Genetics of P2 and Related Phages

GENETICS OF P2 AND RELATED PHAGES

.

1 Elizabeth Bertani and Giureppe Bertani Microbiol Genetics laboratory. Korolinrko Inrtitutet. Stockholm. Sweden

I . Introduction . . . . . . . . . . . . . . . . . . 200 I1. Natural History . . . . . . . . . . . . . . . . . 201 A . Origin of P2 . . . . . . . . . . . . . . . . . 201 B. Host Bacteria for P2 . . . . . . . . . . . . . . 201 C . P2-Related Phages . . . . . . . . . . . . . . . 201 I11. The Free Phage and Its DNA . . . . . . . . . . . . . 202 A . The Phage Particle . . . . . . . . . . . . . . . 202 B . The Phage DNA . . . . . . . . . . . . . . . . 202 C The Physical Map of P2 DNA . . . . . . . . . . . . 204 D . Comparative . . . . . . . . . . . . . . . . . 205 I V . Mutational Types . . . . . . . . . . . . . . . . 207 A. Properties of the Virus Particle . . . . . . . . . . . 207 B. Essential Functions . . . . . . . . . . . . . . . 207 C . Lysogeny . . . . . . . . . . . . . . . . . . 209 D . Comparative . . . . . . . . . . . . . . . . . 211 V . Recombination . . . . . . . . . . . . . . . . . 212 A. Frequency . . . . . . . . . . . . . . . . . 212 B. Different Recombination Mechanisms . . . . . . . . . 212 C . Negative Interference . . . . . . . . . . . . . . 214 D . Recombination Involving the Prophage . . . . . . . . . 214 E . Arrangement of Genes on the P2 Chromosome . . . . . . . 215 F. Comparative . . . . . . . . . . . . . . . . . 217 VI . Replication . . . . . . . . . . . . . . . . . . 217 A . Intracellular Forms of P2 DNA . . . . . . . . . . . 217 B. Point of Origin and Direction of Replication . . . . . . . 218 C . Phage and Bacterial Functions Needed by P2 for Replication . . 218 D . Involvement of Gene A in Replication . . . . . . . . . 219 E . Comparative . . . . . . . . . . . . . . . . . 220 VII . Regulation . . . . . . . . . . . . . . . . . . . 220 A. Functions Involved in Multiplication . . . . . . . . . . 220 B . Factors Affecting Lysogenization . . . . . . . . . . . 221 C. Split-Operon Control of the inl Gene . . . . . . . . . . 222 D . Induction . . . . . . . . . . . . . . . . . . 223 E . Comparative . . . . . . . . . . . . . . . . . 224 VIII . The Lysogenic State . . . . . . . . . . . . . . . . 225 A. Chromosomal Sites . . . . . . . . . . . . . . . 225 B. Chromosite Preference . . . . . . . . . . . . . . 225

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199

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L. ELIZABETH BERTANI AND QIUSEPPE BERTANI

C. Eduction of Host Cell Genes D. Immunity to Superinfection E. Comparative. . . . . References . . . . . .

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230 230 231 232

1. Introduction

Temperate bacteriophages-those that are able to establish a symbiotic relationship (lysogeny) with the host bacteria-are a popular subject of research in molecular genetics. I n such systems, the genetic material of the virus appears in a number of forms: compact and inert in the free phage particle or extended and variously active in the host cell as (a) vegetative phage following infection of a sensitive bacterium; (b) superinfection pre-prophage, following infection of an immune bacterium; (c) prophage, integrated into the host cell chromosome in an established lysogen; and (d) plasmid, in carrier cells. These forms of the genetic material of the virus may be studied with various appropriate physical methods, while genetic theory supplies the thread that connects them. Besides the macromolecular mechanisms found also in virulent bacteriophages, like adsorption to the cell, injection of the nucleic acid, assembly of the progeny virus particles, etc., the lysogenic systems offer the possibility of studying other interesting macromolecular mechanisms, like the insertion of viral DNA into the host cell chromosome, the repression of phage functions in the lysogenic condition, the stabilization of the carrier state, etc. The temperate bacteriophage most widely studied to date is phage A. The temperate phage P2, whose biology and genetics are reviewed here, promises to be an equally rewarding subject for intensive study. I n the first place, P2 appears to be rather different from A in a number of biologically interesting properties : it is not inducible, it interacts with a variety of host chromosomal sites, it recombines very little, etc. Furthermore, A and P2 may be taken to be the prototypes of two main groups of temperate Enterobacteria phages in nature. We isolated from nature (the Los Angeles County Hospital) 42 temperate phages, able to attack Eschem'chiu coli but all dissimilar in some property from each other. Of these, 16 were serologically related to P2, and 12 to A. I n this review attention will be focused on phage P2, but-whenever possible-comparative information on a number of less-well-studied, P2related bacteriophages will be supplied. On some points, more thorough coverage and older references may be found in other reviews (Bertani, 1958; Campbell, 1969; and Calendar, 1970).

GENETICS O F

P2

AND RELATED PHAGES

20 1

II. Natural History

A. ORIGINOF P2 Wild-type P2 is a line of phage isolated from the Lisbonne and Carrere strain of E. coli (G. Bertani, 1951). This bacterial strain, isolated in 1923, is the oldest known lysogen. It carries at least two other bacteriophages, besides P2, which have been called P1 and P3. Phage from this strain was used in very early work. I n more recent times, independent phage isolates from the Lisbonne and Carrere strain have been studied by the Beumers (phage H+, most probably identical to P2, and phage H-, most probably identical to P l ) , who have been interested primarily in the adsorption properties of these phages (see for example, Beumer, 1961) and by Frbdbricq (phage a, most probably identical to P2), who made a first attempt to localize this prophage on the bacterial chromosome (Frbdbricq, 1953). B. HOSTBACTERIA FOR P2 Strains of Shigella were traditionally used to detect the phages of the Lisbonne and Carrere strain: one, called Sh, has been used by us. P2 grows just as well on E. coli C, which is a generally good phage indicator, with no known restriction mechanisms, and is now the standard indicator for this phage. The efficiency of plating of P2 on E. coli K-12 is 5 to 10 times lower: the reason for this has not been studied. On E. coli B, P2 grows well, once it has adapted to the restriction and modification system of this strain (Bertani and Weigle, 1953; see review by Arber and Linn, 1969) . Circumvention of restriction systems and alteration of phage-adsorbing capacity make Salmonella typhimurium LT2 fully sensitive to P1 (B. A. D. Stocker, personal communication) and likewise to P2 (G. Bertani, unpublished). For other aspects of restriction and modification in phage P2 see also Christensen (1964) , Hattman (1964), and Uetake et al. (1964). P2 grows also on some Serratia strains (Bertani et al., 1967), which is interesting because the base ratio of the DNA of P2 differs substantially from that of Xerratia. Details of techniques for growing P2 are found in Bertani and Bertani (1970). The bacterial strains mentioned here will be referred to in the text by their abbreviations, Sh, C, K, B, and Sa (for Serratia).

c. P2-RELATED

PHAGES

Some information is available concerning several temperate phages, independently isolated from nature and related serologically and in other

202

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

ways to P2. These phages are listed in Table 1. The first three were selected from a larger sample for their noninducibility (Jacob and Wollman, 1956). The last seven were selected from our collection mentioned above (Section I ) , because they formed good plaques and were serologically related to P2. The phages listed are all serologically related to P2, and are not inducible by ultraviolet light. Different immunity specificities are represented in this group of phages but their number appears to be limited. These phages are all-unlike the A-related phages testedhelpers for the “satellite” phage P4, discovered by Six (1963). P 4 is a defective phage that can produce mature phage particles only if a helper phage is present in the same cell. The P 4 particles produced are serologically identical to whichever phage was used as helper. It is interesting t o note that phage P1, which differs from P2 in numerous prcjperties (it is larger, has more DNA, is slightly inducible, can transduce, recombines efficiently, does not usually attach to the host chromosome, does not help P4, can grow lytically on rep bacteria, etc.), nevertheless is fairly closely related to P2 in neutralization tests (R = 0.2, see Table 1). The two phages have in fact very similar tail base plates (D. H. Walker, personal communication). Both were isolated from the Lisbonne and Carrere strain, and it might be that a t some point in evolution they came to share some tail genes. I n what follows, however, P1 will not be considered among the P2-related phages.

I l l . The Free Phage and Its DNA

A. THE PHAGE PARTICLE Phage P2 has an isometric icosahedral head (of approximately 60 nm diameter) and a tail (approximately 135 nm long) with a contractile sheath, a base plate, and tail fibers (Anderson, 1960; D. H. Walker, personal communication). The particles are composed of protein (62% by weight) and of DNA (38%) and have a buoyant density of 1.43 to 1.44 in CsCl gradients. Most preparations of P2 contain a small percentage of noninfectious, tailless, or otherwise abnormal particles, separable by density from the main type (D. H. Walker, personal communication). B. THEPHAGE DNA Each particle of P2 contains a piece of double-stranded, nonpermuted DNA about 10-13 p long (depending on how the sample is prepared

GENETICS O F P 2 AND RELATED PHAGES

203

TABLE 1 Temperate Bacteriophages Related to P2

Phagea

Serological relatedness Immunity to P2b specificityC

Notesd

18 186

0.1-0.3 0.01-0.05

W

299

0.1-0.3

P2

EM

W+

1

W

u,

PK P2 H y dis P3

0.1-0.3 1 0.1-0.3

P2 dis P3

h

P4 +D5 +Dl24 +Dl45 +Dl60 +D218 +D252 +D266

0.1-0.3 0.1-0.3 0.1-0.3 0.1-0.3 0.2-0.5 0.1-0.3 0.1-0.3

EM

f,

P4 P3 dis 145 P3

EM

EM EM i,i,

EM

Referencese Jacob and Wollman (1956) Jacob and Wollman (1956) ; Baldwin et al. (1966) Jacob and Wollman (1956); Geisselsoder and Mandel (1970) Glover and Kerseman (1967); Pizer et al. (1968) Jesaitis and Hutton (1963) Cohen (1959) Bertani (1951); D . H. Walker (personal communication) Six (1963); Inman et al. (1971)

k, I

m

P3 dis P2

n

All, except 186, grow on C. All help P 4 (see text), although 186 may do so only to a very limited extent (E. W. Six, personal communication). All are noninducible by UV light (inducibility less than 3 % at a dose that induces more than 90% of bacteria lysogenic for A). With the exception of P4 (Lindqvist and Six, 1971), all are unable to multiply lytically in a rep host (Calendar et al., 1970). Original observations with a P2 specific antiserum a t a K p 2 neutralization constant of 0.3-3.0 per minute. The table lists the values of R = ratio of K for the given phage to K p 2 . Original observations based on tests of all possible combinations: lysogen us. superinfecting phage. Host: C, except for 186. E M signifies that the virus has been studied in the electron microscope. I n all cases the virus particles were practically indistinguishable from those of P2, the only exception being P4. Additional references are given in the text. Immunity specificity not fully tested: 186 is however heteroimmune in respect to P2, P3, P 2 H y dis, and 18 (E. W. Six, personal communication). Phage 186 was grown on K. 0 W+ is probably not the same phage as W of Jacob and Wollman (1956). P 2 H y dis is not a fully independent natural isolate: it arose as a recombinant between P2 and a defective prophage, or other genetic structure, present in B. P4 is a defective phage (see text). i The tail of P4 is identical to that of its helper; the head is smaller. Phage 18 does not form plaques on C(+D124). Phage +Dl24 grows poorly a t temperatures above 37"C, and helps P4 only at 30°C. ln Phage +Dl45 helps P 4 only at 30°C. " Phage P3 does not form plaques on C(+D252). J

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L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

for electron microscopy), molecular weight 22 million daltons, based on a molecular weight for A DNA of 31 million (Mandel, 1967; Inman and Bertani, 1969; A. K. Kleinschmidt, personal communication). This corresponds to approximately 33,000 base pairs or a maximal coding capacity for 30 to 70 different proteins (with an average of 350 to 150 amino acids per protein). Like the DNA of the E . coli host bacterium, P2 DNA contains the four normal bases in approximately equimolar proportions (Bertani and Bertani, 1970). When extracted from the free phage, the DNA is linear, that is, has free ends. These, however, like the ends of phage A DNA, can join reversibly to form rings or dimers, presumably due to the presence of short, complementary, single-stranded terminal segments (cohesive ends) (Mandel, 1967; Inman and Bertani, 1969). The cohesive ends of P2 DNA are much more stable than those of h DNA: in 2 M ammonium acetate, they separate a t 76OC, whereas those of A DNA do so a t 63*C (Itandel and Berg, 1968b; Inman and Bertani, 1969). This reaction may be followed not only by electron microscopy, but also by measurement of DNA infectivity. The “helper” assay, developed by Kaiser and Hogness (1960) for A DNA infectivity has been modified for P2 DNA by Mandel (1967). I n the presence of calcium ions, the DNA itself (without helper) is infective (Mandel and Higa, 1970).

C. THE PHYSICAL MAPOF P2 DNA Heterogeneity of DNA base composition may be demonstrated by a number of methods. It may turn out to be of fairly general occurrence, a t least in viruses and bacteria (see Skalka et al., 1968; Yamagishi, 1970). It possibly reflects evolutionary phenomena where the evolutionary units are functionally organized segments of DNA, rather than independent genes. These questions have been discussed by Hershey (1969). Electron microscopical techniques have been developed for the determination of the position along a piece of double-stranded DNA of those segments presumably richer in adenine and thymine which denature preferentially upon raising the temperature (Inman, 1966), or increasing the alkalinity of the medium (Inman and Schnos, 1970). If the DNA is pure and homogenous, more specifically, if it is not circularly permuted, so that all the molecules have the same base sequence, it is possible to construct from such data denaturation maps like those represented in Fig. 1. One can see that the results with the two methods of denaturation are concordant, that the maps for the two unrelated phages (P2 and A) are quite different, and that the heterogeneity in DNA base pair composition along the phage chromosomes is pronounced.

GENETICS OF

P2

205

AND RELATED PHAGES

C

:;I_ 4

0.6

0.4

I

,_ , _ 2

4

~

8

6

10

12

14

16

18

1 .o-

-

0.8-

0

0.40.4

0.2

0.2-

1

2.5

I

5.0

1 I

7.5

10.0

12.5

15.0

17.5

P h y s i c a l map d i s t a n c e ( m i c r o n s )

F I ~ 1. . Denaturation maps of DNA of phages P2 and A. A. P2, denaturation at 534°C (from Inman and Bertani, 1969). B. P2, denaturation a t high p H (from Schniis and Inman, 1971). C. A, denaturation at 54.0"C (from Inman, 1967). D. h, denaturation at high pH (from Inman and Schnos, 1970).

Thermal denaturation of P2 DNA, followed by means of optical density measurements, gives highly differentiated curves and permits the recognition of a t least three DNA fractions: I, corresponding t o 20% of the DNA mass, with 34% GC content; 11, 16%, with 45% GC content; and 111, 64%, with 58% GC content (Inman and Bertani, 1969).

D. COMPARATIVE The P2-related phages that have been studied with the electron microscope (see Table 1 for references) are morphologically indistinguishable from P2, with the exception of the defective phage P4, which has a smaller head. The straight tail with contractile sheath clearly distinguishes this group of phages from A. Particles of 299 and W+ have

206

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

buoyant densities very close to that observed for P2 (Geisselsoder and Mandel, 1970; Glover and Kersaman, 1967). Particles of P2 Hy dis have a buoyant density slightly higher than that of P2: if this is attributed exclusively to a higher DNA content, the DNA of this phage ought to be 2% in excess of that of P2 (Cohen, 1960). Particles of P2 Hy dis are also more sensitive to heat than P2 (Cohen, 1959), which is consistent with the finding that in several phages (references in Ritchie and Malcom, 1970) and in P2 itself (G. Bertani and D. H. Walker, unpublished) losses of DNA (deletions) increase thermal stability of the particles. The molecular weights of the DNA obtainable from phages 186 and 299 have been estimated at 20 million (Wang, 1967) and 21 million daltons (Geisselsoder and Mandel, 1970), respectively, hence very near the 22 million obtained for P2. Phage P4, on the other hand, contains only about 30% of the amount of DNA typical of a P2 particle (Inman et al., 1971). The DNA molecules of phages 299, 186, and P4 also have cohesive ends (Baldwin et al., 1966; Wang, 1967; Mandel and Berg, 1968a; Inman et al., 1971) and the formation of mixed dimers of P 2 DNA with the DNA of either 299 or 186 has been reported (Mandel and Berg, 1968a,b). Neither of these phages can form dimers with A DNA (Baldwin et al., 1966). Like P2, the cohesive ends of 186, 299, and P2 H y dis DNA begin to separate at temperatures much higher than those required for A DNA (Wang, 1967; Mandel and Berg, 196813; Inman and Bertani, 1969). It is thus likely that these four phages have similar base sequences in their cohesive ends. The cohesive ends of 186 DNA are approximately 17 nucleotides long, hence 5 nucleotides longer than those of A DNA, and partial sequences have been determined (Wu, 1970). Phages 299, 186, and P2 Hy dis may be used equally well as helpers in the infectivity assay for P2 DNA. Since A cannot be used, it has been suggested that the “helping” process involves an interaction between the cohesive ends of the DNA being taken up and of that injected by the helper phage (Mandel and Berg, 1968a). The denaturation map of P2 H y dis (Inman and Bertani, 1969) shows a minor difference in respect to that of P2 near one end. This is consistent with the observation of a small, but definite difference in the melting curves for the two DNAs. The heat denaturation of 299 DNA has been analyzed photometrically (Geisselsoder and Mandel, 1970) : the pattern obtained resembles very much that of P2 DNA. One of the two subfractions of P2 DNA fraction I, however, appears to be missing in 299, and this is consistent with the slightly higher average GC content found for 299 DNA.

GENETICS OF P 2 AND RELATED PHAGES

207

IV. Mutational Types

A. PROPERTIES OF THE VIRUS PARTICLE

P2 requires calcium ions for optimal adsorption. Mutants with altered calcium requirement are known (cui, I , and rd in Table 2 ) . Host range mutants (able to adsorb to resistant mutants of the host bacterium) of P2 have been isolated (G. Bertani, unpublished), but they are rather unstable upon storage. Only one such mutant has been used to any extent (Chase, 1964). Defective virus particles are formed by certain conditional mutants, under nonpermissive conditions (see Section IV, B, 2 ) .

B. ESSENTIAL FUNCTIONS A number of conditional mutants of P2, of both the t s and am (or sus, suppressor sensitive) type, have been isolated and arranged by means of complementation tests into some 18 cistrons (Lindahl, 1969a, 1971). All these genes presumably specify proteins which have to do with some aspect of the lytic multiplication cycle of the phage, including structural proteins for the phage particle. 1. Early Functions

Two “early” essential genes have been identified by Lindahl (1969a, 1970, 1971) : cistrons A and B. Infection with phage carrying a mutation in either of these genes under nonpermissive conditions (high temperature or absence of suppressor) does not result in killing of the bacterial host, which may instead become lysogenic. Furthermore, little if any incorporation of radioactive label into phage DNA can be detected in such cases (E. Ljungquist, personal communication ; Lindqvist, 1971). 2. Late Functions

Phages carrying mutations in other essential genes (cistrons D through H, and J through T: Lindahl, 1969a, 1971) lyse the bacterial host cell

under nonpermissive conditions. In these cases the phage DNA multiplies (E. Ljungquist, personal communication; Lindqvist, 1971) and the formation of incomplete or defective phage progeny particles may be demonstrated by means of in vitro reconstitution (Edgar and Wood, 1966) of active phage particles. I n this test, lysates obtained under nonpermissive conditions of mutants in genes L through Q can give rise to active phage particles when mixed with similar lysates of mutants in genes D through H , J, R , and T (G. Lindahl, personal communication). Since

208

L. ELIZABETH BERTANI AND QIUSEPPE BERTANI

TABLE 2 P2 Mutational Types and Their Abbreviations

am cai C

cc co2 di8

fun

int

1

4J m

old

rd

8af IS

Amber or suppressor sensitive mutants: form plaques only on host bacteria carrying a suppressor gene. A variety of mutations belonging to different cistrons (see text).' Calcium independent in adsorption, but also unstable in the presence of ca1cium.c Mutant cl (or simply c) forms clearer plaques (especially on Sh) than wildtype phage.b I t lysogenizes a t reduced frequency,d but its lysogens are stable,)*eJ even though they have reduced immunity to superinfection.iv' In cistron C.amh Mutants CS and c4 are like c l , from which they differ only quantitatively in lysogenizing capacity and immunity 1evel.j Mutants c6 through c9 form clearer plaques at high temperature (37 to 42°C) than a t 30'C.' Also in cistron C.' Forms slightly clearer plaques than wild-type on citrate agar." Adsorption and stability are unaffected. Lysogenizes normally, but the lysogens produce little or no phage. Affects the activity, the specificity, or the synthesis of the int gene product.= Not a mutation: symbolizes the immunity specificity of the defective prophage present in strain B, which is different from that of P2." P2 Hy dis is the hybrid carrying that specificity. Unable to convert the host bacterium to high sensitivity to 5-fluorouracil.0 Recessive. Unable to lysogenize by itself, although it permits survival of some of the infected cells. Turbid plaques. Recessive: the presence of wild-type phage permits int to lysogenize. Lysogens for intl,~intl4, intd0, intdl, and i n t 3 l ~ produce spontaneously less than 0.1% of the normal amount of phage; lysogens for inti3 and int.90~produce 1 to 30% of the normal amount. Makes plaques larger when combined with rdl .b It is very strongly dependent on calcium for adsorption: it gives no plaques on citrate agar." Forms plaques slightly larger than wild type and has larger burst size.c Adsorption and stability are unaffected. Forms minute p1aques.h Able to lysogenize a recombination defective mutant, lyd, of E. coli Cr; the wild-type phage cannot. Also unable, as prophage, to interfere with the multiplication of phage X.* Mutant rdf (or simply rd) forms smoothly round plaques on Sh.' It is insensitive to high concentrations of citratee; rdda resembles rdl in plaque appearance, but is not necessarily allelic to it. Not a mutation. Symbolizes the genetic change resulting from an interaction (presumably recombination) between P2 and chromosite 11, and affecting the site affinity of the phage.6 Previously symbolized as site specificity SII. Temperature sensitive, generally unable to form plaques a t temperatures from 37 to 42'C. A variety of mutations belonging to different cistrons (see text).h

GENETICS OF

P2

AND RELATED PHAGES

209

TABLE 2 (Continued) vir

Xt

Symbolizes a variety of mutants, all unable to establish lysogeny. Mutant virl is a “weak”a or immunity-sensitive virulent: it gives no plaques on lysogenic indicat0rs.f.k It is recessive: it may become and remain prophage if a second, wild-type prophage is also present.%In cistron C.Osh Mutant virl4 is another weak virulent, isolated however in P2 Hy dis, and therefore sensitive to dis rather than P2 specific immunity.” Mutants virl9 and vir20 (isolate 88 of@)are conditional virulents: lysogenize C to some extent, but not Sh.0 Assigned to cistron Zh.Mutants virSv and vir.@‘ are “strong,”a or immunityinsensitive virulents. They are presumably operator mutations for the early functions A and B.’ Mutant vir82, another strong virulent, is a deletion (G. Bertani and D. H. Walker, unpublished). Mutant vir6 is an “intermediate” virulent: gives plaques on lysogens with variable efficiency dependent on the immunity level of the 1ysogen.f Produces curing of lysogens in high frequency.m Double mutant virl virlSw also behaves as an intermediate virulent. Extratemperate: gives very turbid plaques and lysogenizes much more efficiently than wild

G. Bertani (1953a). * G. Bertani (1954). ~ B e r t a n et i al. (1969). d L . E. Bertani (1959).#Six (1959). f Bertani and Six (1958). L. E. Bertani (1960).h Lindahl (1969a). Lindahl (1971). jL. E. Bertani (1961). kL. E. Bertani (1965).’L. E. Bertani (1968). G. Bertani (1953b). Cohen (1959). 0 Bertani and Levy (1964). P Lindahl (1969b). q Choe (1969). Sironi (1969). a Lindahl et al. (1970). ‘Six (1971). u Six (1966). vL. E. Bertani (1957). G. Bertani (1962). zG. Lindahl and M. G. Sunshine (personal communication). ~

a

@

the mutation 1, which affects adsorption, is located very close to some mutations in cistron G , it is assumed (Lindahl, 1969a) that the genes of the D-J, R, T group specify tail structures, i.e., mutants in such genes can still supply normal phage heads in reconstitution experiments. Conversely, genes of the L-& group would be responsible for the formation of the phage head. Gene K appears to specify a protein required for lysis (G. Lindahl, personal communication). C. LYSOGENY 1 . Prophage Attachment and Detachment

As for other phages, the attachment or “integration” of phage P2 into the bacterial chromosome at specific sites (“chromosites”) requires at least one phage gene product, as shown by the occurrence of int mutants (see Table 2 ) . The fact that lysogens carrying an int prophage (obtained by complementation) are defective in phage production in-

210

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

dicates that this gene is needed also for the excision of the prophage from the host chromosome. Another gene, cox, affecting excision, but not integration of P2, and different in map location from int, has been identified recently (G. Lindahl and M. G. Sunshine, personal communication). The specificity of prophage attachment (i.e., the probability with which a P2 phage is integrated a t one or the other of the available chromosites) is affected by the genetic constitution of the phage. An important role must be played by the DNA base sequence in the “episite,” the segment of phage DNA where the reciprocal exchange with the bacterial chromosome postulated by the Campbell model takes place. The saf variants (see Table 2) very probably represent changes in the episite. 2. Maintenance

Like other temperate phages, P2 produces a specific immunity repressor whose function is to block the transition of the prophage or of a superinfecting phage into vegetative phage. Gene C specifies the repressor and gives a t least three classes of mutants: (a) mutants with reduced lysogenizing ability and imparting, as prophages, low levels of immunity t o superinfection (example: c l ) ; (b) temperature-sensitive mutants, whose lysogens are normal a t 3OoC, but are abortively induced a t high temperatures (example c5) ; and (c) weak virulent mutants, having lost all ability to establish lysogeny, in a.bsence of complementation (example: v i r l ) . Another class of mutants, (example: virl9, vir20) assigned to a gene called 2, are host dependent: they are unable to lysogenize Sh, but do lysogeniee E . coli C. The function of gene 2 in Sh is not clear. 2 mutants complement both C (L. E. Bertani, 1960) and int mutants well (Choe, 1969). Also, they do not affect immunity specificity, since P2 H y dis virZ recombinants may be obtained easily (Cousin, 1963). Among P2 mutants having lost the ability to lysogeniee, one not uncommonly finds the immunity-insensitive type (like vir3). These are probably operator mutants in the early function operon (Lindahl, 1971), and are deletions in many cases (G. Bertani, unpublished). One immunity-insensitive mutant is known which reverts to the immunity-sensitive state (L. E. Bertani and L. Falt, unpublished). 5.

Lysogenic Conversion

Formally this term covers any effect of the prophage on the lypogenic cells, especially if unessential to the maintenance of the lysogenic state. In general, bacteria lysogenic for P2 are more sensitive to 5-fluorouracil

GENETICS OF

P2

AND RELATED PHAGES

211

and its derivatives than the corresponding nonlysogenic strain (L. E. Bertani, 1964). P2 mutants (fun, for fluorouracil nonconverting) which have lost this property, but are otherwise normal, have been isolated. Another property of bacteria lysogenic for P2 is their inability to support the growth of phage A, although adsorption is unaffected. The old mutants of P2 have lost this property. The connection between this effect, and the property by which old mutants have been defined in the first place (their ability to overcome the effects of the Zyd mutation in the host bacterium) is not yet clear. The old’ allele is incompatible with lysogeny in a lyd host even when an already established prophage is introduced by means of bacterial crosses into a Zyd cell from a lyd+ donor lysogen (Sironi, 1969), and this suggests that the old gene is functional also in the lysogenic condition. P2 lysogens also restrict the growth of phage T2 and related phages (Bertani, 1953a; Lederberg, 1957; Smith et al., 1969). This restriction, however, is not affected by old mutations.

D. COMPARATIVE There is very little information on mutation types in P2-related phages. P2 H y dis differs from P2 in that part of the genome which is concerned with immunity and specificity of immunity, as shown by the fact that P2 mutants in cistron C or strong virulent mutants lose such properties in acquiring the dis immunity specificity from the defective prophage of strain B (Cohen, 1959). Nevertheless one can obtain from P 2 H y dis mutant types exactly corresponding to the c, weak and strong virulent mutants of P2 (Cohen, 1959). Heat-inducible mutants have been isolated from phage 186 (Baldwin et al., 1966), and abortively inducible ones from phages 299 (Golub and Zwenigorodsky, 1969) and 18 (Golub and Reshetnikova, 1970). Immunity-insensitive mutants are known for P4 (Lindqvist and Six, 1971). I n general, several of the P2-related phages yield immunity-insensitive mutants easily: this is very common for example with +D145, where such mutants may be found as “spontaneous” plaques in a lawn of the corresponding lysogen. Like P2, prophage W+ also excludes A, and mutants of W+, analogous to the old mutants, have been isolated (Kerszman et al., 1967). The restriction of A in the two systems is different, however. Whereas it is possible to isolate mutants of A that are able to grow on either P2 (Lindahl etal., 1970) or W+ (Glover and Aronovitch, 1967) lysogens, those selected to grow on W+ lysogens are still unable to overcome P2-restriction (Kerszman et aZ., 1967). Like P2, W+ prophage also restricts the

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multiplication of phage T2, but again the two systems are not quite identical in this respect (Pizer et al., 1968). V. Recombination

A. FREQUENCY Genetic recombination of P2 in the course of vegetative multiplication has been studied extensively by Lindahl (1969a,b). P2 has the remarkable property of recombining very little in such experiments, as compared with phages having roughly the same DNA size and burst size. For example, genes at opposite ends of the genetic map of A (which has 30% more DNA than P2) may show recombination frequencies of up to 13%, whereas the maximum observed for P2 is 0.3% under normal conditions. P2 is the exception: the temperate phage P22 of Salmonella, which has as much DNA as A (Thomas, 1966), gives recombination frequencies up to 20-25% (Gough and Levine, 1968) ; phage P1, with twice as much DNA (Ikeda and Tomizawa, 1965) gives up to 6-776 (Scott, 1968). The only other DNA phage known to have very low recombination frequencies (maximum 0.2%) (Tessman, 1965) is 513 and its relative 4x174: these phages have much less DNA than any of the phages mentioned above.

B. DIFFERENT RECOMBINATION MECHANISMS This peculiarity of P2 becomes even more striking when the contributions of the various mechanisms of recombination are analyzed. Genetic recombination in molecular terms is without doubt a complicated reaction sequence. Indeed, in bacteria several mutations are known each of which may reduce drastically the frequency of recombination: these mutations are thought to affect proteins which either are required for the normal recombination process, or may interfere with it as a result of mutation. Some phages are known to carry genes whose products are required for or may be involved in recombination: these products might be homologous to corresponding bacterial gene products, or be highly specific for the phage. Moreover, several temperate phages possess genes whose products are required for the unique reciprocal recombination event between the phage DNA and the host chromosome, that leads t o prophage integration. Recombination of this type (int) can take place also between two phage chromosomes, but is always localized to the episite. Phage is known to use all three recombination pathways: the host

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213

system (called rec), its own system of general (i.e., not site-specific recombination (called red), and the int system. (The use of the word system should not be taken to imply a completely independent reaction pathway in each case.) Using appropriate combinations of mutant phages, it is possible to study the contributions of each of these pathways to the total recombination frequency. Mutations of the red type are not known for P2, but the use of int mutants permits the measurement of the residual recombination, when the int pathway is inactive. One finds (Lindahl, 196913) that the recombination frequency between markers located one on each side of the episite is then reduced as much as one hundredfold, and that the bulk of the recombination observable in P2 between markers a t the extremes of the vegetative map is really due to recombination in the episite. The amount of genetic recombination across the episite which is due to the int pathway is greater for A (2% recombination frequency, Signer et al., 1969) than for P2 (0.3%,Lindahl, 1969b) . Since however this type of recombination is strongly dependent on the amount of the int gene product, and on other conditions, the difference between the two phages in this respect is noticeable, but not tremendously striking. One may also recall that the two phages are both able to lysogenize with good efficiency. Where the difference is really striking instead is in the amount of recombination remaining in the absence of the int pathway as can be already guessed from the figures given above: for P2 the maximum non-int recombination frequency is about 0.03%, for A it may be as high as 10%. When A int red double mutants are used, or when A red is used and the recombination frequency is measured over a segment outside of the episite, the amount of recombination observed is much less than in the red+ control, and is attributed to the operation of the recombination system of the host. Indeed, if in addition a recA host mutant is used, this residual recombination all but disappears. The two contributions, from the host and from the red pathway, although not additive, appear to be of similar magnitude (see Echols and Gingery, 1968). It is remarkable that in P2 the total amount of all non-int recombination is already lower than the residual recombination in due to the host pathway. This phenomenon remains unexplained. One can think of a compartmentalization within the cell during the multiplication process of P2 DNA, so that it would be unusually difficult for P2 DNA molecules of different parentage to meet. Alternatively, the low recombination frequency of P2 could be the result of the greater stability of the cohesive ends of the P2 DNA molecule when paired: it has been suggested (Baldwin et al., 1966) that the formation of circular dimers may be a prerequisite for recombination to take place. P2 DNA might form circular

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monomers immediately following infection, and remain in this form during most of the multiplication cycle, making the formation of dimers relatively rare (Mandel and Berg, 1968a). Alternatively, one could imagine that dimers that are formed fail to monomeriae again later. More generally, a structural constraint might impose a selection against the majority of recombinant classes, which thus would not be recovered as viable phage particles in a standard cross. A limitation of the data discussed in this section is that they were obtained with host C for P2 and host K for A: the possibility of host effects on recombination has not been fully investigated.

C. NEGATIVE INTERFERENCE Negative interference-a common phenomenon in bacteriophage crosses (see Visconti and Delbriick, 1953)-is very strong in P2 (Lindahl, 1969b): the frequency of recombination over a map segment among phage particles having undergone recombination over another map segment may be increased several thousandfold. This suggests that the “resistance” to recombination is not due to some general property of P2 DNA, but rather to an obstacle-the difficulty of meeting for two molecules, or of forming or splitting a dimer-such that, once this is removed, recombination can occur normally over the whole molecule. When one of the two segments used to calculate negative interference includes the episite, the interference observed is not as high (data in Lindahl, 1969a), and-given the limited information available-could even be all attributed to the non-int component of recombination included in measurements of recombination frequency across the episite. Ultraviolet irradiation of the phage used in crosses increases very strongly the frequency of non-int recombination, whereas the factor of increase observed in recombination across the episite is smaller, and may all be due to the effect of UV light on the contribution of the non-int pathway (see Lindahl, 1969a, Fig. 2). Very high negative interference has been observed also in phage S13 (Baker and Tessman, 1967).

D. RECOMBINATION INVOLVING THE PROPHAGE With temperate phages it is possible to perform other types of recombination experiments than the standard cross-mixed infection of a sensitive bacterium-discussed up to this point. One can study the following: (a) recombination between prophages a t allelic chromosites in bacterial

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215

conjugation or transduction experiments ; (b) recombination between a superinfecting phage and the prophage, using as superinfecting phage either an immunity-sensitive or an immunity-insensitive phage ; (c) recombination between two immunity-sensitive phages superinfecting an immune cell; (d) recombination between two prophages, carried by the same host cell, either a t different chromosites, or attached in tandem. Some of these approaches have revealed important information on the prophage state, and will be mentioned again later on. Others have not yet been sufficiently exploited to deserve discussion. Some observations however are relevant to the questions raised in the previous sections. I n bacterial crosses, with P2 in the prophage state in both parent bacteria, the amount of recombination within the prophage appears to be normal, i.e., approximately as expected in proportion t o the DNA length represented by the prophage in the bacterial chromosome (Wiman et al., 1970). Recombination between the prophage and a superinfecting, immunityinsensitive mutant phage has been studied by Chase (1964) and Eastburn (1969), even though their primary concern was the repair of damage produced by UV irradiation in the superinfecting phage due to interactions with the prophage. A great deal of reactivation, as shown by smaller slopes for the survival curves as a function of the irradiation dose, can take place in such a system, if the prophage is closely related genetically to the superinfecting phage. Which chromosite is occupied by the prophage seems to be irrelevant. Relatively high frequencies of recombination accompany the reactivation. The mutation lyd in the host (Sironi, 1969), which reduces very much the amount of recombination in the host cell, does not affect prophage-dependent reactivation (Eastburn, 1969) or even vegetative phage recombination (Lindahl, 1969b).

E. ARRANGEMENT OF GENES ON

THE

P2 CHROMOSOME

A detailed genetic map of P2 has been constructed by Lindahl (1969a,b, 1971). Because of the disproportionately greater amount of recombination a t the episite due to the int recombination pathway, the map, as obtained, shows a long segment, corresponding to 80 to 90% of the total, completely devoid of genes. This is, of course, an artifact, and to obtain a picture which might more adequately represent the real distribution of genes along the DNA, the map of Fig. 2 has been corrected for int recombination, i.e., it represents the map one would obtain if all phage used in the crosses were unable to use the int pathway. The map resembles in some features that known for phage A. I n both phages the genes involved in the synthesis of the structural proteins

216

c-

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

--w

___)

[-

---* episite

W

= 0001 %

recombination frequency

FIQ.2. The genetic map of P2 according to Lindahl (1969a,b, 1970, 1971). The distances represented are based on two-factor crosses. Because of inconsistencies between different sets of crosses (different hosts), the distance T to D may be in error by a factor of 2. The gap in the map represents the episite: and is roughly proportional to the recombination frequency obtainable in a cross between int phages. In crosses between int+ phages this gap would be almost ten times longer than the whole map as given here. The segments with arrows under the map indicate the known transcription units.

are clustered in two groups in the left part of the map (head components to the left, tail components to the right), whereas a few genes that are necessary for phage DNA synthesis ( A and B in P2, 0 and P in A), lie towards the right end of the map, to the right of gene C which specifies the immunity repressor. In both systems, the int gene is immediately to the right of the episite, which, for P2, is between genes D and C (Calendar and Lindahl, 1969). In several other respects the maps of P2 and h appear to differ. T o date no genes comparable to N or red of A have been noted in P2. Attempts to detect a phage-specific, A-like exonuclease in the Pa-related phages 186 and 299 (Shuster et al., 1967) have proved negative. Like N and red, the gene coding for the exonuclease in h. is located in a segment between int and C: it is thus possible that phages of the P2 family do not have the corresponding genes (see also Section VII). Second, the gene thought to be responsible for phage endolysin production in P2, gene K , lies in the left half of the chromosome, whereas the gene specifying endolysin is at the far right in the A map. Third, analogs of the cII and cIII genes, which participate in the establishment of lysogeny in A, have not been reported as yet in P2: the Z gene of P2 clearly does not correspond in its map location to either cII or cIII. Although some of these differences may be the result of insufficient information, it is not unlikely that the gene composition of P2 may be simpler than that of A since the length of DNA available is 30% shorter. A surprising feature of the P2 map is the location of two genes, old and fun, which, in addition to the repressor gene C, appear to be active in the lysogenic state. Both are far from the C gene, and are themselves

GENETICS O F

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217

separated by a run of genes for structural proteins, which ought to be repressed in the lysogenic state. This suggests that there are a t least three operons in P2 which are regularly transcribed in lysogenic cells, whereas only one, containing the C gene, is known for A. Gene Z might belong to the same operon as fun. The orientation of the denaturation map of P2 (Fig. 1) in respect to the genetic map (Fig. 2) is not yet known with certainty. There is some evidence however that the one assumed in the figures is the correct orientation, as discussed by Inman and Bertani (1969) on the basis of differences in denaturation maps and curves between P2 DNA and P 2 H y dis DNA.

F. COMPARATIVE There are no published data on the recombination properties of P2-related phages, with the exception of what has been mentioned already concerning P2 Hy dis. A few am mutants have been isolated from either of phages 4D218 and +D266, and crossed (G. Bertani, unpublished) in strain C. I n both cases the recombination frequencies obtained were quite low. This would suggest that the low recombination frequencies observed in P2 are not a peculiarity of this phage, but may represent a general property of this family of phages. In prophage-dependent reactivation experiments, P2 H y dis prophage may rescue efficiently irradiated superinfecting P2 (Chase, 1964) , and this holds for the inverse phage combination (Eastburn, 1969). I n the same type of experiment,, phages P2, P3, PK, and W+ have been tested in all possible heterologous combinations, without obtaining any detectable reactivation (Eastburn, 1969). When hybrids between two of the phages were tested against one of the parents, however, some reactivation did occur. These results would indicate that this type of reactivation requires a high level of genetic homology between the superinfecting phage and the prophage, and a t the same time strengthens the assumption that P2 H y dis is identical to P2 over a large part of its genome. No prophage dependent reactivation could be observed between P2 and P 4 (Eastburn, 1969). VI. Replication

A. INTRACELLULAR FORMS OF P2 DNA Covalently closed, circular forms of P2 DNA have been found in vivo. Upon infection of a sensitive host, most of the parental DNA

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is converted to a form that sediments more rapidly in sucrose gradients than does DNA extracted from phage particles and is resistant to denaturation in alkali (Calendar et al., 1970; Lindqvist, 1971). This material, which is similar in its properties to the “supercoiled” DNA found following infection with A, also appears following superinfection of an immune host (Lindqvist, 1971), i.e., in the absence of replication. Although the conversion of linear to circular phage DNA may take place prior to replication, the circular structures are also actively involved in DNA synthesis. Radioactive label, added during the multiplication cycle, is incorporated into the rapidly sedimenting form (Calendar et al., 1970; Lindqvist, 1971). Furthermore, in pulse-chase experiments, the labeled circular DNA appears to be later converted to the linear form (E. Ljungquist, personal communication ; Lindqvist, 1971). B. POINTOF ORIGINAND DIRECTION OF REPLICATION Circular forms of vegetative P2 DNA have been observed with the electron microscope by Schnos and Inman (1971), who have applied the technique of denaturation mapping to the study of replication in both P2 and A (Schnos and Inman, 1970). Both the region of the chromosome in which replication begins and the direction i t proceeds can be identified by this technique. Circular, partially replicated phage DNA is first prepared from infected bacteria and then denatured to a limited extent. For each molecule, the pattern of denatured regions compared with the established denaturation map permits the identification of the point that corresponds to the ends of the linear molecule, and of the relative positions of the branched points delimiting the replicated segment. In A, replication begins in the right half of the chromosome, in the region of the P gene, and, surprisingly, appears to proceed in both directions. I n P2, replication begins near one end of the molecule and proceeds in only one direction. If the orientation of the physical with the genetic map of P2 is as suggested in Inman and Bertani (1969), the replication would begin in the region of the A and B genes and proceed to the right.

C. PHAGEAND BACTERIAL FUNCTIONS NEEDED BY P2

FOR

REPLICATION

The observations quoted in Section IV, B, 1 suggest that the products of genes A and B are necessary for normal phage DNA replication. I n addition, P2 is absolutely dependent on at least one host function

GENETICS O F

P2

AND RELATED PHAGES

219

that is not required by A. Denhardt et al. (1967) isolated bacterial mutants ( r e p ) unable to support the multiplication of phage +X174. In these strains, the single-stranded 4x174 DNA is converted to a double-stranded form by synthesis of a complementary strand, but further DNA synthesis is blocked. The same strains are unable to support multiplication of phage P2. Although the fast-sedimenting form of P2 DNA can be found following infection of a rep host, there is no uptake of radioactive label into the phage DNA (Calendar e t al., 1970). Thus, it would appear that P2 and the double-stranded form of +X174 share some step in replication that is not shared with A.

D. INVOLVEMENT OF GENEA

IN

REPLICATION

Lindahl (1970) has reported that mutants in gene A do not complement any other mutants except B mutants and then only when the concentration of salt in the medium is adequate. Lindahl tends to believe that even in this case the complementation found is due to “leakiness.” More surprisingly, gene A mutants cannot be complemented by any other mutants or even by wild-type phage. Following mixed infection under nonpermissive conditions, but adequate salt concentration with an A mutant and a B mutant (or wild-type P2), the yield contains almost exclusively B mutant type (or wild-type P2). The A gene thus appears to code for a protein that cannot be shared between chromosomes, i.e., acts only in cis configuration. Possible explanations for the inability of the product of gene A to act in trans have been discussed by Lindahl (1970) : they include replication of P2 in a “compartment” that is impermeable to the A gene product; rapid inactivation of the A gene protein; synthesis of A gene protein in close proximity to the A gene, followed by rapid binding of the product to the chromosome; or a configurational change in the chromosome of A gene mutants that blocks their replication. Whatever the explanation, the unusual properties of the A gene product could account for the inability of immunity-sensitive phages to replicate in the presence of immunity repressor. In general, when a lysogen is reinfected or superinfected with phage that is homoimmune to the prophage carried by the lysogen, no lysis is observed and no DNA synthesis of the superinfecting phage can be detected (Bertani, 1954; Wolf and Meselson, 1963). Thomas and Bertani (1964) showed that a n immunitysensitive phage did not replicate in a homoimmune lysogen even when an immunity-insensitive derivative of the same phage was actively multiplying in the same cell and all factors necessary for multiplication

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were present. This specific block of replication by the immunity repressor has been demonstrated for both P2 and A, but has never been entirely explained. Assuming that gene A is under direct control of the immunity repressor, an immunity-sensitive phage would not be able to replicate in a homoimmune lysogen because it could neither make its own A gene protein nor utilize that produced by a co-superinfecting, immunityinsensitive phage, since the A gene product acts only in cis. Although this explanation is adequate for the case of P2, an analogous gene having the same behavior as gene A has not yet been demonstrated for A.

E. COMPARATIVE Although the defective phage P4 needs a helper phage to complete its multiplication cycle, it is able to replicate its D N A also in the absence of helper (Lindqvist and Six, 1971). Covalently closed, circular forms, containing either parental or newly synthesized P4 D N A have been detected by sedimentation analysis (Lindqvist and Six, 1971) and observed with the electron microscope (R. B. Inman, personal communication). All the P2-related phages tested so far are, like P2, unable to multiply in rep hosts (see Table 1) with the exception of P4. The latter multiplies normally in such hosts when a helper phage is present, and replicates its D N A even in the absence of helper (Six and Lindqvist, 1971).

VII. Regulation

A. FUNCTIONS INVOLVED IN MULTIPLICATION The early gene B appears to be directly under the control of phageimmunity repressor. It has been shown by complementation experiments (Bertani, 1968) that gene B is expressed following exposure of a P2 c6 lysogen to high temperature, although there was little or no increase in the activity of six other genes (D, F, H, L, M , or 0) concerned with the synthesis of phage structural proteins. Two independent, spontaneous, immunity-insensitive mutations (vir3 and vir24) have been localized by crosses a t a site between the genes C and B (Lindahl, 1971). This site is probably the receptor for the immunity repressor and the operator for the A and B genes. The presumed BA operon would then be transcribed from left to right. I n P2 there is no evidence for a second early function operon controlled by the immunity repressor as observed in A.

GENETICS OF

P2

AND RELATED PHAGES

221

Since phages that have mutations in genes A or B do not lyse or kill the bacteria, it is likely that the expression of late functions in P2 depends in some way on the activity of these genes. The activation of late genes in P2 has not been studied in detail, but from what information is available (Bertani, 1968; L. E. Bertani, unpublished; G. Lindahl, personal communication), there is little or no activation of prophage genes in immune lysogens by superinfecting immunity-insensitive P2 phage. The late genes of P2 prophage can be efficiently activated, however, by phage P4 (Six, 1963; Six and Connelly, 1966; Six and Lindqvist, 1971), although again the mechanism is not yet established. The direction of transcription of genes in the left half of the P2 chromosome has been studied by Lindahl (1971) using polar am mutants. According to this test, if an am mutation in a given gene results in decreased expression of adjacent genes, that gene is assumed to be transcribed first. Of five groups of late genes, there were four that appeared to be transcribed in the rightward direction and one that might be transcribed in the opposite direction. The direction of transcription of the old and fun genes, which are active in the prophage state, has not been determined. B. FACTORS AFFECTING LYSOGENIZATION

It is characteristic of temperate phages that infection of a sensitive cell may have a t least two very different outcomes: lysis of the cell with phage production, or establishment of lysogeny. Although the outcome is probably determined by fluctuations in competing reactions, the mechanisms which lock the infected cell in one or the other pathway have not been completely elucidated. Phage P2 can establish lysogeny in all the bacterial strains that support its growth. Lysogenization occurs very efficiently in C and Sh (5-15% of the infected cells) (Bertani, 1957, 1959, 1962), but rarely in Sa (Bertani et al., 1967). At least in C and in Sh, the probability of establishment of lysogeny is a constant-under a set of cultural conditions-for an infected cell, independently of the multiplicity of infection. This is in contrast to other phages which show the Boyd effect, i.e., lysogenize more efficiently when the bacterium is multiply infected, and suggests that P2 DNA multiplies severalfold in most cells before becoming a stable prophage. When the phage has been irradiated with Uv light, a Boyd effect is noticeable also for P2 (L. E. Bertani, unpublished). It would be desirable to study the effect of multiplicity of infection on lysogenization with the mutants in cistrons A and B , which show

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no or very little DNA replication, but do lysogenize in a non-permissive host. As with other phages, the frequency of lysogenization depends very much on cultural conditions. A transient inhibition of protein synthesis or exposure to acridines in the course of infection increases very strongly the frequency of lysogenization in P2 (Bertani, 1957). A similar effect is obtained if the infected cells are exposed to a small dose of UV (Bertani, 1959). These phenomena are unexplained, but are ripe for a reexamination with more-advanced techniques.

CONTROL OF C. SPLIT-OPERON

THE

int GENE

Although the int gene of P2 occupies approximately the same position on the chromosome as that of A, its expression seems to be controlled in an altogether different way. I n A the int gene is under the control of the immunity repressor. Two experiments suggest, however, that the corresponding gene in P2 is expressed independently of the presence or absence of immunity repressor. First of all, P2 phage, superinfecting an immune lysogen, attaches readily a t the preferred P2 attachment site, provided the superinfecting phage is int+ (Six, 1966; Bertani, 1970). This can be interpreted to mean that the int gene of a superinfecting phage is expressed even when repressor is present. On the other hand, when a P2 c5 lysogen is derepressed by exposure to high temperature, the prophage does not detach efficiently (Bertani, 1968) unless the derepressed lysogen is simultaneously superinfected with wild-type P2. Again, to obtain detachment of the prophage, the superinfecting helper must be id+, suggesting that the prophage is unable to express its int gene even when derepressed. Thus, the expression of the int gene in P2 appears to depend more on the state of integration of the phage, i.e., whether it is superinfecting phage or integrated prophage, rather than on whether repressor is present or not. I n order to explain these observations, it has been proposed (Bertani, 1970) that the int gene in P2 belongs to an operon that extends to either side of the episite and that it is split off from its primary promoter site when the phage integrates into the chromosome. This type of regulatory mechanism is probably of secondary importance in the case of large phages like P2, most of whose genes are controlled by repressor. It might be of greater importance, however, for smaller viruses, such as polyoma or SV40, which have only enough DNA to code for five or six genes. Certainly it would seem more economical than using one gene to make a special regulatory substance to control the expression of the other four or five.

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AND RELATED PHAGES

223

D. INDUCTION One of the properties that P2 and the other related phages have in common is non-inducibility by UV light. P2 has also been tested for induction by other treatments, such as fluorodeoxyuridine (Bertani, 1964), mitomycin C (Levine, 1961), and thymine starvation (L. E. Bertani, unpublished) with negative results. Ultraviolet light induction of phage A has been explained by assuming that some product (“inducing substance”), either a DNA precursor or degradation product, formed as a result of the irradiation, complexes with and inactivates the phage repressor (Goldthwait and Jacob, 1964; Ben-Gurion, 1967; Hertman and Luria, 1967 ; Geissler, 1970). The same sequence of events may also precede the rare, spontaneous production of phage by A lysogens, as shown by the fact that rec- A lysogens are not only no longer induced by UV light, but also have greatly reduced rates of spontaneous phage production (Brooks and Clark, 1967). In the case of P2, however, there is no detectable decrease in the immunity of a lysogen following irradiation with UV light (L. E. Bertani, unpublished). Furthermore rec- P2 lysogens produce as much phage spontaneously as rec+ strains (Calendar, personal communication ; Laffler and Luria, personal communication). Thus, the events that lead to inactivation of the P2 immunity repressor must be quite different and the P2 repressor may be insensitive to the hypothetical “inducing substance.” The latter is consistent with the observation that double lysogens for P2 and A are inducible by UV light (G. Sironi and M. A. Pedrini, personal communication; L. E. Bertani, unpublished). The experiment is complicated by the fact that A can not multiply in the presence of a P2 prophage, but this difficulty was overcome either by using a P2 old mutant instead of wild-type P2 or by using a mutant of A that can circumvent the interference by P2 prophage. This result rules out the possibilities that, for example, the inducing substance is not formed in the presence of a P2 prophage or that P 2 produces so much repressor that there is not adequate “inducing substance” to inactivate all the repressor molecules present, because in these cases the P2 prophage should also protect A from induction. Even if the P2 repressor reacted with the hypothetical “inducing substance,” irradiated P2 lysogens most likely would not produce phage. It is possible to isolate mutants of P2 that make temperature-sensitive repressors (Bertani, 1968). When lysogens carrying such mutants are placed at 42OC, they do not produce phage unless they are superinfected

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L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

with more homologous phage. Similar observations have been made concerning zygotic induction in P2. I n conjugation experiments, the transfer of a P2 c prophage into a nonlysogenic recipient may result in loss of recombinants without any detectable phage production (Kelly, 1963). Thus, inactivation or removal of P2 repressor does not necessarily initiate phage multiplication. As already discussed, this is most likely because the int gene is not efficiently expressed by a P2 prophage. A temperature-inducible derivative of P2 that gives good yields of phage when the lysogen is placed a t 42OC has been isolated (Calendar, personal communication) from the abortively inducible mutant P2 c6; it carries a second mutation that permits phage multiplication following derepression of the lysogen. Although inactivation of the P2 immunity repressor is not sufficient to trigger the conversion of prophage into multiplying phage, P2 lysogens do produce phage spontaneously with a certain low probability. Furthermore, it is known that spontaneous phage production depends on the int gene product, since lysogens carrying int mutant's have reduced phage production. Thus, in P2, spontaneous phage production could result from an accidental lowering of the immunity level, coincidental with some other prophage-localized event, such as exceptional transcription of the int gene. A formally equivalent hypothesis has been proposed by Six (1959) on the basis of the effect of prophage dosage on the rate of spontaneous lysis.

E. COMPARATIVE The defective phage P4 is able to establish itself as prophage even in the absence of helper phage, suggesting that it has all the genes necessary for integration (Lindqvist and Six, 1971). Moreover, stable lysogens or carrier cells may also be obtained following infection of a nonlysogen with P4 virl, an immunity-insensitive mutant of P4 (Lindqvist and Six, 1971). Strains doubly lysogenic for A and W+ phage are inducible: as in the case of P2 described above, the presence of a W+ prophage does not interfere with the induction of A by UV light (Kerszman et al., 1967). Abortive induction of a lysogen carrying a temperature-sensitive clearplaque mutant of phage 299 has been described (Golub and Zwenigorodsky, 1969), and this suggests that split-operon control of integration and excision may also be present in this phage. I n addition, Golub et al. (1970) have isolated a temperature-sensitive mutation in an essential gene, which prevents the loss of immunity a t high temperature in an abortively inducible prophage.

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225

A mutant of phage 186, 186 p , that is temperature inducible and gives good yields of phage, has been reported (Baldwin et al., 1966). VIII. The Lysogenic State

A. CHROMOSOMAL SITES On the basis of linkage between prophage and bacterial markers in crosses, it has been possible to demonstrate that in hosts C and K prophage P2 is attached to the bacterial chromosome (Bertani and Six, 1958; Kelly, 1963). Less-direct evidence indicates that this is true also for Sh (Bertani, 1954), and probably also for Sa (Bertani et al., 1967). P2 can attach as prophage at any of a number of different chromosomal sites, some of which have been precisely localized. The taxonomy of P2 chromosites is summarized in Table 3. It has been found that (1) in C, there is a very strong preference for one chromosomal site, called I, with several “second choice,” but still highly specific (because of demonstrated repeat occupancies) sites; (2) in K , there is no such definite preference-instead, two sites, H and 11, are occupied with about equal probability; (3) site I1 occurs both in C and in K, but H and I are not allelic (the genetic maps of C and K are largely homologous; see Wiman et al., 1970). For Sh there is no evidence from crosses, but results of superinfection experiments (see later) indicate a strong preference for one site, as in C (Bertani, 1954; Bertani and Six, 1958). For all cases where it has been studied (sites I and I1 in C ; sites H and I1 in K ) , prophage P2 appears to insert itself according t o the Campbell model, with the same episite being used in all cases, independently of the chromosite (Calendar and Lindahl, 1969). These experiments localized the episite between genes D and C, i.e., the same segment in the P2 map where int recombination is effective. The orientation of the prophages in respect to the host chromosome is different in the two locations I and 11. B. CHROMOSITE PREFERENCE The mechanism behind the chromosite preference pattern is incompletely understood. Most likely it reflects recognition of base sequences on the chromosome, which must be partly different from one another, by a base sequence on the phage DNA, the specific integration enzymes, or both. If chromosite I in C is replaced with the homologous chromosomal segment of K by transduction (Sunshine and Kelly, 19671, the

10 10 Dl

TABLE 3 Known P2 Chromosites in Eschetichia coli

Chromosite

1.

11.

He

111. IVh V through IX' Ek

Map location Between the histidine operon and methionine gene metG6.d a t about 4 x 0 0 of the E. coli map clockwise, from the conventional origin, tht. Cotransducible with metG, to a small extent also with his.c.bJProphage gene C is near the metG endb Between metE (a methionine gene) and thu (rhamnose)ba t 8s00 to 8 x 0 0 of the map, clockwise, from the origin. Cotransducible with both markers.c*bProphage gene C is near the metE end6 Between shiA (shikimate) and his,c.g,b a t about *$ioo of the map, clockwise, from the origin. Cotransducible in good frequency with his.c-b Prophage gene C is near the his end Between man (mannose) and his, a t about 3%00 of the map, clockwise, from the origind Weakly linked to tr?, (tryptophan) and metEc Not precisely localized Not precisely localized

Independent occurrences studied

Notes

10,a 8," 3,d 6,f Found in C, where it is the preferred site for P2. It is possible that a homologous, but much less 11: 7,jand efficient chromosite exists in K (see Section many others VIII, C.) l , 1,s ~ 4,L 10,~ Found in K and C. This might possibly be the same site studied by Fr6dhricq (1953)for his 14' isolate a

2," 3"

Found in K, where it is occupied about as often &s 1I.c This site could not be distinguished from I in the crosses of Table 4, referencen

1,s lf?,1'

Found in C. Possibly also in Kf

lA 1 each'

Found in C Found in C Found in C. Obtained with P2 suf

lk

The data in the third column must not be taken as a random sample of chromosite occupancies. Moreover, the techniques used in chromosite recognition varied a great deal, and 80 did the reliability of the identification. For chromosite I, data from superinfection experiments are excluded. Bertani and Six, 1958. * Calendar and Lindahl (1969). Kelly (1963).d Wiman, et al. (1970). e Bertani (1962). f Sunshine and Kelly (1967).g M. G.Sunshine (personal communication). A Six (1960).i Six (1966).i Choe (1969). Six (1971).

GENETICS OF

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227

resulting strain, although mostly C, becomes like K in respect to preference, i.e., the phage will lysogeiiize at one of several sites with roughly equal probabilities. The reciprocal situation, where the preferred chromosite I of C is introduced into K, has not yet been studied. There is also evidence for genetic changes in the phage which modify the chromosite preference. Six (1963, 1966, 1971) has shown that some phage which is produced from a prophage established in site I1 differs from the “wild-type” phage, in having an aItered site preference. This phage can be recognized in superinfection experiments in that it establishes double lysogeny more often than wild-type phage. Six was able to show that this difference is not the result of a preexisting phage mutation, but rather the consequence of an interaction between the prophage and chromosite 11. The new property (called saf) is inherited like any other genetic property in the course of vegetative multiplication. One would expect that saf phage has a sequence of bases in its episite partly different from the corresponding sequence in the wild-type phage, and probably more similar, or identical, to the sequence in chromosite 11. The lysogens in site I1 studied by Six did not produce a homogeneous population of phage in respect to the saf property, but rather a mixture of saf and s a p phage. If however one uses saf phage to establish lysogeny in site 11,the lysogens obtained produce pure saf phage. Similarly, heterogeneity of phage produced by a lysogen has been observed with a saf phage lysogenized in site I. Figure 3 presents two models which could explain the observations made to date. Phage produced by K lysogens in site H behaves like phage from site I in respect to site specificity when tested in C ; saf phage may be obtained from K site I1 as well as from C site 11. It should be stressed that these phenomena of site specificity are completely independent of immunity specificity: Six (1971) has repeated many of his experiments using P2 H y dis (which differs from P2 primarily in its immunity specificity) and found superimposable results. In C, as far as experience goes, no singly lysogenic clone has been obtained following infection of sensitive cells that did not have a prophage in site I. Sometimes, however, two prophage copies are established simultaneously, in which case two diff ercnt prophage sites are recognizable in the doubly lysogenic clone obtained, one being I, and the other being a “second choice” site, like I1 (Bertani, 1962). This situation is very different from what one observes in A, where as a rule doubly lysogenic strains carry the two prophages next to each other, in “tandem,” and are rather stable (Calef et al., 1965). Stable tandem dilysogcns are obtained in P2 only when the phage carries an int mutation (Choe, 1969; Bertani, 1971). I n a P2 tandem dilysogen

228

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

A. e-

-

/--

B.

FIG. 3. Possible schemes to explain chromosite preference and formation of suf phage. In both schemes one assumes a simple Campbell model, where the higher the similarity in base sequence between episite and chromosite, the more frequent the integration at that chromosite, and where episite in the wild-type phage and chromosite I are identical in base sequence by definition. According to scheme A (Six, 1966) the integration crossover can take place anywhere within the paired sites. Chromosite I1 differs from chromosite I over a certain stretch; as a consequence chromosite I1 consista of three rmbsegments: a and c, which are identical to the corresponding parts of chromosite I, and b, which is different. When a s a p phage is integrated in chromosite I (or, mutatis nautandis, a suf phage in chromosite 11) it makes no difference where in the site the reciprocal crossover occurs: the phage produced will be always the same.

GENETICS O F

P2

AND RELATED PHAGES

229

one of the two operons containing the int gene is reconstituted through joining of the two prophages, and will thus be active even in the lysogenic state (see Section VII, C ) . If the active operon contains a n int+ allele, the lysogen will be unstable because of int recombination between the prophages (Bertani, 1971). These facts suggest the following possibilities: ( 1 ) that P2 prophage tandems occur quite often in the course of lysogenization, but are rapidly reduced to monolysogens or (more rarely) to two-site dilysogens ; (2) that potential second choice chromosites may exist also for A prophage, but they are never effectively occupied because the second prophage establishes preferentially a tandem which is in this case a stable one (suggested by E. W. Six, personal communication). Similar relationships hold for the results of experiments where a lysogen in site I is superinfected with a homoimmune phage. Here, if the superinfection preprophage establishes itself, it either replaces the existing prophage (the more common event) or i t attaches a t a second-choice chromosite (Bertani, 1954; Bertani and Six, 1958; Six, 1960, 1961). Substitution of the existing prophage (as indicated by the replacement of prophage genetic markers) probably can take place as a result of ordinary recombination between the superinfecting phage and the resident prophage, but the more common event is one requiring int recombination (Bertani, 1970). This suggests that also in homoimmune superinfection a tandem structure is first formed in site I and then segregates to form stable single lysogens, some of which happen to carry the superinfecting ~~

When a snft phage integrates in chromosite I1 (or a s a j phage in site I) the needed reciprocal crossover may occur eithcr in a or in c : when phage is produced by such lysogens, presumably following excision of the prophage by a similar reciprocal crossover, the type of phage produced will depend on where the crossovers occurred. Integration in a and excision in n, or integration in b and excision in b, will give only the original phage type, whereas integration in u and excision in b, or integration in b and excision in a, will produce snf phage. Such lysogens therefore will produce always a mixture of snf and snf' phage. According to scheme B, integration occurs in three steps: (1) a specific enzyme makes single stranded cuts at the ends of episite and of chromosite, (2) exchange of partners takes place between the complementary single stranded ends thus produced (analogous to the cohesive ends of the mature phage DNA), (3) a ligase repairs the cuts, thus inserting covalently the prophage into the host chromosome. Lack of identity in the sequences a t the interacting sites will cause imperfect pairing, hence a hcteroduplex segment, which might then be recognized by degradative and repair enzymes with the final result that one of the two single stranded ends in each pair will be remodelled becoming identical in sequence to its partner. The lysogens obtained in such cases may be of all possible types, i.e., produce only saf phage, only sap phage, or a mixture of the two, and the frequencies of the various types will depend on the particular specificities of the repair enzymes involved.

230

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

phage type, rather than the preexisting type. This possibility is supported by the finding (Six, 1971) that in superinfection of a normal lysogen with saf phage it is possible to obtain dilysogens with the two prophages very near each other, presumably in tandem, but not when both prophage and superinfecting phage are sap, as though tandems of two s a f f prophages were extremely unstable. As in other temperate systems, superinfection of a P2 lysogen with a related heteroimmune phage often leads to curing, i.e., loss of both phages and survival of the bacterium (Cohen, 1959; Six, 1960). It is not a t all clear why curing should be so common in heteroimmune superfection, and very infrequent in homoimmune superinfection.

C. EDUCTION OF HOSTCELLGENES

An interesting property of P2 has been observed in strain K : this is the removal, called eduction, of a piece of host chromosome adjacent to chromosite H (Kelly and Sunshine, 1967). The bacteria which have lost this segment of genome are easily recognizable, because they are histidine-dependent: the histidine operon is in fact very near site H, clockwise from it, on the bacterial chromosome. I n addition to the genes of the histidine operon, in all cases examined, the eductants have also lost two other genes, gnd and rfb, on the other side of the histidine operon (M. G. Sunshine and B. L. Kelly, personal communication). Eduction takes place spontaneously with low probability in bacteria lysogenic for P2 prophage in site H, simultaneous to the loss of the prophage. If the phage is an int mutant, eduction does not occur; if the lysogenic cell is superinfected with homoimmune int+phage, eduction takes place a t a very high frequency (M. G. Sunshine, unpublished). Since the end of the segment lost seems to be constant or nearly constant in all eductants, and it roughly corresponds to where site I should be if K were completely homologous to C, all these data would be easily explained if a site partially homologous to I existed also in K ; eduction could then result from a reciprocal exchange following pairing between one end of the prophage in H and this hypothetical site, with the help of the int recombination pathway (M. G. Sunshine and B. L. Kelly, personal communication).

D. IMMUNITY TO SUPERINFECTION A temperate phage does not give plaques on bacteria lysogenic for the same type of phage: the superinfecting phage is said to be immunity-sensitive. Its DNA becomes a superinfection preprophage, which

GENETICS OF

P2

AND RELATED PHAGES

231

does not multiply and is diluted out, apparently without being rapidly degraded, among the progeny of the superinfected bacterium (Bertani, 1954). The superinfection preprophage P2 is not completely inactive: it synthesizes immunity repressor (gene C) (Bertani, 1965), it converts to fluorouracil-sensitivity (gene fun) (Bertani and Levy, 1964), and it expresses the int gene (Bertani, 1970). I n addition, a small fraction of superinfection preprophages may participate in other reactions: prophage substitution, double lysogenization, and also vegetative multiplication. This last occurs with a small but definite probability for any superinfecting phage particle, which is unable itself, because of mutation, to make immunity repressor during its lifetime as preprophage. Although this probability appears to depend on the amount of immunity repressor present in the cell, it does not seem to be the result of a saturation of the repressor previous to vegetative multiplication of the superinfection preprophage (Bertani, 1965). Within this theoretical framework, the frequency of lytic reactions following superinfection with a weak virulent mutant may be used as a measure of the level of immunity in a lysogenic strain. Other more empirical methods consist in measuring the killing of lysogenic cells following exposure to increasing amounts of superinfecting phage (Bertani, 1961) or in studying the efficiency of plating of the lysogen for a set of P2 mutants having various degrees of immunity insensitivity-"intermediate" virulent mutants (Bertani and Six, 1958; Six, 1963). I n general one finds the following: (1) strains lysogenic for a c mutant have a lower immunity level than lysogens carrying the wild-type prophage; (2) a doubly lysogenic bacterium is more immune than the corresponding singly lysogenic bacterium ; and (3) a monolysogen in site I1 is more immune than the corresponding monolysogen in site I (Six, 1966; B. Ronn, personal communication). Point (1) is obvious and is consistent with gene C being the structural gene for the immunity repressor, point (2) refleck the presence of a double set of prophage genes, and point (3) can also be explained as a gene dosage effect if one considers that the origin of replication of the E. coli chromosome is said to be not far, counterclockwise, from chromosite 11, replication proceeding clockwise. This predicts that, on the average, in an actively multiplying cell population, there will be more than one copy (at most 2) of chromosite I1 for each copy of chromosite I.

E. COMPARATIVE Little is known about lysogeny in P2-related phages; P2 Hy dis and derivatives have been studied most, and, except for immunity specificity, behave very much like P2 (Bertani and Six, 1958; Six, 1960; Six, 1971).

232

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

They cure P2 lysogens efficiently, when superinfecting, and this is reciprocal (Cohen, 1959; Six, 1960). The defective prophage existing in strain B, from which P2 H y dis may be obtained by recombination with P2, has been localized at a site near the histidine operon, clockwise from it (Cousin, 1964). This site, for all we know, could be homologous to site I. Apparently the inducible phage 424 has an attachment site very close if not identical to the site of this defective prophage (Cousin, 1964). Jacob and Wollman (1956) localized a site for each of the prophages 18, 186, and 299 on the bacterial chromosome. PK and 299 resemble P2 in their site specificity in C (Six, 1971) and in their ability to educe the histidine genes (M. G. Sunshine, personal communication). P3 (Bertani and Six, 1958; Kelly, 1963), W+ (Six, 1971), and P4 (M. G. Sunshine, D. Usher, and E. W. Six, personal communication) are known to attach to the host chromosome a t sites different from I ; in the P3 and P4 cases, these sites are also different from 11, 111, and H. Phage 186 does not seem to interact with P2 chromosites: doubly lysogenic bacteria for the two phages may be obtained, and no extensive curing is noted (G. Bertani, unpublished). ACKNOWLEDGMENTS In addition to several colleagues, mentioned in the text, who have allowed us

to refer to some of their still-unpublished results, we wish to thank particularly Dr. Erich W. Six for many discussions during the preparation of this review, Dr. Gunnar Lindahl for supplying extensive mapping data prior to publication, Dr. R. B. Inman for permission to reproduce his in part still-unpublished DNA denaturation maps, and Dr. Richard Calendar, for constructive comments on the manuscript. Our work has been supported by a joint grant from the Swedish Medical and Natural Sciences Research Councils, and the Swedish Cancer Society.

REFERENCES Anderson, T. F. 1960. On the fine structures of the temperate bacteriophages P1, P2 and P22. Proc. Eur. Reg. Conf. Electron Micros., Delft, 1960 2, 1008-1011. Arber, W.,and Linn, S. 1969. DNA modification and restriction. Ann. R e v . Biochem. 38, 467-500. Baker, R., and Tessman, I. 1967. The circular genetic map of phage S13. Proc. Nut. Acad. Sci. U.S. 58, 1438-1445. Baldwin, R. L., Barrand, P., Fritsch, A., Goldthwait, D. A., and Jacob, F. 1966. Cohesive sites on the deoxyribonucleic acids from several temperate coliphages. J . Mol. Biol. 17, 343-357. Ben-Gurion, R. 1967. On the induction of a recombination-deficient mutant of Escherichia coli K-12. Genet. Res. 9,309-330. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293-300.

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Bertani, G. 1953a. Infections bactkriophagiques secondaires des bacGries lysog6nes. Ann. Inst. Pasteur, Paria 84, 273-280. Bertani, G. 1953b. Lysogenic versus lytic cycle of phage multiplication. Cold Spring Harbor Symp. Quant. Biol. 18, 65-70. Bertani, G. 1954. Studies on lysogenesis. 111. Superinfection of lysogenic Shigellu dysentetiue with temperate mutants of the carried phage. J. Bucteriol. 67, 696-707. Bertani, G. 1958. Lysogeny. Advan. Virus Res. 5, 151-193. Bertani, G. 1962. Multiple lysogeny from single infection. Virology 18, 131-139. 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. Bertani, L. E. 1960. Host-dependent induction of phage mutants and lysogenization. Virology 12, 553-569. Bertani, L. E. 1961. Levels of immunity to superinfection in lysogenic bacteria as affected by prophage genotype. Virology 13, 378-379. Bertani, L. E. 1964. Lysogenic conversion by bacteriophage P2 resulting in an increased sensitivity of Escherichia coli to 5-fluorodeoxyuridine. Biochim. Biophys. Acta 87, 631-640. Bertani, L. E. 1965. Limited multiplication of phages superinfecting lysogenic bacteria and its implications for the immunity. Virology 27, 496-511. Bertani, L. E. 1968. Abortive induction of bacteriophage P2. Virology 36, 87-103. Bertani, L. E. 1970. Split-operon control of a prophage gene. Proc. Nut. Acad. Sci. U.S. 65, 331-336. Bertani, L. E. 1971. Stabilization of P2 tandem double lysogens by int mutations in the prophage. Virology (in press). Bertani, L. E., and Bertani, G. 1970. Preparation and characterization of temperate, non-inducible bacteriophage P2 (host: Escherichia coli). J. Gen. Virol. 6, 201-212. Bertani, L. E., and Levy, J. A. 1964. Conversion of lysogenic Escherichia colt by non-multiplying, superinfecting bacteriophage P2. Virology 22, 634-640. Bertani, G., and Six, E. W. 1958. Inheritance of prophage P2 in bacterial crosses. Virology 6, 357-381. Bertani, G., and Weigle, J. J. 1953. Host-controlled variation in bacterial viruses. J. Bacteriol. 65, 113-121. Bertani, G., Torheim, B., and Laurent, T. 1967. Multiplication in Serrutia of a bacteriophage originating from Escherichiu coli: Lysogenization and host-controlled variation. Virology 32, 619-632. Bertani, G., Choe, B. K., and Lindahl, G. 1969. Calcium-sensitive and other mutants of bacteriophage P2. J. Gen. Virol. 5, 97-104. Beumer, J. 1961. Isolement et ktude chez les Shigella des rdcepteurs aux bactkriophages du bacille de Lisbonne. Me'm. Acad. Roy. Med. Belg. 4 (31, 1 4 7 . Brooks, K., and Clark, A. J. 1967. Behavior of X bacteriophage in a recombinationdeficient strain of Escherichia coli. J. Virol. 1, 283-293. Calef, E., Marchelli, C., and Guerrini, F. 1965. The formation of superinfectiondouble lysogens of phage in Escherichiu coli K12. Virology 27, 1-10. Calendar, R. 1970. The regulation of phage development. Annu. Rev. Microbiol. 24, 241-296. Calendar, R., and Lindahl, G. 1969. Attachment of prophage P2: gene order at different host chromosomal sites. Virology 39, 867-881.

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Calendar, R., Lindqvist, B., Sironi, G., and Clark, A. J. 1970. Characterization of REP- mutants and their interaction with P2 phage. Virology 40, 72-83. Campbell, A. M. 1969. “Episomes,” 193 pp. Harper, New York. Chase, M. 1964. Reactivation of phage P2 damaged by ultraviolet light. Ph.D. Thesis in Microbiology, University of Southern California. Choe, B. K. 1969. Integration defective mutants of bacteriophage P2. MoZ. Gen. Genet. 105, 275-284. Christensen, J. R. 1964. Further studies of host-controlled modification of bacteriophages P2 and T1. Virology 24, 270-277. Cohen, D. 1959. A variant of phage P2 originating in Escherichia coli, strain B. Virology 7, 112-126. Cohen, D. 1960. Analyse de I’hybridation du phage P2 et d’un prophage dkfectif d’Escherichia coli B, par la mkthode de centrifugation en gradient de densitk. C.R. Acad. Sci. 250, 946-948. Cousin, D. 1963. Etude d‘un prophage dkfectif de la souche B d’Escherichiu coli. Thirse, Facultk des Sciences, Universitk de Paris. Cousin, D. 1964. Localisation gknetique d’un prophage dkfectif de la souche B d’Escherichia coli. Ann. Inst. Pasteur, Paris 106, 8474366. Denhardt, D. T., Dressler, D. H., and Hathaway, A. 1967. The abortive replication of +X-174 DNA in a recombination-deficient mutant of E. coli. Proc. Nut. Acad. Sci. US. 57, 813-820. Eastburn, J. 1969. Rescue by a prophage of a superinfecting irradiated phage. MS Thesis in Microbiology, University of Iowa, Iowa City, Iowa. Echols, H., and Gingery, R. 1968. Mutants of bacteriophage X defective in vegetative genetic recombination. J . MoZ. Biol. 34, 239-249. Edgar, R. S., and Wood, W. B. 1966. Morphogenesis of bacteriophage T4 in extracts of mutant-infected cells. Proc. Nut. Acad. Sci. U.S. 55, 498-505. Frkdkricq, P. 1953. Transfert gknbtique des propriktks lysogknes chez E. coli. C. R. SOC.Bwl. 67, 2046-2048. Geisselsoder, J., and Mandel, M. 1970. Physical properties of phage 299. Mol. Gen. Genet. 108, 158-166. Geissler, E. 1970. Zum Mechanismus der (W-)-Induktion von Prophagen. Stud. Biophys. Berlin 19, 185-206. Glover, S. W., and Aronovitch, J. 1967. Mutants of baoteriophage lambda able to grow on the restricting host Escherichia coli strain W. Genet. Res. 9, 129-133. Glover, S. W., and Kerszman, G. 1967. The properties of a temperate bacteriophage W+ isolated from Escherichia coZi strain W. Genet. Res. 9, 135-139. Goldthwait, D., and Jacob, F. 1964. Sur le mecanisme de l’induction du dkveloppement du prophage chez les bactkries lysoghes. C. R. Acad. Sci. 259, 661-664. Golub, E. I., and Reshetnikova, V. N. 1970. [A new case of prophage defective induction.] Mikrobiologiya 39, 1046-1050 (in Russian). Golub, E. I., and Zwenigorodsky, V. I. 1969. Defective thermal induction of a non-inducible bacteriophage. Virology 39, 919-921. Golub, E. I., Orlowa, G. G., and Reshetnikova, V. N. 1970. Repressor function of late phage gene. Virology 42, 538-539. Gough, M., and Levine, M. 1968. The circularity of the phage P22 linkage map, Genetics 58, 161-169. Hattman, S. 1964. The control of host-induced modification by phage Pl. Virology 23, 270-271. Hershey, A. D. 1969. Genetics Research Unit, Report of the Director. Carnegie Inst. Washington Year. 67, 555-568.

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Hertman, I., and Luria, S. E. 1967. Transduction studies on the role of a rec+ gene in the ultraviolet induction of prophage lambda. J . Mol. Biol. 23, 117-133. Ikeda, H., and Tomizawa, J. 1965. Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments. J. Mol. Biol. 14, 85-109. Inman, R. B. 1966. A denaturation map of the A phage DNA molecule determined by electron microscopy. J. Mol. Biol. 18, 464-470. Inman, R. B. (1967). Denaturation maps of the left and right sides of the lambda DNA molecule determined by electron microscopy. J. Mot. Biol. 28, 103-116. Inman, R. B., and Bertani, G. 1969. Heat denaturation of P2 phage DNA: compositional heterogeneity. J . Mol. Biol. 44, 533-550. Inman, R. B., and Schnos, M. 1970. Partial denaturation of thymine- and 5-bromouracil-containing X DNA in alkali. J. Mol. Biol. 49, 93-98. Inman, R. B., Schnos, M., Simon, L. D., Six, E. W., and Walker, D. H., Jr. 1971. Structural properties of P4 bacteriophage and P4 DNA. Virology 44, 67-72. Jacob, F., and Wollman, E. L. 1956. Sur le processus de conjugaison e t de recombinasion chez Escherichia coli. I. L’induction par conjugaison ou induction zygotique. Ann. Inst. Pasteur, Paris 91, 48Wi10. Jesaitis, M. A., and Hutton, J. J. 1963. Properties of a bacteriophage derived from Escherichia coli K235. J . E z p . M e d . 117, 285-302. Kaiser, A. D., and Hogness, D. S. 1960. The transformation of Eschen’chia coli with deoxyribonucleic acid isolated from bacteriophage Xdg. J. Mol. Biol. 2, 392-415. Kelly, B. 1963. Localization of P2 prophage in two strains of Escherichia co2i. Virology 19, 32-39. Kelly, B. L., and Sunshine, M. G. 1967. Association of temperate phage P2 with the production of histidine-negative segregants by Escherichia coli. Biochem. Biophys. Res. Commun. 28, 237-243. Kerszman, G., Glover, S. W., and Aronovitch, J. 1967. The restriction of bacteriophage A in Escherichia coli strain W. J . Gen. Virol. 1, 333-347. Lederberg, S. 1957. Suppression of the multiplication of heterologous bacteriophages in lysogenic bacteria. Virology 3, 496-513. Levine, M. 1961. Effect of mitomycin C on interactions bteween temperate phages and bacteria. Virology 13, 493-499. Lindahl, G. 1969a. Genetic map of bacteriophage P2. Virology 39, 839-860. Lindahl, G. 1969b. Multiple recombination mechanisms in bacteriophage P2. Virology 39, 861666. Lindahl, G. 1970. Bacteriophage P2: replication of the chromosome requires a protein which acts only on the genome that codod for it. ViroEogy 42, 522533. Lindahl, G. 1971. On the control of transcription in bacteriophage P2. Unpublished data. Lindahl, G., Sironi, G., Bialy, H., and Calendar, R. 1970. Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2. Proc. Nut. Acad. Sci. U.S.66, 587-594. Lindqvist, B. 1971. Vegetative DNA of temperate coliphage P2. Mol. Gen. Genet, 110, 178-196. Lindqvist, B. H., and Six, E. W. 1971. Replication of bacteriophage P4 DNA in a nonlysogenic host. Virology 43, 1-7. Mandel, M. 1967. Infectivity of phage P2 DNA in presence of helper phage. Mol. Gen. Genet. 99, 88-96. Mandel, M., and Berg, A. 1968a. Cohesive sites and helper phage function of P2, lambda, and 186 DNA’s. Proc. Nut. Acud. Sci. U.S. 60, 265-268.

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