Vol. 129, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 1078-1090 Copyright © 1977 American Society for Microbiology

Isolation and Characterization of Xpleu Bacteriophages MICHELLE GALL DAVIS1 AND JOSEPH M. CALVO*

Section ofBiochemistry; Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

Received for publication 13 September 1976

In the Escherichia coli lysogen HfrH73 described by Shimada et al. (1973), of the enzymes coded for by the leucine operon is synthesized due to an insertion of phage X into cistron leuA. The orientation of X in the chromosome is ara leuDCB XJAN leuA. After heat induction of the lysogen, plaque-forming transducing phages of two types are formed at low frequency. One type (e.g., Xpleu9) transduces leuD, leuC, and leuB strains to prototrophy. The other type (e.g., Xpleu13) transduces leuA strains to prototrophy. Xpleul3 forms lysogens at low frequency (about 0.2%) by integration into the leucine operon. These lysogens are unstable, segregating phage-sensitive clones at high frequency (about 1%). Phages carrying different portions of the leucine operon were formed by aberrant excision after heat induction of strain CV437 (leuA371 Xpleul3). A phage carrying the entire leucine operon (XK2) was constructed by a cross between Xpleu9 and Xpleu13. An analysis of leucine-forming enzyme levels in strains lysogenized with XK2 indicated that leuO and leuP are present and functional inXK2. leu-specific messenger ribonucleic acid fromE. coli hybridizes to the heavy (r) strand of XK2. The leucine operon of XG4 pleuABCD (an S7 derivative of XK2) exists intact on a 7.3 x 106-dalton fragment (XG4EcoRI-B) generated by cleavage with endonuclease EcoRI. Heteroduplexes formed between XG4 and X show a 5.4 x 106-dalton piece of bacterial deoxyribonucleic acid (DNA) replacing a 4.5 x 106-dalton piece of X DNA starting at 0.46 fractional unit on the map of X. Fragment XG4EcoRI-B has about 0.6 x 106 daltons of X DNA from the b2 region at one end and about 1.4 x 106 daltons of X DNA from the int region at the other end. none

Shimada et al. (22) analyzed lysogens of Escherichia coli in which phage X integrated at sites other than the normal attachment site. One such lysogen, strain HfrHAattB B'73 (hereafter referred to as HfrH73), is a leucine auxotroph caused by integration of the prophage into the leucine operon. Upon heat induction of this lysogen, they isolated several specialized transducing phages carrying different portions of the leucine operon. This paper describes the characterization of these phages and of phages we constructed that carry the entire leucine operon. In other reports, these phages are used as sources of leucine operon deoxyribonucleic acid (DNA) in studies of transcription in vivo (M. Davis and J. Calvo, manuscript in preparation), transcription in vitro (F. Berger, S. Wessler, J. Finley, and J. Calvo, manuscript in preparation), and coupled transcription and translation in vitro (G. Schatz, F. Berger, L. Searles, J. French, and J. Calvo, manuscript in preparation). 1 Present address: Department of Bacteriology, University of North Carolina, Chapel Hill, N.C.

MATERIALS AND METHODS Strains. The genotypes of the bacterial and phage strains that were used in this study are shown in Table 1. Growth of bacteria and phage. Tryptone broth (22) and a modified Davis-Mingioli medium (4) were used as rich and minimal media, respectively. When required, amino acids and Casamino Acids were supplemented at 50 ug/ml and 0.12%, respectively. Cultures were grown at 37°C with shaking. Phage were grown either by the method of confluent lysis (17) or by heat induction of lysogens (17). For cultures infected with S+ phage, chloroform was added; after the mixture stood for 15 min at room temperature, cellular debris was removed by centrifugation. Sometimes these phage were concentrated by precipitation with dextran sulfate as described by Wu et al. (29). Phage carrying the S7 marker (19) were isolated as described by Miller (17). Screening Aleu phages for leu genes. Phage lysates were tested by spotting a loopful of a lysate onto a minimal agar plate that had been spread with 0.1 ml of a culture of leucine auxotroph. Recombination was judged positive if either discrete colonies or confluent growth occurred within the spot after incubation for 2 days at 30°C.

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VOL. 129, 1977 Strain

Escherichia coli CSH26 CSH28 CSH73 HfrH B * B' (AcI857) leu strain 73 N205 N205 (XH)1 N205 (XH)2 N205 (XH)3 C600 (P2) CV437

kpleu PHAGES TABLE 1. Strains used in this study Relevant genotype Remarksa A(pro-lac) thi supF his trp A(ara-leu) Alac thi A(gal-bio) leu

XcI857 integrated at leu X lysogen, single X lysogen, double X lysogen, triple

leuA371 A(pro-lac) thi

CV438

leuBO61 A(pro-lac) thi

CV512 CV516 CV514 CV518 CV520 CV522 CV526 CV524 CV528

leuA371 leuBO61 leuB401 leuB201 leuCl71 leuC222 leuDlOl leuD211 leuD141

(X) (X) (X) (X) (X) (X) (X) (X) (X)

Bacteriophage X XcI857 XcIb2

XcIh8O Xvir Ximm434S7 Xcl7cI90 XpleuA 13 XpleuBCD9 XcI857S7 XG1

Amber suppressor

cI857leuABC3 cI857leuBCD9

cI857leuA 13S7

Plaques Spi- phage Prepared from CV512 and CSH26 by P1 transduction Prepared from CV516 and CSH26 by P1 transduction

1079 Source

CSHb strain kit CSH strain kit CSH strain kit M. Gottesmann M. Gottesman M. Gottesman M. Gottesman M. Gottesman M. Gottesman This work

This work

R. R. R. R. R. R. R. R. R.

Middleton Middleton Middleton Middleton Middleton Middleton Middleton Middleton Middleton

M. Gottesman CSH strain kit CSH strain kit D. Wilson J. Roberts M. Gottesman M. Gottesman M. Gottesman CSH strain kit This work

Derived from a cross between XpleuA13 and XcI857S7 XK2 cI857leuABCD This work Derived from a cross between XpleuA13 and XpleuBCD9 XG4 cI857leuABCDS7 Derived from a cross between This work XK2 and XcI857S7 XG3 cI857leuABCD Derived from CV437 (XpleuThis work A 13) from aberrant excision XG5 cI857leuABCDS7 This work Derived from a cross between XG3 and XcI857S7 XG6 This work cI857leuBCD9S7 Derived from a cross between XpleuBCD9 and XcI857S7 XG7 This work cI857keuABC Defective phage derived from CV437 (XpleuA13) by aberrant excision a Spi-, Ability to plate on a P2 lysogen; Ara+, abiility to grow with arabinose as the sole carbon source; Leu+, ability to grow in the absence of leucine. b CSH, Cold Spring Harbor Laboratory.

Phage crosses. Cells grown to log phase in 10 ml of tryptone broth containing 10 mM MgSO4 were infected at a multiplicity of 1 with each phage strain. After incubation for 10 min at room temperature, the samples were shaken at 42°C for 2 h or until lysis was observed. The samples were shaken

with chloroform, and the cellular debris was removed by centrifugation. Lysogenization and transduction frequencies. Lysogenization frequencies were determined as described by Shimada et al. (22). Transduction frequencies were determined by the same procedure,

1080

DAVIS AND CALVO

were plated on minimal plates. Transductants were tested for immunity to A by cross-streaks against XcIb2 and Xvir. Lysogens were tested for a leucine requirement by transferring clones with toothpicks to plates lacking or containing 50 jitg of leucine per ml. Determination of the number of prophages in a lysogen. Single, double, and triple lysogens were distinguished by their sensitivity to Xcl7cI90, a phage that is sensitive to the intracellular concentration of A repressor (22, 26). Chemostat experiments. Cells grown in minimal medium at a generation time of 134 min in a chemostat (4) were harvested after at least three replacement periods. In cases where cells were pulselabeled with [3H]uridine, the culture was decanted rapidly, leaving 25 ml in the cylinder; the pumping rate was decreased by a factor of 10; labeled uridine was added; and 3 min later the cells were harvested as described above. Enzyme assays. Cells were grown in a chemostat or in shake flasks to about 5 x 108/ml, centrifuged at 8,000 x g for 10 min, and washed with 0.10 volume of 0.05 M potassium phosphate buffer (pH 7.5). Pellets, stored frozen, were resuspended in the same buffer (8 ml/g [wet weight]) and disrupted in a Branson Sonifier in two 30-s periods with intermittent cooling. The samples were centrifuged at 8,000 x g for 10 min, and supernatants were assayed immediately or after storage in a freezer. Protein was determined by the method of Lowry et al. (16). a-Isopropylmalate a-ketoisovalerate lyase (coenzyme A acetylating) (EC 4.1.3.12; a-IPM synthase) was assayed as described by Calvo et al. (3), except that resorcinol at 75 mg/ml was used and the incubation period was 1 h at 60°C, instead of resorcinol at 300 mg/ml and incubation at 60°C for 10 min. Also, dilution of samples with 1 M NaOH instead of borate-carbonate buffer diminished the background fluorescence. 2Hydroxy-4-methyl-3-carboxyvalerate:NAD+ oxidoreductase (EC 1.1.1.85; /3-IPM dehydrogenase) was assayed as described by Burns et al. (2). Isolation of DNA. After heat induction of lysogens, phage were pelleted by centrifugation for 3 h at 20,000 x g. Phage pellets were suspended in buffer B (1 ml/g [wet weight] of cells) (17), and a solution of buffer B saturated with CsCl was added to density 1.5 g/cm3. Samples were centrifuged at 12,000 x g for 1 h at 4°C. The floating pellicle was discarded, and the clear supernatant was centrifuged for 20 h at 22,000 rpm in an SW27 rotor. The phage band was removed with a 22-gauge syringe, and the sample was dialyzed overnight at 4°C against three changes of 100 volumes of buffer C (17). DNA was extracted from the phage as described by Miller (17), except that the suspension was heated for 15 min at 60°C and that KCl was added to 1 M (30). Endonuclease digestions and agarose gel electrophoresis. For digestions with endonuclease EcoRI, samples contained 0.5 ,ug of DNA, 1 unit of EcoRI (Miles Laboratories), and 2 ,ul of buffer containing 500 mM tris(hydroxymethyl)aminomethane (Tris)chloride (pH 7.5), 250 mM NaCl, and 25 mM MgCl2. After incubation for 30 min at 37°C, 3 ,ul of 0.1 M ethylenediaminetetraacetate (EDTA) solution con-

except that infected cells

agar

J. BACTERIOL.

taining 0.2% bromophenol blue, 0.2% xylene thymol, and 50% sucrose was added. Samples were loaded onto 0.7% agarose slab gels (28), and electrophoresis was carried out in a buffer containing 50 mM Tris-acetate (pH 8.2), 20 mM sodium acetate, 18 mM sodium chloride, and 2 mM EDTA. Gels were stained in ethidium bromide (1 ,ug/ml) and photographed as described by Sharp et al. (21). DNA was isolated from gels by the homogenization method of Wu et al. (28). Isolation of RNA. Cultures grown in a chemostat or in shake flasks in minimal medium to about 5 x 108 cells per ml were pulse-labeled with [3H]uridine and then poured over 35 ml of frozen crushed medium containing 10 mM sodium azide and 400 ,tg of chloramphenicol per ml. The cells were centrifuged and suspended in 2 ml of a cold solution containing 20 mM Tris-chloride (pH 7.5), 40 mM EDTA, and 200 mM NaCl. RNA was isolated by the method of Ikemura and Dahlberg (12). Ethanol precipitates were dissolved in 1 ml of 2 x SSC (1 x SSC is 0.15 M NaCl0.015 M trisodium citrate) and dialyzed against 300 ml of 2 x SSC. A 25-ml culture containing 5 x 108 cells per ml yielded 450 to 850 ,ug of RNA of specific activity 10,000 to 14,000 cpm/,tg. Separation of DNA strands. X DNA was separated into heavy and light strands by centrifugation with poly(uridylate, guanylate) [poly(U, G); Miles Laboratories] as described by Hradecna and Szybalski (11). Fractions containing the majority of each strand were pooled and dialyzed against three changes of 300 ml of 2 x SSC. The samples were selfannealed by incubation at 60°C for 6 h and were stored at 4°C. DNA-RNA hybridization. The liquid-liquid hybridization procedure of Bovre and Szybalski (1) was employed. Small siliconized tubes containing about 1 ,tg of a single DNA strand, 1 to 20 ,ug of RNA, and phenol-saturated 2 x SSC in a total volume of 0.1 ml were incubated at 65°C for 6 to 18 h. After the mixture cooled slowly to 30°C, 2 ,ug of pancreatic ribonuclease A was added, and the incubation was continued at 30°C for 30 min. Samples were diluted, filtered, dried, and counted as described previously (1). Preparation of heteroduplexes for electron microscopy. Heteroduplexes were prepared by the procedure of Davis et al. (7), with modifications suggested by Chow et al. (5). Renaturation was allowed to proceed until about 50% of the molecules had reannealed. Samples on Parlodion-covered grids were stained in uranyl acetate and shadowed with 80% platinum-20% palladium at an angle of 80. Grids were examined in a Phillips 301 microscope, and photographs were taken on 35-mm film. DNA strands were traced after projection onto a light table, and the lengths of the strands (about 30 inches [ca. 76.2 cm], enlarged) were measured with a map measurer. To some samples, ColEl or pMB9 circular DNA was added as a double-stranded reference length.

RESULTS Position of phage X in the leucine operon of strain HfrH73. Xpleu isolates supplied by M. Gottesman were tested for their ability to

VOL. 129, 1977

Xpleu PHAGES

transduce various leucine auxotrophs to prototrophy (Table 2). Phage strain Xpleul2 (and also Xpleul3) carries at least a portion of leuA, whereas Xpleu6 and Xpleu9 carry all, or portions, of leuBCD. None of the phages transduced an Ara- strain to Ara+. These results suggest that in strain HfrH73, the lysogen isolated by Shimada et al. (22), phage X has inserted into either leuA or leuB. The probable origin of these Xpleu phages is represented diagrammatically in Fig. 1. Strain HfrH73 was assayed for a-IPM synthase an,d ,B-IPM dehydrogenase, enzymes specified by leuA and leuB, respectively. No detectable activity was observed for either enzyme. This result is consistent with the view that phage X has inserted into leuA and that leuB (and leuC and leuD) are intact but not functional, presumably because they have become separated from a functional promoter. Phages XpleuA12 and ApleuAl3 lysogenize by insertion into the leucine operon. Xpleul2 and Xpleul3 form clear plaques at 30 and 37°C. The frequency at which they lysogenize strain

1081

W3350 (Table 3) is about 1% that shown by XcI857. The integration frequency is increased somewhat if the host carries the heteroimmune

phage Xi434.

TABLE 2. Transductions mediated by Xpleu phagesa No. of transductants per plate for

Recipient

phage:

ApleuBCD6 XpleuBCD9 XpleuA12 0 0 2,000

CV512 (leuA371) CV514 (leuB401) 67 39 0 CV516 (leuBO61) 82 39 0 CV518 (leuB201) 45 38 0 CV520 (leuCl71) 45 24 0 CV522 (leuC222) 48 47 0 CV524 (leuD211) 90 46 0 CV526 (leuD101) 42 28 0 CV528 (leuD141) 135 91 0 a About 2 x 108 phage and 2 x 108 bacteria grown to stationary phase in tryptone broth were mixed, incubated for 20 min at 33°C, and diluted 100-fold, and 0.1-ml samples were spread on minimal agar plates. Colonies were counted after incubation for 48 h at 33°C.

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1082

J. BACTERIOL.

DAVIS AND CALVO

TABLE 3. Lysogenization and transduction frequencies of XpleuA12 and XpleuA13 Strain

Phage

Frequency of Lysogens lysogeniza- that were U (%)b tion Ma LeU tion (%)

19 XcI857 CV437 (leuA371) 0.17 XpleuA12 CV437 (leuA371) 0.14 XpleuA13 CV437 (leuA371) 0.32 XpleuA12 CV437 (leuA371) (Xi434) 0.37 XpleuA13 CV437 (leuA371) (Xi434) 14 XcI857 CSH73 A(ara-leu) 0.14 XpleuA13 CSH73 A(ara-leu) 0.13 XK2(pIeuABCD) CSH73 A(ara-leu) 9 XcI857 CSH73 A(ara-leu) (Ai43) 0.67 XpleuA13 CSH73 A(ara-leu) (Xi434) 0.74 XK2(pleuABCD) CSH73 A(ara-leu) (Ai434) a Percentage of cells plated that were lysogens or transductants. b About 30 colonies were tested in each case.

The relationship between lysogeny and transduction was investigated for XpleuA 12 and XpleuA 13. After infection of strain CV437 (leuA371) with phage, a portion of the mixture was spread on minimal agar plates to select for Leu+ transductants, and a portion was spread on tryptone agar plates containing 109 XcIb2 and 109 XcIh8O to select for lysogens. The frequency of lysogens among transductants and the frequency of leu+ derivatives among lysogens were then determined (Table 3). All transductants were lysogens and almost all lysogens were prototrophs. This suggests that Leu+ recombinants do not arise to any significant extent by recombination of a small portion of the phage-mediated genome into the bacterial chromosome (such as in P1- or P22-mediated transduction). Rather, the host becomes Leu+ by integration of the entire phage genome into the host chromosome. Strain CSH73 carries a deletion extending from the arabinose operon through most of the leucine operon. (It is Ara- and lacks detectable a-IPM synthase and ,8-IPM dehydrogenase activities.) CSH73 (XpleuA12) and CSH73 (XpleuA 13) lysogens were prepared and assayed for a-IPM synthase activity. They had no detectable activity. Therefore, the CV437 (leuA371 Xpleu) lysogens described above could not have become Leu+ by integration of the phage into the X attachment site or into any other position outside the leucine operon. The origins of these Leu+ lysogens are diagrammed in Fig. 2. Integration of the phage into the chromosome is portrayed as a reciprocal recombinational event made possible by homology between leuA DNA carried on phage and bacterial genomes. If crossovers occur to the left of mutation leuA371, the resulting lysogen will have a functional leu operon; if crossovers occur

Frequency of transduction (%)a

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to the right of this mutation, the resulting lysogen will remain a leucine auxotroph. The fact that almost all of the lysogens are prototrophs suggests that the leuA gene carried on XpleuA12 and XpleuA13 is attached to a functional promoter. Lysogens formed from XpleuA12 and XpleuA13 are unstable. XpleuA12 and XpleuA13 both have mutation cI857 so that lysogens carrying these phages are induced at high temperature. The frequency of survivors was determined for strains W3350 (XcI857) and W3350 (XcI857 pleuA 12) after incubation at 41°C (Table 4). Both strains were made resistant to X to prevent re-infection of sensitive cells. Survivors were 10,000 times more frequent after heat induction of Xpleu lysogens than normal lysogens. Survivors were sensitive to AcIh8O, indicating that they had lost at least a part of the prophage. Furthermore, these lambda-sensitive heat-resistant cells are present in an uninduced culture and therefore do not require heating to be formed. Construction of X strains carrying the entire leucine operon. Strain CV437 (leuA371) lysogenized with XpleuA13 has the genotype shown in Fig. 2. Upon heat induction of this strain, mutant phage carrying leuAB, leuABC, or leuABCD occasionally arise by aberrant excision. By this method we isolated XG7, a defective phage carrying leuABC and part of leuD (tranduces leuD211 and leuDl01 to prototrophy but not leuD141 or CSH73 Aleu), and XG3, a plaque-forming phage carrying the entire leucine operon (transduces CSH73 Aleu to prototrophy). An alternative method of constructing a XpleuABCD phage is illustrated in Fig. 3. Progeny from a cross between XpleuA 13 and XpleuBCD9 transduced CSH73 A(ara-leu) to

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VOL. 129, 1977

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FIG. 2. Diagrammatic representation of the events leading to lysogenization and transduction of strain CV437 (leuA371) by phage XpleuA13. Integration occurs by a reciprocal recombinational event involving homologous sequences on the phage and chromosome. (a) Crossover to the right of leuA371. (b) Crossover to the left of leuA371. (c) Aberrant excision leading to XG7dleuABC. The latter is drawn to scale. An asterisk indicates that only a portion of a gene is present. Other symbols are defined in the legend to Fig. 1.

prototrophy at low frequency. Phage isolated TABLE 4. Frequency ofsurvival after heat induction of lysogens and purified from one such transductant (designated XK2) transduced CSH73 A(ara-leu) to Cells that surprototrophy at a high frequency. To generate vived incubaLysogen tion at 41°C an intact and functional leucine operon, a re(%)a combinational event (probably int promoted) W3350 (XcI857)Xr 0.0001 must have occurred within very narrow conW3350 (XcI857pleuA 12)Xr 1.0 fines, perhaps at a particular base pair within CSH73 (XcI857K2pleuABCD)Xr 0.87 leuA. The frequencies with which XK2(pleuABCD) lysogenized various hosts (Table 3) and a Samples of diluted cultures were spread on the frequency of survivors after lysogens were tryptone agar plates and incubated at 30 and 41°C. heated at 41°C (Table 4) were similar to values The frequency of survivors is the number of colonies on plates incubated at 41°C divided by the total obtained in experiments involving XpleuA 12. XK2(pleuABCD) carries an intact leucine number of cells plated, the latter determined by the operator and promoter region. Lysogens plating at 300C. CSH73 A(ara-leu) (XK2pleuABCD) and CSH73 A(ara-leu) (XG3pleuABCD) and wild-type genes carried on phages XK2 and XG3 are atstrain CSH62 were analyzed for levels of two tached to the natural leucine operator and promoter. enzymes functioning in leucine biosynthesis (Table 5, experiment 2). The levels of these The effect of the number of leu genes upon enzymes were essentially the same for all three the levels of pathway-specific enzymes was instrains. Furthermore, the addition of leucine to vestigated in several ways. Doubling the numthe growth medium repressed the levels of ber ofleuB genes per chromosome by lysogenizthese enzymes to about the same extent in the ing a prototroph with XK2 had little or no effect three strains. These data indicate that the leu upon leu enzyme levels [Table 5, compare

1084

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J. BACTERIOL.

DAVIS AND CALVO A

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Strain

Sp actb

Growth mediuma

a-IPM

synthase

Expt 1

W3350 W3350 W3350 W3350 W3350 W3350

(XcI857) (XcI857) (XK2) (XK2)

leu+ leu+ leu+ leu+

leu+/XpleuABCD leu+/XpleuABCD

Expt 2

CSH62 CSH62 CSH73 CSH73 CSH73 CSH73 CSH73 Expt 3 CSH73 CSH73 CSH73 CSH73 CSH73 CSH73

leu+ leu+

A(ara-leu)IXpleuABCD A(ara-leu)/XpleuABCD A(ara-leu)/XpleuABCD A(ara-leu)/XpleuABCD

(XK2) (XK2) (XG3) (XG3)

A(ara-leu)

9.6 2.6 10.1 2.5

Minimal Minimal + Leu Minimal Minimal + Leu Minimal Minimal + Leu

Minimal Minimal + Leu Minimal Minimal + Leu Minimal Minimal + Leu Minimal + Leu

(3-IPM

dehydro-

10.1 3.3

0.52 0.19 0.34 0.11 0.31 0.12