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A new effect of the rex gene of phage λ: Premature lysis after infection by phage T1
VIROLOGY
66,
285-290
A New
(1973)
Effect
of the rex after
of Microbiology,
of Phage
Infection
J. R. CHRISTENSEN Depa.rtment
Gene
by Phage JOYCE
AND
A: Premature Tl
M. GEIMAN
University of Rochester, School of Medicine Rochester, New York 14642 Accepted August
Lysis
and Dentistry,
8, 197.9
When phage Tl infects bacteria lysogenic for lambda, lysis is premature and burst sizes are small. This has been shown to be an effect of the lambda rez gene. INTRODUCTION
It was inadvertent,ly discovered in this laboratory that when phage Tl infects hosts that are lysogenic for lambda phage, small burst sizes are obtained. This has been found to be correlated with a significantly reduced latent period in such hosts. We have used a variety of mutant lambda phages to determine what gene or genes are responsible for this effect. MATERIALS
AND
METHODS
Phage. X was from I<. Paigen, X vir from G. Kellenberger, and X ~60 from L. Astrathan. X gOn12g3 was isolated from a C600 lysogen sent by A. Campbell. Bacteria. Table 1 lists the bacterial strains used in t,hese experiments, and their sources. Experimental procedures, The dat,a in Table 1 are derived from a one-step growth curve, modeled after Ellis and Delbriick (1939). The cells were grown in nutrient broth, infected with a low multiplicity of infection (m.o.i.) of Tl in 5 X 10-d M hlgC12, and dilubed into nutrient broth at 37” at time zero. For the experiment in Fig. 3, bacteria were grown with shaking at 37” in tryptone broth plus maltose (1.0 % tryptone, 0.5 % NaCl, 0.1 c’; malt,ose) to approximately 2 X lo* cells per ml. hZgC12 was t#hen added to a final concentration of 0.01 M, and lo-ml aliquots were either infected with one of the lambda stocks or mockinfected by addition of tryptone broth.
Aft,er a 15-min attachment period at 37”, the suspensions were chilled and centrifuged, and the supernat,ant, fluids were saved for assay to determine t’he extent of lambda attachment. The cell pellets were resuspended in the same volume of warm (37”) tryptone wit,hout NaCI? transferred t,o Nephelo flasks (Bellco), and infected with Tl at an m.o.i. of approximately 10. Lysis kinetics were followed by periodic readings in a Klett calorimeter (blue filter). For all other experiments, bacteria were grown with shaking in nutrient brot,h directly in hhe Nephelo flask unt,il a Klett reading of 50-60 was obtained (approximately 2 to 3 X lOa cells/ml). The cells were then infected with Tl and t,he lysis kinetics followed as indicated above. In experiments involving strains lysogenic for prophages with either the cItl or cIs57 mutation, all operations were carried out at 30”; otherwise the temperature was 37”. RESULTS
Typical results presented in Table 2 and in Fig. 1 show the reduced burst size and the early lysis when Tl infects W3350 (X) as compared to the results in W3350. We assume that the reduced burst size is a direct result of the early lysis (Christensen and Tolmach, 1955, Fig. 3). We have used lysis kinetics as the indicator of the phenomenon in all that follows, since these kinetics are more consistent from experiment to experiment than are burst sizes. Early lysis
285 Copyright .%ll rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
286
CHRISTENSEN
AND GEIRlA~
TABLE B.WTERIIL
1
STRMNS USED
Reference
Strain
Skaar and Garen (195Aj PIIichalke (1967) Parkinson (1968) Prepared in this laborator) Campbell (1961) Prepared in this laborat.orJ Drexler (19723” Drexler (1972)” Gussin and Peterson (1972j* Gussin and Peterson (1972)* Gussin and Peterson (1972)* .\dhya and Campbell (1970jr Adhya and Campbell (1970jc Court and Campbell (1972) Dove el al. (1971) Sato and Campbell (1970)+
CSlOO KB3 KB3 (hcZt,wsJn~sj KB3 (Xgonrtstj w3350 w3350 (A) W3350 (hcZtss,sztsN7szlsN~3) w3350 (A SUSQ,3) B219 = W3350 Strr (X CZSU) B235 = W3350 Strr (A cIdez& B189 = W3350 Strr (A czes;rer5a) R734 = W3350 (su~~P~sILsN~susA,,sILsR~~susF~~~~~~~~?~~~~~ R735 = W3350 (susP~susN~s~~sA~,susR~~susF~~~~~~~X) DC50B = AM3100 (X czs5+kd-j SA443 = DC506 deleted from X0 to chlA SA307 = DC506 deleted from chlD to XP
a This reference describes strain KB3 lysogenic for the same prophage. Dr. Drexler prepared t,he W3350 lysogens. b Designated in this reference as a lysogenic derivative of strain 549 (= W3350 StrI). Present designation is from Dr. Gussin (personal communication). c Designated by full genot,ype in this reference. Present designation is from Dr. Campbell (personal communication). d Designated in this reference as strain 307. Present designat,ion is from Dr. Campbell (personal communicationj. TABLE REDUCED Tl PUGE
YIELD
2
ON INFECTION OF A ~LYSOGENIC
Tl phage attached
Host cells Strain
Cells,i’ml
w3350 w3350 (A)
1.6 x 108 1.3 x 108
Tl/ml 1.5 x 109 1.2 x 109
u The 30-min values were approximately
m.0.i. 9.5 9.3
HOST
Tl phage yield at 30 mina Tl/ml 1.6 X lOI0 2.2 x 109
Burst size 100 17
the same as 20-min values.
was also seen in strain KB3 (X cItl SUSJ), but not in IIB3, and also in strain CSlOO, which is a lambda lysogen (data not shown). To begin to define which genes of the prophage are responsible for the effect, experiments were carried out with strains in which large blocks of the prophage had been deleted (see Fig. 2). The results presented in Fig. 3 demonstrate that the block of genes to the left of P in the prophage map are sufficient for the effect (strain SA443 lyses as quickly as strain DC506, which carries a full prophage), but that genes to the right of 0 are dispensable
(st#rain SA307 shows “delayed,” i.e., normal, lysis). This finding rules out the participation, through t,ransactivation, of the lysis genes R and S of lambda, or of any other late genes, in producing early lysis. It may be noted that in comparing strains we have focused our attention on t,he time of initiation of lysis and on the position of the st,eep, earl2: part of the lysis curve. The residual turbidity measured near the end of the lysis curves is based on low Klett readings, 15 or less in most cases, and these data are more subject to experimental errors, such as imperfectly matched Nephelo flasks.
PREMATURE
LYSIS BY
Tt?X
Differences seen between strains in this region of the curve t,ended to be irreproducible. Early lysis as compared with that seen in W3350 was also seen after infection of W3350 (A cIsS7 SUSN, SUSN~B)and W3350 (X .s~&&), ruling out, any participation of the control genes N and Q in the phenomenon (data not shown). The search was further narrowed down to the immunit,y region of lambda by the finding that early lgsis was obt,ained aft,er infection of strain R735, but not after infection of strain R7.74, which is isogenic except that the immunity region of the prophage in the latter strain was derived from phage 434 (data not shown).
I
I
IO MINUTES AFTER
Having ruled out, transactivation of late genes as being involved in the phenomenon, and having shown the importance of the immunity region, it was logical to examine the role of genes cl and rez, parGcularly since they are t,he only genes known to be active in t,he prophage st,ate. We first tried to test the effect of cl by using lysogens carrying prophages with the cItl or cIssi mutation which code for temperature-sensitive repressors. However, heating nonlysogenic control cultSures before Tl infection produced a significant delay in lysis, and so this approach was abandoned. We then turned t,o infect,ing the host bact’eria with lambda mutants 15 min before infection with Tl. In order t.o test the direct effect of cl on lgsis, we chose to use X ce,,, which has a mutat,ion in CI (Kaiser, 1957), and X vZr, which makes normal repressor, but, which is insensitive to repressor action. If repressor were responsible for early lysis, infection by X vir, but not by X ceo, should produce the effect,. In fact, after pre-infection by either phage, Tl lysis is earlier than in a mockinfected control (Fig. 4). Repressor, t#hen, is not required for early lysis. To test the role of the rex gene, st,rains lysogenic for phage mutant in rex were compared with nonlysogenic controls and with strains lysogenic for the rex+ count,erpart, of the mutants. Results with two strains are seen in Fig. 5, and it, is clear that the rex mutations eliminate early lysis. Experiments were also conducted with st#rain KB3 (X gon&, (“go” is an old name for rex). With this strain the lysis curve showed a very slight early lysis, but the kinet,ics were much more like those seen with KB3 than with IiB3 (X cItl sud) (data not shown). Perhaps the go1\12g3 mutation is slightly leaky, despite the fact
I
20 ADDITION
30 OF Tl
FIG. 1. Kinet,ics of lysis when Tl infects a lysogen and a nonlysogenic control. O--O is strain W3350. a----• is strain W3350 (x). Temperature was 37”. imrn434
late
I chl D
N
rex
c1
0 -
-
deleted
in
P
287
GENE OF PHAGE X
Q
functions I
S deleted
R
A in
F
J
m
chl A
SA 443
SA 307 -I
FIG. 2. Map of prophage lambda, showing genes and regions considered in these experiments.
1
IO MINUTES AFTER
ADDITION OF Tl
FIG. 3. Effect of prophage deletions
on the early lysis phenomenon. a--@ is strain DC506 = AM3100 (X c1857ind-). (>---a is strain S-4443 = DC506 deleted from x 0 to chZA. o---l3 is strain SA3Oi = DC506 deleted from chlD to x P. Temperature was 30”. The cl and ijtd mutations are irrelevant, for this experiment.
Fit,, !I Y
MINUTES
, , , IO AFTER
20 ADDITION OF Tl
FIG. 4. Effect of repressor on the kinetics of lysis when Tl was used to infect cells that had been infected 15 min previously by A. 0-0 represents cells infected with X zjir. A--A represent.s cells infected with X c60 O-O represents mockinfected control celIs.
MINUTES AFTER
I
I
I
20
30
40
ADDITION
OF Tl
FIG. 5. Effect
of rer mutations on the kinetics of lysis. O--O is strain W3350. a--O is strain W3350 Strr (X cZB5,). n---n is st.rain W3350 SF (X clss7rexj,). V-V is strain W3350 Sfr’ (X cZsj7rez30a). Temperature was 30”. The sir and cl mutations are irrelevant for this experiment.
that it allows t#he multiplication of T&II phage. Garen (1961) has shown that. high levels of ptIgl+ added to the medium will ovwcome the classical expression of rex: gene acCvitg, of T&II replication. i.e., the inhibition The addition of 0.05M RIg2f within 10 min after infection of lambda lysogens by T&II allows completely normal replication of the T&II to take place. To see whether early lysis by Tl could likewise be prevented by lug2+, we added MgSO, to a final concentration of 0.05 M 4 min after Tl infection of W3350 or of W3350 (A). In neit’her case did Rig”+ have any effect on the kinetics of lysis (data not shown). Either the rex effect being studied here is not reversed by lug?+, or the condit,ions that. lead to early lysis are already est#ablished within 4 min after Tl infection. In these experiments, iJIg”+ cannot be added to the cells before Tl infection, since the concentration used here inhibits Tl attachment almost completely (Puck et al., 1951).
PREMATURE
LYSIS
BY rez GENE
DISCUSSION
In all, lysis kinetics after Tl infection have been st’udied in 14 strains lysogenic for lambda, 2 nonlysogens, and in bacteria freshly infected with 2 strains of lambda. The correlation is complete: whenever there is an act#ive vex gene, there is early lysis, and when t’he rex gene is absent or mutant, Iysis is normal or nearly so. Any role for genes cl, N, 0, P, Q, or any late gene has been excluded. It seems likely that the effect depends solely on the expression of rex. The mechanism of action of the rex gene remains obscure. In the T&II-infected lambda lysogen, at least, t’here is evidence that the presence of rex has an effect at the membrane: level (Buller and Astrachan, 1968), but whether this effrct~ is direct or indirect and how it is relat,ed to the function of the rI1 gene is unclear. The control of lysis in a Tl-infected celI has not been elucidated, but it is not unlikely that two phage genes are involved, as is the case, for example, with T4 (e gene: Streisinger et u,Z.,1961; t gene, Josslin, 1970) and with lambda (R gene: de1 CampilloCampbell and Campbell, 1963; S gene: Harris et al., 1967). In each of these cases, the first gene mentioned codes for a lytic enzyme, act,ing on t,he cell wall, and the second presumably acts t,o modify the membrane in such a way as to allow the l!vsin t,o act (Josslin, 1971). It may be, then, that rex, act,ing directly or indirect,ly at t’he membrane level, can substitute for a Tl gene t,hat is required for lysis, so that a lambda lysogen is primed for lysis even before Tl infection and lysis occurs as soon as a sufficient amount of a Tl Iysin has been produced. In any case, the end result of rex action in Tl infection is quite different from t.he result in T&II infection, since in that case lysis does not occur at, all. ACKNOWLEDGMENTS Drs. L. Astrachan, A. Campbell, A. Doermann, H. Drexler, G. Gussin, G. Kellenberger, and K. Paigen were most generous in supplying phage and bact,erial strains for these experiments. Miss Lorraine Damaduk is thanked for terhnical assist-
OF PHAGE
X
289
ante. Supported in part by grants from the Public Health Service (AI 02781 to J. R. C. and GRSG to the University nf Rochester). REFERENCES ADHPA, S., and CAMPBELL, A. (1970). Crypticogenicity of bacteriophage X. J. 111ol. Biol. 50, 481-490. BULLER, C. S., and ASTRXH~N, L. (1968). Replication of T4rII bacteriophage in Escherichia. coli K-12 (x). J. viral. 2,29&307. CAMPBELL, A. (196lj. Sensit,ive mut,ants of bacteriophage X. T’irology 14, 2232. CHRISTENSEN, J. R., and TOLMACH, L. J. (1955). On the early stages of infection of Esch,erichin coli B by bacteriophage Tl. Arch. Bioch.em. Biophys. 57, 195-207. COURT, D., and C.mmF:LL, A. (1972). Gene regulat.ion in N mutant,s of bacteriophage X. J. I’irol. 9,938-945. DEL CAMPILLO-CAMPBELL, A., and CAMPBELL, A. (1963). Endolysin from mutants of bact.eriophage lambda. Biochem. 2. 342, 485691. DOVE, W. F., INOKUCHI, H., and STEVENS, W. F. (1971). Replication control in phage lambda. 112 “The Bacteriophage Lambda” (-4. I). Hershey, ed.), pp. 747-771. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. DREXLER, H. (1972). Transduct,ion of Gal+ by coliphage Tl. I. Role of hybrids of bacterial and prophage X deoopribonucleic acid. J. Viral. 9, 273-279. ELLIS, E. L., and DELBR~~CK, M. (1939). The growth of bacteriophage. J. GetI. Ph.ysiol. 22, 365-384. GAREN, 9. (1961). Physiological effect,s of rI1 mutations in bacteriophage T4. Virology 14, 151-163. GUSSIN, G. N., and PETERSON, V. (1972). Isolation and properties of re.z- mutants of bacberiophage lambda. J. J’iroZ. 10, 760-765. HARRIS, A. W., MOUNT, D. W. A., FUERST,~. R., and SIMINOVITCH, L. (1967). Mutations in bacteriophage lambda affecting host cell lysis. Virology 32, 553-569. JOS~LIN, R. (1970) The lysis mechanism of phage T4: Mutants affecting lysis. lz’rology 40, 719726. JOSSLIN, R. (1971). Physiological studies on the gene defect. in T4-infected Escherichia coli. Virology 44, 101-107. KAISER, A. (1957). Mutations in a temperate bacteriophage effecting its ability to lysogenize Ederichia co/i. Virology 3, 42-61. hlIcn.ILliE, W. (1967). Erhljte Rekombinationshaufigheit an den Enden des Tl-Chromosoms. Mol. Gerr. Gel/et. 99, 12-33.
290
CHRISTENSEN
J. S. (1968). Genetics of the left arm of t.he chromosome of bact,eriophage lambda. Genetics 59, 311325. PUCK, T. T., GAREN, A., and CLINE, J. (1951). The mechanism of virus attachment to host PARKINSON,
cells. I. The role of ions in the primary
reaction.
J. Exp. Med. 93,65-88. SATO,
K.,
and CAMPBELL,
A. (1970). Specialized
AND
(:EIhIAN
transduction of galactose by lambda phage from a deleted lysogen. T7iroloyy 41, 474-487. SLUR, P. D., and GMEN, -1. (1956). The orientation and extent of gene transfer in Escherichia coli. Proc. :Yat. dcacl. Sci. C’S, 42, 6194624. STREISINGER, C;., hIvIi.11, F., URETER, W. J., MILLER, B., and HORIUCHI, S. (1961). Mutations atlecting the lysozyme of phage T4. c’old Spriug Harbor Symp. Quant. Biot. 26, 2530.
66,
285-290
A New
(1973)
Effect
of the rex after
of Microbiology,
of Phage
Infection
J. R. CHRISTENSEN Depa.rtment
Gene
by Phage JOYCE
AND
A: Premature Tl
M. GEIMAN
University of Rochester, School of Medicine Rochester, New York 14642 Accepted August
Lysis
and Dentistry,
8, 197.9
When phage Tl infects bacteria lysogenic for lambda, lysis is premature and burst sizes are small. This has been shown to be an effect of the lambda rez gene. INTRODUCTION
It was inadvertent,ly discovered in this laboratory that when phage Tl infects hosts that are lysogenic for lambda phage, small burst sizes are obtained. This has been found to be correlated with a significantly reduced latent period in such hosts. We have used a variety of mutant lambda phages to determine what gene or genes are responsible for this effect. MATERIALS
AND
METHODS
Phage. X was from I<. Paigen, X vir from G. Kellenberger, and X ~60 from L. Astrathan. X gOn12g3 was isolated from a C600 lysogen sent by A. Campbell. Bacteria. Table 1 lists the bacterial strains used in t,hese experiments, and their sources. Experimental procedures, The dat,a in Table 1 are derived from a one-step growth curve, modeled after Ellis and Delbriick (1939). The cells were grown in nutrient broth, infected with a low multiplicity of infection (m.o.i.) of Tl in 5 X 10-d M hlgC12, and dilubed into nutrient broth at 37” at time zero. For the experiment in Fig. 3, bacteria were grown with shaking at 37” in tryptone broth plus maltose (1.0 % tryptone, 0.5 % NaCl, 0.1 c’; malt,ose) to approximately 2 X lo* cells per ml. hZgC12 was t#hen added to a final concentration of 0.01 M, and lo-ml aliquots were either infected with one of the lambda stocks or mockinfected by addition of tryptone broth.
Aft,er a 15-min attachment period at 37”, the suspensions were chilled and centrifuged, and the supernat,ant, fluids were saved for assay to determine t’he extent of lambda attachment. The cell pellets were resuspended in the same volume of warm (37”) tryptone wit,hout NaCI? transferred t,o Nephelo flasks (Bellco), and infected with Tl at an m.o.i. of approximately 10. Lysis kinetics were followed by periodic readings in a Klett calorimeter (blue filter). For all other experiments, bacteria were grown with shaking in nutrient brot,h directly in hhe Nephelo flask unt,il a Klett reading of 50-60 was obtained (approximately 2 to 3 X lOa cells/ml). The cells were then infected with Tl and t,he lysis kinetics followed as indicated above. In experiments involving strains lysogenic for prophages with either the cItl or cIs57 mutation, all operations were carried out at 30”; otherwise the temperature was 37”. RESULTS
Typical results presented in Table 2 and in Fig. 1 show the reduced burst size and the early lysis when Tl infects W3350 (X) as compared to the results in W3350. We assume that the reduced burst size is a direct result of the early lysis (Christensen and Tolmach, 1955, Fig. 3). We have used lysis kinetics as the indicator of the phenomenon in all that follows, since these kinetics are more consistent from experiment to experiment than are burst sizes. Early lysis
285 Copyright .%ll rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
286
CHRISTENSEN
AND GEIRlA~
TABLE B.WTERIIL
1
STRMNS USED
Reference
Strain
Skaar and Garen (195Aj PIIichalke (1967) Parkinson (1968) Prepared in this laborator) Campbell (1961) Prepared in this laborat.orJ Drexler (19723” Drexler (1972)” Gussin and Peterson (1972j* Gussin and Peterson (1972)* Gussin and Peterson (1972)* .\dhya and Campbell (1970jr Adhya and Campbell (1970jc Court and Campbell (1972) Dove el al. (1971) Sato and Campbell (1970)+
CSlOO KB3 KB3 (hcZt,wsJn~sj KB3 (Xgonrtstj w3350 w3350 (A) W3350 (hcZtss,sztsN7szlsN~3) w3350 (A SUSQ,3) B219 = W3350 Strr (X CZSU) B235 = W3350 Strr (A cIdez& B189 = W3350 Strr (A czes;rer5a) R734 = W3350 (su~~P~sILsN~susA,,sILsR~~susF~~~~~~~~?~~~~~ R735 = W3350 (susP~susN~s~~sA~,susR~~susF~~~~~~~X) DC50B = AM3100 (X czs5+kd-j SA443 = DC506 deleted from X0 to chlA SA307 = DC506 deleted from chlD to XP
a This reference describes strain KB3 lysogenic for the same prophage. Dr. Drexler prepared t,he W3350 lysogens. b Designated in this reference as a lysogenic derivative of strain 549 (= W3350 StrI). Present designation is from Dr. Gussin (personal communication). c Designated by full genot,ype in this reference. Present designation is from Dr. Campbell (personal communication). d Designated in this reference as strain 307. Present designat,ion is from Dr. Campbell (personal communicationj. TABLE REDUCED Tl PUGE
YIELD
2
ON INFECTION OF A ~LYSOGENIC
Tl phage attached
Host cells Strain
Cells,i’ml
w3350 w3350 (A)
1.6 x 108 1.3 x 108
Tl/ml 1.5 x 109 1.2 x 109
u The 30-min values were approximately
m.0.i. 9.5 9.3
HOST
Tl phage yield at 30 mina Tl/ml 1.6 X lOI0 2.2 x 109
Burst size 100 17
the same as 20-min values.
was also seen in strain KB3 (X cItl SUSJ), but not in IIB3, and also in strain CSlOO, which is a lambda lysogen (data not shown). To begin to define which genes of the prophage are responsible for the effect, experiments were carried out with strains in which large blocks of the prophage had been deleted (see Fig. 2). The results presented in Fig. 3 demonstrate that the block of genes to the left of P in the prophage map are sufficient for the effect (strain SA443 lyses as quickly as strain DC506, which carries a full prophage), but that genes to the right of 0 are dispensable
(st#rain SA307 shows “delayed,” i.e., normal, lysis). This finding rules out the participation, through t,ransactivation, of the lysis genes R and S of lambda, or of any other late genes, in producing early lysis. It may be noted that in comparing strains we have focused our attention on t,he time of initiation of lysis and on the position of the st,eep, earl2: part of the lysis curve. The residual turbidity measured near the end of the lysis curves is based on low Klett readings, 15 or less in most cases, and these data are more subject to experimental errors, such as imperfectly matched Nephelo flasks.
PREMATURE
LYSIS BY
Tt?X
Differences seen between strains in this region of the curve t,ended to be irreproducible. Early lysis as compared with that seen in W3350 was also seen after infection of W3350 (A cIsS7 SUSN, SUSN~B)and W3350 (X .s~&&), ruling out, any participation of the control genes N and Q in the phenomenon (data not shown). The search was further narrowed down to the immunit,y region of lambda by the finding that early lgsis was obt,ained aft,er infection of strain R735, but not after infection of strain R7.74, which is isogenic except that the immunity region of the prophage in the latter strain was derived from phage 434 (data not shown).
I
I
IO MINUTES AFTER
Having ruled out, transactivation of late genes as being involved in the phenomenon, and having shown the importance of the immunity region, it was logical to examine the role of genes cl and rez, parGcularly since they are t,he only genes known to be active in t,he prophage st,ate. We first tried to test the effect of cl by using lysogens carrying prophages with the cItl or cIssi mutation which code for temperature-sensitive repressors. However, heating nonlysogenic control cultSures before Tl infection produced a significant delay in lysis, and so this approach was abandoned. We then turned t,o infect,ing the host bact’eria with lambda mutants 15 min before infection with Tl. In order t.o test the direct effect of cl on lgsis, we chose to use X ce,,, which has a mutat,ion in CI (Kaiser, 1957), and X vZr, which makes normal repressor, but, which is insensitive to repressor action. If repressor were responsible for early lysis, infection by X vir, but not by X ceo, should produce the effect,. In fact, after pre-infection by either phage, Tl lysis is earlier than in a mockinfected control (Fig. 4). Repressor, t#hen, is not required for early lysis. To test the role of the rex gene, st,rains lysogenic for phage mutant in rex were compared with nonlysogenic controls and with strains lysogenic for the rex+ count,erpart, of the mutants. Results with two strains are seen in Fig. 5, and it, is clear that the rex mutations eliminate early lysis. Experiments were also conducted with st#rain KB3 (X gon&, (“go” is an old name for rex). With this strain the lysis curve showed a very slight early lysis, but the kinet,ics were much more like those seen with KB3 than with IiB3 (X cItl sud) (data not shown). Perhaps the go1\12g3 mutation is slightly leaky, despite the fact
I
20 ADDITION
30 OF Tl
FIG. 1. Kinet,ics of lysis when Tl infects a lysogen and a nonlysogenic control. O--O is strain W3350. a----• is strain W3350 (x). Temperature was 37”. imrn434
late
I chl D
N
rex
c1
0 -
-
deleted
in
P
287
GENE OF PHAGE X
Q
functions I
S deleted
R
A in
F
J
m
chl A
SA 443
SA 307 -I
FIG. 2. Map of prophage lambda, showing genes and regions considered in these experiments.
1
IO MINUTES AFTER
ADDITION OF Tl
FIG. 3. Effect of prophage deletions
on the early lysis phenomenon. a--@ is strain DC506 = AM3100 (X c1857ind-). (>---a is strain S-4443 = DC506 deleted from x 0 to chZA. o---l3 is strain SA3Oi = DC506 deleted from chlD to x P. Temperature was 30”. The cl and ijtd mutations are irrelevant, for this experiment.
Fit,, !I Y
MINUTES
, , , IO AFTER
20 ADDITION OF Tl
FIG. 4. Effect of repressor on the kinetics of lysis when Tl was used to infect cells that had been infected 15 min previously by A. 0-0 represents cells infected with X zjir. A--A represent.s cells infected with X c60 O-O represents mockinfected control celIs.
MINUTES AFTER
I
I
I
20
30
40
ADDITION
OF Tl
FIG. 5. Effect
of rer mutations on the kinetics of lysis. O--O is strain W3350. a--O is strain W3350 Strr (X cZB5,). n---n is st.rain W3350 SF (X clss7rexj,). V-V is strain W3350 Sfr’ (X cZsj7rez30a). Temperature was 30”. The sir and cl mutations are irrelevant for this experiment.
that it allows t#he multiplication of T&II phage. Garen (1961) has shown that. high levels of ptIgl+ added to the medium will ovwcome the classical expression of rex: gene acCvitg, of T&II replication. i.e., the inhibition The addition of 0.05M RIg2f within 10 min after infection of lambda lysogens by T&II allows completely normal replication of the T&II to take place. To see whether early lysis by Tl could likewise be prevented by lug2+, we added MgSO, to a final concentration of 0.05 M 4 min after Tl infection of W3350 or of W3350 (A). In neit’her case did Rig”+ have any effect on the kinetics of lysis (data not shown). Either the rex effect being studied here is not reversed by lug?+, or the condit,ions that. lead to early lysis are already est#ablished within 4 min after Tl infection. In these experiments, iJIg”+ cannot be added to the cells before Tl infection, since the concentration used here inhibits Tl attachment almost completely (Puck et al., 1951).
PREMATURE
LYSIS
BY rez GENE
DISCUSSION
In all, lysis kinetics after Tl infection have been st’udied in 14 strains lysogenic for lambda, 2 nonlysogens, and in bacteria freshly infected with 2 strains of lambda. The correlation is complete: whenever there is an act#ive vex gene, there is early lysis, and when t’he rex gene is absent or mutant, Iysis is normal or nearly so. Any role for genes cl, N, 0, P, Q, or any late gene has been excluded. It seems likely that the effect depends solely on the expression of rex. The mechanism of action of the rex gene remains obscure. In the T&II-infected lambda lysogen, at least, t’here is evidence that the presence of rex has an effect at the membrane: level (Buller and Astrachan, 1968), but whether this effrct~ is direct or indirect and how it is relat,ed to the function of the rI1 gene is unclear. The control of lysis in a Tl-infected celI has not been elucidated, but it is not unlikely that two phage genes are involved, as is the case, for example, with T4 (e gene: Streisinger et u,Z.,1961; t gene, Josslin, 1970) and with lambda (R gene: de1 CampilloCampbell and Campbell, 1963; S gene: Harris et al., 1967). In each of these cases, the first gene mentioned codes for a lytic enzyme, act,ing on t,he cell wall, and the second presumably acts t,o modify the membrane in such a way as to allow the l!vsin t,o act (Josslin, 1971). It may be, then, that rex, act,ing directly or indirect,ly at t’he membrane level, can substitute for a Tl gene t,hat is required for lysis, so that a lambda lysogen is primed for lysis even before Tl infection and lysis occurs as soon as a sufficient amount of a Tl Iysin has been produced. In any case, the end result of rex action in Tl infection is quite different from t.he result in T&II infection, since in that case lysis does not occur at, all. ACKNOWLEDGMENTS Drs. L. Astrachan, A. Campbell, A. Doermann, H. Drexler, G. Gussin, G. Kellenberger, and K. Paigen were most generous in supplying phage and bact,erial strains for these experiments. Miss Lorraine Damaduk is thanked for terhnical assist-
OF PHAGE
X
289
ante. Supported in part by grants from the Public Health Service (AI 02781 to J. R. C. and GRSG to the University nf Rochester). REFERENCES ADHPA, S., and CAMPBELL, A. (1970). Crypticogenicity of bacteriophage X. J. 111ol. Biol. 50, 481-490. BULLER, C. S., and ASTRXH~N, L. (1968). Replication of T4rII bacteriophage in Escherichia. coli K-12 (x). J. viral. 2,29&307. CAMPBELL, A. (196lj. Sensit,ive mut,ants of bacteriophage X. T’irology 14, 2232. CHRISTENSEN, J. R., and TOLMACH, L. J. (1955). On the early stages of infection of Esch,erichin coli B by bacteriophage Tl. Arch. Bioch.em. Biophys. 57, 195-207. COURT, D., and C.mmF:LL, A. (1972). Gene regulat.ion in N mutant,s of bacteriophage X. J. I’irol. 9,938-945. DEL CAMPILLO-CAMPBELL, A., and CAMPBELL, A. (1963). Endolysin from mutants of bact.eriophage lambda. Biochem. 2. 342, 485691. DOVE, W. F., INOKUCHI, H., and STEVENS, W. F. (1971). Replication control in phage lambda. 112 “The Bacteriophage Lambda” (-4. I). Hershey, ed.), pp. 747-771. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. DREXLER, H. (1972). Transduct,ion of Gal+ by coliphage Tl. I. Role of hybrids of bacterial and prophage X deoopribonucleic acid. J. Viral. 9, 273-279. ELLIS, E. L., and DELBR~~CK, M. (1939). The growth of bacteriophage. J. GetI. Ph.ysiol. 22, 365-384. GAREN, 9. (1961). Physiological effect,s of rI1 mutations in bacteriophage T4. Virology 14, 151-163. GUSSIN, G. N., and PETERSON, V. (1972). Isolation and properties of re.z- mutants of bacberiophage lambda. J. J’iroZ. 10, 760-765. HARRIS, A. W., MOUNT, D. W. A., FUERST,~. R., and SIMINOVITCH, L. (1967). Mutations in bacteriophage lambda affecting host cell lysis. Virology 32, 553-569. JOS~LIN, R. (1970) The lysis mechanism of phage T4: Mutants affecting lysis. lz’rology 40, 719726. JOSSLIN, R. (1971). Physiological studies on the gene defect. in T4-infected Escherichia coli. Virology 44, 101-107. KAISER, A. (1957). Mutations in a temperate bacteriophage effecting its ability to lysogenize Ederichia co/i. Virology 3, 42-61. hlIcn.ILliE, W. (1967). Erhljte Rekombinationshaufigheit an den Enden des Tl-Chromosoms. Mol. Gerr. Gel/et. 99, 12-33.
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transduction of galactose by lambda phage from a deleted lysogen. T7iroloyy 41, 474-487. SLUR, P. D., and GMEN, -1. (1956). The orientation and extent of gene transfer in Escherichia coli. Proc. :Yat. dcacl. Sci. C’S, 42, 6194624. STREISINGER, C;., hIvIi.11, F., URETER, W. J., MILLER, B., and HORIUCHI, S. (1961). Mutations atlecting the lysozyme of phage T4. c’old Spriug Harbor Symp. Quant. Biot. 26, 2530.