JOURNAL OF BACTERIOLOGY, June 1975, p. 1247-1256 Copyright 0 1975 American Society for Microbiology

Vol. 122, No. 3 Printed in U.S.A.

Genetic and Physical Studies of Lambda Transducing Bacteriophage Carrying the Beta Subunit Gene of the Escherichia coli Ribonucleic Acid Polymerase TOSHIHIKO IKEUCHI, TAKASHI YURA*, AND HIDEO YAMAGISHI Institute for Virus Research* and Department of Biophysics, Faculty of Science, Kyoto University, Kyoto, Japan Received for publication 10 March 1975

The prophage Xc1857 was inserted into the bfe gene located near rif (the structural gene for the subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective X phage particles (Xdrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain Xdrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated Xdrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from Xdrif+-6 and the other from Ximm2' phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage X into the bfe region occurred at the normal cross-over point within the phage attachment site.

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid polymerase (EC 2.7.7.6) of Escherichia coli is known to play a central role in genetic transcription. It consists of at least five subunits (a2fll'cr). The structural gene for the ,B subunit has been identified and mapped on the chromosome, by employing mutants resistant to antibiotics such as rifampin (see references 2 and 12). However, very little is known about the location of the genes for other subunits, although some evidence suggests that the structural gene for the (3' subunit is located in the vicinity of, or perhaps adjacent to, that of the ,B gene (rif) (21, 22). One of the most direct approaches to the structure and function of the ribonucleic acid polymerase genes would be to isolate and characterize transducing phages carrying rif and other neighboring genes that might include the genes for other subunits of the polymerase. Shimada et al. (23) found that X phage could be integrated into many chromosomal sites other than its normal attachment site, attX. Thus, we attempted to insert lambda into the bfe gene that determines synthesis of the re-

ceptor for phage BF23 and colicins El, E2, and E3, since bfe maps very close to rif (15). The resulting lysogen (referred to as bfe lysogen) was induced, and several X transducing phages carrying the wild-type d gene (rif+) were isolated from these lysogens. The results of genetic and physical characterization of these phages are reported in this communication. While this work was still in progess, reports describing the isolation of similar transducing phages from both lambda and 080 lysogens appeared (16, 18). Our results are closely consistent with those reported by Kirschbaum and Konrad (16), although there are some differences; for example, the phages they isolated carry the dominant rif mutation (rifd). We have further studied certain properties of our transducing phages and obtained some interesting results including direct evidence for the structure of DNA isolated from one of them, Xdrif+-6.

MATERIALS AND METHODS Bacterial and phage strains. Bacterial and phage strains used in this study are listed in Table 1. Media. Peptone-glucose medium (2% polypeptone 1247

IKEUCHI, YURA, AND YAMAGISHI

1248

J. BACTERIOL.

TABLE 1. Bacterial and phage strains Bacterial strain B582 KY3302KY3305 KY3101 KY3102 KY5323 KY3121 KY3122 KY3126

Genetic characters HfrH A(gal attA chiA uvrB) thiA HfrH A (gal attA chiA uvrB) thiA bfe (Xc1857 in bfe) HfrH metB arga thiA rif (X); other markers same as in KY3101 F- stv-195 thr leu his metB rha str tsx malA mall; other markers same as in KY5323 (X); other markers same as in KY3121 recA; other markers same as in KY3121

Phage strain

Origin or source

Origin or source

K. Matsubara B582

XcI857 Xclb2

H. Ozeki H. Ozeki

HfrH KY3101

AcI+ Ximm21S7

M. Imai M. Imai

T. Yura et al. (29)

Ximm2'b2

H. Ozeki

KY5323

Aimm43'

H. Ozeki

KY3121

X amber mutants (Aam701, Baml, H. Ozeki and H.

Cam20, Daml5, Eam4, Uam413, Vam438, Gam9, Haml2, Mam87, Iam2, Jam6,

KY3121

Inokuchi

Qam2l, Ram5) KY3361- (X) (Xcl857drif+) KY3366 KL16 Hfr(O-lysA-thyA ---)

see text

BF23

H. Ozeki

B. Low (20)

Plvir

X80

H. Ikeda and J. Tomizawa (13) A. Matsushiro

KY1324

F- his metB ppc argE purD thiA

N. Glansdorff

KY363 W3350

str supE F- lac supE F- gal lac sup+

H. Ozeki H. Ozeki

aLocated at about 78.7 min on the standard genetic map (25).

and 0.5% NaCl, pH 7.2) was generally used for growing bacteria, and tryptone broth with 20 mM MgSO4 for preparation of X phage lysates. Medium E (26) was employed for transduction experiments with Plvir. Solid media usually contained 1.5% agar, and soft agar was peptone-glucose medium containing 0.7% agar. Prophage curing. A sample (0.1 ml) of overnight culture of X lysogens was mixed with 3 ml of soft agar, on which a drop of the hetero-immune phage Ximm434 (1010/ml) was spotted. After overnight incubation at an appropriate temperature, cells from the lytic zone were suspended in broth, diluted, and plated on peptone-glucose agar. In the case of lysogens carrying AcI857, cured clones were obtained by selecting for those that can form colonies at 42 C. Preparation of low-frequency transducing (LFT) lysates. A culture of bfe lysogen was grown in tryptone broth containing 20 mM MgSO4 to reach 100 Klett units (no. 54 filter) at 30 C. After induction at 42 C for 25 min, the culture was returned to 37 C and was aerated for 5 h. The lysates were cleared by centrifugation, and phage particles were concentrated by using polyethylene glycol (PEG6000) as described by Yamamoto et al. (28) with minor modifications. Preparation of high-frequency transducing (HFT) lysates. Cultures of a heterogenote in tryptone broth containing 20 mM MgSO4 were grown to 80 Klett units (no. 54 filter), treated at 42 C for 25 min, and aerated at least for 6 h at 37 C in the presence of mitomycin C (0.3 to 0.5 ug/ml). The lysates were cleared and concentrated by repeated centrifugations or by sedimentation with polyethylene glycol as described above. Transduction with Xdrif+ phages. An overnight culture of a recipient strain in peptone-glucose broth

was centrifuged and suspended in 20 mM MgSO4 at 108 cells per ml. Aliquots (0.1 ml) of this suspension were mixed with 0.1 ml of an appropriate dilution of LFI or HFT lysate, incubated at 37 C for 20 min, and plated on peptone-glucose agar. Plates were incubated at 30 C for 2 h and then at 42 C for 2 days. Temperature-independent colonies thus obtained were scored. Electron microscopy. The formamide technique (7) was employed for analysis of heteroduplex DNA. The spreading solution and hypophase solution used for mounting DNA contained 45 and 17% formamide, respectively. Electrolytes and other conditions were as described by Davis et al. (7). The DNA mounting technique has been modified so that it requires much smaller amounts of DNA and hypophase solution than had previously been employed. A small sample (1.15 ml) of hypophase solution was placed in an indentation on a Teflon block as described by Inman and Schnios (14), and a clean stainless-steel spatula was inserted at an angle of about 450 into the side of the hypophase drop. The spreading solution (0.05 ml) was then applied through a capillary tube onto the surface of the spatula, which was carefully removed after most of the solution has drained down the spatula. Electron micrographs were taken in a JEM-7A at an instrumental magnification of 10,000fold, and were traced on a 10-fold enlarged image with a Nikon 6F projection comparator.

RESULTS Isolation of bfe lysogens. The first step for the isolation of a transducing phage carrying rif was to insert prophage lambda into the bfe gene which is known to be closely linked to rif (Fig. 1). Such a lysogen should be easily selected,

Adrif+ TRANSDUCING PHAGE

VOL. 122, 1975 sup M

argE bfe rif

met B

78

78.5

79

thiA purD

79.5

metA 80

FIG. 1. The genetic map of the argE-purD region of E. coli. The map positions were taken from the standard map of Taylor and Trotter (25).

1249

normally, but the resulting lysates contained at most 1 x 10- 3 plaque-forming particle per induced cell. This low yield of active phage is presumably due to the low efficiency of prophage excision from the bfe region, as has been observed with other sites (23, 24). Also, there was no indication that defective transducing phages were present in these lysates at an unusually high frequency. Thus we have concentrated phage particles from 10 liters of lysate from KY3303, and obtained a high-titer suspension containing 5.2 x 108 plaque formers per ml. This was then used as an LFT lysate for the following transduction experiments. As the recipient strain for transduction of the rif+ gene, we have employed a temperaturesensitive, rifampin-resistant mutant (KY3121) carrying a recessive mutation (stv-195) that primarily affects the # (rif) gene (29; Kawai, Ishihama, and Yura, manuscript in preparation). After infection of the recipient cells by the LFT

since it becomes resistant to BF23 phage by inactivation of the gene(s) essential for synthesis of the specific phage receptor. The parental organism used for this experiment was E. coli K-12, strain B582, carrying a deletion at the attX region (23). An overnight culture of this strain was starved in 20 mM MgSO, solution for 1 h at 37 C and was infected with XcI857 phage at a multiplicity of about 10. After 20 min of adsorption at 37 C, the infected cells were diluted into tryptone broth and were incubated overnight at 30 C. A portion of this culture was plated on peptone-glucose agar seeded with phages BF23 (1.0 x 1010 per plate) TABLE 2. Properties of bfe lysogensa and XcIb2 (1.6 x 109 per plate). After a 2-day incubation at 30 C, colonies appeared at a Hetero-immune curingc frequency of 1.5 x 10-7 per infected cell, and Phage production bfe they were simultaneously resistant to BF23 and after heat sClnsitive No. of cured Lysogen immune to lambda. Most of these colones were Lysogeninduction8 clones tested to BF23 (%) temperature sensitive because they had been lysogenized with XcI857. 20 50 KY3302 1.0 x 104/ml 18 These BF23-resistant, X-immune clones can 50 KY3303 1.5 x 104/ml 10 50 be divided into at least three types: (i) lysogens KY3304 8.6 x 103/ml 10 50 carrying prophage XcI857 at bfe; (ii) lysogens KY3305 1.0 X 104/ml carrying prophage XcI857 at both bfe and some a See text for isolation of bfe lysogeps. other locus (or loci); and (iii) lysogens carrying a I Plaque formers of XcI857 in LFT lysates obtained a prophage XcI857 at site(s) other than bfe, and after induction of bfe lysogens (108 cells/ml) at 42 C separate resistance mutation at bfe. Among 250 for 25 min followed by shaking at 37 C for 5 h. lysogens tested, four were found to be the type 1 See Materials and Methods for conditions of lysogens (bfe lysogens), as shown by curing tests curing. and other experiments described below. Prophage curing experiments were carried out to TABLE 3. Pl transduction with bfe lysogensa see whether some of the cured clones might recover sensitivity to BF23 by reactivation of Co-transduction No. of Arg+ the bfe gene. Indeed, the four lysogens did frequency(%) Donor transductants produce BF23-sensitive clones at high frequentested arg-bfe f arg-rif cies upon curing the XcI857 prophage (Table 2). Further evidence that these four lysogens Non-lysogens carry a prophage at the bfe gene came from 35 B582 103 transduction experiments with phage P1. Co47 64 B582 bfeb 253 transduction frequencies between arg and bfe, bfe lysogens 12 KY3302 12 49 and between arg and rif, were much reduced KY3303 41 10 5 when these lysogens were used as donor (Table KY3304 64 11 3 3). These results as well as those of the curing KY3305 101 11 3 experiments indicate strongly that XcI857 had been integrated into bfe located between arg aThe transduction experiments were carried out and rif (Fig. 1). essentially as described by Lennox (19), except that Transduction of rif+ with LFT lysates. Plvir (13) was employed. The recipient strain was When the prophage of these bfe lysogens were KY3102 (arg bfe+ rif). b Spontaneous bfe derivatives of B582. induced by heat treatment, cell lysis took place I

1250

IKEUCHI, YURA, AND YAMAGISHI

lysate, ture-independent transductants were selected on peptone-glucose agar at 42 C. Since every rif+ transducing phage in the lysate would carry cI857, an excess of XcI+ phage was added simultaneously to the recipient to overcome the temperature sensitivity of the c1857 repressor. The multiplicity of infection for active XcI857 was about 1, and that for XcI+ was about 5. After incubation for 2 days, transductant colonies appeared at a frequency of 1.5 x 10-6 per plaque former in the LFT lysate used. This frequency did not change appreciably when the recipient strain was lysogenic for XcI+. All the temperature-independent transductants were found to be also sensitive to rifampin. This is in agreement with the finding that both temperature sensitivity and rifampin resistance of the recipient strain are due to a single recessive mutation affecting the rif gene. Attempts to find a transductant in which a transducing phage genome had been inserted into the bacterial chromosome were unsuccessful; 100 transductants were purified and checked for their capacity to produce HFT lysates. Cultures of each transductant were treated by heat pulse at 42 C, and were aerated at 37 C in the presence of mitomycin C (0.5 ,ug/ml). None of the lysates contained detectable amounts of rif+ transducing phage. Therefore, all the Ts+ transductants tested presumably represent recombinants in which the rif region of the recipient chromosome had been replaced by the homologous region of the donor rif+ strain. Isolation of rif/rif+ heterogenotes. In view of the results presented above, it was decided to use a recA derivative (KY3126) of strain KY3121 as a recipient in transduction to obtain heterogenotes. In this case, transductants appeared at a frequency of 2.0 x 10-7 per plaque former, which is about 10 times lower than that obtained with the rec+ recipient. The transductants thus obtained were all sensitive to rifampin as expected, and about half of them gave rise to rifampin-resistant clones at a frequency of about 10-5, which is at least 100 times higher than that of spontaneous Rif-R mutation that would occur with an ordinary Rif-S haploid strain. The occurrence of these rifampin-resistant clones presumably represents haploid segregants or homogenotes (rif/rif) with respect to the rif gene, suggesting that those transductants may indeed be heterogenotes. Each of these presumptive heterogenotes carrying recA was then crossed with an Hfr strain (KL16) to obtain rec+ recombinants, because these recA strains could not be induced to produce phage under usual conditions. The

J.- BACTERiIOL.

majority (80%) of the recombinants selected (His+ Str-R) were rec+ with respect to ultraviolet sensitivity. Six independent heterogenotes carrying rec+ were obtained in this way. They are temperature-independent and rifampinsensitive like their recA parents, but can now be induced to produce rif+ transducing phages at much higher frequency than does the bfe lysogen (Table 4). The lysates thus obtained will be called hereafter HFT lysates. Moreover, rifampin-resistant clones appeared at very high frequency (about 10- 2). When these heterogenotes are cured after superinfection with a heteroimmune phage (Ximm434), they become temperature-sensitive and rifampin-resistant as expected if they are indeed lysogenic for a rif+ transducing phage. All these results clearly demonstrate that these strains carry the stv-195 allele on the chromosome and stv+ (rif+) on the X phage genome. The HFT lysates contain active phages of both types, cI+ and c1857, as judged by their plaque morphology (Table 4). These heterogenotes therefore contain at least two prophages integrated into the chromosome. The site of prophage integration may be either at the normal attachment site for X (attX) or at the bfe-rif region, although the result of heteroimmune curing suggests that both a rif+ transducing phage and a helper phage are integrated at the attX site. Transduction experiments with phage P1 were thus performed to see if any prophage is located at the bfe-rif region. Cotransduction frequencies between any of the markers tested are the same whether the donor strain carries the prophage Xdrif+ or not (Table 5). This is in sharp contrast to what was observed with the bfe lysogens (Table 3). On the basis of the results so far presented, the most probable structure of these heterogenotes and of the transducing phages derived from the original bfe lysogen is shown in Fig. 2. The validity of these structures will also be supported by the experiments presented below, and will be considered in detail in Discussion. Separation of the defective rif+ transducing phage from active phage. The HFT lysates from these heterogenotes were then examined by CsCl density gradient equilibrium centrifugation to determine the buoyant density of rif+ transducing phage relative to that of normal phage X (27). The result obtained with one of the heterogenotes is shown in Fig. 3. The defective rif+ transducing phage (Xdrif+-6) formed a peak at a density much lighter than that of normal phage X. It is also apparent from the optical density profile that the number of transducing phage particles present in the ly-

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Xdrif+ TRANSDUCING PHAGE

VOL. 122, 1975

TABLE 4. Active and transducing phages in HFT lysatesa Active phage (P)b

T/P

Transduction titer (T)c

Source of HFT lysate C1857

cI+

KY3361 KY3362 KY3363 KY3364 KY3365 KY3366

1.9 x 3.0 x 1.3 x 4.7 x 1.5 x 3.4 x

109 107 108 108 108 109

3.3 1.7 3.0 6.0 4.9 1.6

x x x x

108

5.5 4.0 1.0 4.0 3.0 6.0

108 107 107 x 108 x 109

x 105 x 105 X 104 x 105 x 105 x 105

2.5 2.0 5.5 7.5 4.7 1.2

x x x x x x

10-i

10-3 10-' 10-4 10-4 10-4

See Materials and Methods for preparation of HFT lysates. b Phage titers (per milliliter) were determined by using cells of strain W3350 as indicator. c Transduction was scored on the X-sensitive (Mal+) derivative of KY5323. a

TABLE 5. Pl transduction with heterogenotes" Co-transduction Donor Donor

KY3122b KY3361 KY3362 KY3363

KY1364 KY3365 KY3366

~No.

of Arg+tested transductants ductant tested

100 104 103 90 72 84 103

No. of Pur+ trans-

Cotasuin

fo-req suecyti)of

frequency ()ductants frequency (%) of totastcinested arg-rif arg-purD tetdpurD-rif 49 50 53 52 50 48 47

10

28

75

12 10 11 10 10 13

11 13 16 16 11 15

73 85 88 88 73 80

The donor heterogenotes carry argE+ rif purD+, whereas the recipient strain (KY1324) carries argE rif+ purD. Transduction was carried out as in Table 3. b The control strain which does not carry Xdrif+ as prophage.

a

sate is about half of that of the normal phage X despite the rather low rif+ transducing activity (bfe) rif arg (bfe) obtained (Table 4). From a series of experiJ AR C1857 inT ments with several reference phages, the buoyant density of the transducing phage Xdrif+-6 (obtained from KY3362) was estimated to be about 1.495. The densities of two other transA R A R ducing phages, Xdrif+-14 (from KY3364) and Xdrif+-17 (from KY3366), were also determined C1857 C cl |t by similar experiments (Table 6). n int Marker rescue experiments with Xdrif+ phages. To find out the extent of deletion of the AclI+ (helper) Adrif + phage genes from these transducing phages, Xdrif+ particles were purified by three cycles of CsCl density gradient centrifugations, and were used to infect cells of strain KY363 or W3350 he terogenot e (A) together with each of the X amber mutants to for appearance of wild-type recombinants look int C1857 from each cross. As summarized in Table 6, the late genes located between, and including, J heterogenote ( B) and A are deleted in the genome of Xdrif+-6, whereas the segment spanning J and D seems to gal ioc4 lbi c in t be deleted from Adrif+-14 and Xdrif+-17. On the FIG. 2. The proposed genetic structure of bfe lyso- basis of these results and the buoyant densities gen, Xdrif+ transducing phage, and rif/Xdrif+ hetero- obtained above, the length of the bacterial portion of the genome has been estimated for genote.

bfe lysogen

ir f

gal

bio

1252

J. BACTrERIOL.

IKEUCHI, YURA, AND YAMAGISHI

each Xdrif+ phage (Table 6). According to these calculations, Xdrif+-14 carries the host chromosomal fragment whose length is approximately 0.1 min longer than that of Adrif+-17. Since both of the Adrif+ phages carry the functionally intact rif+ gene, it may be surmised that at least Xdrif+-14 carries a fair portion of the neighboring genes located between rif and thiA. Heteroduplex mapping of Adrif+-6 DNA.

lo

Il

3

2 I

cP

IV

The direct mapping of the bacterial DNA fragment on the Xdrif+ genome was carried out with one of the transducing phages obtained above. DNA extracted from purified X-drif+-6 was hybridized with that of Ximm21, and the resulting heteroduplex DNAs were observed under electron microscope. An electron micrograph of a typical heteroduplex molecule is shown in Fig. 4, and is schematically represented in Fig. 5. The substitution of the immunity region present in the reference DNA (Ximm2t) can be clearly located on the "right arm" of the A genome ("imm21loop"). The much larger substitution loop shown between xl and x2 represents substitution of the phage DNA by the bacterial DNA and is referred to as "rif loop". The calibration technique employed was based on that originally used by Davis and Parkinson (6), and the sizes and locations are presented in terms of the fractional length of the wild-type A DNA. The published values (4) for X3 and (0.710 and 0.796, respectively) served as the internal reference points. Fourteen heteroduplex molecules were thus examined, and the results are given in Table and Fig. 5. The lengths of duplex DNA for the regions C and A were obtained by converting the actual measurements into fractional lengths, setting the length for E (0.204) as a standard. The end points of the rif loop (x, and x2) are thus calculated to be 0.035 and 0.575, respectively. The location of x, is very close to the reported position of the gene A (5). These results comx4

10

Fraction

Number

FIG. 3. CsCI density gradient equilibrium centrifugation of an HFT lysate. CsCl was added to a concentrated HFT lysate obtained from KY3362 to the final density of 1.50 g/cm -3, and the centrifugation was carried out at 25,000 rpm for 18 h at 4 C in a Spinco ultracentrifuge. Fractions were collected from the bottom of the tube, and were assayed for rif+ transduction with KY3122 as recipient and for plaque formers with W3350. TABLE 6. Genetic constitution of Xdrif+ phage genome Adrif+ (No.) 6 14 17

Q

Wild-type recombinants obtained in crosses with A amber mutantsa + A

Estimated Buoyant

densityb

++. 1.495 - -1-- - .+ + + -+ + + 11512 1.501 + + + + +. - _- _ __

DNA length"

(A unit) 0.891

(0.928) 1.034 0.941

Estimated length of bacterial DNA segmente % (min)

0.48

(0.52) 0.53 0.43

0.54

(0.58) 0.59 0.48

aAbout 107 cells of strain KY363 or W3350 were simultaneously infected with purified Xdrif+ (about 105 particles) and each of the A amber mutants as indicated (about 3 x 107 phages), and were plated with 2 x 10' cells of W3350. Plates were incubated overnight at 37 C and were scored for wild-type recombinants. Cells infected with each parental phage alone were similarly plated as controls. + or - indicate presence or absence, respectively, of the recombinant plaques for each cross. At least two independent experiments were performed for each cross. b The densities were determined by CsCl density gradient equilibrium centrifugation employing X+, X80, and Ximm2tb2 as internal references. CsCl was added to HFT lysates to the final density of 1.50 g/cm3, and the centrifugation was carried out at 23,000 rpm for 20 h at 20 C in a Spinco L3 ultracentrifuge with a SW50.1 rotor. Fractions were collected and assayed with appropriate indicator bacteria by the procedure described in the legend to Fig. 3. cThe values were calculated from the buoyant density as described by Bellet et al. (1). ' The values are given both in minutes and in percentages, assuming that the total E. coli genome is 2.5 x 109 daltons and corresponds to 90 min of the genetic map (25). The values were calculated from the results of marker rescue experiments and from the map distances cited in reference 5. The values in parentheses were obtained from the results of the heteroduplex study.

~~~~

1253

Xdrif+ TRANSDUCING PHAGE

VOL. 122, 1975

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with that of the marker rescue therm~~~Ore,th agree pletely publshed valu wit the orx.0.5)isn demonstrting thatmost of te late excllentfo agrement experiment th phge ttahmet ste 6).Thia ar ha ben rplaed y te (0574 onthelef gene

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1254

IKEUCHI, YURA, AND YAMAGISHI ri- loop mm2'- loop 0540

Bi

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o0

3

3~~2

0~to135

0 468

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0o204

Q

0035)

0575)

0710)(0796)

FIG. 5. A schematic representation of the heteroduplex DNA molecules formed between DNAs from Xdrif+-6 and Ximm21. Capital letters represent DNA regions, whereas small x letters indicate end points of each substitution loop. Values represent fractional length or position from the left end of the molecule given in X unit. See text for explanation. TABLE 7. Fractional length of various regions of heteroduplex DNA formed between DNAs of Adrif+-6 and of Ximm2' Reference

Estimated fractional

region measuredaRa employeda

Region

C A B2 B2

E E

BI

D2 D2

D,I

B1

D2

lengthb Avg

SDC

0.135 0.035 0.468 0.466 0.535 0.039

0.004 0.003 0.016 0.049 0.058 0.004

aSee Fig. 5 for these regions. bThe values were obtained from measurements of 14 complete heteroduplex molecules and are presented in A unit. c SD, Standard deviation.

originally inserted into the bfe region by a recombinational event involving the normal attachment site on the A genome presumably by the int function. In view of the buoyant density of Xdrif+-6 particles obtained above (Fig. 3 and Table 6), the shorter strand (B2) of the rif loop must represent the bacterial DNA and the longer strand (B,) corresponds to the phage DNA missing from the genome of Xdrif+-6. The length of B2, was then determined by using the values for B1 (0.540) or D2 (0.086) as internal reference (Table 7). Although standard deviations of these estimates for single-strand regions are much larger than those for double-strand regions, the two values of B2 obtained agree very well with each other. The length of B1 (0.535) as estimated by D2 as a reference also agrees with that employed above as a standard (0.540).

J. BACTrERIOL.

Similarly, the estimated value of Dl (0.039) coincides with that (0.038) reported by other workers (4). The total molecular lengths of Xdrif+-6 and Ximm21 obtained by simple summation of the fractional lengths of each region are 0.928 and 0.950, respectively. This value for Xdrif+-6 is in fair agreement with that calculated from its buoyant density. DISCUSSION To isolate a lambda phage that can specifically transduce the rif and its neighboring genes, we have obtained four independent "bfe lysogens" in which prophage XcI857 has been inserted into the bfe region of the E. coli chromosome. The results of prophage curing experiments and of P1 transduction experiments strongly suggest the location of the prophage to be within the functional region of the bfe gene(s). All the bfe lysogens exhibited similar properties, suggesting that the site of prophage insertion might be the same among these lysogens. Induced lysates of one of these lysogens contained phages that can transduce the rif gene or the arg genes (data not presented) located clockwise or counterclockwise to bfe, respectively (Fig. 1). In view of the structure of Xdrif+ genome as studied by marker rescue experiments as well as by electron microscopy, the prophage A must have been integrated at the bfe region by the classical mechanism proposed by Campbell (3) with the direction of prophage shown in Fig. 2. A direct measurement of the end points of the bacterial DNA inserted into the Xdrif+ genome demonstrated that the prophage A was integrated into bfe by a recombinational event involving the normal attachment site on the phage genome. The integration was presumably mediated by the int function, though this point has not been examined directly. Induction of such a lysogen yielded LFT lysates containing rather low titer of active phage. This probably reflects an inefficient excision of the prophage DNA from bfe, as was observed previously with other lysogens carrying A prophage at several unusual sites (23, 24). The recipient strain KY3121 that we have employed for isolation of Xdrif+ phage carries a mutation (stv-195) at the rif(stv) gene which directs the synthesis of the temperature-sensitive, rifampin-resistant d subunit of RNA polymerase (29; Kawai et al., manuscript in preparation). All temperature-independent transductants obtained are shown to be sensitive to rifampin, when the recA as well as rec+ recipient was used. These results together with those of the curing experiments with the hetero-

VOL. 122, 1975 TRANSDUCING PHAGE Vdrif+

genotes lead us to conclude that the transduction involves the rif gene and not other genes that might phenotypically suppress the temperature-sensitive mutation. Conversely, all temperature-sensitive mutations that can be rescued by Xdrif+ phage were found to map very close to the rif region of the chromosome (unpublished results). Heterogenotes carrying both the wild-type and the mutant rif genes can be obtained, provided that the recipient carries a recA mutation which prevents genetic recombination between the bacterial portion of the Xdrif+ genome and the host chromosome. That these heterogenotes are in fact doubly lysogenic for both normal active phage and a defective rif transducing phage was demonstrated, after each heterogenote was converted to rec+ by conjugation with an Hfr rec+ strain. Since the rif+ gene which was inserted into the Xdrif+ prophage appears to be normally expressed, in contrast to most viral genes that are repressed by the cI repressor, a bacterial rather than viral promoter seems to be responsible for transcription of the rif+ gene. Available evidence suggests that both Xdrif+ and a helper phage are integrated into the host chromosome at the normal attachment site for X (attX) in all the heterogenotes studied, in contrast to the results reported by Kirschbaum and Konrad (16). Although the relative position of the two prophage genomes was not examined directly, it appears more likely- that Xdrif+ is located on the side of the gal genes and a helper phage on that of the bio genes (Fig. 2), for the following reasons. (i) Since a part of the attachment site carried by rdnift originates from the bacterial bfe, this attachment site presumably has a relatively low affinity to the int gene product, making the int-promoted integration of Xdrif+ to the bio side less likely. (ii) Heteroimmune curing of the heterogenotes (KY3361 and KY3362) failed to give any lysogens carrying Xdrif+ but not the helper phage; all temperature-sensitive clones obtained (70 from each strain) were found to have lost both rifampin sensitivity and X immunity simultaneously. Assuming that the above prophage order is correct, the two alternative structures may be considered (Fig. 2). Recombination between Xdrif+ and a helper phage, presumably mediated by the phage red function, would be required to obtain the structure B, but is not necessarily required to obtain the structure A. Four of the six heterogenotes tested produced more active phage with the cI+ repressor than those with the cI857 reprssor (Table 4). These heterogenotes may have the prophage structure

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B, if we assume that prophage excision by the int-xis function gives the dimer circle which in turn results in the formation of mature phage genomes by the terminase action (8, 9, 10, 11). Similarly, the two other heterogenotes that give more cI857 phage than cI+ phage may have the prophage structure A. The results of marker rescue experiments, as well as of electron microscopy of the heteroduplex DNA molecules, clearly demonstrate that most of the late genes of X were replaced by the bacterial genes located between bfe and rif. These results, taken together with the observed buoyant density of Xdrif+ phages, enable us to calculate the length of the bacterial segment of the Xdrif+ genome. It was estimated to vary between 0.48 and 0.59%, or between 0.43 and 0.53 min equivalent, of the E. coli chromosome (Table 6). Our major concern is whether any of the Xdrif+ phages carries the bacterial genes that can code for ribonucleic acid polymerase subunits other than d3. Kirschbaum and Scaife (17) recently reported genetic evidence which suggests that one of the Xdrifd phages they tested carries the genes for both (3 and (3' subunits. Although direct experiments are under way to answer this question, a number of temperature-sensitive and amber mutants affecting, or (' subunits of the polymerase have already been shown to be converted to the wild-type phenotype upon infection with Adrif+ phage (unpublished results). Systematic analyses of these bacterial mutants as well as of Xdrif+ transducing phages should contribute to our understanding of the structure and function of RNA polymerase genes in E. coli. ACKNOWLEDGMENTS We are grateful to K. Shimada, H. Ozeki, and H. Inokuchi for providing us of bacterial and phage strains used and for valuable discussions during this study. Helpful discussions and advice of M. Imai, T. Nagata, and other members of this Institute are gratefully acknowledged. We are also indebted to W. Ginoza for his kind help in preparation of the manuscript. LITERATURE CITED 1. Bellet, A. J. D., H. G. Busse, and R. L. Baldwin. 1971. Tandem genetic duplications in a derivative of phage lambda, p. 501-513. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, New York. 2. Burgess, R. R. 1971. RNA polymerase. Annu. Rev. Biochem. 40:711-740. 3. Campbell, A. 1962. Episomes. Adv. Genet. 11:101-145. 4. Chow, L. T., and N. Davidson. 1974. Electron microscope study of the structure of Xdv DNA. J. Mol. Biol. 86:69-89. 5. Davidson, N., and W. Szybalski. 1971. Physical and chemical characteristics of lambda DNA, p. 45-82. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, New York.

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6. Davis, R. W., and S. Parkinson. 1971. Deletion mutants of bacteriophage lambda. III. Physical structure of att4,. J. Mol. Biol. 56:403-423. 7. Davis, R. W., M. N. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. p. 413-428. In S. P. Colowick and N. 0. Kaplan (eds.). Methods in enzymology, vol. 21D. Academic Press. Inc., New York. 8. Emmons, S. W. 1974. Bacteriophage lambda derivatives carrying two copies of the cohesive end site. J. Mol. Biol. 83:511-525. 9. Feiss, M., and A. Campbell. 1974. Duplication of the bacteriophage lambda cohesive end site: genetic studies. J. Mol. Biol. 83:527-540. 10. Freifelder, D., C. Lawrence, and E. E. Levine. 1974. Requirement for maturation of Escherichia coli bacteriophage lambda. J. Mol. Biol. 83:503-509. 11. Gottesman, M. E., and M. B. Yarmolinsky. 1968. The integration and excision of the bacteriophage lambda genome. Cold Spring Harbor Symp. Quant. Biol. 33:735-747. 12. Heil, A., and W. Zillig. 1970. Reconstitution of bacterial DNA-dependent RNA-polymerase from isolated subunits as a tool for the elucidation of the role of the subunits in transcription. FEBS Lett. 11:165-171. 13. Ikeda, H., and J. Tomizawa. 1965. Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments. J. Mol. Biol. 14:85-109. 14. Inman, R. B., and M Schnis. 1970. Partial denaturation of thymine- and 5-bromouracil-containing X DNA in alkali. J. Mol. Biol. 49:93-98. 15. Jasper, P., E. Whitney, and S. Silver. 1972. Genetic locus determining resistance to phage BF23 and colicins El, E, and E, in Escherichia coli. Genet. Res. Camb. 19:305-312. 16. Kirschbaum, J. B., and E. B. Konrad. 1973. Isolation of a specialized lambda transducing bacteriophage carrying the beta subunit gene for Escherichia coli ribonucleic acid polymerase. J. Bacteriol. 116:517-526. 17. Kirschbaum, J. B., and J. Scaife. 1974. Evidence for a A transducing phage carrying the genes for the ,B and 6' subunits of Escherichia coli RNA polymerase. Mol. Gen. Genet. 132:193-201.

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18. Konrad, B., J. Kirschbaum, and S. Austin. 1973. Isolation and characterization of 408 transducing bacteriophage for a ribonucleic acid polymerase gene. J. Bacteriol. 116:511-516. 19. Lennox, C. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190-206. 20. Low, B. 1968. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 60:160-167. 21. Matzura, H., S. Molin, and 0. Maalfe. 1971. Sequential biosynthesis of the ,i and ,B' subunits of the DNAdependent RNA polymerase from Escherichia coli. J. Mol. Biol. 59:17-25. 22. Nakamura, Y., and T. Yura. 1973. Localization of the structural gene for the ,B' subunit of RNA polymerase in Escherichia coli. Biochem. Biophys. Res. Commun.

53:645-652. 23.Shimada, K., R. A. Weisberg, and M. E. Gottesman. 1972. Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J. Mol. Biol. 63:483-503. 24. Shimada, K., R. A-. Weisberg, and M. E. Gottesman. 1973. Prophage lambda at unusual chrdmosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12. J. Mol. Biol. 80:297-314. 25. Taylor, A. L., and C. D. Trotter. 1972. Linkage map of Escherichia coli strain K-12. Bacteriol. Rev. 36:504-524. 26. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: Partial purification and some properties. J. Biol. Chem. 218:97-106. 27. Weigle, J., M. Meselson, and K. Paigen. 1959. Density alterations associated with transducing ability in the bacteriophage lambda. J. Mol. Biol. 1:379-386. 28. Yamamoto, K. R., B. M. Alberts, R. Benzinger, L. Lawhorne, and G. Treiber. 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40:734-744. 29. Yura, T., K. Igarashi, and K. Masukata. 1970. Temperature-senstive RNA-polymerase mutants of Escherichia coli, p. 71-89. In L. Silvestri (ed.), RNA polymerase and transcription, North-Hollwnd Publishing Co., Amsterdam.

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-depe...
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