Phage P1 mutant with decreased abortive transduction

VIROLOGY

118,329~344 (1982)

Phage Pl Mutant with Decreased YOSHIHIRO Deparhent of Genetics,

Hyogo

Received

July

Abortive

YAMAMOTO

College of Medicine, i&

Transduction

1981; accepted

Nishinomipa, De&

Hyogo

66$, Japan

11, 1981

One amber mutant, named sz&O, of phage Pl with decreased abortive transduction was isolated from PlCM&-1, a thermo-inducible mutant of PlCM, after hydroxylamine mutagenesis. When sus50 phage is grown in Su- host, defective phage particles are produced at a rate 670 times that of infective phage particles and, in addition, many head particles containing sus50 DNA and many phage tails are also produced. In generalized transduction by sue50 mutant phage, the frequency of abortive transduction per infective phage decreased to about one-tenth that of PlCMets-1 sus+ phage, while that of stable transduction appeared to be about 10 times higher than that of PlCMck-1 sus+ phage. Stimulation of stable transduction by uv irradiation was not observed. From revertant analysis of the sus50 mutant, it is suggested that sus50 might be a single mutation, forming defective phage particles as well as decreasing abortive transduction. INTRODUCTION

Bacteriophage Pl can transfer various genetic elements from donor to recipient bacterial cells by generalized transduction (Lennox, 1955). A lysate of Pl phage contains two kinds of phage particles. One is an infective phage particle which carries a phage genome and the other is a transducing particle which carries a fragment of bacterial chromosome or other genetic element (Ikeda and Tomizawa, 1965a). Only transducing particles are responsible for Pl-mediated generalized transduction. Two kinds of transductants, called stable and abortive, are observed in generalized transduction of chromosomal genes of donor bacteria. Roughly 10% of transduced fragments which have recombined with the recipient chromosome participate in stable transduction, while the rest mediate abortive transduction (Gross and Engelsberg, 1959; Ozeki and Ikeda, 1968). Since stable transduction is dependent on recombination functions of the recipients, no stable transduction takes place in recombination-deficient mutants (Clark and Margulies, 1965; Howard-Flanders and Theriot, 1966), and only abortive transduction is observed. Phage mutants which have increased or

decreased transduction abilities were isolated in SalmoneUa phage P22 (Schmieger, 1971, 1972), and in phage Pl (Wall and Harriman, 1974). However, little is known about the mechanism of formation of abortive transductants in recipient bacterial cells. This paper describes the isolation of a Pl mutant which alters the phage yield and the transduction mode. This mutant will be useful to clarify the relation between stable and abortive transduction by phage Pl. MATERIALS

AND METHODS

Phage strains. Bacteriophage PlCM, Pl wir, and Plkc were obtained from Dr. J. Tomizawa. Phage Xind- was the gift of Dr. H. Ogawa. PlCMcts-1 was a thermo-inducible mutant of PlCM, isolated by the same method as Dr. J. L. Rosner (1972). Bacterial strains. Bacterial strains used are listed in Table 1. Media L-broth, L-agar, and L-soft agar were used for growth of recipient cells and for plating of Pl phage. L-broth contained 1% polypeptone, 0.5% yeast extract, and 0.5% NaCl. The pH was adjusted to 7.4. L-agar or L-soft agar contained L-broth and 1 or 0.5% agar. 329

00.42~6322/82/060329-16$02.00/o Copyright 0 1982 by Academic Preac., Inc. All right-8of repmductionin any form -ed

YOSHIHIRO

336

YAMAMOTO

TABLE

1

BACTERIAL STRAIN@ Strains

Characters

C600 594 CGoothy594thyc600( hind--) 594( Xind-) W3633 N23-76 JCL557

SuII leu thr lac Tl-r gal str-r thy- derivative of C66Q thy- derivative of 594 Lysogenic for Xix& Lysogenic for Xindtrp gal str-r recA42 trp gal arg str-r leu his arg met lac gal xyl ma1 Tl-r str-r recA1 leu his arg met lac gal xyl ma1 Tl-r str-r trp pro his metE ilv mtl ma1 ara gal lac Tl-r str-r recA1 gal str-r

JC1569 ABZ3’77 594m.K

Use

Source

Permissive host Nonpermissive host Radioisotope labeling Radioisotope labeling Donor of transduction Donor of transduction Recipient for transduction Recipient for transduction Recipient for transduction

Appleyard (1954) Lederberg (1956) Ogawa et al. (1968) Ogawa et al. (1968) This paper This paper Lederberg (1956) Ogawa et al. (1968) Clark et al. (1966)

Recipient for transduction

Clark et al. (1966)

Recipient for transduction

Obtained from Dr. E. A. Adelberg

Isolation of PlCM&-1

Isolated by Dr. H. Ogawa by Pl transduction

a All bacteria strains used in this -naner _ except Xi& Dr. H. Ogawa.

TCG medium (Tris-Casamino acidsglucose medium, Kozinski and Szybalski, 1959) was used for labeling of phage DNA with radioisotopes. Davis minimal medium contained KzHPOl 7 g, KH2P04 2 g, MgS04, 0.1 g, (NH&SO4 1 g, sodium citrate 0.5 g, and glucose 2g/liter. Minimal agar is Davis minimal medium with 1.5% agar. The concentration of various supplements were amino acid, each 20 pg/ml; Casamino acids (Difco, vitamin-free), 0.2%; thiamine, 1 rg/ml. X agar contained 1% polypeptone, 0.25% NaCl, and 1.2% agar. X soft agar contained 1% polypeptone, 0.25% NaCl, and 0.7% agar. X agar and X soft agar were used for transduction of Xind- prophage. Dilution buffer, 10 mM Tris-HCl (pH 7.4), 1 mM MgCIB, 5 g NaCl, and 10m5g gelatin per liter, was used for dilution and suspension of both phage and bacterial cells. TEN buffer, 20 mM Tris-HCl (pH 8.0), 1 mAf EDTA, and 10 mM NaCl, was used for transfection of Pl phage DNA. Saline citrate buffer contained, 0.15 M NaCl and 0.015 M sodium citrate, and the pH was adjusted to 7.2.

lysogen were kindly supplied by Dr. J. Tomizawa

and

Plating of Pl phage. Phage Pl was adsorbed to late log-phase culture bacteria, plated on L-plates containing 2.5 mM CaC12, and incubated at 42” unless otherwise noted. Preparaticm of PI phage &sates. Lysogens of PlCMcts-1 or amber mutants were grown at 30” in L-broth until 2-3 X lo* cells/ml. The culture was incubated for 30 min at 42’ and then for an additional 6090 min at 37”. After treatment with CHC&, the bacterial debris was removed by centrifugation, and stored at 4”. By this procedure phage titer of PlCMcts-1 SW’ reached about 2-5 X lO’/ml. Mutagenesis. A O.l-ml aliquot of PlCM&s-l phage (phage titer about lO”/ml) was added to 0.9 ml of 0.5 M solution of hydroxylamine hydrochloride made up in Lbroth and brought to pH 7.8 with NaOH. After incubation at 37” for 24 hr, a lOOfold dilution was made into L-broth containing 10% (v/v) acetone. The mixture was then stored at 5” for additional 24 hr and excess acetone was removed with aspiration at 0”. Phage survival was reduced between lo-’ and 10m4 during the above procedure. Selection of sus mutants. Sus mutants of

PHAGE

PlCMcts-1 were isolated by the double layer method (Studier, 1969). Mutagenized PlCMcts-1 phages were adsorbed to C600 log-phase culture, and plated onto L-plates with 2.5 ml L-soft agar. After the agar hardened, 594 log-phase%dture in 2.5 ml L-soft agar were poured gently onto these plates. After incubation overnight, turbid plaques which formed on these double layer plates were saved and their ability to form plaques on C600 but not on 594 was confirmed. Then purification procedures to form a single plaque on C600 and to characterize each mutant phage were repeated twice. Isolation of Pl lysogens. The lysogens of PlCMcts-1 and of isolated sus mutants were made as follows: Pl phage lysates were spotted on host bacteria spread on L-plates containing chloramphenicol at 25 pg/ml, and colonies formed at 30” were purified. Purified colonies were then tested for phage production and colony formation with chloramphenicol at 42 and 30”. If necessary, these purified bacteria were tested further for Pl immunity by the cross-streak test for Plkc and Pl vir phages at 30”. Complementation spot-test. About 5 X lo7 cells of 594 Su- log-phase culture were plated on L-plates containing 2.5 mM CaCl,. About 10 ~1 of one Pl sus mutant lysate, roughly lo8 infective phages/ml, was spotted and after the liquid was absorbed by the plate, about 10 ~1 of another sus mutant lysate at the same concentration was spotted on the same place. Then plates were incubated overnight. Transduction. Transduction of trp, his, and arg genes were made as follows: recipient bacterial cells were grown in Lbroth to a concentration of 2-4 X lo8 cells/ ml, collected by centrifugation, suspended in L-broth containing 2.5 mlMCaC& to give 2 X log/ml, and infected by Pl phage at 37’ for 30 min. The infected cells were washed twice with dilution buffer and suspended in the same volume of dilution buffer. An aliquot of the infected cell suspension was spread on suitable plates for selection of transductants. Plates were incubated at 37” for 2 days. To avoid Pl lysogenization, the multiplicity of infection was kept under 0.05. As abortive trans-

Pl MUTANT

331

ductants formed on selective plates were too small to count even after 2 days incubation, they were enlarged with a stereoscopic microscope, Olympus model SZ, and were counted while illuminated from below. Transduction of Xiru- prophage was done as follows: recipient cells 594 Su(PlCM) in late log-phase in L-broth were collected by centrifugation, and suspended with L-broth containing 2.5 mM CaClz at a concentration of about 2 X log/ml. They were infected by Pl phage lysate at 37’ for 30 min. After appropriate dilution, the infected cells were plated on X plates with 2.5 ml X soft agar containing 594 (PlCM) log-phase cells. Plates were incubated at 3’7” overnight. Since free phages of Xindin lysogens and in Pl phage lysates never reached lO’/ml, they did not affect the transduction frequencies. PurQication of Pl phage particles. About 5-400 ml phage lysates were centrifuged at 25,000 rpm for 90 min at 5”, and pellets were suspended in 2 ml of L-broth. When phage DNA was labeled with radioisotope, the suspension was treated with DNase 50 pg/ml and RNase 50 pg/ml at 3’7” for 30 min. Then the suspension was layered on top of a CsCl step gradient, and centrifuged at 25,000 rpm for 60 min at 5”. Densities of CsCl steps were 1.7 g crnm3 (0.2 ml), 1.5g cmp3 (0.5 ml), and 1.3 g crnm3 (1.5 ml) in the tube of a Beckman SW50.1 rotor. CsCl solution was buffered with 10 mM Tris-HCI (pH 7.4) and contained 0.7% Casamino acids to prevent the decrease of phage infectivity. Pl phage particles of normal size formed a band between 1.5 and 1.3 g crnd3 CsCl steps, and Pl head particles in sus50 lysate sediment a little faster than normal phage particles. In order to separate clearly Pl phage particles and head particles, phage particles collected by CsCl step gradient centrifugation were further centrifuged in a CsCl density gradient. They were suspended in about 3 ml of CsCl solution buffered with 10 mM Tris-HCl (pH 7.4) and containing 0.7% Casamino acids, and the density of this CsCl solution was adjusted exactly to 1.47 g cmp3. Centrifugation was carried out at 25,000 rpm for 18 hr at 5” in a Beckman SW50.1 rotor. Samples were

332

YOSHIHIRO

taken from the bottom of the tube, and fractions containing Pl phage particles were dialyzed overnight at 4’ against dilution buffer containing 0.7% Casamino acids. Sucrose density gradient centr$ugaticm. Purified Pl phage particles were layered on the top of 4.5 ml sucrose gradient, 5% (w/v) to 20% (w/v) in dilution buffer with 0.7% Casamino acids, in the tube of a Beckman SW50.1 rotor. They were centrifuged at 20,000 rpm for 35 min at 5”. Extract&m of DNA. Purified Pl phage particles were treated with 0.2% sodium dodecyl sulfate at 50” for 10 min, and DNA was extracted with redistilled phenol saturated with saline citrate buffer (pH 7.2) containing 2 mM EDTA by rotation method (Tomizawa and Anraku, 1964). Then Pl DNA solution was dialyzed at 5” for 48 hr against saline citrate buffer (pH 7.2) with 2 mM EDTA, or against TEN buffer (pH 8.0). Transfection of Pl DNA. The procedure was a modification of that of Mandel and Higa (1970). JC1557 recBC late log-phase culture in L-broth (5-8 X lO*/ml) were centrifuged, washed once with an equal volume of cold 50 miV CaCl, solution, and resuspended in a half volume of cold 30 mM CaClz solution. After 10 min at 0”, the bacterial suspension was centrifuged, and suspended finally in one-tenth volume of cold 30 mM CaClz solution. Kept at 0” for 15 min, these competent cells were infected by l-2 pg Pl DNA in about 10 ~1 TEN buffer at 0” for 15 min, and an adequate portion of infected bacterial cells was added to 2.5 ml L-soft agar with 2.5 mlM CaClz which had been kept at 50” previously. After 2 min incubation in L-soft agar at 50”, indicator bacteria were added to soft agar and plated on L-plates with 2.5 mM CaC12. Plates were incubated at 42 or 37” overnight. Electron microscopic observation. For observation by electron microscope, the PTA-negative staining method was used (Brenner and Horne, 1959). Purified phage particles in dilution buffer were mixed with an equal volume of 2% (w/v) phosphotungstate solution previously adjusted to pH 7.0 with 0.1 N NaOH. After 30 set, the mixture was put on carbon-collodion-

YAMAMOTO

coated grids and excess solution was removed with blotting paper. Observations were made at a magnification of 30,000. Labeling of phage DNA with radiois~ topes. 3H-labeled Pl phages were prepared as follows: thymine-requiring bacteria lysogenic for Pl phage were cultured in TCG medium containing thymidine 20 pg/ml at 30” until the cell concentration reached 23 X lO*/ml. Then bacterial cells were collected by centrifugation, and suspended with TCG medium containing thymidine 4 @g/ml and rH]thymidine (2 &i/gg). They were incubated at 42” for 30 min and then at 37” for additional 60-90 min until lysis. After treatment with CHCL, bacterial debris was removed by centrifugation at 5”. azP-labeled Pl phages were prepared in the same manner as aH-labeled phage, except that the medium used was TCG medium containing phosphorus 2 pg/ml and [3zP]phosphorus (5 &i/pg). Disc gel electrophoresis of HindHI digested Pl DNA. Pl DNA in 10 mM TrisHCI (pH 7.4) containing 1 mMEDTA was heated at 65” for 10 min, and diluted in the reaction buffer (10 miV Tris-HCl pH 7.6,50 mMNaC1, 10 mM MgClz, and 14 mM dithiothreitol) to give the concentration of about 2-4 pg DNA/W ~1. Hind111 endonuclease was added to DNA solution at higher than 2 units/gg Pl DNA. After incubation at 37” for 30 min, one-fifth volume of the dye solution (0.2% (w/v) xylene cyanol, 50% (w/v) sucrose, and 2.5 miU EDTA) was added and 20 ~1 of the sample was loaded on the top of 0.7% (w/v) agarose gel column (0.6 X 12 cm). Electrophoresis was carried out at 80 V for 3 hr at room temperature with 90 mM Tris-borate (pH 8.3), 2.5 m&f EDTA, and 0.5 pg/ ml ethidium bromide. DNA was visualized under short wave length ultraviolet light. Hind111 endonuclease was obtained from Boehringer Mannheim. RESULTS

Isolation

of

sm50

Mutant

Rosner (1972) isolated a thermo-inducible mutant of Pl phage, named PlCMcZrlOO, from a recA- lysogen of PlCM

PHAGE Pl MUTANT LlENSITYkm")

10

FRACTION NUMBER

15

20

25

30

FRACTIONNUMBER

FIG. 1. C&l density gradient centrifugation of sus50 mutant. V-labeled sus56 mutant phages were centrifuged in CsCl solution at 25,996 rpm for 18 hr at 5’ with ‘H-labeled, uv-irradiated PlCMcks-1 sus+ phages. In order to count infectivities of PlCMck-1 sue+ phage, about 10’ phages of nonirradiated PlCM&-1 sue+ phages were also added. Indicator bacteria for counting infectivity of sus56 and PlCM&-1 sus+ phages were 0 and 594, respectively. Recipients for transduction of Xi& prophage and trp gene were 594(PlCM) and W3623(PlCM), respectively. (a) sus56 mutant phages produced in C696 SuII+ host. Total number of fractions was 53. (b) sus56 produced in 594 Su- host. Total number of fractions was 54. 0 0, mP radioactivities; 0 0, ‘H radioactivities; -, plaque-forming phages of eus50; - - -, plaque-forming phages of PlCMcts-I sus+; AA, X&z& prophage transduction; A __ A, trp transduction.

phage (Kondo and Mitsuhashi, 1964). By the same method, a thermo-inducible mutant was isolated from 594 recA- lysogen of PlCM, and named PlCMds-1. Pl sus mutants were isolated from this phage, since it is easy to isolate and maintain its lysogens because of its resistance to chloramphenicol, and also easy to get phage lysates because of its thermo-inducibility. Mutagenized PlCMcts-1 phage (0.5 M hydroxylamine, pH 7.8, in L-broth) were plated by the double layer method using C600 and 594 as &I+ and Su- host bacteria, respectively. From turbid plaques formed on the plates at 42”, each phage was purified twice and tested for growth in Su+ and Su- host bacteria. Ninety-four amber mutants of PlCMcts-1 were isolated. These mutants were roughly classified according to complementation tests by double spots of phage lysates on 594 Su- bacterial lawn. To select phage mutants with altered transduction frequencies, lysate of 31 amber mutants (one amber mutant from each

presumed complementation group) and of two amber mutants with weak suppressor sensitivities were prepared from 594 Sulysogens in TCG medium by thermal induction. Using these lysates, transduction frequencies of trp gene were measured. Lysates were mixed with W3623 Trp(PlCM) cells which had been suspended in TCG medium, and they were plated on minimal agar plates supplemented with 0.2% Casamino acids after phage adsorption at 37” for 30 min under 2.5 mM CaClz. After incubation at 37” for 2 days, Trp+ transductants were scored. Among these 33 mutants, one mutant of weak suppressor sensitivity, named .su.s50, had a high transduction frequency of trp gene. With its weak suppressor sensitivity, infective phages were produced at about 10’ and 106/ml in C600 SuII+ and 594 Su- host bacteria by thermal induction of lysogens, respectively. Phage production and generalized transduction by sus50 mutant phage were further investigated.

334

YOSHIHIRO

-1 -g

-I-

a

30 z

15

FIG. 2. Sedimentation profiles of peak II and peak I of sus50 mutant in neutral sucrose density gradients. =P-labeled peak II and peak I fractions of Fig. lb were sedimented with *H-labeled PlCMcts-1 su8+ phages at 20,ooO rpm for 35 min at 5”. Samples were taken from the bottom of the tube. (a) Peak II of ~~50. Total number of fractions was 44. (b) Peak I of ~950. Total number of fractions was 38. 0 __ 0, 9 radioactivities; 0 0, aH radioactivities.

Phage Production

by sus50 Mutant

32P-labeled sus50 phage particles were prepared in C600 SuII+ (Xind-) or 594 Su(X&d-) host bacteria and purified as described under Materials and Methods. They were mixed with uv-irradiated (500 J me2, this dose was sufficient to lose all infectivities of the phages) 3H-labeled PlCMcts-1 sus+ and nonirradiated PlCMc&l su& phages, and centrifuged at 25,000 rpm for 18 hr at 5” in CsCl solution. After fractionation, radioactivities, infectivities, and transducing abilities were measured with each fraction. Since %P-labeled sus50 phage particles were added much more than nonirradiated PlCMcts-1 sus+ phages, they could be counted independently with C600 SuII+, while nonirradiated PlCMcts1 SU.S+phages were scored with 594 Su-. The transduction of Xind- prophage was measured with 594 Su- (PlCM), while that of trp gene was measured with W3623 Trp(PlCM). Figures la and b show these results. The peaks of 3H radioactivities and infectivi-

YAMAMOTO

ties of PlCMcts-1 susf phages were found to coincide, and the density was found to be about 1.47 g crnd3 as previously reported (Ikeda and Tomizawa, 1965a), while BP radioactivities formed two peaks both in Figs. la and b. Henceforth, the higher density fractions and the lower density fractions are called peak I and peak II, respectively. In Fig. la radioactivities of peak I were about one-half of those of peak II, while the former were about five times as much as the latter in Fig. lb. The density of peak I formed in both Figs. la and b seemed to be nearly the same, and was calculated to be about 1.558 g cme3. No infectivity of Pl phage and transduction of hind- prophage and trp gene markers was found in peak I fractions. It was reported previously that lysates of Pl phage contained at least three morphological variants with respect to head size (Walker and Anderson, 1970), but the density of peak I of sus50 phage seemed to be different from those of Pl morphological variants. In Fig. lb, 32P radioactivities of peak II formed a 0.004 g crnw3heavier peak in density than 3H radioactivities and infective particles of PlCMcts-1 sust phage. Analysis

of Peak I of sus5O Mutant

Peak I and peak II fractions of su.350 in CsCl density gradient centrifugation were further analyzed by neutral sucrose gradient centrifugation with [3H]P1CMcts-l sust phages as a reference. The sedimentation profiles were presented in Figs. 2a and b, respectively. As seen, in Fig. 2a, 32p radioactivities of peak II seemed to sediment at nearly the same speed as [3H]P1CMcts-l SUS+ phages. However, 32P radioactivities of peak I seemed to sediment about 1.26 times faster than PlCMcts-1 sus+ phages in Fig. 2b. The sedimentation profile observed in Fig. 2a suggested that peak II fractions might contain phage particles of sus50 mutant. But peak I fractions might contain head particles of Pl phage, considering that head particles of bacteriophage lambda sediment faster than normal phage particles in sucrose density gradient centrifugation (Weigle, 1966).

PHAGE Pl MUTANT

Next, peak I and peak II fractions were analyzed directly by electron microscopic observation. Phage particles seen in the electron microscope are presented in Figs. 3a and b. Figure 3a shows phage particles observed in peak II fractions of aus50 mutant, and they seemed to have nearly the same size and shape as PlCMcts-1 su-s+ phages. The size of these heads was measured to be 85 X 95 nm, and the length of tails was about 220 nm. These data agreed well with those previously reported (Walker and Anderson, 1970). In Fig. 3b, particles just like Pl phage heads were observed, and the size of them was revealed to be nearly the same as Pl phage heads. As expected from the sedimentation profile of peak I fractions, they were found to contain head particles of sue50 mutant phage. The empty heads observed

335

in Fig. 3b are thought to be a preparation artifact occurring during the dialysis. When the purification process of CsCl step gradient centrifugation was omitted, small phage particles of Pl (Ikeda and Tomizawa, 1965c) and their head particles were also observed by electron microscopy, but in more slowly sedimenting fractions in neutral sucrose gradient centrifugation. Anal&s of DNA Extracted Fractions

from Peak I

DNAs were extracted from =P-labeled peak I and peak II fractions of sus50 mutant by the SDS-phenol method, and analyzed by neutral and alkaline sucrose density gradient centrifugation. v-labeled DNA extracted from peak I and peak II fractions sediment at the same speed

FIG. 3. Electron micrograph of peak II and peak I fractions of sus50 mutant. (a) Peak II. (b) Peak I. The bar represents 100 nm.

YOSHIHIRO

336

a

b

c

d

e

f

FIG. 4. Elution patterns of peak II and peak I DNA fragments digested by Hind111 restriction endonuclease. Arrows indicate discrete bands in agarose gel after electrophoresis. (a) and (f) PlCMets-1 sus+ DNA. (b) Peak II DNA. (c) Peak I DNA. (d) PlCMcts1 eus+ DNA + peak II DNA. (e) PlCMds-1 sus+ DNA + peak I DNA.

as rH]DNA of PlCM&-1 su.s+ phage in both neutral and alkaline sucrose gradients (data not shown). In order to investigate the constitutions of DNA extracted from peak I and peak II fractions of sus50 mutant, they were further analyzed by agarose gel electrophoresis. Figure 4 shows the elution profiles of these DNAs after digestion by restriction endonuclease HindIII. DNA of peak I, peak II of sus50 mutant, and PlCMcts-1 sus+ digested with Hind111 endonuclease formed five bands in agarose gel electrophoresis. Pl phage DNA, however, formed four bands after digestion by Hind111 in agarose gel electrophoresis (B&hi and Arber, 1977). This difference may depend on the different phages, PlCMcts-1 and Plc1225ts. Next, the mix-

YAMAMOTO

ture of sue50 peak I or peak II DNA with PlCMds-1 sus+ DNA was digested by Hind111 endonuclease, and eluted in agarose gel electrophoresis in the same ways. As seen in Fig. 4, still five discrete bands were formed. These results showed that most of DNA extracted from peak I fractions, i.e., head particles of sus50 mutant, should be Pl phage DNA as well as peak II DNA, and the size of peak I DNA should also be the same as them. Infectivities of peak I and peak II DNA were tested by DNA transfection. Competent cells of JC1557 recBC treated with CaC& were infected by peak I, or peak II DNA of sus50. As seen in Table 2, infectivities of peak I and peak II DNAs were observed, although their frequencies were about one-fourth and one-fifth of that of PlCMcts-1 SUS+ phage DNA, respectively. Plaques formed by transfection of peak I and peak II DNAs were further analyzed. All of them were revealed to be those of sus50 mutant phage from the complementation spot test. All above results suggest that most of the head particles in peak I fractions of su.950 should contain ~~~750infective DNA. Defective Phage Production tant in Su- Host

by sus50 Mu-

-3H-labeled phage particles were prepared by thermal induction of W3110 Suthy- bacteria lysogenic for PlCMcts-1 sus+ or sus50 mutant. And also in the same medium containing rH]thymidine, XC1857 phage particles were prepared by the induction of W3110 thy- lysogen at the same TABLE TRANSFECTION

OF DNA EXTRACTED AND PEAK I OF swm

“The DNA.

number

FROM PEAK II

Infectivity per pg DNA”

DNA PlCMcts-1 Peak II Peak I

2

sus+

of plaques

405 + 26 105 f 11 73 -t 13 formed

per

microgram

PHAGE Pl MUTANT TABLE DEFECTIVE

PHAGES

337

3 IN ~~50

LYSATE

SH count (cpm)

Infective phages

?I3 count/ infective

Pl/ XC1857”

PlCMcts-1 sus+

XC1857

1.58 x lo5 1.03 x lo6

3.3 x lo7 1.04 x 108

4.79 x 10-a 9.96 x 10-a

2.07

XC1857 sus50

5.35 x 106 1.07 x lo5

8.2 X 10’ 1.76 X 10”

6.52 X lo-’ 6.08

Phage

Defective phage$

451 933

“The relative ratio of [8H]thymidine incorporation per one infective phage of Pl to XC18.57. *Defective phages existed in Pl lysate per one infective phage estimated from values of ‘H radioactivities of Pl per XC18.57.

time, and they were used for the reference of rH]thymidine incorporation into phage DNA. Phage mixture of Pl and lambda were collected by centrifugation at 5” at 25,000 rpm for 90 min and purified by CsCl step gradient centrifugation, by which peak I fractions of sus50 mutant were removed. Either pair of 3H-labeled phages, PlCMcts-1 su..s+ and XCI857, or sus50 and XC1857, was added to CsCl solution, and both samples were centrifuged at 25,000 rpm for 18 hr at 5”. The amount of defective phages in peak II fractions of sus50 mutant could be estimated by the ratio of 3H radioactivity to the amount of infective phages of sus50 to C600 SuII+. Results were summarized in Table 3. The rate of rH]thymidine incorporation of PlCMcts-1 SU.S+phage was revealed to be about two times that of XC1857 phage, as expected from their molecular weights (Ikeda and Tomizawa, 1965a; Davidson and Sybalski, 1971). The ratio of the amount of 3H radioactivity to that of infective phages of sus50 mutant was found to be about 450 in one experiment and 890 in another (data not shown). Therefore, the amount of defective phage particles might be calculated as about 670-fold (mean value of two experiments) higher than that of infective phages in ~~50 mutant phage lysate produced in Su- host bacteria. Another experiment to estimate the amount of defective phage particles in sus5O mutant lysate was performed by specialized transduction of the chloram-

phenicol resistance gene, which lies on PlCMcts-1 phage genome. Each phage lysate obtained from su-350 lysogen of C600 SuII+ or of 594 Su- was mixed with C600 (Plkc) bacteria and chloramphenicol-resistant colonies were counted after plating and incubation. Since Pl lysogens contain only one prophage per host chromosome (Ikeda and Tomizawa, 1968), it is conceivable that this superinfection of Pl phage to Pl lysogen might result in the exclusion of prelysogenized Pl phage or the formation of a recombinant between superinfecting Pl phage and prelysogenized one. As seen in Table 4, frequencies of specialized transduction of chloramphenicol resistance gene by sus50 lysates produced in Su+ and Su- hosts were found to be about 2-fold and 800-fold higher than that by PlCMcts-1 SW+ phage lysate, respectively. All chloramphenicol-resistant colonies formed by infection with ~~50 produced in Su- host carried Pl immunity simultaneously. About 60% of chloramphenicol-resistant colonies were found to have both sus50 mutation and temperature sensitivity, which are probably derived from the exclusion of Plkc phage. The rest of the transductants are thought to be recombinant lysogens, because about 44% of them seemed to have sus50 mutation alone and others to have temperature sensitivity of cts-1 alone. From the experiment of [3H]thymidine incorporation, defective phage particles were estimated to be about 670-times more abundant than infective phages in sus50 lysate

338

YOSHIHIRO

YAMAMOTO

TABLE

4

SPECIALIZED TRANSDUCTION OF CHLORAMPHENICOL RESISTANCE GENE BY et&O MUTANT=

Phage PlCMck-1 sus50 SW50

sue+

Host*

Infective dwe input

CM-r”

CM-r/ infective

Ratiod

594 C600 594

1.39 x lo4 6.5 X lo6 1.71”X lo3

2.25 X 10r 2.19 x lo6 2.23 x lo6

0.162 0.337 129

1.00 2.08 796

a Recipient for transduction was C6OO(Plkc). Pl infective phages of the number noted above infected to about 4 X 10’ cells of C6OO(Plkc). * Host bacteria which were used for phage lysate preparation by thermal induction. ’ Chloramphenicol-resistant colonies formed on L-plates containing chloramphenicol 40 pg/ml after incubation for 48 hr at 30”. d Relative ratio of CM-r colonies per one infective input phage when that of PlCM c&l eu& was decided to be 1.00.

produced in Su- host. This value is in good agreement with that expected from the above specialized transduction experiments. These defective phages produced in Su- host are thought to be able to adsorb and inject their DNA, but not to grow in even SuII+ host bacteria. Transduction by as50 Mutant For the sus50 mutant, the yield of infective phages by thermal induction, transduction of Xind- prophage, and stable and abortive transduction of various markers were examined with the results summarized in Table 5. On the whole, transduction frequencies by sus50 lysate seemed to be higher than those by PlCMcts-1 .sus+, because of the existence of many defective phages in ~24.~50lysate. When transduction frequencies by sus50 lysate produced in Su- host were divided by 670, the mean value of defective phages in its lysate, these corrected values represented in parentheses (l/670) in Table 5 were found to be not much higher than those by PlCMcts-1

su.s+.

In the transduction of Xin& prophages, almost all of them should form plaques in recipient cells. Then, in order to compare transduction frequencies, the ratios of transduction frequencies of various chromosomal markers to those of Xin& prophage were calculated, and are also listed in Table 5. Stable transduction of the trp

gene by sus50 lysates produced in SuII+ and Su- hosts were about lo- to 20-times higher than those by PlCMcts-1 sus’, and an increase of stable transduction was also observed for the his and arg genes. On the contrary, abortive transduction of the trp gene by sus50 lysate was relatively lower than that by PlCMcts-1 aus+. This characteristic was also observed in abortive transduction of his or arg gene (data not shown). In transduction by sus50 mutant, stable transductants seemed to increase and abortive transductants to decrease whether the donor is Su+ or Su- bacteria. Therefore, it is suggested that most of transducing particles of sus50 containing trp, his, or arg gene might form stable transductants, while most of those of PlCMcts-1 su.s+ form abortive transductants. All of the stable transductants were sensitive to chloramphenicol and have no immunity to Pl phage. When transduction was done using recA- bacteria as recipients, no stable transductants were formed in sue50 transduction as well as PlCMct.s1 sus+. These data indicate that stable transductants should not be formed by specialized transduction of sus50 mutant. Ultraviolet light irradiation of transducing particles prior to infection increases the frequency of stable transduction, while abortive transduction frequency is decreased (Garen and Zinder, 1955; Benzinger and Hartman, 1962; Wall and Har-

PHAGE

Pl MUTANT

riman, 1974). Stable transduction of trp gene by PlCMcts-1 SW’ and ~50 mutant phage lysate irradiated by ultraviolet light are shown in Fig. 5. In transduction by PlCMcts-1 sus+, the amount of stable transductants was increased slightly by irradiation with a small uv dose, and then decreased linearly at a higher uv dose. However, in transduction by sus50 mutant, no stimulation of stable transductank was observed, the amount decreasing linearly with increasing dose. This also supports the fact that almost all the transducing particles in su.s50 lysate should form stable transductants. Revertant

Anal&s

grow in 594 Su- bacteria, were isolated. Frequencies of Ain& prophage transduction, stable and abortive transduction of trp gene were measured with these four SW+ revertants, with results shown in Table 6. Frequencies of stable and abortive transduction of trp gene were also expressed as the ratio to those of Xirw? prophage transduction in each phage lysate. The production of infective phages by thermal induction of lysogens was recovered in all four revertants in both SuII+ and Su- host bacteria. Frequencies of stable transduction of trp gene were decreased compared to suA0 mutant, and were nearly the same as those of PlCMcts1 sus+, while those of abortive transduction were found to increase to the same rate as PlCMcts-1 sus+ phage. Therefore, it may be concluded that abortive trans-

of s.4~50 Mutant

Plsus50 was mutagenized with hydroxylamine, and four revertants, which could TABLE

THE PHAGE PRODUCTION

Pl titer

Abortive

FREQUENCIES”

OF ~~50

m-s+

MUTANTS sus50

or SU+b

su-

SU+”

su-c

( 1/670)d

C600

2.8 X lo9

2.4 X 10’

1.2 x lo8

1.1 x 106

594 (PlCM)

1.4 x 1o-4 (l.OO)e

4.6 X 1O-5 (1.W

5.4 x lo-’ w-w

7.3 x 1o-s (1.W

(1.1 x 10-5)

trp

W3623

2.9 x lo+ (0.021)

1.6 X 1O-6 (0.035)

2.6 X 1O-4 (0.48)

4.6 X 1O-3 (0.W

(6.9 X 10-6)

tw

AB2277

3.0 x 1o-6 (0.021)

2.2 x 1o-6 (0.047)

1.8 X lo-’ (0.W

3.1 x 1o-a (0.42)

(4.6 X lo-+)

his

PC1557

2.6 X 1O-6 (0.019)

1.4 x 1o-6 (0.030)

1.0 x 10-d (0.19)

2.2 x 10-s (0.30)

(3.3 x 10-6)

a3

JC15.57

1.0 x 1o-6 (0.007)

1.5 x 1o-6 (0.032)

3.4 x lo-” (0.W

2.1 x 1o-3 (0.29)

(3.1 x 10-6)

trp

W3623

2.7 X 1O-5 (0.192)

1.8 X 1O-5 (0.391)

3.1 x 1o-5 (0.057)

4.5 x lo-” (0.062)

(6.7 X 1O-7)

trp

N23-‘76

2.9 x 1o-5 (0.207)

2.9 x 1o-5 (0.436)

1.8 X lo-’ (0.033)

3.2 X lo-’ (0.044)

(4.8 X 1O-7)

(per

Xi&

Stable

Indicator recipient

5

AND TRANSDUCTION PlCMds-1

Transduction marker

339

ml)

recA

a All transduction frequencies listed above show the number of transductants per one infective b Phage lysate used for transduction was prepared in C600 SuII+ (Xind-) bacteria. c Phage lysate used for transduction was prepared in 594 Su- (Xind-) bacteria. d Corrected values of transduction frequencies of sus50 lysate prepared in 594 Su-. “This value in parentheses shows the ratio of transduction frequency of trp, his, or arg gene that of Xind- prophage transduction in each lysate.

phage.

marker

to

YOSHIHIRO

340

UV

IRRADIATION

(x 102Jmi2

YAMAMOTO

(data not shown), and so head particles should not be formed in them.

)

Olllrl DISCUSSION

0.1

-

L

1

FIG. 5. Stable transduction after uv irradiation. Phage lysates were irradiated by ultraviolet light, and then irradiated phages were infected to W3623 Trplog-phase culture bacterial cells. The relative numbers of Trp+ transductants formed by each uvirradiated phages to those by nonirradiated phages were plotted. The survival of plaque-forming ability was decreased with the increase of irradiated uv dose, and the survival at 300 J/m2 was found to be about 0.01 in each phage lysate. 0 0, PlCMcte-1 sus+ phage produced in C666 SuII+; 0 0, PlCM&-1 sue+ phage produced in 594 Su-; A A, sue50 produced in C666 SuII+; A A, ewe56 produced in 594 su-.

duction was recovered completely in these aus+ revertants. When N23-76 recA- bacteria were used as recipients in transduction by sus+ revertant phages, abortive transductants of Trp+ were observed at the transduction frequency of about lop5 regardless of the difference of donor bacteria between SuII+ and Su- (data not shown). Furthermore, these four revertants seemed to produce little or no defective phage particles in Su- host bacteria by thermal induction, measuring defective phage formation by the specialized transduction of chloramphenicol resistance gene (data not shown). In CsCl density gradient centrifugation analysis of phage particles of these four revertants produced in Suhost, peak I fractions were not observed

Mutation aus50 was found to affect frequencies of generalized transduction. However, the apparent increase of transduction frequencies of .~~..a50should not be derived from an increase of the amount of transducing particles, but from the existence of many defective phage particles in its lysate. The relative ratio of defective phage particles to infective phages was measured by two different ways, i.e., rH]thymidine incorporation and specialized transduction of chloramphenicol reexperiments of rH]sistance. From thymidine incorporation into phage DNA, defective phage particles were found to be about 670-times more abundant than infective phages in su.s50 lysate produced in Su- host bacteria. From specialized transduction of chloramphenicol resistance gene, which lies on phage genome, defective phage particles were estimated to be about 800-fold higher than infective phages in sus50 lysate produced in Su- host. Thus, the relative ratio of defective to infective phages in the sus50 lysate are in fairly good agreement in these two experiments. In sus50 lysate produced in Su+ host bacteria, the ratio of defective phage particles to infective phages was found to be about 3.2 from [3H]thymidine incorporation, and to be about 2.0 from specialized transduction. These results show that defective phage particles of sus50 mutant should be produced not only in Su- but also in Su+ host bacteria. When =P-labeled phage particles of sue50 were centrifuged in CsCl density gradient, they formed two bands: peak I for the heavier band and peak II for the lighter band, respectively. Infectivities and transduction abilities were found only in peak II fractions. When phage particles produced in Su- host were analyzed, peak II was found to be about 0.004 g crnm3 heavier than PlCMcts-1 sus+ phages. As infectivities of sus50 mutant are found in a little lighter density fractions, peak II is thought to be made up of defective

PHAGE

Pl MUTANT

TABLE TRANSDUCXION

Donor C600 (Aim?-)

594 (hind-)

Phage

FREQUENCIES

341

6

OF AMBER+ REVERTANT~ OF sus50

Pl titer (per ml)

Transduction’ frequency of xi?&

x X X x x x

Ratio of transduction xi?&

frequencyb

trp-st.c

trp-ab.d

PlCMck-lsus+ .!3u&o 50b-1 5Ob-2 5Ob-3 5Ob-4

2.1 9.6 2.1 1.7 1.9 2.4

x X x x x X

lo9 10’ 109 lo9 lo9 10’

6.3 6.2 2.3 3.8 3.3 2.2

lo-’ lo-” 1O-5 10-S 10-S 1o-5

1.00 1.00 1.00 1.00 1.00 1.00

0.052 0.61 0.091 0.061 0.067 0.059

0.34 0.079 0.19 0.17 0.22 0.15

PlCMcts-lsus+ sue50 50b-1 50b-2 5Ob-3 50b-4

1.9 6.4 2.2 1.8 2.0 2.2

x X x x x x

lo9 105 10s lo9 109 log

1.56 X lo-’ 2.4 X lo-’ 4.3 x 10-e 3.8 X 10” 3.2 X lo-’ 4.2 X lo-’

1.00 1.00 1.00 1.00 1.00 1.00

0.025 0.50 0.11 0.105 0.14 0.067

0.26 0.040 0.21 0.29 0.28 0.13

‘Transduction frequencies were estimated to be the number of formed transductants per one infective phage. Recipient for Xind- prophage transduction was 594(PlCM). * The relative ratio of transduction frequency of stable or abortive transduction of trp gene to that of Xindprophage in each phage lysate. ’ Stable transduction of trp gene. Recipient was W3623 Trp-. d Abortive transduction of trp gene. Recipient was W3623 Trp-.

phage particles produced in Su- host for the most part. However, no difference was observed in their structures at least by electron microscopic analysis. The increase of the density of sus50 defective phage particles may be caused by the lack of some proteins involved in mature phage coats. Peak I in heavier fractions having a density of about 1.558g cm-3 was found to have no infectivity of Pl and transduction ability, and was revealed to be composed of Pl head particles by experiments of sucrose density gradient centrifugation and electron microscopic observation. DNA extracted from peak I fractions were identified with sus50 phage DNA by the digestion pattern with Hind111 endonuclease and by DNA transfection. Head particles in peak I were produced about five times as much as defective phage particles in peak II in Su- host bacteria, while they were produced about one-half in Su+ host. This suggests that the formation of head particles should be consistent with that of

defective phage particles, and that both of them should be due to the ~24.~50mutation. From experiments of specialized transduction of chloramphenicol resistance of ~24.~50mutant, defective phage particles in peak II fractions are thought to maintain their abilities for adsorption to host bacterial cells and for injection of their DNAs. However, they seem to lose abilities for growth and phage production in ‘infected cells. It was reported that amber mutants of gene 7, 16, and 20 in Salmon&u phage P22 produced defective phages which could adsorb to host cells, but could not inject their DNAs (Botstein et al., 1973; Poteete and King, 1977). Mutation sus50 should not be equivalent to these amber mutations in P22 phage. No cut or gap was found in DNAs extracted from sus50-defective phage particles by the analysis of both neutral and alkaline sucrose gradient centrifugation (data not shown). In agarose gel electrophoresis, the size of DNAs of su..s50-defec-

342

YOSHIHIRO

tive phage particles in peak II appeared to be the same as of PlCMcts-1 aus+. Therefore, defective phages may be produced in the phage maturation process. Further, electron microscopic observation revealed that many long tails without head joining were produced in ~7.~~50lysate, and some of them were found to be about lo-fold longer than normal phage tails (data not shown). This suggests that sue50 mutant may have a defect(s) in the formation process of phage tails, and/or in the joining process of head and tail components, which should cause the formation of long tails. Features of sus50 mutant shown in generalized transduction of various markers are summarized as follows. (1) Abortive transduction is extremely decreased compared with stable transduction. The ratio of abortive to stable transduction is about 0.08, while that of PlCMcts-1 sus+ is about 10. (2) The stimulation of stable transduction by uv irradiation of phage lysate before transduction is not observed. (3) Only abortive transduction is found when recipients are recA- bacteria, but at a decreased rate compared with PlCMct.91 sus+. (4) Stable transductants formed are found to be nonlysogenic with respect to ~50 mutant phage. (5) Frequencies of stable and abortive transduction of amber+ revertants are nearly the same as those of PlCMc&l sus+. From these results, the sue50 mutation seems to change the ratio of abortive to stable transduction, and almost all transducing particles are thought to form stable transductants in Ret+ recipient cells. Stable transduction by the ~24.~50mutant should depend on Ret function of recipient bacteria, but should not be derived from specialized transducing phages in its lysate. Phage mutants with increased transduction frequencies have been isolated as HT mutants in P22 phage (Schmieger, 1971, 1972) and as HTF mutants in Pl phage (Wall and Harriman, 1974). However, the sue60 mutant is thought to be

YAMAMOTO

different from these mutants. The phage production of HTF mutants is normal and the burst sizes of them are the same as that of Pl tir phage, while sus50 mutant produces many defective phage particles and the burst size of sus50 is much lower than that of PlCMcts-1 sus+ (data not shown). HFT mutants show the stimulation of stable transduction after uv irradiation, while in the sus50 mutant no stimulation is observed. Because synthesis of host chromosomal DNA is not shut off after Pl phage infection except by Pl wir phage (Ikeda and Tomizawa, 1965a), attempts to measure the relative amount of host DNA packaged into sue50 transducing particles directly by differential labeling of host and PI phage DNAs with different radioisotopes were unsuccessful. It has been revealed that E. coZi DNA is bound to protein molecules in Pl transducing fragments (Ikeda and Tomizawa, 1965b). However, transducing fragments of sus50 mutant could not be labeled differentially, so it is not known whether they are bound to protein molecules or not. It was considered that abortive transduction is a negative episode in Pl generalized transduction, and the transducing materials to which apparently nothing has happened are participating in abortive transduction (Ozeki and Ikeda, 1968). If all the transducing fragments would first form the abortive state in recipient cells, and later some of them would convert to the stable state by recombination, in sus50 transduction some kind of stimulation of recombination between transducing fragments and recipient chromosome might occur, or the abortive state might not be established in recipient cells. Considering that abortive transduction of su.s50 is decreased in recA- recipients compared with PlCMcts-1 SU.S+,the abortive state of s~s50 transducing fragments may be unstable, and failure to maintain the abortive state may lead to conversion to the stable state in Ret+ recipients, while in recA- recipients this failure may cause the loss of most transducing fragments. As revertants of sus50 recover abilities not only for the production of infective phages in Su- host but also for that of

PHAGE

Pl MUTANT

abortive transductants, the sus50 mutation should be responsible for both the production of defective phage particles and the decrease of abortive transduction. Transducing fragments of sus50 produced in Su+ host bacteria also cannot form abortive transductants, although infective phages can be produced in Su+ bacteria at a rate not much lower than PlCMcLs-1 aus+. This suggests that the requirement for sus50 gene function for phage growth may be less than that for the stabilization of abortive fragments. The reason why such morphological defects as those leading to failures in head and tail joining process observed in sus50 mutant decrease abortive transduction is not known. However, it may be that some phage coat protein, which should be needed for the phage growth in host bacteria, might be necessary in the stabilization of abortive fragments. It is considered to be certain that some phage protein, which is lost from defective phage particles of the ~7.4~50 mutant, has an essential role for stabilization of abortive fragments in recipient bacteria. As binding proteins to transducing fragments should enter into recipient cells with transducing fragments, they might be necessary for abortive transduction, especially in the stabilization process of transducing fragments. Two-point crosses of sus50 with other amber mutants, of which map positions have been decided by prophage deletion mapping (Scott, 1970, 19’73, 1975; Walker and Walker, 1975, 1976), revealed that sus50 mutation might be defined between linkage cluster IV and V. However, direct deletion mapping of ~24.~50mutation was found to be impossible due to its weak suppressor sensitivity. In generalized transduction by phage Pl, the frequency of stable transduction is very low (usually under 1O-5 per infective phage), because most transducing fragments form abortive transductants. However, whether the donor is Su+ or Su-, most transducing fragments of sus50 mutant form stable transductants. In addition, when a sus50 lysate prepared in Suhost is used for transduction, the frequency of stable transduction is suffi-

343

ciently high because of the existence of many defective phage particles, which cannot grow in recipient cells and cannot interfere with the formation of transductants. Therefore, the sus50 mutant should be useful for transduction of markers which have been difficult to select or score. ACKNOWLEDGMENTS I would like to thank Drs. H. and T. Ogawa for their advice and support in the performance of this work. And I am very grateful to Dr. H. Ozeki and Dr. H. Ogawa for their critical reading of the manuscript. REFERENCES APPLEYARD, R. K. (1954). Segregation of lysogenicity during bacterial recombination in E. coli K-12. G.metics 39,429-439. B~CHI, B., and ARBER, W. (1977). Physical mapping of BglII, BamHI, EcoRI, Hind111 and PstI restriction fragments of bacteriophage Pl. Mol. Gen Genet. 153,311-324. BENZINGER, R., and HARTMAN, P. E. (1962). Effects of ultra-violet light on transducing phage P22. viroZogg 18, 614-626. BOTSTEIN, D., WADDELL, C. H., and KING, J. (1973). Mechanisms of head assembly and DNA encapsulation in SolmoneUa phage P22. I. Genes, proteins, structures and DNA maturation. J. MoL BioL 80, 669-695. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Bimhim. Biophys. Acta 34,103114. CLARK, A. J., CHAMBERLINE, M., BOYCE, B. P., and HAWORD-FLANDERS, P. (1966). Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichiu coli K12. J. Mol. Bid 19,442-454. CLARK, A. J., and MARGULIES, A. D. (1965). Isolation and characterization of recombination deficient mutants of E. coli K12. Proc. Nat. Ad Sci USA 53,451-459. DAVIDSON, N., and SZYBALSKI, W. (1971). Physical and chemical characteristics of lambda DNA. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 45-82. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. GAREN, E., and ZINDER, N. D. (1955). Radiological evidence for partial genetic homology between bacteriophage and host bacteria. firology 1, 347-376. GROSS,J., and ENGELSBERG, E. (1959). Determination of the order of mutational sites governing L-arabinose utilization in Escherichia coli B/r by transduction with phage Plbt. Virology 9, 314-331. HARRIMAN, P. (1972). A single-burst analysis of the

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YAMAMOTO OZEKI, H., and IKEDA, H. (1968). Transduction mechanisms. Annu. Rev. Gem& 2.245478. POTEETE, A. R., and KING, J. (1977). Functions of two new genes in salmanella phage P22 assembly. Vi?-o&y 76.725-739. ROSNER, J. L. (1972). Formation, induction, and curing of bacteriophage Pl lysogens. Vi* 49,679689. SCHMIEGER, H. (1971). A method for detection of phage mutants with altered transducing ability. Mel Gen. Gem& 110,378-381. SCHMIEGER, H. (1972). ‘Phage P22 mutants with increased or decreased transduction abilities. MoL Gem Gaet. 119.75-88. SCOIT, J. R. (1970). A defective Pl prophage with a chromosomal location. Vi40,144-151. SCOT, J. R. (1973). Phage Pl cryptic. II. Location and regulation of prophage genes. Piirdogy 53,327-336. SCOTT, J. R. (1975). Superinfection immunity and prophage repression in phage Pl. Virology 65,173178. STUDIER, F. W. (1969). The genetics and physiology of bacteriophage T7. Vi39, 562-574. TOMIZAWA, J., and ANRAKU, N. (1964). Molecular mechanism of genetic recombination in bacteriophage. II. Joining of parental DNA molecules of phage T4. J. Mol. BioL 6,516540. WALKER, D. H., JR., and ANDERSON, T. F. (1970). Morphological variants of coliphage Pl. J. ViroL 5,765-782. WALKER, D. H., JR., and WALKER, J. T. (1975). Genetic studies of coliphage Pl. I. Mapping by use of prophage deletions. J. Vi& 16,525-5&f. WALKER, D. H., JR., and WALKER, J. T. (1976). Genetic studies of coliphage Pl. III. Extended genetic map. J. Vi& 20, 177-187. WALL, J. D., and HARRI~~AN, P. D. (1974). Phage Pl mutants with altered transducing abilities for Esckerichia coli Virology 59,532-544. WEIGLE, J. (1968). Studies on head-tail union in bacteriophage lambda. J. MoL Bid 33,483-489.