Multiple steps during the interaction between coliphage lambda and its receptor protein in vitro

72, 182-194

VIROLOGY

Multiple

(1976)

Steps during

the Interaction between Coliphage Its Receptor Protein in Vitro

MICHBLE Dbpartement de Biologie Molhdaire,

ROA’

AND

DOROTHEA

Lambda

and

SCANDELLA*

Znstitut Pasteur, 75015 Paris, France; and Abteilung Biozentrum, 4056 Bowel, Switzerland

Mikrobiologie,

Accepted February 6,1976 Phage lambda and its purified receptor protein interact in vitro even when the phage is not inactivated: in the absence of detergent the receptor is relatively insoluble and it leads to the formation of phage aggregates. Under conditions where the phage is inactivated by the receptor, only a small fraction (about 30%) of its DNA becomes sensitive to nucleases. Ejection of the DNA apparently is almost complete upon sucrose gradient centrifugation since clear separation of ghosts and DNA can be obtained. It is, however, possible to recover from the gradients some inactive phage particles which have not yet ejected their nucleic acid. The different steps occurring in uitro, i.e., reversible interaction, phage inactivation, and DNA ejection, are correlated with the first steps of phage infection in vivo.

mixture. This requirement is not shown, however, either if the phage carries a host range mutation allowing it to grow on a given class of lambda resistant mutants (Appleyard et al., 1956) or if the receptor is extracted from some strains of E. coli other than K12 or from some strains of Shigella. Conditions have been described (Randall-Hazelbauer and Schwartz, 1973) where phage inactivation is accompanied by ejection of the phage nucleic acid. Results presented herein demonstrate that DNA ejection is in fact the end of a complex process including at least three different steps.

INTRODUCTION

Bacteriophage adsorption requires the presence of specific receptors on the bacterial surface (for a review, see Lindberg, 1973). Information about the early steps of phage infection should be obtained by studying the interactions occurring in vitro between the phage and its receptor. Particularly suitable for such a study is coliphage lambda, one of the best known bacteriophages (for instance, see Hershey, 1971). Its receptor, a protein of the outer membrane of Escherichia coli, was recently partially purified and characterized (Randall-Hazelbauer and Schwartz, 1973). Inactivation of phage lambda by its receptor in vitro presents some unusual features (Randall-Hazelbauer and Schwartz, 1973; Schwartz and Le Minor, 1975). Wild-type phage is inactivated by receptor extracted from E. coli K12 only if a saturating amount of chloroform, or 10 to 20% ethanol, is present in the incubation

MATERIALS

It should be recalled that ZamB, the structural gene for lambda receptor, is located in one of the two maltose operons constituting the malB region of E. coli genetic map (Hofnung et al., 1974; Hofnung, 1974). The following strains of E. coli K12 are from the collection of Drs. Hofnung and Schwartz, and were previously described (Hofnung et al., 1974): C600; CR63, a ZamB 182

Q 1976 by Academic Press, Inc. of reproduction in any form reserved.

METHODS

Bacterial and Phage Strains

’ Author to whom reprint requests should be addressed. 2 Present address: Department of Biochemistry, State University of New York, Stony Brook, New York 11794.

Copyright All rights

AND

PHAGE

LAMBDA-RECEPTOR

mutant used for plating host range mutants of lambda; Hfr G6, the standard lambda-sensitive strain used as a source of lambda receptor; pop 1732, a spontaneous lambda-resistant mutant of HfrG6 carrying deletion MBA101 which encompasses the whole m&B region, including ZamB. Strain YMC (Zac 1 carries a sup F mutation and is used to plate ACI,,,susS,. Pop72 and pop79 are spontaneous lambda resistant mutants (maZT) of C600 (ACl,,,h,,) and C600 (XCIRB~susS,), respectively. Strains 921 (peZ+) and 2127 (pel ) have been described by Scandella and Arber (1974). Pop 154 is a derivative of E. coli K12 carrying the maZB region of ShigeZZa sonnei 3070. This strain was constructed as follows. First a Mal- Thr- Leu- Met+ His+ recombinant was selected from a cross between the maZB his Hfr strain pop 1732 and the F- strain 921 which is met thr Zeu Zuc supE hsdS,. This recombinant was then transduced to Mal+ using a stock of phage Pl grown on ShigeZZa sonnei 3070 (Schwartz and Le Minor, 1975). Pop 154, one of the resulting transductants, is thr Zeu Zac supE hsdS,, and is sensitive to phage lambda. The receptor extracted from this strain, like that extracted from ShigeZZa sonnei 3070 inactivates wild-type lambda in the absence of chloroform or ethanol, i.e., is of the R* type. The phage strains $80 vir, hC1857, and ACI,,,sus S, are from the laboratory collection. Strain ACI,,,h,,, isolated during the course of this work, is a spontaneous host range mutant of ACIs5, obtained by plating on CR63. Since only the host range characteristic of the phage is relevant in this work, ACZ8,,sus S, will be called “wildtype” and ACI,,,h,, will be called “host range mutant.” Strain ACI hpl was described by Scandella and Arber (1974). Preparation

of Radioactive

Phage

“2P-ZabeZed phage. Phosphate is precipitated out of ML medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) by adding to it 0.6 ml of 17 M NH,OH, and 1 ml of 1 M MgSO, per 100 ml. After neutralization

INTERACTION

183

the supernatant is supplemented with 6.10w4 M phosphate and used to grow the lysogenic strains. Thermoinduction of the prophage and preparation of the lysates are done according to classical techniques (Miller, 1972; Scandella and Arber, 1974). Ten microcuries of carrier-free [32Plphosphate (CEA, France) are added for each milliliter of culture, just before thermoinduction. When convenient, the lysates are concentrated by ultrafiltration over an XMlOO A Amicon membrane. Purification of the phage is done using two successive centrifugations in CsCl, one in a block gradient, the other in a continuous gradient (see Miller, 1972). The purified stocks are dialyzed extensively against lo-’ M MgS04, 10e2 M Tris, pH 7.5 (TM buffer). “Prepurified” phage is a stock which is further purified on a sucrose gradient, prior to an experiment, in order to remove free DNA or protein originating from particles which burst spontaneously during storage. YS-labeled phage. It was prepared as above except that the label was carrierfree [35Slsulfate (10 &i/final ml from CEA, France) and that the growth medium had the following composition: to a minimal salt medium (13.6 g of KH2P04, 2 g of NHgCl, 0.2 g of MgC12,6H2O, 0.005 g of FeCl,, 0.01 g of (NHJ2S04, per liter) was added a mixture of all natural amino acids except methionine and cysteine, at a concentration of 0.1 g/liter each, as well as 5 mglliter of thiamine and 4 g/liter of glucose. 13HlZeu-ZabeZed phage. In some of the early experiments 13H11eu was used to label the phage proteins, rather than 3sS. In these cases [3Hlleu (1 Cilmmole, from CEA, France) was used at a final concentration of 10 Z.&i/ml. The medium was M63 (13.6 g of KH,POg, 2 g of (NH,), SO4, 0.2 g Of MgS04, 7H@, 0.005 g Of FeSO,, 7H#, per liter) supplemented with 0.5 g/liter of casamino acids (Difco), 0.1 g/liter of threonine, 5 mg/liter of thiamine, and 4 g/liter of glucose. Titration of the phage stocks was done as usual except that plating of ACZ,,~us S, with YMC was done on MacConkey agar (Difco) where it yielded larger plaques.

184

ROA

AND

SCANDELLA

The proportion of plaque-forming units (PFU) among total particles as determined by AZ60varies from 20 to 50% depending on the preparations. (A correspondance of 8.7 x 10” total particles/ml for one absorbance unit is used, according to Schwartz, 1975). Preparation of Receptor R-receptor, purified from HfrG6, was a gift of Dr. M. Schwartz (Randall-Hazelbauer and Schwartz, 1973; Schwartz and Le Minor, 1975). In the fraction used, about 70% of the protein is receptor, the rest being mainly composed of a single contaminating protein. (Purified receptor preparations were previously reported to yield three bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two of the bands were later shown to correspond to two different conformations of the receptor, while the third band corresponds to a contaminant protein). The fraction contained 2 x 1013 active receptor molecules per milliliter, as determined from the stoichiometric inactivation of a host range mutant of lambda. R* receptor was extracted from pop 154. Extraction and purification procedures were as described for R-receptor. A similar degree of purity was achieved, and the final preparation contained 1013 active molecules per milliliter, R and R* receptors were extensively dialyzed against 10V2 M Tris, pH 7.5, at the end of the purification procedure, and kept frozen at -15”. Incubation of Phage with Receptor All incubations were in 10e2 M MgS04, lo+ M Tris, pH 7.5, at 37”, and lasted 15 min. The amounts of phage and receptor used are indicated for each experiment but were always such that there was at least a twofold excess of receptor over phage particles. Except when wild-type phage was mixed with R-receptor, at least 99% of the phage was inactivated at the end of the incubation period. Sucrose Gradient Centrifugation The 5-20% sucrose gradients were all in 10m2 M MgS04, low2 M Tris, pH 7.5. In

addition, when indicated, they contained 1% sodium cholate (Schuchardt). The lOO,ul sample was deposited on the top of the gradient, itself poured over a l-ml layer of saturated CsCl. All centrifugations were performed at 4” in a SW50 L rotor, using a Beckman Model L3-50 centrifuge. The speed and duration of centrifugation are indicated for each experiment. In some of the gradients $80 vir phage, which does not react with lambda receptor, was used as a marker. It was titered on strain pop 1732. The gradients were collected from the bottom, with four drops per fraction. N&ease Treatment The nuclease treatment is a 15 min incubation at 37”, in the presence of 50 pglml of pancreatic DNAse (Worthington), and 5 pg/ml of venom phosphodiesterase (Worthington). Phenol extracted DNA from 32Plabeled phage was used as a control, at a concentration similar to that present in the experimental tubes. In all cases, 95 to 98% of the control DNA was rendered soluble in 10% trichloracetic acid (TCA). Counting of Radioactivity Protein and nucleic acid were precipitated in the sample by adding 10% TCA, at 4”. The samples were filtered on nitrocellulose filters (Schleicher and Schuhl), the filters were washed with 5% TCA, dried, transferred in 10 ml of Bray’s solution (Bray, 1960), and counted in an Intertechnique scintillation counter. Bray’s solution was used instead of toluene-popop solution because of the quenching due to cholate, which precipitates in the presence of TCA. In the double-label experiments the 32P spillover to the 35S channel (about 6%) and the 35S spillover to the 32P channel (less than 0.1%) were neglected. Recovery of the 32P label from the gradients was generally close to 100%. Recovery of the protein label t3H or YS), on the other hand, was somewhat variable (60-100%) apparently due to the preferential loss of empty particles. Electron Microscopy Samples were deposited on carboned formvar-coated grids which had been submitted to a glow discharge. They were

PHAGE

LAMBDA-RECEPTOR

185

INTERACTION

then fixed with 3% formaldehyde, negatively stained with 2% phosphotungstic acid, and immediately examined under a Siemens Elmiskop 101 electron microscope. RESULTS

I. Aggregation of Wild-Type coli K12 Receptor

Phage

by E.

Addition of receptor purified from E. coli K12 (thereafter called R-receptor) to wildtype phage lambda, has a dramatic effect on the sedimentation of the phage particles in a sucrose gradient (Fig. la and b). The pattern of radioactivity as well as of infectivity is heterogeneous but corresponds only to material heavier than untreated phage. Essentially identical numbers of PFU are recovered from the gradients shown in Fig. la and b. As seen in Fig. 2a and b, about 50% of the phage particles present in the heavy fractions are associated in rosettes. The material at the center of the rosettes is presumably composed of aggregates of receptor molecules, known to be strongly hydrophobic. (Randall-Hazelbauer .and Schwartz, 1973). About 90% of the particles in these fractions are apparently intact, i.e., still filled with DNA. When 1% sodium cholate is added to the reaction mixture and to the gradient, all the radioactivity sediments as free phage (Fig. lc), as would be expected if the receptor aggregates are dissociated by detergent. These results demonstrate that even though wild-type phage lambda is not inactivated by R-receptor, it still interacts with it. Receptor-induced aggregation of phage lambda was not found in other experiments (Randall-Hazelbauer and Schwartz, 1976; McKay and Bode, 1976). However, the degree of phage aggregation is likely to depend on several factors such as the absolute concentration of receptor, the degree of purity of the receptor preparation, and the relative concentration of phage and receptor. II. Analysis of Phage-Receptor Mixtures under Conditions of Phage Inactivation When chloroform or ethanol is added to mixtures of wild-type lambda and R-recep-

IO FRACTION

20 NUMBER

3(

FIG. 1. Aggregationof wild-type phage by R-receptor. Prepurified 35S-labeled wild-type lambda was used (3 x lOlo PFU, 1.5 x lo4 cpm). Incubation with receptor and sucrose gradient centrifugation were as indicated in Materials and Methods, except that the phage was titered on aliquots from each fraction before TCA precipitation. The gradients were centrifuged for 30 min at 18,000 rpm. (a) Phage alone. (b) Phage incubated with 2 x 10” molecules of R-receptor. (c) Phage incubated with 2 x 10” molecules of R-receptor. One percent sodium cholate is present in the incubation mixture and in the gradient. W (cpm/fraction), O-O-O; plaque-forming units (PFU/ml), O-0-0. The arrow indicates the location of @OV used as a marker. Sedimentation is from right to left in all gradients. When receptor alone is centrifuged in the presence of detergent, all the activity is found in the three top fractions. For unknown reasons, in the absence of detergent, no activity could be recovered from the gradient.

tor, 95-99% of the phage gets inactivated. The same result is observed, in the absence of chloroform or ethanol, either if the phage carries a host range mutation, or if

FIG. 2. Electron micrographs of wild-type phage lambda aggregated after interaction with receptor. Sucrose gradients similar to the one presented in Fig. lb were made. The heavy fractions (numbers 6 to 8) were collected, dialyzed one night against TM buffer, concentrated, and negatively stained. (a) and (b) Phage preincubated with R-receptor. (c) Phage preincubated with R* receptor. The bar represents 0.2 pm. 186

PHAGE

LAMBDA-RECEPTOR

the receptor (called R* receptor) is extracted from a strain like Shigella sonnei 3070. Most of the experiments were performed in these two last conditions. The results obtained in both cases were essentially identical and are reported together. As a source of R* receptor, we used a strain of E. coli K12 in which the structural gene for the receptor was transduced from Shigella sonnei 3070. When phage and receptor are mixed in the presence of 1% sodium cholate about 90% of the phage particles are seen under the electron microscope to be associated with some material, presumably receptor, at the tip of their tail (Fig. 3a and b). About 60 to 75% of the particles are empty. This type of mixture was analyzed on sucrose gradients containing 1% sodium cholate, using phage particles labeled in the DNA (32P) or the proteins ([3H]1eu or 35S) (Figs. 4 and 5). Most of the 32P label migrates independently of the protein label, suggesting separation of DNA and empty particles (ghosts). Verification that the protein label corresponds to apparently intact ghosts was obtained by electron microscopy (not shown). However, a fraction (10 to 35% depending on the experiments) of both labels, in relative proportions identical to those present in the untreated phage mixture, sediments at the same rate as intact phage particles. The infectivity of these apparently intact particles, standardized to their radioactivity, is less than 1% of that of untreated phage. Therefore these particles do not constitute a random fraction of the phage stock. Either they constitute a specific fraction unable to react with the receptor and thus inactive, or they are particles which reacted with receptor but did not eject their DNA. The experiment reported below suggests that the latter is true. When the incubation of phage with receptor, as well as the centrifugation, are performed in the absence of detergent, (Fig. 6) most of the DNA label can still be seen at the top of the gradient, but the rest of it, as well as most of the protein label, sediments faster than untreated particles. Therefore, as otherwise confirmed by electron microscopy (Fig. 2c), the empty parti-

187

INTERACTION

cles as well as the apparently intact particles referred to above, are bound to receptor aggregates. III.

N&ease Sensitivity beled Material after Interaction

of the 32P-LaPhage-Receptor

In the experiment illustrated in Fig. 7, a mixture of 32P- and 35S-labeled host range phage previously incubated with R-receptor was centrifuged in a sucrose gradient, and half of each fraction was treated with pancreatic DNAse and venom phosphodie&erase. As expected, most of the 32P material which remains at the top of the gradient is rendered TCA-soluble by the action of the nucleases, and is therefore free DNA. On the other hand, the 32P material sedimenting as complete phage (about 10% of the total in this experiment) is partially sensitive to the nucleases, about 65% of it being rendered TCA-soluble. Such a result is obtained neither with complete phage particles, the DNA of which is totally resistant to nucleases (Fig. 7b), nor with free DNA, which is totally solubilized by them. When the sample is treated with nucleases before layering on the gradient, no TCAinsoluble 32P material is found at the top, but the ratio of 32P to YJ material in the fast sedimenting component is the same as in complete phage particles (data not shown). When the fast sedimenting component is dialyzed and centrifuged a second time in the absence of receptor, most of the DNA is then found on the top of the gradient. This result is illustrated in Fig. 8 in the case of wild-type lambda and R* receptor. These different results suggest that the particles which did not eject their DNA prior to the first centrifugation and therefore sedimented as intact phage, are rather unstable. Some of them release their DNA during the collection of the fractions or their incubation with nucleases, and all of them do so upon a second centrifugation. IV. Conditions DNA

Affecting in Vitro

the Release

of

The experiments presented so far, which all involved a centrifugation step or EM,

188

ROA

AND

SCANDELLA

FIG. 3. Electron micrographs of wild-type phage lambda in the presence 4 x 10”’ PFU/ml incubated with 2 x 10” active molecules of R* receptor/ml receptor added. The bar represents 0.2 Frn.

of 1% sodium cholate. and stained. (c) Phage

(a) and (b) control: no

PHAGE

LAMBDA-RECEPTOR

suggest that 70 to 90% of the phage DNA is immediately released into the medium when receptor inactivates the phage; however, when a preincubated mixture of phage and receptor is treated with nucleases, only 15 to 35% of the DNA is rendered TCA-soluble (Table 1). Therefore centrifugation of the phage-receptor mixture somehow enhances the ejection of DNA. Other conditions were sought which would have a similar effect. As shown in Table 1, addition of a high concentration of salt (10-l it4 NaCl) either before or after phage inactivation increased to 65% the fraction of DNA sensitive to nuclease. Whatever the conditions used, this fraction did not increase significantly upon prolonged incubation, and never reached 90% as often observed in sucrose gradients. V. In Vitro Effects of Phage and Bacterial Mutations which Alter DNA Znjection in Vivo

Lambda is unable to inject its DNA into an E. coli host carrying a pel mutation but certain lambda mutants, called Ahp, regain the ability to inject into pel bacteria (Scandella and Arber, 1974). In order to determine if in vitro DNA ejection is affected by these mutants, we tested the ability of receptor extracts from pelt and pel cells to inactivate and cause ejection from A and hhp. Crude receptor extracts were prepared by cholate-EDTA treatment of whole cells and assayed with host range lambda in the absence of chloroform, as described by Randall-Hazelbauer and Schwartz (1973). Essentially identical receptor activities were found in extracts of peZ+ (strain 921) andpel (strain 2127) bacteria. Inactivation of wild-type lambda by pel extracts requires chloroform as is the case with pel’ extracts. Furthermore, the hhp mutants, selected for their ability to grow on pel strains, differ from the classical Ah type host range mutants selected for their ability to grow on some ZamB strains: the former require chloroform like wild-type A to be inactivated by R-receptor, while the latter do not. Wild-type phage lambda, labeled with =P, was mixed with extract from a pel

189

INTERACTION

IO

0.5 73 ; E E 61 Pi= 0.50

0.25

IO FRACTION

20 NUMBER

30

FIG. 4. Reaction of host range lambda with Rreceptor in the presence of cholate. A mixture of prepurified 13Hlleu-labeled particles (7 x 10” PFU, 2.5 x lo3 cpml and prepurified 32P-labeled particles (8 x 10’ PFU, 5 x lo3 cpm) was used. Sodium cholate was present in the incubation mixture as well as in the sucrose gradients. Centrifugation was at 36,000 rpm during 1 hr. (a) Phage alone; (b) phage incubated with 8 x lOlo molecules of R-receptor. 3H (cpml fraction), O-O-O; 3zP cpm/fraction), O-0-0.

strain in the presence of chloroform, and the mixture was analyzed on cholate-containing sucrose gradients. A profile similar to that shown in Fig. 4b was obtained. A typical experiment in which nuclease sensitivity was tested after phage-receptor interaction gave the following fraction of acid-soluble 32P counts: pel+ receptor 36%, pel receptor 28%, and no receptor 7%. The experiments in this section demonstrate that in all the properties tested the pel receptor behaves in vitro like R-receptor extracted from a pel+ strain, and Ahp behaves like wild type lambda. DISCUSSION

The first conclusion which can be drawn from this work is that wild-type phage lambda interacts with R-receptor, even if it is not inactivated by it. Schwartz (1975) reached the same conclusion by demonstrating that R-receptor protects wild-type

190

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IO FRACTION

20 NUMBER

FIG. 5. Reaction between wild-type lambda and R* receptor in the presence of cholate. A mixture of “S-labeled particles (8 x log PFU, 1.2 x lo5 cpm) and 32P-labeled particles (9 x lo9 PFU, lo5 cpm) was used. Cholate was present in the incubation mixture and in the gradients. The centrifugation was for 90 min at 18,000 rpm. (a) Phage alone; (b) phage incubated with 5 x lOlo molecules of R* receptor. YS (cpm/fraction), O-O-O; 32P (cpm/fraction), O-0-0.

lambda against inactivation by R* receptor. In the experiments demonstrating that R-receptor aggregates wild-type phage (Figs. 1 and 2a and b) the specific infectivity of the aggregates is the same as that of free phage. This result, suggesting that the aggregates dissociate before titration, is in agreement with the demonstration by Schwartz (1975) that the phage receptor complex is reversible and very unstable in lop2 M MgS04, the solution used for all phage dilutions. Since the sucrose gradients also contain 10e2 M MgS04, the cosedimentation of phage with the receptor aggregates must be due to the retention effect, as recently discussed by Silhavy et al. (1975). This interpretation would also account for the fact that upon electron microscopic examination only a fraction of the phage particles present in the fast sedimenting material is actually engaged in an aggregate. The next conclusion is that, in condi-

tions where the phage is inactivated by receptor, complete ejection of phage DNA does not immediately follow phage-receptor interaction. Experiments are described in which at least 99% of the plaque-forming units are inactivated, about 90% of the physical particles are bound to receptor (electron microscopy), and where only 10 to 30% of the phage DNA has become accessible to nucleases. Do most particles eject only part of their DNA, or do some particles eject all their DNA while the others remain full? Both possibilities are compatible with the results presented here. Electron microscopy only revealed two classes of particles: either filled or empty. However, since the fraction of empty particles is definitely greater than 30% (Fig. 3a and b), a large fraction of them must have lost their DNA during preparation of the grids. The fact that centrifugation increases the amount of ejected DNA would be more

20

IO FRACTION

NUMBER

FIG. 6. Reaction of host range lambda with Rreceptor in the absence of cholate. A mixture of prepurified %S-labeled particles (lo9 PFU, 5 x lo3 cpm) and prepurified 32P-labeled particles (8 x lo9 PFU, 1.2 x 10’ cpm) were incubated with 8 x 1O’O molecules of R-receptor. Centrifugation was for 90 min at 18,000 rpm. (a) Phage alone; (b) phage with receptor. Y3 (cpm/fraction), O-O-O; 3zP (cpmlfraction), O-0-0.

PHAGE

LAMBDA-RECEPTOR

191

INTERACTION

0.25

FRACTION

NUMBER

IO FRACTION

20

30

NUMBER

FIG. 7. Effect of nucleases on the products of phage-receptor interaction. A mixture of host range mutants of lambda (W label: 2 x 10y PFU and 1.8 X 10’ cpm; 32P label: 4 x lOlo PFU and 8 x lo3 cpml was used. Cholate was present in the incubation mixture and the sucrose gradients. Centrifugation was for 90 min at 18,000 rpm. One-half of each fraction was precipitated and counted as usual while the other half was treated with nucleases as indicated in Materials and Methods. (a) Phage alone; untreated control. (b) Phage alone; nuclease-treated fractions. (c) Phage incubated with 8 x 1O’0 molecules of R-receptor; untreated control. (d) Phage incubated with 8 x lOlo molecules of R-receptor; nuclease-treated fractions. “S kPm/ fraction), O-O-O; :vzP(cpm/fraction), O-0-0.

easily explanable if most particles had ejected part of their DNA which could be pulled out due to the differential sedimentation properties of DNA and particles. However, the fact that 10 to 35% of the particles still sediment as intact phage, is difficult to reconcile with the above interpretation, unless these particles only ejected a very small segment of DNA. At any rate, the results suggest that the phage particles are heterogeneous since some eject all or most of their DNA upon interaction with receptor, while the others eject none or very little. Conceivably, the source of this heterogeneity would be related to the way the DNA is packaged into the head, perhaps because of a different content of the particles in ions or polyamines (Bode and Harrison 1973; Harrison and Bode, 1975). Since only a fraction of the phage particles in a given stock is active (15 to 50% depending on the stock), it is tempting to argue that the plaque-forming units are the particles which release

all or most of their DNA immediately upon interacting with receptor. There is, however, no evidence that this is so. The results presented here also give some information about the end products of the overall reaction. DNA and ghosts can be separated on sucrose gradients but the possibility remains that some specific phage proteins are released at the same time as DNA, perhaps even attached to it, as found in other systems (Krahn, O’Callaghan, and Paranchych, 1972; Jazwinski, Lindberg, and Kornberg, 1975; Jazwinski, Marco, and Kornberg, 1975). In fact, a small amount of protein label is often found at the top of the gradients, together with free DNA, but this material was not analyzed. On the other hand, the sedimentation properties of the ghosts in the absence of detergent, as well as results obtained by electron microscopy, suggest that the ghosts remain attached to the receptor, once the DNA has been ejected. This is in agreement with the demon-

ROA

AND

SCANDELLA

1.0

0.5 ; E ,” a :: 1.0

0.5

IO FRACTION

20 NUMBER

30

FIG. 8. DNA ejection from particles in an intermediary state. 3*P-labeled wild-type lambda particles (1.5 x 10IDPFU, 8 x lo5 cpm) were used. Incubation and centrifugation were in the presence of cholate. Centrifugation was for 90 min at 18,000 rpm. Patterns similar to those of Fig. 5 were obtained. The fractions containing the fast sedimenting 3zPlabeled material were pooled, and dialyzed for 4 hr against Tris-magnesium buffer. Aliquots from the resulting solutions were layered on cholate containing sucrose gradients, and centrifuged for 90 min at 18,000 rpm. (a) Phage incubated alone. Fractions 14 to 16 of the first gradient were pooled, dialyzed, and lo5 cpm of this solution was layered on the second gradient. (b) Phage incubated with 5 x 1Ol0 molecules of R* receptor. Fractions 14 to 17 containing 1.25 x lo5 cpm were pooled and dialyzed. An aliquot containing 4 x lo4 cpm was put on the second gradient without any addition of receptor. 32P (cpm/ fraction), O-0-0.

stration that inactivation of phage by receptor is a stoichiometric reaction (Randall-Hazelbauer and Schwartz, 1973). The interaction between phage lambda and its receptor in vitro can be formally described by the following sequence of reactions: phage + receptor =G [phage-receptor]

(1)

[phage-receptor]

(2)

[phage-receptor]’

+ [phage-receptor]’

+ phage DNA + [ghost-receptor]

(3)

When wild-type lambda and R-receptor are incubated in buffer, the interaction remains limited to Reaction (1). Reaction (2) corresponds to the inactivation of the PFU present in the stock. It occurs upon addition of chloroform or ethanol. It also occurs in the absence of these chemicals either if the phage is a host range mutant, or if the receptor is of the R* type. While the host range mutation is known to be located in the phage gene J, which probably codes for the tail fiber (Buchwald and Siminovitch, 1969; P. Kiihl, personal communication), the R* characteristic is shown in this work to be coded by gene ZamB, the structural gene of the receptor (see Materials and Methods). Finally Reaction (3) occurs to some limited extent spontaneously but can be favored by various treatments. The problems regarding this reaction were discussed above. Preliminary evidence suggests that the reactions observed in vitro reflect the early steps of phage infection in Go. The adsorption of phage lambda was recently shown to involve a reversible step bearing many similarities with Reaction (1) (Schwartz, 1976). On the other hand, bacterial pel mutants adsorb lambda irreversibly, but the phage cannot inject its DNA. (Scandella and Arber, 1974). These mutants are shown here to have a normal receptor, as assayed in vitro, and their mutation is not linked to ZamB (Scandella, unpublished). It would therefore seem that in uiuo Reaction (3) requires the action of at least one other bacterial product. The role of this product could be envisioned differently depending on the interpretation regarding the in vitro ejection experiments. If most phage particles eject part of their DNA upon reaction with receptor, the pel product could participate in pulling the rest of the DNA out of the particle and through the bacterial envelope. This type of reaction was indeed suggested to occur in other systems (Labedan et al., 1973; Jazwinski, Lindberg, and Kornberg, 1975; Jazwinski, Marco, and Kornberg, 1975). On the other hand, the ejection observed in vitro may be an artifact, phage-receptor interaction simply leading to a destabilization of the particle. In this case, the pel

PHAGE

LAMBDA-RECEPTOR TABLE

EFFECT Conditions

OF VARIOUS

1

FACTORY

of incubation

ON DNA

EJECTION

Averageper-

Percentage of hydrolysis after nuclease treatment6

centage of remaining PFU

0 +

13 32

100 0.2

R* receptor

Normal Normal

193

INTERACTION

10% Sucrose 10% Sucrose

added added

at 0 min at 0 min

0 +

16 36

100 0.5

10-l M NaCl 10-I M NaCl

added added

at 0 min at 0 min

0 +

20 67

80 0.3

10-l M NaCl 10-l M NaCl

added added

at 15 min just at 15 min just

0 +

27 65

93 0.1

10-l M NaCl Nuclease Nuclease Nuclease Nuclease Nuclease Nuclease Nuclease Nuclease

added added added added added added added added added

0 + 0 + 0 + 0 +

14 58 14 69 11 69 22 75

100 1.5 97 0.1 90 0.2 95 0.1

at 0 at 15 at 15 at 30 at 30 at 60 at 60 at 90 at 90

before before

nuclease nuclease

addition addition

min min min min min min min min min

o Normal conditions of incubation are as follows: 32P-labeled wild-type lambda (1.25 x 10’” PFU/ml final) is incubated at 37” with or without R* receptor (5 x lOlo molecules/ml), in presence of 2 x 10m3 M MgSO, and 5 x IO-:$ M Tris-HCI, pH 7.5. After 15 min (unless otherwise indicated), ZOO-~1 aliquots are withdrawn and treated with nucleases, while control 200-~1 aliquots are similarly incubated, in the absence of nucleases. Both aliquots are then assayed for PFU and radioactivity. b Each number is the average from two to five determinations performed with the same phage stock and the same receptor preparation.

product could trigger DNA ejection, which would then proceed spontaneously. At any rate, the pel product probably acts on Reaction (3) and not, or not only on Reaction (2). Indeed, the classical host range mutants, (Xh type), known to proceed spontaneously through Reaction (2) in vitro, still do not inject their DNA in pel mutants (Scandella and Arber, 1974; Scandella and Arber, 1976). Furthermore, introduction of an R* receptor into a pel strain does not suppress the Pel phenotype (Roa, unpublished results). Therefore the occurrence of Reaction (2) in E. coli K12 may be stimulated by the action of still another component in addition to the receptor and the pel product. Alternatively R-receptor may take in uiuo a conformation which it assumes in vitro only in the presence of chloroform or ethanol.

ACKNOWLEDGMENTS We thank Maxime Schwartz and Werner Arber for many useful discussions, Raymond Hellio for introducing M.R. to electron microscopy, and Maxime Schwartz for his help in preparing the manuscript. The interpretation of our results benefited from a discussion with Vernon Bode and Donna MacKay who, independently, performed work similar to ours. This work was supported by grants from the National Institutes of Health, the Centre National de la Recherche Scientifique, and the Delegation G&&ale a la Recherche Scientifique et Technique. D.S. thanks the European Molecular Biology Organization for a short term fellowship during the course of her stay at the Institut Pasteur. REFERENCES APPLEYARD, R., MACGREGOR, J., and BAIRD, K. (1956). Mutation to extended host range and the occurrence of phenotypic mixing in the temperate

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