Vol. 130, No. 1 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 354-365 Copyright ©D 1977 American Society for Microbiology

Isolation and Characterization of Plaque-Forming AdnaZ+ Transducing Bacteriophages JAMES R. WALKER,* JOAN M. HENSON, AND CHONG S. LEE Departments of Microbiology* and Zoology, The University of Texas at Austin, Austin, Texas 78712 Received for publication 13 September 1976

The Escherichia coli dnaZ gene, a deoxyribonucleic acid (DNA) polymerization gene, is located 1.2 min counterclockwise from purE, at approximately min 10.5 on the E. coli map. From a lysogen with XcI857 integrated at a secondary attachment site near purE, transducing phages (XdnaZ+) that transduced a dnaZts (A+) recipient to temperature insensitivity (TS+) were discovered. Three different plaque-forming transducing phages were isolated from seven primary heterogenotes. Genetic tests and heteroduplex mapping were used to determine the length and position of E. coli DNA within the X DNA. Complementation tests demonstrated that the deletions in all three strains removed both att P and the int gene, i.e., DNA from both prophage ends. Heteroduplex mapping confirmed this result by demonstrating that all three strains had deletions of X DNA that covered the b2 to red region, thereby removing both prophage ends. Specifically, the deletions removed A DNA between the points 39.3 to 66.5% of X length (measured in percent length from the left end of A phage DNA) in all three strains. The three strains are distinct, however, because they had differing lengths of host DNA insertions. These phages must have been formed by an anomalous procedure, because standard X transducing phages are deleted for one prophage end only. In Xgal and Xbio strains, the deletions of A DNA begin at the union of prophage ends (i.e., position 57.3% of A length) and extend leftward or rightward, respectively (Davidson and Szybalski, in A. D. Hershey [ed.], The Bacteriophage Lambda, p. 45-82, 1971). Models for formation of the AdnaZ+ phages are discussed. sites. One such lysogen contained A integrated nearpurE (i.e., near dnaZ). The availability of this lysogen and the usefulness of transducing phages for host mapping and studies on host gene expression encouraged a search for AdnaZ+ phages. This paper describes the isolation and characterization of AdnaZ+ transducing phages from the lysogen with K integrated near purE. From seven primary heterogenotes, three different plaque-forming AdnaZ+ phage strains were isolated. These three had differing lengths of host insertions but similar A DNA deletions. The deletions removed both prophage ends, in contrast to deletions in standard Agal or Abio strains.

The Escherichia coli dnaZ gene is one of the four known genes that code for products necessary for deoxyribonucleic acid (DNA) polymerization (10). In addition to participating in E. coli chromosome replication, the dnaZ product also is required for growth of phages M13, OX174, and A, but not for T7 or T4 growth (30, 32). In the cases of M13 and kX174, the dnaZ product participates in vivo in parental replicative-form formation and replication and in single-strand synthesis (17; W. G. Haldenwang and J. R. Walker, unpublished data). Haldenwang and Walker (17) suggested that the dnaZ product participates in the polymerization phase of M13 replicative-form synthesis, rather than in initiating synthesis. Wickner and Hurwitz (36) demonstrated that the dnaZ protein participates in vitro in elongation of DNA during 4X174, M13, and ST-1 replicative-form synthesis from primed single-strand DNAs. Mapping studies demonstrated that dnaZ is cotransducible with purE, which is located near min 12 on the E. coli chromosome map (10). Shimada et al. (25) described X lysogens that contain prophages at secondary attachment

MATERIALS AND METHODS Conventions. The transducing phages that carry the wild-type dnaZ gene will be designated XdnaZ+, in contrast to the usual convention, to permit the distinction in future publications from derivatives that carry defective dnaZ genes, e.g., XdnaZ-. Prophage left and right ends refer to the left and right ends of X when integrated at att BOB'. Media. X was grown in yeast extract-tryptone (YET) broth (20) containing 0.5% NaCl and 10-3 M 354

XdnaZ+ PHAGES

VOL. 130, 1977

MgSO4 or on plates of soft- and bottom-agar media of the same composition with 0.6 and 1.2% agar, respectively. YET plates contained 0.5% yeast extract, 1% tryptone, 0.5% NaCl, 10-3 M MgSO4, and 1.8% agar. Phage P1 was grown on media described by Rosner (24), and P1 transductants were selected on the medium of Caro and Berg (6). Tryptone broth contained 1% tryptone, 0.5% NaCl, and 0.2% maltose (26). EMBO plates were as described by Gottesman and Yarmolinsky (14). Strains. Bacterial and phage strains are listed in Table 1. Strain 2eOlc was the usual host for X; RH288 was used as the permissive host for X strains that carry the susS7 mutation. Growth and purification of phages. P1 was prepared according to the procedure of Rosner (24). X replication was induced in strain 81 by mixing 10 ml of culture grown at 300C to 3 x 108 cells/ml with 10 ml of liquid soft agar; the mixture was distributed to three bottom-agar plates and incubated at 40°C overnight. The soft agar was collected, mixed with 5 ml of CHCl3, and centrifuged.

355

2eOlc as host. The bacteria were grown to 3 108 cells/ml at 37°C, 108 phages/ml were added, and the mixture was incubated at 42°C for 30 min and then x

shifted to 37°C until lysis. XcII2002 and Xbio275 were similarly but at 37°C. XcI857susS7 was grown lytically on strain 2eOlc after infection at a multiplicity of 6 (2). Growth was at 370C after an initial heat shock at 420C. Xi434cTts56susS7 was prepared by a similar procedure, except that the host, lysogen W3101(Xi434cIts56susS7), was superinfected at a multiplicity of 2. XcI857dnaZ+17susS7 was prepared by lytic infection of strain 2eOlc. Strain 2eOlc was grown at 370C 108 cells/ml, and phages were added at a to 5 multiplicity of infection (MOI) of 2. The culture was mixed with an equal volume of broth at 420C and incubated at 420C for 30 min, followed by 4 h of incubation at 370C. The cells were lysed by chloroform without concentration. This procedure gave titers of about 2 x 1010 phages/ml; other procedures routinely used for preparation of other susS7 muX replication was induced in primary heterogentants resulted in very low titers with this phage. Stocks of about 1010 phages/ml were concentrated otes, presumed to be of genotype dnaZts(X+) (OcI857dnaZ+), as follows: the cultures were grown by centrifugation in a Beckman 6OTi rotor at 40C at 39,000 rpm for 2.3 h and resuspension in 0.005 volto 3 x 108 cells/ml, resuspended in X dilution buffer (33), irradiated with an incident ultraviolet dose of ume of X dilution buffer. Phages were purified in two 300 ergs/mm2, resuspended in YET broth at 42°C step gradients in CsCl followed by one equilibrium and incubated for 12.5 min, and shifted to 370C until gradient (31). Phages collected from gradients were dialyzed against 0.01 M tris(hydroxymethyl)amilysis occurred (about 2 h). nomethane (Tris) (pH 8.0) containing 0.01 M MgSO4. XcI857 was grown as described by Thomas and Phage cross. XcI857dnaZ+17susS7 was prepared Davis (31). by infecting strain RH288 (3 x 108 cells/ml) at an The XcI857dnaZ+ phages that do not form lysoMOI of 1 each with XcI857susS7 and XcI857dnaZ+17. gens efficiently were first grown lytically from sinThe culture was incubated at 420C for 30 min and gle plaques, with strain 2eOlc as host, by the softagar overlay method (33). This yielded titers of then at 370C for 2 h. Progeny were plated on strain about 3 x 109 plaque-forming units (PFU)/ml. These RH288, and individual plaques were tested for the phages then were used as inocula for further cultisusS7 marker and transducing ability. vation in liquid medium. P1 transduction. P1 transduction was performed The XcI857dnaZ+ phages, XcI857int6, and by the procedure of Willetts et al. (37). Lysates were XcI857b2 were grown in liquid cultures with strain grown in the donor strain twice before use. ProtoTABLE 1. Principal strains Strain

Bacteria AX727 AX727(X+)

X156 2eOlc RH288 81(AcI857) AB2487

W3101(Xi434cIts56susS7) Phage Plvira A+ XcI857 XcI857int6 XcI857b2 XcI857susS7 Abio275 a ts, Temperature sensitive.

grown

x

Characteristicsa

F- lac gal str dnaZts2O16 F- leu proC purE trpE thi ara lacY galK xyl mtl tonA tsx azi str supE44 F- thr leu thi lac str

supF Hfr(gal-att BOB'-bio)A F- thr leu thi proA his thyA drm recA mtl xyl ara galK lacY str tsx sup-37 sup+

of referSourceence

Filip et al. (10) H. Chu B. Bachmann

M. Malamy R. Gayda M. Gottesman B. Bachmann L. Reichardt

E. Moody

D. Kaiser D. Kaiser R. Weisberg R. Weisberg L. Reichardt W. Szybalski

356

WALKER, HENSON, AND LEE

trophic transductants were selected on minimal medium at 300C; dnaZ+ transductants were selected by plating infected AX727 cells on minimal medium, incubating for 3 h at 300C, and then shifting the plates to 420C. A transduction in quantitative experiments. The transduction procedure was essentially that of Shimada et al. (26). Infected cells of strain AX727 (X+) dnaZts2016 were plated on YET plates and incubated at 300C for 3.5 h and then shifted to 420C to select TS+ transductants. Spot test for AdnaZ+ transduction. Individual plaques were resuspended in 0.5 ml of X dilution buffer and chloroformed. Sterile lysates (109 to 1010 PFU/ml) and the resuspended phages from plaques were tested by transferring 0.05 ml to the surface of YET plates spread with 2 x 107 AX727(X+) cells. The plates were incubated at 30°C for 3.5 h and then at 420C. Positive tests were recognized by the growth of several hundred colonies of the AX727(X+) indicator (when lysates were used) and 10 to 40 individual TS+ colonies (when resuspended plaques were tested). Genetic test for gam+ activity. Phages to be tested for gam+ were plated on 2eOlc (recA+) and AB2487 (recA) at 400C. Genetic test for int+ activity. The procedure used was essentially that of Gottesman and Yarmolinsky (14). Strain 2eOlc cells were infected at an MOI of 10 for single infections and an MOI of 5 (of each phage) for mixed infections. After a 25-min adsorption period at 300C, the cells were diluted and plated on YET plates for incubation at 300C. Colonies were picked from the YET plates, transferred to EMBO plates spread with 2 x 109 XcII2002 particles, and incubated at 300C. Immune clones formed large, white entire colonies; nonimmune clones formed faint purple colonies with fried-egg-like peripheries. Electron microscopy. Electron microscopy of heteroduplexes was done essentially according to Davis et al. (8). The absorbance at 260 nm of all phage stocks was 2.0 to 2.5. Contour length measurements. DNA was prepared from the phages by diluting 25 ,ul of phage stock into 375 gl of 50% formamide containing 0.01 M Tris (pH 8.0) and 0.001 M ethylenediaminetetraacetate (EDTA). This solution was dialyzed against the same formamide solution for 2 to 3 h at room temperature. Formamide was then removed by dialysis at 40C against 0.5 M ammonium acetate containing 0.01 M Tris (pH 8.0) and 0.001 M EDTA. The DNA solution then was heated at 450C for 30 min to permit cyclization. Electron microscope grids were prepared by the aqueous technique. The spreading solution contained 0.5 ammonium acetate, 0.1 mg of cytochrome c per ml, and 0.1 volume of the DNA solution. The hypophase was 0.25 M ammonium acetate. Photographs of circular DNA molecules were taken at the same magnification for all phage DNAs, using a Hitachi 7H electron microscope. Molecules were traced in a Nikon shadowgraph, and contour lengths were measured with a HewlettPackard calculator equipped with a digitizer. All contour lengths were normalized to that of XcI857susS7 DNA.

J. BACTERIOL.

Heteroduplex preparations. DNAs were denatured directly from the phages. The denaturation mixture contained 40 ,ul of 0.02 M EDTA, 5 ,ud of 1 N NaOH, and 2 to 3 ,i of Xi434cts56susS7 phage stock and XcI857susS7 or one of the XcI857dnaZ+ strains. After 15 min at room temperature, the mixture was neutralized by adding 15 ,ul of 1 M Tris (pH 8.0). Renaturation was achieved by annealing at room temperature for at least 3 h in the presence of 50% formamide. For the formamide technique, the spreading solution contained 50% formamide, 0.1 M ammonium acetate, 0.01 M Tris (pH 8.0), 0.1 mg of cytochrome c per ml, and 0.1 volume of the annealed DNA. The hypophase was 20% formamide in 0.01 M Tris (pH 8.0) and 0.001 M EDTA. Heteroduplexes of interest were identified by the presence of the single-strand loops formed in the immunity region of the Xi434 and X DNA heteroduplex. Heteroduplex molecules were photographed, and lengths of various regions were measured as described above. RESULTS Position of dnaZ on the E. coli map. Earlier mapping by P1 demonstrated that dnaZ is cotransducible with purE but did not determine

the sequence of dnaZ with respect to other markers in this region (10). To determine this sequence, phage P1 was grown on strain AX727(proC+ tsx+ dnaZts purE+) and used to transduce a proC tsx dnaZ + purE recipient (X156) to purE+ and proC+ at 300C. The transductants were purified and tested for the unselected tsx and dnaZ markers (Table 2). purE and dnaZ were 4.5% co-transduced; purE and tsx were not co-transducible (less than 1%). proC and tsx were closely linked (64.5% co-transduction), but dnaZ did not co-transduce withproC (less than 0.53%). For the reciprocal cross, P1 was grown on strain X156 (proC tsx dnaZ+ purE) and used to transduce strain AX727 (proC+ tsx+ dnaZts purE+) to dnaZ+ (Table 2). Analysis of the unselected markers showed that dnaZ and purE co-transduced at the level of 6.6% and dnaZ and tsx at the level of 1.2%, and that dnaZ andproC were not co-transducible (less than 0.4%). Inasmuch as dnaZ is co-transducible with tsx and purE, but tsx and purE are not co-transducible, dnaZ must be situtated between tsx and purE. From the Wu (38) formula, dnaZ is 1.5 min clockwise from tsx and 1.2 min counterclockwise from purE (Fig. 1). According to the current linkage map (1), tsx and purE are located at min 9 and 11.7, respectively, which means that dnaZ is at min 10.5. Isolation of AdnaZ+ transducing phages. The availability of a lysogen with XcI857 inserted nearpurE prompted a search for XdnaZ+ transducing phages. A lysate from strain 81

VOL. 130, 1977

357

XdnaZ+ PHAGES

TABLE 2. Map position of dnaZ Donor n Rec

AX727

X156

Selected

No.

oftrans--

marker

lected

purE+

132

Unselected marker

Transduc-

unselected marker

proC tsx dnaZ proC+ 191 tsx dnaZ purE AX727 dnaZ+ X156 258 proC tsx purE a AX727 proC+ tsx+ dnaZts purE+; x156 proC tsx dnaZ+ purE

(a)

purE I

8.7 9.0

10.5

11.7

att int y N

RA

J

BOP'

(c)

30

40

_-

K

J

1.3 0.28

1.2 6.6

1.5 1.2

0 6 120 0 0 0 3 17

dnaZ

gal

(b)

4.5 64.5

0

proC tsx a I I

duction

Map distance (min) between selected and unselected markers

Co-tran-

tants wih

att bio

POB'

50 1

otl

60 aa

P6Pintxisexojey

,

70 a

cm

b2

dnaZ

bio72 gal FIG. 1. Genetic maps. (a) E. coli chromosome dnaZ region. The numbers represent minutes. (b) The X prophage map (7). (c) X phage map from Davidson and Szybalski (7). The numbers above the map represent percentage of X length from the left end. The extent of the deletion of X genes in the three XdnaZ+ transducing phages is indicated by the dashed line. The extents of the deletions of X genes in Xbio72 and in Xgal strains are indicated by the thin solid lines.

was used to transduce strain AX727(A+)dnaZts to TS+ on YET agar without added NaCl. After infection at an MOI of 25, TS+ transductants were isolated with frequencies of 3 x 10-9/recipient (corrected for reversion frequency, which was 2 x 10-9/recipient) and 2 x 10-10/infecting PFU. A total of seven TS+ transductants (presumed primary heterogenotes) was isolated. They

were purified and induced to yield high-frequency-transducing lysates. The lysates (about 1010 PFU/ml at 40°C on host 2eOlc) that, by spot tests, transduced strain AX727(X+) to TS+ were plated to yield individual plaques. The phages from isolated plaques were resuspended in buffer, chloroformed, and tested for transducing ability by the spot test. Three of the seven primary heterogenotes yielded plaque-forming

358

WALKER, HENSON, AND LEE

J. BACTERIOL.

transducing phages; the other four presumably contained defective transducing phages.

General properties of the plaque-forming AdnaZ+ phages. The plaque-forning transducing phages (XcI857dnaZ+2, XcI857dnaZ+17, and XcI857dna.Z+18) were purified and grown by lytic infections to yield lysates of 5 x 109 to 1010 PFU/ml. These phages formed clear plaques at 400C and slightly turbid plaques at 300C, which indicates that they carry the cI857 mutation and that the deletion of X DNA did not remove the cI gene. They transduced strain AX727(X+)dnaZts to TS+ on YET plates with a frequency of 7 x 10-3 to 1.9 x 10-2 transductants/PFU after infection at multiplicities from 0.1 to 0.2. When the recipient was not a lysogen (AX727 dnaZts), the frequency was only 3 x 10-s to 4 x 10-5 transductants/PFU; presumably these transductants arose by recombination between the host and phage-carried dnaZ regions. Genetic tests for the presence of genes on the prophage left (gam and int). X normally integrates into the host chromosome at the prophage site POP' (Fig. 1). During transducingphage formation, incorporation of host genes to the left of the prophage is accompanied by deletion of the right prophage attachment site, P, and additional X DNA from the prophage right. Incorporation of host genes to the right of the prophage is accompanied by deletion of the left prophage attachment site, P', and additional A DNA from the prophage left (Fig. 1) (3, 7). Transducing phages that delete non-essential genes from the b2 or the int-gam region would be nondefective on wild-type hosts. A priori, the XdnaZ plaque-forming phages would be expected to have deletions of either the prophage left or right end. To determine if the deletions removed the prophage left end, the transducing phages were assayed for the presence of gam+ and int+ genes by genetic tests. Zissler et al. (40) demonstrated that functional red and 8 genes or a functional gam gene is essential for plaque formation on recA

hosts. Therefore, deletions of A that extend from the left through red and into gam would render the transducing phages unable to plate on a recA host. Xbio275, deleted for the gam gene (as well as red and (; 29), did not grow on a recA host. All three transducing phages formed plaques, however, on a recA strain (Table 3). Therefore, the deletion of A DNA did not extend into the gam gene when the XcI857dnaZ+ chromosomes were formed (Fig. 1). A mutants that lack functional int activity do not lysogenize efficiently (12, 13, 39), but a Xint mutant can be complemented by Xint+ (14). The XcI857dnaZ+ transducing phages were tested for the presence of the int+ gene by determining if the transducing phages could complement Xint6. All three phages lysogenized inefficiently after single infections and failed to provide int+ function after mixed infection with Aint6 (Table 4). The deletion of A genes, therefore, abolished int activity. This finding suggested that the KdnaZ+ strains were formed by deletions of the prophage left end to remove att P' and int. Genetic test for presence of the prophage right (att P). Inasmuch as the genes on the prophage left were deleted, it was anticipated that the XdnaZ+ phages would carry the prophage right end (i.e., att POA) (A designates nucleotides other than P, P', B, or B'.) Infecting Kbio (att POB') and Ab2 (att AOP') undergo int-promoted recombination and lysogenize efficiently, although neither phage alone does so (22). Therefore, ability to complement Kb2 for eficient lysogenization provides an assay for att P, as a part of att POB'. The three KdnaZ phages were tested for complementation with Ab2 by measuring the frequency of stable lysogenization. None complea

a

TABLE 4. Complementation tests for int+ and attP of transducing phages

a

TABLE 3. Efficiency ofplating of transducing phages on a recA host Efficiency of platinga

on:

A strain

Abio275 XcI857dnaZ+2 XcI857dnaZ+17

2eOlc recA+

AB2487 recA

1.0

0.96

1.0