VIROLOGY

94,

10-23 (1979)

Studies

MICHAEL

on the initial Interactions of Bacteriophage with its Host Ceil, Salmonella anatum

McCONNELL,‘,~

Department

ANDREA REZNICK,

AND

of Molecular Biology and Microbiology, Tufts University Boston, Massachusetts 02111 Accepted November

cl5

ANDREW WRIGHT School of Medicine,

17, 1978

The initial interaction between bacteriophage G5 and its host cell, Salmonella anatum involves at least three steps including adsorption, enzymatic cleavage of surface polysaccharide (O-antigen) and ejection of DNA. The DNA ejection function exhibits the most stringent pH and temperature requirements and is also divalent cation-dependent. DNA ejection can be completely inhibited under conditions which are permissive both for adsorption and 0-polysaccharide depolymerization. Phage attachment to cells shows the least dependence upon physical conditions, whereas O-antigen depolymerization appears to be of intermediate sensitivity. Several mutants of bacteriophage cl5 with mutations in the tail gene were isolated which were abnormal in one or more of the above steps. The phenotypes of the mutants support the kinetic data in showing the initial attachment of phage to cells does not require depolymerization of O-antigen and that depolymerization of O-antigen is necessary but not sufficient for triggering release of phage DNA. In addition, the mutants prove that the tail protein of phage t I5 is responsible for initial attachment of phage to cells and for depolymerization of cellular O-antigen. Tail protein also appears to be directly involved in DNA release from the phage capsid. INTRODUCTION

anatum lipopolysaccharide consists of mannosylrhamnosylgalactose trisacBacteriophage that infect gram-negative charide repeating units joined together by Enterobacteriaceae must contend initially with a multilayered cell envelope, the outer- a-glycosidic linkages. It is cleaved by e15 most layer of which is a membrane consist- phage into fragments which contain rhaming primarily of phospholipid, protein, and nose as their sole reducing sugar, thus indilipopolysaccharide. The 0-polysaccharide cating that the phage-associated enzyme is (O-antigen) portion of lipopolysaccharide an endorhamnosidase (Robbins and Uchida, serves as an initial cell surface receptor 1962; Kanegasaki and Wright, 19’73;Takeda for several such bacteriophages. Within re- and Uetake, 1973). In this paper, we present biochemical and cent years, 0-polysaccharide-specific phages such as coliphage flR8and Salmonella phages genetic studies which were conducted for P22, e34,g341, and cl5 have all been shown the purpose of better understanding the to possess virion-associated, O-polysac- function of 0-polysaccharide hydrolysis in charide-hydrolyzing enzymes (Takeda and the early interactions of phage e15with its Uetake, 1973, 19’75; Kanegasaki and host cell. Our data on physiological requireWright, 1973; Iwashita and Kanegasaki, ments for phage adsorption, O-polysac1973, 1975, 19’76a, 1976b; Israel, et al., charide hydrolysis (depolymerization), and 19’72;Reske, et aZ., 1973; Prehm and Jann, DNA ejection suggest the following: that 1976). The 0-polysaccharide portion of Sal- initial attachment of phage to cells does not require depolymerization of O-antigen, and 1 To whom requests for reprints should be addressed. that O-antigen depolymerization is neces2 Present Address: Department of Biology, Point sary but not sufficient for the initiation of Loma College, San Diego, Calif. 92106. DNA release from the phage capsid. Studmonella

10

0042-6822/79/050010-14$02.00/O Copyright All rights

0 1979 by Academic Press, of reproduction in any form

Inc. reserved.

INITIAL

INTERACTIONS

OF BACTERIOPHAGE

ies on mutants of bacteriophage e15support these hypotheses and also show that a single 125,000 MW polypeptide in the cl5 virion (the depolymerase or tail protein) is responsible for initial attachment of phage to host cells and depolymerization of host cell O-antigen. In addition , the tail protein appears to cooperate directly with other phage proteins to trigger release of DNA from the phage capsid. MATERIALS

Bacteria

AND METHODS

and Phage

The wild-type

bacterial strain, SalAl, and bacteriophage e15vir have been described previously (Wright, 1971). Bacterial strain 37AZSu+ is a proline auxotroph of strain Al which carries an amber suppressor (isolated by Dr. Michael Malamy). Escherichia coli DlO (Gesteland, 1966) was provided by Dr. Paul Leibowitz. Temperature-sensitive phage strains tsll, ts53, and ts82 and nonsense mutant strain am2 were derived from hydroxylamine-mutagenized EX5vir (Hall and Tessman, 1966) using enrichment techniques for mutants defective in attachment to cells at high temperatures (see below). Double mutant phage strains carrying combinations of these missense and nonsense mutations were isolated by mixedly infecting 37A2Su+ cells at permissive temperature (30”) with pairs of phage mutants, then screening the progeny for nonsense mutants using a double overlay plating technique (Studier, 1969) and anaerobic incubation conditions which increase plaque size (McConnell and Wright, 1975). Nonsense mutants were then screened for temperature sensitivity in order to identify strains carrying both nonsense and missense mutations.

monella

Chemicals

anatum

and Enzymes

Carrier-free H,32P04 (300 Ci/mg), carrier-free H,35S0, (43 Ci/mg), and [3H]deoxythymidine (20 Ci/mmol) were obtained from New England Nuclear. 14C-Labeled glucose/fructose mixture (200 mCi/mmol) was prepared as previously described (Rob-

cl5 WITH

S. anatum

11

bins and Uchida, 1962). Alkaline phosphatase and P-galactosidase used as molecular weight markers on sucrose gradients were from Sigma. DNase I was from Worthington Biochemicals. Cesium chloride (biological grade) was from Schwartz/Mann. All other chemicals were standard products of reagent grade. Microbiological

Techniques

Many of the conditions for growth of cells and growth and assay of phage have been previously described (Wright, 1971; Wright and Barzilai, 1971). The growth media used were LB broth (Bertani, 1951), LSTG (Hershey, 1955), M9 casamino acids (MSCA) (Smith and Levine, 1964), and LCGZO (Botstein, 1968). The following methods were used for isolation and genetic characterization of phage mutants altered in their O-antigen depolymerizing ability. Bacteriophage l l5vir was mutagenized with hydroxylamine then grown in either S. anatum 37A2Suf, a host cell carrying an amber suppressor, or in S. anatum Al, an Su- host, at 30” to allow expression of mutations. Phage mutants with defects in irreversible attachment to S. anatum were enriched for by successive addition of S. anatum Al cells at 38” to remove normally adsorbing phage. Temperature sensitive mutants, present among the unadsorbed phage, were recognized by their inability to form plaques at 37” (nonpermissive temperature) on 37A2Suf indicator cells. Growth in the Su- host (S. anatum Al) allowed the isolation of nonsense mutations in the O-antigen depolymerase (presumed to be the tail) gene. After adsorption (as above) the unadsorbed phage, including defective particles, were complemented in vitro with purified O-antigen depolymerase protein (tail), then tested for plaque-forming ability on Su+ and Su- host cells. One amber mutant, am2, was isolated in this way. Preliminary assays showed that most of the phage mutants isolated by these methods were altered in their ability to depolymerize O-antigen: many exhibited abnormally low depolymerization rates, while a

12

MCCONNELL,

REZNICK.

few appeared to hydrolyze O-antigen at faster than normal rates. Several phage mutants were chosen for further study on the basis of their O-antigen depolymerization phenotypes. In vivo complementation studies were carried out by mixedly infecting Al cells at 30” with various combinations of the missense and nonsense phage mutants (final m.o.i. = 10) and incubating for 10 min at 30“ to allow adsorption to cells. Anti-@ serum was added (final K = 1) and after an additional 10 min at 30”, cultures were diluted 105-fold into prewarmed LB broth at 37”, incubated for 130 min then treated with chloroform. Plaque-forming units were assayed on 37AZSu+ cells at 30”. Complementation was determined by comparing yields in mixed infections with yields from single infections involving the two mutants of interest. Recombination experiments were performed in the following manner: 37AZSu+ cells were infected with pairs of phage mutants allowing 10 min in LB broth at 30” for adsorption (final m.o.i. = 10). Antil l5 serum was added (final K = 1) and cultures were incubated for an additional 10 min at 30”. Cultures were diluted lOOO-fold and incubated for 3 hr at 30”. Lysates were treated with chloroform and plaqueforming units were measured under permissive conditions (30” on 37AZSu+ cells) and nonpermissive conditions (37” on Al cells). The number of wild-type recombinants was multiplied by 2, corrected for background, and divided by the total number of progeny phage to obtain the recombination frequency. Three-factor crosses involved mixed infection of cells with a double mutant phage (missense and nonsense mutation) and single mutant phage (missense and nonsense). The procedure was identical to that described above except that recombinants were identified by plating either at 37” on 37S2Suf cells (for detection of recombination between two missense mutations) or at 30” on Al cells (for detection of recombination between two nonsense mutations). Recombinants were then screened for the presence of the unselected nonsense or mis-

AND WRIGHT

sense markers, respectively, in order to determine the linear order of the three markers in question. Preparation Bacteria

of Radioactively-Labeled and Phage

S. anatum Al cells used as substrate for assay of phage depolymerizing activity were labeled with [14C]glucose as before (Kanegasaki and Wright, 1973), with the following modifications: after growth of cells in LB broth at 37” in the presence of [‘“Clglucose (1 $X/ml), sufficient 2 N HCI was added to reduce the pH to between 1 and 2. Five minutes later, cells were collected by centrifugation and washed three times with 5 mfl4 Tris-Cl buffer (pH 7.8) containing 0.1 mM MgSO,. Labeled cells were stored at -20” and used within 2 weeks after preparation. Phage e15vir and its missense and nonsense mutant derivatives were labeled with 35Sby addition of carrier-free H,35S04 (25 $X/ml) to S. anatum Al cells growing in low sulfate LSTG, 20 min after infection with phage (m.o.i. = 20). r3H]Thymidinelabeled l l5vir phage were prepared by addition of [3H]thymidine (50 @X/ml) plus deoxyadenosine (250 pg/ml) to S. anatum cells growing in MSCA, following infection with phage (m.o.i. = 20). 32P-Labeled phage were prepared by adding H332P04(20 pCi/ml) to S. anatum Al cells that had been grown to midlog phase in LCG20 medium and resuspended in low phosphate medium (LCG0.5) in the presence of phage (m.o.i. = 20). Labeled phage were purified from cell lysates by centrifugation on CsCl block gradients, followed by sedimentation on 5-20% sucrose gradients. Typical specific activity values for the various phage preparations are: 35S-85vir, 2 x 10e5 dpm! PFU; [3H]thymidine-85vir, 2.5 x lo+ dpm/PFU; and 32P-e15vir, 3.5 x 10e4 dpm/PFU. Experimental

Assays

Adsorption. The kinetics of adsorption of wild-type and mutant phage particles to S. anatum cells were determined by mixing

INITIAL

INTERACTIONS

OF BACTERIOPHAGE

exponentially growing cells with 35S-labeled phage (m.o.i. = lo), removing samples at various time intervals and measuring the amount of labeled material retained on membrane filters. Filters were washed three times with 5 ml of cold LB broth. The radioactivity retained on membrane filters in the presence of E. coli DlO cells was used as a control for nonspecific sticking. The background was usually about 5% of the total radioactivity in a sample. A second method for measuring phage adsorption involved mixing phage and cells together and measuring the decrease in free phage. At various time intervals, samples of the phage/cell suspension were diluted loo-fold into chloroform-saturated, Tris-Cl buffer (5 mM, pH 7.8) containing 10 miW EDTA. The samples were further diluted and mixed with S. anatum Al indicator cells on LB plates to determine the number of free plaque-forming units remaining. The kinetics of deDepolymerixation. polymerization of O-antigen were measured by mixing [14C] glucose-labeled S. anatum cells with e’+ir phage (m.o.i. = lo), stopping the reaction by acidification (pH between 1 and 2) and measuring the radioactivity remaining in the supernatant after removal of the cells by centrifugation. In a typical experiment at 37” (m.o.i. = lo), the depolymerization reaction was complete within 5 to 10 min and a maximum of about 12% of the 14C-labeled cellular material was solubilized. Controls for nonspecific release of radioactivity included incubating 14C-labeledS. anatum cells either with buffer alone, or with P22, a phage that infects S. typhimurium. In either case, about 2% of the 14C radioactivity was found in the supernatant. DNA ejection. S. anatum cells were mixed with 32P-E15virphage (m.o.i. = 0.5) in 5 mM Tris-Cl buffer (pH 7.8) containing 0.1 mM MgS04. At regular time intervals, 0.7-ml samples were transferred into tubes along with 0.2 ml of a 0.1 M Tris-Cl buffer (pH 9.0) containing 0.05 M EDTA, Triton X-100 (5 pg/ml), and [3H]thymidine-labeled E15vir phage (20,000 cpm). The samples were chilled, sonicated for 45 see to break open the infected cells, then mixed with

c+ WITH

S. unatum

13

DNase 1 (0.1 ml; 2 mg/ml in 0.2 M MgCl,) and incubated at 37” for 20 min. Reactions were terminated by the addition of 1 ml of cold 10% TCA. Precipitates were collected on Whatman GF/C filters and washed three times with 4 ml of cold 5% TCA. 3H and 32P radioactivity on the dried filters was measured in a scintillation counter using Bray’s scintillation fluid. In typical experiments carried out at 37”, the reaction is complete within about 10 min, at which time 70-80% of the 32Pradioactivity is sensitive to DNase I. The [3H]thymidine-labeled phage serve as an internal control for release of phage DNA during sample processing. Usually, not more then 10% of the [3H]thymidine phage DNA label added to an EDTA-treated sample containing S. anatum Al cells and 32P-labeledphage was solubilized during the sonication and DNase treatments. At least 80% of the [3H]thymidine label behaved like phage DNA in the ejection assay (results not shown). The following formula was used for calculating the percentage of phage DNA ejection at time t: Percentage DNA ejection control 3H/32Pratio at time t x 100 = 100 * experimental 3H/32P [ ratio at time t

1

Putification

of Phage Depolymerase

Two techniques were used in the purification of phage depolymerase protein, both of them involving the same initial steps. Cells (either S. anatum Al or 37A2Suf) were grown at 30” in 250 ml of LSTG medium (supplemented with 10 pg/ml of proline) to about 4 x lo8 cells/ml, then diluted with an equal volume of LSTG-proline medium containing enough phage to give a final eoncentration of 2 x log PFU/ml (m.o.i. = 10). Infected cultures were incubated at 30 for 90 min and harvested prior to lysis by cooling on ice and centrifuging for 10 min at 12,000 g. The infected cells were resuspended in 10 ml of cold 5 m&f Tris-Cl buffer (pH 7.8) containing 0.1 mM MgSO,

14

MCCONNELL,

REZNICK,

and 0.1% azide, sonicated (four periods of 1 min each), and centrifuged for 15 min at 12,000g and 4” in order to remove the cellular debris. The supernatant was decanted and centrifuged at 4” for 2 hr at 81,000 g in order to pellet whole phage particles. At this point, the supernatant containing soluble cell and phage proteins was treated in either of two ways to obtain the depolymerase protein. (a) Purified S. anatum Al lipopolysaccharide was added to the above lysate at a final rhamnose concentration of 100 pg/ml. After incubation for 15 min at 4”, the sample was centrifuged at 81,000 g for 2 hr, pelleting the lipopolysaccharide with adsorbed phage depolymerase. SDS-polyacrylamide gel analysis of the resuspended lipopolysaccharide pellet revealed a single polypeptide that migrated with the mobility of phage depolymerase. Under these purification conditions, the depolymerase enzymatic activity measured in the resuspended lipopolysaccharide pellet was 20% of the total activity initially present in the original high-speed supernatant. (b) Protein precipitating from the 81,000g supernatant at 30% ammonium sulfate (w/w) was further fractionated by sucrose gradient (5-20%) sedimentation essentially as described by Kanegasaki and Wright (1973), using /3-galactosidase and E. coli alkaline phosphatase as standards. The purity of depolymerase in the peak fraction of sucrose gradients was about 10% as estimated by SDS-polyacrylamide gel analysis. Such preparations were free of whole phage particles, as indicated by the absence of phage capsid protein on gels. Depolymerase prepared by this method was used in experiments involving tail addition to tailless e15phage mutants.

AND WRIGHT

gradient gels, using the discontinuous buffer system of Laemmli (1970). Unlabeled proteins were detected by staining with Coomassie brilliant blue. Labeled proteins were detected by radioautography using Kodak Blue-Brand X-ray film. RESULTS

Physical Parameters for Adsorption, Depolymerixation of O-Antigen, and Ejection of Phage DNA

The pH and temperature requirements for initial attachment of phage to cells (adsorption), depolymerization of cellular O-antigen by phage, and ejection of phage DNA were examined for phage e15vir, using the three biochemical assays described under Materials and Methods. The rate of association of 35S-labeled phage particles with cells was used as a measure of adsorption. Depolymerization of polysaccharide O-antigen was assayed by measuring the phage catalyzed release of soluble oligosaccharide products from 14Clabeled cell envelopes. DNA ejection from 32P-labeled phage was assayed by measuring the radioactivity solubilized by pancreatic DNase in sonicated extracts of infected cells. pH effects. The optimal pH for adsorption of G5 phage particles to S. anatum Al cells is pH 7, whereas the maximum rate of O-antigen depolymerization occurred at a pH of 5.3 (Fig. 1). Each data point in Fig. 1 was derived from a separate experiment in which the reaction rate was calculated from the initial linear range of the reaction (O-100 see). In every case, the reaction rate for both adsorption and depolymerization appeared to extrapolate back to time zero (data not shown). While both processes are pH dependent, the data indicate that Analysis of Proteins by Gel Electrophoresis depolymerization of O-antigen is considerably more sensitive to pH variation than Proteins were denatured by boiling for 3 is phage adsorption to cells. min in sample buffer containing 1% SDS, The DNA ejection mechanism is much 1% P-mercaptoethanol, 10% glycerol, 55 more sensitive to pH (Fig. 2). The fastest mJ4 Tris-Cl (pH 6.8), and pyronin Y marker reaction rate for DNA ejection was obdye. Samples were routinely electro- served at pH 7.0, with marked decreases phoresed on 5-15% SDS-polyacrylamide occurring either in slightly acidic (pH 6.0)

INITIAL

31, LL

4

INTERACTIONS

,

,

,

,

,

5

6

7

8

9

OF BACTERIOPHAGE

]

PH

FIG. 1. l Phage adsorption and O-antigen depolymerization as a function of pH. All reactions were carried out at 31”. Assays for adsorption of %-labeled l %ir phage and depolymerization of ‘V-labeled cellular O-antigen are described under Materials and Methods. Tris-Cl buffers were used for the higher pH range, while Tri-maleate buffers were used for the lower pH range. The reaction rates at pH 4 were determined by using citrate-phosphateborate buffers at pHs of 4 and 6 and normalizing the data with respect to that obtained with Tris-maleate buffer at pH 6. All of the buffers contained 0.1 mM MgSO,.

@ WITH S. anatum

15

much greater extent than was adsorption, suggesting that O-antigen hydrolysis may not be necessary for initial attachment of phage to cells. The average Q10 value for depolymerization over the range of 5 to 50” was 2.3, which is within the expected range for a thermochemical reaction involving alteration of covalent bonds. The maximum rate of DNA release from phage P incubated with S. anatum Al cells in Tris-chloride buffer (pH 7.8) occurred at 30” (Fig. 4). Reaction rates dropped off sharply at temperatures above 45” and below 20”. At 5”, some DNA ejection occurred, but only after a lag of more than 20 min (data not shown). Lag periods were also observed at other temperatures that were tested. Only at 30” was there no detectable lag preceding DNA ejection (Fig. 4, inset). When phage and cells were mixed together at 20” and assayed biologically for phage inactivation using chloroform treatment of infected cells and titration of the remaining free phage, all of the phage could be “rescued” up to 4 min after the addition of S. anatum Al host cells simply by diluting the reaction mix with a buffer that contained 10 mM EDTA (data not shown). The 4 min lag period observed in this experiment was similar to that observed at 20” using the biochemical assay for DNA ejection (Fig. 4). The studies discussed above suggest that pH and temperature are of relatively little importance for initial attachment of phage to cells, as compared with depolymerization and DNA ejection. Furthermore, there are pH and temperature conditions that completely inhibit DNA ejection while still permitting adsorption and depolymerization of these to occur. One interpretation results is that adsorption, depolymerization, and DNA ejection are part of a dependent pathway. The properties of the phage mutants discussed below support this hypothesis.

or slightly basic (pH 8.2) medium. Little or no DNA ejection occurred either at pH 5.0 or at pH 8.6 and above, conditions that were permissive for both adsorption and O-antigen depolymerization. Temperature effects. The rate of phage adsorption to cells varied by a factor of about 3 between 5 and 50”, with maximal adsorption rates occurring in the 40-50 range (Fig. 3). The average Q10 value for adsorption of c15vir phage was 1.3 when calculated over the temperature range of 5 to 42”. This value is typical for a reaction in which the rate is limited only by diffusion (Giese, 1973). The maximum rate of adsorption (40-50” range) ‘had a calculated rate constant of about 1.2 x lo+ ml/min, which is within the range of values reported in other virus-host cell adsorption studies (Adams, 1959; Fukasawa, et al., 1964). In contrast to phage adsorption, phagespecific depolymerization of O-antigen is strongly temperature dependent. The reac- Phage Mutants with Defects in Early tion rate for depolymerization varied by Stages of Infection 35-fold over the range of 5-60”, with a maximum in the 40-50” range (Fig. 3). A series of phage mutants, tsll, ts53, At 5”, depolymerization was inhibited to a ts62, ts82, and am2, unable to adsorb to

16

MCCONNELL,

REZNICK,

5

AND WRIGHT

IO

15

20

TIME (mid FIG. 2. The effects of pH on the kinetics of ejection of DNA by cl5 phage in the presence of S. anatum cells. The reaction conditions were similar to those described in Fig. 1. The assay for l g5DNA ejection is described under Materials and Methods. The inset in this figure showsthe relative DNA

ejection rates at different pHs calculated from the linear portion of each of the curves.

cells at high temperature were isolated as described under Materials and Methods. Two- and three-factor crosses gave the map order shown in Fig. 5. As shown below, the mutants have different phenotypes but all are located in the same gene since they show no complementation for one another. Phage am2, a tail gene nonsense mutant with a null phenotype. When phage am2 was grown in an Su+ host (am2.Su+) and used to infect S. anatum Al cells, normal cell lysis resulted and phagelike particles were produced which behaved like wild-type phage during purification on CsCl and sucrose gradients. These particles were however 10,000 times less efficient than wild-type phage in plaque-forming ability, and exhibited a pleiotropically negative phenotype with regard to their ability to interact with the cell surface of S. anatum. Such particles are not retained with cells

IO 20

30 40

50 60

TEMPERATURE

FIG. 3. cl5 Phage adsorption and O-antigen depolymerization as a function of temperature. Assays for adsorption of YS-labeled phage and depolymerization of %-labeled O-antigen were carried out as described under Materials and Methods. All reactions were done in 5 m44 Tris-Cl buffer (pH 7.8) containing O.l d MgSO+ l %ir Phage and S. anatum Al cell suspensions were incubated separately for 5 min at reaction temperature, then mixed to initiate the reaction (final m.o.i. of 10).

INITIAL

INTERACTIONS

OF BACTERIOPHAGE

TIME

17

ei5 WITH S. anatum

(MIN)

FIG. 4. The effects of temperature on the rate of DNA release form l L5phage in the presence of S. anatum cells. DNA ejection was measured as described under Materials and Methods, except that 10 m44 cyanide was included in the mixture used for terminating the reaction. Phage and cells were preincubated separately for 5 min at reaction temperature, then mixed together to initiate the reaction (m.o.i. 0.1). The inset in this figure shows the relationship between the lag in DNA ejection and the reaction temperature.

on filter disks, they fail to hydrolyze O-antigen, and they are unable to eject their DNA (Fig. 6). Growth of am2 phage on 37A2Su+ cells at 30” restores their ability to absorb to cells, hydrolyze O-antigen, and eject DNA (Fig. 6).

TlME

(MINI

FIG. 5. Biochemical characterization of amZ.Su+ and

amB.Su- phage particles. Phage particles were assayed for adsorption, O-antigen depolymerization, and DNA ejection at 38” in 5 mM Tris-Cl buffer (pH 7.8) containing 0.1 miI4 MgSO,. Symbols: 0, ame.&-; X , am2*Su+.,, 0 l ?ir.

Analysis of 35S-labeledphage particles on SDS-polyaerylamide gels (Fig. 7) reveals that a 125,000 MW polypeptide that is present in wild-type phage and in am2 phage grown in 3’7A2Su+ cells at 30” (anL’.Su+> is missing from am2 phage particles grown on Al cells (am2.Su). This polypeptide has the same electrophoretic mobility on gels as partially purified O-antigen depolymerase extracted from Al cells during the late

anW5u-l am Its&

il 18%

1 f

Wt

Isli

I

r j

q 6,

FIG. 6. SDS-Polyacrylamide gel profile of 35Slabeled am2.Su+ and am2.Su phage particles. Phage were labeled with 35Sduring growth on S. anatum Al or S. anatum 37A2Suf cells at 30” as described under Materials and Methods, disrupted by boiling in buffered SDS and electrophoresed on 5615% SDSpolyacrylamide gradient gels.

18

MCCONNELL,

REZNICK,

AND WRIGHT

FIG.7. Purification of cl5 O-antigen depolymerase by affinity binding to lipopolysaccharide. The procedure used for purification of depolymerase by affinity binding to lipopolysaccharide is described under Materials and Methods. stages of infection with l l5vir phage (Kanegasaki and Wright, 1973; unpublished data). While this evidence indicates strongly that the am2 mutation is in the structural gene for the O-antigen depolymerase, there remains the possibility that normal depolymerase is synthesized in am2-infected Al cells but is unable to attach to phage capsids because of the am2 defect. This possibility was tested by using S. anatum Al lipopolysaccharide to precipitate free depolymerase protein from lysates of phage-infected cells (see Materials and Methods). Figure 8 demonstrates the specificity of this technique for phage depolymerase protein in a lysate obtained by infecting S. anatum Al cells with phage l l5vir. When lysates of am2-infected Al cells were treated in the same manner, no polypeptides were detected

Ill

c

in the lipopolysaccharide pellet (data not shown). Electron microscopy of amB.Su- particles showed capsids lacking a normal tail structure (data not shown). These particles were similar to P22 9- phage (gene 9 is the tail gene of phage P22) grown in a nonsuppressing strain of S. typhimurium (Israel et al., 1967). In summary, the am2 mutation is in the structural gene for the O-antigen depolymerase and the protein product of this gene is the major structural component of the phage tail. Phage which lack the tail protein are unable to interact with the cell surface. Temperature-sensitive phage tail gene mutants altered in O-antigen depolymerixation. Mutants tsll, ts53, and ts82, when grown at 30”, produce viable progeny IYS

_,'

tsl, 1562

ts53 + om2mlIO

ls62

X(m)

I

-

FIG.8. Genetic map of temperature bacteriophage l . The recombination units shown represent average values from several experimental determinations. The order of markers tsll, ts62, ts53, t&32, am2, and am10 was confirmed by three-factor crosses. Other markers include the following: int, integration function; lys, a lytic function required for release of progeny phage; X(am), an uncharacterized nonsense mutation; and C, an immunity function. The C (immunity) marker is probably a missense mutation within the structural gene for the phage repressor protein, since &%ir (C) does not form plaques on S. netilzgton an EI5 lysogen of S. anatum. Complementation groups were determined as described under Materials and Methods.

INITIAL

INTERACTIONS

OF BACTERIOPHAGE

phage that can be tested for their ability to adsorb to cells, hydrolyze O-antigen, and eject DNA. Such studies revealed nearly identical patterns of behavior for mutants ts53, t&2, and ts82. Figure 9 presents data obtained using phage mutant ts53. The ts53 phage particles adsorbed normally but hydrolyzed O-antigen at less than wild-type efficiency and showed a longer lag prior to initiation of DNA ejection, which then proceeded at a slower than normal rate. For ts53 as well as ts62 and ts82 (data not shown), slow rates of O-antigen depolymerization and DNA ejection were observed whether the incubation temperature was 30 or 38”. Thus it seems unlikely that these abnormalities are responsible for the failure of the mutants to form plaques at 37”. It is more likely a temperaturesensitive defect in head-tail (depolymerase) assembly which results in low plaqueforming ability at 37”. All three mutants produce particles that are deficient in tail protein during growth at 37” (data not shown). Perhaps of greater interest is the fact that each of three different mutants with decreased O-antigen depolymerase activity exhibited a longer lag period prior to the initiation of DNA ejection and a

cl5 WITH

S. anatum

slower rate of DNA ejection once the process had been initiated. A tail gene mutant defective in DNA ejection. Phage tsll produces a low yield of plaque-forming units per infected cell at 37” and is not complemented in viva by nonsensemutant am2. Unlike mutants ts53, ts62, and ts82, tsll phage particles formed at 37” contain a normal amount of depolymerase, or tail protein; however, when partially purified wild-type and tsll depolymerases were prepared from cell lysates by ammonium sulfate precipitation and sucrose gradient sedimentation (see Materials and Methods), the specific activity of the tsll depolymerase in the O-antigen depolymerization assay was only 2% of that for wild-type protein (data not shown). This is consistent with the genetic evidence for the tsll mutation being located in the gene for tail protein (depolymerase). Whereas partially purified tsll depolymerase exhibits low enzymatic activity, whole tsll phage particles hydrolyze O-antigen more rapidly than do wild-type phage. When assayed in Tris-Cl buffer (pH ‘7-8) containing 0.1 ti MgS04, tsll phage particles adsorbed reversibly, depolymerized O-antigen more rapidly and in

TIME (MIN)

FIG. 9. Biochemical

19

characterization of ts53 phage particles. Phage were grown in S. an&urn Al cells at permissive temperature (30”); the assays were the same as those described in Fig. 6. Symbols. ., 0 ts53,,, 0 @vir.

20

MCCONNELL,

REZNICK,

larger quantities than Pvir phage and were completely blocked in DNA ejection (Fig. 10A). Figure 10B shows the results of similar assays carried out in LB broth at 30 and 38”. At 30” tsll phage behaved normally in all three assays, while at 38” they showed reversible adsorption, an abnormally high initial rate of O-antigen depolymerization, and a block in DNA ejection. Thus, both high temperature (38”) and minimum buffer conditions are sufficient to induce pleiotropic changes in the behavior of tsll phage particles, and DNA ejection appears to be most adversely affected.

TIME

AND WRIGHT DISCUSSION

Genetic and biochemical studies have been conducted for the purpose of better understanding the initial interactions that occur between bacteriophage P and its host cell, Salmonella anatum. The data suggest a dependent pathway leading to infection which involves initial attachment of phage via tail protein to O-antigen on the cell surface (adsorption), depolymerization of O-antigen by the phage tail protein, and ejection of phage DNA. Several phage mutants have been isolated which appear to be defective in different stages of the

(MIN)

TIME (mid

FIG.10. Biochemical characterization of tsll phage particles. Phage were grown in 5’. anatum Al at permissive temperature (307, then assayed for adsorption, depolymerization, and DNA ejection either in Tris-Cl buffer (A) or in LB broth (B). Symbols: X, tsll; 0, 8Vir.

INITIAL

INTERACTIONS

OF BACTERIOPHAGE

pathway. All of the mutations map in the same cistron as the am2 mutation. The am2 mutation (and therefore the other mutations as well) clearly lies within the gene for the phage tail protein, as indicated by the following evidence. Nonsense mutant phage am2, when grown on a non suppressing host cell completely lacks phage tail. When such particles are analyzed on SDSpolyacrylamide gels, a single polypeptide with molecular weight equal to that of known depolymerase protein purified in free form from infected cell lysates is missing. No depolymerase-like protein can be isolated from lysates of am2-infected, nonsuppressing cells using the technique of affinity binding to lipopolysaccharide. The phage tail protein must be present in order for phage to interact with the cell surface. Am2 phage particles grown on a nonsuppressing host are unable to adsorb to cells, depolymerize O-antigen, or eject their DNA. When such phage are grown on bacteria that suppress nonsense mutations, the tail protein is restored and the phage regain all of the above functions. There is both biochemical and genetic evidence for initial attachment of phage occurring independently of O-antigen depolymerization. Adsorption of cl5 phage occurs efficiently even at a temperature of 5”, a condition that is strongly inhibitory for O-antigen depolymerization. Furthermore, temperature-sensitive phage mutants ts53, ts62, and ts82, all depolymerize O-antigen at less than wild-type efficiency, yet are able to attach to cells with normal kinetics. Depolymerization of O-antigen appears to be a necessary prerequisite for ejection of phage DNA. Under all conditions so far tested, the reaction kinetics for depolymerization extrapolate back to time zero (not shown); in contrast, lag periods were routinely observed to precede the initiation of DNA ejection, suggesting a temporal arrangement in which depolymerization of O-antigen precedes ejection of phage DNA. In addition, phage mutant ts53 (also ts62 and ts82) exhibits a decreased O-antigen depolmerizing activity and a correspondingly increased lag period prior to the initiation of DNA ejection.

d5 WITH

S. anatum

21

Depolymerization of O-antigen is not enough to guarantee ejection of phage DNA. Conditions such as alkaline or acidic pH are permissive for both adsorption and O-antigen depolymerization, but totally nonpermissive for phage DNA ejection. The situation for e15is therefore similar to that of phage P22, which under acidic conditions hydrolyzes the O-antigen of Salmonella typhimurium lipopolysaccharide without being inactivated (Eriksson and Lindberg, 1977). Presumably, prolonged incubation of e15or P22 phage with their respective host cells at low pH would eventually result in desorption of phage particles from the cell surface due to complete hydrolysis of the O-antigen. Reversible adsorption interactions between phages and their host cells have been recognized for many years. Reversible adsorption of T-phages toE. coli cells was demonstrated by incubating phage and cells in salt-free culture medium (Hershey et aZ., 1944), at low temperature (Garen and Puck, 1951), or in medium that had been depleted of necessary divalent cations (Kozloff and Henderson, 1955). More recent studies suggest that bacteriophage X interacts with its E. coli cell surface receptor protein via a temperature-dependent mechanism that is potentially reversible (Randall-Hazelbauer and Schwartz, 1973; Schwartz, 1975; MacKay and Bode, 1976). The properties of phage mutant tsll appear to define a role for tail protein in the DNA ejection mechanism of bacteriophage e15. When incubated in Tris buffer or in LB broth at 38”, tsll phage particles depolymerize O-antigen at a rate that is more rapid than that of wild-type phage, yet fail to eject their DNA. The phage eventually becomes detached from the cell surface. Takeda and Uetake (1973) suggested that the process of DNA ejection anchors cl5 phage to the cell surface in a manner that precludes further O-antigen depolymerization. Phage tsll at nonpermissive conditions appears unable to eject its DNA and may therefore continue to depolymerize until the local concentration of O-antigen remaining on the cell surface is insufficient to maintain its attachment. It appears than that an interaction between the phage tail and the cell surface,

22

MCCONNELL.

REZNICK,

distinct from either adsorption or O-antigen depolymerization but dependent on both, is required for DNA ejection. Whether it is a direct effect in which the tail itself triggers ejection or an indirect effect is unclear. A series of experiments involving in vitro complementation of P2.2 heads with tails suggest that the tail protein of phage P22, like that of G5, performs a function in addition to adsorption and O-antigen depolymerization which is required for DNA ejection (Israel, 1975). In the case of P22, there is evidence implicating the involvement of additional virion proteins (the products of genes 16 and 20) in DNA ejection and transport through the cell envelope (Hoffman, 1973). Similarly, we have results (unpublished data) which suggest the involvement of at least one other phage protein in the DNA ejection process of phage e15. The above mechanism for irreversible adsorption of e15phage to S. anatum cells predicts the existence of two classes of S. anatum mutants that would be resistant to infection. One class would be lacking or altered in O-antigen, and hence be unable to absorb phage. Such “rough” mutants are known to occur at high frequency. A second class would perhaps be altered in an R-core or lipid A component of the lipopolysaccharide that is required by cl5 phage for irreversible attachment and ejection of phage DNA. Putative S. anatum mutants of this kind have been recently isolated by Kanegasaki and Tomita (1976). In addition, we have isolated a mutant of S. anatum, the lipopolysaccharide of which contains O-antigen, but which exhibits altered phage-inactivating capacity in vitro (McConnell and Wright, 1978). ACKNOWLEDGMENT We thank Dr. Ruth Griffin-Shea for performing the electron microscopy studies, and Dr. A. L. Sonnenschein and Dr. J. T. Park for critical readings of the manuscript. The research was supported by Grant GM15837 from the National Institutes of Health. M. R. M. was a predoctoral trainee for the National Institutes of Health. REFERENCES ADAMS, M. H. (1959). “Bacteriophages.” Wiley-Interscience, New York.

AND WRIGHT

BERTANI, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by lysogenic E. coli. J. Bacterial.

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BOTSTEIN, D. (1968). Synthesis and maturation of phage P22 DNA. I. Identification of intermediates. J. Mol. Biol. 34, 621-641. ERIKSSON,U., and LINDBERG, A. (19’77).Adsorption of phage P22 to Salmonella typhimurium. J. Gen. Viral. 34, 207-221. FUKASAWA, T., and SAITO, S. (1964). The course of infection with T-even phages on mutants of E. coli K12 defective in the synthesis of uridine diphosphoglucose. J. Mol. Biol. 8, 175-183. GAREN, A., and PUCK, T. (1951). The first two steps of the invasion of host cells by bacterial viruses. J. Exper.

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GIESE, A. (1973). “Cell Physiology,” 4th ed., pp. 253-257. Saunders, Philadelphia. GESTELAND, R. F. (1966). Isolation and characterization of ribonuclease I-deficient mutants of Escherichia coli. J. Mol. Biol. 16, 67-84. HALL, D., and TESSMAN, I. (1966). T4 mutants unable to induce deoxycytidylate deaminase activity. Virology

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HERSHEY, A. (1955). An upper limit to the protein content of the germinal substance of bacteriophage T2. Virology 1, 108-127. HERSHEY, A., KALMANSON, G., and BRONFENBRENNER,J. (1944). Coordinate effects of electrolyte and antibody on the infectivity of bacteriophage. J. Immunology 48, 221-239. HOFFMAN, B. (1973). “A Phage P22 Head Protein Which Performs an Essential Early Function.” Ph.D. dissertation, University of Michigan, Ann Arbor. ISRAEL, V., ANDERSON, T., and LEVINE, M. (1967). In vitro morphogenesis of phage P22 from heads and baseplate parts. Proc. Nat. Acad. Sci. USA 57, 284-291. ISRAEL, V., ROSEN, H., and LEVINE, M. (1972). Binding of bacteriophage P22 tail parts to cells. J. Viral. 10, 1152-1158. ISRAEL, V. (1975). Role of tail protein in P22 infection. In “74th Annual Meeting of the Society for Microbiology.” S273, p. 259. IWASHITA, S., and KANEGASAKI, S. (1973). Smooth specific phage adsorption: endorhamnosidase activity of tail parts of P22. Biochem. Biophys. Res. Commun.

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IWASHITA, S., and KANEGASAKI, S. (1975). Release of O-antigen polysaccharide from S. newington by phage c34.Virology 68, 27-34. IWASHITA, S., and KANEGASAKI, S. (1976a). Enzymic and molecular properties of base plate parts of bacteriophage P22. Eur. J. Bioehem. 65, 87-94. IWASHITA, S., and KANEGASAKI, S. (1976b). Deacetylation reaction catalyzed by Salmonella phage ~341 and its base plate parts. J. Biol. Chem. 251, 5361-5365.

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KANEGASAKI, S., and TOMITA, T. (1976). Mutants of 8. anatum which block the phage l 1j infection at early stages. J. Baeteriol. 127, 7-13. KANEGASAKI, S., and WRIGHT, A. (1973). Studies on the mechanism of phage adsorption: Interaction between phage elj and its cellular receptor. Virology 52, 160-173. KOZLOFF, L., and HENDERSON, K. (1955). Action of complexes of the zinc group metals on the tail protein of bacteriophage T2r+. Nature (London) 176, 1169-1171. LAEMMLI, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MACKAY, D., and BODE, V. C. (1976). Binding to isolated phage receptors and DNA release in vitro. Virology

72, 167-181.

MCCONNELL, M., and WRIGHT, A. (1975). An anaerobic technique for increasing bacteriophage plaque size. Virology 65, 588-590. MCCONNELL, M., and WRIGHT, A. (1979). Variation in the structure and phage-inactivating capacity of S. anatum lipopolysaccharide as a function of growth temperature. J. Bacterial., in press. PREHM, P., and JANN, K. (1976). Enzymatic action of coliphage a8 and its possible role in infection. J. Viral.

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PUCK, T., and LEE, H. (1954). Mechanism of cell wall penetration by viruses. I. An increase in host cell permeability induced by bacteriophage infection. J. Exp. Med. 99, 481-494. RANDALL-HAZELBAUER, L., and SCHWARTZ, M. (1973). Isolation of the bacteriophage lambda receptor fromE. coli. J. Bacterial. 116,1436-1446.

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RESKE, K., WALLENFELS, B., and JANN, K. (1973). Enzymatic degradation of 0-antigenie lipopolysaccharide by coliphage 08. Eur. J. Biochem. 36, 167-171. ROBBINS, P., and UCHIDA, T. (1962). Studies on the chemical basis of the basis conversion of O-antigens in the E-group Salmonellae. Biochemistry 1, 323-335.

SCHWARTZ,M. (1975). Reversible interaction between coliphage lambda and its receptor protein. J. Mol. Biol. 99, 185-201. SMITH, H., and LEVINE, M. (1964). Two sequential repressions of DNA synthesis in the establishment of lysogeny by phage P22 and its mutants. Proc. Nat. Acad. Sci. USA 52, 356-363.

STENT, G. (1963). “Molecular Biology of Bacterial Viruses,” pp. 88-115. Freeman, San Francisco. STUDIER, F. (1969). The genetics and physiology of bacteriophage T7. Virology 39, 562-574. TAKEDA, K., and UETAKE, H. (1973). In vitro interaction between phage and receptor lipopolysaccharide: A novel glycosidase associated with Salmonella phage @. Virology 52, 148-159. TAKEDA, K., and UETAKE, H. (1975). Adsorption of Salmonella phage e34and receptor splitting endoglycosidase. Ann. Rep. Inst. Virus Res. (Kyoto) 17, 23-24.

WRIGHT, A. (1971). Mechanism of conversion of the Salmonella O-antigen by bacteriophage $4. J. Bacterial.

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WRIGHT, A., and BARZILAI, N. (1971). Isolation and characterization of non-converting mutants of bacteriophage es4.J. Bacterial. 105, 937-939.