Chromosome mobilization in rec-merodiploids of Escherichia coli K12 following infection with bacteriophage λ

VIROLOGY

39, 467-481

(1969)

Chromosome

Mobilization K12

in Ret-Merodiploids

Following KIYOSHI

Institute

Infection MIZUUCHI

for Protein

with AND

Bacteriophage

TOSHIO

Research, Osaka University, Accepted

July

of Escherichia

co/i

X’

FUKASAWA Osaka,

Japan

1, 1969

Two recombination systems (Int and Red) directed by bacteriophage x have been studied with regard t,o the ability to promote recombination events involving the host chromosome by making use of the chromosome mobilization experiment of Signer and Beckwith (1966). The transfer of a chromosomal marker from a ret-/F/gal-atth merodiploid into an F- cell has been shown to be significantly enhanced as a result of infection of the merodiploid with X phage prior to the mating. Various X strains have been tested for the ability to enhance the chromosome mobilization. The phage strains studied include hint, kred, Xintred, Xgal, hbio, Xb2, Abared, and sus mutants in the early cistrons, N, 0, P, and Q. The X-induced mobilization appears to be due mostly to Int and partly to Red. When the mobilization is stimulated by Int, the phage genome must be integrated in the merodiploid; this suggests that Int is incapable of promoting recombination between two copies of the bacterial allA in the merodiploid. In the case of Red-promoted mobilization, phage integration is unnecessary; it is inferred that Red stimulates a recombination act between the episome and the host chromosome, probably at the homology region. Among the mutants in the early cistrons studied, an N defective is unable to promote mobilization. The deficiency is complementable by a co-infecting X defective both in Int and Red. INTRODUCTION

It has been established that there exist two types of recombination systems directed by the genome of bacteriophage X or 480, as evidenced by the isolation of mutant phage defective in either of these systems (Zissler, 1967; Franklin, 1967) : (1) Int mediates a site-specific recombination, called integrative recombination, which involves integration of the viral genome in the bacterial chromosome (Gingery and Echols, 1967; Gottesman and Yarmolinsky, 1968; Echols et al., 1968; Weil and Signer, 1968). (2) Red promotes a general recombination which plays a role in the vegetative recombination of the viral genomes (Franklin, 1967; Echols and Gingery, 1968; Signer and 1 This work was supported by research grants GM1435342 of Nat,ional Institut,e of General Medical Sciences, U. S. Public Health Service, and Ministry of Education, Japan. 467

Weil, 1968). The genesresponsible for these systems are named int and red, respectively. It is not yet clear, however, whether these two systems are capable of stimulating recombinations between the bacterial chromosomes. We have attempted to answer this question by making use of the ‘Lchromosome mobilization” phenomenon described by Signer and Beckwith (1966). A recombination-deficient (ret-) strain of Escherichia coli K12 harboring an F’ factor rarely transfers chromosomal markers to an F- cell (Clowes and Moody, 1966). The transfer of a chromosomal gene (chromosome mobilization), however, is enhanced if a ret- cell harboring an F’ containing the attachment site for phage $430 (F’try-attss) is infected with that phage before mating (Signer and Beckwith, 1966). This phenomenon has been interpreted to mean that a site-specific recombination is stimulated be-

keen the chromosome and t,hc cpisomc by a phage-directed diffusible system, resulting in conversion of the M- 1:’ merodiploid to an Hfr state. Recently, we have reported (Alizuuchi and l~ukasawa, 1967, presented at the sixth anrmal meeting of Japanese Society of Biophysics held in Osaka) that it is possible to construct a system analogous to that, of Signer and Beckwith for X phage; following infection with A phage, a ret-/ F’gal-a& merodiploid shows an enhanced transfer of a chromosomal gene into an Fcell. With this system, we have carried out a series of experiments that permit us to draw the following conclusions: (1) Both systems Int and Red stimulate chromosome mobilization. (2) When chromosome mobilization is promoted by Int function, the genome of the infecting phage must be in-

tegrated in the merodiploids. (3) Rcd-promot,ed chromosome mobilization is not’ tic-companied by the irkegration of phage genome. Thus Tve suggest that the Red system acts directly on bacterial chromosomes to stimulate some step of the general recombination, whereas the Int system promotes a site-specific recombination which involves a “hybrid region” composed of viral and bacterial chromosomes. We shall also describe an experiment suggesting that these gene functions are positively controlled by another viral function encoded by the N gene. MATERIALS

AND

METHODS

Bacterial strains and F’ factor. The bacterial strains and F’ fact,or used are all derived from E. coli strain K12 and are listed

TABLE

1

I~ESCRIPTIONS OF BACTERIAL STRAINS AND F’ FACTOR USED Strain and F’ factore (1) (2) (3)

A437 PL437 Jc1569

(4) PL437rec (5) W3623rec (6) MFRl (7) MFR2 (8) C600 (9) u155 (10) UlGOgalrec (11) FI’

Character SU+, su, leu-, Tl,51, su-, SC, su-, MFRl

Source

leu-, thr-, thi-, pro-, gal-, bio-, strr thi-, gal-, bio-, str’ his-, arg-, met-, lac-, gal-, xyl-, mtl-, strr, recAthi-, gal-, bio-, str’, recAgalT-, try-, str’, recAproC-, galK-, strP, spc’, X’ lysogenic for Xhy (i”, hBO)

SW+‘, leu-, thr-, thi-, T1,5r SC, his-, trysu+, gaZK-, recA- carrying F’suII-, sue+, nicA+, gal+,

a cryptic atth, bio+

prophage

and/or

reference

From Dr. S. E. Lurin This paper From Dr. T. Watanabe; Clark et al. (1966) This paper Tomizawa and Ogawa (1967) This paper Phage hhy is the gift of Dr. H. Ozeki Appleyard (1954) From Dr. A. Campbell This paper From Dr. T. Watanabe; Eisen et al. (1968).

a (1) The bio-gal segment is derived from W602 (Rothman, 1965). (2) The bio-gal segment was transduced by phage Pl from A437 into PL225 (F-su-, thi-, str’ with a deletion of gal-nicA segment; our unpublished isolate). (4) The ret marker was introduced from Jc1569 essentially by the method of Hertman (1967). (6) Starting from AT2059 (F-proC-; gift of Dr. A. L. Taylor), other markers were introduced successively either by spontaneous mutations (for spc’, str’, h’) or mutagenesis with ethylmethane sulfonate (for galK-). (10) The ret marker was introduced into a galKderivative of U160 (FischerFantuzzi, 1967) from Jc1569 as in PL437rec. The cryptic phage, Xcry(A-J) contains cistjrons A to J. All strains are F- (female) derivatives of Escherichia coli K12. su+: permissive for xsus mutants. su-, suII-: nonpermissive for Xsus mutants. gal-, Zac-, mtl-, zyl-: unable to utilize D-lactose, n-mannitol, u-xylose as a carbon source. arg-, his-, Zeu-, met-, pro-, thr-, fry-, /hi-, bio-: requiring arginine, histidine, leucine, methionine, proline, threonine, tryptophan, thiamine, biotin for growth. &A+, sue+: wild-type allele of nicA-, sue- (requiring nicotinic acid, succinate). str’, sprr, A’, TI ,5r: resistant to streptomycin, spectinomycin, phage X, phages Tl, T5. recA-: recombination deficient. a&: attachment, site for prophage X. Xhy: hybrid phage between X and &O. iA: immunity specific for X. h*o: host range specific for 480.

X-INDUCED

CHROMOSOME

MOBILIZATION

469

fhr leu I/ 0 90

FIG. 1. Chromosomal map of Escherichia coli K12. The associated time scale (figures inside t,he circle) is in conformit,y with Taylor and Trotter (1967). Genetic loci contained in two F’ factors, F,’ (F’gal-atlh) (see Eisen et al., 1968) and F’try (see Signer and Beckwith, 1966), are shown in the lines beside the expanded arcs outside the circle. For the abbreviations, see the footnote to Table 1.

in Table 1. A chromosomal map of the strain K12 shown in Fig. 1 includes the markers pertinent to the present work. Bacteriophages: The X mutations used in various combinations were the following: (1) Suppressor-sensitive mutations susAll , susGg, SUSN~,susOa?susPa, and susQzl (Campbell, 1961); (2) CT857 (Sussman and Jacob, 1962); (3) the b2 deletion (Kellenberger ef al., 1961); (4) i&2 (Gingery and Echols, 1967) and in16 (Gottesman and Yarmolinsky, 1968) causing defects in integration of the viral genome in host chromosomes; and (5) red14 (Echols and Gingery, 1968), resulting in a defect in vegetative recombination; this is identical to red114 (Signer et al., 1968). Two classes of transducing h used carry deletions of viral cistrons; cistrons A to J are missing from Xdg, while i&A (and also exo in somecases)are missing from three nondefective bio transducing phages namely Xpb123, Xpb212, and Xpb316 (see below). Phages 434hy (Kaiser and Jacob, 1957), 480 (Matsushiro, 1963), and a hybrid phage between 480 and X (named Xhy in this paper) were alsoused. Phage Xhy shows the integration specificity to at& (Mr. A. Oka, personal

communication), the immunity of X (ii), and the host range of 480 (hso). A clear-plaque mutant, XC6o, was used in measurements of lysogenization frequency of various X by the method of Gottesman and Yarmolinsky (1968). A map of X phage is shown in Fig. 2. Media. Tryptone broth containing 1% Bacto-tryptone (Difco), 0.5 % NaCl, lop3 M MgClz , and 1 pg of thiamine-HCl per milliliter was generally used to grow bacteria that served as recipient cells for various X phages. Tryptone media containing agar (Eiken) at 0.7 % and at 0.4 % were used for plaque counting of phages as bottom layers and top layers, respectively. Nutrient agar for colony counting of bacteria contained 1% meat extract (Kyokuto), 1% Polypeptone (Daigo), 0.25 % NaCl, and 1.0% agar (Eiken). EMT agar was composed of 0.04% eosine Y (Merck) and 0.0065% methylene blue (Merck), and 1% agar (Eiken) in Tryptone broth. BTB-gal agar contained 0.002 % bromothymol blue and 2 % galactose in nutrient agar. All the above media were adjusted to pH 7 with NaOH. Penassay broth (Difco) was used on preparation of recipient cells in bacterial crosses.The selec-

470

MIZUUCI

IT ANI‘, FUKASAW.4 general

recombinotlcn late

phoge

head

phage

,J,

integration

tail

regulation

DNA

I

h ..-

protein 1

turn

on

lysozyme I

b2

/ bio

hpb316

FIG. 2. Vegetative map of bacteriophage X. The functional clustering of the genes is indicated above the map after Gingery andEchols (1967) and alsoSigneret al. (1968).Also shownis the presumedextent of deletions in several transducing phages (Xpb and Xdg).

tion media for assays of recombinants or transductants basically consisted of the minimal medium M63 (Jacob and Wollman, 1961) solidified with 1.5% Bacto-agar (Difco) and supplemented with 5 pg of thiamine-HCl per milliliter, 200 pg of 2,3,5triphenyltetrazolium chloride per milliliter, and appropriate amino acids and sugars. For isolation of pro+spc’ recombinants, 0.2 % glucose and 100 pg spectinomycin per milliliter were added into the above minimal medium. For gal+spc’ recombinants, 0.2 % galactose, 100 gg spectinomycin per milliliter, and 50 pg proline per milliliter were added. For bio+ transductants, the minimal medium was supplemented with 0.1% vitamin-free casamino acids and 0.2% glucose. Tris-buff ered minimal medium supplemented with appropriate amino acids and sugar was used to prepare cell extracts for X exonuclease assay. TM was composed of 1O-2M Tris-HCl buffer (pH 7.4) and 10V3M MgClz . X-buffer contained 1O-2 M MgCl, and 0.002 % gelatin in TM. Phage lysates and phage assays. Stock lysates: thermal induction was used to prepare stock lysates of C1857phages (Sussman and Jacob, 1962); mitomycin induction (Otsuji et al., 1959) for Cl+ phages; and infection of sensitive cells for hpb, hint2, Xint6, Xb2,

and XC&. High titer lysates, except for Xdg, were prepared by vegetative growth in strain C600, and the phage was purified through differential centrifugations. To prepare the lysate of Xdg, a log-phase culture of a mitomycin sensitive strain, W3llO Mbr (Imae, 1968) doubly lysogenic for X and Xdg was induced by mitomycin at a concentration of 0.2 pg/ml in Tryptone broth. The lysate, after concentration, was subjected to the CsCl fractionation according to Kaiser and Hogness (1960) to free Xdg of X. The high titer lysates were stored in suspensions of X-buffer. The Xdg was assayed by scoring the number of transductants formed on the recipient cells of PL437 on BTB-gal agar. Assays of other phages were as described by Gottesman and Yarmolinsky (1968). Test for various phenotypes of A. In the construction of various recombinant phages, int and b2 markers were scored by the spot test of Zissler (1967) using EMT plates. The int phenotype was determined essentially by the method of Gottesman and Yarmolinsky (1968), and the b2 character by measurement of particle densities using the standard X and Xb2 as references. The red character was determined by a spot test for the ability of a phage carrying a sus

h-INDUCE11

CHltOlMOSOME

mutation to rescue the wild-type allele from a ret- cryptic lysogen (see Echols and Gingery, 1968); the two markers C&357 and susAll or susGg were introduced into the phage strain to be tested. The phage lysate was spotted onto a Tryptone agar plate covered with two layers of soft agar (2 ml for each) ; the top layer received 0.1 ml of a fully grown culture of the sufrec-Xcry(A-J) cells (U160gaZrec), and the second layer received 0.1 ml of a fully grown culture of the su- sensitive cells (Ul55). The plate was incubated at 39” for about 18 hours. The red+ phage produced clear plaques and the red- phage turbid plaques. Frequencies of the vegetative recombination were determined according to Echols and Gingery (1968). Isolation of bio transducing phages. Plaqueforming bio transducing x strains, originally described by Wollman (1963), have been isolated by Manly, Signer, and Radding (1969). We isolated, through the following procedures, similar phage strains which were characterized and used in the present work. An LFT lysate with a titer of 2 X 10” PFU per milliliter was prepared by inducing a culture of strain W3110 Mbr lysogenic for X, with mitomycin (0.2 Hg/ml). Recipient cells for transduction were prepared from a fully grown culture (1 X lo8 cells per milliliter) of a bio- strain, A437; the cells were washed with TJI and starved for 1 hour in 10e2 M ,1JgC12 before use. Two-tenths milliliter of the LFT lysate was mixed with 0.S ml of the recipient culture. After an adsorption period, O.l-ml aliquots of the mixture were spread onto enriched minimal plates for bio transduction. Transductant colonies were present at a frequency of 3 X 10eg per PFU. Twelve out of 24 bio+ colonies gave rise to HFT lysates. Five independent lysates thus obtained were found to contain plaque-forming particles that transduced the bio+ marker; appropriate dilutions of these lysates were mixed in Tryptone top agar with the bio- cells and poured onto Tryptone plates. Well-isolated plaques were tested for bio transducing ability; cells at the plaque centers were transferred by means of a platinum wire onto enriched minimal plates on which only bio+ cells will grow. Some of the plaques tested were found to

MOBILIZATION

471

have bio+ cells in their centers. Starting from such plaques, three independent lysates, designated Xpb123, 212, and 316 were established after at least two cycles of singleplaque isolations. Preparation of cell extract for X exonuclease. Strain U155 was grown to 2 X IO8 cells per milliliter in a Tris-buffered minimal medium supplemented with 0.1% vitamin-free casamino acids, 20 pg of tryptophan per milliliter, 20 pg of histidine per milliliter, and 0.25 % maltose. Cells were collected by centrifugation, suspended in 355 volume of lob2 ,$I MgC12, and aerated at 37” for 15 min. One milliliter of the cell suspension was mixed with 1 ml of a phage lysate to give a mult,iplicity of infection of 2.5. The mixture was incubated at 37” for 15 min for adsorption and then brought to a total volume of 20 ml by adding prewarmed medium with glucose as a carbon source. The culture was incubated at 37” with vigorous shaking for 35 min and poured into an ice-cold centrifuge tube containing chloramphenicol to a final concentration of 100 pg/ml. The following procedures were conducted below 5’. The phage-infected cells were collected by centrifugation at 3000 g for 20 min. The cells were resuspended in 6 ml of 0.05 M glycylglycine-NaOH buffer (pH 7.0) and disrupted by sonication in Branson Sonifier (at 20 kc for 30 set X 2). A clear supernatant sample obtained through centrifugation at 10,000 g for 30 min was used as the enzyme preparation. dssay of X exonuclease. This was carried out essentially according to Little, Lehman, and Kaiser (1967) : Assay mixture was composed of 50 ~1 of 3H-labeled E. coli DNA (260 rnp OD unit of 2.5 or 1.73 X lo6 cpm/ ml), 30 pl of 0.025 M MgClz , 20 ~1 of 1 M glycine-KOH buffer (pH 9.4), 10 ~1 of enzyme preparation, and Hz0 to a total volume of 300 ~1. 3H-labeled DNA was prepared through the phenol extraction, after treatments with lysozyme, detergent, and ribonuclease, from thymine-requiring bacteria of E. coli K12 grown in presence of thymine-6-T(n) (5 pg or 5 PCi per milliliter). The reaction was allowed to proceed at 37” and was halted by adding 0.2 ml of bovine serum albumin solution (5 mg/ml) and 0.5 ml of ice-cold 10% trichloroacetic acid. The

tubes \vere kept in ice for at least 20 min tion ~vas pipet,ted intao i.S ml of a recipient culture contained in a 50-ml flask. A O.l-ml and then centrifuged at 3000 rpm for 5 min aliquot n-as also transferred from a phagein the cold. A O.&ml aliquot of the supernatant fract,ion was transferred to a vial, infected donor culture int’o 10 ml of Tryptone neutralized by 0.1 ml of 2 iI1 Tris solution, broth with a fe\v drops of chloroform for and mixed with 10 ml of Bray’s scintillntor determinat,ion of unadsorbed phage. Mixed solution. The radioactivity was determined cultures \vere shaken very gently in a Gyroin Beckman liquid scintillation spectrometer tory water bath shaker at 33” for 60 min. One milliliter of a mating mixture was then LS 250. Standard transduction of bio marker. From pipetted into an ice-cold tube and agitated an overnight culture of &o- strain (PI,437 for 45 seeon a Vortex mixer to interrupt the or PL437rec) in Tryptone broth, cells were mating. In each sample, gal+spc’ and pro+collected, washed once with TM, and re- spc’ recombinant colonies were scored on the suspended in 10e2 M AIgClz to give a cell appropriate selection media described above. density of 1 to 2 X log per milliliter. The The plates were incubated at 33” for 2 or 3 cell suspension was aerated at 37” for 30 min days before counting of recombinant colobefore infection. The infection mixture connies. tained 0.5 ml of bacterial suspension, 0.1 ml RESULTS of transducing lysate (about 2 X lo6 PFU), and 0.1 ml of helper lysate (about 5 X log Characterization of bio Transducing Phages PFU) or 0.1 ml of X-buffer (for the control The hpb strains used in the present work without helper). After an adsorption period were all defective in Int and some in Red, of 30 min, O.l-ml aliquots or appropriate in agreement with the results of Manly et al. dilutions were spread onto enriched minimal (1969). They were unable to establish stable plates for bio+ transductants. The plates lysogeny in a sensitive host; the deficiency were incubated at 33” for about 40 hours in lysogenization was not complemented by until transductant colonies developed to be point mutants in intA gene, such as kint2 or counted. Xint6. A typical result is shown in Table 2. Chromosome mobilization. Two ret- merodiploids, W3623rec/Fl’ or Jc1569/F:, were TABLE 2 used as donor strains, and an F-proC-yalHELPING EFFECT OF VAIUOUS Xbio (Apb) IN Xrspcr (MFRl) or its lysogens for Xhy LYSOGENIZATION OF XintGa (MFR2) were used as recipients. (1) A donor Frequencyof stablelysogens Helperphage culture was grown overnight at 37” with per 100infectedcells aeration in Penassay broth. The culture was diluted 30 times with Tryptone broth conNone <0.005 Xpb123C1857 <0.005 taining 0.5% of maltose. The tube mas hpb212CI857 <0.005 shaken until the cell density reached about Xpb316Cr857
x-INDUCED TABLE COMPLEMENTATION

hredB

MOBILIZATION

473

3

OF V.~RIOUS Xbio IN GICNERAL RECOMBINATIOW

Crosses

Xpb123slLsGsC1857 susAnC1857 Xpb212susG&&57 susA,,Cr857 Xpb316susG&1857 susAnCr857 Xpb123sl&&I857 Xpb212susG&1857 Xpb316susG&I857

CHROMOSOME

TABLE (kpb)

WITH

X EXONUCLIMSE

ACTIVITY

INFECTION

Percent A-G recombination

X Xb2red14

2.4

X Xb2red14

2.6

X Xb2red14

2.4

X Xb2 susAll X Xb2 susAll X Xb2 susA1,

2.3 1.9 2.0

a A log-phase culture (1 X lo* per milliliter) of PL437rec was washed with TM and starved in 10-z M MgCl, for 30 min. The cells were infect.ed with the indicated parental phages at a multiplicity of exposure of about 10 for each. After 20 min for adsorption at 37”, free phage was removed by centrifugation. The infected cells were aerated for 90 min at 39” in Tryptone broth, into which then a few drops of chloroform was added. The Iysates were centrifuged at 10,000 9 for 20 min. On the supernatant samples, total progeny phages and sus+ recombinants were scored on C600 and on U155, respectively. Numbers in the table are (plaques on U155) X lOO/(plaques on CSOO).

With respect to vegetative recombination, Xpb316 is normal while Xpb123 and Xpb212 are defective, as judged by the spot test for red phenotype whose procedures are described under Materials and Methods; Xpb123 or Xpb212 bearing the su.sG mutation were unable to rescue susG+ marker from the ret- cryptic lysogen, whereas Xpb316susG did so as efficiently as Xred+susG. The deficiency of Xpb123 and Xpb212 in Red function Kas complementable by Xredl4 (Table 3), suggesting that redB (in which red14 maps; seeSigner et al., 1968) was normal in those strains of Xpb. Xpb316 was as capable of directing synthesis of A exonuclease upon infection of a qensitive cell (Kern and Weissbach, 1963) as the wild-type phage, but Xpb123 and hpb212 were not (Table 4). Since the exonuclease is known to be involved in X-directed Red system (see Signer et al., 1968), we assume the Red-deficiency both in Xpb123

Infecting

phage

None hpb123CI857 Xpb212CI857 Xpb316Cr857 XC1857

WITH

4 INDUCED VARIOUS

AFTISR Xa

Exonuclease

activity

240* 240 300 1570 15fiO

u Log-phase cells of skain U155 were infected with the indicated phage at a multiplicity of infection of 2.5. The infected culture was aerated at 37” for 35 min in an enriched minimal medium. Cells were collected by centrifugation, resuspended in 0.05 M glycylglycine-NaOH buffer (pH 7.0), and disrupted by sonication. A clear supernatant fraction obtained by centrifugation at 10,000 g for 30 min was used as the enzyme preparation. The exonuclease activity was determined by the method of Little, Lehman, and Kaiser (1967). For detailed procedures see Materials and Methods. b Figures represent radioactivity (cpm) of 3Hlabeled Escherichia coli DNA rendered soluble in acids in 10 min per milligram of protein.

and Xpb212 to be derived from defects in the ezo gene. All the Xpb phages were helped by Xb2 in lysogenization of a ret-bio- cell (data not presented) ; this finding suggests that these phages contain the b2 region, which is believed to contain a region required structurally for normal lysogenization. Considering the above results on the basis of the Campbell model (1962), we suggest that our isolates of bio transducing h phages carry deletions as shown in Fig. 2. Involvement of Int and Red Functions in ChromosomeMobilization (Table 5) Whenrec-/F’gaZ-utth merodiploids (W3623rec/F1’) were mated to F-proC-gal-X’spc’ cells immune to X (MFR2), the frequency of transfer of a chromosomal (nonepisomal) marker (proC+) was about 2 X 1OP per F’ donor cell under our experimental conditions. In parallel experiments, the merodiploids were infected prior to the mating by X phages with or without point mutations in red and/ or int genes. As seen in the table, a redBmutant @e&4) promoted the chromosome

474

MIZUUCIII

APiD

mobilization as effectively as the wild type. Thus, the freyuency of p/o+ recombinants increased 60 to 120 times over the uninfected control. A point mut,ant in intA gent (kir2tJ) TABLE CHROMOSOME TION

Expt. NcLa

5

MOBILIZATION WITH

FOLLOWIN(:

V.UlIOLX

Phage Red -__~__

.~ 1

2

3

INWC-

PHAGIS

None hint2Ch857 Xred14C1857 Xint2red14Ci857 XC1857

+ +

22 10.6 154 4.31 2630 5.70 4' 3.43 1340: 3.52,

0.21 3.57 46.2 0.12 38.0

+ +

15 20 19 175 1340

0.18 0.78 0.91 7.15 64.6

-

-t

None Xpb123Ci857 Xpb212Ci857 Xpb316Cr857 XC1857 None

-

68’3

l-

+

8.35 2.53 2.12 2.45 2.08

21 11.2 288 13.2

/ 0.19 2.2

a In experiments 1 and 2, a log-phase culture (2 X lo8 per milliliter) of st)rain W3623 rec/F1’ (ret-gal-/F’gaZ-atth) was washed with TM and starved in 10e2 M MgCl, for 30 min. The cells were infected with the indicated phage to give a multiplicit,y of infection of 5. After an adsorption period of 15 min at 33”, the mixture was diluted lo-fold into a log-phase cult,ure (2 X lOa per milliliter) of strain MFR2 (F-g&proC-Xrspcr with Xhy (i”h80) prophage). After 60 min for mating at 33”, the mixt,ure was blended and plated for recombinants on spectionomycin medium. Experiment 3 was carried out as above except that the recipient strain used was a 2’1,P derivative of MFRl (F-g&proC-X’spcr) and that the multiplicity of infection was 4. For detailed procedures, see Materials and Methods. b, c b and c represent pro+spc’ and gal+spcr recombinants respectively; values are expressed as frequencies per lo6 donor cells. d Ratios of pro+ recombinant number to gal+ (pro+/gaP) were found to be reasonably constant for a given phage from an experiment to another, even though the numbers of the respective recombinants varied in a rather wide range. One may therefore take the ratio as a “normalized” value to represent the extent of phage-promoted chromosome mobilization.

FUKASAWA

also caused an increase of chromosome mobilization. So enhancement of the chromosome mobilization was observed in the case of a double mutant, ~int&dl4. These results suggest that the function encoded by the int gene plays a major role in enhancement of the chromosome mobilization, and that the function of the red gene also cont,ributes the enhancement but less efficiently and independently of the int gene function. Essentially similar result,s were obtained with three nondefective bio transducing X (Xpb) which have deletions of the viral genome, as shown in Fig. 2, either only in intA (Xpb316) or in intA and exo, but not redB (Xpb123, Xpb212). Qualitatively similar results were obtained when another retmerodiploid Jc1569/Fr’ was used in place of W3623rec/Fr’ (data not presented). When the X-related phage 480 was used as infecting phage, chromosome mobilization was also stimulated to an extent comparable to that of the M-deficient phages, as shown in Table 5. Since $80 has no attachment site on the F’ factor, the observed stimulation is presumably due to the general recombination system directed by the ~$80 genome (see Franklin, 1967). A slight but significant increase of chromosome mobilization was also observed by Signer and Beckwith (1966) in a ret-/F’lac merodiploid infected with $80 or X, whose attachment sites are not present on that episome. We suggest therefore that both Int and Red stimulate recombination acts between the host chromosome and an episome, resulting in the enhanced chromosome mobilization. Lysogenization

Ability

of ~pb Used

The Xpb strains used in the preceding experiments were tested for lysogenization ability in ret- or ret+ cells either with or without prophage 434hy. In these experiments (Table 6), our attention was focused on whether the X-directed Red function by itself can mediate the general recombination either at bio or at the prophage regions. In the first place, all the phages tested were unable to conduct bio transduction in a sensitive ret- cell (PL437rec) unless the Int func-. tion was supplied by co-infection with helper

x-INDUCED

CHROMOSOME

phages. The results supported the conclusion that these bio transducing phages are defective in Int system. In a control experiment, these phages produced transductants upon infection of a nearly isogenic ret+ strain (PL437). Similar findings for nonlysogenic ret- or ret+ recipients have been reported by Manly et al. (1969) for their isolates of bio transducing X. Xpb316, which has the normal Red system, was unable to produce transductants in the ret- cell irrespective of the presence or absence of a heteroimmune prophage. These results suggested that the Red function cannot accomplish recombination between the Xpb genome and the host chromosome of the ret- cell although homology regions are available in bio or in 434hy regions. Requirement for Integration of an Infecting Genome for Chromosome Mobilization .

X

Two types of deletion mutants of X phage, Xb2 and Xdg, were used in this series of experiments (Table 7). These phages are known to lysogenize sensitive hosts rarely even though they have no defects in the TABLE

6

TRANSDUCTION OF BIOTIN MARKER NONDEFECTIVE Xbio (Xpb)” Recipient*: T%%llSducing &age

Xpb123 kpb212 Xpb316

rec-

“i%z


<0.005 <0.005 <0.005,

WITH

ret-(434hy) ~__ X+

NlXle

24.1 26.4 27.9

<0.005 <0.005 <0.005

ret+ NOtE

7.0 6.3 17.0

a A fully grown culture in Tryptone broth (2 X 108 cells/ml) of the indicated recipient was washed with TM and starved in 1c2 M MgClz for 30 min. Five-tenths milliliter of the starved culture was mixed with 0.1 ml (2 X lo6 PFU) of the indicated transducing phage lysate and 0.1 ml (5 X 109 PFU) of the indicated helper lysate. After an adsorption period of 30 min, the mixture was plated on enriched minimal medium for bio+ transductants. b reef and ret- recipients are PL437 and PL437 Tee, respectively. c Xint, and X+ are Xint2C1857, and X&857, respectively. d Values represent frequencies of bio+ transductants per lo3 PFU of transducing phage.

475

MOBILIZATION TABLE CHROMOSOME MOBILIZATION TION WITH VARIOUS Phage

%?

4

None Xb2 Xdg* x

5

None XbLsusAll Xb2red14susAnC&57 XC1857

7 FOLLOWING INFECDELETION PHAQES~ pro+

(a&

P(m+$gj

18 312 275 1280

8.93 4.10 3.82 2.61

0.20 7.60 7.19 49.2

29 175 17 714

13.0 3.30 3.48 1.27

0.22 5.31 0.50 56.5

a Log-phase cells of strain W3623rec/F,’ were infected with the indicated phage to give multiplicities of infection of 5 in experiment 4, or 7 to 8 in experiment 5. The infected culture was mixed with a log-phase culture of strain MFR2 for mating. For other procedures and presentation, see Table 4. b Bn approximate Xdg particle titer was estimated by multiplying the transductant-forming unit (TFU) by a factor of 5 according to Kaiser and Hogness (1960), which gave a cross section comparable to that of those authors. TFU was assayed by scoring gal+ colonies in a starved culture of PL437 infected with appropriate dilution of the lysate in presence of X+ helper (input multiplicity of 10). For other information, see Materials and Methods. Contaminating X particles in the lysate were estimated to be less than lyO of Xdg particles.

intA gene. The deficiency in lysogenixation is believed to be due to structural abnormalities in the regions involved in the integrative recombination (see Signer, 1968). As shown in Table 7, both Xb2 and Xdg have the ability to enhance chromosome mobilization, but less effectively than the wild type; the efficiencies are comparable to those of the int- phages (see also Table 5). The observed chromosome mobilization induced by Xb2 (or Xdg) may be accounted for by the Red function because a red- derivative of Xb2 was found incapable of enhancing chromosome mobilization at all. These experiments strongly suggest that an efficient integration of the infecting phage genome is a prerequisite for Int function to promote the chromosome mobilization, but not for Red. This conclusion was further supported by the exneriments to be nresented next.

ThI3I,I:, IXPLUCNCE

OF I~ICNITY

IN MATING

I-: bx2k~1x~x5 -

Infecting phage on donor cells

Function involved

~_~.~~~ son immune

~ pd

Xred14Ci857 Xin.t2CI857 Xred14intXi857

Int Red Ilone

8 C)N ‘~oIiSl;HV~:l~~'

51 Hi 15

.ai(X lo-"]

0 .n9 1.x 1.19

-

~~~~- P$

(y;

5.5 (i .3 1.3

C~~rto~Osor~n;

MOBILI~ATI~S”

Recipien? Immune

pro

12x0 119 25

1’ was infected with the indicated a A log-phase culture of strain W3623rec/F plicity of infect,ion of F. The infected culture was mixed with log-phase culture ents for mating. For other procedures and presentation, see Table 4. b Strains MFRl and MFR2 (Xhy(iAhso)-lysogenic derivative of MFRl) were immune recipients, respectively.

(X

ml

lo-“)

“(g+:“;;;-

3.37 3.96 3.39 phage to give of the indicated used as nonimmune

38.1 3.8 0.7 a multirecipiand

a FIG. 3. Diagrammatic representation of a presumed mechanism of Int-promoted chromosome mobilization (see the text). The arrow in the F’ represents the point where the genetic transfer begins during conjugation. Thefirst step shows the integration of the infecting X genome in the F’ at atth, which generates two kinds of “hybrid” attachment sites at the right (R) and left (L) ends of the prophage; the filled parts represent segments derived from viral attachment site (atti”), and the open parts represent those from the bacterial attachment site (ullhB). The second step represents the integration of the F’ carrying prophage X int,o the bacterial chromosome. When “L” and att xn are involved in the recombination (a), the resulting Hfr would transfer the prophage genome prior to proC+ during conjugation, whereas when “R” and attABare involved (b), the propha.ge genome would be among the last markers to be trans)fnrred.

476

X-INDUCED TABLE LINKED WITH

TRANSFER

proC+

IN

Infecting pha e on donor ccl “;s

9

OF INFECTING CHROMOSOME

PHAGE GENOME MOBILIZATIOW

Number of colonies carrying infecting phage gemxne among proC+ recombinants tested “Nonimmune”

Xred14CI857 Xint2CI857 Xred14int2C1857

CHROMOSOME

8 (52)* 0 (52) 0 (50)

“I~UUlll~”

33 (50) 0 (52) 0 (50)

a The recombinant colonies were those obtained in the experiments of Table 8. After two cycles of single colony isolation, each recombinant was inoculated into 1 ml of Tryptone broth. The tubes were then incubated overnight at 33”. One loopful of each culture was spotted onto Tryptone plates covered with 2.5 ml of Tryptone top agar containing a drop of fully grown culture of C600. (These cells do not adsorb hBo phage which preexisted in MFRS.) When recombinants were originated from the immune recipient, 5 pg of mitomycin was added into top agar in addition to the bacteria. The plates were incubated overnight at 42”. Confluent lysis observed at a spot was taken as indication that the culture was lysogenie for hh phage. b Figures in parentheses represent numbers of

coloniestested. In the experiments of chromosome mobilization just described (except experiment 3 in Table 5), a X-resistant and immune strain (MFR2) was used as the recipient. When instead a nonimmune derivative of MFR2 was employed as recipient (Table 8), an efficiency of the Int function to promote the chromosome mobilization appeared to be decreased to the same level as that of Red; the decreasemay be accounted for by assuming that zygotic induction (see Jacob and Wollman, 1961) eliminated those nonimmune recipients that received the X genome along with the pro+ gene (seeFig. 3). On the other hand, Red-promoted chromosome mobilization seemed unchanged by the immunity in the recipient cells. These results predicted that, when the chromosome mobilization was promoted by Int, the genome of the infecting phage would be recovered in the pro+ recombinants at a considerable frequency, reflecting the close linkage of proC to alth (see Taylor and Trotter, 1967). This prediction was substantiated in an

MOBILIZATION

477

analysis of the recombinant colonies (Table 9) ; more than 60 % pro+ recombinants, after two single-colony isolations, were found to carry the infecting phage genome when the recombinants resulted from the mating of MFR2 with Xredl4-infected merodiploids. Eight out of 52 recombinant colonies tested also carried the phage genome in the mating of MFRl. In the case of Red-promoted chromosome mobilization, no recombinant colonies carried the infecting phage genome under precisely the same conditions. E$ect of Mutations in L‘Early” Cistrons on ChromosomeMobilization In the experiments presented in Table 10, four Xphageswhich bear suppressor-sensitive mutations in so-called early cistrons (Eisen et al., 1966) were tested for ability to promote chromosome mobilization. A mutant in the N cistron seemed not to enhance the chromosome mobilization. Mutants of 0 or P cistrons promoted mobilization with efficiencies slightly lower than those of the wildtype or XsusQ: 0 and P mutants are known to be defective in DNA replication, which would lower the frequency of integration upon infection of the merodiploids (see Brooks, 1965). This may explain the low efficiencies of mobilization by these mutants. We suggest, therefore, that some function(s) directed by N cistron is a prerequisite for promotion of chromosome mobilization, but that none of the functions of 0, P, or Q cistrons are directly involved. TABLE EFFECT

OF MUTATIONS CHROMOSOME Phage

Xs~sN,C1857 XsztsOsC1857 XsusP&1857 XsusQz,C1857 XC1857

10 IN EARLY FUNCTIONS MOBILIZATION”

pro+

6.6 126 258 960 G54

gal+ (X 10-9

1.41 0.64 1.63 1.29 1.22

ON

yrof/gal’ (X 109

0.47 19.6 15.8 74.2 53.6

a Log-phase cells of strain W3623rec/Fl’ were infected with the indicated phage t,o give a multiplicity of infection of 6. The infected culture was mixed with a log-phase culture of st.rain MFR2 for mating. For other procedures and presentation, see Table 4.

478

MIZUUCHI TABLK

COMPLEMENTATION IN

Phage

None kmN,C1857 + Xint2red14CI857 XC1857

FUKASAWA

recombination acts on the bacterial genomesin the j’ec- cells has been studied by the chromosomemobilization technique, originally devised for 480 by Signer and Beckwith (1966). We have demonstrated that transfer of a ehromosomal marker (pro(Y) from a Tech/F’gaZ-atthmerodiploid into an Fcell was enhanced by infection of the merodiploid with X prior to the mating. The enhanced transfer strongly suggests a role of recombinations involving bacterial chromosomes and promoted by the above viral systems, becausea X phage deficient in both systems did not show any mobilizationenhancing action. Furthermore, our experiments have demonstrated that Int and Red independently exert a stimulatory effect on mobilization, but in distinctive manners, which will be discussedseparately. ing

11

BETWEEN

CHROMOSOME

AXD

XsusN

AND

Xinlred

MOBILIZ.ATIOJV

pro+

tXgqy-5) fig++;;

55 1290

12.4 2.77

1390

2.12

0.44 46.7 65.5

a Log-phase cells of strain W3623rec/F1’ were infected with the indicated phages to give multiplicities of infection of 2.3 for ~susN~C1857, 2.3 for Xint2red14CI857, and 5 for XC1857. The infected culture was mixed with a log-phase culture of

strain MFR2 for mating. For other procedures and presentation, see Table 4.

There are three possible interpretations for the failure of the N mutant in promoting the chromosomemobilization: (1) The product encoded by N cistron, besidesthat of int or red, plays an essential role in stimulating a recombination between episome and chromosome in the merodiploid. (2) A polar effect of the amber mutation in N cistron may interfere with the expression of the genes (i.e., int and red) whose functions are required for the recombination. (3) The product of N cistron positively controls the expression of the genes involved in the recombination. The experiments shown in Table 11 eliminated the possibility that a polar effect of the mutation in XSUSNTinterfered with the expression of int and probably also of red. The results indicate that successful complementation took place between XSU.SNT and knt2redl4 in chromosome mobilization; mixed infection with both mutants promoted chromosome mobilization at an efficiency comparable to that of the wildtype phage. DISCUSSION

The frequency of recombination events involving the bacterial chromosome is reduced to a considerable extent in ret- cells of E. coli K12, in which, however, the X genome undergoes recombination at a frequency comparable to that in ret+ cells by action of two distinctive viral systems, namely, Int and Red. The question whether or not these systems are capable of stimulat-

Chromosomemobilization Stimulated by the Int System The Int system stimulates a site-specific recombination between viral genomes in a ret- bacterium. Thus two X phages, both carrying a defect in a red gene, recombine with each other at a specific region (attx”) which maps between b2 and intA in the viral genome (Echols et al., 1968; Weil and Signer, 1968). On an assumption that attx” is identical to the attachment site for X in the bacterial chromosome (c&x”), the chromosome mobilization promoted by Int might be thought to be due to a direct recombination between the episome and the chromosome at attxB. If this were the case, the chromosome mobilization promoted by Int would not necessarily be accompanied by integration of the infecting phage genomes in the ret-/F’gaZ-atth merodiploid. On the contrary, our results strongly suggest that the integration of infecting phage genome is a prerequisite for the chromosome mobilization. This is evidence that attx+ and attAB are nonidentical to each other as the “recognizable site” for Int system; a similar conclusion has been reached independently by other workers (Fischer-Fantuzzi, 1967; Gingery and Echols, 1968; and see Signer, 1968). We assume, therefore, that the Intpromoted mobilization is a result of a sitespecific recombination involving attAB and a

X-INDUCED

CHROMOSOME

“hybrid region” of attAB and attx4 (see Fig. 3). Such a region would be generated by insertion of X genome into either the episome or the chromosome. In this context, we can make an additional comment on the data of Table 8. When a red- x was used as an infecting phage, a frequency of proC+ transfer to a X-immune recipient strain was almost 10 times higher than that determined with an isogenic nonimmune recipient. Such large differences were observed reproducibly in several independent experiments. This result implies that most of the merodiploids transferring proC+ also transfer X genomes into the F- cells. This would be expected under the following two conditions: (1) the X genome is inserted preferentially into either the episome or the chromosome; and (2) the recombination promoted by Int takes place exclusively in either one of the hybrid regions (L or R in Fig. 3). If either condition is not met, the ratio of the recombinant frequency determined with MFR2 to that with MFRl would not exceed 2. These predictions are now being tested.

MOBILIZATION

479

may be explained in two ways: Either Red does initiate recombination on the host chromosome in bio transduction as it does in chromosome mobilization, but for some reason the recombination product cannot be detected, or there is something special about the chromosome mobilization. For example; an F function expressed upon mating of the merodiploid might cooperate in recombination between episome and chromosome. More information is needed for an understanding of the relationship between the bacterial and the viral systems of general recombination. Involvement of N Gene in ChromosomeMobilization

Mutations in gene N are known to lead to multiple defects in various X functions, including the synthesis of p-protein and X exonuclease (Eisen et al., 1966; and see Radding and Echols, 1968). In this paper we have shown a failure of a .susN mutant of X to promote the chromosome mobilization. Apparently red and int are under the control of the N gene. The successful complementaChromosome Mobilization Stimulated by Reel tion of the N mutant by a red-i&- phage System shown in Table 11 has ruled out the possibilRecent studies with various red- mutants ity that the control of the N gene over the of X have revealed that the bacterial re- int gene be accounted for by a polar effect of combination (Ret) system stimulates general the amber mutation studied. Recent experirecombination of X genomes, although not ments of Radding and Echols (1968) supas effectively as Red system (seeEchols and port the idea that the N gene product, Gingery, 1968; Signer and We& 1968). Our probably a protein, positively controls the results on the Red-promoted chromosome synthesis of P-protein and also X exonuclease. mobilization have suggestedthat Red system These two proteins have been suggested to exerts its action on bacterial genomes to be constituents of Red system (seeSigner et stimulate a direct recombination between al., 1968). Our results may therefore be exthe episome and chromosome at homologous plained, like those of Radding and Echols, regions in the ret- merodiploid. This finding, by assuming that not only red but also int are positively controlled by the N gene however, does not constitute evidence that Red system by itself can complete the host product. On the other hand, it is known that chromosome recombination in the ret- cell. N mutants can lysogenize sensitive hosts at In fact, we have also shown that a red+ bio a frequency much higher than int- phage but transducing phage (Xpb316) is unable to lower than N+ (Brooks, 1965; Gottesman accomplish bio transduction in a ret- host and Yarmolinsky, 1968). It seemsclear that irrespective of presence or absence of 434hy N mutants make a low level of Int product. prophage; the finding may be interpreted to We may assumethat this level of Int is inimply that Red is unable to accomplish a sufficient for promoting the chromosome recombination involving bacterial chromo- mobilization, which presumably require an some in the ret- cell. The apparent discrep- eficient integration in a limited period. The ancy between the two types of experiments possibility that the N gene-directed protein

MIZUUCHI

4so is directly mobilization

ASD

involved in the chromosome remains t,o be excluded. ACKNOWLEDGMENTS

We wish to thank a number of workers, cited in the text, who have generously furnished bacteria and phage. We are indebted to Dr. Harrison Echols for making his manuscripts available to US prior to publication and for commlmicating unpublished observations. REFERENCES R. K. (1954). Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39, 440452. BROOKS, K. (1965). Studies in the physiological genetics of some suppressor-sensitive mutants of bacteriophage X. Virology 26, 489-499. CAMPBELL, A. (1961). Sensitive mutants of bacteriophage X. Virology 14, 22-32. CAMPBELL, A. (1962). Episomes. Advan. Genet. 11, APPLEYARD,

101-145. M., BOYCE, R. P., and P. (1966). Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K12. J. Mol. Biol. 19, 442454. I &OWES, R. C., and MOODY, E. E. M. (1966). Chromosomal transfer from “recombinationdeficient” strains of Escherichia coli K-12. Genetics 53, 717-726. ECHOLS, H., and GINGERY, R. (1968). Mutants of bacteriophage h defective in vegetative genetic recombination. J. Mol. Biol. 34, 239-250. ECHOLS, H., GINGERY, R., and MOORE, L. (1968). Integrative recombination function of bacteriophage X: Evidence for a site-specific recombination enzyme. J. Mol. Biol. 34, 251-260. EISEN, H. A., FUERST, C. R., SIMINOVITCH, L., THOMAS, R., LXNBERT, L., PEREIRA DA SILV~, L., and JACOB, F. (1966). Genetics and physiology of defective lysogeny in K12(h) : Studies of early mutants. Virology 30, 224-241. EISEN, H. A., SIMINOVITCH, L., and MOHIDF,, P. T. (1968). Excision of lambda prophage : Effec t,s on host survival. Virology 34, 97-103. FIsCHI~R-FANTUZZI, L. (1967). Integration of X and Xb2 genomes in nonimmune host bacteria carrying a X cryptic prophage. Virology 32, 18-32. FRANKLIN, N. C. (1967). Delet,ions and functions of the center of the 480-X phage genome. Evidence for a phage function promoting genetic recombination. Genetics 57, 301-318. GINGERY, R., and ECHOLS, H. (1967). Mutallts of bacteriophage X unable to integrate into the host CLbRK,

A. J., CHSMBERLIN,

HOWARD-FLANDERS,

FUKASAWA chromosome. Proc. Snf(. .icad. Sci. C-.S. 58, 1507-1514. GISGERY, It., and ECHOLS, II. (1968). Iut,egration, excision, and transducing particle genesis by bacteriophage X. Cold Spring Harbor Symp. Quunt. Biol. 33, 721-727. GOTTESX~S, M. E., and ~~~~~~~~~~~~~ M. B. (1968). Integration-negative mutants of bacteriophage lambda. J. AVfoZ. Biol. 31, 487-505. HERTM.IN, I. M. (1967). Isolation and characterization of a recombination-deficient Hfr strain. J. Bacterial. 93, 58(t583. IMAF,, Y. (1968). Mitomycin C-sensitive mutant of Escherichia coli K-12. J. Bacterial. 95,1191-1192. Jacoe, F., and WOLLM~X, E. L. (1961). “Sexuality and the Genetics of Bacteria.” Academic Press, New York. KAISER, A. D., and HOGNESS, D. S. (1960). The transformation of Escherichia coli with deoxyribonucleic acid isolated from bacteriophage Xdg. J. Mol. Biol. 2, 392415. KAISER, A. D., and JACOB, F. (1957). Recombination between related temperate bacteriophages and the genetic control of immunity and prophage localization. Virology 4, 509-521. KELLENBERGER, G., ZICHICHI, M. L., and WI”IGLE, J. (1961). A mutation affecting the DNA content of bacteriophage lambda and its lysogenizing properties. J. Mol. Biol. 3, 399-408. KORN, D., and WEISSRXH, A. (1963). The effect of lysogenic induction on the deoxyribonucleases of Escherichia coli K12h I. Appearance of a new exonuclease activity. J. Biol. Chem. 238, 33903394. LITTLE, J. W., LEHMBN, I. R., and KAISER, A. D. (1967). An exonuclease induced by bacteriophage X I. Preparation of the crystalline enzyme. J. Biol. Chem. 242, 672-678. M.~NLY, K., SIGNER, E. R., and RADDING, C. M. (1969). Nonessential functions of bacteriophage

X. Virology 37, 177-188. A. (1963). Specialized transduction of tryptophan markers in Escherichia coli K12 by bacteriophage $80. Virology 19, 475-482. OTSUJI, N., SEKIGUCHI, M., IIJIM.~, T., and TXAGI, Y. (1959). Induction of phage formation in the lysogenic Escherichia coli K-12 by mitomycin C. Nature 184, 1079-1080. RIDDING, C. M., and ECHOLS, H. (1968). The role of the N gene of phage x in the synthesis of two phage-specified proteins. Proc. Natl. Scad. Sci. MSTSUSHIRO,

U.S. 60, 707-712. J. L. (1965). Transduction studies on the relation between prophaTe and host chromosome. J. Mol. Biol. 12, 892-912. SIGNER, E. R. (1968). Lysogeny: The integration problem. dnn. Rev. Microbial. 22, 451-488.

ROTHMAN,

x-INDUCED

CHROMOSOME

E. R., and BECKWITH, J. R. (1966). Transposition of the lac region of Escherichia coli III. The mechanism of attachment of bacteriophage 680 to the bacterial chromosome. J. Mol. Biol. 22, 33-52. SIGNER, E. R., ECHOLS, H., WEIL, J., RADDIHG, C. M., SHULMAN, M., MOORE, L., and MANLY, K. (1968). The general recombination system of bacteriophage h. Cold Spring Harbor Symp. Quant. Biol. 33, 711-714. SIGNER, E. R., and WEIL, J. (1968). Recombination in bacteriophage X I. Mutants deficient in general recombination. J. Mol. Biol. 34, 261-271. SUSSMAN, R., and JACOB, F. (1962). Sur un systbme de repression thermosensible chez le bacthrioi phage X d’Escherichia coli. Compt. Rend. Acad. Sci. 254, 1517-1519. TAKANO, T. (1966). Behavior of some episomal SIGNER,

MOBILIZATION

481

elements in a recombination-deficient mutant of Escherichia coli. Japan. J. Microbial. 10, 201210. TAYLOR, A. L., and TROTTER, C. 1). (1967). Revised linkage map of Escherichia coli. Bacterial. Rev. 31, 332-353. TOMIZAWA, J.-I., and Ocawa, T. (1967). Effect of ultraviolet irradiation on bacteriophage lambda immunity. J. Mol. Biol. 23, 247-263. WEIL, J., and SIGNER, E. R. (1968). Recombination in bacteriophage X. II. Site-specific recombination promoted by the integration system. J. Mol. Biol. 34, 273-279. WOLLMAN, E. L. (1963). Transduction spbcifique du marqueur biotine par le bactCriophage X. Compt. Rend. Acad. Sci. 257, 4225-4226. ZISSLER, J. (1967). Integration-negative (int) mutants of phage X. Virology 31, 189.