Vol. 17, No. 2
JOURNAL OF VIROLOGY, Feb. 1976, p. 675-677 Copyright i 1976 American Society for Microbiology
Printed in U.S.A.
Sonic Fragility of the Head-Tail Bond of Bacteriophage P22 MARK RUDDEL AND VANCE ISRAEL* Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30902
Received for publication 1 August 1975
The binding of tail parts to the head of phage P22 is normally irreversible, but after adsorption to host cells sonication releases many of the tail parts intact. This release is dependent on the power of sonication, but is independent of the length of sonication from 2 to 32 s. This phenomenon has been used to show that the upper limit of the number of P22 particles that can bind to a cell is no lower than 700. The phage P22 particle is a relatively simple structure. It consists of an icosahedral head containing one major protein species (1), a short neck to which are attached six identical tail or base plate parts (2), and a long slender spike (1), which also appears to be attached to the neck. Purified tail parts can be added in vitro to heads that lack them (but contain the neck and spike) to form active particles. This reaction goes rapidly to completion, at which point the following relationship holds: 3.3 log (7) = 2.3 log (H) + log (PFU). In this equation, H refers to structures containing the spike and neck, as well as the main capsid protein, and T refers to phage equivalents of tail parts. The equation is valid when T < H (2). A priori considerations suggest that tail parts are bound to heads irreversibly in the mature P22 particle. If there were an equilibrium between tail parts and heads, it is clear that it would have to be far on the side of binding. If this were not true it would be impossible to assay the phage biologically, since at low dilutions the tail parts that were detached would not be able to reassociate with heads. However, it is conceivable that tails can spontaneously dissociate from heads with a very low probability. Such an occurrence could be detected by forcing the equilibrium towards dissociation. We have attempted to do this. Approximately 109 whole phage were allowed to stand at room temperature for several days in the presence of 2 x 1011 heads carrying a temperature-sensitive mutation in gene 9. Any tail part that detached from the phage would immediately reassociate with a head. This should eventually result in a dramatic lowering of the titer of the phage, since when heads are in excess the number of PFU is strongly dependent on the number of tails (2). The titer of phage incubated with heads dropped no more than the
control (Table 1), and this was true in the case of in vitro reconstituted phage, as well as normal phage. In another attempt to detect spontaneous reversibility, 1011 phage carrying an amber mutation in gene 12 were incubated with 101 heads bearing a temperature-sensitive mutation in gene 9. In this case, an increase in the titer of 12+9- phage would indicate that tail parts had become detached from the mature phage particle. This test would be positive if at least 0.6% of the mature phage each lost one spike. The test was negative, and we conclude that under physiological conditions this is an irreversible reaction (Table 2). This tight binding of the tail to the neck appears to be altered when the phage adsorbs to a cell. Mature free phage are quite resistant to sonication, but after adsorption sonication proved effective in separating the tails from the rest of the phage (Fig. 1). Recovery is a function of the power of sonication; at the highest setting used, about 20% of the tails could be recovered, as opposed to less than 1% in unadsorbed controls. The addition of chloroform to adsorbed cells did not free tails. This phenomenon, which we refer to as the sonic fragility of the head-tail bond, was investigated further by observing the effect of the time of sonication on the release of tails. The length of sonication had no effect over a range of 2 to 32 s (Fig. 2). Therefore, all of the releasable tails must be released almost instantaneously. To account for this result, we speculated that sonic fragility may not result from an alteration in the bonding between the tail and the head. Rather, it might result from the phage being held rigidly in place by the 0 antigens to which it is bound (5). If the 0 antigens themselves can assume many positions, only some of which render the head-tail bond sonically fragile, then the first
675
676
J. VIROL.
NOTES
TABLE 1. Effect of heads on phage titer Phage
Additionb
Day
PFU x 10-' Day Day
-20/ Day
z
typea0a
a
a
a
1
3
5
7
0
c+ c+
am heads
2.1 2.0 3.0
1.7 1.7 2.5
1.6 1.5 2.6
r_/
Reconstituted ts9 Reconstituted ts9
2.0 1.9 3.2
am heads
3.1
3.3
3.0
2.9
0
0/ w /o
aPhage were suspended in 1 ml of buffered saline (0.9% NaCl, 0.0067 M KPO4, pH 7.0) and allowed to stand at room temperature. c+ is wild-type phage P22. Reconstituted ts9 are phage made by reacting excess tails with heads bearing the temperature-sensitive mutation ts9.1 (gene 9 is the structural gene for tail protein). The reconstituted phage were purified by CsCl density gradient centrifugation. Purification of heads, tails, and phage is described elsewhere (3). bam heads are heads carrying the amber mutation 9-amN8. TABLE 2. Lack of activation of heads by phage Additional
PFU/mlb
109 ts9 heads .... ... 109 ts9 heads plus 1011 am phage ...
3.2 x 104 3.5 x 104
aHead and phage were allowed to stand overnight at 37 C in 1 ml of buffered saline. The am phage (12-amN80) were purified twice in CsCl denisty gradients. Gene 12 is an early gene necessary for DNA synthesis (4). bPFU were assayed at 25 C on strain 18, a derivative of Salmonella typhimurium LT-2, which is nonpermissive for phage bearing an amber mutation (3).
blast of sonic power may remove all eligible tails but prevent any other 0 antigens from assuming the correct position. These considerations led us to sonicate adsorbed phage and then allow them to "recover" by centrifuging and resuspending them. When this was done (Table 3), an additional quantity of tails, roughly equal to the first, could be recovered. Although this result is consistent with the idea that it is the arrangement of the 0 antigens themselves that is responsible for sonic fragility, it does not rule out the possibility that the binding between the neck and tail is indeed altered. We have also investigated the relationship between the multiplicity of infection and sonic fragility. The number of tails recovered is proportional to multiplicity up to about 700 phage/ cell (Fig. 3), at which point recoverable tails
(I) -j
20
40
30
50
70
60
80
POWER SETTING FIG. 1. Tail release as a function of power of sonication. S. typhimurium strain 18 was grown to log phase, washed once, and starved for 30 min in M-9 salts at a concentration of 5 x 106 cells/ml (3). P22-13-amlOlts9.42cl was added at a multiplicity of infection of 1 and allowed to adsorb for 10 min. Aliquots (3 ml) were sonicated for 15 s in an ice bath at different settings of a Sonic 300 dismembrator (Artek), using the large probe. Each sample was then chloroformed and centrifuged for 6 min at 12,000 x g, and the supernatant was assayed for tails. Tail assays were carried out as previously described (3), except that the final PFU were assayed at 40 C, which is nonpermissive for phage carrying the ts9.42 allele. Strain 192, a su+ derivative of strain 18, was used for plaque assays. The heads used in these assays carried amber mutations in genes 9 and 13 lysiss gene) (1). Symbols: (- ) Tails recovered from adsorbed phage; (0-----0) tails recovered from free phage. 15
p z
w
0)
0
4
8
12
16
20
24
28
32
TIME (SECONDS)
FIG. 2. Tail release as a function of sonication time. Conditions were similar to those in Fig. 1. Aliquots (3 ml) were sonicated at a setting of 60 for the times indicated, and the samples were analyzed for tails, as in Fig. 1.
VOL. 17, 1976
NOTES
TABLE 3. Recovery of sonic fragility following centrifugation and resuspensiona Treatment
No. of tails released
No sonication ...............
1 x 106
5-s sonication' 1st ........... 2nd ...................... 3rd ...................... Total ......................
7 x 10' 4.8 x 106 4.8 x 106 1.7 x 109
10-s sonication ..............
4.2 x 10'
15-s sonication .............
4.6 x 108
aConditions were the same as in Fig. 1. A power setting of 60 was used. t After the first 5-s sonication, the cells were centrifuged at 12,000 x g for 6 min and resuspended in the original volume. A sample of the supernatant was saved for assay. This process was then repeated two more times.
level off. The number 700 would, therefore, seem to be a lower limit for the number of P22 virions that can adsorb to the cell surface, and may indeed be the upper limit of phage that can adsorb effectively. From the data presented here, we may conclude that adsorption is a necessary condition for sonic fragility. On the basis of other work (V. Israel, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, S273, p. 259; manuscript in preparation), it is clear that adsorption is not sufficient to cause this phenomenon. As judged by their density in CsCl all of the freed heads are empty,
677
C) LLJ
o:1 LLU > 10 -J 100 -J LU)
-J
1.0_
10
100
1000
10000
PHAGE PER CELL ADDED
FIG. 3. Tail release as a function of multiplicity of infection. Conditions were similar to those described for Fig. 1 except that the cells were resuspended at a concentration of 106 cells/ml. Sonications were carried out for 15 s at a power setting of 60.
indicating that sonic fragility exists after ejection (data not shown). What is not clear at the moment is whether ejection is necessary for this phenomenon to occur. This matter is now under investigation. LITERATURE CITED 1. Botstein, D., C. Waddell, and J. King. 1973. Mechanism of
head assembly and DNA encapsulation in Salmonella phage P22. J. Mol. Biol. 80:669-695. 2. Israel, J. V., T. F. Anderson, and M. Levine. 1967. In vitro morphogenesis of phage P22 from heads and base plate parts. Proc. Natl. Acad. Sci. U.S.A. 57:284-291. 3. Israel, J. V., H. Rosen, and M. Levine. 1972. Binding of bacteriophage P22 tail parts to cells. J. Virol. 10:1152-1158. 4. Levine, M., and C. Schott. 1971. Mutations of phage P22 affecting DNA synthesis and lysogeny. J. Mol. Biol.
62:53-64. 5. Lindberg, A. A. 1973. Bacteriophage receptors. Annu. Rev. Microbiol. 26:205-241.
JOURNAL OF VIROLOGY, Feb. 1976, p. 675-677 Copyright i 1976 American Society for Microbiology
Printed in U.S.A.
Sonic Fragility of the Head-Tail Bond of Bacteriophage P22 MARK RUDDEL AND VANCE ISRAEL* Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30902
Received for publication 1 August 1975
The binding of tail parts to the head of phage P22 is normally irreversible, but after adsorption to host cells sonication releases many of the tail parts intact. This release is dependent on the power of sonication, but is independent of the length of sonication from 2 to 32 s. This phenomenon has been used to show that the upper limit of the number of P22 particles that can bind to a cell is no lower than 700. The phage P22 particle is a relatively simple structure. It consists of an icosahedral head containing one major protein species (1), a short neck to which are attached six identical tail or base plate parts (2), and a long slender spike (1), which also appears to be attached to the neck. Purified tail parts can be added in vitro to heads that lack them (but contain the neck and spike) to form active particles. This reaction goes rapidly to completion, at which point the following relationship holds: 3.3 log (7) = 2.3 log (H) + log (PFU). In this equation, H refers to structures containing the spike and neck, as well as the main capsid protein, and T refers to phage equivalents of tail parts. The equation is valid when T < H (2). A priori considerations suggest that tail parts are bound to heads irreversibly in the mature P22 particle. If there were an equilibrium between tail parts and heads, it is clear that it would have to be far on the side of binding. If this were not true it would be impossible to assay the phage biologically, since at low dilutions the tail parts that were detached would not be able to reassociate with heads. However, it is conceivable that tails can spontaneously dissociate from heads with a very low probability. Such an occurrence could be detected by forcing the equilibrium towards dissociation. We have attempted to do this. Approximately 109 whole phage were allowed to stand at room temperature for several days in the presence of 2 x 1011 heads carrying a temperature-sensitive mutation in gene 9. Any tail part that detached from the phage would immediately reassociate with a head. This should eventually result in a dramatic lowering of the titer of the phage, since when heads are in excess the number of PFU is strongly dependent on the number of tails (2). The titer of phage incubated with heads dropped no more than the
control (Table 1), and this was true in the case of in vitro reconstituted phage, as well as normal phage. In another attempt to detect spontaneous reversibility, 1011 phage carrying an amber mutation in gene 12 were incubated with 101 heads bearing a temperature-sensitive mutation in gene 9. In this case, an increase in the titer of 12+9- phage would indicate that tail parts had become detached from the mature phage particle. This test would be positive if at least 0.6% of the mature phage each lost one spike. The test was negative, and we conclude that under physiological conditions this is an irreversible reaction (Table 2). This tight binding of the tail to the neck appears to be altered when the phage adsorbs to a cell. Mature free phage are quite resistant to sonication, but after adsorption sonication proved effective in separating the tails from the rest of the phage (Fig. 1). Recovery is a function of the power of sonication; at the highest setting used, about 20% of the tails could be recovered, as opposed to less than 1% in unadsorbed controls. The addition of chloroform to adsorbed cells did not free tails. This phenomenon, which we refer to as the sonic fragility of the head-tail bond, was investigated further by observing the effect of the time of sonication on the release of tails. The length of sonication had no effect over a range of 2 to 32 s (Fig. 2). Therefore, all of the releasable tails must be released almost instantaneously. To account for this result, we speculated that sonic fragility may not result from an alteration in the bonding between the tail and the head. Rather, it might result from the phage being held rigidly in place by the 0 antigens to which it is bound (5). If the 0 antigens themselves can assume many positions, only some of which render the head-tail bond sonically fragile, then the first
675
676
J. VIROL.
NOTES
TABLE 1. Effect of heads on phage titer Phage
Additionb
Day
PFU x 10-' Day Day
-20/ Day
z
typea0a
a
a
a
1
3
5
7
0
c+ c+
am heads
2.1 2.0 3.0
1.7 1.7 2.5
1.6 1.5 2.6
r_/
Reconstituted ts9 Reconstituted ts9
2.0 1.9 3.2
am heads
3.1
3.3
3.0
2.9
0
0/ w /o
aPhage were suspended in 1 ml of buffered saline (0.9% NaCl, 0.0067 M KPO4, pH 7.0) and allowed to stand at room temperature. c+ is wild-type phage P22. Reconstituted ts9 are phage made by reacting excess tails with heads bearing the temperature-sensitive mutation ts9.1 (gene 9 is the structural gene for tail protein). The reconstituted phage were purified by CsCl density gradient centrifugation. Purification of heads, tails, and phage is described elsewhere (3). bam heads are heads carrying the amber mutation 9-amN8. TABLE 2. Lack of activation of heads by phage Additional
PFU/mlb
109 ts9 heads .... ... 109 ts9 heads plus 1011 am phage ...
3.2 x 104 3.5 x 104
aHead and phage were allowed to stand overnight at 37 C in 1 ml of buffered saline. The am phage (12-amN80) were purified twice in CsCl denisty gradients. Gene 12 is an early gene necessary for DNA synthesis (4). bPFU were assayed at 25 C on strain 18, a derivative of Salmonella typhimurium LT-2, which is nonpermissive for phage bearing an amber mutation (3).
blast of sonic power may remove all eligible tails but prevent any other 0 antigens from assuming the correct position. These considerations led us to sonicate adsorbed phage and then allow them to "recover" by centrifuging and resuspending them. When this was done (Table 3), an additional quantity of tails, roughly equal to the first, could be recovered. Although this result is consistent with the idea that it is the arrangement of the 0 antigens themselves that is responsible for sonic fragility, it does not rule out the possibility that the binding between the neck and tail is indeed altered. We have also investigated the relationship between the multiplicity of infection and sonic fragility. The number of tails recovered is proportional to multiplicity up to about 700 phage/ cell (Fig. 3), at which point recoverable tails
(I) -j
20
40
30
50
70
60
80
POWER SETTING FIG. 1. Tail release as a function of power of sonication. S. typhimurium strain 18 was grown to log phase, washed once, and starved for 30 min in M-9 salts at a concentration of 5 x 106 cells/ml (3). P22-13-amlOlts9.42cl was added at a multiplicity of infection of 1 and allowed to adsorb for 10 min. Aliquots (3 ml) were sonicated for 15 s in an ice bath at different settings of a Sonic 300 dismembrator (Artek), using the large probe. Each sample was then chloroformed and centrifuged for 6 min at 12,000 x g, and the supernatant was assayed for tails. Tail assays were carried out as previously described (3), except that the final PFU were assayed at 40 C, which is nonpermissive for phage carrying the ts9.42 allele. Strain 192, a su+ derivative of strain 18, was used for plaque assays. The heads used in these assays carried amber mutations in genes 9 and 13 lysiss gene) (1). Symbols: (- ) Tails recovered from adsorbed phage; (0-----0) tails recovered from free phage. 15
p z
w
0)
0
4
8
12
16
20
24
28
32
TIME (SECONDS)
FIG. 2. Tail release as a function of sonication time. Conditions were similar to those in Fig. 1. Aliquots (3 ml) were sonicated at a setting of 60 for the times indicated, and the samples were analyzed for tails, as in Fig. 1.
VOL. 17, 1976
NOTES
TABLE 3. Recovery of sonic fragility following centrifugation and resuspensiona Treatment
No. of tails released
No sonication ...............
1 x 106
5-s sonication' 1st ........... 2nd ...................... 3rd ...................... Total ......................
7 x 10' 4.8 x 106 4.8 x 106 1.7 x 109
10-s sonication ..............
4.2 x 10'
15-s sonication .............
4.6 x 108
aConditions were the same as in Fig. 1. A power setting of 60 was used. t After the first 5-s sonication, the cells were centrifuged at 12,000 x g for 6 min and resuspended in the original volume. A sample of the supernatant was saved for assay. This process was then repeated two more times.
level off. The number 700 would, therefore, seem to be a lower limit for the number of P22 virions that can adsorb to the cell surface, and may indeed be the upper limit of phage that can adsorb effectively. From the data presented here, we may conclude that adsorption is a necessary condition for sonic fragility. On the basis of other work (V. Israel, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, S273, p. 259; manuscript in preparation), it is clear that adsorption is not sufficient to cause this phenomenon. As judged by their density in CsCl all of the freed heads are empty,
677
C) LLJ
o:1 LLU > 10 -J 100 -J LU)
-J
1.0_
10
100
1000
10000
PHAGE PER CELL ADDED
FIG. 3. Tail release as a function of multiplicity of infection. Conditions were similar to those described for Fig. 1 except that the cells were resuspended at a concentration of 106 cells/ml. Sonications were carried out for 15 s at a power setting of 60.
indicating that sonic fragility exists after ejection (data not shown). What is not clear at the moment is whether ejection is necessary for this phenomenon to occur. This matter is now under investigation. LITERATURE CITED 1. Botstein, D., C. Waddell, and J. King. 1973. Mechanism of
head assembly and DNA encapsulation in Salmonella phage P22. J. Mol. Biol. 80:669-695. 2. Israel, J. V., T. F. Anderson, and M. Levine. 1967. In vitro morphogenesis of phage P22 from heads and base plate parts. Proc. Natl. Acad. Sci. U.S.A. 57:284-291. 3. Israel, J. V., H. Rosen, and M. Levine. 1972. Binding of bacteriophage P22 tail parts to cells. J. Virol. 10:1152-1158. 4. Levine, M., and C. Schott. 1971. Mutations of phage P22 affecting DNA synthesis and lysogeny. J. Mol. Biol.
62:53-64. 5. Lindberg, A. A. 1973. Bacteriophage receptors. Annu. Rev. Microbiol. 26:205-241.
