Inheritance of prophage P2 in bacterial crosses

VIROLOGY

6,

357-381 (1958)

Inheritance

of Prophage G. BERTANI

Department

of

P2 in Bacterial

Crosses’

AND E. S1x2

Medical Microbiology, University of Southern California of Medicine, Los Angeles 33, California, and Los Angeles County General Hospital

School

Accepted May 18, 1958

Prophage P2 may be located on the genetic chromosome of the host cell coli strain C) by crossing genetically marked lysogenic strains. At lysogenization, prophage P2 establishes itself at a specific locus (location I or preferred location). In the process of double lysogenization by means of superinfection of a singly lysogenic strain, the second prophage establishes itself at a different locus. Two such second-choice locations are recognized. In superinfection of doubly lysogenic strains, substitution of the prophage in location I occurs more often than substitution of the prophage in the second-choice location. A temperate phage closely related to P2, but belonging to a different immunity class, behaves like P2 in respect to location choice. In doubly lysogenic strains the prophage in the preferred location contributes more heavily than the other prophages to the phage yield of the lysogenic cells. (Escherichia

INTRODUCTION

Work with the temperate phage X (Lederherg and Lederberg, 1953; Appleyard, 1954; Wollman, 1953; Jacob, 1955; Lennox, 1955) indicates that from the point of view of formal genetics the relationship of prophage x to the bacterial cell that carries it is not different from that of a welllocalizable genetic marker. The only exception to this rule is the phenomenon of zygotic induction (Jacob and Wollman, 1956; Jacob, 1955), whereupon a nonlysogenic cell receiving that part of the bacterial 1 Supported by grants from the National Science Foundation (G-3841) and from the Unit,ed States Public Health Service (E-1839). Part of the work presented here was done while the authors were at the California Institute of Technology, Division of Biology, their work being aided by a grant from the Nat,ional Foundation for Infantile Paralysis. * Fellow of the Deutsche Forschungsgemeinschaft. Present address: MaxPlanck-Institut fur Biologie, Abt. Weidel, Ttibingen, West Germany. 357

358

BERTANI

AND

TABLE ABBREVIATIONS

rd 1 b

= = = = ks = Hy =

Hfr P2r s’, SI UriTl ,try+

VW P@I (-) T, CT

1

USED IN THIS ARTICLE

Phage properties round large plaque-type temperate mutants of phage P2 (see Bertani, 1954) blue clear 1 dismune in respect to P2 (see Cohen, 1958) hybrid [between P2 and a prophage carried by E. coli B: see Cohen (1958)] Bacterial

ArgF+, F-

SIX

properties

= requiring arginine for growth = fertility types (see Cavalli et al. 1953; Hayes, 1953a) = high frequency of recombination (see Cavalli et al., 1953; Hayes, 1953a) = unable to adsorb P2 = streptomycin-sensitive and -resistant, respectively = requiring uridine for growth = resistant to phage Tl and requiring tryptophan for growth (one mutation) = wild-type allele = lysogenic for phage P2 = lysogenic for P2, with prophage in location I = not lysogenic for PI = followed by symbol of character(s) = frequency of transfer or cotransfer of the character(s)

chromosome containing X prophage in the course of a bacterial cross or of a transduction experiment often lyses as though it had been infected by a X phage. Similar work with the temperate phage P2, unrelated to X, is reported in this paper. The questions asked are essentially the following: (a) Can prophage P2, like X, be considered the equivalent of a chromosomally localizable bacterial genetic marker? (b) Are independently established P2 prophages allelic to one another? (c) Are the two PW prophages present in doubly lysogenic bacteria inherited together, as seems to be the case for X (Appleyard, 1954), or are they differently localized on the bacterial chromosome? (d) Is there zygotic induction with prophage P2, which is not inducible by ultraviolet light, as with X, which is inducible by ultraviolet light? For a definition of terms used in this paper and for a more general dis-

INHERITANCE

OF

PROPHAGE

Pd

IX

BACTERIAL

CROSSES

359

cussion of the problems considered, the reader is referred to a recent review by Bertani (1958). See Table 1 for abbreviations used. MATERIAL

AND

METHODS

Phages and bacteria. The temperate phage Pf?, its plaque-type mutants rd, 1, b, and c, its bacterial hosts, strains Sh and Xh/s of Xhigella dysenterise, and the techniques and media usually employed in the study of this material have been described before (Bertani, 1954). In addition, P.2 Hy dis, a hybrid between phage Pd and a prophage carried by strain B of Bscherichia coli (Cohen, 1958) was used. The bacterial strains used are listed in Table 2. They are all derivatives of either strain Sh of Xhigella dysenteriae, or strains C (n-0. 122, Kationa,l Collection of Type Cultures, London) or K-12 of Xscherichia coli. The derivative strains were obtained in various manners: (a) by Iysogenization. From a phage spot or plaque on a sensitive strain, a lysogenic clone is isolated from the secondary growth. Such strains are in general quite stable. (b) by superinfection of a lysogenic strain. This type of experiment has been the subject of a previous report (Bertani, 1954). In general, several cell generations are allowed to pass between superinfection and plating out for colonies. Some of these colonies are prophage-substituted clones (they carry the superinfecting phage type, in place of the pre-existing prophage); others are doubly lysogenic (they carry both the pre-existing prophage and the superinfecting type). Sometimes, in the process of substitution, genetic recombination occurs. If the superinfected strain is already doubly lysogenic, substitution of either one of the two prophages or triple lysogenization occurs. It should be noted that these experiments involve several hours of growth for the superinfected culture, at a low cell concentration to avoid readsorption of the phage present in the culture. In quantitative experiments (those of Table 6, for example) to avoid excessive fluctuations in sampling, care was baken that the total number of bacteria in the culture or series of cultures to be eventually examined for phage types carried was never below 104. (c) by prophage recombination in a doubly lysogenic strain. New combinations of prophage types are obtained in this manner. Prophage recombination occurs spontaneously. Its frequency (of the order of 10e3 per cell generation) can be increased by irradation of the cells with ultraviolet light. (d) by contact with an F+ strain. F+ and F- are symbols pertaining to

360

BERTANI

AND

TABLE LIST OF DERIVATIVE

c-02 c-2 c-3

F+ (P2) F+ (P2) Ff F- Arg-

C-6

F- Arg-

1’1, try-

C-8

F- Arg-

Tl, try-

C-10 c-13 c-14 C-16 c-23

FFFFF-

C-01

C-26 C-27 C-28 c-29 c-31

C-84 c-41

F+ FFF+

C-67

F-

C-63

C-64 C-66 C-67 C-72 K-9 K-lf

C-2, by lysogenization C-2, by lysogenization C, by exposure to F+ C, by mutation; isolated by Dr. M. Lieb C-9, by spontaneous mutation to resistance to phage Tf C-5, by mutation, following irradiation with ultraviolet light C-8, by lysogenization C-8, by lysogenization C-8, by lysogenization C, by lysogenization C-14, by superinfection

Sr

C-8, by lysogenization C-2, by lysogenization C-8, by lysogenization C-2, by lysogenization C-19, by lysogenization C-16, by exposure to F+ C-28, by prophage recombination following irradiation with ultraviolet light C-27, by superinfection C-8, by mutation C-14, by superinfection C-60, by spontaneous prophage recombination C-28, by mutation

(PR rd 1 c)(Pd) Arg- Tl, try- Sr Pdr Arg- Tl , try- Sr (P2) (P2 c) (P2 c) (P2 rd I)

ArgTi ,trySr (P2 rd b) (P2 c) P2r F+ (P2 Hy dis) F- UriF+ UriF+ (P2 c) F- Arg- Tl ,try- Sr Plr (P2) _---___----__---F+ Hfr

C-2, by lysogenization C, by mutation C-64, by exposure to F+ From a cross C-14 X C-66 C-13, by mutation ------------------

U’f)(~) (X) (plus other

STRAINS USED Origin

Arg- Tl, try- ~9 (P2 c) Arg- Tl, try- Sp (P2) Arg- Tl, try- S’ (P2 c) (P2) Arg- Tl, tr?/- Sr (P2 rd b) u9 c) F- Arg- Tf , try- Sr (P3) F+ (P2 rd I c) F- Arg- Tf ,try- L? (P2 rd 1) F+ (P2 c) F- Arg- Tl , try- Sp (P2) (PS) F+ (P2) F- Arg2’1, try- Sr (P2 rd b) (P2 rd c)

c-51 C-63 C-66

C-60

2

BACTERIAL

Genetic symbol

strai

SIX

markers)

-

K-12, by lysogenization derivative of A @, prototrophic K-12, obtained from Dr. W. Hayes, who got it by recombination from his originally methionineless Hfr strain (Hayes, 1953b)

INHERITSKCE

OF

PROPHAGE

TABLE Strain

Genetic symbol

Sh-2 Sh-6

(P2 rd) (P2 rd 1)(P2 rd)

Sh-10 Sh-17 Sh-37 Sh-39

(P2 rd L)(PZ)

Sh-65

(PZ)(PZ

Sh-58

(P2 c)(PZ

Sh-59 Sh-60

(P2 l)(PZ rd 1) (P2 rd 1)(P2 1)

Sh-62

(I'2 rd)(PZ

bw (P2 rd I) (PZ)(I'2 7-d 1)

rd I)

rd 1) PZr

1)

Pb

IN

BACTERIAL

CROSSES

361

P-Continued Origin

Sh, by lysogenization Sh-IO, by prophage recombination following irradiation with ultraviolet light Sh-37, by superinfection wit,h P2 Sh, by lysogenization Sh, b,v lysogenization Sh-17, by superinfection with P2 ~1 1 (experiment of Fig. 2, Bertani, 1954). Sh-17, l)y superinfertion with I’2 rd 1 (experiment h, Table 1, Bertani, 1954). Sh, by lysogenization with P2 c, followed by superinfect,ion with P2 rd 1 (experiment b, Table 1, Bertani, 1954), and mut,ation to P2' Sh-39, after superinfection with P2 1 Sh-62, by prophage recombinatjion following irradiation wit.h ultraviolet light Sh-2, by superinfection wit.h I’2 I

different “sexual” conditions in bacteria (Cavalli et al. 1953; Hayes, 1953a). Genetic recombination can be demonstrated in F- X Ff and (to a lesser extent) in F+ X Ff crosses, not in F- X F- crosses. F- cells exposed to F+ cells become F+ themselves, with high frequency. To make a strain F+, it was grown together with F+ cells (usually of strain K-12) and then reisolated, taking advantage of some other different)ial property of the original F- strain. In our material (using LB medium) Ff cultures always showed a tendency to clump (see Maccacaro and Comolli, 1956). (e) by bacterial cross. This consists in mixing cells of two bacterial strains (not both F-) so that contacts between cells and genetic recombination may occur. When the recombinants are rare, as in all our experiments, selective media must be employed. For general information on bacterial crosses see Wollman et al. (1957). (f) by mutation. For general techniques in isolating bacterial mutants, see Lederberg (I 950) and Witkin (1950).

362

BERTANI

AND

SIX

Standard cross. Much of the work to be reported involves one type of bacterial cross, performed as follows. Standing cultures in LB medium or in nutrient broth of the two parental strains, usually having reached a titer of 8 X 10’ to 2 X lo8 per milliliter, are centrifuged. The cells are resuspended in a nonnutrient medium (saline, or phosphate buffer at pH 7), centrifuged and resuspended twice more, the final volume being only xc to +dQc of the original volume. The two concentrated cultures are mixed in approximately 1: 1 ratio, and plated at various dilutions by spreading on the surface of a synthetic agar medium (7 g K,HI’Od , 2 g KHsP04, 0.5 g Na-citrate.5Hz0, 1 g (NH&Sod, 0.1 g MgS04 , 1 g glucose, 14 g Difco-agar, 1 g asparagine, 250 mg streptomycin, 1 liter distilled water). The plates are incubated 48 hours at 37”. The colonies obtained are picked (preferentially from the least-crowded plates) and streaked out on nutrient agar. From each streak, one subcolony is picked and tested for its nonselected genetic markers. In a few instances, the colonies obtained in a cross were tested directly. The frequency of genetically mixed clones which are recovered by the simplified technique, is usually less than 15 %. The parent strains in the standard cross are an F- Arg- Tl , try- ST strain (eit:her C-8 or a derivative of it) and an F+ Arg+ Tf , try+, x8 strain. The selective markers in the standard cross are thus the normal alleles of the requirements for arginine and for tryptophan, and resistance to streptomycin. The two parental strains, of course, will differ for other, unselected properties, varying from cross to cross. At the highest concentrations of parental bacteria plated (log per plate), up to 1000 recombinant colonies per plate were obtained. Analysis of bacterial crosses. The genetic studies to be reported here were not carried on to the point of building a genetic map of the bacterial host cell. The results of the various bacterial crosses are therefore formally expressed as frequencies of transfer of the various unselected markers. The frequency of transfer of a given unselected marker is expressed as the fraction of colonies obtained from a certain F- X F+ or F- X Hfr cross, which carry the allele that was present in the Ff or Hfr parent. The frequency of transfer of a marker is of course a function of the linkage relationships of this marker to the selective markers. The frequency of cotransfer of any two or more unselected markers, is given by the fraction of colonies that carry the F+ alleles for each of the two or more unselected markers considered. When the frequency of cotransfer of two markers is significantly different from the product of the

INHERITANCE

OF

PROPHAGE

Pd

IN

BACTERIAL

CROSSES

363

frequencies of transfer of the two markers under the same selective conditions, there is evidence for linkage between the two markers.3 Testing lysogenic colonies for type(s) of phage carried. In many instances the streptomycin method previously described (Bertani, 1954) was used. When optimal differentiation of plaque types was needed, the phage being tested was preadsorbed to the indicator bacteria (3 drops of indicator bacteria with 0.0025 M CaClz added) at 37” for approximately 8 minutes, and then plated. The streptomycin technique could not be applied to; f&e recombinant colonies obtained in the standard cross, which are necessarily streptomycin-resistant. With such isolates the sample of lysogenic bacteria to be tested for type of phage carried was first exposed to 58-60’ for 30 minutes (in LB broth with NaCl and MgS04 added to reach 2% and 1O-4 M concentrations respectively) to kill the majority of the bacteria. Most of the phage survived such a treatment. Properties of doubly lysogenic strains. Strains doubly lysogenic for PW have two properties, both described before, which are of diagnostic importance. (a) There are some mutants of Pz? which can form plaques on singly lysogenic strains, but do not, or do so with an extremely low efficiency, on doubly lysogenic strains. By spotting a loopful of a culture on agar coated with a preparation of one such mutant, it is possible to distinguish very easily singly from doubly lysogenic strains (Fig. 1). This test permits the recognition of strains carrying a double dose of genetically identical prophagex. The method works very well with derivatives of strain Sh: nine independently isolated singly lysogenic strains and five partially or completely independently isolated (by superinfection of a singly lysogenic strain) doubly lysogenic strains showed no exception in their behavior toward ten different virulent mutants of PZ, two of which gave plaques on the singly, but not on the doubly lysogenic strains 3 The correctness of this statement is not independent of the type of recombination mechanism assumed. The statement is certainly valid if the elementary recombination event.s are randomly distributed along the genetic material, assumed t,o be one-dimensional. Accepting the interpretation of F- X F+ crosses given k),v Jacob and Wollman, one has to assume further that many di$erent Hjr clones exist in the F+ cultures used [compatible with the findings of Jacob and Wollman (1957)], and that t)heir relative frequencies do not vary very much from culture to culture (compatible with the relatively small fluctuations in the frequencies of transfer observed in repeats of the same cross; see Results section).

364

BERTANI

AND

SIX

FIG. 1. Spots produced by growth of loopfuls of cultures of various derivatives of strain Sh on agar coated with several thousand particles of phage Pi? vi+ (a spontaneous intermediate virulent mutant, of Pi?). (a) Nonlysogenic; (b) singly lysogenic; (c) doubly lysogenic.

(“intermediate” mutants), four or all strains (“strong” mutants), and the other four on none of the lysogenic strains (“weak” mutants). The size and number of plaques obtained in such tests seem to be affected by the genetic markers of the phage(s) carried by the lysogenic strains, but, as far as derivatives of strain Sh are concerned, this effect is not large enough to make less certain the classification. This cannot be said, however, of derivatives of strain C, where perhaps other complicating factors occur. It is sufficient to say that the method can be usefully applied to derivatives of strain C, but it gives unequivocal results only when the genetic background of the host cell and the genetic markers of the phage carried are not varied. (b) Strains doubly lysogenic for PZ only exceptionally (with certain combinations of phage markers?) liberate phages of the two carried types in 1: 1 ratio : usually one of the two types predominates in the phage yield (Bertani, 1956). In many experiments this property of doubly lysogenic isolates was routinely scored. The results of these observations are given at the end of the next section.

INHERITANCE

OF

PROPHAGE

Ps$? IN

BACTERIAL

CROSSES

365

RESULTS

In standard crosses involving strain C-13 as the F-, lysogenic parent, and strain K-12 as the F+, nonlysogenic (for phage P2) parent, approximately 30% of the colonies obtained are not lysogenic for P2 (Table 4, experiment b). Such a frequency of transfer is not independent of the markers used to select for recombinant colonies, as shown by performing the same C-13 X K-12 cross on partially supplemented agar, to use either only Tl , try+ and ST, or only Rrg+ and ST, as the selective markers (Table 3, experiment b). Substitution of the F+ strain with a known Hfr strain (Table 4, experiment c, and Table 3, experiment c) does not seem to alter the results of the cross, except for the fact that the Tl , try locus appears to be possibly within the region of high frequency of transfer typical of the Hfr used. TABLE BACTERIAL

EXperi men

Parents

3

CROSSES ON PARTIALLY

-

Unsld&~d

SUPPLEMENTED AGAR~~

Analysis of colonies obtained on minimal agar supplementedwith tryptophan ___~

Analysis of colonies obtained on minimal agar supplemented with arginine ..____--

(1’2 c) C-1

T(-) = 2/48 = 0.04 Among Tf ,try+ colonies: T(-) = 13/34 = 0.38 Frequency of 7’1, try+ colonies = approximately 0.01

T(-) = l/85 = 0.01 Among nrg+ colonies: T(-) = ‘i/25 = 0.28 Frequency of A rg+ colonies = approximately 0.01

Wh (-)

T(-) = 16/189 = 0.085 Colony yield = 5 X 10-e of K-12 input cells

T(-) = z/l91 = 0.01 Colony yield = 5 X 10m6 of K-12 input cells

T(-) = s/95 = 0.03 Colony yield = 2 X lo-” K-16 input cells

T(-) = o/95 Colony yield = 3 X lo-” of K-16 cells

a

-b

F- C-13 F+ K-12

C

F- C-13 Hfr h’-16

I-VW1

-I

C-1

of

u Crosses were performed by the standard technique, except for supplementing the minimal agar with tryptophan or arginine. The supplement concentration in the agar was 20 pg/ml in experiments a and b, 10 rg/ml in experiment a. As a it was possible in experiment, a to di&nguish on the plates consequence, Srg+ !l’f ,try+ colonies, since they grew larger than the iirg1’1 ,try+ or Arg+ Tf , try colonies. Appropriate controls indicated that the number of spontaneous reversions to either Arg+ or 7’1, try+ was well below the number of colonies due to recombination.

366

BERTANI

AND

TABLE STANDARD

-

EXperimen1t

BACTERIAL

Parents

CROSSES

Unselected markers

SIX

4

INVOLVING

SINGLY

LYSOGENIC

Analysis of colonies obtained on minimal medium

a

F- C-10 F+ K-12 ------

(PZ cl C-1 ----~---~

T(-)

= so/%

b

F- C-13 F+ K-12

VW1 C-1

T(-)

= 37/114 = 0.32

c

F- C-13 Hjr K-16 -. __----_--------

(WI C-1

T(-)

= 12/49 = 0.25

d

F- C-8 F+ K-9 _-------~------

C-1 VW

T(P2)

.~

--e --

F- C-10 F+ c-2 -. ___---

f

-. g ---

-.

h _--.

= 0.31

= 24/80 = 0.30

T(-)

= ‘to/98 = 0.40

= 30/81 = 0.37

F- C-13 F+ C-2

(WI C-1

T(-)

F- C-8 F+ C-34

C-1

T(P2)

= 46/99 = 0.46

T(P2)

= 31/100 = 0.31

T(P2)

= 42/100 = 0.42

F- C-61 F+ C-01

_ _---~-__------F- C-61 F+ C-02

i

(P2 c) C-1 __-------

STRAINS’”

WI C-1

WI (-)

--

uw

F- C-51 F+ C-63* -. __----___------

(-) (PZ Hy dis)

T(PZ Hy dis)

k

F- C-13 F+ C-63* -. __----__-------

VW1 (P2 Hy dis)

T(Pf

1

F- C-28 F+ C-29

(P2 rd 1 +) (P2 + + c)

T(PZ + + c) = 47 (including two colonies carrying a recombinant prophage)/lOO = 0.47

m

F- C-31 F+ C-27

(PZ + + +)I (P2 rd 1 c)

T(Pd

j ---

-

Hy dis)

= 5/20 = 0.25

= 36/100 = 0.36

rd 1 c) = 45 (including two colonies carrying a recombinant prophage)/lOO = 0.45

INHERITANCE

OF

PROPHAGE

Pd

IN

BACTERIAL

CROSSES

367

TABLE 4--Continued

T

Experi. men1

Parents

F- c-13 F+ c 27

(P2 + + +)I

F- C-28 F+ C-27

T(P~ rd 1 c) = 21 (including seven colonies carrying a recombinant prophage)/80 = 0.26

(P2 rd 1 c)

F- C-72 F+ (r-34 P

Analysis of colonies obtained on minimal medium

Unselected markers

(P2 rd 1 +) (P2 rd 1 c)

/ Nonlysogenic

= O/236

I-Nonlysogenic

= O/200

-i

a Crosses were performed by the standard technique. In experiments h, i, n, o, p, exceptionally, the colonies were tested directly without previous purification. As a consequence, in experiment n 6 colonies were found to produce more than one kind of phage; such colonies were then streaked out, and one subclone from each streak was re-examined for type of phage carried. Each of these subclones liberated only one kind of phage. Experiment c (involving an Hfr parent) gave a yield of recombinant colonies not higher than usual. * For a peculiarity of C-63 see footnote 4 in text.

Substitution of the F- with C-10, which differs from C-13 in that it carries a plaque-type mutant of PW, and represents a different lysogenization event, does not seem to alter the results of the cross (Table 3, experiment a, and Table 4, experiment a). The experiments described above suggest that (a) the presence vs. absence of P2 prophage can be considered to be a localizable genetic marker of the bacterium, and (b) the location of such a marker is the same for the two independent lysogenization events that occurred in C-13 and C-10. The chromosomal location of prophage P2 in strain C-13 will be considered the standard or preferred location of P2, or location I. The conclusion above is confirmed by the other crosses presented in Table 4. These involve ten independent lysogenization events, as one can reconstruct from the list of strains used (Table 2). Each event is the result of infection of a cell, a derivative of either K-12 (one case, K-9), or C (nine cases), with either P2 or any one of several coimmune mutants of P2, or the dismune P2 Hy dis. Of the ten independent lysogenization events, four had occurred in F- strains, and six in F+ strains. Standard crosses4 of three types in respect to carried P2 4 The crosses involving strain c-63 (experiments properly be considered to be of the standard type,

j and k, Table 4) may not since it was later found that

368

BERTANI

AND

SIX

were performed: (A) between a lysogenic F- and a nonlysogenic F+, (B) between a nonlysogenic F- and a lysogenic F+, and (C) between a lysogenie F- and a lysogenic Ff of independent origin. A series of five crosses of type (A) involved two independent lysogenization events (Table 4, experiments a, b, c, e, and f), and gave a transfer, frequency between 0.25 and 0.40, the weighted average being 0.34. A series of five crosses of type (B) involved five independent lysogenization events (Table 4, experiments d, g, h, i, and j), and gave transfer frequencies between 0.25 and 0.46, the weighted average being 0.37. The six crosses of type (C) involved six independent lysogenization events (Table 4, experiments k, 1, m, n, o, and p), three of which had been already tested in experiments of types (A) or (B).The transfer frequencies in the four crosses where they were measured, varied between 0.26 and 0.47, the weighted average being 0.39, and nonlysogenic or doubly lysogenic recombinants were not found. In two crosses of type (C) recombinant colonies were simply scored for lysogeny(rather than for type of phage carried) : no nonlysogenic recombinants were found. These results again suggest that in each case the two prophages were located at allelic sites. Most of the crosses of Table 4 involve strains able to adsorb P2. One may ask whether in crosses of type (B), infection on the plate, leading to lysogenization, may not simulate transfer of Pi? prophage. This is, however, unlikely to have produced a major aberration in the results since similar transfer frequencies were obtained when the F- strain used was unable to adsorb P2 (Table 4, experiments h and i). Tables 5 and 7 also contain experiments with F- strains unable to adsorb P2 which confirm the validity of the statement above. From the data presented in Table 4 one may very well suspect that the frequency of transfer of prophage P2 in crosses involving a derivative of E. coli C as the F- and a derivative of E. coli K-12 as the F+ is slightly, but significantly, lower than in crosses involving only derivatives of h’. coli C. Such a difference could be due among other possibilities, to zygotic induction of prophage X (carried by the various derivatives of E. coli K-12 used here), and possible linkage between X and P2 prophages. The similarity of values of frequency of transfer of P2 prophage in crosses of types (A) and (B), makes it unlikely that appreciable zygotic induction occurs for phage P2. such a strain had developed a nutritional requirement, which would not be satisfied by the selective medium used in the crosses. It is not known when this requirement arose in the history of the strain.

TABLE STANDARD Parents

a

F- c-14 F+ C-63

BACTERIAL

CROSSES

5

INVOLVING

STRAIN

Unselected markers (final interpretation)

Analysis of colon~;die~~ined

F- (‘-14

CT = b/99

(PI C)I (P.2 ch

r(P2 Hy dis) = 33/80 = 0.41 CT = l/80 = 0.012 __-_-_-----------

(P2 Hy dis)r

(-)

f

--

-R

r(P% +) = 7/20 = 0.35 CT = 2/20 = 0.1 Nonlysogenic = O/336

l-

CT = 3/100

F+ C-2 F- C-68 F+ C-34

(P2 C)I (P2 (P2 +)I (-)

Nonlysogenic = O/181 T(t??) = 155/181 = 0.85

F- C-69 F+ C-27 i

P2’ P2*

C)II

-

i- (P2 C)I (P2 c),, P2? (P2 rd 1 C)I (-)

F- C-61 F+ C-67

(-, P2r (P2 c)n P2”

h

F- C-72 F+ C-67

(P2 +)I

= 0.03

Nonlysogenic = O/198 T(P2’) = 16/99 = 0.16

PB

T(P2 C) = 7/54 = 0.13 T(P2’) = 5/54 = 0.09 CT = 4154 = 0.07

--

--

= 0.04

(P2 C)I (P2 C)II C-1 C-1

--e

on minimal

(PI C)I (P.9 cl11 C-1 C-1

(P2 C)I w C)II (P2 +)I C-1 d

C-14 AND DERIVATIVES~

_---------------(-)

(P2

(-) C)II

T(-)I = %/loo = 0.39 T(P2 C) = lo/l~ = 0.1 CT = 2/100 = 0.02

P2r P28

T(P2’) = 44/440 = 0.10 CT(~% and (-)I) = 18/440 = 0.041 c~(P2~ and (P2 c)) = 22/440 = 0.050 (!T(PZ*, (-)I, and (P% c)) = 9/440

= 0.0039 ---i -

-

F- C-69 F+ C-67

(P2

C)I

-

(P2

Nonlysogenic

C)II

= O/140

a Crosses were performed by the standard technique. All the colonies in experiments d and i, and 140 in experiment c were tested for lysogeny without previous purification. In experiment h, 340 colonies were tested for the P2’ marker (by streaking across a virulent mutant of P2 able to lyse both singly and doubly lysogenic bacteria) without previous purification; those which appeared to be Pgn were then streaked out, and a subclone of each was tested for type or types of phage carried. Note that the P2r mutants are independent isolations (see Table 2) and thus need not be allelic. 369

370

BERTANI

AND

SIX

Besides the ten independent lysogenic isolates mentioned above, one more, strain C-14, was tested in standard crosses and gave aberrantly low values for the transfer frequency of prophage P2 (Table 5, experiments a and d). It was at first thought (Bertani, 1956) that in such a strain the establishment of the prophage had occurred at a chromosomal site different from that commonly occupied by prophage Pd. It was later found that in crosses between such a strain (or its Pdr derivatives) and strains carrying a prophage P2 in location I (Table 5, experiments b, c, e, and f), no nonlysogenic recombinants were ever obtained, a result incompatible with the above interpretation. It was then thought that in strain C-14 perhaps both location I and a second site might be each occupied by a P2 prophage, thus making the strain doubly lysogenic for two genetically ident,ical prophages. On this assumption, it was possible to isolate a new strain carrying one prophage in the new location and no prophage in location I, by crossing strain C-66, a uridine-requiring mutant of E. coli C, made F+, to strain C-14 (which is F- Arg- Tl , try- Sr, lysogenic for P2 c, and uridine-independent), selecting for prototrophs. Among these (ST lysogenic, 2; Ss lysogenic, 2; ST nonlysogenic, 43; Ss nonlysogenic, 77) an 33 clone was found, C-67, which, upon testing (Table 5, experiments g, h, and i), behaved as expected of a singly lysogenic strain, carrying the prophage in the new location (location II). In standard crosses, location II is transferred independently of location I, but not of a marker, P2?, affecting the ability of the cells to adsorb P2 (Table 5, experiments g and h), a result which is to be attributed to linkage relationships between location II and one of the P2? loci. Location I appears to be transferred independently of such a locus. The frequency of transfer for location II in standard crosses (Table 5, experiments g and h; Table 7, experiments b, c, and d) varied between 0.02 and 0.19 (0.10 and 0.19, if one neglects the cross involving K-12 as a parent), the weighted average being 0.12. The interpretation offered for the case of strain C-1.4 is consistent with the results from superinfection experiments described below. In a previous paper (Bertani, 1954) it was shown that superinfection of a lysogenic cell with phage closely related to the phage type carried may result in the replacement of the carried type with the superinfecting type (prophage substitution) or in the establishment of a doubly lysogenie condition. The latter event occurred in general much more rarely than the former: by pooling all data of Table 1 in the paper referred to above, one obtains 5 cases of double lysogenization as compared with

EX-

C-53

c-14 ----

Strain superinfected

(P2 + + C)II

(P2 + + C)I

/ (P2 c),(PZ C)Il ---__-_-_~ ~ (P2 + + +)I (P2 + + cl,,

I

_-

/(PZ Id 1 (.)I

Pha:c types carried

-

i jP2 rd 1 +

P2 +

P2 rd h +

p2+++

PZftC

Superinfecting phage

-__---

25

7

13

----

9

----

----

9

kdtiplicity of superinfection

-

17

ii

14

14

18

tions elapsed before testing

Genera-

OF SUPERIXFECTION

691

tested

colonies

T-tLi1

160

iii

I

~ 535

i

!

obtained

(P2 c)

(P2 + + c)

/ (P2 rd I f) (P2 + + c) 1 (P2 + + +) (P2 rd l +) i (P9 + + c) (P2 + + C)

~ (P2 f)

(P2 ul b +)

(P2 + + f) (P,P rd I c) (PZ + + f)

Iysogenic

5)

4

;

3

7 c+ 1)

11 (+ 1

2 (+ 1)

2 (see text)

1

colonies

Triply

of eareptional

2 1 1

and numbers

(PZ rd + +) (P2 + + cl (P2 + + f) (I?2 + 1 +I (P2 rd + c) (P2 + 1 +) (P~/xJ+ +)(P2rdl+) (P*9 + I +) (P2 + 1 +)

Types

ESPERIMENTW

I 357

-

”

* All types of colonies differing from the superinfected type are listed in the last column of the table. The figures in parentheses are the numbers of colonies carrying more than one type of phage (in experiments b and c) or more than two types of phage (in experiments a and c), and which upon segregation gave origin to stable exceptional types besides the original superinfected type. In experimentsa and e all the exceptional colonies producing only one type of phage were tested for immunity level, and were classified as doubly lysogenic on this basis. It should be noted that some of the exceptional types obtained in experiment a may have been produced by spontaneous prophage recombinat.ion. This however does not affect our conclusions. The same applies to the last class of exceptional types in experiment e.

b

periITlent

-

RESULTS

% r

g $ E

B

2 $

? 2 m

2

2

% $ 8

s?

$

i F;

2

372

BERTANI

AND

SIX

78 cases of substitution. Such experiments involved strains of Shigella representing five independent lysogenization events. It was also found (Bertani, 1956, Table 2) that in superinfection experiments of doubly lysogenic strains two types of prophage substitution could occur: (a) replacement of the prophage that was first established, and (b) replacement of the prophage that was established in the course of double lysogenization, and that the former occurs much more frequently than the latter. Table 6 gives additional superinfection experiments. Experiment a supports the above statement for superinfection of doubly lysogenic Shigella, by extending the previously published data (Bert,ani, 1956, Table 2; strain used there was Sh-6) to another doubly lysogenic strain of independent origin. Experiment b shows that what usually happens in superinfection of singly lysogenic Shigella strains also occurs for the common type of lysogenic strains of E. coli C (that is a strain carrying one prophage in location I). Experiments c and d, show that upon superinfection &rain C-14 gives the results that one would expect of a doubly lysogenic strain carrying a prophage in location I and another at a different location, and not what one would expect of a singly lysogenic strain carrying its prophage in location I. A later argument (page 375, and Table 9) will indicate that all cases of prophage substitution in experiments c and d involved location I. Experiment e contains a case of prophage substitution in location II. Pooling the data of experiments c, d, and e, the ratio (cases of substitution in location II): (cases of substitution in location I) is equal to l/16, in good agreement with similar ratios established for doubly lysogenic Sh strains. A doubly lysogenic strain C-23 obtained in the last-mentioned experiments, and its derivative, C-41, were tested in standard crosses (experiments b, c, and d, Table 7) to nonlysogenic strains: the transfer frequencies obtained for the two prophages are consistent with the idea that the prophage type acquired in the superinfection experiment established itself in location I. Similarly consistent with this idea are the allelism tests of experiments i and a of Table 7. The latter, comparing the four prophages (all independently established) in the two doubly lysogenic strains C-41 and C-50, furthermore reveals that location II is not allelic with the location (hereafter called location III) occupied by the second prophage of strain C-50, as shown by the recovery of both singly and triply lysogenic strains. Crosses of C-50 and its derivative C-56 to nonlysogenic strains (Table 7, experiments e, f, g, and h) show that location III gives in standard crosses a transfer frequency varying

TABLE

7

Analysis

markers

Unsrlrrtcd

01 colox~~dy~ained

____~

(Z’2rdb), (z’srdc),, (Pd rd / (:)I (--),I

______~~ (P2 /xzb +)I

C

(-)

(P2 d

C-1

All

_ (1’2 + + c)11

T(__)I = 4O/IW = 0.40 T(--_),, = 19/1oo = 0.10 f?P = 7/100 = 0.07

(P2 rd + c),*

T(_), = 39/100 = 0.39 l’(__)II = lS/lOO = 0.16 C’T = 4/1uO = 0.04

C-1

T(Z’Brr/Ic) = 41/75= 0.55 T(PZ + + +) = 13/75 = 0.57 CT = 21/75 = 0.28

C-1 C-1 (I’2 rd Ic)l (I’2 f

+ +)III

I-

c-1

T(PZ rtll c) = 81/180 = 0.45 T(P2 + + +) = 60/1X0 = 0.33 CT = 35/180 = 0.10

(-)

(P.2 rcl 1 c), (I’2

+ + f),,,

,r(Z% rd 1 c) = 37/100 = 0.37 T(Z’d + + +) = 12/100 = 0.w c1’ = 1!)/100 = 0.19 ~__~__~_ ~~___.__~__

(-) (-) (P2 Id 1 C)I (1% + + +)I11

/ (-)

T(Z?z + + c) = 31/w T(Z’~ ~(1 I + ) = 26/!)9 (‘T = 8/W = 0.0s ~__~__~_______

(-1

(Z’2 + + (:)I (P2 /Y/l +),I,

-I

(38)

T(_), = 37/M = 0.39 T(__)II = Z/D5 = 0.02 <:* = o/o5

(-)

6 +)I

____-

colonies \I-PI’R lysogenic, and classifiable on the basis of the phage types carried as: t,riply lysogcnir = 16, doubly Iysogenir = 19, and singly lysogrnia (fol either Z’s rd b, or Z% rd I c, or I9 rrl I) = 3 ~__~____~

(-),,, (Z’Z),,,

(Z’2rdO+),(Z’2++c)11 (-) (-)

I,

on minimal

(I’2 rd b +) (1’2 + + C),I (M Id I ‘:)I (_),I

Nonlysogenio

=

= 0.31 = 0.26

O/200

-

(1 Crosses were performed by Lhe standwd technique. Since strain C-60 and its dcrivat,ives show a particularly high frcqucncy of prophage recombination, the I*‘+ cultures in experiments e, f, g, and h were not pure us Lo the prophage types. Nevertheless, knowing t,he frequencies of the various prophuge recombination it was possible Lo clnssify the few colonies carrying recombinant classes, pmphages with little uncertainty. In cxpcriment. n, to the limitation above, is added the wnnplication caused by recombination between itllelic prophages in the bacterial cross, and the difficulty wit.h which plaques of PZ rrl I nnd P2 rrl b can he distinguished; the data of KUC~ an experiment therefore are poor eslimxt,es of trurlsfer frequencies. In experiment i the rccornl~inant colonies were tested for lysogeny wit,hout, previous purification. 373

374

BERTANI

AND

SIX

between 0.26 and 0.57, the weighted average being 0.38, thus very similar in value to that typical of location I. The two prophage sites however appear to be transferred independently of one another. The existence of at least three prophage locations for P2 had been already suggested by the recovery of rare triply lysogenic strains upon superinfection of doubly lysogenic strains. These are described below. In experiment a of Table 6 some triply lysogenic colonies were obtained in the form of one isolate (A) which even after reisolation liberated three types of phages : P2 rd + +, P2 + 1 +, and P2 + + c (approximately 53, 25, and 22 % of the phage yield, respectively, neglecting a small and variable percentage of other types accounted for by prophage recombination), and another isolate which was apparently a mixed clone, since it gave upon reisolation, two types of true-breeding colonies: one type (B) liberating P2 rd + +, P2 + 1 c, and P2 + + c (approximately 35, 22, and 43 %, respectively), and another type (C) liberating phages P2 rd + +, P2 + 1 +, and P2 + + c (approximately 34, 19, and 47 %, respectively). The three strains A, B, and C were carried on slants for a year through three or four transfers, and then were streaked out, and several colonies of each tested for type of phage carried. For type A, 6 out of 9 colonies were still detectably triply lysogenic and still liberated the same three kinds of phages as the original isolate in approximately the same proportions. For type B only 1 colony out of 9, and for type C only 1 colony out of 10 tested were still detectably triply lysogenic. In each case the other colonies were liberating only two types of phage, presumably as a consequence of prophage recombination. One can conclude from these preliminary observations that the triply lysogenic condition may be established, and is fairly stable, but not as stable as the doubly lysogenic condition. It should be emphasized that the data are consistent with the idea that the triply lysogenic condition, considered as the presence of three prophages per nucleus, is in itself as stable as the singly or doubly lysogenic condition, except for the fact that the frequency of recombination between prophages giving origin to partial homozygosis of the prophages is higher in triply than in doubly lysogenie strains. In standard crosses involving two singly lysogenic strains each carrying an appropriately marked prophage in location I (Table 4, experiments 1, m, and n) a small proportion (approximately 2 %) of isolates carried a recombinant prophage. The frequencies of the various recombinant types (Table 8) do not show, as far as the limited data go, any striking

INHERITANCE

OF

PROPHAGE

Pg

TABLE PROPHAGE

RECOMBINATION

m, in Table

n, in Table

THE

4

4

4

BACTERIAL

CROSSES

375

8 COURSE

Prophages carried by parent strains

Experiment

1, in Table

IN

IN

OF BACTERIAL

CROSSEST

Types and numbers of isolates obtained in the cross and carrying a recombinant prophage

Fm F+

U=2 rd 1 +)I

w+++)

1

(P2 + + cl1

(Pa rd 1 c)

1

FFf

(P2 + + +)I (P2 rd 1 C)T

(P2 + + c) (P2 rd 1 +) (P2 + 1 +I (P2rd++)

9 3

FFf

(P2 + + +)I

(P2rdl

(P2 rd 1 C)I

(P2 + + c)

+)

: 4 2

a In experiment 1, and for 100 colonies in experiment m, the colonies obtained from the cross were streaked out once, and a subcolony picked and tested for phage type from each streak. In experiment n, and for 199 additional colonies (not included in Table 4) in experiment m, the colonies obtained from the cross were directly tested for type of phage carried: those which appeared to produce more than one type of phage (approximately 1O70 of the total) were streaked out, and one or more subcolonies from each streak were analyzed for type of phage carried. No st,able doubly lysogenic clones were obtained in these experiments. All occurrences of recombinant prophages are reported in the present table.

asymmetry in recombination and confirm the previous observations as to the greater map distance between P2 genes c on the one hand, and rd and 1, on the other, than between rd and 1 (Bertani, 1954). It has been stated before (Bertani, 1956) that bacteria doubly lysogenie for P2 do not usually liberate the two types of phage carried in a 1: 1 proportion, but that the prophage carried in the preferred location contributes more than the other prophage to the phage yield. The data of Table 9 extend and confirm the previously published data, both for Sh strains, and for all the doubly lysogenic E. coli C strains used in the cross or superinfection experiments reported in this paper. It should be pointed out that various factors not usually controlled in these experiments will contribute to variations in the proportions of the two phage types liberated by doubly lysogenic bacteria: physiological conditions of the culture, differential readsorption of the two phages, differential variations in efficiency of plating. In spite of this variability, it seems possible (a) to use the rule as a diagnostic method for recognizing

376

BERTANI

AND

TABLE VARIATIONS

of strain(s)

tested

tested

2

Sh(P2

9

IN THE PROPORTIONS OF PHAGE TYPES LYSOGENIC STRAINS”

Yfbper Constitution cultures 10

SIX

+ +)

LIBERATED

Strain number or experiment in which strains originated

Sh-39

(P2 rd 1)

Sh-10

Sh(P.2 rd I) (P2 + +)

R

Kumher of plaques examined per culture

1.13

44-133

1.28 -I-

2

Sh(Pz

+ +)

(I3 rd 1)

BY DOUBLY

796~1Q62 -I-

Sh-65

1.38

1113-1818

1.17

36331373

1.26

1129-1467

-I-

3

Sh(P2

Sh-69

+ I) (PZ rd 1)

.I.

Sh-60

3

Sh(P2

rd I) (P2 + I)

5

C(P2 +)I

(PR

ch

Expt.

c, Table

5

8

C(P2 +)I

(P2

cl11

Expt.

h, Table

5

>l

for every isolate

Plaques not counted

8

C(P2 rd b +)I

Expt.

c, Table

6

>I

for every isolate

Plaques not counted

3

C(P2 +)I

Expt.

d, Table

6

(P2 + + C)II

(P2 C)II

53-158

2.7

3.4

24-56

1.4

89-319

2.9

97-171 140-315

-I-

10 6

C(P2 rd b +)I C(P2 +)I

(P2 + + C)II

C-23 C-63

(P2 ch

-I-

5 C(P2 rd 1 +)I -__-------------C(P2 + + +)r 1 _____-_-------------------1

(P2 + +

ch

(P2 rd 1 +)I[

C(P2 rd I C)I (P2 + + +)III

Expt.

e, Table

6

3.5

Expt.

e, Table

6

3.4

162 -1.

i Expt.

b, Table

6

i

4.7

546

a The cultures used were all from single colonies. Where no collection number is given, each culture represents an independent recombination or substitution event. For the Sh strains, the prophage first listed is also the one first established, and therefore assumed to be in the preferred location. R is the ratio (number of plaques of the phage type carried in the preferred location): (number of plaques of the other phage type). Recombinant-type plaques (usually a few per cent of the total) were not included in the evaluation of R or in the totals given. When more than one culture was tested, the R value given is the average of the R values separately estimated for each culture.

INHERITANCE

OF

PROPHAGE

P.%

IN

BACTERIAL

CROSSES

377

prophage location with the great majority of the combinations of phage markers tested in our experiments, and (b) to investigate further variations in the proportions of phages liberated as expressions of the number, location, or genetic structure of prophages in the cell as exemplified by the observations reported below. A Sh (P2 rd + +)(Pd + + c) isolate obtained by superinfection of ShB2 with P2 c, and thus presumably still carrying P2 rd in the preferred location, when tested for phage-type ratio gave values varying between 1 and 1.4 in favor of P2 c. Another isolate carrying the same two phages, obttlined from Sh-58 by spontaneous prophage recombination, and thus presumably ca,rrying P2 c in the preferred location, gave values varying between 1.6 and 5.1 in favor of P2 c. This need not be considered an exception to the rule, since the ratio is much more in favor of P2 c when the latter is in the preferred location. It appears that PW rd is a poor mutant altogether and is likely to compete poorly with PW c in its intracellular multiplication. One may ask whether all variations in the values of R (Table 9) between strains having apparently identical prophage structure are completely due to physiological differences in the growth or adsorption of the various phage types involved. This is not the case. A Xh(P2 + + +)(P2 rd I +) spontaneous derivative of Sh-58 gave an R value of 3.5 (average of two single colony cultures) which compares well with the value found for the parent strain of 4.3 (average of two cultures; see Bertani, 1956, Table X), and not at all with the value found for the independently isolated Sh-55 (Table 9). CONCLUSIONS

AND I~ISCUSSIOK

The data presented support the following inferences. Prophage P2 may be localized on the genetic chromosome of the host cell. At lysogenization, prophage P2 is established as a rule at one particular site on the chromosome (preferred location or location I). The prophage however can be established also at different sites on the chromosome (locations II and III, or second-choice locations). Under the selective conditions of our standard cross, involving derivatives of E. coli C, the best estimates for the frequencies of transfer of locations I, II, and III, are 0.41, 0.15, and 0.38, respectively. In standard crosses involving F- C and F+ K parents, the estimates for locations I and II are 0.32 and 0.02, respectively. Location II has been shown to differ from location I also in its linkage to an unselected bacterial marker. The similarity of the transfer frequency values for locations I and III is

378

BERTANI

AND

SIX

surprising; unfortunately we are unable at this time to interpret it as anything other than a coincidence. It should be noted that some standard crosses were also performed with strains lysogenic for phage P3 (of Bertani, 1951) and a transfer frequency similar to that observed for location I was found. In both cases, location I vs. III, and PZ location I vs. P3, the two prophages appeared to be inherited independently of one another. Tests for allelism between P2 location III and P3 have not been done as yet. P3 cross reacts serologically with P2, but the two are not, or not usually, liberated together in the same burst when produced by a strain lysogenic for both. Strains lysogenic for P3 are normally sensitive to lysis by P2, and vice versa (independently of the location of P2), that is, the two phages give no cross-immunity. For location I, there is no evidence that it is located in the region of the chromosome which is controlled by the Hfr mutant of Hayes (see Hayes, 1953b; Wollman et al., 1957). For the other locations this has not been tested. No differences with respect to location choice were found for the various mutants of P2 used, all of which give cross-immunity with P2 (they are coimmune with 1’2). This applies also for phage P2 Hy dis (Cohen, 195S), which is the result of recombination between P2 and a hypothetical prophage carried by E. coli B, and which does not give cross-immunity with PW (it is dismune with respect to P2). In other words, prophages imparting different specificities of immunity may be established in the same chromosomal location. An alternative, but less simple, view is that although PW and P2 Hy dis are usually located in the same region of the chromosome, they actually occupy different sites, so close to one another that their separation is beyond the sensitivity of our method. Even if it were so, the other fact remains, i.e., that prophages imparting identical specificities of immunity can be established at different locations. These conclusions bear on the interpretation of the mechanism of immunity to superinfection, as pointed out before (Bertani, 1956, 1958). If specificity of prophage location may vary independently of specificity of immunity, it seems very unlikely that immunity to superinfection may come about because a specific chromosomal site is occupied by the prophage, and thus is forbidden to the superinfecting phage. The two identical P2 prophages demonstrated for strain C-14 may have established themselves either simultaneously or at different times, in the course of the development of the plaque from which the lysogenic

INHERITANCE

OF

PROPHAGE

Pg

IN

BACTERIAL

CROSSES

379

strain was fished, as a consequence of superinfection. Simultaneous establishment of two prophages does occur sometimes in Sh infected with P2 (unpublished) and in K-12 derivatives infected with X (Lieb, 1953) No cases of single lysogenization at location II or III were observed. If however lysogenizing phages infecting a sensitive cell show the same degree of preference for location I as prophage-substituting phages in superinfection of established lysogenic strains, it is not surprising not to have observed any such case in our limited sample of (11) independent lysogenization events. There is no doubt, however, that established prophages in location II or III are perfectly stable even when location I is unoccupied. There are several remarkable differences between the behavior of P2 and that of X. (a) There is no evidence for zygotic induction of P2. This correlates well with the lack of inducibility by ultraviolet light of strains lysogenic for Pd. A similar correlation was noted for other phages (*Jacob and Wollman, 1956). A discussion of possible mechanisms of induction will not be attempted here. (b) Only one chromosomal location is known for phage X. Even in crosses involving doubly lysogenic strains it would appear that both X prophages are located at the same site or at very closely linked sites (Appleyard, 1954). (c) Several phages serologically related to X which do not give crossimmunity with it in superinfection have been shown to occupy different chromosomal sites (Jacob and Wollman, 1957). Kaiser and Jacob (1957) have crossed one of these phages (phage 434) to phage X, to obtain a hybrid indistinguishable from X except for its specificity of immunity which is still typical of phage 434. This hybrid phage does not attach itself at the chromosomal site used by X. This contrasts with our finding that P2 Hy dis can attach itself at a site indistinguishable from location I of P2. In evaluating the significance of this difference between the two systems, one has to note that different recombination techniques were used (bacterial crosses, in our case, transduction by Kaiser and Jacob) and that X and phage 434 give zygotic induction when transferred as prophages from a donor to a recipient, nonimmune cell, although Kaiser and Jacob minimized induction in their experiments by transducing at low temperature. Chromosomal localization of a phage which is probably identical to

380

BERTANI

AND

SIX

PZ has been previously attempted by l%d&icq (1953). He used, however, different host strains and different selective markers. No comparison with our results will be tried here. Another phage able to occupy more than one chromosomal site has been reported (Fischer-Fantuzei et al., 1956). Its relationship to Pd is not known. REFERENCES APPLEYARD, R. K. (1954). Segregation of lambda lysogenicit,y during bacterial recombination in Escherichia coli K 12. Genetics 39, 429-439. BERTANI, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacterial. 62, 293-300. III. Superinfection of lysogenic BERTANI, G. (1954). Studies on lysogenesis. Shigella dvsenteriae with temperate mutants of the carried phage. J. Bacterial. 67, 696-707. BERTANI, G. (1956). The role of phage in bacterial genetics. Brookhaven Symposia in Biol. no. 8, 50-57. BERTANI, G. (1958). Lysogeny. Advances in Virus Research 6, 151-193. CAVALLI, L. L., LEDERBERG, J., and LEDERBERG, E. M. (1953). An infective factor controlling sex compatibility in Bacterium coli. J. Gen. Microbial. 8, 89-103. COHEN, 1). (1958). A variant of phage PZ originating in Escherichia coli, strain B. Virology in press. FISCHER-FANTUZZI, L., RITA, G., and RUSSI, M. (1956). l’rimi risultati sulla trasmissione genetica di profagi in E. coli K 12. Nuovi ann. igiene e microbial. 7, (5). FRI?D&RICQ, I’. (1953). Transfert g&&ique des propri&s lysogknes chez E. coli. Compt. rend. sot. biol. 147, 2046-2048. HAYES, W. (1953a). Observations on a transmissible agent determining sexual differentiation in Bacterium coli. J. Gen. Microbial. 8, 72-88. HAYES, W. (1953b). The mechanism of genetic recombination in Escherichia coli. Cold Spring Harbor Symposia Quant. Biol. 18, 7.593. JACOB, F. (1955). Transduction of lysogeny in Escherichia coli. Virology 1, 207-220. JACOB, F., and WOLLMAN, E. (1956). Sur le processus de conjugaison et, de rem combinaison chez Escherichia coli. I. L’induction par conjugaison ou induction sygotique. Ann. inst. Pasteur 91, 486-510. JACOB, F., and WOLLMAN, E. (1957). Analyse des groupes de liaison gCn&ique de differentes souches donatrices d’Escherichia coli K12. Compt. rend. 246, l&101843. KAISER, A. I)., and JACOB, F. (1957). Recombination between related temperate bacteriophages and the genetic control of immunity and prophage localization. Virology 4, m-521. LEDERBERG, E. M., and LEDERBERG, J. (1953). Genetic studies of lysogenicity in Escherichia coli. Genetics 38, 51-64. LEDERBERG, J. (1950). Isolation and characterization of biochemical mutants of

INHERITANCE

OF

PROPHAGE

Pi?

IN

BACTERIAL

CROSSES

381

bacteria. In Methods in Medical Research (R. W. Gerard, ed.), Vol. 3, pp. 5-22. Year Book Publishers, Chicago. LENNOX, E. S. (1955). Transduction of linked genetic characters of the host by bacteriophage Pl. Virology 1, 19&206. LIEB, M. (1953). Studies on lysogenieation in Escherichia coli. Cold Spring Harbor Symposia Quant. Biol. 18, 71-73. MACCACARO, G. A., and COMOLLI, R. (1956). Surface properties correlated with sex compatibility in Escherichia coli. J. Gen. Microbial. 16, 121-132. WITKIN, E. M. (1950). Bacterial mutations involving resistance to destructive agents. In Methods in Medical Research (R. W. Gerard, ed.), Vol. 3, pp. 23-36.

Year Book Publishers,

Chicago.

WOLLMAN, E., JACOB, F., and HAYES, W. (1957). Conjugation and genetic recombination in Escherichia coli K-12. Cold Spring Harbor Symposia Quant. Biol. 21,

141-162. WOLLMAN, E. (1953). Sur le dbterminisme Pasteur 84, 281-293.

g&&tiyue

de la lysogdnie. Ann. inst.