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Inheritance of prophage P2 in superinfection experiments
VIROLOGY
14, 229-233 (1961)
Inheritance
of Prophage
P2 in Superinfection ERICH
Department
of Bacteriology, Accepted
SIX
University
State
Experiments’
of Iowa,
Iowa City,
Iowa
February 24, 1961
Genetically marked P2 phage was used to superinfect cells of Escherichia coli (or Shigella dysenteriae) which were lysogenic for P2 and hence immune to the superinfecting phage. The genetic incorporation of the superinfecting P2 was studied by examining the progeny of the superinfected cells. The results obtained are in agreement with the previously established rule that genetic incorporation occurs most frequently at the preferred location (I) either by prophage addition or by prophage substitution. Besides complete substitution, partial (= single marker) substitution was also observed, but at a lower frequency than complete substitution. The frequency of genetic incorporation was found to increase linearly with the multiplicity of superinfection for small multiplicities. The probability of genetic incorporation of a superinfecting phage is decreased by the presence of a prophage in location I; this hindrance does not depend on the immune specificity of such prophage. It is assumed that in immune cells multiplication of prophage precursors (= preprophages) is prevented. The probability of genetic incorporation of a preprophage in the absence of steric hindrance (5%) compares well with the probabilities found in other systems for the genetic incorporation of bacterial markers in transduction experiments. Weak virulent mutants of P2 and P2 Hy dis can become prophages, but only in cells already lysogenic for a temperate phage of the same immunity class. INTRODUCTION
The noninducible temperate phage P2 and its relationship to its normal hosts Escherichia coli strain C and Shigella dysenteriae strain Sh have been studied for several years (see Bertani, 1958). These investigations have led to the following conclusions: ‘The work described in this paper was begun at the Division of Biology, California Institute of Technology and was continued at the following institutions : Department of Medical Microbiology, University of Southern California; Max-PlanckInstitut fur Biologie (Tubingen, Germany), and Department of Biology, University of Rochester. Parts of this work were done under a fellowship of the Deutsche Forschungsgemeinschaft and later under a fellowship of the Max-Planck-Gesellschaft. Further support was received from a grant of the National Foundation for Infantile Paralysis, from National Science Foundation grant G-3841, and from United States Public Health Service grants E-1839 and E-2862.
(1) P2 can become attached as a prophage at any one of several sites (I, II, etc.) of the bacterial chromosome. For E. coli C, these sites can be recognized and separated by means of bacterial crosses (Bertani and Six, 1958). (2) If a nonlysogenic cell is infected with P2, one site on the bacterial chromosome (location I) is preferred for the attachment of P2 as a prophage. (3) Superinfection of such lysogenic cells with P2 leads, with a rather small probability, to the establishment of the superinfecting phage as prophage, either by replacing a pre-existing prophage in location I (= prophage subsitution) or by attaching at a previously empty site (= prophage addition or double lysogenization) . Under these conditions, substitution is more frequent than double lysogenization. (4) A temperate “dismune” variant of P2
220
SUPERINFECTION
EXPERIMENTS
was found, which belongs to an immunity class different from that of P2 and its plaque-type mutants (G. Bertani, 1957). It was shown by Cohen (1959) that this dismune phage results from a genetic interaction between P2 and coli B ; the variant phage was designated P2 Hy dis. (5) The same attachment site in coli C (location I) that is preferred by P2 is also preferred by P2 Hy dis (Bertani and Six, 1958; Six, 1960). The purpose of this investigation is to gain more insight into the process of genetic incorporation of a superinfecting phage in cells that carry one (or more) prophages belonging to the same immunity class as the superinfecting phage and hence are immune to the superinfecting phage. In particular, the following questions were asked: (a) How does the multiplicity of the superinfecting phage affect the frequency of genetic incorporation? (b) How do the prophages present in the superinfected cell affect the frequency of genetic incorporation of the superinfecting phage?
WITH
221
P2
judgment, limited in its validity by the number of detectable genetic markers. Since the number of available P2 markers is small, it shall not be implied that complete and partial genetic incorporation are necessarily two different processes. A “probability of genetic incorporation” will be introduced, defined as: g = G/M This definition is, of course, useful only if G is proportional to M, and assumption which for small M is supported by the experimental findings reported in this paper (generally, one could define g = dG/dM) . The probability g can be interpreted in the following way:
g = 4W)a’ M
= Wv)g’
where g’ is the probability for a superinfecting phage to become incorporated into the genome of one bacterial nucleus within one generation time; v is the number of nuclei per cell, probably v = 2. OL is a parameter which depends on the fate of the superinfecting phage in the bacteria: If geDefinition and Interpretation of netic incorporation can only happen imSome Terms mediately after superinfection, then OL= 1. The two primary parameters that are If the superinfecting phage-prior to its measured in the superinfection experiments genetic incorporation-enters a state (as to be described are: suggested by Bertani, 1953, 1954) where it M, the multiplicity of superinfection = is carried without multiplication or loss average number of superinfecting phages (being diluted out), and if the probability per cell. of genetic incorporation per generation cycle G, the frequency of genetic incorporation is constant = g’, then a = 2. A value Q > 2 of the superinfecting phage, i.e., the fre- would be expected if some multiplication quency, among the progeny of the super- occurred prior to genetic incorporation. infected cells, of cells carrying a prophage Since no attempt will be made to measure CL newly introduced by superinfection (de- or V, g may be taken as a convenient estermined after a sufficient number of timate for g’. growth cycles has been allowed to give a The probability of genetic incorporation rather complete segregation with respect as defined this way (as g) must not be conto the new prophage). If the new pro- fused with the probability that a superinphage apears to be genetically identical fecting phage will be finally incorporated with the superinfecting phage, the genetic into Some bacterial genome. The latter incorporation is termed “complete.” “Parquantity may be 1 when g’ < 1, namely in tial” incorporation is the case in which the case of “slow” incorporation due to the not all markers (but at least one) of the “diluting out” of the preprophage. The value superinfecting phage appear in the new I/g (or l/g’) measures then the average prophage. It should be noted, however, number of bacterial growth cycles after that the completeness of genetic incorpo- which genetic incorporation occurs. G and ration always rests on rather arbitrary g with the subscripts A, S, or P are used to
222
ERICH
SIX
described before (Bertani and Six, 1958; Six, 1959) ; other are listed in Table 1. Testing for prophages carried by lysogenie cells was done by one of the following methods : (1) Assay of a growing culture for the free phage by plating with a suitable indicator strain. If the lysogenic cells were streptomycin sensitive, they were selectively killed by adding streptomycin upon plating (“streptomycin method,” see Bertani, 1951). If the cells were streptomycin resistant, they were selectively killed by heating the culture for 20 minutes at 58” (see Bertani and Six, 1958). (2) Immunity assay by spotting a loopful of the growjng lysogenic culture on a L agar plate see&i Jith L, suitable virulent phage. This had to be done (using P2 Hyl dis vir14) in order to detect the presence of P2 Hyl dis prophage in streptomycin-resistant cells, since the heat sensitivity of P2 Hyl dis (Cohen, 1959) did not allow application of the 58” treatment. Superinfection experiments. (1) Superin fection was carried out in mass culture Many of these experiments were done as described by Bertani (1954) : the lysogenic cells were grown in L broth to reach 5 x 107/ml and superinfected at 37” in L broth containing 5 x 10W3M CaCIz . An adsorption period of 10-20 minutes was allowed to assure adsorption of more than 50% of the phage input. Then the cells were diluted l:lOO, with L broth containing a sufficient
denote the type of genetic incorporation: A for addition of the new prophage without loss of the old one(s) , S for prophage substitution (complete substitution if distinguishable from partial substitution), P for partial substitution. Phages and prophages belonging to the same immunity class are called “homoimmune.” Those belonging to different immunity classes “heteroimmune” (Six, 1959 j . MATERIALS
AND
METHODS
Phages. The temperate phage P2, its plaque-type mutants (markers: rd = round; 1 = large; c = clear) ; and its virulent mutants: virl (= weak virulent), vir” (= strong virulent 1 have been described before, together with the general techniques and the media employed in the study of P2-like phages (G. Bertani, 1954.. 1957). Furthermore, P2 Hyl dis, a phage heteroimmune with respect to P2 :dnd a weak virulent mutant, P2 Hyl dis vi@* have ‘already been described (Cohen, 1959). Bacteria. (1 j .‘CP the plating bacterium the nonlysogenic, streptomycin-resistant indicator strain Sh/s (described before: Bertani, 1951) was :-ed as well as two lyso‘q: Sh-76, carrying genie derivatives c P2 Hy’ dis, and Sh-, , v *rying P2 as prophage. (2) All lysogenic !bi?lins used for the superinfection experiments were derivatives of either E. coli strain t’: or of S. dysenteriae strain Sh. Many of these strains have been TABLE LIST Strain Sh-36 Cb C-86 C-208 C-216 C-217
Lysogenic
OF STRAINS
NOT
state
1
PREVIOUSLY Origin
Remarks
-
(P2 rd hz)
(WI (-)1&‘2
rd 1)~ (P2 rd 1)1 (P2 Hyl dis)1(P2 (P2 Hyl dish(P2
DESCRIBEDU
Lysogenization Cross c-55 x Partial curing Superinfection Superinfection
rd Z)U rd 1)~
a By Bertani and Six (1958) or sensitive. b C-55 = C(P2)1 (P2 rd Z)n , Ftained by G. Bertani (unpublished). c P2 Hy* dis is like P2 Hy’ dis, B. It belongs to the same immunity It is probably a recurrence of the
Six (1959). strain
All
derived
strains from
of C-2 C-66* of C-71 of C-86 of C-86 listed
C-53
here
hz = hazy (for experiment
no.
(for (for (for
experiment experiments experiments
no. 16) nos. 12, 13) nos. 12, 13)e
prototrophic,
streptomycin
are F+,
by superinfection.
C-55
4)
and C-86
were
ob1
as it was also obtained by interaction between P2 and E. coli strain class as P2 Hyl dis. On Sh/s it gives larger plaques than P2 Hyl dis. character “large” mentioned by Cohen (1959).
SUPERINFECTION
EXPERIMENTS
amount of anti-P2 serum to neutralize unadsorbed phage and 5 x 10d3 M Caf + in order to prevent desorption. The cells were then grown at 37” in aerated L broth, keeping the titer below 5 X 107/ml, by diluting with fresh medium, until a sufficient number of generations (about 10) had elapsed. Streaks were then made on nutrient agar plates from which colonies were picked later for investigation of their lysogenic state (L’colony assay method”). If these isolates were found to be lysogenie for the phage type used for superinfection, it was assumed in general that a stable incorporation of the superinfecting phage had occurred. In some instances exceptional doubly lysogenic isolates were observed, which liberatedI’ the’? i.tiw phage type at a lower frequency than could be expected for stable doubly lysogenic clones. These isolates were restreaked and up to ten subcloneswere retested. These subclones often included some that no longer carried the new prophage. Subclones from doubly ,ysogenic isolates liberating the new phage Lype with the expected frequency showed no such segregation. (2j Since the low frequency of prophage substitution in cells superinfected with homoimmune phages necessitates the isolation and testing of a large number of colonies derived from a superinfected culture, the frequency of genetic incorporation of the superinfecting phage was in some cases estimated simply by measuring the frequency of the superinfecting phage type among the total free phage in a growing culture of superinfected cells (“free-phage assay method”). The standard procedure was as described under (I), except that (in experiments 1 to 5) the cell titer in the growth tubes was kept between 8 x lo* and 9 x 106/ml in order to minimize reinfection. Finally, flasks containing 100 ml L broth were inoculated with 2 x lo7 cells, and these cultures were grown again at 37”, but unaerated, to reach 5 x 106/ml. Then the cells were killed by adding streptomycin and the culture was assayed for free phage. Even with a final cell number of 5 x 10s in t.he flasks, sampling errors should become critical if the frequency of prophage incorporation is as low as 10e3, since the rate
WITH
P2
223
of spontaneous lysis is about 2 x lo-” per cell per generation time (Six, 1959). No special precautions had to be taken (in experiments nos. 9 and 10) when the expected frequencies of incorporation were much higher. For experiments nos. 9 to 18 no anti-P2 serum was available. Hence, the cells were diluted from the adsorption tube into L broth and grown to reach 5 x 107/ml. Then they were washed once in order to reduce the free-phage titer by a factor of 30 and then diluted into LS broth. LS broth was then used as growth medium in order to reduce the chances for reinfection. The multiplicity of superinfection was calculated from the phage input and the cell input applying the following dorrections: (1) The phage input was corrected for the percentage of unadsorbed phage, either determined from assays of free phage or assumedto be ,.O%. (2) The cell titer as determined by colony counts was multiplied by aafactor of 1.4 in order to take into accouo~:the existence of cell aggregates (doublets, triplets, etc.) in the culture (Six, 1959). The streptomycin UWA was dihydrostreptomycin sulfate, eitl,. ti+orn Eli Lilly and Company or a gift. tirorn the Farbwerke Hochst. The chlorar .:henicol used was a gift from Parke, Dav s,and Company. For ultraviolet irazdiation the bacteria were washed and resuspendedin chilled saline and placed under a 15-watt GE “germicidal” tube (effective wavelength 254 rnp) . Photoreactivation was minimized by use of dim yellow light during the irradiation and for 90 minutes afterward. Media. Unless otherwise mentioned, the bacteria were grown in nutrient broth. L broth contains 10 g Bacto-Tryptone (Difco), 5 g Bacto-Yeast-Extract (Difco), 10 g NaCl, and 1 g glucose per liter of water. LS broth has the same composition except that it contains only 2 g of NaCl. RESULTS
A. Superinfection of Cells Carrying a Prophage in Location I Because of the preference of P2 for location I, in these experiments substitution of the prophage in location I is expected to be
ERICH
SIX
0 20
FIG. 1. Experiment no. 1. The frequency of prophage substitution G, vs. the multiplicity of superinfection M for Sh-36 = Sh(P2 rd hz), superinfected with P2 wild type. C = superinfection in the presence of chloramphenicol(5 X 10“ g/ml). Free phage assay 1 day after the sunerinfection experiments. The curve inserted at the right represents the initial part of the lamer curve on a-different scale.
FIG. 2. Spontaneous inactivation of free phages in experiment no. 2. t = Time of phage assay (t = 0 : streptomycin added to growing cultures). Between assays the cultures were kept in a refrigerator. Average values of the two cultures for M = 10.3. The total titer is practically identical with the P2 rd 1 c titer. The P2 titer for t = 0 was extrapolated from the four measurements at t > 0. 0: P2rdZc; x: P2.
the most likely process. Only “free-phage assays” were done; results are interpreted in terms of prophage substitution, assuming that the addition of a new prophage OCcurred at a negligible frequency. The main purpose of these experiments in
(A) was to measure Gs , the frequency of prophage substitution, as a’function of M, the multiplicity of the superinfecting phage and to calculate the probability of prophage substitution (gs) . The influence of such variables as the genetic markers of the phage, the presence of a second prophage, and the treatment with chloramphenicol or ultraviolet light on the frequency of prophage substitution was explored. The findings are summarized in Figs. 1 and 3 and in Table 2. They show that for superinfection of Sh cells (Fig. 1, experiment no. 1) as well as of C cells (Fig. 3, experiments nos. 2-7) Gs increases linearly with M for M < 10, leveling off at higher multiplicities. The linearity of the increase justifies the use of the ‘parameter gs . The causes of the final leveling off remain unknown. One reason may be a selective death of cells superinfected with more than some critical number of phages, Free-phage assays in experiment no. 2 (superinfection of C-27 = C (P2 rd 1 c) r with P21 wild type) revealed ‘a slow spontaneous inactivation of both phage types: P2 rd 1 c and ‘P2 wild type (Fig. 2). During the fir-St 40 hours the titer of the P2 rd 1 c phage decreased faster
SUPERINFECTION
0
2
4
6
8
EXPERIMENTS
IO
I2
WITH
I4
I6
P2
I8
225
20
22
24
M FIG. 3. The frequency of prophage substitution G, vs. the multiplicity of superinfection M for different lysogenic strains superinfected with P2 (free phage assay). Solid line: X: Experiment no. 2 (C-27) ; A : Experiment no. 4 (Cb) ; 0 : Experiment no. 5 (C-27) ; l : Experiment no. 5 (C-59); 0 : Experiment no. 6 (C-27, unirradiated). Broken line: f: Experiment no. 2 (C-27, superinfected in the presence of chloramphenicol, 25 X lo-’ g/ml).
than that of the wild-type phage, while after 40 hours the titers for both phage types decreased at about the same rate. This differential decay of P2 rd 1 c may increase the observed ratio of P2: P2 rd 1 c by a factor of almost 4.5. In the other experiments somewhat different inactivation rates were observed, but P2 wild type was always more stable than P2 rd 2 c. Since a comparable differential decay was also found for P2 rd hz as compared to P2, it is assumedthat the rd marker is responsible for the lower stability of the phages; therefore, the recombinant P2 rd I c+ should show the same stability as P2 rd 1 c, and P2 rd+ Z+ c the same as P2 wild type. Except for experiment no. 1, all results given are corrected for the differential rd decay as observed for the P2 rd 1 c phage. In experiment no. 1, the free-phage assay was done several hours after the 1ysogeni.c cells had been killed with streptomydin. Since at that time it was not known that rd phages are more unstable than rd+, the
measured Gs values may be too high (at all M values tested) by a factor of about 2. For this reason, the gs value was not calculated for this experiment. For the prophage substitution in singly lysogenic strains of E. coli C, a gs value of 1.2 x 1O-3 per phage ‘is obtained from the data of experiments nos. 2, 3, 5, 6, and 7 as plotted in Fig. 3. This value represents a mean of the corresponding values compiled in Table 2. In experiment no. 3, Gs was determined for a Sh strain comparable to C-27: Sh-74 = Sh (P2 rd I c) superinfected with P2 wild type. The value obtained for gs is about 4 times as high as for the C strain. No significant difference in the Gs values was observed whether the cells were grown in the presence or in the absenceof anti-P2 serum. While in most experiments strain C-27 = C(P2 rd 2 c) was superinfected with P2 wild type, the markers were reversed in experiment no. 4: C (P2)r was superinfected with P2 rd Z c. The gs value was not sig-
226
ERICH
-
Expt. no.
RENJLTS
T Strain I -C-27
OF
TABLE 2 SUPERINFECTION EXPERIMENTS,
-
Remarks
t
rd I C)I
3
Sh-74
--
N I
____
+ Chloramphenicol
-_
M I
~_ = C(P2
SIX
I
GROCP -
- -
Ns PJ PP
rd 1 c)
43 1.4 1933 20 2.751701 20 5.4 1524 2010.3 2082
8 9 16 29
43 1.4 2347 20 2.751825 20 5.4 1315 2010.3 1177
5 10 18 33
0 7.6
1011
1 i 0 3
i: 6
: ;
37
; 1 2
4
-4
4
3
0 -116 I 7.6
4 5
-Cb -C-27
No anti-P2
_-
= C(P2)r (no correction for rd decay)
6 __7
-C-27 -C-27 -
2
(: -1
23
2
2
38
3
1
11
2
0
17
2
0
21
4
2
--
rd 1 c)r -90
C-59
’3684 I
serum 0
= C(P2
= C(P2 rd 1 c)r (P2 rd 1 C)III
_1unirradiated UV; surv.
50 Set = 0.30
WI Set UV; survivorsc = 1.5 x .10-s
6.3 011.7 -90 11.7 023.6 -90 23.6
7020 2603 7764 2406 6033
0 4.5 4.5 0 8.9 -90 8.9 0 18 0 -90 18
2313 5915 1959 4925 1969 5315
0 0.9 0 0.9
4391’ 492
4 4
0 0.9
1686
14
-90
_-
----
I
I
-
03 x g .03x g,‘A
103
x
RPB
-~
l= Sh(P2
i PI
Aa. *
0”
_- --
:
_ii
0 1 Ii
1.2 1.2 1.25 0.9
0.3 0.3
E
i::
0:4 0.3
::;
0.1 0.5 0.2 0.1
E 0:1 0.2
4.8 5.0
0.5 0.3
0.5 0.3
::3’
0”::5 -__.
0.6 1.3
1.4 1.4
::; 0.8 0.9 0.7 0.7 1.3 1.3 :.; 0:7 0.9 1.1 9.5 --9.1
n0::
---
0.3 0.25 0.3 0.0
0.14 0.06 0.07 0.06 0.06 0.04
0.07 0.07 0.07 0.03 0.01 0.04
0.24 0.15 0.14 0.10 0.14 0.10
00::4 0.0 0.0 0.07 0.04
0.27 9.5 2.3
-l--
0.0 2.4 7.1
I
- a Symbols: nG : number of generation times elapsed between superinfection and addition of streptomycin t : time in hours when free phage assay was done after cessation of growth M : multiplicity of superinfection N: total number of plaques scored Ns : number of plaques revealing total prophage substitution NP* : number of plaques revealing partial prophage substitution (loci rd and 1 together) Npn : number of plaques revealing partial prophage substitution (locus c only) gs : probability of total prophage substitution gp, : probability of substitution of loci rd and 1 gps : probability of substitution of locus c b 911 g-values (except for experiment no. 4) are corrected for r&decay, if t > 0. The superinfecting phage was wild-type P2 except for experiment no. 4, where P2 rd 1 c was used. c 4 rd I+ c plaques (gp = 1.9 X 1OW) and 2 rd 1+C+ plaques (gp = 0.9 X 10-a) were also observed.
nificantly influenced by this reversal (Table 2, Fig. 3). In experiment no. 5 the value of gs was compared for the singly lysogenic strain C-27 and a doubly lysogenic strain C-59 = C (P2 rd I c) i (P2 rd I c) rrr. Assuming prophage substitution in location I to be the
most likely event (Six, 1960), no significant difference was found between the gs values for the two strains. In these experiments recombinant-type prophages were always observed at a lower frequency than prophages of the superinfecting type. At low multiplicities, the ratio
SUPERINFECTION
EXPERIMENTS
of P2 rd+ 1+ c to P2 rd+ 1+ c+ (or P2 rd 1 c+ to P2 rd 1 c in experiment no. 4) may be as high as 1:4; at higher multiplicities, 1:lO. These figures do not include the recombinant types found after superinfection of the doubly lysogenic strain C-59. For the reciprocal recombinant type no estimate can be attempted because of the complications due to the rd decay. Pooling all data from experiments nos. 2, 3, 4, 5 (only C-27), and 6 (only unirradiated) in Table 2, there are 32 instances of a substitution at the rd 1 loci and 32 cases of the reciprocal substitution at the c locus. The average value of g, for the rd 1 loci is about 75% of the average g, of the c locus (3.6:4.8; ratio of sums of corrected g, values in Table 2). Experiments nos. 1 and 2 include preliminary results of the effects of chloramphenicol on the frequency of prophage substitution. Chloramphenicol at a concentration of 5 X low6 g/ml led to a marked increase of the frequency of prophage substitution in Sh (P2 rd hz) at a multiplicity of 14 (see Fig. 1). At a concentration of 25 X 10W6 g/ml of chloramphenicol (experiment no. 2: Table 2 and Fig. 3) an effect was found mainly at the higher multiplicities. Preliminary experiments (nos. 6 and 7) showed that exposure of the lysogenic cells to ultraviolet light prior to superinfection leads to a marked increase of the probability both of complete substitution and of partial substitution. At the higher ultraviolet dose the probability of partial substitution is considerably higher than that of complete substitution. Although this may be an indication that partial and complete substitution are caused by two different mechanisms, too little is known about the genetic map of P2 to warrant any definite conclusion at the present time. B. Superinfection of Cells Carrying a Prophage in Location II The presence of a P2 rd 1 or P2 c prophage in location II provides the lysogenic cells with immunity without keeping the preferred location I occupied. The purpose of the experiments nos. 8 to 13 was to study the genetic incorporation of a superinfecting
WITH
P2
227
phage into immune cells where the preferred location I was unoccupied, to compare the incorporation probabilities under such conditions with those found when location I was occupied by a prophage that was either homoimmune or heteroimmune with respect to the superinfecting phage, and to study the effect of the presence of a prophage in location II on prophage attachment to location I. The results of the colony assays for experiments nos. 8, 9, 12, and 13 indicate that when location II is occupied by a prophage, location I is still preferred for the incorporation of the superinfecting phage; among 1598 colonies examined not one showed incorporation at location II. The average of all g values for a homoimmune phage superinfecting C ( - ) r (P2) rI cells is 5.6 x lo-” (Table 3)) a value much larger than the g values found for superinfection of C(P2)r cells or Sh (P2) cells. Furthermore, the g values do not decrease for M > 1, indicating that several phage superinfecting the same cell act independently of each other in “trying” to give rise to prophage. A plot of all G values vs. M (Fig. 4) reveals that the relationship bet.ween G and M is not quite linear, but can be represented by G = 0.046 X M1.24. This result depends mainly on experiment no. 9. The other experimental points may be represented fairly well by G = 0.055 x 1LIl.O. It should be noted that all these experiments (with the exception of no. 8) were done without antiP2 serum; if an intern1ediat.e virulent phage had been present in the P2 c lysate used in experiment no. 9, this would account for the higher g values found at higher multiplicities.” Experiments nos. 12, 13, 14, and 8 allow a comparison between strains whose location II was occupied by a P2 prophage while location I was either free (C-67 and C-86) or occupied with a heteroimmune prophage. For comparison, strains were also included whose location I was occupied by a P2 pro* The plates used in experiment no. 9 for the free phage assay actually showed some clear phages distinguishable from P2 c plaques, which were probably caused by phage contamination.
228
ERICH TABLE RESULTS
Exnt. no.
Strain
8
C-67
OF SUPERINFECTION -
Lysogenic
I9
C-86
10
C-86
11
C-86
(--hU'2
I-
(-)1(P2
rd
I)II
rd 011
(- hE’2 rd Z)II
11 2.6 -- -. --
100 --
15 0.0 15 0.21 15 0.5 15 1.9 15 5.7 -- _.--
065 0.1: 3 2.1 436 0.6, 5 3.3 355 1.6 2.9 947 10.7 5.5 50649.2 8.6 --_-
200 199 99 --
25 25 25 --
0.5 1.5 .2.8
686 1.5 037 10.9 902 19.0
3.1 7.1 6.8
4.4 7.8
984 30.0 078 42.7
6.9 5.6
M
no
25 25 --
_I12
--13
-14
C-86 C-216 C-208 C-81 --C-86 C-216 C-217 C-208 c-34 C-81 --c-70
C-h@‘2 rd h
I (P2 Hyl
dis)I(P2
rd I)Ir
0’2 rd Z)I(- III (P2)1(P2
Hyl
dis)rr
I-
(-- h(P2 rd 1111 (P2 Hyl dis)I(P2 rd Z)n (P2 Hy* dis)1(P2 rd I)11 P2 rd UI(-)II
w%(-III (P2),(P2
-I
(P2 Hyl
Hyl dis)I(P2
dis)n c)n
GROUP
&J -__ -__-_--
state
cl11
3 EXPERIMENTS
-
C-11 w
SIX
-.
NO
--
G Stable (%)
gj
G
G Total (%I
lstagble) (%I
11
2
13
4.2
1.5 10.0 42.4 ---
O.! l.! 3.f
2.0 11.6 45.5
2.6 5.2 7.4
19 3 0 0 ---
5 3 0 5
24 6 0 5
3.8 0.8 0.0 0.0
34.3 1.1 1.1 3.75 2.5 1.1 ---
1.4 4.1 0 0 2.t 0
4.6 0.25 0.3 0.7 0.35 0.2
0 -
0.0
--
-.
I-
14 14 14 14 --
-.
5.0 3.9 5.9 10.2
100 100 100 100 --
13 13 13 13 13 13 --
7.5 4.4 4.2 5.2 7.1 G.3 -. --
70 90 90 80 80 90
12 -
1.9
-
Ba. *
-
-I
100 -
0
-
Q Symbols : no : number of generations elapsed between superinfection and assay M: multiplicity of superinfection N, : total number of plaques scored G: frequency of incorporation of the superinfecting phage g: probability of incorporation of the superinfecting phage N, : total number of colonies scored seg: segregating different subclones b The superinfecting phage was P2 c except for experiments nos. 8 and 14, where P2 rd 1 was used. All G and g values concern incorporation at location I. Among all colonies tested no case of incorporation elsewhere was found, and it was assumed that the results of the free-phage assays also reflect the frequency of incorporation at location I.
whereas their location II was either free (C-208 and C-34) or occupied by the heteroimmune prophage P2 Hyl dis (C-81). The results, compiled in Table 3, indicate that the presence of a heteroimmune prophage in location I reduces the g values to the levels found for substitution of a homoimmune prophage in location I; g seems to be unaffected by the prophage in location phage
II. These experiments demonstrate, therefore, that the probability of incorporation is governed by steric factors rather than by the immunity class of the prophage in the preferred location. The fact that the g values for substitution in location I are somewhat higher than the ones reported for the experiments in group A may be due to different environmental
SUPERIT\TFECTIOT\T EXPERIMENTS
WITH P2
229
I-
lo-‘-e IO++-Q ’
k= I.24
FIG. 4. The frequencyG of incorporationof a superinfectingphageat a free location I in cells carrying a prophagein locationII vs. the multiplicity M of the superinfectionin a “log log plot.” Line k = 1: G = C X M (C = 0.055); Line k = 1.24:G = C X Ml,” (C = 0.046).
Pointsfrom measurements by colony assays:+ (R) : Experimentno. 8; l : Experimentno. 9; X : Experiment nos.12and 13; + (V) : Experimentno. 18.Points from measurements by free phageassays:0: Experimentno. 9; 0 : Experiment no. 10; A: Experiment no. 11.In experimentno. 8, C-67= C(-)1(P2 C)II wassuperinfectedwith P2 rd 1.In experimentsnos. 9 to 13,C-86= C(-)r(P2 rd l)rI wassuperinfectedwith P2 c. In experimentno. 18,C-86was superinfectedwith P2 vir’. conditions (change of laboratory), different experimental techniques (free-phage vs. colony assays; presence vs. absence of antiP2 serum), or different prophage stabilities in the strains employed. Another aspect of the results obtained in t,hese experiments may be mentioned. The progeny of lysogenic cells (carrying prophages of one immunity class only) that survive superinfection with a heteroimmune phage are often cured of their prophages (Cohen, 1959). Sometimes only one prophage is lost from doubly lysogenic cells (Six, 1960). No curing has been observed as a result of superinfection with homoimmune phage. In experiments nos. 12 to 14 the superinfecting phage was homoimmune with respect to one prophage and heteroimmune with respect to the other. Among a total of 570 colonies examined, no stable isolate showed curing. In experiment no. 13, one colony was found which upon restreaking gave a subclone that had lost the prophage heteroimmune with respect to the superin-
fecting phage. This indicates that the curing mechanism may be affected by the immunity of the superinfected cells. C. Superinfection of Lysogenic Cells with Wealc Virulent Phage Weak virulent phages lyse all infected nonlysogenic cells, and are unable to lyse lysogenic cells carrying a homoimmune prophage. The purpose of the experiments nos. 15 to 19 was to test whether the incorporation of weak virulent phages into lysogenic recipients can occur. The results presented in Table 4 may be summarized as follows. No genetic incorporation of the weak virulent phage is found if the superinfected cells are not immune (experiments nos. 15a and 16). Incorporation can occur if the superinfected cells are homoimmune (experiments nos. 15b, 17, 18, 19). Apparently this genetic incorporation is quite stable: from 14 isolates C(P2 Hyl dis vir14) (P2 Hyl dis) obtained in experiment no. 19 a total of 140 subcloneswas tested; all showed
ERICH
230
TABLE RESULTS Expt. no.
Strain
15a 16 17 15b 19 18
C-83 c-71 c-70 C-81 C-81 C-86
1OOC 77.3d 97 88.5 90.0 64 a Symbols A, B = S = (-) = no = Pi = N = seg. = g = b Note that at the location c Growth found to be d Only 60 P2 c, 1 only
4
OF SUPERINFECTION
EXPERIMENTS,
stage of strain (@II
Superinfecting
Lysogenic (Ah
(-- )I (P2 Hy’ 0’2 rd I)103 (P2 Hyl (P2)r(P2 (PZh(P2 (-hU’2
Frequencies
(A) 03)
SIX
(-)@I 14.3d 2 1.5 0
(S) 031 0 Od 1 0 7.0 36
P2 P2 P2 P2 P2 P2
dis)n ch dis)r (P2 c)rr Hyr dis)rr Hyr dishi rd l11r
(yc)
of different (A)(-) 0 1.3d 0 5.5 0.5 0
lysogenic (A) (3 0 Od 0 2.5 0 0
vir’ Hy’
GROUP phage
S
dis virl"
vi+ vir'
Hvl
dis vi+
vi;'
types c-1
C-1
no
M
N
40 10 15 18 11 17
3.5 4.9 2.1 6.0 22.4 4.0
100 100 100 200 200 109
carrying”: seg.
(9
7.0 0 1.0 0 -
: old prophages in locations I and II superinfecting phage and new prophage no prophage number of generation times elapsed between superinfection and multiplicity of superinfection total number of colonies tested segregating different subclones probability of incorporation of S (S)(B) and (A)(S) do not necessarily indicate that the virulent of the old prophage which was lost. was observed only after an unusually long incubation period. P2 resistant. colonies liberating P2 rd 2 were retested for the presence of P2 P2 rd 1 (= C-%X3), 1 only P2 c.
both prophages P2 Hyl dis vir14 and P2 Hyl dis. One subclone was grown in LS broth for at least 30 generations, then 20 new subclones were tested. Again all showed both phage types. In experiments nos. 17 to 19 the preference for genetic incorporation at location I was again observed. The g values for the probability of genetic incorporation by substitution or addition are in good agreement with those found for superinfection with temperate phage. When strain C (P2) r (P2 Hyl dis) rI was superinfected with P2 virl (experiment no. 15b) incorporation of P2 vi+ was coupled with loss of the prophage in location II
CQ
0 0 0 0 0 0
0 0 0 1 2.5 0
0.5 0.1 0.3 9.0
assay
phage One
(S) became colony
attached
was tested
c; 58 had both;
and
P2 rd I and
rather than in location I, as would be expected from the rule of “preference of location I.” This apparent exception indicates once more that the continued presence of a homoimmune “temperate” prophage is a necessary condition for the prophage incorporation of a weak virulent phage. The g value (0.14%) is lower than for substitution in location I. One isolate C (P2) (P2 vi+) from this experiment was crossed with a nonlysogenic F- strain (C-51 ; Bertani and Six, 1958)) in an attempt to derive a strain carrying only P2 vi+. Among 240 recombinants, none was lysogenic for P2 vi+ only, although some (2.5%) carried both P2 and P2 vi+. Another
SUPERINFECTION
EXPERIMENTS
attempt to obtain a strain C(P2 vi+) by crossing an F- strain lysogenic for P2 virl and P2 rd 1 with a nonlysogenic F+ strain also failed. These findings again indicate the requirement for presence of a temperate homoimmune prophage for the incorporation of a weak virulent phage. DISCUSSION
In a discussion of the experiments3 it is important to note that the incorporation of P2 and its dismune variants P2 Hy dis occurs preferentially at one site of the bacterial chromosome called location I. This has been found for lysogenization of nonlysogenic cells with either P2 or P2 Hy dis (Bertani and Six, 1958), for genetic incorporation of either P2 or P2 Hy dis into lysogenic cells carrying only prophages heteroimmune with the superinfecting phage (Six, 1960)) and for the incorporation of P2 into lysogenic cells carrying a homoimmune prophage in location I, either alone or with a second P2 prophage in another location (Bertani 1954; Bertani and Six, 1958). The results reported here are in agreement with the rule of preference for location I, especially since it could be shown that in cells carrying only P2 in location II genetic incorporation of the superinfecting phage occurred by addition of a new prophage, without loss of the P2 in location II. This was found not only for the incorporation of P2 but also for P2 Hy dis. The reason for the preference for -location I remains unknown. It is possible that the different attachment sites found so far possess different degrees of affinity for P2 and P2 Hy dis. Another explanation may be an inhomogeneity of the phage lysates in the sense that the majority of phage are able to attach themselves only at one location (I), whereas a minority of phage have affinity to different sites as a result of some genetic event (mutation or recombination with the bacterial genome). A similar behavior has been found meanwhile to occur ‘In the discussion the symbol to indicate any phage with the erties of phage P2, as contrasted phage P2 Hy dis.
P2 will be used immunity propwith those of
WITH
P2
231
for another episome, the sex factor F (Jacob and Adelberg, 1959). Experiments are under way to examine this question for P2. The following conclusions can be drawn concerning the incorporation of superinfecting P2 at location I in cells carrying one or more P2 prophages: The probability of addition of a new prophage at an empty location I is about 40 times higher than the probability of substitution at location I. This difference is apparently due to a steric hindrance caused by the presence of a prophage at t’he preferred location. This steric hindrance does not depend on immune specificity because it is exerted equally by a homoimmune or a heteroimmune prophage at location I. There is no indication that the substitution of a location I prophage is hindered by the presence of a second prophage in another location. The full additivity of the incorporation chances of phages in multiple infection for multiplicities up to 10 supports the hypothesis (Bertani, 1953) that no multiplication of the genome of the superinfecting phages occurs in immune cells. It follows that the probability of incorporation of a phage should reflect the affinity of the prophage attachment site(s) in the bacterial chromosome for the nonincorporated genetic determinants of the infecting phage or “preprophage.” (Note that this definition of the “preprophage” does not specify an inability to multiply.) The data presented here do not reveal any significant difference for the probability of incorporation of P2 carrying the c or the c+ markers. Since this pair of alleles affect the frequency of lysogenization (L. E. Bertani, 1959), the difference in this frequency cannot be due to a difference in the affinity of the attachment site for phage with the c or the cf allele. This conclusion is supported by the results with weak virulent mutants. Although they have completely lost their ability to lysogenize, these mutants can become prophages and be transmitted as such over many cell generations if another prophage of the homoimmune temperate type is present. If this temperate prophage is eliminated by genetic recombination in a bacterial cross or by another superinfection (Six, unpublished)
232
ERICH
the virulent prophage, no longer stabilized, will be lost, either by lysis or by curing. The possibility that the virulent prophage attaches itself to the pre-existing prophage rather than to the bacterial chromosome is made unlikely by the observation of steric hindrance for the genetic incorporation of P2 virl. The observed failure of a P2 Hy dis prophage to stabilize P2 vi+ indicates instead that immunity provides the necessary stabilization. A weak virulent phage appears to possess only the passive immune specificity; i.e., it recognizes the immunity of a lysogenic cell but cannot itself produce immunity. This conclusion is in agreement with the interpretation of Jacob and Campbell (1959) for the case of hC (a weak virulent X, comparable to P2 vi+). Another group of observations (Campbell, 1957) may also be quoted in support of the views presented here. From the data on gal-transduction it is possible to estimate the probability of genetic incorporation of the transducing phage Agal, which is unable to multiply by itself in a nonlysogenic cell, but can do so in mixed infection with A. The probability for gal transduction in single infection of nonlysogenic (or lysogenic) cells is only about one-twentieth of the probability for transduction in mixed infection of nonlysogenic cells. The ratio indicates a g value of 0.05, the same as that found for attachment of P2 to a free location I. The probability of gal transduction in mixed infection of cells lysogenic for h is as low as the probability in single infection; this supports the hypothesis that immunity prevents the multiplication of preprophages. For general transduction by phage Pl or P22 the ratio of the frequency of stable transduction to the frequency of stable plus abortive transductions may be interpreted as the probability of genetic incorporation of the transduced chromosomal piece. Although there is a rather wide variation in the available data’ (Ozeki, 1956, 1959), the ratio of the transduction frequencies is of the same order of magnitude as the g values calculated for P2 and Agal. While the incorporation of a phage as pro-
SIX
phage is certainly not the same as the integration of a transduced fragment into the bacterial genome, the similarity of the incorporation probabilities may reflect a similarity in these processes. In conclusion some further remarks may be made concerning the role of immunity. The observation that steric hindrance, while affecting the genetic incorporation of a phage, is not related to the immune specificities of the phages and prophages involved lends support to the notion that immunity must be caused by a specific, prophage-controlled “immunity substance” (Bertani, 1956) or “repressor” (Jacob and Campbell, 1959) produced in the lysogenic cells. At the time being, it seemssufficient to assumethat the mechanism of immunity involves only the prevention of multiplication of the phage genome. The lack of phage production in immune cells would be a consequence of this hypothesis. Upon infection of nonimmune cells, multiplication of the phage genome need not occur only in those cells that produce phage and lyse. There is evidence (see discussion in Luria et al., 1958) that in cells that become lysogenic multiplication of the phage genome precedes the establishment of the prophage. In particular, this view is supported for P2 by the experiments of L. E. Bertani (1957). It seemsreasonable to assume that infection of nonlysogenic cells with P2 leads to the formation of a pool of 100 or more preprophages. A g value of 0.05, as found for free location I, is then high enough to assure lysogenization of almost all cells surviving the infection as has indeed been observed (L. E. Bertani, 1957). ACKNOWLEDGMENT Dr. G. Bertani and Dr. A. Campbell have contributed greatly to this work through many stimulating discussions. REFERENCES BERTAX, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacterial. 62, 293-300. BERTANI, G. (1953). Lysogenic versus lytic cycle of phage multiplication. Cold Spring Harbor Symposia
Quant.
Biol.
l&65-70.
SUPERINFECTION
EXPERIMENTS
G. (1954). Studies on lysogenesis. III. Superinfection of lysogenic Shigella dysenteriae with temperate mutants of the carried phage. J.
BERTANI,
Bacterial.
67,696707.
G. (1956). The role of phage in bacterial genetics. Brookhaven Symposia in Biol. No. 8,
BERTANI,
50-57. G. (1957). A dismune mutant of temperate phage P2. Bacterial. Proc. (Sot. Am. Bacteriologists), p. 38. BERTANI, G. (1958). Lysogeny. Advances in Virus Research 5, 151-193. BERTANI, G., and SIX, E. (1958). Inheritance of prophage P2 in bacterial crosses. Virology 6, 357-381. BERTANI, L. E. (1957). The effect of the inhibition of protein synthesis on the establishment of lysogeny. Virology 4,53-71. BERTANI, L. E. (1959). The effect of ultraviolet light on the establishment of lysogeny. Virology 7,92-111. CAMPBELL, A. (1957). Transduction and segregation in Escherichia coli K12. Virology 4,366384. COHEN, D. (1959). A variant of phage Pb originatBERTANI,
WITH
P2
233
ing in Escherichia coli, strain B. Virology 7, 112126. JACOB, F., and ADELBERG, E. A. (1959). Transfert des caracteres genktiques par incorporation au facteur sexuel d’Escherichia coli. Compt. rend. mad.
xi.
249,189-191.
F., and CAMPBELL, A. (1959). Sur le systeme de repression assurant l’immunite chez les bacteries lysogenes. Compt. rend. acad. sci. 248, 3219-3221. LURIA, S. E., FRASER, K., ADAMS, J. N., and BURROUS, J. W. (1958). Lysogenization, transduction, and genetic recombination in bacteria. Cold Spring Harbor Symposia Quant. Biol. 23, 71-82. OZEKI, H. (1956). Abortive transduction in purine requiring mutants of Salmonella typhimurium. Carnegie Inst. Wash. Publ. No. 612,97-166. OZEKI, H. (1959). Chromosome fragments participating in transduction in Salmonella typhimuJACOB,
rium.
Genetics
44,457-470.
E. (1959). The rate of spontaneous lysis of lysogenic bacteria. Virology 7,328-346. SIX, E. (1960). Prophage substitution and curing in lysogenic cells superinfect.ed with hetero-immune phage. J. Bacterial. 80,728-729. SIX,
14, 229-233 (1961)
Inheritance
of Prophage
P2 in Superinfection ERICH
Department
of Bacteriology, Accepted
SIX
University
State
Experiments’
of Iowa,
Iowa City,
Iowa
February 24, 1961
Genetically marked P2 phage was used to superinfect cells of Escherichia coli (or Shigella dysenteriae) which were lysogenic for P2 and hence immune to the superinfecting phage. The genetic incorporation of the superinfecting P2 was studied by examining the progeny of the superinfected cells. The results obtained are in agreement with the previously established rule that genetic incorporation occurs most frequently at the preferred location (I) either by prophage addition or by prophage substitution. Besides complete substitution, partial (= single marker) substitution was also observed, but at a lower frequency than complete substitution. The frequency of genetic incorporation was found to increase linearly with the multiplicity of superinfection for small multiplicities. The probability of genetic incorporation of a superinfecting phage is decreased by the presence of a prophage in location I; this hindrance does not depend on the immune specificity of such prophage. It is assumed that in immune cells multiplication of prophage precursors (= preprophages) is prevented. The probability of genetic incorporation of a preprophage in the absence of steric hindrance (5%) compares well with the probabilities found in other systems for the genetic incorporation of bacterial markers in transduction experiments. Weak virulent mutants of P2 and P2 Hy dis can become prophages, but only in cells already lysogenic for a temperate phage of the same immunity class. INTRODUCTION
The noninducible temperate phage P2 and its relationship to its normal hosts Escherichia coli strain C and Shigella dysenteriae strain Sh have been studied for several years (see Bertani, 1958). These investigations have led to the following conclusions: ‘The work described in this paper was begun at the Division of Biology, California Institute of Technology and was continued at the following institutions : Department of Medical Microbiology, University of Southern California; Max-PlanckInstitut fur Biologie (Tubingen, Germany), and Department of Biology, University of Rochester. Parts of this work were done under a fellowship of the Deutsche Forschungsgemeinschaft and later under a fellowship of the Max-Planck-Gesellschaft. Further support was received from a grant of the National Foundation for Infantile Paralysis, from National Science Foundation grant G-3841, and from United States Public Health Service grants E-1839 and E-2862.
(1) P2 can become attached as a prophage at any one of several sites (I, II, etc.) of the bacterial chromosome. For E. coli C, these sites can be recognized and separated by means of bacterial crosses (Bertani and Six, 1958). (2) If a nonlysogenic cell is infected with P2, one site on the bacterial chromosome (location I) is preferred for the attachment of P2 as a prophage. (3) Superinfection of such lysogenic cells with P2 leads, with a rather small probability, to the establishment of the superinfecting phage as prophage, either by replacing a pre-existing prophage in location I (= prophage subsitution) or by attaching at a previously empty site (= prophage addition or double lysogenization) . Under these conditions, substitution is more frequent than double lysogenization. (4) A temperate “dismune” variant of P2
220
SUPERINFECTION
EXPERIMENTS
was found, which belongs to an immunity class different from that of P2 and its plaque-type mutants (G. Bertani, 1957). It was shown by Cohen (1959) that this dismune phage results from a genetic interaction between P2 and coli B ; the variant phage was designated P2 Hy dis. (5) The same attachment site in coli C (location I) that is preferred by P2 is also preferred by P2 Hy dis (Bertani and Six, 1958; Six, 1960). The purpose of this investigation is to gain more insight into the process of genetic incorporation of a superinfecting phage in cells that carry one (or more) prophages belonging to the same immunity class as the superinfecting phage and hence are immune to the superinfecting phage. In particular, the following questions were asked: (a) How does the multiplicity of the superinfecting phage affect the frequency of genetic incorporation? (b) How do the prophages present in the superinfected cell affect the frequency of genetic incorporation of the superinfecting phage?
WITH
221
P2
judgment, limited in its validity by the number of detectable genetic markers. Since the number of available P2 markers is small, it shall not be implied that complete and partial genetic incorporation are necessarily two different processes. A “probability of genetic incorporation” will be introduced, defined as: g = G/M This definition is, of course, useful only if G is proportional to M, and assumption which for small M is supported by the experimental findings reported in this paper (generally, one could define g = dG/dM) . The probability g can be interpreted in the following way:
g = 4W)a’ M
= Wv)g’
where g’ is the probability for a superinfecting phage to become incorporated into the genome of one bacterial nucleus within one generation time; v is the number of nuclei per cell, probably v = 2. OL is a parameter which depends on the fate of the superinfecting phage in the bacteria: If geDefinition and Interpretation of netic incorporation can only happen imSome Terms mediately after superinfection, then OL= 1. The two primary parameters that are If the superinfecting phage-prior to its measured in the superinfection experiments genetic incorporation-enters a state (as to be described are: suggested by Bertani, 1953, 1954) where it M, the multiplicity of superinfection = is carried without multiplication or loss average number of superinfecting phages (being diluted out), and if the probability per cell. of genetic incorporation per generation cycle G, the frequency of genetic incorporation is constant = g’, then a = 2. A value Q > 2 of the superinfecting phage, i.e., the fre- would be expected if some multiplication quency, among the progeny of the super- occurred prior to genetic incorporation. infected cells, of cells carrying a prophage Since no attempt will be made to measure CL newly introduced by superinfection (de- or V, g may be taken as a convenient estermined after a sufficient number of timate for g’. growth cycles has been allowed to give a The probability of genetic incorporation rather complete segregation with respect as defined this way (as g) must not be conto the new prophage). If the new pro- fused with the probability that a superinphage apears to be genetically identical fecting phage will be finally incorporated with the superinfecting phage, the genetic into Some bacterial genome. The latter incorporation is termed “complete.” “Parquantity may be 1 when g’ < 1, namely in tial” incorporation is the case in which the case of “slow” incorporation due to the not all markers (but at least one) of the “diluting out” of the preprophage. The value superinfecting phage appear in the new I/g (or l/g’) measures then the average prophage. It should be noted, however, number of bacterial growth cycles after that the completeness of genetic incorpo- which genetic incorporation occurs. G and ration always rests on rather arbitrary g with the subscripts A, S, or P are used to
222
ERICH
SIX
described before (Bertani and Six, 1958; Six, 1959) ; other are listed in Table 1. Testing for prophages carried by lysogenie cells was done by one of the following methods : (1) Assay of a growing culture for the free phage by plating with a suitable indicator strain. If the lysogenic cells were streptomycin sensitive, they were selectively killed by adding streptomycin upon plating (“streptomycin method,” see Bertani, 1951). If the cells were streptomycin resistant, they were selectively killed by heating the culture for 20 minutes at 58” (see Bertani and Six, 1958). (2) Immunity assay by spotting a loopful of the growjng lysogenic culture on a L agar plate see&i Jith L, suitable virulent phage. This had to be done (using P2 Hyl dis vir14) in order to detect the presence of P2 Hyl dis prophage in streptomycin-resistant cells, since the heat sensitivity of P2 Hyl dis (Cohen, 1959) did not allow application of the 58” treatment. Superinfection experiments. (1) Superin fection was carried out in mass culture Many of these experiments were done as described by Bertani (1954) : the lysogenic cells were grown in L broth to reach 5 x 107/ml and superinfected at 37” in L broth containing 5 x 10W3M CaCIz . An adsorption period of 10-20 minutes was allowed to assure adsorption of more than 50% of the phage input. Then the cells were diluted l:lOO, with L broth containing a sufficient
denote the type of genetic incorporation: A for addition of the new prophage without loss of the old one(s) , S for prophage substitution (complete substitution if distinguishable from partial substitution), P for partial substitution. Phages and prophages belonging to the same immunity class are called “homoimmune.” Those belonging to different immunity classes “heteroimmune” (Six, 1959 j . MATERIALS
AND
METHODS
Phages. The temperate phage P2, its plaque-type mutants (markers: rd = round; 1 = large; c = clear) ; and its virulent mutants: virl (= weak virulent), vir” (= strong virulent 1 have been described before, together with the general techniques and the media employed in the study of P2-like phages (G. Bertani, 1954.. 1957). Furthermore, P2 Hyl dis, a phage heteroimmune with respect to P2 :dnd a weak virulent mutant, P2 Hyl dis vi@* have ‘already been described (Cohen, 1959). Bacteria. (1 j .‘CP the plating bacterium the nonlysogenic, streptomycin-resistant indicator strain Sh/s (described before: Bertani, 1951) was :-ed as well as two lyso‘q: Sh-76, carrying genie derivatives c P2 Hy’ dis, and Sh-, , v *rying P2 as prophage. (2) All lysogenic !bi?lins used for the superinfection experiments were derivatives of either E. coli strain t’: or of S. dysenteriae strain Sh. Many of these strains have been TABLE LIST Strain Sh-36 Cb C-86 C-208 C-216 C-217
Lysogenic
OF STRAINS
NOT
state
1
PREVIOUSLY Origin
Remarks
-
(P2 rd hz)
(WI (-)1&‘2
rd 1)~ (P2 rd 1)1 (P2 Hyl dis)1(P2 (P2 Hyl dish(P2
DESCRIBEDU
Lysogenization Cross c-55 x Partial curing Superinfection Superinfection
rd Z)U rd 1)~
a By Bertani and Six (1958) or sensitive. b C-55 = C(P2)1 (P2 rd Z)n , Ftained by G. Bertani (unpublished). c P2 Hy* dis is like P2 Hy’ dis, B. It belongs to the same immunity It is probably a recurrence of the
Six (1959). strain
All
derived
strains from
of C-2 C-66* of C-71 of C-86 of C-86 listed
C-53
here
hz = hazy (for experiment
no.
(for (for (for
experiment experiments experiments
no. 16) nos. 12, 13) nos. 12, 13)e
prototrophic,
streptomycin
are F+,
by superinfection.
C-55
4)
and C-86
were
ob1
as it was also obtained by interaction between P2 and E. coli strain class as P2 Hyl dis. On Sh/s it gives larger plaques than P2 Hyl dis. character “large” mentioned by Cohen (1959).
SUPERINFECTION
EXPERIMENTS
amount of anti-P2 serum to neutralize unadsorbed phage and 5 x 10d3 M Caf + in order to prevent desorption. The cells were then grown at 37” in aerated L broth, keeping the titer below 5 X 107/ml, by diluting with fresh medium, until a sufficient number of generations (about 10) had elapsed. Streaks were then made on nutrient agar plates from which colonies were picked later for investigation of their lysogenic state (L’colony assay method”). If these isolates were found to be lysogenie for the phage type used for superinfection, it was assumed in general that a stable incorporation of the superinfecting phage had occurred. In some instances exceptional doubly lysogenic isolates were observed, which liberatedI’ the’? i.tiw phage type at a lower frequency than could be expected for stable doubly lysogenic clones. These isolates were restreaked and up to ten subcloneswere retested. These subclones often included some that no longer carried the new prophage. Subclones from doubly ,ysogenic isolates liberating the new phage Lype with the expected frequency showed no such segregation. (2j Since the low frequency of prophage substitution in cells superinfected with homoimmune phages necessitates the isolation and testing of a large number of colonies derived from a superinfected culture, the frequency of genetic incorporation of the superinfecting phage was in some cases estimated simply by measuring the frequency of the superinfecting phage type among the total free phage in a growing culture of superinfected cells (“free-phage assay method”). The standard procedure was as described under (I), except that (in experiments 1 to 5) the cell titer in the growth tubes was kept between 8 x lo* and 9 x 106/ml in order to minimize reinfection. Finally, flasks containing 100 ml L broth were inoculated with 2 x lo7 cells, and these cultures were grown again at 37”, but unaerated, to reach 5 x 106/ml. Then the cells were killed by adding streptomycin and the culture was assayed for free phage. Even with a final cell number of 5 x 10s in t.he flasks, sampling errors should become critical if the frequency of prophage incorporation is as low as 10e3, since the rate
WITH
P2
223
of spontaneous lysis is about 2 x lo-” per cell per generation time (Six, 1959). No special precautions had to be taken (in experiments nos. 9 and 10) when the expected frequencies of incorporation were much higher. For experiments nos. 9 to 18 no anti-P2 serum was available. Hence, the cells were diluted from the adsorption tube into L broth and grown to reach 5 x 107/ml. Then they were washed once in order to reduce the free-phage titer by a factor of 30 and then diluted into LS broth. LS broth was then used as growth medium in order to reduce the chances for reinfection. The multiplicity of superinfection was calculated from the phage input and the cell input applying the following dorrections: (1) The phage input was corrected for the percentage of unadsorbed phage, either determined from assays of free phage or assumedto be ,.O%. (2) The cell titer as determined by colony counts was multiplied by aafactor of 1.4 in order to take into accouo~:the existence of cell aggregates (doublets, triplets, etc.) in the culture (Six, 1959). The streptomycin UWA was dihydrostreptomycin sulfate, eitl,. ti+orn Eli Lilly and Company or a gift. tirorn the Farbwerke Hochst. The chlorar .:henicol used was a gift from Parke, Dav s,and Company. For ultraviolet irazdiation the bacteria were washed and resuspendedin chilled saline and placed under a 15-watt GE “germicidal” tube (effective wavelength 254 rnp) . Photoreactivation was minimized by use of dim yellow light during the irradiation and for 90 minutes afterward. Media. Unless otherwise mentioned, the bacteria were grown in nutrient broth. L broth contains 10 g Bacto-Tryptone (Difco), 5 g Bacto-Yeast-Extract (Difco), 10 g NaCl, and 1 g glucose per liter of water. LS broth has the same composition except that it contains only 2 g of NaCl. RESULTS
A. Superinfection of Cells Carrying a Prophage in Location I Because of the preference of P2 for location I, in these experiments substitution of the prophage in location I is expected to be
ERICH
SIX
0 20
FIG. 1. Experiment no. 1. The frequency of prophage substitution G, vs. the multiplicity of superinfection M for Sh-36 = Sh(P2 rd hz), superinfected with P2 wild type. C = superinfection in the presence of chloramphenicol(5 X 10“ g/ml). Free phage assay 1 day after the sunerinfection experiments. The curve inserted at the right represents the initial part of the lamer curve on a-different scale.
FIG. 2. Spontaneous inactivation of free phages in experiment no. 2. t = Time of phage assay (t = 0 : streptomycin added to growing cultures). Between assays the cultures were kept in a refrigerator. Average values of the two cultures for M = 10.3. The total titer is practically identical with the P2 rd 1 c titer. The P2 titer for t = 0 was extrapolated from the four measurements at t > 0. 0: P2rdZc; x: P2.
the most likely process. Only “free-phage assays” were done; results are interpreted in terms of prophage substitution, assuming that the addition of a new prophage OCcurred at a negligible frequency. The main purpose of these experiments in
(A) was to measure Gs , the frequency of prophage substitution, as a’function of M, the multiplicity of the superinfecting phage and to calculate the probability of prophage substitution (gs) . The influence of such variables as the genetic markers of the phage, the presence of a second prophage, and the treatment with chloramphenicol or ultraviolet light on the frequency of prophage substitution was explored. The findings are summarized in Figs. 1 and 3 and in Table 2. They show that for superinfection of Sh cells (Fig. 1, experiment no. 1) as well as of C cells (Fig. 3, experiments nos. 2-7) Gs increases linearly with M for M < 10, leveling off at higher multiplicities. The linearity of the increase justifies the use of the ‘parameter gs . The causes of the final leveling off remain unknown. One reason may be a selective death of cells superinfected with more than some critical number of phages, Free-phage assays in experiment no. 2 (superinfection of C-27 = C (P2 rd 1 c) r with P21 wild type) revealed ‘a slow spontaneous inactivation of both phage types: P2 rd 1 c and ‘P2 wild type (Fig. 2). During the fir-St 40 hours the titer of the P2 rd 1 c phage decreased faster
SUPERINFECTION
0
2
4
6
8
EXPERIMENTS
IO
I2
WITH
I4
I6
P2
I8
225
20
22
24
M FIG. 3. The frequency of prophage substitution G, vs. the multiplicity of superinfection M for different lysogenic strains superinfected with P2 (free phage assay). Solid line: X: Experiment no. 2 (C-27) ; A : Experiment no. 4 (Cb) ; 0 : Experiment no. 5 (C-27) ; l : Experiment no. 5 (C-59); 0 : Experiment no. 6 (C-27, unirradiated). Broken line: f: Experiment no. 2 (C-27, superinfected in the presence of chloramphenicol, 25 X lo-’ g/ml).
than that of the wild-type phage, while after 40 hours the titers for both phage types decreased at about the same rate. This differential decay of P2 rd 1 c may increase the observed ratio of P2: P2 rd 1 c by a factor of almost 4.5. In the other experiments somewhat different inactivation rates were observed, but P2 wild type was always more stable than P2 rd 2 c. Since a comparable differential decay was also found for P2 rd hz as compared to P2, it is assumedthat the rd marker is responsible for the lower stability of the phages; therefore, the recombinant P2 rd I c+ should show the same stability as P2 rd 1 c, and P2 rd+ Z+ c the same as P2 wild type. Except for experiment no. 1, all results given are corrected for the differential rd decay as observed for the P2 rd 1 c phage. In experiment no. 1, the free-phage assay was done several hours after the 1ysogeni.c cells had been killed with streptomydin. Since at that time it was not known that rd phages are more unstable than rd+, the
measured Gs values may be too high (at all M values tested) by a factor of about 2. For this reason, the gs value was not calculated for this experiment. For the prophage substitution in singly lysogenic strains of E. coli C, a gs value of 1.2 x 1O-3 per phage ‘is obtained from the data of experiments nos. 2, 3, 5, 6, and 7 as plotted in Fig. 3. This value represents a mean of the corresponding values compiled in Table 2. In experiment no. 3, Gs was determined for a Sh strain comparable to C-27: Sh-74 = Sh (P2 rd I c) superinfected with P2 wild type. The value obtained for gs is about 4 times as high as for the C strain. No significant difference in the Gs values was observed whether the cells were grown in the presence or in the absenceof anti-P2 serum. While in most experiments strain C-27 = C(P2 rd 2 c) was superinfected with P2 wild type, the markers were reversed in experiment no. 4: C (P2)r was superinfected with P2 rd Z c. The gs value was not sig-
226
ERICH
-
Expt. no.
RENJLTS
T Strain I -C-27
OF
TABLE 2 SUPERINFECTION EXPERIMENTS,
-
Remarks
t
rd I C)I
3
Sh-74
--
N I
____
+ Chloramphenicol
-_
M I
~_ = C(P2
SIX
I
GROCP -
- -
Ns PJ PP
rd 1 c)
43 1.4 1933 20 2.751701 20 5.4 1524 2010.3 2082
8 9 16 29
43 1.4 2347 20 2.751825 20 5.4 1315 2010.3 1177
5 10 18 33
0 7.6
1011
1 i 0 3
i: 6
: ;
37
; 1 2
4
-4
4
3
0 -116 I 7.6
4 5
-Cb -C-27
No anti-P2
_-
= C(P2)r (no correction for rd decay)
6 __7
-C-27 -C-27 -
2
(: -1
23
2
2
38
3
1
11
2
0
17
2
0
21
4
2
--
rd 1 c)r -90
C-59
’3684 I
serum 0
= C(P2
= C(P2 rd 1 c)r (P2 rd 1 C)III
_1unirradiated UV; surv.
50 Set = 0.30
WI Set UV; survivorsc = 1.5 x .10-s
6.3 011.7 -90 11.7 023.6 -90 23.6
7020 2603 7764 2406 6033
0 4.5 4.5 0 8.9 -90 8.9 0 18 0 -90 18
2313 5915 1959 4925 1969 5315
0 0.9 0 0.9
4391’ 492
4 4
0 0.9
1686
14
-90
_-
----
I
I
-
03 x g .03x g,‘A
103
x
RPB
-~
l= Sh(P2
i PI
Aa. *
0”
_- --
:
_ii
0 1 Ii
1.2 1.2 1.25 0.9
0.3 0.3
E
i::
0:4 0.3
::;
0.1 0.5 0.2 0.1
E 0:1 0.2
4.8 5.0
0.5 0.3
0.5 0.3
::3’
0”::5 -__.
0.6 1.3
1.4 1.4
::; 0.8 0.9 0.7 0.7 1.3 1.3 :.; 0:7 0.9 1.1 9.5 --9.1
n0::
---
0.3 0.25 0.3 0.0
0.14 0.06 0.07 0.06 0.06 0.04
0.07 0.07 0.07 0.03 0.01 0.04
0.24 0.15 0.14 0.10 0.14 0.10
00::4 0.0 0.0 0.07 0.04
0.27 9.5 2.3
-l--
0.0 2.4 7.1
I
- a Symbols: nG : number of generation times elapsed between superinfection and addition of streptomycin t : time in hours when free phage assay was done after cessation of growth M : multiplicity of superinfection N: total number of plaques scored Ns : number of plaques revealing total prophage substitution NP* : number of plaques revealing partial prophage substitution (loci rd and 1 together) Npn : number of plaques revealing partial prophage substitution (locus c only) gs : probability of total prophage substitution gp, : probability of substitution of loci rd and 1 gps : probability of substitution of locus c b 911 g-values (except for experiment no. 4) are corrected for r&decay, if t > 0. The superinfecting phage was wild-type P2 except for experiment no. 4, where P2 rd 1 c was used. c 4 rd I+ c plaques (gp = 1.9 X 1OW) and 2 rd 1+C+ plaques (gp = 0.9 X 10-a) were also observed.
nificantly influenced by this reversal (Table 2, Fig. 3). In experiment no. 5 the value of gs was compared for the singly lysogenic strain C-27 and a doubly lysogenic strain C-59 = C (P2 rd I c) i (P2 rd I c) rrr. Assuming prophage substitution in location I to be the
most likely event (Six, 1960), no significant difference was found between the gs values for the two strains. In these experiments recombinant-type prophages were always observed at a lower frequency than prophages of the superinfecting type. At low multiplicities, the ratio
SUPERINFECTION
EXPERIMENTS
of P2 rd+ 1+ c to P2 rd+ 1+ c+ (or P2 rd 1 c+ to P2 rd 1 c in experiment no. 4) may be as high as 1:4; at higher multiplicities, 1:lO. These figures do not include the recombinant types found after superinfection of the doubly lysogenic strain C-59. For the reciprocal recombinant type no estimate can be attempted because of the complications due to the rd decay. Pooling all data from experiments nos. 2, 3, 4, 5 (only C-27), and 6 (only unirradiated) in Table 2, there are 32 instances of a substitution at the rd 1 loci and 32 cases of the reciprocal substitution at the c locus. The average value of g, for the rd 1 loci is about 75% of the average g, of the c locus (3.6:4.8; ratio of sums of corrected g, values in Table 2). Experiments nos. 1 and 2 include preliminary results of the effects of chloramphenicol on the frequency of prophage substitution. Chloramphenicol at a concentration of 5 X low6 g/ml led to a marked increase of the frequency of prophage substitution in Sh (P2 rd hz) at a multiplicity of 14 (see Fig. 1). At a concentration of 25 X 10W6 g/ml of chloramphenicol (experiment no. 2: Table 2 and Fig. 3) an effect was found mainly at the higher multiplicities. Preliminary experiments (nos. 6 and 7) showed that exposure of the lysogenic cells to ultraviolet light prior to superinfection leads to a marked increase of the probability both of complete substitution and of partial substitution. At the higher ultraviolet dose the probability of partial substitution is considerably higher than that of complete substitution. Although this may be an indication that partial and complete substitution are caused by two different mechanisms, too little is known about the genetic map of P2 to warrant any definite conclusion at the present time. B. Superinfection of Cells Carrying a Prophage in Location II The presence of a P2 rd 1 or P2 c prophage in location II provides the lysogenic cells with immunity without keeping the preferred location I occupied. The purpose of the experiments nos. 8 to 13 was to study the genetic incorporation of a superinfecting
WITH
P2
227
phage into immune cells where the preferred location I was unoccupied, to compare the incorporation probabilities under such conditions with those found when location I was occupied by a prophage that was either homoimmune or heteroimmune with respect to the superinfecting phage, and to study the effect of the presence of a prophage in location II on prophage attachment to location I. The results of the colony assays for experiments nos. 8, 9, 12, and 13 indicate that when location II is occupied by a prophage, location I is still preferred for the incorporation of the superinfecting phage; among 1598 colonies examined not one showed incorporation at location II. The average of all g values for a homoimmune phage superinfecting C ( - ) r (P2) rI cells is 5.6 x lo-” (Table 3)) a value much larger than the g values found for superinfection of C(P2)r cells or Sh (P2) cells. Furthermore, the g values do not decrease for M > 1, indicating that several phage superinfecting the same cell act independently of each other in “trying” to give rise to prophage. A plot of all G values vs. M (Fig. 4) reveals that the relationship bet.ween G and M is not quite linear, but can be represented by G = 0.046 X M1.24. This result depends mainly on experiment no. 9. The other experimental points may be represented fairly well by G = 0.055 x 1LIl.O. It should be noted that all these experiments (with the exception of no. 8) were done without antiP2 serum; if an intern1ediat.e virulent phage had been present in the P2 c lysate used in experiment no. 9, this would account for the higher g values found at higher multiplicities.” Experiments nos. 12, 13, 14, and 8 allow a comparison between strains whose location II was occupied by a P2 prophage while location I was either free (C-67 and C-86) or occupied with a heteroimmune prophage. For comparison, strains were also included whose location I was occupied by a P2 pro* The plates used in experiment no. 9 for the free phage assay actually showed some clear phages distinguishable from P2 c plaques, which were probably caused by phage contamination.
228
ERICH TABLE RESULTS
Exnt. no.
Strain
8
C-67
OF SUPERINFECTION -
Lysogenic
I9
C-86
10
C-86
11
C-86
(--hU'2
I-
(-)1(P2
rd
I)II
rd 011
(- hE’2 rd Z)II
11 2.6 -- -. --
100 --
15 0.0 15 0.21 15 0.5 15 1.9 15 5.7 -- _.--
065 0.1: 3 2.1 436 0.6, 5 3.3 355 1.6 2.9 947 10.7 5.5 50649.2 8.6 --_-
200 199 99 --
25 25 25 --
0.5 1.5 .2.8
686 1.5 037 10.9 902 19.0
3.1 7.1 6.8
4.4 7.8
984 30.0 078 42.7
6.9 5.6
M
no
25 25 --
_I12
--13
-14
C-86 C-216 C-208 C-81 --C-86 C-216 C-217 C-208 c-34 C-81 --c-70
C-h@‘2 rd h
I (P2 Hyl
dis)I(P2
rd I)Ir
0’2 rd Z)I(- III (P2)1(P2
Hyl
dis)rr
I-
(-- h(P2 rd 1111 (P2 Hyl dis)I(P2 rd Z)n (P2 Hy* dis)1(P2 rd I)11 P2 rd UI(-)II
w%(-III (P2),(P2
-I
(P2 Hyl
Hyl dis)I(P2
dis)n c)n
GROUP
&J -__ -__-_--
state
cl11
3 EXPERIMENTS
-
C-11 w
SIX
-.
NO
--
G Stable (%)
gj
G
G Total (%I
lstagble) (%I
11
2
13
4.2
1.5 10.0 42.4 ---
O.! l.! 3.f
2.0 11.6 45.5
2.6 5.2 7.4
19 3 0 0 ---
5 3 0 5
24 6 0 5
3.8 0.8 0.0 0.0
34.3 1.1 1.1 3.75 2.5 1.1 ---
1.4 4.1 0 0 2.t 0
4.6 0.25 0.3 0.7 0.35 0.2
0 -
0.0
--
-.
I-
14 14 14 14 --
-.
5.0 3.9 5.9 10.2
100 100 100 100 --
13 13 13 13 13 13 --
7.5 4.4 4.2 5.2 7.1 G.3 -. --
70 90 90 80 80 90
12 -
1.9
-
Ba. *
-
-I
100 -
0
-
Q Symbols : no : number of generations elapsed between superinfection and assay M: multiplicity of superinfection N, : total number of plaques scored G: frequency of incorporation of the superinfecting phage g: probability of incorporation of the superinfecting phage N, : total number of colonies scored seg: segregating different subclones b The superinfecting phage was P2 c except for experiments nos. 8 and 14, where P2 rd 1 was used. All G and g values concern incorporation at location I. Among all colonies tested no case of incorporation elsewhere was found, and it was assumed that the results of the free-phage assays also reflect the frequency of incorporation at location I.
whereas their location II was either free (C-208 and C-34) or occupied by the heteroimmune prophage P2 Hyl dis (C-81). The results, compiled in Table 3, indicate that the presence of a heteroimmune prophage in location I reduces the g values to the levels found for substitution of a homoimmune prophage in location I; g seems to be unaffected by the prophage in location phage
II. These experiments demonstrate, therefore, that the probability of incorporation is governed by steric factors rather than by the immunity class of the prophage in the preferred location. The fact that the g values for substitution in location I are somewhat higher than the ones reported for the experiments in group A may be due to different environmental
SUPERIT\TFECTIOT\T EXPERIMENTS
WITH P2
229
I-
lo-‘-e IO++-Q ’
k= I.24
FIG. 4. The frequencyG of incorporationof a superinfectingphageat a free location I in cells carrying a prophagein locationII vs. the multiplicity M of the superinfectionin a “log log plot.” Line k = 1: G = C X M (C = 0.055); Line k = 1.24:G = C X Ml,” (C = 0.046).
Pointsfrom measurements by colony assays:+ (R) : Experimentno. 8; l : Experimentno. 9; X : Experiment nos.12and 13; + (V) : Experimentno. 18.Points from measurements by free phageassays:0: Experimentno. 9; 0 : Experiment no. 10; A: Experiment no. 11.In experimentno. 8, C-67= C(-)1(P2 C)II wassuperinfectedwith P2 rd 1.In experimentsnos. 9 to 13,C-86= C(-)r(P2 rd l)rI wassuperinfectedwith P2 c. In experimentno. 18,C-86was superinfectedwith P2 vir’. conditions (change of laboratory), different experimental techniques (free-phage vs. colony assays; presence vs. absence of antiP2 serum), or different prophage stabilities in the strains employed. Another aspect of the results obtained in t,hese experiments may be mentioned. The progeny of lysogenic cells (carrying prophages of one immunity class only) that survive superinfection with a heteroimmune phage are often cured of their prophages (Cohen, 1959). Sometimes only one prophage is lost from doubly lysogenic cells (Six, 1960). No curing has been observed as a result of superinfection with homoimmune phage. In experiments nos. 12 to 14 the superinfecting phage was homoimmune with respect to one prophage and heteroimmune with respect to the other. Among a total of 570 colonies examined, no stable isolate showed curing. In experiment no. 13, one colony was found which upon restreaking gave a subclone that had lost the prophage heteroimmune with respect to the superin-
fecting phage. This indicates that the curing mechanism may be affected by the immunity of the superinfected cells. C. Superinfection of Lysogenic Cells with Wealc Virulent Phage Weak virulent phages lyse all infected nonlysogenic cells, and are unable to lyse lysogenic cells carrying a homoimmune prophage. The purpose of the experiments nos. 15 to 19 was to test whether the incorporation of weak virulent phages into lysogenic recipients can occur. The results presented in Table 4 may be summarized as follows. No genetic incorporation of the weak virulent phage is found if the superinfected cells are not immune (experiments nos. 15a and 16). Incorporation can occur if the superinfected cells are homoimmune (experiments nos. 15b, 17, 18, 19). Apparently this genetic incorporation is quite stable: from 14 isolates C(P2 Hyl dis vir14) (P2 Hyl dis) obtained in experiment no. 19 a total of 140 subcloneswas tested; all showed
ERICH
230
TABLE RESULTS Expt. no.
Strain
15a 16 17 15b 19 18
C-83 c-71 c-70 C-81 C-81 C-86
1OOC 77.3d 97 88.5 90.0 64 a Symbols A, B = S = (-) = no = Pi = N = seg. = g = b Note that at the location c Growth found to be d Only 60 P2 c, 1 only
4
OF SUPERINFECTION
EXPERIMENTS,
stage of strain (@II
Superinfecting
Lysogenic (Ah
(-- )I (P2 Hy’ 0’2 rd I)103 (P2 Hyl (P2)r(P2 (PZh(P2 (-hU’2
Frequencies
(A) 03)
SIX
(-)@I 14.3d 2 1.5 0
(S) 031 0 Od 1 0 7.0 36
P2 P2 P2 P2 P2 P2
dis)n ch dis)r (P2 c)rr Hyr dis)rr Hyr dishi rd l11r
(yc)
of different (A)(-) 0 1.3d 0 5.5 0.5 0
lysogenic (A) (3 0 Od 0 2.5 0 0
vir’ Hy’
GROUP phage
S
dis virl"
vi+ vir'
Hvl
dis vi+
vi;'
types c-1
C-1
no
M
N
40 10 15 18 11 17
3.5 4.9 2.1 6.0 22.4 4.0
100 100 100 200 200 109
carrying”: seg.
(9
7.0 0 1.0 0 -
: old prophages in locations I and II superinfecting phage and new prophage no prophage number of generation times elapsed between superinfection and multiplicity of superinfection total number of colonies tested segregating different subclones probability of incorporation of S (S)(B) and (A)(S) do not necessarily indicate that the virulent of the old prophage which was lost. was observed only after an unusually long incubation period. P2 resistant. colonies liberating P2 rd 2 were retested for the presence of P2 P2 rd 1 (= C-%X3), 1 only P2 c.
both prophages P2 Hyl dis vir14 and P2 Hyl dis. One subclone was grown in LS broth for at least 30 generations, then 20 new subclones were tested. Again all showed both phage types. In experiments nos. 17 to 19 the preference for genetic incorporation at location I was again observed. The g values for the probability of genetic incorporation by substitution or addition are in good agreement with those found for superinfection with temperate phage. When strain C (P2) r (P2 Hyl dis) rI was superinfected with P2 virl (experiment no. 15b) incorporation of P2 vi+ was coupled with loss of the prophage in location II
CQ
0 0 0 0 0 0
0 0 0 1 2.5 0
0.5 0.1 0.3 9.0
assay
phage One
(S) became colony
attached
was tested
c; 58 had both;
and
P2 rd I and
rather than in location I, as would be expected from the rule of “preference of location I.” This apparent exception indicates once more that the continued presence of a homoimmune “temperate” prophage is a necessary condition for the prophage incorporation of a weak virulent phage. The g value (0.14%) is lower than for substitution in location I. One isolate C (P2) (P2 vi+) from this experiment was crossed with a nonlysogenic F- strain (C-51 ; Bertani and Six, 1958)) in an attempt to derive a strain carrying only P2 vi+. Among 240 recombinants, none was lysogenic for P2 vi+ only, although some (2.5%) carried both P2 and P2 vi+. Another
SUPERINFECTION
EXPERIMENTS
attempt to obtain a strain C(P2 vi+) by crossing an F- strain lysogenic for P2 virl and P2 rd 1 with a nonlysogenic F+ strain also failed. These findings again indicate the requirement for presence of a temperate homoimmune prophage for the incorporation of a weak virulent phage. DISCUSSION
In a discussion of the experiments3 it is important to note that the incorporation of P2 and its dismune variants P2 Hy dis occurs preferentially at one site of the bacterial chromosome called location I. This has been found for lysogenization of nonlysogenic cells with either P2 or P2 Hy dis (Bertani and Six, 1958), for genetic incorporation of either P2 or P2 Hy dis into lysogenic cells carrying only prophages heteroimmune with the superinfecting phage (Six, 1960)) and for the incorporation of P2 into lysogenic cells carrying a homoimmune prophage in location I, either alone or with a second P2 prophage in another location (Bertani 1954; Bertani and Six, 1958). The results reported here are in agreement with the rule of preference for location I, especially since it could be shown that in cells carrying only P2 in location II genetic incorporation of the superinfecting phage occurred by addition of a new prophage, without loss of the P2 in location II. This was found not only for the incorporation of P2 but also for P2 Hy dis. The reason for the preference for -location I remains unknown. It is possible that the different attachment sites found so far possess different degrees of affinity for P2 and P2 Hy dis. Another explanation may be an inhomogeneity of the phage lysates in the sense that the majority of phage are able to attach themselves only at one location (I), whereas a minority of phage have affinity to different sites as a result of some genetic event (mutation or recombination with the bacterial genome). A similar behavior has been found meanwhile to occur ‘In the discussion the symbol to indicate any phage with the erties of phage P2, as contrasted phage P2 Hy dis.
P2 will be used immunity propwith those of
WITH
P2
231
for another episome, the sex factor F (Jacob and Adelberg, 1959). Experiments are under way to examine this question for P2. The following conclusions can be drawn concerning the incorporation of superinfecting P2 at location I in cells carrying one or more P2 prophages: The probability of addition of a new prophage at an empty location I is about 40 times higher than the probability of substitution at location I. This difference is apparently due to a steric hindrance caused by the presence of a prophage at t’he preferred location. This steric hindrance does not depend on immune specificity because it is exerted equally by a homoimmune or a heteroimmune prophage at location I. There is no indication that the substitution of a location I prophage is hindered by the presence of a second prophage in another location. The full additivity of the incorporation chances of phages in multiple infection for multiplicities up to 10 supports the hypothesis (Bertani, 1953) that no multiplication of the genome of the superinfecting phages occurs in immune cells. It follows that the probability of incorporation of a phage should reflect the affinity of the prophage attachment site(s) in the bacterial chromosome for the nonincorporated genetic determinants of the infecting phage or “preprophage.” (Note that this definition of the “preprophage” does not specify an inability to multiply.) The data presented here do not reveal any significant difference for the probability of incorporation of P2 carrying the c or the c+ markers. Since this pair of alleles affect the frequency of lysogenization (L. E. Bertani, 1959), the difference in this frequency cannot be due to a difference in the affinity of the attachment site for phage with the c or the cf allele. This conclusion is supported by the results with weak virulent mutants. Although they have completely lost their ability to lysogenize, these mutants can become prophages and be transmitted as such over many cell generations if another prophage of the homoimmune temperate type is present. If this temperate prophage is eliminated by genetic recombination in a bacterial cross or by another superinfection (Six, unpublished)
232
ERICH
the virulent prophage, no longer stabilized, will be lost, either by lysis or by curing. The possibility that the virulent prophage attaches itself to the pre-existing prophage rather than to the bacterial chromosome is made unlikely by the observation of steric hindrance for the genetic incorporation of P2 virl. The observed failure of a P2 Hy dis prophage to stabilize P2 vi+ indicates instead that immunity provides the necessary stabilization. A weak virulent phage appears to possess only the passive immune specificity; i.e., it recognizes the immunity of a lysogenic cell but cannot itself produce immunity. This conclusion is in agreement with the interpretation of Jacob and Campbell (1959) for the case of hC (a weak virulent X, comparable to P2 vi+). Another group of observations (Campbell, 1957) may also be quoted in support of the views presented here. From the data on gal-transduction it is possible to estimate the probability of genetic incorporation of the transducing phage Agal, which is unable to multiply by itself in a nonlysogenic cell, but can do so in mixed infection with A. The probability for gal transduction in single infection of nonlysogenic (or lysogenic) cells is only about one-twentieth of the probability for transduction in mixed infection of nonlysogenic cells. The ratio indicates a g value of 0.05, the same as that found for attachment of P2 to a free location I. The probability of gal transduction in mixed infection of cells lysogenic for h is as low as the probability in single infection; this supports the hypothesis that immunity prevents the multiplication of preprophages. For general transduction by phage Pl or P22 the ratio of the frequency of stable transduction to the frequency of stable plus abortive transductions may be interpreted as the probability of genetic incorporation of the transduced chromosomal piece. Although there is a rather wide variation in the available data’ (Ozeki, 1956, 1959), the ratio of the transduction frequencies is of the same order of magnitude as the g values calculated for P2 and Agal. While the incorporation of a phage as pro-
SIX
phage is certainly not the same as the integration of a transduced fragment into the bacterial genome, the similarity of the incorporation probabilities may reflect a similarity in these processes. In conclusion some further remarks may be made concerning the role of immunity. The observation that steric hindrance, while affecting the genetic incorporation of a phage, is not related to the immune specificities of the phages and prophages involved lends support to the notion that immunity must be caused by a specific, prophage-controlled “immunity substance” (Bertani, 1956) or “repressor” (Jacob and Campbell, 1959) produced in the lysogenic cells. At the time being, it seemssufficient to assumethat the mechanism of immunity involves only the prevention of multiplication of the phage genome. The lack of phage production in immune cells would be a consequence of this hypothesis. Upon infection of nonimmune cells, multiplication of the phage genome need not occur only in those cells that produce phage and lyse. There is evidence (see discussion in Luria et al., 1958) that in cells that become lysogenic multiplication of the phage genome precedes the establishment of the prophage. In particular, this view is supported for P2 by the experiments of L. E. Bertani (1957). It seemsreasonable to assume that infection of nonlysogenic cells with P2 leads to the formation of a pool of 100 or more preprophages. A g value of 0.05, as found for free location I, is then high enough to assure lysogenization of almost all cells surviving the infection as has indeed been observed (L. E. Bertani, 1957). ACKNOWLEDGMENT Dr. G. Bertani and Dr. A. Campbell have contributed greatly to this work through many stimulating discussions. REFERENCES BERTAX, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacterial. 62, 293-300. BERTANI, G. (1953). Lysogenic versus lytic cycle of phage multiplication. Cold Spring Harbor Symposia
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G. (1954). Studies on lysogenesis. III. Superinfection of lysogenic Shigella dysenteriae with temperate mutants of the carried phage. J.
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50-57. G. (1957). A dismune mutant of temperate phage P2. Bacterial. Proc. (Sot. Am. Bacteriologists), p. 38. BERTANI, G. (1958). Lysogeny. Advances in Virus Research 5, 151-193. BERTANI, G., and SIX, E. (1958). Inheritance of prophage P2 in bacterial crosses. Virology 6, 357-381. BERTANI, L. E. (1957). The effect of the inhibition of protein synthesis on the establishment of lysogeny. Virology 4,53-71. BERTANI, L. E. (1959). The effect of ultraviolet light on the establishment of lysogeny. Virology 7,92-111. CAMPBELL, A. (1957). Transduction and segregation in Escherichia coli K12. Virology 4,366384. COHEN, D. (1959). A variant of phage Pb originatBERTANI,
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ing in Escherichia coli, strain B. Virology 7, 112126. JACOB, F., and ADELBERG, E. A. (1959). Transfert des caracteres genktiques par incorporation au facteur sexuel d’Escherichia coli. Compt. rend. mad.
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F., and CAMPBELL, A. (1959). Sur le systeme de repression assurant l’immunite chez les bacteries lysogenes. Compt. rend. acad. sci. 248, 3219-3221. LURIA, S. E., FRASER, K., ADAMS, J. N., and BURROUS, J. W. (1958). Lysogenization, transduction, and genetic recombination in bacteria. Cold Spring Harbor Symposia Quant. Biol. 23, 71-82. OZEKI, H. (1956). Abortive transduction in purine requiring mutants of Salmonella typhimurium. Carnegie Inst. Wash. Publ. No. 612,97-166. OZEKI, H. (1959). Chromosome fragments participating in transduction in Salmonella typhimuJACOB,
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E. (1959). The rate of spontaneous lysis of lysogenic bacteria. Virology 7,328-346. SIX, E. (1960). Prophage substitution and curing in lysogenic cells superinfect.ed with hetero-immune phage. J. Bacterial. 80,728-729. SIX,