Molec. gem Genet. 140, 123--135 (1975) © by Springer-Verlag 1975

Isolation of Phage P2-186 Intervarietal Hybrids and 186 Insertion Mutants

Clifford Bradley, Ong Peng Ling and J. Barry Egan

Department of Biochemistry, The University of Adelaide, Adelaide, South Australia

Received May 7, 1975

Summary. Intervarietal hybrids formed between coliphages P2 and 186 have been isolated and their preliminary genetic characterization described. Three insertion mutants of 186 have also been isolated.

Introduction

The value to phage studies of the availability of phage hybrids and of phage deletions is exemplified by the roles that hybrid phage and deletion phage have played in the study of the coliphage ,~. Our interest in the "noninducible" eoli- phages P2 and 186 led us therefore to consider the feasibility of isolating P2-186 hybrid phage and 186 deletion phage.

As evidenced in the lambdoid series of eoliphages, the ability of two lambdoid parental phages to cohere via their cohesive ends appeared to be a primary requirement for their intervarietal recombination (Baldwin et al., 1966). Re- combination between P2 and 186 was conceivable as the two phages were known to cohere (Mandel and Berg, 1968)--in fact it has recently been shown that their cohesive ends differ in only 2 of the 19 residues forming the cohesive end (Padmanabhan and Wu, 1972; Murray and Murray, 1973)--and the two phages share homologous sequences (Skalka and Hanson, 1972; ¥ounghusband and Inman, 1974). Other pertinent criteria also appeared favourable--thus both phage have genomes approximately the same size (Bert~ni and Bertani, 1971) with a similar genetic distribution of functions (Hocking and Egan, manuscript in preparation), both phage appear morphologically similar by electron microscopy (Bertani and Bertani, 1971) and both phage can infect Eseherichia coli strain K-12. We wish now to report the successful isolation of P2-186 hybrid phage.

We have also isolated heat resistant mutants of 186, in an at tempt to obtain deletion mutants. We instead exposed a heat resistance marker in 186, and among the heat resistant mutants recovered three insertion mutants, but no deletion mutants.

Materials and Methods

Bacterial and Phage Strains The genotype and source of each bacterial and phage strain are listed in Tables 1 and 2.

Media and Bullets T broth: 1% bacto tryptone, 0.5% NaC1. L broth: 1% bacto tryptone, 0.5% yeast extract, 1% NaC1.

3 Molec. gem Genet.

124 C. Bradley etal.

Table 1. BacteriM strains

Collection Relevant Genotype Origin or reference number phenotype

E. coli K12

594 Su- F- galK ga i t str Weigle (1966) C600 Su+ F- thr leu thi lac touA supE tsxE Appleyard (1954) S26Rle Su + Hfr C rel touA T2 r ThoA supD Garen etal. (1965) H12R8A Su + Hfr C tel tonA T2 r phoA supF Garen etal. (1966) E756 Su + (P2) C600 (P2 lg) this laboratory

E. cell C

C1055 Su- F+ thr leu xan his str Wiman etal. (1970) C1757 Su + F+ ary T1 r trp str supD Sunshine etal, (1971) C1792 Su + F+ arg T1 r trp his str supF Sunshine etal, (1971) E691 Su- (P2) C1055 (P2) 1K. Mandel E903 Su- (186) ]~+ thr leu xan str (186) This laboratory--by

conjugation

Table 2. Bacteriophage strains

Relevant phenotype Genotype Origin or reference

P2 wild type P2 lg from LG 106, Sunshine (1972) t)2 amD P2 virl amD6 Sunshine etal. (1971) P2 ainu P2 virl amU25 Sunshine etal. (1971) P2 amJ P2 virl amJ21d Sunshine etal. (1971) P2 amL P2 (lg ?) amL308 c5 dell Bertani (Stockholm) P2 amL int P2 (lg ?) amL308 c5 dell intl Bertani (Stockholm) 186 wild type 186 Jacob and Wollman (1956) 186 t i ts 186 t i ts 186p Baldwin et al. (1966) 186 am5 186 t i ts am5 This laboratory 186 am7 186 t i ts am7 This laboratory 186 am17 186 clts am17 This laboratory 186 am26 186 clts am26 This laboratory Hyl,2,3,4,5 1)2.186 Hyl,2,3,4,5 This work Hy2 amJp2 P2.186 I-Iy2 amJp21005 This work

In some instances 0.2% glucose (LG), 2 mM CaC12 (LGC) or 80 mMMgSO, (LGM)were added.

Agar plates contained 1.2% bacto agar, and soft agar for phage assays contained 0.7% bacto agar.

Phage diluent (TM buffer) : 0.01 M Tris l, pI-I 7.2, 0.01 M MgSO,. Phage storage buffer: 1 M NaC1, 0.01 M Tris, pH 7.2, 0.08 M MgS04, 10 Elg/ml bovine

serum albumin. All solutions were sterilized by autoclaving.

Phage A s s a y s 0.1 ml of the phage dilution was incubated with 0.2 ml indicator bacteria in logarithmic

phase (5 × 10 a cfu/ml) at 37 ° C for 20 rain, then 3 ml of T soft agar (at 45 ° C) added, mixed,

1 Abbreviations used: Tris-Tris (hydroxymethyl) amino methane EDTA-ethylene diamine tetraaeetate; eol0-efficiency of plating; tin-colony forming units; pin-plaque ~orming units.

P2-186 Hybrids and 186 Insertion Mutants 125

and poured over a base agar plate. For P2 phage assays the base agar was LC agar and the indicator bacteria were grown in LC broth, while T agar and T broth were used in phage 186 assays. All P2 and hybrid phage assays were performed on E. eoli C strains even for phage carrying the K modification, since P2-K gave better plaques and a higher eop on C strains than on K strains.

Phage Stocks

P2 Derivatives were stocked from liquid infection of E. coli K12 strains. A 15 ml cu]ture in LGC broth in logarithmic phase at A60 o = 0.04 was inoculated with a plaque, and incubated at 37°C with aeration until lysis. EDTA (0.004 M) was added just prior to lysis. For larger volumes a 590 ml culture in a 2 litre flask at A~00 = 0.5 was inoculated with 1011 pfu and incubated with aeration at 37°C until lysis.

186 Derivatives carrying the vir allele were stocked from liquid infection as above, except that L broth was used, and no EDTA was added. Phage carrying the clts allele were stocked by heat induction. The lysogen was incubated at 30°C in L broth to A600~ 0.8, heated at 45°C for 10 min, and incubated at 37°C with aeration until lysis. The hybrids were stocked by this method. Often no significant drop in optical density to indicate lysis was observed, but rather a plateau. Such cultures were harvested at 4 hours, with yields greater than 4 × 10 ~° pfu/ml. In spite el the presence of a P2 tail, the addition of EDTA was not necessary for high yields; in fact, the hybrids were sensitive to the addition of EDTA. !~or Hy2 yields were low, but were increased 100 fold by the presence of 0.08 M MgSOa in the medium. I t was presumed to have a stabilizing effect on hybrid phage, and was routinely added for the preparation of all hybrid stocks.

Plate Stocks were used in the heat inactivation studies. Approximately 10~-10 e pfu were plated with 10 s bacteria on fresh, wet T agar plates, and incubated overnight at 37°C. Plates were chilled, flooded with 3 ml TM buffer, and harvested after 2 hours at 4°C.

High Titer Stocks. Phage preparations were centrifuged at 7600 g, 10 rain, 4°C to pellet the bacterial debris, then 10200 g, 16 hours, 4°C to pellet the phage. The pellet was resus- pended in storage buffer, and stored over CHC1 a. Most preparations however were purified by equilibrium centrifugation in CsC1 (~ = 1.43 gem -a, 30000 rpm, 15 hours, 4°C, Beckman Ti50 rotor). In such cases the phage pellets were resuspended in TM buffer. I t may be of practical interest that original at tempts to band the hybrids failed because of the presence of 0.08 M Mg++, even with prolonged centrifugation. There was no loss in phage viability, and the phage did band when the Mg++ concentration was reduced to 0.01 M.

Analytical Ccntri/ugation

Phage were sedimented to equilibrium (44090 rpm, 48 hours, 25 ° C) in CsC1 in a Beckman Model E ultracentrifuge, equipped with a photo-electric scanner (courtesy Dr. Paul Day, Department of Physical and Inorganic Chemistry., University of Adelaide). The phage densities were calculated with reference to the iso-concentration point (Ifft, Veer and Vine- grad, 1961), and with reference to 2 clts857 (~ ~ 1.490 g c m -3, Davidson and Szybalski, 1971) as an internal standard (Belier, Busse and Baldwin, 1971).

Results

Isolation o] Hybrids

Fig . 1 r eco rds r e l e v a n t sec t ions of t h e gene t i c m a p of P2 (Lindahl, 1971;

S u n s h i n e et al., 1971) a n d 186 ( H o c k i n g a n d E g a n , m a n u s c r i p t in p r epa ra t i on ) . I f an Su+ s t r a i n is co in f ee t cd w i t h P2 a n d 186 am, t h e n t h e a p p e a r a n c e of P2-186

r e c o m b i n a n t s in t h e l y s a t e cou ld in t h e o r y be d e t e c t e d as p l a q u e fo rmer s on

Su- (P2) i n d i c a t o r bac te r i a , s ince p r o p h a g e i m m u n i t y selects aga ins t t h e P2

pa r en t , a n d S u - se lects aga in s t t h e 186 pa r en t . H o w e v e r t h e 186 a m b e r m u t a n t s

t e s t e d h a d a d e t r i m e n t a l e f fec t on t h e S u - i n d i c a t o r l a w n w h e n p l a t e d a t t h e

126 C. Bradley et al.

P2 d U D art int c B

186 7 26 17 att int (c $)

Fig. 1. Genetic maps of P2 and 186, giving the order of genes or alleles involved in the present study. For P2, genes U and D are tail genes belonging to one polarity group, and gone J is a tail gene of another polarity group, gene B is an early gene, gene c the immunity gene, att the attachment site and int the gene for prophage integration. For 186, am7 and am26 are mutations in different genes belonging to the same polarity group, am17 and am5 are mutations in early genes mapping on either side of the att site. The int gene has not been mapped genetically, but is physically close to the c gene (Younghusband et al., 1975). The

order of genes within parenthesis has not been established

high concentrations anticipated for the parental phage in the isolation procedure. E. cell C does not adsorb 186 (Woods and Egan, 1974) and the use of E. cell C(C2) avoided this problem. On this indicator 1)2 immunity insensitive mutants were present in the parental P2 stocks at a frequency of 5 X 10 -9. E. cell C(P2) was therefore used as indicator, selecting for a phage capable of adsorbing to E. coli C, but insensitive to P2 immunity.

I t was observed in preliminary experiments tha t the titers of progeny 186 and P2 appearing in the lysate after eoinfection were both severely reduced, whereas in control cultures, involving infection with either P2 or 186, only P2 showed the reduction. This reduction is considered to reflect readsorption of progeny to cell debris (Bertani and Bertani, 1970) and in practice is avoided by the addition of phosphate (or citrate or EDTA) to remove the Ca ++ ions necessary for adsorption. Although 186 has no apparent cation requirement for adsorption, we found tha t the addition of 0.01 M potassium phosphate 30 min after infection permitted the complete recovery of both 186 and P2 in the lysate of a coinfected culture, which suggests the existence of a second phosphate-sensitive inactivating agent besides adsorption.

Recombination between P2 and 186

E. coli K12 Su + (strain C600) was coinfectcd with 132 and 186 eIts am7 at a multiplicity of at least 1O for each parent and incubated with aeration at 37°0. At 30 mins 0.01 M potassium phosphate was added and at 60 mins the lysate was plated on E. cell C Su-(P2) to select for recombinants. Assays of P2 on E. cell C and of 186 am on E. coli K12 Su+(P2) gave the number of parental phage in the lysate. Presumptive recombinant plaques appeared at a frequency of 5 x 10 -8, ten fold higher than the frequency of appearance of immunity in- sensitive mutants in the control culture infected with P2 alone. No ple~ques appeared on the selective indicator from the 186 am7 infected control culture.

Five plaques were isolated from the test plate and purified through repeated single plaque isolations. These presumptive hybrid phage were confirmed as different from either parental phage, as they were ~ble to plate on both E. cell K12 (P2) and E. cell C. The five isolates, whose genomes were expected to be composed of P2 left segment and 186 right segment, have been termed Hyl through tIyS.

P2-186 Hybrids and 186 Insertion Mutants

Table 3. Marker rescue of 186 genes

127

Bacterial indicator

Efficiency of plating of 186 vir am mutants

am17 am26 am7

Su+ 1.0 1.0 1.0 Su- 2.3 × 10 -~ ~3.9 × 10 -s 5.9 × 10 -s Su- (186) 2.1 ~( 10 -1 2.3 × 10 -3 2.0 × 10 -3 Su- (Hyl) 2.4 × 10 -1 5.9 × 10 -6 ~3.1 × 10 -s Su- (Hy2) 2.0 × 10 -1 6.1 × 10 -G ~3.1 × 10 -s Su- (Hy3) 2.4 × 10 -1 6.1 × 10 -6 3.1 × 10 -s Su- (Hy4) 2.0 × 10 -1 4.1 × 10 -3 3.1 × 10 -3 Su- (HyS) 1.9 × 10 -1 1.5 × 10 -6 ~3.1 × 10 -s

The 186 am mutants each carried the immunity insensitive mutation vir2. These phage were assayed on the different indicator bacteria strains with incubation at 30 ° C as each of the hybrid lysogens carried the 186 clts marker. The strains used were E. col/ K12 C600 Su+ and 594 Su-.

Genetic Composition o/the Hybrids

Immunity. Each h y b r i d p la tes on a P2 lysogen bu t no t on a 186 lysogen; P2 phage pla tes on the h y b r i d lysogen, while 186 does not. The five hybr id s therefore possess t he 186 i m m u n i t y region, which is as expec ted because of t he select ion imposed a t the i r isolat ion, b u t no t the P2 i m m u n i t y region. Poss ib ly i t is a p p r o p r i a t e to record here t h a t P2 p la tes a t a 15% (approx.) efficiency on 186 ]ysogens, and on the lysogens of the five hybr ids , compared wi th i ts eop on the non- lysogen.

186 Genes. The presence of 186 genes represen ted b y the a m b e r mu ta t i ons 17, 26 and 7 to the lef t of art was inves t iga ted b y m a r k e r rescue exper iments . I n these exper iments the eop of a v i ru len t de r iva t ive of the 186 a m b e r m u t a n t s on Su - s t ra ins lysogenic for the h y b r i d phage was de t e rmined re la t ive to the p la t ing on Su + bac ter ia . As can be seen in Table 3 t he eop on Su- (Hybr id) was 102-105 fold h igher t h a n backg round (eop on Su-) for am26 and am17, b u t a t backg round for amT. W e concluded t h a t each h y b r i d possessed the am + alleles of m u t a t i o n s am17 and am26 b u t no t t h a t of amT. The high p la t ing efficiency for 186 am17 on Su-(186), 100-fold h igher t h a n the eop of e i ther the am26 or am7 mutan t s , sugges ted to us the poss ib i l i ty t h a t th is gene is no t under repressor control .

The 186 att-int region is to the r igh t of am17 (Fig. 1), and some of the hybr ids were t e s t ed for the i r s i te of in teg ra t ion on the bac te r ia l chromosome. Al though the 186 p rophage is UV inducib le i t is no t zygot ica l ly inducible , and i ts s i te of in teg ra t ion can be de t e rmined b y conjuga t iona l mapp ing (Woods and Egan, 1972). A lysogenic der iva t ive , Hf r K L 1 6 (I ty2) , was i so la ted and con juga ted wi th a F-pheA his str s t ra in . Of the phe+ r ecombinan t s i sola ted 78 % were lysogenic for Hy2 , while 50 % of the his+ r ecombinan t s were lysogenic. This l inkage to the phe si te is ind ica t ive of in tegra t ion a t t he 186 si te (Woods and Egan, 1972), and confirms the presence of t he 186 att-int region on the H y 2 genome. Similar r esu l t s were ob ta ined wi th H y 3 and Hy4.

128 C. Bradley etal.

Table 4. Marker rescue of P2 genes

Bacterial indicator

Efficiency of plating of P2 amber mutants

amD6 am U25 am J214

Su + (186~Its) 1.0 1.0 1.0 Su- (186tits) 1.2 X 10 -v 1.1 × 10 -~ 4.0 × 10 -7 Su- (IIyl) 1.3 × 10-4 3.7 × 10-4 1.2 × 10 -a Su- (Hy2) 2.1 × 10-4 6.4 × 10-4 2.0 × 10 -8 Su- (Hy3) 1.3 × 10-4 4.1 × 10-4 1.5 × 10 -a Su- (Hy4) 1.4 × 10 -4 3.6 × 10 -4 1.5 × 10 -8 Su- (Hy5) 8.4 × 10 -v 2.0 × 10-4 9.4 × 10-4

The P2 amber mutants used were the virl derivatives of P2 amD6, P2 amU25 and 1)2 am J214. Firl cannot establish lysogeny, but is immunity sensitive. All assays were incubated at 30 ° C, as the hybrids each carry the 186 cIts mutation. The strains used were E. coli K12 C600 Su + and 594 Su-.

P 2 Genes. The presence of P2 genes J, U, and D was investigated by marker rescue experiments. In those experiments the cop of P2 amber mutants on Su- strains lysogenic for the hybrid phage was determined relative to their cop on Su + bacteria. As can be seen in Table 4 for each hybrid the alleles am J214, amU25 and amD6 were rosened at frequencies of 103-104 ovor background, except for the rescuo of amD6 from Hy5 which was 7 fold ovor background. We concludod that the fivo hybrids possossed the wild type allele of each of these mutations.

As only a low isolates of the Hfr lysogens woro studied by conjugation, the absenco of evidence above for hybrid integration at the P2 H location (Calendar and Lindahl, 1969) could not be taken as indicative of tho absence of the P2 att.int region in the hybrids. Bertani (pers. comm.) has eheckod Hy2 and Hy5 for int m function and finds it absent. We investigated the possible presence of att~2 in these two hybrids by two sets of recombination experiments. In the first set of experiments 1)2 amber mutants were crossed with the hybrids, and the frequency of P2 immune wild type recombinants determined. I t was argued that if attp2 was present on Hy2, as anticipated from the physical studies (Ymmghusband, Egan and Inman, 1975), and not on Hy5, then the frequency of wild type rocombinants would reflect int mediated recombination for Hy2, some 100 fold higher than the 1)2 general recombination available for Hy5 (Lindahl, 1969). The results in Table 5 show the same low level of recombination for both Hy2 and Hy5 in crosses with P2amJ . Although the recombination frequency was symptomatic of general P2 recombination (~--0.01%) it is possible that int mediated recombination was operative but inefficient. Therefore the role played by the intp~ gone product was investigated by comparing the frequency of wild type recombinants for both Hy2 and Hy5 in crosses with P2 a m L and 1)2 amL int. The results (Table 5) show again a low recombination frequency, apparently independent of the intp2 gene product, although the recombination frequency was 10 fold higher for Hy2 than for Hy5. However the actual number of plaques counted was very low, for the technical reasons indicated in the legend of Table 5.

P2-186 I-Iybrids and 186 Insertion Mutants

Table 5. Grosses between P2 amber and hybrid derivatives

129

Cross Number of plaques Recombination frequency c

Recombinants Progeny (%) x 10 --~ x 10 -8

tty2 x P2 amJ 4 a 210 0.0028 tty5 × P2 amJ 4 a 226 0.0026 Hy2 × P2 amL 90 b 280 0.046 Hy2 X P3 amL int 60 b 380 0.022 Hy5 × P2 amL l0 b 315 0.004 Hy5 x P2 amL int 25 b 412 0.008

To 0.2 ml (4 X l0 s cfu/ml) of a logarithmic phase culture of E. coli K12 Su+ (strain $26) in LGC broth was added 0.1 ml of each parental phage (8 x 109 pfu/ml of LGC). After 20 rain incubation at 37°C (80% adsorption for P2, 65% for hybrids) the infected culture was diluted 10 a fold in LGM broth (104 fold in later experiments) and incubated with aeration at 37 ° C for a further 70 rain. Aliquots (0.1 ml) were assayed on E. coll C Su- (186) at 37 ° C to score wild type recombinant plaques (the plating efficiency of P2 vir at 37 ° C on this strain is 15% compared with the non-lysogenic parent as indicator), and diluted a further 104 fold and assayed on E. coli C Su + (C1757) at 30 ° C to score progeny. The assay for progeny was incubated at 30 ° C so that the proportion of Hy2 (turbid plaques because of the 186cIts allele) and P2 virl (clear plaques) in the burst could be monitored. In the burst of 100-200 phage/cell the ratio of hybrid to P2 was approximately 1:4, although in control cultures infected with a single parental phage the burst sizes for hybrid and P2 were approximately equivalent. These control cultures gave a negligible background on the selective indicators. a Average of 5 plates, 0.1 ml of 10 -a dilution assayed. b Average of 2 plates, 0.1 ml of 10 -4 dilution assayed. Although the number of recombinant plaques is low, lower dilutions to give higher plaque numbers/plate could not be used for two reasons. Firstly, to avoid the loss of progeny phage after lysis of P2-infected cells, the lysate must be diluted at least 1000 fold. EDTA could not be added to prevent this loss as Hy2 is sensitive. In fact, the presence of Mg ++ during lysis was necessary to stabilize Hy2. Secondly, plating of low dilutions on the selective indicator adversely affected the quality of the indicator lawn. c In the calculation of the recombination frequency the number of recombinant plaques has been multiplied by 7 to correct for the lowered plating efficiency of P2 virl on E. coli C Su- (186). The recombination frequency = 2 × 7 x wild type × 100/progeny.

The quest ion of Hy2 possessing attp2 was pursued in a second set of ex- per iments , in which the reciprocal recombinant , the 186 i m m u n e recombinant , was assayed. An amber m u t a n t of Hy2 was isolated and by plate tests shown unable to complement P2 a m J bu t able to complement 132 amber m u t a n t s in genes F, K, L and P. For the present s tudy polar i ty considerations were neglected, and the allele considered to reside in gene J. The m u t a n t was te rmed t t y2 amJp2lO05. This m u t a n t was crossed with P2amD and with P2amB. I t was argued in this case t h a t the recovery of 186 i m m u n e wild type recombinants would be m a n y fold greater in the cross with P 2 a m B t h a n with P2amD only if attp2 were present on Hy2 to permi t int mediated recombinat ion. The results in Table 6 indicate t ha t with 132 araB the frequency of wild type recombinants in the crosses Hy2 amJp2 X P 2 a m B and P2amD × P 2 a m B was the same, 200 fold higher t h a n in the cross Hy2 amJp2 X P2amD, support ing the conclusion t ha t attpa is present on Hy2.

130 C. Bradley et al.

Table 6. Recovery of the 186 immune recombinant in crosses involving tty2

Number of plaques

I~ecombinants Progeny × 10 -5 × 10 -s

Recombination frequency

Hy2 amJp2 × P2 arab 570 265 0.44% I-Iy2 amJp2 × P2 amD 3 260 0.002% P2 araB × P2 amD 545 275 0.40%

The recombination experiments were performed as described in the legend of Table 5. Wild type recombinant phage were assayed on E. coli C Su- at 37 ° C, and progeny phage assayed on E. coli C Su + (C1757) at 30 ° C to monitor the proportion of hybrid (turbid plaques) and P2 (clear plaques) phage in the burst. Recombination frequency = 2 × wild type × 100/progeny.

Isolation o] Heat Resistant Mutants

The relative resistance of deletion mutants to inactivating agents such as heat and chelating chemicals has provided a powerful means of selection for the isolation of such mutants (Parkinson and Huskey, 1971). A similar approach was investigated with 186 in an attempt to isolate mutants of 186 deleted for nonessential genes. However 186 mutated to heat resistance, which subverted the selection procedure for deletion isolation. Nevertheless, as will now be de. scribed, the procedure did yield some insertion mutants.

Heat Inactivation. Coliphage 186 cIts was found to be completely resistant to the conditions of heat or pyrophosphate that rapidly inactivated ~ (Parkinson and Huskey, 1971). However, heating the phage at 65°C led to its rapid in- activation to a 10 -4 survival during the first hour, followed by a slower rate of inactivation over the next 4 hours (Fig. 2 a).

The survivors after 1 hour incubation were used as inoculum for a plate stock of phage. The plate stock was harvested in TM buffer, concentrated by high speed centrifugation and resuspension in TM buffer, and heated at 65°C for 1 hour. This first cycle material was significantly more heat resistant than the original stock. The cycle of heating and stocking was repeated six times. As can be seen in Fig. 2b, the sixth cycle material was appreciably more heat resistant than the original stock. This sixth cycle material will be referred to as the mixed heat resistant (MHR) stock.

Isolation o/ hr (Heat Resistant) Mutants. The MHR stock was expected to contain a number of different genotypes, each giving a heat resistant phenotype. The stock was plated for single plaques, and the phage from three plaques of different morphology isolated.

hrl formed a plaque similar to that of 186 eIts. hr2 formed a plaque at 30°C with a large turbid centre and a clear irregular

edge, and at 37°C a smaller clearer plaque. hr3 formed a clear plaque at both 30°C and 37°C.

Isolation o] Density Mutants. The three isolates, hrl, hr2, hr3 and the MHR stock were centrifuged to equilibrium in CsC1 together with 186e + wild type phage, and the fractions collected were assayed for plaques at 37°C. For hrl,

10-1

2a

z !0-2

10 -3 I J -

~10 -4 : >

N 10 -5 03

10-6

P2-186 Hybrids and 186 Insertion Mutants 131

i S Z 3'0 68 TIME (hours) TINE (rnin)

2b

40-3 k wild e

Fig. 2a and b. Heat inactivation o/186 clts. Stocks of 186 cIts at 10 TM pfu/ml in TM buffer were assayed at different times after heating at 65°C. In Fig. 2b, survivors after 1 hour at 65°C were used as inoeulum in generating a plate stock. The plate was harvested in TM buffer, concentrated by centrifugation, resuspended in TM buffer, and a 10 TM pfu/ml stock heated at 65 ° C with assays after 30 min and 1 hour. The survivors after 1 hour were used to generate

the second cycle stock, and the process repeated through six cycles

hr2 and MHR, the peak of clear plaques, due to the cIts allele present in the hr phage, was coincident with the peak of turbid plaques due to the 186c+ marker phage, and so showed no density difference. However hr3 showed a reproducible density shift, with the unexpected fact that it was more dense than the wild type. As our immediate concern was for the isolation of deletion mutants a fraction from the the light side of the MHR peak was collected and plated for single plaques. Ten plaques (hr11-20) were isolated and purified through repeated single plaque isolation. Also, 250 plaques were tested for integration defectiveness (Gottesman and Yarmolinsky, 1968) and two int- mutants (hr21, hr22) isolated. Finally the light fraction was plated at high concentration on a 186 lysogen to select for virulent mutants, but none were detected.

Some Properties of the hr Mutants. The heat resistance of the different mutants was determined and the results appear in Table 7. Mutants hr12 and hr15 were the most heat resistant and therefore were expected to possess the largest dele- tions. Stocks of hr12, hr15, the known density mutant hr3, and 186cIts were centrifuged to equilibrium in the analytical ultracentrifuge with 2 as a known density marker. Mutants hr12 and hr15 showed no shift in density and banded at a density of 1.429 g c m -a, while hr3 banded at 1.433 g c m -a, confirming the earlier result in the preparative ultracentrifuge.

With this unexpected result, other characteristics of the mutants were ex- amined so that a representative selection could be examined by heteroduplex analysis in the electron microscope (¥ounghusband et al., 1975). These properties

132 C. Bradley et al.

Table 7. Some properties of the hr mutants

Heat resistance a Other properties

186tits 4 × 10 -~ - - 186hr3 2 × 10 -5 clear plaque eI mutant b 186hrll 6 × 10 -5 clear plaque cI mutant b 186hr12 4 × 1 0 - 2 - -

186hr13 1 × 10 -a - - 186hr14 6 × 10 .4 no lysis c 186hr15 1 × 10 -1 - - 186hr16 4 × 10 -3 rapid lysis c 186hr17 4 × 10 -3 - - 186hr18 3 × lO -2 - - 186hr19 2 × 10 -5 in t - art +d 186hr20 2 × 10 -2 - - 186hr21 4 × 10 -4 in t - art + d 186hr22 3 × 10 -4 in t - a~t +d

a Survival after 3 hours incubation at 65 ° C in TM buffer. b Clear plaque mutants of 186 fall into two complementation groups, cI and ¢/ / (Huddles ton, 1970). 186 cIts, because of its heat inducible phenotype, defines the d group, presumably coding for the represser, hr3 and h r l l complement mutants of the c l i group, but not members of the cI group. c Lysogens formed with the hr mutants were heat induced. The hrlg lysogen did not lyse, while hr16 lysed significantly earlier than the control cIts lysogen. The other lysogens lysed if anything later than the control. d The ability to integrate was determined by the method of Gottesman and Yarmolinsky (1968). In a coinieetion with 186 cI, hr19, hr21 and hr22 each formed lysogens, suggesting that each mutant possesses the art region. However, the result could have been due to the formation of the double lysogen (186 hr) (186 cl), with art and int provided by 186 cI, and represser by the hr mutant. Such double lysogens would have been heat inducible. The above lysogens yielded no phage upon induction, indicating that single lysogens of the hr mutant were present, and that each hr mutant possesses the art region. :Furthermore, this result indicated the need of an active int product for induction.

h a v e been t a b u l a t e d (Tab le 7), a n d t h e fo l lowing m u t a n t s were se lec ted for

h e t e r o d u p l e x s tud ie s :

hr3 a c I m u t a n t w i t h a k n o w n d e n s i t y shif t .

hr11 a c I m u t a n t .

hr12, hr15 m o s t h e a t r e s i s t a n t m u t a n t s .

hr16 a r a p i d lysis m u t a n t .

hr19 a n in~- m u t a n t .

T h e resu l t s a re r e p o r t e d in t h e c o m p a n i o n p a p e r ( Y o u n g h u s b a n d et al., 1975).

Suff ice i t to s ay he re t h a t hr3, hr11 a n d hr19 p r o v e d to be i n se r t i on m u t a n t s ,

whi le no change f r o m wi ld t y p e D N A was d e t e c t e d w i t h t h e r e m a i n i n g hr m u t a n t s .

D i s c u s s i o n

F r o m cells i n f ec t ed w i t h co l iphage P2 a n d 186 we h a v e i so l a t ed a t a fre-

q u e n c y of a p p r o x i m a t e l y 10 -7 , p h a g e t h a t possess t h e i m m u n i t y r eg ion of 186

P2-186 Hybrids and 186 Insertion Mutants 133

and the P2 host range ability to Flate on E. cell C. From both genetic and physical (Younghusband, et al., 1975) evidence the recombinant phage possess a P2-186 hybrid chromosome, and the five recombinant phage isolated have been termed H y l , Hy2, Hy3, Hy4, Hy5.

At present we consider the genetic composition of the hybrids to be

<--P2 186-+ Hy2 . . . . . . U + D+ art+ (int) 26 + 17+ art c + . . . . . Hy5 . . . . . . . . . . U + D+ 26 + 17 +a t t e + . . . . .

The rescue of the am26 + allele at a lower frequency from Hy5 than from Hy2 suggests tha t a smaller length of 186 DNA to the left of am26 exists in Hy5 than in the other hybrids, which is confirmed by heteroduplex analysis (Young- husband et al., 1975). The P2 contribution to Hy5 is also less than its contribution in the other hybrids since the frequency of recovery of the amD6 allele is 200-fold lower with Hy5. Furthermore, it appears that the 1)2 contribution of Hy5 terminates very close to the amD6 allele since the recovery of amD6+ is only 7-fold over background, while for an allele in the contiguous gene U, the recovery is 1400-fold over background.

Our experiments to determine the presence of attp2 in Hy2 produced con- flicting results, with the assay for the 186 immune recombinant producing ex- cellent evidence for the presence of attp2 , while our initial experiments assaying for the reciprocal P2 immune recombinant suggested the opposite. The physical evidence necessitates the presence of attp2 on Hy2 and we expect the P2-186 junction point to occur in the intp2 gene (Younghusband et al., 1975). The directionality of the int mediated recombination remains an enigma.

The other remarkable aspect of the genetic compositions of Hy2 and Hy5 is the indication of unequal crossing over and suggests that the hybrids were formed by illegitimate recombination (Franklin, 1971). This suggestion is further supported by the fact that the crossovers are in regions of relative non-homology (Younghusband et al., 1975).

Our attempts to isolate deletion mutants as density mutants from a popula- tion of heat resistant phage was confused by the availability to the phage of a presumably single point mutation to heat resistance. For example mutant hr15 shows a 104 fold higher survival in our heating conditions than wild type, but shows no detectable density change nor any chromosome deletion in heteroduplex analysis. Other hr isolates display intermediate values of heat resistance. Of the 6 hr mutants investigated by hcteroduplex analysis, only three had detectably altered chromosomes, but were insertion mutants rather than deletions. These insertion mutants must also carry a second mutation in the "hea t resistance" gene to accommodate the observation that they are more heat resistant than the wild type and not more sensitive. We have termed these phage hr3 ins1, hr11 ins2, and hr19 ins3. They each possess an altered phenotype (besides heat resistance) which is most probably due to the insertion, so that the site of the insertion locates the genes involved. Hr3 ins1 and hr11 ins2 are clear plaque mutants, defective in the represser cI gene, while hr19 ins3 is an int mutant. Heteroduplex analysis thus permits the probable location of the cI and int genes (Younghusband et al., 1975). Also the hybrids have been valuable in providing the probable

134 C. Bradley et al.

loca t ion of the 186 genes represen ted b y alleles am26 and a m l T , and poss ib ly t he P2 gene D, and new hybr ids cur ren t ly under inves t iga t ion pe rmi t fu r the r mapp ing in t he t a i l regions of P2 and 186.

The hybr id s are also p rov ing beneficial in o ther areas. Thus H y 5 D N A has p roved v e r y convenien t in resolving ea r ly / l a t e m R N A popula t ions of P2 (Sunshine and Sauer, 1975) and we are cur ren t ly using H y 2 and H y 5 to isola te P2 m R N A specific to the H y 2 - H y 5 in t e rva l (nominal ly t he att , ( int) in terval ) in co l labora t ion wi th B jo rn Lindqvis t . H y 5 is i tself of in te res t in la te gene control as i t poss ib ly represents a case of 186 control of l a te P2 gene expression.

Acknowledgements. This work was supported by a research grant from the Australian Research Grants Committee. The authors appreciate the generous interest of the P2ologists, especially Rich Calendar for his constant encouragement.

References Appleyard, R. K. : Segregation of new lysogenic types during growth of the doubly lysogenic

strain derived from Escherichia coli K-12. Genetics 39, 440-452 (1954) Baldwin, R. L., Barrand, P., Fritsch, A., Goldthwait, D. A., Jacob, F. : Cohesive sites on the

deoxyribonucleic acids from several temperate coliphages. J. melee. Biol. 17, 343-357 (1966)

Bellet, A. J. D., Busse, H. G., Baldwin, R. L. : Tandem genetic duplications in a derivative of phage lambda. In: The Bacteriophage Lambda. (A. D. Hershey, ed.), p. 501-513. Cold Spring Harbor Laboratory 1971

Bertani, G. : Deletions in P2. Circularity of the genetic map and its orientation relative to the DNA denaturation map. Melee. gen. Genet. 136, 107-137 (1975)

Bertani, L.E. , Bertani, G.: Preparation and characterization of temperate, noninducible bacteriophage P2 (host: Escherichia coli). J. gen. Virol. 6, 201-212 (1970)

Bertani, L. E., Bertani, G. : Genetics of P2 and related phages. Advanc. Genet. 16, 199-237 (1971)

Davidson, N., Szybalski, W.: Physical and chemical characteristics of lambda DNA. In: The Bacteriophage Lambda (A. D. Hershey, cd.), p. 45-82. Cold Spring Harbor Laboratory 1971

Franklin, N. C. : Illegitimate recombination. In: The Bacteriophage Lambda. (A. D. Hershey, ed.), p. 175-194. Cold Spring Harbor Laboratory 1971

Garen, A., Garen, S., Wilhelm, R. C. : Suppressor genes for nonsense mutations. I. The Su-1, Su-2 and Su-3 genes of Escherichia coli. J. melee. Biol. 14, 167-178 (1965)

Gottesman, M.E., Yarmolinsky, M.B.: Integration-negative mutants of bacteriophage 2. J. melee. Biol. 31, 487-505 (1968)

Huddleston, V.: Coliphage 186 control el the lysogenic response. B. Se. (Honours) thesis, University of Adelaide, 1970

Ifft, J. B., Veer, D. H., Vinograd, J. : The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge. J. phys. Chem. 65, 1138-1145 (1961)

Jacob, F., Wollman, E .L . : Sur los proeessus de conjugaison et de recombinaison chez Escherichla coll. I. L'induction par conjugaison ou induction zygotique. Ann. Inst. Pasteur 91, 486-510 (1956)

Kelly, B. : Localization of P2 prophage in two strains of Escherichia coli. Virology 19, 32-39 (1963)

Lindahl, G. : Genetic map of bacteriophage P2. Virology 89, 839-860 (1969) Lindahl, G. : On the control of transcription in bacteriophage P2. Virology 46, 620-633 (1971) Mandel, M., Berg, A.: Cohesive sites and helper phage function of P2, 4, and 186 DNAs.

Prec. nat. Acad. Sci. (Wash.) 60, 265-268 (1968) Murray, K., Murray, N. E.: Terminal nucleotide sequences of DNA from temperate coli-

phages. :Nature (Lend.) 243, 134-139 (1973)

P2-186 Hybrids and 186 Insertion Mutants 135

Padmanabhan, R., Wu, R. : Nueleotide sequence analysis of DNA IV. Complete nucleotide sequence of the left-hand cohesive end of coliphage 186 DNA. J. molec. Biol. 65, 447-467 (1972)

Parkinson, J. S., Huskey, R . J . : Deletion mutants of bacteriophage lambda I. Isolation and initial characterization. J. molec. Biol. 56, 369-384 (1971)

Skalka, A., Hanson, P. : Comparisons of the distribution of nucleotides and common sequences in deoxyribonucleic acid from selected bacteriophages. J. Virol. 9, 583-593 (1972)

Sunshine, M. : Dependence of eduction on P2 int product. Virology 47, 61-67 (1972) Sunshine, ~[., Thorn, M., Gibbs, W., Calendar, R., Kelly, B.: P2 phage amber mutants:

characterization by use of a polarity suppressor. Virology 46, 691-702 (1971) Sunshine, M. G., Sauer, B. : A bacterial mutation blocking P2 phage late gene expression.

Proc. nat. Acad. Sci. (Wash.) manuscript submitted. Weigle, J. : Assembly of phage lambda in vitro. Proc. nat. Acad. Sci. (Wash.) 55, 1462-1466

(1966) Wiman, M., Bertani, G., Kelly, B., Sasaki, I.: Genetic map of Eschesichia coli strain C.

Molec. gem Genet. 107, 1-31 (1970) Woods, W. H., Egan, J. B. : Integration site of noninducible coliphage 186. J. Bact. 111,

303-307 (1972) Woods, W. H., Egan, J. B. : Prophage induction of noninducible coliphage 186. J. Virol. 14,

1349-1356 (1974) ¥ounghusband, H. B., Inman, R. B. : Base sequence homologies between bacteriophage P2

and 186 DbTAs. Virology 62, 530-538 (1974) Younghusband, H. B., Egan, J. B., Inman, R. B. : Characterization of the DNA from bacterio-

phage 1)2-186 hybrids and physical mapping of the 186 chromosome. Molec. gen. Genet. 149, 101-110 (1975)

Communica ted by W. Arber

Dr. J. Barry Egaa Dr. Clifford Bradley Dr. Ong Peng Ling Dept. of Biochemistry The University of Adelaide Adelaide, South Australia, 5001