Host specificity of DNA produced by Escherichia coli

J. Mol. Biol. (1965) II, 238-246

Host Specificity of DNA Produced by Escherichia coli IV. Host Specificity of Infectious DNA from Bacteriophage Lambda DAISY

Drrssorxt

AND WERNER ARBER

Institute of Molecular Biology, University of Geneva, Switzerland (Received 10 September 1964) DNA extracted with phenol from bacteriophage A gives rise to phage production after uptake by helper-infected, competent recipient cells (Kaiser, 1962). Under our experimental conditions, the number of infective centres obtained by infection of Escherichia coli K12 is about 10- 3 per phage equivalent of DNA from A·K or from A·K(Pl). But on KI2(Pl) recipient cells only A·K(Pl) DNA infects with an efficiency of 10- 3 , while A·K DNA gives about 100 times less infective centres. The same factor of restriction for A'K is found in controls done by infection of the competent cells with intact phage particles instead of the phage DNA. Similarly, restrictions displayed by Kl2 against phage grown on E. coli B or E. coli C and those displayed by B against phage grown on K12 or C are found to hold true for DNA preparations. E. coli C accepts all tested A DNA with about the same efficiency. We conclude that the phenol extraction does not affect the host specificity of the phage DNA. One-cycle growth of A initiated by infection of K12 with A'K(PI) DNA confirms this result: the parental Pldirected host specificity is transferred into the phage progeny, and it is found only in such phage particles that also inherit one strand of the infecting DNA molecule. The stability of the association of DNA with its host specificity is further revealed by its resistance to various physical, chemical and enzymic treatments of the A DNA. It is significant with respect to the understanding of the mechanism of competence of bacteria for infection with A DNA that only non-restricted phage acts as a good helper.

1. Introduction In earlier studies on the host-controlled modification of bacteriophage lambda, we have defined "host specificity of DNA" as a principle imparted to the phage DNA by the bacterial host cells (Arber & Dussoix, 1962). A definite host specificity is, in general, required for successful infection. For example, when lambda phage grown on strain KI2 of Escherichia coli (= A·K) infect PI-lysogenic derivatives of KI2, the phage are restricted, that is, most of the infected cells degrade the phage DNA following its injection (Dussoix & Arber, 1962). Thus, a host specificity appears to be required in cells carrying PI prophage. Only in a small proportion of "exceptional" KI2(PI) cells is A·K allowed to grow; some of the progeny phage from these cells acquire the capacity to grow without restriction in the new host. The phage is said to have undergone host-controlled modification; that is, the phage DNA has acquired the host specificity of KI2(PI).

t Present address: Department of Biochemistry, Stanford University, Palo Alto, California, U.S.A. 238

HOST SPECIFICITY OF INFECTIOUS ,\ DNA

239

Host specificity appears to be carried by the phage DNA, since upon growth of A·K(P1) for one cycle in strain K12 the parental host specificity is transferred to those

progeny particles which receive one or both strands of the parental DNA. Phage containing only newly synthesized DNA exhibit exclusively the host specificity characteristic of the new host (Arber & Dussoix, 1962). These observations made it attractive to test whether purified phage DNA retains the specificity imparted to it by the host in which it was produced. The experimental conditions for infection of E. coli with A DNA have been worked out by Kaiser & Hogness (1960) and Kaiser (1962). They involve making the recipient cells competent by infection with A or A-related "helper" phage previous to their mixture with the A DNA. These techniques are here applied for the infection of various strains of E. coli with DNA extracted from stocks of phage ,\ grown on each of the various hosts. It is shown that at least those DNA molecules which are biologically active have maintained the host specificity which they had in the intact phage particle. These experiments provide more direct evidence that the determinants involved in host specificity are physically incorporated into the phage DNA. Preliminary results of these studies have been reported by Arber (1962), while more detailed information on some of the experiments was given by Dussoix (1964).

2. Materials and Methods Bacterial strains of E. coli K12, strain C 600 (Appleyard, 1954); B, strain Be 251 (Arber & Lataste-Dorolle, 1961); C (Bertani & Weigle, 1953), and lysogenic derivatives of these strains.

Bacteriophages Phage A, wild type (Kaiser, 1957) and its mutants c (clear plaque; Jacob & Wollman, 1954), and b2 (buoyant density mutant; Kellenberger, Zichichi & Weigle, 1960). Phage ,\i 4 3 4 , a hybrid between ,\ and phage 434 with the immunity region from 434 (Kaiser & Jacob, 1957). Phage PI adapted to K12 (see Arber, 1960).

Media Tryptone broth and synthetic M9a medium have been described previously (Arber & Dussoix, 1962). P medium: 0·02 M-KH 2P04, 0·015 M-(NH4)2S04' pH 7·1; supplemented after sterilization with 0·001 M-MgS0 4, 2x 10- 6 M-FeS04, 0·5 vol. % glycerol. For strain C 600 and its derivatives this medium was further supplemented with 0·1 mg/ml. L-leucine, 0·1 mg/ml. L-threonine and 0·01 mg/ml. thiamine. TCM medium: 0·01 M.triS, 0·01 M-CaC1 2, 0'01 M-MgS0 4, pH 7 to 8. TM medium: 0·01 M-tris, 0·001 M-MgS0 4, pH 7·4. Infection procedure Infectious DNA was extracted from purified phage stocks (lOll/mI.) in TM medium by shaking gently for 1 min with an equal vol. of water-saturated phenol at pH 7 to 8. After centrifugation the aqueous phase was exhaustively dialysed against TM medium. Appropriate dilutions of the DNA stocks were made in TCM medium; Ovl-ml. portions thereof were mixed with 0·2 ml. competent bacteria (see below) and incubated for 30 min at 37°C. Following addition of DNase (1 fLg/ml.), the mixtures were incubated for another 5 min and plated without further dilution. In order to obtain competent cells of strain K12, aerated cultures were grown in P medium to saturation (about 10 9 bacteria/ml.}, The bacteria were then resuspended in 0-01 M.MgS0 4 at a concentration of 2 X 109/m l. and infected with ,\i434 helper phage at a multiplicity of 2 to 10 phages/cell. After 8 min of adsorption at 37°C, an equal vol. of cold

240

D. DUSSOIX AND W. ARBER

TOM medium (pH 7'4) was added; the complexes were sedimented in the cold and resuspended in cold TOM medium (pH 8) at a concentration of 4 X 10 9 bacteriajml. Deviations from this procedure, in particular with strains Band C, are indicated in the experimental section.

3. Results I nfedion with DNA from non-restricted and restrided phage When DNA preparations from phages A·K, A·K(PI), A·B and A'C were assayed for infectivity on competent Kl2and KI2(PI) host bacteria, the results shown in Table 1

TABLE

I

Infection of strains K12 and K12(Pl) with DNA extracted from phage A Infective centres per 10 7 phage-equivalents, assayed on K12(Pl)('\i 434) (a) Infection with phage DNA

,\·K '\·K(Pl) ,\·B

x-o

7400 12700 31 23

76 11000 6 3

(b) Infection with intact phage particles

,\·K '\·K(Pl) ,\·B '\·C

6·2 X 106 5·5 X 106 2·4 X 10 4 2'5 X 10 4

4·4 X 10 4 4·7 X 106 1·9 X 10 4 1·3 X 10 4

Helper phage: ,\i434·K for K12(,\i434), multiplicity of infection, 2·6; ,\i 434·K(Pl) for K12(Pl) ('\i 4 3 4 ) , multiplicity of infection, 2·0. The infection with phage DNA was carried out as described

in the Materials and Methods section. The infection with appropriate dilutions of intact phage was done for 15 min at 37°C in 0·01 M-MgS0 4 subsequent to the 8-min adsorption period of the helper phage.

were obtained. The efficiency of successful infection (= plaques per phage-equivalent of DNA) was about 10- 3 for DNA from A·K infecting Kl2 cells and for DNA from A·K(PI) infecting Kl2 or KI2(PI) cells. In all other instances plaques were also obtained, but only with efficiencies of!0 - 5 to 10- 6. When the experiment was repeated under identical conditions using intact phage particles in place of infectious DNA, the relationships between the efficiencies of plating on different hosts were as above, although the absolute efficiency of plating values were greater by a factor of approximately 103 • This result indicates that the host bacteria exert a restriction on infectious DNA which is both qualitatively and quantitatively similar to that observed upon infection with intact phage of various host specificity type. Similar experiments were carried out using strains Band C as recipient bacteria, although experimental conditions have not yet been found for obtaining cells of these strains with a competence as high as that reported for Kl2 above. The results (Table 2)

HOST SPECIFICITY OF INFECTIOUS ,\ DNA TABLE

241

2

Infection of strains K12, Band 0 with DNA extracted from phaqe ): Host strain Helper phage

KI2('\i4 3 4 )

B(Ai434)

,\i 4 3 4 ·K

,\i 4 3 4 · B

C('\i 4 3 4 ) ,\i 4 3 4 ·K

Infective centres per 4 X 109 phage-equivalents of DNA from'\·K

A·B A'C

6900 140 124

29 2800 11

2800 7510 6050

The host bacteria were grown in Tryptone broth to saturation, starved in 0·01 M.MgS0 4 • infected with 10 helper phages/cell and resuspended in TCM pH 7·1 at a concentration of 3 X lOa bacteria/ml. The procedure of infection was as described in Materials and Methods.

show that strain 0 accepts DNA from '\·K, ,\·B and ,\·0 with equal efficiencies of infection. Strain B accepts ,\·B DNA, but restricts ,\·K DNA and ,\·0 DNA. The pattern of restriction of infecting ,\ DNA is thus the same as that previously observed with intact phage particles (Arber & Dussoix, 1962). It should be noted that the restriction is less pronounced in competent cells than on Tryptone-grown "standard" indicator cells (Arber & Dussoix, 1962). This difference is probably due to the different physiological condition of the cultures and, in particular, to the pre-infection with helper phage. Role of the helper phage in infection with ,\ DNA When infections were carried out using ,\i 4 34 helper phage grown on various host strains, it was found that restricted helpers do not make the cells competent for infection with ADNA of either non-restricted or restricted host specificity type (Table 3). For example, on K12(Pl) hosts, only ,\i43 4 ·K (P l ) is a good helper, whereas on K12 both ,\i43 4·K and Ai 43 4·K(Pl) are good helpers. A·B and ,\·0 do not act as good helpers on K12. Controls carried out with intact phage showed that pre-infection of the host with restricted helper does not affect the reproduction of the infecting phage. The effectiveness of the helper does not appear to be much influenced by the presence or absence of a '\i 4 34 prophage which renders the recipient cell immune (Table 3). One-cycle growth of'\ initiated by infection of competent cells with ,\ DNA In an experiment similar to that described in Table I, competent KI2('\i 43 4 ) bacteria were exposed to either '\·K(Pl) DNA or ,\·K DNA for 30 minutes at 37°0. The mixtures were then treated with DNase, diluted 50-fold in Tryptone broth and aerated for 60 minutes at 37°0. After the lysates had been chloroformed, the titres of progeny phage particles were assayed on K12('\i 43 4 ) and K12(Pl)('\i 434 ) indicator bacteria. Parallel control experiments were carried out with intact phage particles in place of phage DNA. The results (Table 4) were similar regardless of whether the one-cycle growth was initiated by intact phage particles or by free ,\ DNA molecules. In particular, it was found that the PI-directed host specificity of the parental '\·K(Pl) DNA is transferred into the phage progeny, as evidenced by the ratio of plaque titres measured on PI-lysogenic and PI-sensitive cells.

242

D. DUSSOIX AND W. ARBER TABLE

3

Role of the helper phage in infection with ..\ DNA

Infectious DNA or intact phage

Infective centres per 10 7 phage-equivalents, assayed on Helper phage] K12~

(a) Example of an infection with non-restricted DNA >.i434·K >'i434·K(Pl) >.i 434·B .\i434·C >.i434·K >'i434·K(Pl) >.i 434·B >.i 434·C none

12700 19900 118 215 5·5xl06 6·5x 106 6·0 X 106 6·0x 10 6 7·0X 106

7100 8900 10 6 3·2X 10 6 4·2X 10 6 4·5x 106 6·0X 106 6·0x 106

138 11000 96 201 5·5X 108 4·7 X 10 8 4·3 X 10 6 7·0Xl08 7·0X 108

(b) Example of an infection with restricted DNA M434·K >'i434·K(Pl) >.i 434·B >.i434·C >.i434·K >'i4 34 ·K (P l ) >.i434·B >.i434·C none

31 30 27 67 2·4x 10 4 3'Ox 10 4 1·lxl05 1·5 X 104 3·8X 10 3

133 119 92 60 8·8x 105

7·3 X 105 6·8xl05 1'9x105 5'8Xl03

4

6 1 9 1·4Xl08 1·9 X 10· 1·6 X 108 9·3 X lOll 1'4Xl0a

t Multiplicity of infection between 1·8 and 3·6. ~

Infection of helper-infected K12, but KI2(M434) indicator added at the time of the plating.

TABLE

4

One-cycle growth of..\ initiated by infection of strain K12 (..\i434 ) with DNA from A·K(Pl) orA·K Lysates Competent cells infected with

>.·K(Pl) DNA >"K(Pl) phage >.·KDNA >.·Kphage

Number of infecting phage or DNA phageequivalentsjml.

8xl07 1·6x106 4 X 107 1·6xl06

Infective oenteesjml. assayed before lysis on KI2(>.i 43 4 )

5·0X 10 4 7·5 X 105 2·9 X 10' 8·3 X 105

Number of plaquesjml., assayed on Ratio (a)/(b) KI2(Pl) (a)

KI2(Ai434) (b)

1·6xl03 8·4 X 10· 10 ' 103

1·0xlD5 1·lxl07 9·2 X 10' 1·lxl07

(M434)

1·6 X 10- 2 7·6 X 10- 3 10-' 10-'

The average burst size appears to be quite low, perhaps as a consequence of still incomplete phage reproduction at the time of the addition of chloroform. The efficiency with which parental host specificity appears in progeny phage particles is thus relatively low.

HOST SPECIFICITY OF INFECTIOUS>. DNA

243

By the use of deuterated A·K(Pl) DNA (Table 5), it was shown that the parental host specificity is transferred into the progeny jointly with the parental DNA. In the experiment shown in Fig. 1, the one-step lysate was centrifuged to equilibrium

TABLE

5

One-cycle growth of'\ initiated by infection of etrain. K12(.:\i 43 4 ) with deuterated A·K(Pl) DNA (A·K(PI) DNA is extracted from phage grown in M9a medium prepared with heavy water} Injection (35 min at 37°0) Titre of baoteriajml. Multiplicity of infection of M434·K helper phage Infecting DNA, phage-equivalentsjml, One.cycle growth. injected complexe8 diluted 20-Jold in Tryptone broth Infective centresjml., assayed before lysis on K12(Pl)('\i 03 4 ) K12(M434)

Lysate, assayed on (a) K12(Pl)(Ai43 4 ) (b) K12(W 3 0 ) Ratio of titres (a)/(b) Control of efficiency of plating with ,\·K phage stock: ratio (a)/(b)

5·6 X 10 9

4 4·6 X 10 1 0

8·7 X 10 4 2·3 X 10& 2·7 X 10 4 1·9 X 10 6 1·4 XlO- 2 2 X 10- 0

in a CsC! density-gradient, and the collected fractions were assayed for total phage on KI2(Ai43 4 ) and for phage with PI-specificity type on KI2(PI)(Ai 43 4 ) . On the PIlysogenic indicator only phage particles with semi-conserved parental DNA molecules showed activity. The phages with entirely light, newly synthesized DNA grew only on the indicator non-lysogenic for Pl. Similar experiments were carried out with competent cells of strain C. Again "·K and A·K(Pl) host specificity was transferred into the phage progeny upon one cycle of phage growth after infection of the host with "·K DNA and A·K(Pl) DNA, respectively. Stability of the aesociaiioti of DNA with its host .specificity The fact that free infective DNA exhibits the same host specificity as intact phage provides an opportunity to examine effects of various treatments on the principles conferring host specificity and on their association with the DNA molecule. ADNA was submitted to the following treatments. (1) Variation in pH between 3 and 11, either during or after DNA extraction; (2) variation of ionic strength up to 3 M-NaCl; (3) phenol extraction in presence of sodium lauryl sulphate (0'4 or 2%) or 0·15 ror-potassium trichloroacetate, pH 7·5; (4) incubation with trypsin, chymotrypsin or crude protease; (5) heating for 10 min at 80°C in presence of 50 iLg/ml. RNase, in 0·15 M-NaCl0·015 M-Bodium citrate.

244

D. DUSSOIX AND W. ARBER

106 ,---

-

-

-

-

-

-

-

-

40

-

-

-

-

-

-

-..

70

Fraction no. FIG. 1. Density distribution of one-step lysate obtained after infection of competent KI2(>.i 4 3 4 ) with deuterated >.cb 2+·K(Pl) DNA (see Table 5). One ml, of the lysate was centrifuged in a total volume of 3 ml. of CsCI to density equilibrium, then 95 fractions were collected into 0·5 ml, Tryptone broth each through a hole punched into the bottom of the tube, and the fractions assayed on KI2(.\i 4 3 4 ) and KI2(Pl)(Ai434) indicators. Titrations obtained on KI2(PI)(>.i 434) were plotted uncorrected for the efficiency of plating of restricted .\·K which was 2 X 10- 4 on the indicator culture involved. The position of the peak obtained on KI2(PI )(>.i4 3 4 ) corresponds to that of phage particles with semiconserved DNA molecules (Arber & Dussoix, 1962).

Mter each treatment the DNA was tested for infectivity on non-restricting and restricting hosts, and sometimes a one-cycle transfer experiment was carried out on a non-restricting host. In general, the infectivity was unchanged by the above treatments. Under some conditions, for example at low or high pH, the absolute infectivity of the DNA preparation was decreased, but in all our experiments we found that those DNA molecules which showed infectivity had also maintained their original specificity.

HOST SPECIFICITY OF INFECTIOUS ,\ DNA

245

Invariance of the buoyant density of DNA from A grown on different hosts The buoyant densities of DNA from wild type A·K, A·K(Pl), A·B and A'C were compared with that of Micrococcuslysodeikticus DNA by centrifugation to equilibrium in a CsCI density-gradient in the Spinco analytical ultracentrifuge. All A DNA preparations showed the same buoyant density to within 0·001 g/cm3 •

4. Discussion Competence for infection with A DNA is obtained by pre-infection of the recipient cells with A or A-related helper phage. The role taken by the helper phage is still not clear. Restricted phage is known to adsorb and to inject its DNA (Dussoix & Arber, 1962), but it is ineffective as helper. On the other hand, the helper functions are exerted with similar efficiency by non-restricted phage infecting sensitive or immune recipient cells. One might conclude that an essential step is carried out by the helper after the injection of its DNA and independently of whether the replication of its DNA is inhibited by the immunity. The results presented in this paper confirm the close association which we had found to exist between phage DNA and the principles that provide it with host specificity (Arber & Dussoix, 1962). Although our experiments indicate the presence of the host specificity only of those DNA molecules which are biologically active, it seems doubtful that this is a unique fraction of the population. If it were, one would have to assume that a close correlation existed, for any given molecule, between the presence of host specificity on one hand and a high probability of penetrating into a competent cell and initiating phage growth on the other hand. One might then expect that these steps were not completed by the DNA molecules without biological activity because of the supposedly absent or incomplete host specificity. As a consequence of such a restriction one might expect the DNA to be degraded upon contact with the recipient cells. However, experiments carried out with 32P-Iabelled DNA preparations did not give any evidence for such a breakdown, nor were we able to measure uptake of label by the non-restricting competent recipient culture. The one-cycle growth experiments confirm the results of Kaiser & Rogness (1960) and Kaiser (1962) that the growth of Ais initiated, as a rule, by infection with unbroken DNA molecules. They show also that, upon infection with ADNA, the parental DNA strands have the same chance to be transferred intact into a progeny phage particle as when Agrowth is initiated by phage infection. The finding that DNA without a required host specificity shows no, or only very low, biological activity may be relevant to studies of DNA synthesized in vitro using purified enzymes. Assays for biological activity of such DNA, which would not be expected to carry host specificity determinants, may not be meaningful unless carried out in recipient strains which show no restriction to any infecting DNA. The following conclusions regarding the still unknown nature of the host specificity determinants can be gathered from experimental observations of this and previous papers. (1) The host specificity of DNA is not destroyed by phenol extraction and various physical, chemical or enzymic treatments, in particular with proteolytic enzymes and with RNase. (2) Host specificity of an infecting phage DNA molecule is transferred into the onestep phage progeny jointly with the parental DNA material; thereby it does not seem to interfere with DNA replication or to induce alterations in the genetic message. 17

246

D. DUSSOIX AND W. ARBER

(3) Host specificity is not located at a single spot on the DNA molecule, as shown by the loss of parental host specificity in molecules undergoing break and join recombination (see Arber & Dussoix, 1962, who used genetic and density labelling). (4) Host specificity is imparted to non-replicating as well as replicating DNA (Arber & Dussoix, 1962). (5) DNA's with different host specificity do not show measurable differences in buoyant density. The work was supported by a grant from the Swiss National Foundation for Scientific Research. REFERENCES Appleyard, R. K. (1954). Genetics, 39, 440. Arber, W. (1960). Virology, 11, 273. Arber, W. (1962). Path. microbiol; 25, 668. Arber, W. & Dussoix, D. (1962). J. Mol. Biol. 5, 18. Arber, W. & Lataste-Dorolle, C. (1961). Path. microbiol, 24, 1012. Bertani, G. & Weigle, J. J. (1953). J. Bact. 65, lI3. Dussoix, D. (1964). These de doctorat no. 1376, Faculte des Sciences, Univeraite de Geneve, Dussoix, D. & Arber, W. (1962). J. Mol. Biol. 5, 37. Jacob, F. & Wollman, E. L. (1954). Ann. Inst. Pasteur, 87, 653. Kaiser, A. D. (1957). Virology, 3, 42. Kaiser, A. D. (1962). J. Mol. Biol. 4, 275. Kaiser, A. D. & Rogness, D. S. (1960). J. Mol. Biol. 2, 392. Kaiser, A. D. & Jacob, F. (1957). Virology, 4, 509. Kellenberger, G., Zichichi, M. L. & Weigle, J. (1960). Nature, 187, 161.