VIROLOGY 21, 30-35 (1963)

On the Host-Controlled Modification of Bacteriophage A’

WERNER ARBER, STANLEY HATTMAN,” AND DAISY DUSSOIX

Biophysics Laboratory, University of Geneva, Switzerland

Accepted May 10, 1963

Phage X.C, grown on Escherichia coli strain C, is restricted on E. coli strains K12, K12(Pl) and B251, and its DNA is broken down after injection into these bacterial strains. Phage X.K or X.K(Pl)-that is, grown on K12 or Kl2(Pl)-is accepted by C, but undergoes host controlled modification upon reproduction in C (Weigle and Bertani, 1953). In a one-cycle growth of x.K(Pl) on E. coli C, parental DNA and host specificity undergo linked transfer into the progeny. Thus, host specificity is a prop- erty of the DNA molecule, as was previously shown for phage X in another host system (Arber and Dussoix, 1962). Host specificity is serially transferable, and the probability of transfer to the progeny is constant for any given DNA strand under given experimental conditions.

INTRODUCTION

The DNA of bacteriophage h.K (Pl) , grown on Pl-lysogenic Escherichia coli K12 (Pl) , is endowed with a principle which is independent of the genetic content of the A phage genome but which is determined by the genetic information of the host (includ- ing the information carried by the Pl pro- phage) . This principle has been called “host specificity of DNA” (Arber and Dussoix, 1962). It is physically linked to the DNA molecule as demonstrated by the joint transfer of parental host specificity and DNA material into the phage progeny pro- duced after infection of K12 with h.K(Pl). In such a one-cycle growth the parental host specificity is not multiplied; the newly synthesized DNA molecules are endowed with another host specificity, that deter- mined by nonlysogenic K12 cells. Phage h is said to have undergone “host-controlled

‘This investigation was supported by a grant from the Swiss National Foundation for Scientific Research.

‘Trainee under Training Grant 2G-602 of the U.S.P.H.S. to the Department of Biology, Massa- chusetts Institute of Technology. Present address : Department of Biology, Massachusetts Institute of Technology, Cambridge 39, Massachusetts.

modification” (Luria, 1953). Modified phage h.K, grown on K12 bacteria, is no longer accepted by K12(Pl), and its DNA is degraded shortly after its injection in K12(Pl) cells (Dussoix and Arber, 1962). Host specificity is the only known difference between restricted h.K DNA and nonre- stricted A. K(P1) DNA, and it is probably also the principle recognized as acceptable or not by the bacterial host. The chemical nature of host specificity is still unknown, as is the mechanism by which differences in host specificity of infecting DNA are recog- nized.

It would be desirable to know whether other host-controlled modifications of phage besides the one described above can also be explained by differences in host specificity of DNA. Bertani and Weigle (1953) and Weigle and Bertani (1953) found host-con- trolled modification to occur with phage h grown successively in E. coli K12 and E. coli C. Phage A. K is accepted by both strains K12 and C, but phage h. C is re- stricted on K12 while nonrestricted on C. The present paper shows (1) that restricted h.C DNA is degraded in both E. coli K12 and E. coli B cells and (2) that nonre- stricted phage h. K12 (Pl) , upon one-cycle

30

MODIFICATION OF PHAGE h 31

TABLE 1 MATERIALS AND METHODS

EFFICIENCY OF PLATING OF PHAGE X VARIANTS ON DIFFERENT HOST STRAINS”,~

Bacterial and bacteriophage strains and

I

the media and techniques employed were

Phage variant

Efficiency of plating on host strain the same as those previously described (Arber and Dussoix, 1962; Dussoix and

K12 K12(Pl) B2.51 C Arber, 1962). Table 1 gives the efficiencies of plating

1 2 x 10-E 10-4 1 obtained for the different h variants if plated 1 1 10-d 1 under standard conditions on different host

4 x 10-d 7 x 10-7 1 1 strains. 4 x 10-b 4 x lo-’ 2 x lo-’ 1

RESULTS

X.K X.K(Pl) X.B x.c

a Indicator bacteria grown in aerated tryptone broth to about, 4 X 108 cells per milliliter and starved in 0.01 M MgSO, ; preadsorption of phage X for 15 minutes at 37°C.

b From Arber and Dussoix (1962).

gr0wt.h in C, transfers its K12(Pl) -deter- mined host specificity jointly wit.h the pa- rental DNA. Phages carrying transferred DNA in a semiconserved molecule are found, upon a second cycle of growth on C bacteria, to give rise again to transfer of both the original parental host specificity and DNA material. Efficiencies of transfer are compared for infecting phage DNA la- beled on both or only one strand with the host specificity principle.

Breakdown of DlVA from Infecting A.C in E. coli Strains Kl2, K12(Pl), and B251

Phage h. C (genotype &) labeled with P32 at low specific activity (0.5 atoms of P32 per phage particle) was thoroughly pu- rified and then allowed to adsorb to various recipient strains: restricting K12 (= strain C600), K12(Pl) (= st,rain C6OO(Pl)), or B251 and nonrestricting C. At various times after the mixing of phage and bacteria ali- quots were analyzed for their content in cold-acid-soluble P32. It can be seen from the results in Table 2 that x.C DNA is de- graded in all restricting strains and that this breakdown occurs rather fast, as had been found previously for the DNA of h.K ad-

TABLE 2

BREAKDOWN OF P32-L~~~~~~ X.c DNA AFTER INFECTION OF RESTRICTING HOSTS~

Host Strain Restricting K12 Restricting K12(Pl) Restricting B251 Nonrestricting C

CPM % CPM % CPM % CPM %

Adsorption mix- 25,100 27,300 24,000 27,650 tures, total P3*

Acid-soluble P32 after incubation for:

1 minute 1,508 6 1,560 5.7 1,460 6.1 1,670 6.0 8 minutes 12,300 49 7,040 26 10,650 44 2,380 8.6

11 minutes 16,800 67 13,300 49 19,900 83 2,330 8.4 18 minutes 20,600 83 17,000 62 19,300 80 2,380 8.6 25 minutes 19,600 78 18,700 69 18,800 78 2,330 8.4

a Bacteria were grown in tryptone broth to a concentration of 109 cells per milliliter, starved at the same concentration in 0.01 M MgSO, for 1 hour at 37”C, and infected with P32-labeled x.C at a multi- plicity of about one phage per cell at zero time. Ten minutes later the adsorption mixtures were diluted twofold in tryptone and further incubated at 37°C with aeration. Aliquots taken at various times re- ceived carrier DNA and were treated with cold perchloric acid (final concentration 3%). The counts of P32 activity are all adjusted to the initial concentration; statistical errors of counts are of the order of f3.5Yo.

32 BRBER, HA4TTMAS, AND DUSSOIX

sorbed to the restricting K12(Pl) or B251 hosts (Dussoix and Arber, 1962).

Joint Transfer of Parental Host Specificity and DNA upon One-Cycle Growth of x.K(Pl) in E. coli C

A deuterated X. K(P1) phage stock was prepared by growth of A c&f on E. coli K12 (Pl) (strain W3350 (Pl) ) in synthetic M9a medium which had been prepared with 99.7% DZO. The low proportion of Pl phages were inactivated by treatment of the stock with anti-P1 serum. The phage was then adsorbed at a multiplicity of in- fection (m.o.i.) of 0.18 phages per cell to a culture of E. coli C. The bacteria had pre- viously been grown to a concentration of log cells per milliliter in H medium supple- mented with 0.6% casamino acids, and had then been starved in 0.01 M MgS04 by 1 hour of aeration at 37°C. After a 15-minute adsorption period the bacteria were chilled, washed twice by centrifugation, and resus- pended in fresh H medium again supple- mented with casamino acids. After 2 min- utes of incubation at 37”C, free phage and infective centers were assayed on three dif- ferent indicators: K12, K12(Pl), and C. The lysate was chloroformed at 50 minutes and assayed on the same indicator bacteria. It can be seen from the results in Table 3 that the host specificity of the parental X. K (Pl) phage is transferred to the progeny with good efficiency.

An aliquot of the lysate was centrifuged in a CsCl density gradient; the density fractions were collected and assayed again on the three different indicator strains. The

result of this analysis is shown in Fig. 1. All the phages carrying parental host specificity (the one controlled by the K12 genome and the one added by the PI prophage) are found in the fraction of phages with semi- conserved DNA; but no phages have en- tirely new DNA associated with parental host specificity. This result corresponds to what has previously been found for Pl-de- termined host specificity upon growth of A. K (PI) in K12 cells. The efficiency of plating of parental /\.K(Pl) was the same on K12 and K12(Pl) indicators, but it is obvious from Fig. 1 that more semicon- served phages grow on K12 than on K12(Pl). This fact suggest,s that the host specificity determined by the K12 genome has a slightly greater probability to be transferred than the one determined by the K12 chromosome and Pl prophage together.

Semiconserved phage of drop number 20 (Fig. 1) was dialyzed against 0.01 M MgS04 and then used for a second cycle of growth on C bacteria. This second-cycle lysate was centrifuged in a CsCl density gradient, and the fractions were assayed on K12, K12(Pl), and C indicators (Fig. 2). Phages with still intact host specificity from the first parental phage stock again all banded with phages carrying semiconserved, heavy parental DNA.

Eficiency of Transfer of Host Specificity upon One Cycle Growth of Phage with Conserved or Semiconserved DNA

A stock of deuterated x cbz+ . K(P1) was grown for one cycle on K12 bacteria (strain CSOO) under the same conditions as de-

TABLE 3

ONE-CYCLE GROWTH OF DEUTERATED X.K(Pl) ON Escherichia coli C IN Hz0 MEDIUMS

Parameter K12

Indicator strain

K12(Pl) C

Free phage after washing per ml Infective centers ml per (plaque

titer before lysis) Progeny phage per ml

Corrected for x.C Average burst size

1.2 x 105 9.6 X lo4 1.1 x 10’ 7.4 x 106 3.2 x 10’

6.3 X lo6 4.7 x 106 1.7 x 109 5.6 x 106

0.5 0.6 53

a Multiplicity of infection = 0.18.

MODIFICATION OF PHAGE x 33

FIG. 1. Density distribution of lysate obtained by one cycle of growth of deuterated x.K(Pl) on E. coli C (Table 3) and centrifuged in CsCl gradient. Collected fractions (drops) were assayed on the indicator strains C, K12, and K12(Pl). The values are plotted without correction for plating efficiency of x.C on the various indicators. Parental, nonadsorbed phages band with a peak at drop number 6, phages with semiconserved DNA at drop num- ber 19, and phages with entirely new DNA at drop number 24. The centrifugation tube showed a slight disturbance in density gradient at drop number 14. PFU = plaque-forming units.

FIG. 2. Density distribution in a CsCl gradient of a lysate obtained by a second cycle of growth of semiconserved phages (drop number 20 of Fig. 1) on E. coli C. Collected fractions were assayed on indicator strains C, K12, and K12(Pl). The values are plotted without correction for plating efficiencies of x.C on the various indicators. Phages with semiconserved DNA peak at drop number 27, phages with entirely new DNA at drop number 32. In the latter region most of the plaque formers on K12 are accounted for by XC growing with a probability of about 5 X lo-’ on K12.

scribed for the experiment of Fig. 1. The semiconserved x.K(P~) phage, was used to lysate was centrifuged in a CsCl density infect K12, and the efficiency of transfer of gradient, and the fractions with phages con- Pl-directed host specificity was measured. taining semiconserved DNA were collected This efficiency was compared with that and dialyzed against Tris buffer with 0.001 found in another aliquot of the same bac- M MgS04. This preparation, enriched in teria infected with parental h.K (Pl) phages

34 ARBER, HATTMAN, AND DUSSOIX

TABLE 4

TRANSFER OF HOST SPECIFICITY FROM CONSERVED OR SEMICONSERVED DNA OF X.K(Pl) UPON GROWTH ON K12 (STRAIN W311O)O

Parameter Dilution of initial Enriched stock of X.K(Pl) phage stock semiconserved phage

Phage titer on K12 Phage titer on K12(Pl) M.o.i. total M.o.i. semiconserved x.K(Pl)

Growth medium Free phage after washing

Assayed on K12(Pl) Infective centers per ml

Assayed on K12 Assayed on K12(Pl)b

Input X.K(Pl) (= number of infec- tive centers on K12 multiplied by the fraction of x.K(Pl) in in- fecting phage stock, i.e., 0.35)

Progeny phage per ml Assayed on K12 Assayed on K12(Pl)

Burst size X.K Transfer frequency of Pl-directed

host specificity progeny X.K(Pl)

= input LK(P1)

3.7 x 106 2.6 x 106 3.7 x 10” 9 x 105(=35Ye)

5 x 10-a 4.5 x 10-a 1.3 x 10-a

.--

M9a Tryptone M9a Tryptone

4 x 102 5 x 102 10’ 10’

6 X 10” 9.5 x 10’ 4 x 10’ 7.2 X 10’ 3.5 x 104 2.7 X lo4 1.5 x 104 1.2 x 104

1.4 x 10’ 2.5 X IO4

7.6 X lo6 1.1 x 10’ 5.5 x 106 8.5 x 106 2.5 x 104 3.8 x 10” 3.3 x 103 5.1 x 103

126 116 138 118

0.42 0.40 0.23 0.20

a Bacteria were grown in the indicated growth medium to a concentration of lo9 cells per milliliter, concentrated twice, starved in 0.01 M MgSOd , and infected at low m.o.i. with the indicated phage preparations. After 15 minutes’ adsorption at 37”C, 3 volumes of fresh growth medium were added and the mixtures were aerated for 5 minutes at 37°C and chilled. The bacteria were washed twice by centrif- ugation, resuspended in fresh growth medium at a concentration of 2 X lo7 bacteria per milliliter. Free phage and infective centers were assayed after 2 minutes of incubation, and the lysates were chloroformed 50 minutes later and assayed on K12 and K12(Pl) indicators.

6 The infective centers assayed on K12(Pl) consist mostly of irregular plaques; their number is not very significant because of the big number of uninfected K12 cells on the plates.

labeled in both DNA strands with the initial host specificity (Table 4). Under the experi- mental conditions used, phage with semi- conserved DNA had a probability of about 0.2 to give rise to a progeny particle with parental host specificity, but this same probability was about 0.4 for phages with conserved parental DNA. This result sug- gests that (1) both DNA strands of an in- fecting phage have the same probability to be integrated into progeny phage particles and (2) a DNA strand transferred in a single cycle of growth into a progeny phage shows still the same probability to be trans- ferred a second time upon a new growth cycle.

DISCUSSION

The present investigations extend our ear- lier findings that host-controlled modifica- tion of phage x is due to specific changes on the phage DNA molecule (Arber and Dus- soix, 1962). It is now certain that host-con- trolled modifications occurring with phage x in a set of different strains (derivatives of E. coli K12, K12 (Pl), C, and B) all imply changes in the host specificity of DNA. Host specificities determined by different genetic information of the host are not com- pletely overlapping, as suggested by the fol- lowing effects. The plating efficiency of phage h. C is higher if assayed on K12 than on K12(Pl). The latter strain has an addi-

MODIFICATION OF PHAGE x 35

tional restriction for h. C besides that di- rected by K12 cells, and this Pl-directed re- striction prevents also the acceptance of A.K by K12(Pl) cells. On the other hand, host specificity of DNA from h.K (Pl) is transferred to the progeny upon phage growth on C cells. But transfer of KlB-di- rected host specificity is slightly more fre- quent than joint transfer of both host spec- ificities, directed by the K12 genome and by the Pl prophage, respectively. This differ- ence can be explained in one of the following ways. According to how far the host speci- ficity extends on the phage genome, genetic recombinants between the infecting parental DNA molecule and one of its progeny mole- cules may either lose or maintain the pa- rental host specificity in a transfer experi- ment. Or, host specificity may sometimes disappear during DNA replication by some unknown mechanism. If the latter alterna- tive should prove correct, experiments using host specificity as biological label of DNA should give results quantitatively different from experiments in which DNA is labeled by a physical method.

The finding that host specificity of DNA is transferable serially together with trans- ferred DNA into progeny phage particles im- plies that both strands of the original DNA molecule have the same probability to re-

appear in a semiconserved DNA progeny molecule. Polarity does not seem to influ- ence the rate of transfer, as first and second generation transfer occurs with practically stable probability. Our experiments do not show whether both strands of a single DNA molecule are found in two distinct progeny phages produced in one singly infected cell. Single-burst experiments recently performed by Fox (1963) suggest that both strands of an infecting x phage sometimes do appear in the progeny from a one-cycle growth ex- periment.

In phages with semiconserved DNA, the newly synthesized DNA strand never ac- quires the host specificity of the infecting parental phage; otherwise one should find in the second progeny some phages with en- tirely new (light) DNA with the parental host specificity.

REFERENCES

ARBER, W., and DUSSOIX, D. (1962). J. Mol. Biol. 5, B-36.

BERTANI, G., and WEIGLE, J. J. (1953). J. Bac- teriol. 65, 113-121.

DUSSOIX, D., and ARBER, W. (1962). J. Mol. BioZ. 5,37-50.

Fox, E. (1963). Ph.D. Thesis, Harvard University. LURIA, S. E. (1953). Cold Spring Harbor Symp.

Quant. BioZ. 18,239-244. WEIGLE, J. J., and BERTANI, G. (1953). Ann. Inst.

Pasteur 84, 175.