VIROLOGY

77,

306-318

(19771

Isolation and Characterization of Specialized Transducing Bacteriophages for the recA Gene of Escherichia co/i KEVIN Department

of Biophysics

and

McENTEE Theoretical

Biology Chicago, Accepted

WOLFGANG

AND

and Department Illinois 60637 October

EPSTEIN

of Biochemistry,

University

of Chicago,

29,1976

Lysogens ofEscherichia coli have been isolated in which hc1857 is integrated near the recA locus in a gene required for sorbitol utilization (srl). These secondary-site lysogens produce lysates containing plaque-forming specialized transducing phages which carry part of the srl locus and the recA gene. Genetic evidence, DNA heteroduplex examination, and restriction enzyme analysis demonstrate that the bacterial DNA substitution occurs in the b2 region of the transducing i\. One such recA transducing phage, designated AprecA, contains approximately 3% less DNA than the parental A.

hprecA (McEntee, 1976). This phage was obtained from a strain in which bacteriophage A is integrated near the recA gene in a locus (srl) affecting sorbitol utilization. In this paper, we describe the isolation of lysogens in which he1857 is integrated into the srl locus and present a biophysical characterization of the XprecA phage, which carries part of the srl locus in addition to the recA gene.

INTRODUCTION

The product of the recA gene of Escherichia coli performs an important role in several cellular processes. Homologous recombination in recA mutants is reduced to less than 0.001% of that in recA+ strains (Clark, 1973). Furthermore, recA- mutants are exceedingly sensitive to killing by uv or X irradiation and alkylating agents such as mitomycin C (Clark and Margulies, 1965). Repair of DNA is impaired in these mutants and much of the damaged DNA is hydrolyzed to acid-soluble material (Clark et al., 1966). In recAmutants, uv treatment neither produces mutations (Miura and Tomizawa, 1968) nor induces prophages (Hertman and Luria, 1967). These pleiotropic properties result from mutations in a single gene near min 58 on the revised linkage map of E. coli (Bachmann et aZ., 1976). The isolation of amber (Mount, 1971) and temperaturesensitive (Lloyd et al., 1974) recA- mutations indicates that the gene product is a protein. Although much information exists on the physiology and genetics of recA- mutants, nothing is known about the biochemical properties and function of the recA gene product. To identify the recA gene product and study it biochemically, we have isolated a specialized recA transducing phage,

MATERIALS

AND

METHODS

Bacterial strains and bacteriophages. The strains and phages used in this study are listed in Table 1. Bacteriological media. KML complex medium and K115 minimal medium have been described (Epstein and Kim, 1971). Solid minimal medium contains 10 g of the appropriate sugar per liter, except that sorbitol (Pfanstiehl) minimal medium contains 3 g/liter. Nitrofurantoin (Sigma) plates contain 2 mg of drug/liter of KML medium. Indicator medium consists of 10 g of the appropriate sugar and 40 g of MacConkey agar base (Difco) per liter. Isolation of A inserted into the srl locus. A slight variation of the method of Shimada et al. (1972) was employed to isolate lysogens with A inserted in the srl region. Strain KM2134 was grown to a density of 7 x 10’ cells/ml in KML medium supple306

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0042-6822

recA

TRANSDUCING TABLE

BACTERIALAND

1

BACTERIOPHAGE

Relevant Strains” AB2462 AB2463 KM100 KM2134 C6OOS’ KL16-99 KM197 KM21352146

307

PHAGES

STRAINS

genotype

Sourceb

KLF8/MA50 KA197 KM3000 KM601

F-pro arg his thr leu recAl2 supE strA thi F- pro arg his thr leu recAl3 supE strA thi F- srl-1 cysC 1ysA argA mtl strA thi F- mtl 1ysA argA thi (gal-atth-bio) deletion F- thr leu lac tonA supE thi strA Hfr KL16 recA1 thi F- srl-1 thyA pheA cysC thi mtl nalA KM2134 lysogenized at srl; at least several are independent tyrA pyrD thi his trp thyA recA1 mtl xyl malA A' galK strAIF143 Same as KLF43iKL259, but with the F143-1 episome pyrB argG metB 1ysA his leu mtl xyl malA A’ galK lac tonA supEiF160 thr leu lac mtl thi ma1 A' cysD lysAlF108 Hfr KL16 thi pheA KM2136(A at srl) (hpgal8)lFB F- thr leu recA1 strA tonA lac supE

KM2199 KM155 H560

F- mtl recA99 (gal-atth-bio) E. coli C his (P2) F+ endol polA strA

KLF43/KL259 KLF43-A/KL259 NF306

Phages hcI857 hcI857susS7 AC1 hcIh80 hpgal8 Apro Aimm434b515b519

See Table

deletion

K. B. Low

CGSC CGSC CGSC CGSC CGSC KL16-99 x thyA of C6OO.S’

R. M. R. R. suppressors

and are

is from the laboratory University.

mented with biotin (0.1 mg/liter) and maltose (4 g/liter). The cells were harvested, washed once with 0.17 M NaCl, and resuspended in 0.2 vol of 10 mM MgSO,. After shaking at 37” for 60 min, the cells were infected with A~1857 at a multiplicity of approximately 8. The suspension was chilled on ice for 10 min and then incubated with agitation for 45 min at 30”. The cells were diluted into KML medium and, after several generations of growth, were spread on sorbitol MacConkey plates which had been seeded with hc1 and hcIh80 (approximately log of each phage). After 2-3 days incubation at 30”, the plates were

mutant

strA

4

a All bacterial strains lack nonsense wise indicated. @ Where no source is shown, the strain c Coli Genetics Stock Center at Yale

CGSC’ CGSC

sensitive collection

Gayda E. Gottesman Weisberg Weisberg

to streptomycin or was constructed

and

A unless for this

otherwork.

examined and white colonies were picked to sorbitol and to glucose indicator plates. Colonies which gave a negative sorbitol indicator reaction, but gave a positive glucose reaction, were saved for further testing. Sorbitol selection and scoring. The linkage of recA to srl-1 was determined by Plkc transduction using srl+ as the selected marker. Misleading results can be obtained in these crosses when selecting or scoring the srl marker. Strains which are mtl+ srl- will grow on sorbitol, although more slowly than srl+ strains. This problem can be avoided by using mtl- strains

308

McENTEE AND EPSTEIN

as Plkc donors. When this is not possible, the distinction between mtl+ srl- and srl+ can be made on MacConkey indicator plates. The former class of recombinants is white or pink on sorbitol MacConkey medium, while the latter appear red. Another difficulty in the selection is cross-feeding of the srl~ recipients by srl+ recombinants. In addition, the frequency of unstable heterozygotes obtained in the srl+ selection was unusually high and contributed to the low linkage measurements obtained in early experiments. Upon repurification to obtain pure srl+ recombinants, Plkc cotransduction of srl-1 with several recA alleles ranges from 70 to 90% (see Results). Determination of prophage orientation. The orientation of the prophage was determined by chromosome mobilization. Strains KM21352146 are gal- because of the gal-atth-bio deletion. These A lysogens were transduced to gal+ with Apgal8. Because both gal homology and attA are deleted in these recipients, gal+ transductants result from homologous recombination between Apgal8 and the prophage. The location of Apgal8 at srl was confirmed by Plkc transduction of the gal+ derivatives to srl+. All srl+ transductants (80/80) lost the ability to ferment galactose. To mobilize the chromosome from this transposed gal region, the F8 episome was introduced. The resulting F’ strains are capable of mobilizing chromosomal markers from two separate origins: One of these sites is determined by homology of the gal region of F8 with Apgal8, while the other is determined by residual homology of F8 with the chromosome near nadA. Mobilization of markers near srl was tested by selecting for phe+ nalA and thy+ nalA recombinants in 30-min matings at 30” with strain KM197. Chromosome mobilization by the F8 episome in Apgal8 transductants is a general method for determining the orientation of A at unusual prophage sites not near gal. Other genetic techniques. Transduction with Plkc and isolation of A’ strains were performed as described by Miller (1972). Matings were performed by mixing 0.2 ml of a recipient culture in KML medium containing 4 x 10” cells/ml with an equal

volume of donor culture at the same cell density, incubating without agitation at 30 for a sufficient length of time (usually 30 to 60 min) to obtain the desired recombinants, and plating serial dilutions on selective medium. To isolate cured derivatives of strains with A integrated at srl, the strains were made A’ to prevent killing by reinfection. The Ar derivatives were grown in KML medium at 30” to log cells/ml and volumes of 1 ml were spread on sorbitol MacConkey plates incubated at 42”. The EDTA sensitivity of phages was tested as described by Shimada et al. (1973). Measurements of lysogenization frequency were performed as described by Shimada et al. (1975). UV survival measurements were done as previously described (McEntee, 1976). Heteroduplex mapping. Phage were purified in CsCl step density gradients. Disruption of the phage, denaturation of the DNA, formamide spreading, and shadowing were done essentially as described by Davis et al. (1971), except that for spreading the hypophase was 17% formamide and the hyperphase was 50% formamide. An RCA EMU-4 electron microscope was used to photograph heteroduplex molecules. +X174 RF molecules were added to all preparations. Molecules were traced at a final magnification of at least 200,000 and measured with a Keuffel and Esser map measurer. The locations of the b515 and 6519 deletions, att, and the imm434 substitution are from Davidson and Szybalski (1971). EcoRI restriction enzyme analysis. CsCl purified phage were disrupted by dialysis against a solution containing 50% formamide, 100 mM Tris-Cl (pH 8.5), and 10 mM EDTA for 16 hr at room temperature followed by dialysis against a solution containing 100 miJ4 Tris-Cl (pH 7.5), 0.1 mM EDTA overnight at 4”. Phage DNA (2 pug) was digested at 37” for 2 hr in a total mixture (0.1 ml) which contained 6 mM Tris-Cl (pH 7.8), 6 mM MgCl*, 1 mM dithiothreitol, and 3 units of EcoRI endonuclease. The exonuclease-free enzyme was the gift of K. Agarwal. The reaction was terminated by addition of EDTA to 50 mM and heating for 2 min at 80”. Each sample

recA

TRANSDUCING

was made 20% (w/v) in glycerol and 0.02% (w/v) in bromophenol blue. Electrophoresis in 0.7% agarose slab gels (20 x 20 cm) was done at 4” for 16 hr with 40 mM Trisacetate (pH 7.6) buffer at an applied field of approximately 2 V/cm. Gels were stained for 20 min in a solution of 1 pg/ml of ethidium bromide and photographed under uv light. The molecular weight of the restriction fragment unique to AprecA (fragment R) was calculated from the mobilities of h restriction fragments of known molecular weight (Thomas and Davis, 1975). RESULTS

A Insertion

into the srl Locus

Several mutants defective in sorbitol metabolism were isolated and characterized in this laboratory (W. Epstein and E. Jackson, unpublished observations). One of these mutations, d-1, was located at min 58 on the E. coli genetic map (Bachmann et al., 1976). The gene order cysCsrl-recA is deduced from three-factor transductional crosses (Table 2, Fig. 1). The donor recA allele is inherited at high frequency when srl+ is the selected trait (Table 2). In several experiments, cotransduction frequencies of 70-95% have been observed for several recA mutations and srl-1 in this laboratory and in the laboratory of A. J. Clark (A. J. Clark and M. Kotewicz, personal communication). We have exploited this high linkage between recA and d-1 to isolate a recA transducing phage from a secondary-site lysogen in which A is integrated into the srl locus. Strain KM2134 was lysogenized with A (see Materials and Methods). Approximately 1 lysogen in 2000 did not ferment sorbitol. Because sorbitol is a substrate for the phosphoenolpyruvate-de-

Donor

AB2462 AB2462 KL16-99

(recAl2) (recA1)

KM100 KM100 KM100

Plkc

COTRANSDUCTION

Recipient

Selected marker

(cysC (cysC (cyst

d-1 1 d-1 1 srl-1)

cyst+ srl+ srl+

309

PHAGES

pendent phosphotransferase system (PTS) and A is known to integrate frequently into the pts locus (Shimada et al., 19731, it was expected that many phenotypically sorbito1 negative (Srll) lysogens would be pts mutants. The Sri- lysogens were scored on glucose MacConkey medium to distinguish pts- mutants from the true srl- mutants. Approximately 5% of the lysogens tested were positive for growth on glucose (pts+). In three experiments, a total of 12 srl- mutants was obtained. Six of these lysogens were arbitrarily selected for further study. Three genetic tests were performed to map the sorbitol mutation of these lysogens and to demonstrate that the sorbitol defect is the result of A insertion into the srl locus: (i) Complementation tests were performed with four episomes which extend from 1ysA to different points in the cysC-pheA region. All lysogens tested were complemented for growth on sorbitol by F143 and F108 but not by the F143-1 or F160 episomes (Fig. 1). This complementation pattern is identical to that observed with the srl-1 point mutation. (ii) Hfr strain KA197 was mated with three of the srl-1 lysogens. This Hfr strain transfers the srl region early; all (77177) srl+ strrecombinants obtained in a 30-min mating at 30” had lost A immunity and temperature sensitivity. (iii) The srl- mutation and A immunity are 100% linked in transductional crosses as well. When a mtl~ srl+ strain was used as donor, all (3201320) srl+ recombinants had lost A immunity and were capable of growth at high temperature. However, when Plkc grown on a mtl+ srl+ strain was used, a fraction of the Sri’ transductants retained the prophage. All of these A-immune transductants were shown to be mtl+ srl- by testing on indica-

TABLE 2 OFCYSC,

d-l,

AND recA

Number scored

160 75 64

Recombinants srlrecA -

srlrecA +

srl+ recA -

sr1+ recA L

0

149

8 56 57

3 19 7

310

McENTEE

tor media (see Materials Orientation

AND

EPSTEIN

and Methods).

pheA + recombinants per 0.1 ml of mating mixture were obtained, but no thy+ recombinants were obtained. Spot matings confirmed that cysC+ is not transferred early in crosses with strain KM3000, consistent with the order shown in Fig. 2.

of h in the srl Locus

Chromosome mobilization by the F8 episome in a hpgal8 transductant of strain KM2137 was used to determine the orientation of the resident prophage in the strain (see Materials and Methods). The prophage orientation shown in Fig. 2 predicts that FS will transfer as early markers pheA and other genes counterclockwise to the phage. In a 30-min mating at 30” between strain KM3000 and thyA- pheArecipient strain KM197, approximately 300

--

IysA \

IhyA argA 1 /

61

60

cyst

srl

red \/

In 59

56

--077

tyrA , 57

isolation of srl and Phages

pheA

y 56--

F 143

F 143-I F 160

______ - _____ - - - - - - ---

Transducing

Thermal induction of strain KM2136 produces low-titer lysates, approximately lop4 phagellysogen. Phages capable of transducing srl were detected among plaque-forming phage by plating the lysate on strain KMlOO. Two hundred forty turbid plaques on this host were picked and cloned on sorbitol MacConkey plates seeded with XCI. Fifty-two independent lysogens were Srl+ as judged by a positive indicator reaction as well as by their ability to grow on sorbitol minimal medium. Three of the Srl+ lysogens were grown in liquid. Following thermal induction, each of the lysogens produced plaque-forming transducing phages which complement the srl-1 mutation of strain KMlOO. Typically, lo-20 transducing phages were released per lysogenic cell. Specialized recA transducing derivatives were identified among the plaqueforming phage by picking turbid plaques formed on several recA- hosts (AB2462, AB2463, and KM6011 and testing for

- 0.81

F IO6

recA

_--

FIG. 1. A genetic map of the recA region of E. c&i. The gene locations and minute scale are from the revised E. coli linkage map (Bachmann et al., 1976). Linkage data shown are from Table 2. The length of each of the episomes is based upon complementation of the markers shown; dashed segments represent uncertainty in the extent of the episome. The representation of F143-1 is based upon the failure of this episome to complement d-1, recA, pheA, and tyrA mutations.

XprecA +

4

thyA argA cysC b2 J ”

A R cl ‘,,’ In\

IhyA orgA cysC

J b2 J

A R cl int

.--------F8

SJ ,_.~_..~

srl-I recA pheA I”+ ’ -f”

srl-I recA pheA / A R cl I”+ , . . . . . . ~.... _,-.._i ..__._.

;

FIG. 2. Orientation of h integrated at srl and mobilization of represents the orientation of the prophage at srl. Connected arrows which generate hprecA. The formation of a double lysogen incorporating tion by FS of chromosomal markers to the right of the prophages, are including the location and orientation of gal on this phage has been orientation of the gal region on the F8 episome is from Low (1972).

chromosomal markers. The top line at the top indicate the sites of cutting hpgul8, and the resultant mobilizashown below. The structure of hpguld reported by Nissley et al. (1971). The

recA

TRANSDUCING

growth in the presence of the radiomimetic agent nitrofurantoin (2 pg/ml). Approximately 1% of the turbid centers tested produced colonies on the nitrofurantoin medium. Several lysogens isolated in this way were tested for sensitivity to killing by uv irradiation. All were found to be uv resistant and recombination proficient in transductional crosses (McEntee, 1976) and in conjugal crosses with Hfr strains which do not transfer the recA+ gene. Nine independent Ret+ lysogens were grown in liquid and thermally induced; each of the strains produced high-frequency transducing lysates (phage titer of log/ml) which conferred recombination proficiency and resistance to uv to recAmutants. No segregation of recombination proficiency and uv resistance was detected among several dozen phages tested from these lysates. Both uv resistance and recombination ability were lost simultaneously upon curing the recA- lysogen at 42”. A second screening method, based upon the ability of the phages to restore recombination to recA- hosts, was used to isolate four additional recA transducing derivatives, The low-frequency transducing lysate was plated on strain AB2462 and lysogens of this recA- mutant were tested for the ability to form thr+ leu+ proi str- recombinants in conjugal crosses. Each of TABLE RESTORATION

OF uv

Host

RESISTANCE

AND

Relevant

RECOMBINATION

311

PHAGES

the four Ret+ lysogens isolated by this technique produces phage which restore uv and nitrofurantoin resistance to recA strains in addition to complementing the recombination defect. This result is consistent with the notion that the recombination and repair functions of recA are due to the expression of a single gene. Isolation of Amber Phage hprecA99

3 BY AprecA99

IN

sup+ supE supE

(B) Recombinationb KL 16-99

recA1

sup+

AB2462

recAl2

supE

SUPPRESSING

recA

STRAINY

Prophage AcI857

recA1 recAl2 recA1

Transducing

Mount (1971) has described a mutation in the recA gene which is suppressed by nonsense suppressors. Strain amber KM2199, which carries the recA99 mutation, was lysogenized with hcI857susS7 and a Sri- lysogen was obtained as previously described. From this strain, a AprecA99 transducing phage was isolated. This phage complements the uv sensitivity and recombination deficiency of recAstrains which carry amber suppressors (AB2462, AB2463, KM601) but fails to complement the recA- defects of sup+ recA- strains (Table 3). All phage isolated which transduce the recA gene, representing at least four independent isolations, complement the srl-1 mutation of strain KMlOO. In the remainder of this paper, we present a genetic and biophysical characterization of one deriva-

genotype

(A) uv resistancea KL 16-99 AB2462 KM601

recA

Survival after 4 x 10-G 5 x 10-S 3 x 10-S

0 0

ADred

hored 200

erglmm’ of irradiation 7 x 10-S 0.59 0.40 0.45 0.20 0.32

bgl recombinantsIlO 5 pro+ recombinantsIlO 159

Plkc 469 Plkc 132

n The recA host lysogenic for the indicated phage was grown in K115 medium containing 50 mg of required amino acids per liter and irradiated as described under Materials and Methods. The dose delivered was approximately 200 erg/mmz. Results are presented as a fraction of the initial viable cell titer that survived irradiation. b The recAhost lysogenic for the indicated phage was grown in KML medium at 30”, infected with Plkc at a multiplicity of infection between 0.1 and 0.3, plated on appropriate selective medium, and incubated at 30”.

312

McENTEE

tive of he1857 designated hprecA, transduces the recA + gene. Site of Lysogenization by hprecA

AND

which

Specialized transducing phages carrying hybrid attachment sites (AOP’ or POA’) may insert into the host chromosome via homologous recombination dependent upon the function of the host recA gene or by int promoted site-specific recombination at the bacterial attA site. Phage integration by homologous recombination depends upon the extent of homology between the transducing variant and the bacterial chromosome, while int-dependent insertion of the phage depends upon the structure of the particular hybrid attachment site carried by the transducing derivative (Shimada et al., 1975). Three lines of evidence suggest that the principal site of AprecA insertion is at or near atth: (1) A AprecA lysogen of gal- recAstrain AB2462 was transduced to gal + with PMc grown on strain KMlOO. Among 160 gal+ recombinants, seven (4%) had lost A immunity and regained the uv sensitivity of the parental recA- strain. Since attA is cotransduced with gal by Plkc, this result is consistent with insertion of XprecA at or near at& (2) Evidence which argues against a prophage location in the recA region of the E. coli chromosome was obtained in conjugal crosses between a AprecA lysogen of strain KL16-99 and recipient strain KMlOO. Selecting cysC+ str- recombinants results in 37% coinheritance of the donor recA- mutation. This linkage is identical to that observed when the nonlysogenic donor strain KL16-99 is used. The linkage value is not altered when the recipient is lyso-

EPSTEIN

genized with A to prevent zygotic induction. The normal transmission of the donor recA- allele and the failure to detect zygotic induction in matings of 30-min duration indicate that the prophage is not transferred as an early marker in conjugal crosses with AprecA lysogens of strain KL16-99. Since the recA gene is transferred as an early marker by this Hfr strain, we conclude that AprecA does not lysogenize by homologous recombination in the recA region of this strain. (3) The frequency with which AprecA lysogenizes a host is markedly affected by the presence of the bacterial atth region. Infection of strain KM100 (attA+) by AprecA results in 27% lysogenization. Under identical conditions (see Methods), A infection yields approximately 50% lysoFor atth deletion strain genization. KM2134, the lysogenization frequencies are 0.4 and 0.2% for AprecA and A, respectively. Thus AprecA, like A, shows significantly lower lysogenization efficiency when the atth site is deleted. This evidence strongly suggests that AprecA lysogenizes in the atth region of the E. coli chromosome. Structure

TABLE TEST

Phage recA + (KM1001 AC1857 hpFOh

AprecA

(1.0) (1.0) (1.0)

4 OF ApprecA

OF SPI PHENOTYPE

Host

of AprecA

The observation that AprecA lysogenizes at or near attA implies that the int gene of the phage is intact. To test for loss of genes in the early region of the phage, the plating efficiency of AprecA on a p&A- strain and on a P2 lysogenic host was examined (Table 4). The plating efficiency of AprecA is similar to Ac1857; it is phenotypically Spi+ and plates with normal efficiency on a polA1 mutant, indicating that the gam

(efficiency

of plating

1.0 CO.002 0.8 which

to recA+)”

P2 lysogen

recA

(AB2462)

* Relative plating efficiency was determined at 37”. h The hpro is a plaque-forming pro transducing phage gam, i.e., Spi- (R. Weisberg, personal communication).

relative

is deleted

(KM1551

polA (H560)

Isolation and characterization of specialized transducing bacteriophages for the recA gene of Escherichia coli.

VIROLOGY 77, 306-318 (19771 Isolation and Characterization of Specialized Transducing Bacteriophages for the recA Gene of Escherichia co/i KEVIN D...
5MB Sizes 0 Downloads 0 Views