Mol Gen Genet (1982) 186:497—500
`
`MUG
`
`© Springer—Verlag 1982
`
`Mechanism of pBR322 Transduction Mediated
`by Cytosine-Substituting T4 Bacteriophage
`
`Hideo Takahashi and Hiuga Saito
`Institute of Applied Microbiology, University of Tokyo, Bunkyo—ku, Tokyo, Japan
`
`Summary. A cytosine—substitution type mutant of bacterio-
`phage T4 (T4dC phage) has been shown to mediate the
`transfer of plasmid pBR322. The transduction frequency
`was around 10‘2 per singly infected cell at low multiplicity
`of infection. The transductants contained either a monomer
`
`or multimers of pBR322. The transducing capacity of T4dC
`phage was resistant to methylmethanesulfonate treatment.
`The results of Southern blotting experiments have indicated
`that the pBR322 DNA exists as head—to—tail concatemers
`in the transducing particles. The mechanism of transfer of
`pBR322 mediated by T4dC phages is discussed
`
`Introduction
`
`Bacteriophage T4 is one of the most intensively studied
`Escherichia coli phages and contains glucosylated hydroxy—
`methylcytosine (glu-HMC) in its DNA in place of cytosine.
`Isolation of mutants termed T4dC, which incorporate cyto-
`sine instead of HMC, has made it possible to analyze the
`role of glu—HMC in the T4 genome (Snyder et al. 1976).
`T4dC phages have mutations in gene 42(dCMAP hydroxy—
`methylase), gene 56(dCTPase), denB(endonuclease IV), and
`alc (=unf). When T4dC phages are grown in a restriction-
`deficient, non amber—suppressing host, the progeny phage
`particles contain cytosine-substituted DNA, which is sus-
`ceptible to restriction endonucleases in vivo and in vitro
`(Velten et al. 1977; Wilson et al. 1977;'Takahashi et al.
`1978).
`Recently, Wilson et al. (1979) reported that one of the
`T4dC phages, T4GT7, had a capacity to transduce plasmids
`as well as chromosomal markers. Little is known about
`the mechanism of T4GT—mediated transduction. Since the
`
`T4 genome is circularly permuted and encapsulated in a
`headful mode (Streisinger et al. 1964; Wood and Revel
`1976), it would be quite interesting to know the physical
`state of the pBR322 genome in T4 phage particles.
`In this paper we analyze the mechanism of T4dC phage-
`mediated transduction.
`
`Materials and Methods
`
`top—agar. Tryptone medium contained 1.0%
`with 2.5 ml
`Bactotryptone (Difco, Detroit, USA), 0.5% NaCl, 0.1%
`glucose, and 1.5% Bactoagar (Difco). Escherichia coli B40
`(supD) and B834(r’ m‘ sup°) were described in a previous
`paper (Takahashi et al. 1978). B834 was used for propaga-
`tion of cytosine-substituted T4 phages and as the recipient
`of transduction. Bacterial strains carrying pBR322 (Bolivar
`et al. 1977) were constructed by CaCl2—treated transforma~
`tion.
`
`Cyl0sine—Substiluzed T4 Phages ( T4dC) and Phage Prepara~
`tion. T4dC(+) phage has point mutations
`in gene
`42(amC87), gene 56(amE51), denB(s19), and alc(unf39)
`(Takahashi
`et al.
`1978;
`Takahashi
`et al.
`1979).
`T4dC(NB5060) phage has a deletion in the rII—a’enB—ndd
`region (NB5060) beside triple mutations of amC87, amE51
`and alc(Wilson et al. 1979). T4dC phages were grown in
`B834 carrying pBR322 by the method described in previous
`papers (Takahashi et al. 1978; Takahashi et al. 1979).
`Phage particles were purified by differential centrifugations.
`
`Cleavage of DNA. Cleavage of DNA was performed as
`described in a previous paper (Takahashi et al. 1979). Re»
`striction endonucleases were purchased from Takara Shuzo,
`Co., Kyoto, Japan. Agarose gel electrophoresis was per~
`formed on a horizontal
`slab gel
`(0.7 cm x 14.5 cm x
`13.5 cm) with 0.7% agarose (Seakem). The electrophoresis
`buffer consisted of 0.09 M Tris—0.09 M boric acid, pH 8.2,
`and 2.5 mM EDTA. Southern transfer (Southern 1975) and
`hybridization were done according to the method described
`in ‘Manual for Genetic Engineering’ (Davis et al. 1980).
`Hybridization was performed in a buffer consisting of
`0.9 M NaCl,
`50 mM Na2HPO4—NaH2PO4,
`pH 7.0,
`5 mM EDTA, 0.3% SDS, 100 pg/ml denatured and soni~
`cated calf thymus DNA, and “P-labeled pBR322 DNA
`at 38° C for 20 h. The probe DNA was prepared by labeling
`HaeIII~c1eaved pBR322 DNA with T4 DNA polymerase
`and oc32P—dATP according to the method of O’Farrell et al.
`(1980). Specific radioactivity of the probe was around
`107cpm per pg. Nitrocellulose filters of Schleicher and
`Schuell, Co. were used for DNA transfer.
`
`Bacterial Strains and Media. M9S medium (Takahashi et al.
`1975) was used for growing bacterial strains. Titration of
`bacteria and phage was done on tryptone plates overlayed
`
`Transduction by T4dC Phage. For transduction of pBR322,
`T4dC phages were grown in B834 carrying pBR322. Puri-
`fied phages were diluted with M9 buffer (Takahashi 1978)
`and mixed with recipient cells grown in M9S medium. Usu-
`Mylan v. Genentech
`Offprint requests to: H. TakahashiMylan V. Genentechally, 100 pl recipient cells (5 X 107 viable cells) were mixed
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`

`
`498
`
`with the same volume of T4dC phage suspension. After
`incubation at 37° C for 7 min for adsorption, the infected
`cells were centrifuged and washed once with M9 buffer.
`Portions of the infected cells were plated on tryptone agar
`plates containing 10 ug/ml ampicillin. Transduction fre-
`quencies are presented as Amp’ transductants per singly
`infected cell.
`
`Isolation and Detection of Plasmid DNA. Rapid detection
`of plasmids from colonies was performed as follows: A
`medium sized colony was suspended in 10 pl TE buffer
`(10 mM Tris—HCl, pH 7.5 and 10 mM EDTA) containing
`100 pg/ml egg-white lysozyme and incubated for 5 min on
`ice. To this were added 2 pl 2 N NaOH and 1 pl 10% SDS.
`The mixture was incubated at 37° C for 30 min. The solu-
`
`tions were neutralized with 2 ul 2N Tris-base and mixed
`with 3 pl 4 N NaCl. Samples were analyzed by agarose gel
`electrophoresis. For isolation of plasmid DNA on a prepar-
`ative scale, the alkaline method mentioned above was scaled
`up. In this case, a cleared lysate was prepared by centrifug-
`ing the neutralized NaCl-containing sample at 30,000 rpm
`for 2 h. The cleared supernatant solution was treated with
`an equal volume of chloroform-phenol mixture (1 :1).
`Nucleic acids were precipitated with ethanol and treated
`with RNAse and proteinase K. Finally plasmid DNA was
`separated by CsCl-EtBr centrifugation.
`
`MMS Treatment of T4dC Phage. Methylmethanesulfonate
`(MMS, Eastmann, Co.) was added to 0.1 M Tris—HCl solu-
`tion containing 109 plaque forming units (p.f.u.) of T4dC
`phage. After various times of 37° C incubation, aliquots
`were diluted with M9S medium containing 0.2 M NaZS2O3
`to inactivate MMS. The pBR322 transducing capacity was
`determined by infecting B834 cells at m.o.i. =0.01 and plat-
`ing on tryptone agar plates containing 10 pg/ml ampicillin.
`
`Results
`
`1. Transduction ofpBR322 Mediated by T4dC Phages
`
`To examine the transducing capacity of T4dC phages, two
`T4dC phages, T4dC(+) and T4dC(NB5060), were propa-
`gated in B834 carrying pBR322. When the lysates were used
`to infect B834, ampicillin resistant (Amp‘) colonies were
`obtained. Niether wild-type T4 grown in B834 carrying
`pBR322 nor T4dC phages grown in B834 without pBR322
`gave rise to any Amp‘ colonies. The transduction frequency
`by T4dC(NB5060) grown in B834 carrying pBR322 was
`around 2><10”2 Amp’ transductants per viable cell at a
`low multiplicity of infection (m.o.i.), which was 5-fold high-
`er than that obtained with T4dC(+) phage. As shown in
`Fig. 1, the number of transductants increased with increas-
`ing m.o.i. in the range of 0.0001—0.5. At a higher m.o.i.,
`the number of transductants decreased, presumably because
`of the killing effects of viable T4 phage particles. The linear
`relationship between the input phage particles and the
`number of transductants suggests that one transducing par-
`ticle is capable of giving rise to one transductant.
`
`2. Occurrence of Multimers in pBR322 Transductan ts
`
`To analyze plasmids in T4dC-mediated transductants,
`Amp‘ transductants were purified and the plasmid DNA
`derived from them was examined by agarose gel electro-
`
`1o“
`
`1
`
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`U=
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`Multiplicity
`
`Of
`
`0.1
`0.01
`Infection
`
`1
`
`Fig. 1. Effect of multiplicity of infection on T4dC-mediated
`pBR322 transduction. T4dC(NB5060) phage propagated in either
`B834 carrying pBR322 9-0;) or B40 carrying pBR322
`(;o—) was used as the donor phage. A culture of B834 was
`infected with T4dC phage at
`increasing m.o.i.
`ranging from
`0.0001—1.0. The number of pBR322 transductants was determined
`by plating on ampicillin-containing plates
`
`<— Trimer
`
`<— Dimer
`
`<- Monomer
`
`
`
`Fig. 2A—C. Agarose gel electrophoresis of plasmids from T4dC-
`mediated transductants. The DNA samples were electrophoresed
`through
`a
`0.7% agarose
`gel. A Standard
`pBR322 DNA
`(monomer), B partially purified plasmid DNA from a transductant
`containing dimer plasmids, and C partially purified plasmid DNA
`from a transductant containing trimer plasmids
`
`phoresis. Out of 40 Amp‘ transductants examined, 23 clones
`were shown to contain the monomeric form of pBR322,
`and the remaining clones contained either dimers or trimers
`of pBR322. These multimers had a tendency to segregate
`to monomers of pBR322 (Fig. 2). After 20 generations in
`broth culture in the presence of ampicillin, more than half
`the multimers kept the original configuration. In contrast,
`when pBR322 was introduced by CaCl2-transformation, all
`Ampr transformants tested contained monomers. Therefore
`we suspected that the unusually high occurrence of multi-
`meric forms in T4dC—mediated transductants might be due
`to multimeric forms of the pBR322 genome in the transduc-
`ing particles.
`
`

`
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`
`45
`30
`15
`I ncubation Time (min)
`
`60
`
`Fig. 3. Effect of MMS on viability and transducing capacity of
`T4dC phage. T4dC(NB5060) phage propagated in B834 carrying
`pBR322 was treated with 0.02 M MMS at 37° C. At times of incu~
`bation, the plaque-forming units (fo—) and transducing capac~
`ity (—-o—) were determined
`
`3. Methylmethanesulfonate (MMS) Resistance
`of the Transducing Particles
`
`MMS inactivates phages by interacting with their DNA.
`To test the MMS sensitivity of T4dC transducing particles,
`T4dC(NB5060) phage propagated in B834 carrying pBR322
`was treated with 0.02 M MMS at 37° C. Surviving fractions
`of plaque—forming units and the transducing capacity were
`determined. As shown in Fig. 3, the transducing capacity
`remained constant up to 120 min at which time the plaque-
`forming units were reduced by 1075. The transducing ca-
`pacity was resistant also to ultraviolet irradiation (data not
`shown). These results suggest that the pBR322 DNA in
`the transducing particles is physically very stable.
`
`4. Head-T0—Tail Multimeric Form 0fpBR322
`in the Transducing Particles
`
`To analyze the physical structure of pBR322 molecules in
`the T4dC transducing particles, DNA from T4dC(NB5060)
`
`499
`
`phage grown in B834 carrying pBR322 was examined by
`agarose gel electrophoresis. No visible DNA band corre-
`sponding to either CCC or linear forms of pBR322 was
`detected when the intact DNA was analyzed on the gel.
`However, when the DNA was cleaved with BamHI endonu—
`clease, a DNA band having identical mobility with that
`of the pBR322 linear form was observed (Fig. 4,
`lane B
`and D). Since T4 phage DNA has only one site for BamHI
`(Takahashi et al. 1979; Wilson et al. 1980), the DNA band
`was conceivably derived from DNA of a larger size, pre-
`sumably head—to—tail concatemers of pBR322 DNA.
`To confirm the presence of multimeric forms of pBR322
`molecules in the transducing particles, Southern hybridiza-
`tion experiments were performed. A 32P—labeled pBR322
`probe hybridized only with high molecular weight DNA
`when the intact DNA was used (Fig. 4, lane a). However,
`when the T4dC DNA was treated with restriction endonu—
`
`cleases BamHI, Pstl or Ball, which cleaves pBR322 DNA
`only once, then the pBR322 probe hybridized extensively
`with the band corresponding to the linear monomer of
`pBR322 DNA (Fig. 4, lanes b, e, f, and g). On the other
`hand, the probe hybridized only with high molecular weight
`DNA when the DNA was treated with KpnI endonuclease
`which has no cleavage site in pBR322 (Fig. 4, lane c and
`(1). These results indicates that the pBR322 DNA in the
`transducing particles exists as a head—to-tail concatemer and
`not integrated in the T4dC genome. Moreover, the intact
`size of DNA from the transducing particles had an identical
`size to that of DNA from the viable T4dC phage particles
`(Fig. 4, lane a). This result indicates that the transducing
`particles contain the same amount of DNA as the viable
`T4dC phage particles. Since the size of T4 phage DNA
`encapsulated in the phage particles is 166 kilobases (kb)
`(Wood and Revel 1976), the size of concatemieric DNA
`encapsulated in the transducing particles was estimated to
`be about 38 monomer equivalents of pBR322 DNA.
`
`Discussion
`
`T4dC phages grown in a suppressor-negative host Carrying
`pBR322 were shown to transduce the plasmid at a high
`frequency. The frequency was around 1072 per singly in-
`fected cell. Procedures for T4dC phage-mediated transduc-
`tion are quite simple and fast. A number of pBR322 deriva~
`tives into which DNA fragments from various origins have
`
`
`
`Fig. 4. Hybridization of 32P—labelcd pBR322
`DNA to restriction fragments of DNA from
`T4dC(NB5060) phage grown in B834
`carrying pBR322. The DNA fragments were
`separated by agarose gel electrophoresis and
`visualized by fluorescence with ethidium
`bromide (A—G). “P-labeled pBR322 DNA
`was hybridized to a Southern imprint of the
`fragments and hybrids were detected by
`autoradiography (a—g). A and a, intact
`DNA; B and b, BamHI digest; C and c,
`Kpnl digest; D and d, Kpnl digest; E and
`e, Ball digest; F and f, PstI digest; G and
`g BamHI digest. D (d) and G (g) samples
`were from the same DNA preparation. The
`arrow indicates the position of pBR322
`linear monomers
`
`

`
`500
`
`been cloned, were also transduced at frequencies equivalent
`to that of pBR322 (Takahashi, Noguchi, and Saito, unpub-
`lished results). Since T4 phage particles can be stored for
`a long period without significant loss of transducing capaci-
`ty, the T4dC—mediated transduction of plasmids can be used
`for preservation as well as transfer to plasmids into new
`hosts.
`
`The T4 phage genome is circularly permuted and has
`a terminal redundancy. The phage DNA is encapsulated
`by a headful mode (Streisinger et al. 1964; Wood and Revel
`1976). The results in this paper have shown that pBR322
`DNA exists as head—to—tail linear multimers in the transduc-
`
`ing particles. Moreover, the transducing particles were esti-
`mated to contain about 38 monomer equivalents of
`pBR322 DNA. DNA of pBR322 mostly exists in the CCC
`form in its host cells. Therefore, intramolecular recombina-
`tions are a prerequisite for producing linear multimers of
`pBR322 in the transducing particles and returning to CCC
`monomers in the recipient cells. The recA function of the
`recipient cells is essential for this interconversion (Taka-
`hashi and Saito, manuscript in preparation).
`T4dC phages grown in a suppressor—plus host carrying
`pBR322 were also able to transduce pBR322 although the
`transduction frequency was lower
`than that by T4dC
`phages grown in a suppressor-minus host carrying pBR322.
`DNA from T4dC phage grown in a suppressor—plus host
`carrying pBR322 was resistant to the restriction endonucle-
`ases Ec0RI, SalI, and Pstl (data not shown). Therefore,
`it
`is
`likely that both cytosine and HMC-containing
`pBR322 DNA in the transducing particles have been repli-
`cated by the T4 replication system, presumably in the mode
`of rolling circle (Streisinger et al. 1964; Wood and Revel
`1976).
`So far we have not succeeded in isolating transducing
`particles free of viable T4dC phage particles. However, the
`viable T4 particles can be selectively inactivated by MMS
`treatment or UV—irradiation. As viable T4 phage particles
`kill the cells infected with transducing particles, this selec-
`tive inactivation of viable phage particles is convenient for
`T4dC-mediated transduction.
`
`References
`
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`Crosa JH, Falkow S (1977) Construction and characterization
`of new cloning vehicles. II. A mltipurpose cloning system. Gene
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`taining T4 DNA. Proc Natl Acad Sci USA 73:2098—3102
`Southern EM (1975) Detection of specific sequences among DNA
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`separated by gel electrophoresis.
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`23:503—517
`Streisinger G, Edgar RS, Denhardt GH (1964) Chromosomal
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`Takahashi H (1978) Genetic and physiological characterization of
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`Takahashi H, Shimizu M, Saito H, Ikeda Y (1979) Studies of
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`bacteriophage
`containing
`cytosine-substituted
`DNA(T4dC phage). II. Cleavage of T4dC DNA by endonucle-
`ase SalI and BamHI. Mol Gen Genet 168 :49—53
`Velten J, Fukada K, Abelson J (1977) In vitro construction of
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`fragments from bacteriophage T4. Gene 1:93—106
`Wilson GG, Tanyashin VI, Murray NE (1977) Molecular cloning
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`Wilson GG, Young KY, Edlin GJ (1979) High-frequency general-
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`
`Acknowledgments. The authors are grateful to Dr. G.G. Wilson
`for generously supplying us with a phage strain. This work was
`supported in part by Grant-in-Aid for Scientific Research from
`Ministry of Education, Science and Culture of Japan.
`
`Communicated by K. Isono
`
`Received January 26 / April 2, 1982