. The precipitate was removed by centrifugation, and the polyamines were quantitated as the fluorescent dansyl derivatives (11) as described by Dion and Cohen (9). The phosphate determinations were performed as described by Ames and Dubin (1). Phage DNA synthesis was measured by following the incorporation of ['H ]thymine into trichloroacetic acid-precipitable material as previously described (2). Preparation of phage particles. Polyamine and phosphate determinations were made on phage particles prepared in the following manner. The cells were grown and infected as described above; 2 h after infection the cells were lysed with chloroform and the majority of the cellular debris was removed by centrifuging at 5,000 x g for 5 min. The phage were then collected by centrifuging at 25,000 x g for 2 h and resuspended in 0.4% NaCl. The remainder of the cellular debris was removed by a low-speed centrifugation, and the phage were then washed three times in 0.4% NaCl. As will be described in Results, care was taken to avoid any treatment which might have resulted in a loss of polyamines from the phage head.
233
RESULTS Effect of canavanine on the intracellular pool of polyamines. Previously, we demonstrated that canavanine results in about an 80% reduction of T4 DNA synthesis (2, 7). To determine if canavanine could be responsible for this reduction by interfering with the intracellular polyamine pools, the accumulation of the polyamines was examined after treatment with canavanine. The cells were divided into several portions after infection. A portion of the culture was treated with 130 ,g of canavanine per ml at 5 min postinfection (PI) and, at 25 min PI, portions of the canavanine-treated cultures were treated with 200 Mg of arginine per ml. These conditions are similar to those described for inhibiting DNA synthesis, and they are sufficient for the induction and formation, of lollipops (2). Canavanine dramatically inhibited the intracellular accumulation of putrescine and spermidine (Fig. 1). Putrescine accumulation was inhibited to a greater degree than was spermidine. As was shown to be the case for DNA synthesis, arginine readily stimulated the accumulation of the polyamines. It should be noted that, whereas canavanine did inhibit the accumulation of polyamines, no new polyamines that might possibly have been derived from canavanine were detected using the thinlayer chromatography system described (9). In some experiments, the acetylated forms of putrescine and spermidine were detected, but these could be readily converted to putrescine and spermidine by acid hydrolysis. The reduced accumulation of putrescine and spermidine could result from either decreased synthesis or increased leakage from the cell. During the course of the above described experiments, the cell-free medium was examined to determine if canavanine induced an increased excretion of polyamines from the cells. However, only a negligible amount of the polyamines was present in the medium until late in the infection (80 to 100 min PI). This increase was probably due to cell lysis. It was demonstrated that canavanine did not prevent the transport of external polyamines. At 65 min PI in the presence of 1 mM putrescine, untreated cells contained 97 nmol of putrescine per ml of cell culture, and canavanine-treated cells contained 82 nmol of putrescine per ml of cell culture. In the presence of 1 mM spermidine, the untreated cells contained 55 nmol of spermidine per ml of cell culture, and canavanine-treated cells contained 52 nmol of spermidine per ml of cell culture. Effect of exogenous polyamine on cana-
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234
E \
-o
ECL
-I
0.
0
E
E
T ime Post Infection ( Min]
FIG. 1. Effect of a canavanine pulse on the accumulation of polyamines in T4-infected cells. At 5 min PI, 130 gg of canavanine per ml was added and 200,g of arginine per ml was added at 25 min PI to a portion of the treated cells. ARG, arginine; CAN, canavanine.
vanine-inhibited T4 DNA synthesis. Our data indicate that canavanine inhibits polyamine synthesis in T4-infected cells but that exogenously added polyamines can increase the intracellular concentration of polyamines. If the reduction of intracellular polyamine levels were responsible for the inhibition of DNA synthesis by canavanine, exogenously added polyamines should result in a stimulation of DNA synthesis. To examine this possibility, a portion of an infected culture was first treated with 130 ug of canavanine per ml at 5 min PI. (We have shown previously that the stimulation of DNA synthesis after the addition of arginine was more efficient if canavanine was added at 5 min PI [2].) At 15 min PI, portions of the canavaninetreated culture were treated with various combinations of putrescine, spermidine, and spermine as well as with ornithine and arginine (Fig. 2). Arginine (100 ug/ml) resulted in a strong stimulation of DNA synthesis. Ornithine (100 ug/ml) resulted in a moderate stimulation. If the ornithine concentration was reduced to 50 ,g/ml, the stimulation was reduced about 50% (not shown). The polyamines were added individually and in various combinations at concentrations from 1 to 10 mM, but no stimulation of DNA synthesis was observed. The data indicate that polyamine depletion cannot be the sole reason for the canavanineinduced inhibition of DNA synthesis. No stimulation by polyamines could be demonstrated. The stimulation by ornithine was not as strong as the stimulation by arginine. The ornithine stimulation was probably the result of conversion of ornithine to arginine rather than conversion to putrescine. This result was not unex-
12
/ I
x
E10 _/ ~~No CAN-O/
0.
e-ARG 15_
8
E
O/ /0
0
6
-.--~~ORN 15'
// 0
1 I 40 20) 0*/ 2/
A"'~~~'~
6
0
60
80
PA 15'
1012
0
Add. 20
40
100
120
Time Post Infection [ Min]
FIG. 2. Effect of polyamines on the stimulation of DNA synthesis. At 5 min PI, a portion of the infected culture was treated with 130 tig of canavanine per ml, and at 15 min PI additions were made as noted. The arginine and ornithine were added at 100 gg/ml. The polyamines (putrescine, spermidine, and spermine) were added individually and in various combinations at concentrations from 1 to 10 mM. ARG, arginine; CAN, canavanine; PA, polyamines; No Add, no additions.
pected, since we demonstrated previously (2) that the inhibition by canavanine and the stimulation by arginine were both sensitive to
POLYAMINE POOLS AND HEAD MORPHOGENESIS
VOL. 15, 1975
chloramphenicol, indicating a requirement for protein synthesis. We have also noted that a temperature-sensitive mutant (P36A) in the DNA polymerase (gene 43) has acquired a greater sensitivity to canavanine at permissive temperatures than the wild-type phage. Normally, canavanine reduces the phage yield about 200-fold but the mutant yield was reduced about 5,000-fold by canvanine. This suggests that canavanine may interfere with DNA synthesis by its incorporation into proteins essential for DNA synthesis. Effect of canavanine and arginine on the polyamine content of the phage head. We have shown that canavanine affects the polyamine content of the infected cell. The effects of canavanine and arginine on the polyamine content of the phage head are shown in Table 1. Cells were infected, and then at 10 min PI portions of the cells were treated with either canavanine or arginine. Cells treated with canavanine were further treated with arginine at 25 min PI (conditions which result in the formation of lollipops). The addition of arginine to infected cells resulted in a decrease in the total amount of phosphate neutralized by polyamines. However, increasing concentrations of arginine resulted in an increase in the amount of polyamines in the virus. In each case in which only arginine was added to the cultures, the ratio of putrescine to spermidine was essentially constant. The ratio was identical with that of the untreated culture. The addition of canavanine markedly influenced the ratio of putrescine to spermidine within the phage head. However,
235
the amount of phosphate neutralized by the polyamines was dependent on the amount of arginine added after the addition of the canavanine. The addition of canavanine affected only the amount of putrescine relative to the spermidine but did not affect the neutralization of the phosphate. Canavanine clearly affects the polyamine content within the phage head. Based upon the amount of DNA in the lollipop heads, approximately 25% of the polyamines in the canavanine-treated cultures should have been derived from the lollipop heads. Attempts were made to quantitate the polyamines in purified lollipops, but they were unsuccessful. Unfortunately, the lollipops were very permeable to ions, as evidenced by their resistance to osmotic shock with sodium sulfate (8). The head construction may be analogous to mutants of T4 (T401) which are resistant to osmotic shock (4). T401 particles were shown to be very susceptible to losing their polyamines during normal purification procedures (1). The purification of lollipops from phage of normal head length involves the use of CsCl-D2O-glycerol gradients (8), and we have found that this procedure leads to the loss of the polyamines from the heads. Usually several cycles through the gradients are required for achieving high purity lollipops, but after just one gradient the vast majority of the polyamines are removed from the lollipop heads. Phage of normal head lengths lost some polyamines, but their polyamine content was higher than that of the lollipops, as would be expected. It should be noted that small amounts of both
TABLE 1. Effect of arginine and canavanine on the polyamine (PA) content of T4a Polyamine
PutrescineSpermidine Treatment_______scine_SpermiPtrscn
% ofPi neutralized
of Pi
PA
nmol/tmol of Pi
% of Total PA
165
62.8
97.7
37.2
1.7
62.3
nmol/umol % of Total
Untreated
Ratio of putrescine to spermidine
by PA
Arginine (20 ,gg/ml)
94.2
62.8
55.9
37.2
1.7
35.6
Canavanine (130 jg/ml) Arginine (20 ug/ml)
55.6
44.2
70.2
55.8
0.8
32.2
119.6
64.5
65.8
35.5
1.8
42.6
86.2
50.1
86.0
49.9
1.0
43.0
139.5
64.2
77.6
35.7
1.8
51.2
Arginine (100jig/ml)
Canavanine (130,ug/ml) Arginine (100jig/ml) Arginine (200 Ag/ml)
aInfected cells were treated with canavanine at 10 min PI. Arginine was added to canavanine-treated cultures at 25 min PI and to untreated cultures at 10 min PI. The phage were collected at 3 h PI. Pi, Phosphate.
236
BOLIN AND CUMMINGS
putrescine and spermidine are detectable in the lollipops, but the results were variable and quantitatively were meaningless. Alternate procedures of purification using nonionic gradients have been attempted, but an adequate enrichment of lollipops was not achieved.
J. VIROL.
We were unable to establish any causal relationship between the polyamine reduction in the presence of canavanine and the inhibition of DNA synthesis by canavanine or the induction of lollipops. Although exogenously added polyamines are concentrated by canavanine-treated cells, polyamines are unable to stimulate DNA synthesis. Although we were unable to accurately measure the polyamines in the lollipops, they do contain both putrescine and spermidine, and no new polyamine analogue derived from canavanine was noted. We feel that it is likely that polyamines play a role in the induction of lollipops. Canavanine does not appear to affect the neutralization of the phosphates in the viral DNA, although the ratio of putrescine to spermidine is affected. If polyamines were involved in the formation of the lollipop particle, they would most likely influence the packaging of the DNA, and it now appears that the formation of the lollipop precursor (3) as well as the normal proheads (12) occurs prior to any involvement of the viral DNA.
DISCUSSION We have described the effects of canavanine on the intracellular polyamine pool of T4infected cells and the effects of canavanine and arginine on the polyamine content of the T4 head. The addition of canavanine to T4infected cells results in a decreased synthesis of putrescine and spermidine and, since it has been demonstrated that the polyamine content of the phage head reflects the polyamine content of the infected cell (1), it can be assumed that a change in the polyamine content of the head reflects a similar change within the cell. The data concerning the effects of canavanine and arginine on the polyamine content of the phage head have interesting implications concerning the effect of canvanine upon polyamine ACKNOWLEDGMENTS synthesis. It was shown that arginine depresses the amount of polyamines within the phage We thank I. Fukuma, T. Sakai, and R. Torget for helpful head relative to the untreated sample but that advice and instruction in performing the polyamine assays. reported here was supported by grant GB-38447 lower concentrations of arginine depress the fromThethework National Science Foundation and by Public Health extent than do a to levels greater polyamine Service grant ST01GM02219 from the National Institute of higher concentrations of arginine. This suggests General Medical Sciences. that the lower concentrations of arginine are LITERATURE CITED depressing ornithine production, which in turn inhibits the amount of putrescine and spermi- 1. Ames, B. N., and D. T. Dubin. 1960. The role of polyamines in the neutralization of bacteriophage dedine synthesized from ornithine. Increased conoxyribonucleic acid. J. Biol. Chem. 235:769-775. centrations of arginine are apparently stimulat- 2. Bolin, R. W., and D. J. Cummings. 1974. Structural ing the production of polyamines via the agmaaberrations in T-even bacteriophage. IV. Parameters of tine to putrescine pathway. In each case the induction and formation of lollipops. J. Virol. 13:1368-1377. ratio of putrescine to spermidine remained the R. W., and D. J. Cummings. 1974. Structural same, indicating that the conversion of putres- 3. Bolin, aberrations in T-even bacteriophage. V. Effects of of cine to spermidine is independent the argicanavanine on the maturation and utilization of spenine concentration. However, canavanine cific gene products. J. Virol. 13:1378-1391. clearly must have prevented the synthesis of 4. Brenner, S., and L. Barnett. 1959. Brookhaven Symp. Biol. 12:86. putrescine from ornithine by normal feedback 5. Cohen, S. S., and A. Raina. 1967. Some interrelations of repression, but canavanine or canavanine derivnatural polyamines and nucleic acids in growing and atives also stopped the synthesis of putrescine virus infected bacteria, p. 157-182. In H. J. Vogel, J. 0. Lampen, and V. Bryson (ed.), Organizational biosynby the agmatine pathway, possibly by competthesis. Academic Press Inc., New York. ing with arginine or agmatine for active sites on 6. Couse, N. L., D. J. Cummings, V. A. Chapman, and S. S. the enzymes. Curiously, the presence of the DeLong. 1970. Structural aberrations in T-even bactecanvanine must not have prevented the converriophage. I. Specificity of induction of aberrations. Virology 42:590-602. sion of the previously existing putrescine to N. L., P. Haworth, W. Moody, and D. J. Cumspermidine, since phage from lysates treated 7. Couse, mings. 1972. Intracellular events in canavanine-treated with canavanine had far more phosphate neuT4-infected Escherichia coli. Virology 50:765-771. tralized with spermidine than did phage from 8. Cummings, D. J., V. A. Chapman, S. S. DeLong, and N. L. Couse. 1973. Structural aberrations in T-even bacteuntreated or arginine-treated lysates. This sugriophage. III. Induction of "lollipops" and their partial gests that the levels of agmatine may be incharacterization. Virology 54:245-261. volved in the control of the synthesis of spermi- 9. Dion, A. S., and S. S. Cohen. 1972. Polyamines in the dine from putrescine. synthesis of bacteriophage deoxyribonucleic acid. I.
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POLYAMINE POOLS AND HEAD MORPHOGENESIS
Lack of dependence of polyamine synthesis on bacteriophage deoxyribonucleic acid synthesis. J. Virol. 9:419-422. 10. Dion, A. S., and S. S. Cohen. 1972. Polyamines in the synthesis of bacteriophage deoxyribonucleic acid. II. Requirement for polyamines in T4 infection of a polyamine auxotroph. J. Virol. 9:423-430. 11. Dion, A. S., and E. J. Herbst. 1970. Polyamine changes during development of Drosophila melanogaster. Ann. N.Y. Acad. Sci. 171:723-734. 12. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80:575-599. 13. Luftig, R. B., W. B. Wood, and R. Okinaka. 1971. Bacteriophage T4 head morphogenesis. On the nature
14.
15. 16. 17.
237
of gene 49-defective heads and their role as intermediates. J. Mol Biol. 57:555-573. Morris, D. R., and K. L. Koffron. 1969. Putrescine biosynthesis in Escherichia coli. Regulation through pathway selection. J. Biol. Chem. 244:6094-6099. Morris, D. R., and A. B. Pardee. 1965. A biosynthetic ornithine decarboylase in Escherichia coli. Biochem. Biophys. Res. Commun. 29:697-702. Morris, D. R., and A. B. Pardee. 1966. Multiple pathways of putrescine biosynthesis in Escherichia coli. J. Biol. Chem. 241:3129-3135. Simon, L. D. 1972. Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope: T4 head morphogenesis. Proc. Nat. Acad. Sci. U.S.A. 69:907-911.
JOURNAL OF VIROLOGY, Feb. 1975, p. 232-237 Copyright © 1975 American Society for Microbiology
Canavanine-Mediated Depletion of Polyamine Pools in Escherichia coli: Effect on Head Morphogenesis and DNA Synthesis REX W. BOLIN* AND DONALD J. CUMMINGS Department of Microbiology, University of Colorado Medical Center, Denver, Colorado 80220 Received for publication 12 September 1974
We have found that L-canavanine inhibited the synthesis of polyamines in T4-infected Escherichia coli. These polyamines are known to be required for T4 DNA synthesis and may be involved in phage morphogenesis. The new data indicate that the inhibition of polyamine synthesis is not primarily responsible for the L-canavanine-mediated inhibition of DNA synthesis nor does it seem to be involved in the induction of lollipops. L-Canavanine does influence the relative amounts of putrescine and spermidine found in the phage particle, but it does not influence the amount of DNA phosphate neutralized by polyamines.
It has been previously shown that when a T-even bacteriophage-infected cell is exposed to canavanine followed by an arginine chase a viable monster phage particle termed a "lollipop" is produced. The lollipops usually represent about 3% of the phage particles and, due to their abnormally large size, up to 30% of the assembled proteins (8). The addition of canavanine to T4-infected cells markedly inhibits viral DNA synthesis and the virus yield (2, 7). Net RNA and protein synthesis remain unaffected. However, the cleavages of the head proteins P22, P23, P24, and IPIII are inhibited and two additional proteins, P20 (head protein) and P12 (tailplate protein), do not appear (3). Canavanine also induces the appearance of highermolecular-weight forms of P10 (tailplate protein) and P18 (sheath protein) in addition to the normal forms. Polyheads similar to those produced by mutants defective in gene 20 are produced in the presence of canavanine. Addition of arginine to canavanine-treated infected cells results in a stimulation of the viral DNA synthesis, a partial restoration of the phage yield, and the appearance of the monster particles. The induction of the lollipops requires a phage late function and, under the most favorable conditions, a 3-min exposure to canavanine (2). The length of the lollipop depends to some extent on the length of the exposure to canavanine. Our data indicate that canavanine induces the formation of lollipops by interfering with a step in the virus prohead assembly which is critical for the control of normal head length (3). The block allows the accumulation of a lollipop precursor which is converted into a lollipop upon the addition of arginine and the restoration of some vital function. Several possi232
ble events could be affected by canavanine. The termination of the elongation events during head assembly possibly involves a cleavage; therefore, canavanine may be incorporated into proteins such that either the cleaving enzymes are inactive or the substrate proteins are not susceptible to cleavage. The inhibition of DNA synthesis could be responsible, although there is evidence that the normal proheads are formed prior to the packaging of the DNA (12, 13, 17). The purpose of this paper is to investigate a third possibility. Canavanine is an analogue of arginine and, since arginine is a potential precursor to polyamines, it is possible that canavanine interferes with the viral processes by interfering with the polyamine accumulation in the infected cells. In Escherichia coli, polyamines are synthesized by two independent pathways (15, 16). In the absence of exogenous arginine, the principle pathway involves the decarboxylation of ornithine, a precursor to arginine, directly to putrescine (14), which can be converted to spermidine. In the presence of exogenous arginine, the majority of the polyamines arise from the conversion of arginine to agmatine, which is then converted to putrescine. Canavanine could interfere with the polyamine pathways by (i) acting like arginine to repress the ornithine to putrescine pathways or (ii) possibly by being converted to an agmatine analogue which is converted to the putrescine analogue which might impair normal polyamine function. Following T4 infection of E. coli, the amounts of both polyamines increase dramatically in the cell (5). The polyamines have been shown to neutralize approximately 50% of the DNA phosphates in the phage head, although the phage
VOL. 15, 1975
POLYAMINE POOLS AND HEAD MORPHOGENESIS
polyamine content is dependent on the polyamine content of the bacteria (1) which is a function of the growth conditions. T4 DNA synthesis is dependent on the presence of polyamines (10), but the accumulation of polyamines is not coupled to DNA synthesis (9). High concentrations of polyamines have been shown to increase the production of smallheaded phage in T4 infections and to result in aberrant tail attachment (6). In this report we have investigated the possibility that polyamines may be responsible for the canavanine-induced inhibition of T4 DNA synthesis as well as for the induction of lollipops. Our data indicate that canavanine does reduce the polyamine pools in the infected cell. However, the reduction in the polyamine content is probably not primarily responsible for the inhibition of DNA synthesis or for the induction of lollipops. MATERIALS AND METHODS Growth conditions. E. coli B was grown to 2 x 108 cells/ml in minimal medium supplemented with 5 Ag of an amino acid supplement per ml (6) and then infected with T4B at a multiplicity of infection of five phage per bacterium. Tryptophan at 10 gg/ml was added prior to infection to enhance adsorption. The bacteria were superinfected 7 to 9 min later with the same amount of virus. The infected cells were incubated at 37 C with aeration. The polyamines (Calbiochem), L-canavanine sulfate (Calbiochem), and L-arginine were added as noted in the text. Assays. For estimation of intracellular polyamines, 2.0-ml samples of cells were removed from cultures as noted in the text and filtered through membrane filters (0.45-Mum pore size, type HA; Millipore Corp.). The filters were washed with 6.0 ml of medium and then were placed in 1.0 ml of ice cold 0.2 N perchloric acid for 1 h. The precipitate was removed by centrifugation, and the polyamines were quantitated as the fluorescent dansyl derivatives (11) as described by Dion and Cohen (9). The phosphate determinations were performed as described by Ames and Dubin (1). Phage DNA synthesis was measured by following the incorporation of ['H ]thymine into trichloroacetic acid-precipitable material as previously described (2). Preparation of phage particles. Polyamine and phosphate determinations were made on phage particles prepared in the following manner. The cells were grown and infected as described above; 2 h after infection the cells were lysed with chloroform and the majority of the cellular debris was removed by centrifuging at 5,000 x g for 5 min. The phage were then collected by centrifuging at 25,000 x g for 2 h and resuspended in 0.4% NaCl. The remainder of the cellular debris was removed by a low-speed centrifugation, and the phage were then washed three times in 0.4% NaCl. As will be described in Results, care was taken to avoid any treatment which might have resulted in a loss of polyamines from the phage head.
233
RESULTS Effect of canavanine on the intracellular pool of polyamines. Previously, we demonstrated that canavanine results in about an 80% reduction of T4 DNA synthesis (2, 7). To determine if canavanine could be responsible for this reduction by interfering with the intracellular polyamine pools, the accumulation of the polyamines was examined after treatment with canavanine. The cells were divided into several portions after infection. A portion of the culture was treated with 130 ,g of canavanine per ml at 5 min postinfection (PI) and, at 25 min PI, portions of the canavanine-treated cultures were treated with 200 Mg of arginine per ml. These conditions are similar to those described for inhibiting DNA synthesis, and they are sufficient for the induction and formation, of lollipops (2). Canavanine dramatically inhibited the intracellular accumulation of putrescine and spermidine (Fig. 1). Putrescine accumulation was inhibited to a greater degree than was spermidine. As was shown to be the case for DNA synthesis, arginine readily stimulated the accumulation of the polyamines. It should be noted that, whereas canavanine did inhibit the accumulation of polyamines, no new polyamines that might possibly have been derived from canavanine were detected using the thinlayer chromatography system described (9). In some experiments, the acetylated forms of putrescine and spermidine were detected, but these could be readily converted to putrescine and spermidine by acid hydrolysis. The reduced accumulation of putrescine and spermidine could result from either decreased synthesis or increased leakage from the cell. During the course of the above described experiments, the cell-free medium was examined to determine if canavanine induced an increased excretion of polyamines from the cells. However, only a negligible amount of the polyamines was present in the medium until late in the infection (80 to 100 min PI). This increase was probably due to cell lysis. It was demonstrated that canavanine did not prevent the transport of external polyamines. At 65 min PI in the presence of 1 mM putrescine, untreated cells contained 97 nmol of putrescine per ml of cell culture, and canavanine-treated cells contained 82 nmol of putrescine per ml of cell culture. In the presence of 1 mM spermidine, the untreated cells contained 55 nmol of spermidine per ml of cell culture, and canavanine-treated cells contained 52 nmol of spermidine per ml of cell culture. Effect of exogenous polyamine on cana-
J. VIROL.
BOLIN AND CUMMINGS
234
E \
-o
ECL
-I
0.
0
E
E
T ime Post Infection ( Min]
FIG. 1. Effect of a canavanine pulse on the accumulation of polyamines in T4-infected cells. At 5 min PI, 130 gg of canavanine per ml was added and 200,g of arginine per ml was added at 25 min PI to a portion of the treated cells. ARG, arginine; CAN, canavanine.
vanine-inhibited T4 DNA synthesis. Our data indicate that canavanine inhibits polyamine synthesis in T4-infected cells but that exogenously added polyamines can increase the intracellular concentration of polyamines. If the reduction of intracellular polyamine levels were responsible for the inhibition of DNA synthesis by canavanine, exogenously added polyamines should result in a stimulation of DNA synthesis. To examine this possibility, a portion of an infected culture was first treated with 130 ug of canavanine per ml at 5 min PI. (We have shown previously that the stimulation of DNA synthesis after the addition of arginine was more efficient if canavanine was added at 5 min PI [2].) At 15 min PI, portions of the canavaninetreated culture were treated with various combinations of putrescine, spermidine, and spermine as well as with ornithine and arginine (Fig. 2). Arginine (100 ug/ml) resulted in a strong stimulation of DNA synthesis. Ornithine (100 ug/ml) resulted in a moderate stimulation. If the ornithine concentration was reduced to 50 ,g/ml, the stimulation was reduced about 50% (not shown). The polyamines were added individually and in various combinations at concentrations from 1 to 10 mM, but no stimulation of DNA synthesis was observed. The data indicate that polyamine depletion cannot be the sole reason for the canavanineinduced inhibition of DNA synthesis. No stimulation by polyamines could be demonstrated. The stimulation by ornithine was not as strong as the stimulation by arginine. The ornithine stimulation was probably the result of conversion of ornithine to arginine rather than conversion to putrescine. This result was not unex-
12
/ I
x
E10 _/ ~~No CAN-O/
0.
e-ARG 15_
8
E
O/ /0
0
6
-.--~~ORN 15'
// 0
1 I 40 20) 0*/ 2/
A"'~~~'~
6
0
60
80
PA 15'
1012
0
Add. 20
40
100
120
Time Post Infection [ Min]
FIG. 2. Effect of polyamines on the stimulation of DNA synthesis. At 5 min PI, a portion of the infected culture was treated with 130 tig of canavanine per ml, and at 15 min PI additions were made as noted. The arginine and ornithine were added at 100 gg/ml. The polyamines (putrescine, spermidine, and spermine) were added individually and in various combinations at concentrations from 1 to 10 mM. ARG, arginine; CAN, canavanine; PA, polyamines; No Add, no additions.
pected, since we demonstrated previously (2) that the inhibition by canavanine and the stimulation by arginine were both sensitive to
POLYAMINE POOLS AND HEAD MORPHOGENESIS
VOL. 15, 1975
chloramphenicol, indicating a requirement for protein synthesis. We have also noted that a temperature-sensitive mutant (P36A) in the DNA polymerase (gene 43) has acquired a greater sensitivity to canavanine at permissive temperatures than the wild-type phage. Normally, canavanine reduces the phage yield about 200-fold but the mutant yield was reduced about 5,000-fold by canvanine. This suggests that canavanine may interfere with DNA synthesis by its incorporation into proteins essential for DNA synthesis. Effect of canavanine and arginine on the polyamine content of the phage head. We have shown that canavanine affects the polyamine content of the infected cell. The effects of canavanine and arginine on the polyamine content of the phage head are shown in Table 1. Cells were infected, and then at 10 min PI portions of the cells were treated with either canavanine or arginine. Cells treated with canavanine were further treated with arginine at 25 min PI (conditions which result in the formation of lollipops). The addition of arginine to infected cells resulted in a decrease in the total amount of phosphate neutralized by polyamines. However, increasing concentrations of arginine resulted in an increase in the amount of polyamines in the virus. In each case in which only arginine was added to the cultures, the ratio of putrescine to spermidine was essentially constant. The ratio was identical with that of the untreated culture. The addition of canavanine markedly influenced the ratio of putrescine to spermidine within the phage head. However,
235
the amount of phosphate neutralized by the polyamines was dependent on the amount of arginine added after the addition of the canavanine. The addition of canavanine affected only the amount of putrescine relative to the spermidine but did not affect the neutralization of the phosphate. Canavanine clearly affects the polyamine content within the phage head. Based upon the amount of DNA in the lollipop heads, approximately 25% of the polyamines in the canavanine-treated cultures should have been derived from the lollipop heads. Attempts were made to quantitate the polyamines in purified lollipops, but they were unsuccessful. Unfortunately, the lollipops were very permeable to ions, as evidenced by their resistance to osmotic shock with sodium sulfate (8). The head construction may be analogous to mutants of T4 (T401) which are resistant to osmotic shock (4). T401 particles were shown to be very susceptible to losing their polyamines during normal purification procedures (1). The purification of lollipops from phage of normal head length involves the use of CsCl-D2O-glycerol gradients (8), and we have found that this procedure leads to the loss of the polyamines from the heads. Usually several cycles through the gradients are required for achieving high purity lollipops, but after just one gradient the vast majority of the polyamines are removed from the lollipop heads. Phage of normal head lengths lost some polyamines, but their polyamine content was higher than that of the lollipops, as would be expected. It should be noted that small amounts of both
TABLE 1. Effect of arginine and canavanine on the polyamine (PA) content of T4a Polyamine
PutrescineSpermidine Treatment_______scine_SpermiPtrscn
% ofPi neutralized
of Pi
PA
nmol/tmol of Pi
% of Total PA
165
62.8
97.7
37.2
1.7
62.3
nmol/umol % of Total
Untreated
Ratio of putrescine to spermidine
by PA
Arginine (20 ,gg/ml)
94.2
62.8
55.9
37.2
1.7
35.6
Canavanine (130 jg/ml) Arginine (20 ug/ml)
55.6
44.2
70.2
55.8
0.8
32.2
119.6
64.5
65.8
35.5
1.8
42.6
86.2
50.1
86.0
49.9
1.0
43.0
139.5
64.2
77.6
35.7
1.8
51.2
Arginine (100jig/ml)
Canavanine (130,ug/ml) Arginine (100jig/ml) Arginine (200 Ag/ml)
aInfected cells were treated with canavanine at 10 min PI. Arginine was added to canavanine-treated cultures at 25 min PI and to untreated cultures at 10 min PI. The phage were collected at 3 h PI. Pi, Phosphate.
236
BOLIN AND CUMMINGS
putrescine and spermidine are detectable in the lollipops, but the results were variable and quantitatively were meaningless. Alternate procedures of purification using nonionic gradients have been attempted, but an adequate enrichment of lollipops was not achieved.
J. VIROL.
We were unable to establish any causal relationship between the polyamine reduction in the presence of canavanine and the inhibition of DNA synthesis by canavanine or the induction of lollipops. Although exogenously added polyamines are concentrated by canavanine-treated cells, polyamines are unable to stimulate DNA synthesis. Although we were unable to accurately measure the polyamines in the lollipops, they do contain both putrescine and spermidine, and no new polyamine analogue derived from canavanine was noted. We feel that it is likely that polyamines play a role in the induction of lollipops. Canavanine does not appear to affect the neutralization of the phosphates in the viral DNA, although the ratio of putrescine to spermidine is affected. If polyamines were involved in the formation of the lollipop particle, they would most likely influence the packaging of the DNA, and it now appears that the formation of the lollipop precursor (3) as well as the normal proheads (12) occurs prior to any involvement of the viral DNA.
DISCUSSION We have described the effects of canavanine on the intracellular polyamine pool of T4infected cells and the effects of canavanine and arginine on the polyamine content of the T4 head. The addition of canavanine to T4infected cells results in a decreased synthesis of putrescine and spermidine and, since it has been demonstrated that the polyamine content of the phage head reflects the polyamine content of the infected cell (1), it can be assumed that a change in the polyamine content of the head reflects a similar change within the cell. The data concerning the effects of canavanine and arginine on the polyamine content of the phage head have interesting implications concerning the effect of canvanine upon polyamine ACKNOWLEDGMENTS synthesis. It was shown that arginine depresses the amount of polyamines within the phage We thank I. Fukuma, T. Sakai, and R. Torget for helpful head relative to the untreated sample but that advice and instruction in performing the polyamine assays. reported here was supported by grant GB-38447 lower concentrations of arginine depress the fromThethework National Science Foundation and by Public Health extent than do a to levels greater polyamine Service grant ST01GM02219 from the National Institute of higher concentrations of arginine. This suggests General Medical Sciences. that the lower concentrations of arginine are LITERATURE CITED depressing ornithine production, which in turn inhibits the amount of putrescine and spermi- 1. Ames, B. N., and D. T. Dubin. 1960. The role of polyamines in the neutralization of bacteriophage dedine synthesized from ornithine. Increased conoxyribonucleic acid. J. Biol. Chem. 235:769-775. centrations of arginine are apparently stimulat- 2. Bolin, R. W., and D. J. Cummings. 1974. Structural ing the production of polyamines via the agmaaberrations in T-even bacteriophage. IV. Parameters of tine to putrescine pathway. In each case the induction and formation of lollipops. J. Virol. 13:1368-1377. ratio of putrescine to spermidine remained the R. W., and D. J. Cummings. 1974. Structural same, indicating that the conversion of putres- 3. Bolin, aberrations in T-even bacteriophage. V. Effects of of cine to spermidine is independent the argicanavanine on the maturation and utilization of spenine concentration. However, canavanine cific gene products. J. Virol. 13:1378-1391. clearly must have prevented the synthesis of 4. Brenner, S., and L. Barnett. 1959. Brookhaven Symp. Biol. 12:86. putrescine from ornithine by normal feedback 5. Cohen, S. S., and A. Raina. 1967. Some interrelations of repression, but canavanine or canavanine derivnatural polyamines and nucleic acids in growing and atives also stopped the synthesis of putrescine virus infected bacteria, p. 157-182. In H. J. Vogel, J. 0. Lampen, and V. Bryson (ed.), Organizational biosynby the agmatine pathway, possibly by competthesis. Academic Press Inc., New York. ing with arginine or agmatine for active sites on 6. Couse, N. L., D. J. Cummings, V. A. Chapman, and S. S. the enzymes. Curiously, the presence of the DeLong. 1970. Structural aberrations in T-even bactecanvanine must not have prevented the converriophage. I. Specificity of induction of aberrations. Virology 42:590-602. sion of the previously existing putrescine to N. L., P. Haworth, W. Moody, and D. J. Cumspermidine, since phage from lysates treated 7. Couse, mings. 1972. Intracellular events in canavanine-treated with canavanine had far more phosphate neuT4-infected Escherichia coli. Virology 50:765-771. tralized with spermidine than did phage from 8. Cummings, D. J., V. A. Chapman, S. S. DeLong, and N. L. Couse. 1973. Structural aberrations in T-even bacteuntreated or arginine-treated lysates. This sugriophage. III. Induction of "lollipops" and their partial gests that the levels of agmatine may be incharacterization. Virology 54:245-261. volved in the control of the synthesis of spermi- 9. Dion, A. S., and S. S. Cohen. 1972. Polyamines in the dine from putrescine. synthesis of bacteriophage deoxyribonucleic acid. I.
VOL. 15, 1975
POLYAMINE POOLS AND HEAD MORPHOGENESIS
Lack of dependence of polyamine synthesis on bacteriophage deoxyribonucleic acid synthesis. J. Virol. 9:419-422. 10. Dion, A. S., and S. S. Cohen. 1972. Polyamines in the synthesis of bacteriophage deoxyribonucleic acid. II. Requirement for polyamines in T4 infection of a polyamine auxotroph. J. Virol. 9:423-430. 11. Dion, A. S., and E. J. Herbst. 1970. Polyamine changes during development of Drosophila melanogaster. Ann. N.Y. Acad. Sci. 171:723-734. 12. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80:575-599. 13. Luftig, R. B., W. B. Wood, and R. Okinaka. 1971. Bacteriophage T4 head morphogenesis. On the nature
14.
15. 16. 17.
237
of gene 49-defective heads and their role as intermediates. J. Mol Biol. 57:555-573. Morris, D. R., and K. L. Koffron. 1969. Putrescine biosynthesis in Escherichia coli. Regulation through pathway selection. J. Biol. Chem. 244:6094-6099. Morris, D. R., and A. B. Pardee. 1965. A biosynthetic ornithine decarboylase in Escherichia coli. Biochem. Biophys. Res. Commun. 29:697-702. Morris, D. R., and A. B. Pardee. 1966. Multiple pathways of putrescine biosynthesis in Escherichia coli. J. Biol. Chem. 241:3129-3135. Simon, L. D. 1972. Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope: T4 head morphogenesis. Proc. Nat. Acad. Sci. U.S.A. 69:907-911.
