removed from the growth medium, converting ... conversion of restricting cells into special cells is ..... derivedfrom A by the equation: St/So = [PSht(ODt)]/.
JOURNAL OF VIROLOGY, Dec. 1968, p. 1368-1373 Copyright © 1968 American Society for Microbiology
Vol. 2, No. 12 Printed in U.S.A.
Loss of Host-controlled Restriction of x Bacteriophage in Escherichia coli Following Methionine Deprivation' ROBERT J. GRASSO2 AND KENNETH PAIGEN Department ofExperimental Biology, Roswell Park Memorial Institute, Buffalo, New York 14203
Received for publication 29 July 1968
X Bacteriophages produced in Escherichia coli C (designated as X * C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict X C. These infections yield modified progeny phage (designated as X K) which, unlike X * C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified X -K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of X -C and X -K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for X * C but not for X K. Thus, retention of the Pl-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine. -
Host-controlled variation in bacteriophages consists of two distinct phenomena: restriction and modification (4, 12). Host-controlled restriction enables a bacterial cell to prevent the successful replication of a phage which had grown previously in a different host. For example, phage X propagated in Escherichia coli C (designated as X C) plates with an efficiency of approximately 10-4 on E. coli K-12, whereas X grown in E. coli K-12 (designated as X .K) plates with an efficiency of one. In host-controlled modification, a bacterial host imparts an altered host range to phage particles produced within it. For example, X .K, but not X C, carries K-host specificity, rendering the phage insensitive to the restriction mechanism of E. coli K-12. Modification represents a nongenetic, and as yet unidentified, change in the ' Taken in part from a dissertation submitted by R. J. Grasso to the State University of New York at Buffalo in partial fulfillment of the requirements for the Ph.D. degree. ' Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. -
02139.
structure of the phage deoxyribonucleic acid (DNA; reference 3). Although the majority of cells in a restricting K-12 bacterial population are capable of preventing the successful growth of X -C, a few "special" cells which have lost their capacity to restrict, but maintain their capacity to confer host specificity and produce X K progeny, occur at low frequencies (4, 14). The frequency of special cells can be increased by exposing the restricting host to ultraviolet light (4, 11) or elevated temperatures (6, 8, 17, 19), or by growth into stationary phase (11, 16). In a previous paper (5), we showed that the expression of K-12-restriction is regulated by the presence of alanine, leucine, and methionine during bacterial growth and that the removal of methionine from the growth medium caused a rapid 30- to 50-fold increase in the frequency of special cells. We report here the existence of a mutant strain of E. coli K-12 in which the special cell frequency increases 500- to 3,000-fold when methionine is removed from the growth medium, converting most of the population into special cells. This
1368
-
VOL. 2, 1968 conversion
1369
METHIONINE AND RESTRICTION OF PHAGE X
of restricting cells into special cells is
not reversed by subsequent methionine supple-
mentation. Furthermore, the special cells formed after methionine removal are incapable of undergoing cell division for at least 4 hr after methionine is returned to the culture. MATERIALS AND METHODS Microorganisms. In most experiments, Escherichia coli K-12 F+ (thi), designated as Q81 in our laboratory, was the restricting host. Q81 is a derivative of E. coli K-12 HfrH (met thi) strain 15-161 (18) which has since become F+ and prototrophic for methionine. E. coli strain C and E. coli K-12 strain W3350 (gal-i, gal-2), designated as K-12, were provided by J. J. Weigle. Strain K12(PI) was constructed by lysogenizing strain W3350 with P1 kc obtained from S. Lederberg. E. coli B strain Bc 251 mal+ X9 was supplied by W. Arber. The phage double mutant Xch, which yields clear plaques (7) and has an extended host range (1), was used. Lysates of X- C were prepared in E. coli C, and X -K lysates in K-12 as previously described (5). Media. Minimal medium consisted of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-chloride (pH 7.4), 0.003 M MgSO4, 0.001 M KH2PO4, 0.0015 M NH4Cl, 0.007 M NaCl, 10-6 M FeCl3, 0.01%17 gelatin, and 0.5% of glucose. In all experiments with strain Q81, minimal medium was supplemented with 6 X 10-6 M thiamine-HCl. The 20 L-amino acids listed in Table 1, first column, were used as supplements at a final concentration of 25 ,g/ml each and were prepared as previously described (5). Tris-magnesium buffer consisted of 0.05 M Tris-chloride (pH 7.4) and 0.01 M MgSO4. Tryptone broth consisted of 1% tryptone (Difco) and 0.085 M NaCl, adjusted to pH 7.4 with 1 N NaOH. For tryptone plates. 1.5% agar was added to the broth for the bottom layer and 0.65%cO agar for the top layer. PSI determinations. The probability of a successful infection (PSI) of X on a given host is defined as the ratio of successful infections obtained on that host to the number of infected cells. Since the multiplicities of infection were 99%, the number of infected cells was calculated from the number of input phage particles as determined by titering on the host used to prepare the lysate. Portions of restricting cultures were centrifuged, and the pellets were resuspended to approximately 5 X 109 cells per ml in Tris-magnesium, mixed with X, and plated for infective centers as previously described (5), with the restricting host grown in tryptone broth as indicator. Medium shift procedure. Starting with a 1:100 dilution of fresh preadapted overnight cultures, cells were grown at 37 C with aeration into log phase (approximately 2 X 108 cells per ml) in minimal medium supplemented with either 20 amino acids or methionine alone. After harvesting the cells by centrifugation at 4 C, pellets were resuspended in ice-cold Tris-magnesium, centrifuged again, and finally suspended in appropriate medium to the original cell density. Unless stated otherwise, the PSI was determined after the culture was aerated in the new medium at 37 C for 90 min.
TABLE 1. Loss of host-controlled restrictio following methionine deprivationa PSI X 103 L-Amino acid removed from growth medium
None ................... 20 Amino acids .......... Methionine .............. ala, arg, asp, asp-N, cys, glu, glu-N, gly, his, ile, leu, lys, phe, pro, ser, thr, trp, tyr, valb .
Without
With
chloramphenicol
cheloram
0.4 625 500
0.5
580
0.2-1.1
a Log phase Q81 was grown in minimal medium supplemented with 20 amino acids (PSI X 103 of X C = 0.5) and divided into two portions. One portion remained untreated, the other was treated with 20 ,Ag of chloramphenicol per ml, which immediately stopped growth. After 10 min, both cultures were subjected to a medium shift. In the case of the treated culture, the wash fluid contained chloramphenicol. Cultures were then aerated at 37 C in the indicated medium for 90 min before the PSI of X-C was determined. b Alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, respectively.
Bacterial viability and growth. Multiplicities of infection were calculated from viable cell counts determined by the soft-agar-overlay method. Doubling times of growing bacteria were estimated from turbidity measurements at 550 nm by using a Zeiss spectrophotometer (PMQII). RESULTS Loss of host-controlled restriction after methionine deprivation. When Q81 that had grown in minimal medium supplemented with 20 amino acids was transferred to medium without amino acids, a rapid 500- to 3,000-fold increase in the frequency of special cells occurred (Fig. 1, curve A). The presence of amino acids prevented this near-total loss of restriction (curve B). These manipulations had no effect on the probabilities of successful infections by nonrestricted X * K, which were close to unity (curve C). To determine whether it was the removal of a specific amino acid which caused this loss of restriction, Q81 was grown in minimal medium supplemented with 20 amino acids and then singly deprived of each amino acid. Table 1 (second column) shows that restriction was lost when methionine alone was removed from the growth medium. The removal of each of the other 19 amino acids was without effect. Separate experiments showed that when the 19 amino acids other than methionine
1370
GRASSO AND PAIGEN 0
* !
*
were
_i
simultaneously removed from the growth
medium, loss of restriction did
c
not occur, and
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I that, when Q81 was grown in minimal medium
_*
o~supplemented with only methionine and subse~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I quently deprived of this amino acid, restriction
_
/A
-II
was again almost entirely lost. From these observations, we conclude that the rapid increase in special cell frequencies which occurs in strain Q81 after the removal of 20 amino acids from the growth medium is due specifically to the removal of methionine. To examine whether the loss of restriction which follows methionine deprivation requires de novo protein synthesis, Q81 was deprived of methionine in the presence of chloramphenicol. Restriction was also lost under this circumstance, eliminating the possibility that any mechanism requiring protein synthesis, as for example derepression of the methionine pathway (15), is responsible for this effect. Physiological separation of restriction and
LOG -2 PSI
-.
.
B
*
-4
-60
J. VIROL.
-30
0 TI
BEFORE
MEDIUM
SHIFT
120
30 60 90 [ME IN MINUTES AFTER MEDIUM SHIFT
modification. Although
loss of restriction
a
occur-
red after methionine was removed from the growth medium, the ability to confer K-host specificity
FIG. 1. Loss of host-controa /led restriction following amino acid deprivation. PSI c)f XA C (0) canid X K (X) were determined on Q81 culttures conitainling miitinial medium supplemented with 2,0 amino aciCds; PSI of X- C (0) andX -K (D) were determined on Q81 cailtures containing unsupplementi red minimal mediiln.
was
not
affected. This
was
demon-
strated by the plating efficiencies of progeny obtained after a single cycle of growth of X C in strain Q81 in a methionine-deficient environment ah methionin
sTain
(Table
2)
Thus,
special
ece
cells
envion
formed
en
after
methionine removal behave similarly to the
TABLE 2. Single-cycle growth of X- C in Q81 in the presence and absence of methionliine1 Methionine present througisout expt 'rime
No. of infective centers titered on E. coli K-12
E. cs/i C
5 1.7 X 10' 1.7 X 10' 50 100 120 3.4 X 106 3.4 X 10' 160 200 200 Avg burst size
1Ratio K-12,(
1.0 1.0
'Methionine absent throughout expt
NNo. of infective centers titered on .
coli K 12
F. csli C
2.2 5.2 1.0 1.2
X 10' 2.4 X X 104 6.0 X X 106 1.2 X X 106 9.0 X 1.0 X 106 2.0 X
46
46
104 10' 10 10106
R
Ratio
K-i2Y'C
dethionine absent at zero-time No. of infective centers titered on E. coli K-12
E. coli C - K-12/C'
0.91 0.87 3.8 X 104 6.0 X 0.83
1O4 0.63
1.30 0.50 6.0 X 106 7.2 X 106
270
Ratio
0.83
300
a Log phase Q81 was grown in minimal medium supplemented with methionine. The PSI of X C plated on this culture was 6 X 10-4. The culture was then deprived of methionine for 90 min, at which time the PSI of X- C had increased to 5 X 10-1. The culture was then divided into two portions. One portion received 25 j,g of methionine per ml; the other portion remained untreated. After 10 min, both cultures were centrifuged, and the pellets were resuspended at a cell density of >5 X 109 per ml in their respective media and infected with 6.8 X 106 X. C particles. After 10 min, a sample from each culture was diluted 200-fold into respective prewarmed media (taken as zero-time; 3.4 X 104 infected complexes per ml). After 40 min, 25 ,ug of methionine per ml was added to a portion of the methionineless culture. At the indicated times, samples were plated on E. coli strains K-12 and C. To express differences in host specificity of progeny phage, the ratio of the titer on E. coli K-12 over E. coli C is given.
METHIONINE AND RESTRICTION OF PHAGE X
VOL. 2, 1968
special cells normally present at low frequencies in restricting cultures. Kinetics of restriction loss and recovery. To examine whether the conversion of restricting cells to special cells was readily reversible, methionine was returned shortly after its removal from Q81 cultures, and the time required to reach the original low special cell frequency was estimated (Fig. 2). Removing methionine from a restricting culture containing 20 amino acids abolished restriction (curve A), but the presence of methionine prevented this from occurring (curve D). The addition of methionine to a culture shortly after its removal immediately stopped any further loss of restriction, but a long period of log-phase growth was required to reestablish the original frequency of special cells (curve C). When methionine and chloramphenicol were added simultaneously, restriction loss also ceased, but the 0o-
0~~~~ B
LOG -2
=c
PSI
-4 0
30
60
90
120
150
MINUTES AFTER MEDIUM SHIFT FIG. 2. Loss of host-controlled restriction following
methionine deprivation. Log phase Q81 was grown in minimal medium supplemented with 20 amino acids prior to a medium shift. The culture was then divided and aerated at 37 C in minimal medium containing either 20 amino acids (0) or 19 amino acids minus methionine (0). At 35 min, the methioninleless culture was divided into three portions. One portion received 25 lig ofmethionine per ml (O); the second portion received 25 l.g of methionine per ml plus 20 ,ug of chloramphenicol per ml (*), which stopped growth; the third portion was allowed to continue incubationt untreated (0).
1371
special cell frequency remained constant at that particular level over a period of several hours (curve B). The return of methionine to a deprived culture did not immediately restore a low special cell frequency, whether or not chloramphenicol was present. Thus, the conversion of a restricting cell into a special cell is not readily reversible when methionine again becomes available. From this we conclude that the size of the methionine pool is probably not a critical factor in maintaining reduced special cell frequencies. Restriction was recovered quite slowly after methionine was returned to the medium (Fig. 2, curve C), suggesting that the special cells formed after methionine removal cannot divide and are diluted out by growth of the remaining restricting cells. To test this hypothesis directly, Q81 was deprived of methionine until 66% of the total cell population was converted into special cells. Methionine was then added back and the probability of successful infections of X C and growth of the culture were followed over the next several hours. Figure 3A confirms the slow recovery of host-controlled restriction. During this time, the total number of special cells remained constant (Fig. 3B). From this result, it appears that special cells cannot divide to give rise to two special daughter cells. Two possibilities remain, either that special cells cannot divide at all, or that when a special cell divides it gives rise to one special daughter cell and one restricting daughter. These alternatives predict different rates of growth of the culture after methionine is restored to the medium. The solid curves in Fig. 3C are theoretical and show the expected increases in turbidity if (I) both the restricting and special cells commenced growth at the maximal rate (doubling time, 45 min), and (II) if only the restricting cells divided (34%7 of the population) but not the special cells. The experimental points fall directly on curve II. We therefore conclude that the special cells formed after the removal of methionine from the growth medium are incapable of cell division for at least 4 hr after methionine is returned to the culture. Response of the B- and P1-restriction systems after methionine removal. We previously found strain B of E. coli to differ from K-12 in that growth in the presence of amino acids did not reduce the special cell frequency (5). However, after E. coli B was deprived of methionine, it showed a 500- to 1,000-fold increase in the frequency of special cells for both X C and X K (Table 3). In the case of K-12 (P1), the ability to restrict X K was not lost when methionine was removed from the culture medium. However, an increased probability of successful infection of X C did occur after K-12 (P1) was deprived of methionine, indicating that the removal of this
1372
GRASSO AND PAIGEN O
J. VIROL.
TABLE 3. Response o.f the B- and P1-restriction systems following methionine deprivationa
=
PSI X 103 Host strain
LOG -I PSI
L-Amino acid removed from growth medium X.C
=
_
B
None 20 Amino acids Alanine
Arginine Methionine 19 Amino acids (except methio-
0.3 100 0.3 0.3 262 0.4
X-K
0.3 114 0.3 0.3 138 0.4
nine) St/o
jOo-o-
K-12(P1)
None 20 Amino acids Alamine
Arginine Methionine 19 Amino acids (except methionine)
10.0 a
0.0005 0.03 0.0004 0.0004 0.06 0.0009
0.9 1.0 0.9 0.9 0.9 1.1
Log phase cultures of strains B and K-12(P1)
were grown in minimal medium supplemented with 20 amino acids. Prior to the medium shifts, the PSI X 103 of both X- C and X- K on strain B was 0.3; of X-C on strain K-12(P1) it was 0.0004 and of X-K on strain K-12(P1) it was 0.7.
O.D.550
1.0
0 100 200 300 MINUTES AFTER METHIONINE SUPPLEMENTATION
FIG. 3. Recovery of host-controlled restriction and growth after methionine supplementation. Log phase Q81 was grown in minimal medium supplemented with methionine and deprived of this amino acid for 150 min. At zero-time, 25 ,ug of methionine per ml was added back to the culture, and the PSI of X- C and turbidity of the culture were determined over a 4-hr period. B was derivedfrom A by the equation: St/So = [PSht(ODt)]/ PSIo[(ODo) ]. The numbered curves in Care theoretical; the points are experimental.
amino acid probably abolishes the expression of K-12-restriction without affecting the ability of the lysogen to exert P1-restriction. Thus, the expression of the P1-restriction system is unaffected by the same nutritional factors which alter the expression of the B- and K-12-restriction systems. DIscussIoN
In the E. coli K-12 strain studied, the conversion of restricting cells into special cells is specifically induced by removing methionine from the growth medium and does not require the synthesis of a new protein. This conversion is irreversible
in any given cell, but the formation of new special cells ceases immediately upon the return of methionine to a deprived culture. These observations suggest that methionine itself assumes a critical role in the maintenance of a functional restriction mechanism. Growth of a phage for a single cycle in Q81 cultures composed mainly of special cells showed that, although the ability to restrict was lost, their ability to modify progeny phage was unimpaired. Thus, cells can lose their ability to restrict and still retain their ability to modify DNA, either by exposure to methionine deprivation or after mutation (9, 20). Our results differ from the observations of Arber (2) who reported that, when auxotrophs of E. coli K-12 were infected with 'X K and starved for methionine during a portion of the latent period, the progeny phage lacked Khost specificity. This apparent discrepancy may derive from the fact that Arber's results were obtained with methionine auxotrophs. Strain Q81 is a methionine prototroph and, after the medium shift, may be able to maintain a sufficient intracellular concentration of this amino acid to allow developing X particles to acquire K-host specificity. Recently, Meselson and Yuan (13) have identified two endonucleases in E. coli that are
VOL. 2, 1968
METHIONINE AND RESTRICTION OF PHAGE X
responsible for the K- and Pl-restrictions of phage X. Linn and Arber (10) have identified cell fractions responsible for the B- and P1restrictions of phage fd. This is probably the same enzymatic activity which acts on phage X, since the activity is missing in mutants unable to restrict X. Both laboratories showed that these enzymes require S-adenosylmethionine (SAM) and adenosine triphosphate, but not methionine, for activity. It is possible that the K-12 and B enzymes are inactivated or inhibited in a cell unable to maintain its SAM pools. Since the P1restriction system is not lost after methionine deprivation, it appears, if this is the mechanism, that the Pl-restriction enzyme does not require the presence of SAM to maintain activity. The most striking property of the special cells formed after the removal of methionine from the growth medium of Q81 cultures is their inability to reproduce. The kinetic experiments show that, after methionine is restored to a deprived culture, the special cells do not divide and are diluted away by the growth of the remaining restricting cells in the population. This implies that the frequency of special cells in balanced log-phase cultures reflects the possibility that a restricting cell will divide and give rise to one restricting cell and one special cell. If the loss of K-endonuclease activity is the mechanism by which special cells arise, it suggests that the function of this enzyme is required for cell division. ACKNOWLEDGMENTS
This work was supported by National Science Foundation grant GB-5974, and by Public Health Service training grant CA-5061 from the National Institute of Cancer Research. LITERATURE CITED 1. Appleyard, R. K., J. F. McGregor, and K. M.
Baird. 1956. Mutation to extended host range and the occurrence of phenotypic mixing in the temperate coliphage lambda. Virology 2:565576. 2. Arber, W. 1965. Host-specificity of DNA produced by Escherichia coli. V. The role of methionine in the production of host specificity. J. Mol. Biol. 11:247-256. 3. Arber, W., and D. Dussoix. 1962. Host specificity of DNA produced by Escherichia coli. I. Hostcontrolled modification of bacteriophage X. J. Mol. Biol. 5:18-36. 4. Bertani, G., and J. J. Weigle. 1953. Host con-
5. 6.
7. 8.
9.
1373
trolled variation in bacterial viruses. J. Bacteriol. 65:113-121. Grasso, R. J., and K. Paigen. 1968. The effect of amino acids on host-controlled restriction of lambda phage. Virology 36:1-8. Holloway, B. W. 1965. Variation in restriction and modification of bacteriophage following increase of growth temperature of Pseudomonas aeruginosa. Virology 25:634-642. Kaiser, A. D. 1957. Mutations in a temperate bacteriophage affecting its ability to lysogenize Escherichia coli. Virology 3:42-61. Lederberg, S. 1965. Host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli. Virology 27:378-387. Lederberg, S. 1966. Genetics of host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli. J. Bacteriol. 91:1029-
1036. 10. Linn, S., and W. Arber. 1968. Host specificity of DNA produced by Escherichia coli. X. In vitro restriction of phage fd replicative form. Proc. Natl. Acad. Sci. U.S. 59:1300-1306. 11. Luria, S. E. 1953. Host-induced modification of viruses. Cold Spring Harbor Symp. Quant. Biol. 18:237-244. 12. Luria, S. E., and M. L. Human. 1952. A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol. 64:557-569. 13. Meselson, M., and R. Yuan. 1968. DNA restriction enzyme from E. coli. Nature 217:11101114. 14. Paigen, K., and H. Weinfeld. 1963. Cooperative infection by host-modified lambda phage. Virology 19:565-572. 15. Rowbury, R. J., and D. D. Woods. 1961. Further studies on the repression of methionine synthesis in Escherichia coli. J. Gen. Microbiol. 24: 129-144. 16. Schell, J., and S. W. Glover. 1965. The nature of the restriction of host-modified phages by nonaccepting hosts. Antonie van Leeuwenhoek J. Microbiol. Serol. 31:470-471. 17. Schell, J., and S. W. Glover. 1966. The effect of heat on host-controlled restriction of phage X in Escherichia coli K(Pl). J. Gen. Microbiol. 45:61-72. 18. Tatum, E. L. 1945. X-ray induced mutant strains of Escherichia coli. Proc. Natl. Acad. Sci. U.S. 31:215-219. 19. Uetake, H., S. Toyama, and S. Hagiwara. 1964. On the mechanism of host-induced modification. Multiplicity activation and thermolabile factor responsible for phage growth restriction. Virology 22:202-213. 20. Wood, W. B. 1966. Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J. Mol. Biol. 16:118-133.
