Nonsense mutants of the lipid-containing bacteriophage PR4

VIROLOGY126, 600-613 (1983)

Nonsense Mutants of the Lipid-Containing Bacteriophage PR4 T R I S H A N E L L D A V I S AND J O H N E. C R O N A N , JR. 1

Department of Microbiology, University of Illinois, Urbana, Illinois 61801, and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510 Received November 11, 1982;accepted January 25, 1983 Thirty-three nonsense mutants of phage PR4 representing 12 complementation groups were isolated. One or two mutants of each group were grown on a suppressor-negative (Su-) host and characterized by the following criteria (i) proteins synthesized, (ii) level of phage DNA synthesis, and (iii) ability to assemble particles. We determined the protein and phospholipid compositions of the particles assembled in an Su- host, the presence of DNA in the particles, and the ability of the particles to adsorb to host cells. Finally each complementation group was tested for the ability to lyse an Su- host. We have identified one protein required for DNA synthesis, five proteins required for proper assembly of the protein coat and lipid membrane of the phage, two proteins required for stable insertion of DNA into the virion, a protein required for adsorption, a protein required for attachment of the adsorption protein to the virion, and a phage-encoded lytic enzyme. INTRODUCTION

d o u b l e - s t r a n d e d , l i n e a r D N A molecule of 14,500 b a s e p a i r s ( D a v i s et al., 1982). M u l l e r a n d C r o n a n (1983) have modified t h e c o m p o s i t i o n of t h e p h a g e l i p i d s by altering the host phospholipid composition. These s t u d i e s s h o w e d t h a t a s s e m b l y of t h e p h a g e m e m b r a n e does n o t r e q u i r e a specific p h o s p h o l i p i d c o m p o s i t i o n . To s t u d y t h e role of t h e p h a g e p r o t e i n s in t h i s m o r p h o g e n e s i s we h a v e i s o l a t e d a n d c h a r a c t e r i z e d a collection of n o n s e n s e m u t a n t s of the phage.

The lipid-containing b a c t e r i o p h a g e s provide a s i m p l e s y s t e m to s t u d y t h e a s s e m b l y of a f u n c t i o n a l b i o l o g i c a l m e m b r a n e . PR4 is one of a g r o u p of l i p i d - c o n t a i n i n g p h a g e s t h a t can infect t h e well c h a r a c t e r i z e d h o s t s E s c h e r i c h i a coli a n d S a l m o n e l l a t y p h i m u r i u m . These p h a g e s a r e specific f o r bact e r i a l s t r a i n s h a r b o r i n g a p l a s m i d of t h e P, N, or W i n c o m p a t i b i l i t y g r o u p s ( W h i t e a n d Dunn, 1978). The v i r i o n of PR4 is an i c o s a h e d r a l p a r ticle w i t h a d i a m e t e r of 65 nm ( B r a d l e y a n d R u t h e r f o r d , 1975). I t c o n s i s t s of a p r o t e i n c o a t e n c l o s i n g a m e m b r a n e of p h o s p h o l i p i d a n d p r o t e i n ( L u n d s t r S m et al., 1979; D a v i s et al., 1982). The p h o s p h o l i p i d c o m p o s i t i o n of t h e v i r i o n is q u a l i t a t i v e l y s i m i l a r to t h a t of t h e host. H o w e v e r , PR4 is e n r i c h e d in p h o s p h a t i d y l g l y c e r o l a t t h e e x p e n s e of p h o s p h a t i d y l e t h a n o l a m i n e ( D a v i s et al., 1982). The v i r i o n c o n t a i n s 14 p r o t e i n s . The m a j o r s t r u c t u r a l p r o t e i n of t h e c a p s i d m a k e s up 80% of t h e v i r i o n p r o t e i n ( D a v i s et al., 1982). The g e n o m e is a

MATERIALS AND METHODS

P h a g e Strains, B a c t e r i a l Strains, a n d Plasmids P h a g e PR4 a n d p l a s m i d RP1 w e r e des c r i b e d p r e v i o u s l y ( D a v i s et al., 1982). P l a s m i d pLM2 ( M i n d i c h et al., 1976) is a d e r i v a t i v e of RP1 a n d c a r r i e s k a n a m y c i n r e s i s t a n c e a n d a m b e r m u t a t i o n s in t h e genes f o r a m p i c i l l i n a n d t e t r a c y c l i n e res i s t a n c e . P l a s m i d pTD1, d e r i v e d f r o m pLM2, c a r r i e s genes c o n f e r r i n g r e s i s t a n c e to t e t r a c y c l i n e (10 m g / l i t e r ) a n d k a n a m y c i n (60 m g / l i t e r ) a n d an a m b e r m u t a tion in t h e gene f o r a m p i c i l l i n r e s i s t a n c e . P l a s m i d s RP1, pLM2, a n d pTD1 w e r e

1To whom reprint requests should be addressed. Both authors are currently at the first address. 0042-6822/83 $3.00 Copyright 9 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

600

NONSENSE

MUTANTS OF PHAGE PR4

transferred by conjugation as described (Bradley, et al., 1980). Bacterial strains are listed in Table 1. Strain TD67 was made by transferring the RP1 plasmid to strain MX554 (Oeschger and Wiprud, 1980). Strain W1485 (Coli Genetic Stock Center) was transduced with P1 grown on a strain with TnlO inserted near recA (the gift of N. Kleckner). Strain TD64 was selected as a tetracycline resistant (10 mg/liter), uv-sensitive (200 ergs/ mm 2) transductant. Plasmid RP1 was transferred to strain TD64 giving strain TD76. Strain TDl14 is a thymine-requiring derivative of strain M4131 (Gardner et al., 1974) obtained by trimethoprim selection (Miller, 1972). Strain T D l l 6 is strain M4131 containing RP1. Strain TD127 is strain LE392 (from L. Enquist) containing RP1. Strain TD125 is strain WFK.pJH2 carrying plasmid pTD1. The plasmid pJH2 contains the R (endolysin) genes of phage under transcriptional control of the lactose operon operator (Garrett et al., 1981). When grown in media lacking glucose, strains containing plasmid pJH2 accu-

601

mulate the endolysins and are readily disrupted by a freeze-thaw procedure. Transductions using P l v i r were performed as described previously (Cronan et al., 1972). Media and Buffers Rich broth (RB), low phosphate (LP), and LPC media as, well as ~, dilution buffer were previously described (Davis et al., 1982). TB medium contained 10 g tryptone, 5 g NaC1, 1 mmol MgSOa, and 1 mg thiamine/liter. Medium M9C was medium M9 (Miller, 1972) supplemented with 1 g/liter casein hydrolysate (vitamin and salt free, from ICN), 4 g/liter glucose, and 1 mg/ liter thiamine. For labeling with [2all]glycerol, cells were grown in medium LPF (LP medium supplemented with 10 m g / m l casein hydrolysate and 1 mg/liter thiamine). For labeling with [4,5-SH]leu cine, cells were grown in LPL medium which was a modified LP medium containing 2 mM MgSOa, 10 mg/liter CaC12, 0.1 m M FeSOa (added for growth of Pseudomonas aeruginosa), 4 g/liter glucose, 1 mg/ liter thiamine, a 200-fold dilution of the

TABLE 1 BACTERIAL STRAINS Strain

Relevant characteristics

Source

E. coli 1(-12 TD6 TD17 TD67 TD76 TD114 TDl16 TD125 TD127 TD130

Su § (supE), c a r r i e s p l a s m i d RP1 Su-, c a r r i e s p l a s m i d RP1 Su § (supD), c a r r i e s p l a s m i d RP1 Su-, recA, c a r r i e s p l a s m i d R P 1 Su , thy- (low r e q u i r e m e n t ) , c a r r i e s p l a s m i d RP1 Su , c a r r i e s p l a s m i d RP1 Su-, c a r r i e s p l a s m i d s p J H 2 a n d pTD1 Su + (supE, supF) c a r r i e s p l a s m i d RP1 Su , thy (low r e q u i r e m e n t ) , / e u - , carries p l a s m i d RP1

D a v i s et al., 1982 D a v i s et al., 1982 This study This study This study This This This This

study study study study

Salmonella typhimurium 7155 TDll8 MS1550.pLM2

Su + (supE) 7155, c a r r i e s p l a s m i d pLM2 Su-, uvrB, carries p l a s m i d pLM2

D. Botstein This paper Y o u d e r a i n a n d S u s s k i n d 1980 a n d Mindich et al., 1976

Su , c a r r i e s p l a s m i d pLM2

M i n d i c h et al., 1976

Pseudomonas aeruginosa PAOI.pLM2

602

DAVIS AND CRONAN

a m i n o acid m i x t u r e given below, a n d 50 m g / l i t e r each of g l u t a m i n e , tyrosine, and t r y p t o p h a n . F o r labeling w i t h [35S]m e t h i o n i n e , cells w e r e g r o w n in L P M med i u m which was L P m e d i u m s u p p l e m e n t e d w i t h 4 g / l i t e r glucose, 1 m g / l i t e r t h i a m i n e , 2 m g / l i t e r biotin, the a m i n o acid m i x t u r e diluted 50-fold, and 200 m g / l i t e r each of leucine, isoleucine, t r y p t o p h a n , and t y r o sine. The a m i n o acid m i x t u r e c o n t a i n e d 10 m g / m l of each of the following: alanine, a s p a r t i c acid, g l u t a m i c acid, glycine, histidine, lysine, p h e n y l a l a n i n e , proline, serine, and threonine. All a m i n o acids w e r e the L form.

Phage Propagation P h a g e were g r o w n and t i t e r e d essentially as described (Davis et al., 1982). C h a n g e s in the p r o c e d u r e s are described below. P l a t e l y s a t e s of the a m b e r m u t a n t s w e r e r o u t i n e l y p r e p a r e d using s t r a i n TD6 as the host. The p l a t e s w e r e s c r a p e d into l a m b d a dilution buffer s u p p l e m e n t e d with 10 m M M g S Q and a c r y s t a l of D N a s e w a s added. Stocks w e r e cleared by c e n t r i f u g a t i o n and dialyzed e x t e n s i v e l y a g a i n s t eit h e r 50 m M Tris-HC1 ( p H 8.0) c o n t a i n i n g 0.1 M NaC1 or 50 m M sodium p h o s p h a t e buffer ( p H 8.0) c o n t a i n i n g 0.1 MNaC1. The dialyzed stocks w e r e sterilized by filtration t h r o u g h a p o l y c a r b o n a t e filter (Nucleopore) and stored at 8-10 ~

Mutagenesis Procedure P h a g e PR4 w a s m u t a g e n i z e d with N - methyl - N' - nitro - N - nitrosoguanidine (NTG) using the following procedure. Cells (Su § g r o w i n g in RB or TB w e r e infected w i t h PR4 and a e r a t e d for 15 m i n a t 37 ~ N T G (100 m g / m l ) w a s dissolved in acetone and t h e n added to the culture a t a final c o n c e n t r a t i o n of 1 m g / m l . Cells w e r e aerated for 10 min and t h e n collected by cent r i f u g a t i o n or filtration, washed, resuspended in f r e s h m e d i u m , and d i s p e n s e d into several flasks which w e r e s h a k e n for 4-6 hr. Cultures w e r e t i t e r e d a n d the plaques w e r e picked onto a lawn of an Su § s t r a i n and onto a lawn of an S u - strain. P l a q u e s t h a t w e r e clearly l a r g e r on the Su + s t r a i n t h a n on t h e Su- s t r a i n w e r e

picked and s t r e a k e d . P h a g e t h a t still g r e w m o r e vigorously on t h e Su § s t r a i n w e r e plaque purified at l e a s t twice, t h e n stocks w e r e p r e p a r e d a n d retested. A n y p h a g e m u t a n t h a v i n g an efficiency of p l a t i n g (e.o.p.) on an S u - s t r a i n decreased by less t h a n a f a c t o r of 104 c o m p a r e d to the e.o.p. on an Su § s t r a i n w a s not c h a r a c t e r i z e d f u r t h e r . M u t a n t s w e r e isolated at 30, 33, and 37~ however, all the nonsense m u t a n t s characterized plaqued at 37 ~ and thus all infections w e r e done at 37 ~. A l t h o u g h m u t a n t s were isolated on s t r a i n s c a r r y i n g supD (TD67), supE (TD6), or supE and supF (TD127), all m u t a n t s isolated, except m u t a n t 97 of g r o u p 6, g r e w on s t r a i n TD6 which c a r r i e s only supE. M u t a n t 97 required supF for g r o w t h . M u t a n t s 43 a n d 44 w e r e in the s a m e c o m p l e m e n t a t i o n g r o u p and were isolated f r o m the s a m e m u t a g e n i z e d p h a g e stock; t h e r e f o r e t h e y could be siblings. Similarly, m u t a n t s 56 and 65 could be siblings as could m u t a n t s 100 and 101. None of the o t h e r m u t a n t s are siblings. M u t a n t s w i t h n u m b e r s g r e a t e r t h a n 91 w e r e isolated using a slightly modified p r o c e d u r e in which the stocks w e r e p l a t e d at several dilutions on s t r a i n TD127 (supE, supF) a f t e r r e m o v a l of the m u t a g e n . A f t e r incubation at 33 ~ o v e r n i g h t , plates showing a l m o s t confluent lysis were scraped, resuspended, and cleared of debris as described (Davis et al., 1982). These stocks w e r e t h e n t i t e r e d a n d plaques were picked to identify a m b e r m u t a n t s as described above.

Complementation Analyses The c o m p l e m e n t a t i o n p r o c e d u r e s of H o w e et al. (1979) w e r e modified for PR4. A m b e r m u t a n t s [107-10 s p l a q u e - f o r m i n g units (PFU)] w e r e m i x e d with 0.1 ml of an o v e r n i g h t culture of s t r a i n TD17 (Su-) or TD76 (Su , recA) a n d 2.5 ml of 0.6% a g a r in RB a n d t h e n p l a t e d on antibiotic medium 3. Diluted a m b e r m u t a n t l y s a t e s (10 s P F U / m l ) were s p o t t e d onto the p l a t e s a n d onto control plates c o n t a i n i n g u n i n f e c t e d Su cells. P l a t e s w e r e i n c u b a t e d at 37 ~ I t w a s concluded t h a t two p h a g e m u t a n t s c o m p l e m e n t e d each o t h e r if lysis occurred in the doubly infected spot but not in the

NONSENSE MUTANTS OF PHAGE PR4 singly infected controls. All m u t a n t s having n u m b e r s less t h a n 90 were checked with each o t h e r and used both as the phage in the agar overlay and as the phage spotted on the overlay. M u t a n t s with n u m b e r s g r e a t e r t h a n 90 were checked with one m e m b e r of each c o m p l e m e n t a t i o n group except group 8. In general, i n t e r p r e t a t i o n of the results was very s t r a i g h t f o r w a r d . The few ambiguities were f u r t h e r examined by the liquid c o m p l e m e n t a t i o n test. In the liquid complementation test strain TD17 (Su-) or s t r a i n TD76 (Su , recA) was g r o w n to 50 K l e t t units (1 K l e t t unit equals a p p r o x i m a t e l y 5 • 106 cells/ml) in RB. The bacterial culture (0.1 ml), phage (each mut a n t h a d an m.o.i, of 8), and ~ dilution buffer (for a final volume of 0.12 ml) were mixed and incubated for 20 min at 37 ~ A n t i - P R 4 a n t i s e r u m (final k ~ 0.5) was added to i n a c t i v a t e u n a d s o r b e d phage and the m i x t u r e was incubated for an additional 10 min at 37 ~ The m i x t u r e was then diluted 100-fold in RB and shaken at 37 ~ for 90 min. The cultures were t h e n t i t e r e d on s t r a i n s TD6 (Su +) and TD17 (Su-) or TDl16 (Su). M u t a n t s were assigned to the same comp l e m e n t a t i o n group if the b u r s t f r o m the mixed infection was no more t h a n two-fold g r e a t e r t h a n the combined b u r s t s f r o m the single infections. The exceptions to this rule were the m e m b e r s of group 2, m u t a n t s 9, 26, 76, and 88. Although m u t a n t s 9 and 26 clearly did not complement, all o t h e r pairwise combinations of the m u t a n t s in group 2 produced bursts 5- to 20-fold g r e a t e r t h a n the combined b u r s t s f r o m single infections (burst size was from 0.2 and 2 p h a g e / b a c t e r i u m ) . Only 10% of the phage produced in each mixed infection were r e c o m b i n a n t s or r e v e r t a n t s (capable of m a k i n g plaques on an Su- strain). Mut a n t s 9, 26, 76, and 88 were placed in the same c o m p l e m e n t a t i o n group because all were defective in P2 and were similar in all o t h e r characteristics. The basis of the c o m p l e m e n t a t i o n between the m e m b e r s of group 2 is not understood. P e r h a p s it represents i n t r a g e n i c c o m p l e m e n t a t i o n (Ullm a n and Perrin, 1970). It is also possible (although unlikely) t h a t m u t a n t s 9, 76, and 88 each have lesions in different genes all of which are required for synthesis of P2.

603

Characterization of Particles Assembled in Amber Mutant Infections The protein compositions of the particles assembled in an Su host by m u t a n t s of c o m p l e m e n t a t i o n groups 5-12 were det e r m i n e d by [35S]methionine labeling followed by electrophoresis of the appropriate fraction f r o m the sucrose gradients. S t r a i n PAOI.pLM2 was grown in LPM medium (plus F e S O 0 to 50 K l e t t units and infected (m.o.i. of 15). A f t e r 15 min of furt h e r incubation, 40 m C i / l i t e r of [35S]methionine (40 m C i / m g ) was added for a furt h e r 45 min at which time 5 m M E D T A and 0.01 m g / m l egg-white lysozyme were added to d i s r u p t any unlysed cells. A f t e r addition of MgSO4 and DNase, the lysates were sedimented on 5-20% (w/v) sucrose g r a d i e n t s made up either in 50 m M T r i s HC1 (pH 8.0) c o n t a i n i n g 0.1 M NaC1 or in 50 m M sodium p h o s p h a t e buffer (pH 8.0) containing 0.1 MNaC1. S e d i m e n t a t i o n was p e r f o r m e d in a Beckman SW41 r o t o r at 76,400 g (25,000 rpm) for 50-60 min at 20 ~ The a p p r o p r i a t e fractions were pooled and e i t h e r dialyzed and lyophilized or TCA precipitated and t h e n analysed by electrophoresis (Davis et aL, 1982). F o r d e t e r m i n a t i o n of the phospholipid compositions of particles made in an Suhost, one m e m b e r of each group 6 t h r o u g h 12 was used to infect (m.o.i. = 8) a portion of a culture of s t r a i n TD17 grown for at least five g e n e r a t i o n s in m e d i u m LPC supplemented with 50 m C i / l i t e r ~2Pi (167 mCi/ retool). A f t e r 2 h r the lysates were cleared and the phage were i m m u n o p r e c i p i t a t e d with antibody to PR4 (10 #1 a n t i s e r u m / m l of lysate). The cells infected with group 11 were lysed 3 h r a f t e r infection with 1 m M EDTA, 15 ttg/ml lysozyme, and then the phage were i m m u n o p r e c i p i t a t e d . The imm u n o p r e c i p i t a t e f r o m each sample was washed twice, c a r r i e r cells were added, and the phospholipids were e x t r a c t e d (Davis et al., 1982). The phospholipids were separ a t e d by one-dimensional t h i n - l a y e r chrom a t o g r a p h y and the a p p r o p r i a t e areas of silica gel were scraped f r o m the plates and counted (Davis et al., 1982).

Assay for Lysis of Host Each group was checked for its ability to lyse an Su host. A culture of s t r a i n

604

DAVIS AND CRONAN

TD17 growing in RB medium was infected (m.o.i. of 10). After incubation for 2 hr at 37 ~ the optical density (in Klett units) was read. Lysates with turbidities t ha t had decreased to <35 Klett units were considered lysed, whereas those >60 Klett units were considered not to have lysed.

Antibody Preparation and Immunoprecipitation Antibodies were prepared as described by Livingston (1974). To immunoprecipitate phage or cellular debris, the appropriate antiserum and antigen were mixed and incubated at 8 ~ for at least 20 min. Then protein A covalently coupled with Sepharose CL-4B (usually 0.3 mg/td of serum) was added as a slurry. The Sepharose beads settle quickly, so the mixture was gently vortexed every 5 min for 20 min and then stored overnight at 8% The antigen-antibody-protein A-Sepharose bead complex was pelleted in an Eppendorf centrifuge. RESULTS AND DISCUSSION

Identification of PR4 Proteins Synthesized in an Irradiated Host Before characterizing the nonsense mutants, we identified the proteins synthesized by wild type PR4 infecting a uv-irradiated uvrB host. We detected 20 proteins synthesized by infected uv-irradiated cells but not by the uninfected uv-irradiated host. Four proteins (P1A, P5A, P8A, and P l l B ) were synthesized both early and late during infection whereas the other 16 proteins were synthesized only late in infection (Fig. 1). Seven of the late proteins (P1, P3, P4, P6, PT, P10, and P13) were identified as structural components of the m a t u r e virion (Davis et al., 1982). Each of these proteins comigrated with a virion protein in SDS-gel electrophoresis. These proteins are clearly PR4 coded because phage nonsense mutations resulted in both the loss of the protein from infected cells and the loss of the protein from the noninfectious particles assembled in an Suhost (see below). The major ( ~ 8 0 % ) capsid protein P2 was identified in infected cells by the large amount synthesized as well as by its characteristic electrophoretic mobility. As expected, mutants lack-

ing this protein accumulated no particles in an Su- host. We were assisted in identifying several of the proteins made in infected cells by use of phage mutations t h a t altered the electrophoretic mobilities (in SDS-gels) of certain proteins. Cells infected with phage carrying such a mutation did not synthesize one of the wild type phage proteins but synthesized an extra protein not found in wild-type-infected cells. In each case, the extra protein was identified as a modified version of the missing wild type phage protein because the extra protein and the wild type protein (i) had similar mobilities on SDS-gels, (ii) were made in similar quantities, (iii) were synthesized at the same time during the infectious cycle, and (iv) had very similar methionine peptide patterns [upon SDS-gel electrophoresis after partial proteolysis with either V8 protease or chymotrypsin (Cleveland et al., 1977)]. The change in mobility was seen in both Su- and Su § hosts and therefore was not due to a nonsense mutation. Protein P5 comigrated with a virion protein and a m u t a n t t hat synthesized a P5 with an altered mobility assembled particles with a similarly shifted protein thus allowing positive identification. Protein PSA comigrated with the virion protein P8 but was not P8 because m u t a n t 47 which produces a P8A with an altered electrophoretic mobility (as well as bearing a nonsense mutation in P6) assembled particles containing P8 with a normal mobility (a minor band corresponding to P8 can be seen in cells infected with m u t a n t 47). Similarly P9A was not the virion protein P9 because m u t a n t 40, which carries an amber mutation in the gene encoding P9A, assembled P9 containing particles in an Su- host. Proteins P12A and P14A comigrated with virion proteins P12 and P14 and thus may be virion proteins but in the absence of suitable m ut ant s were not f u r t h e r analyzed. The minor virion protein P l l was obscured by a protein synthesized by the irradiated host. The 20 phage-specific proteins identified in infected cells plus the 3 virion proteins (P8, P9, and P l l ) that were obscured by other proteins account for 93% of the cod-

NONSENSE MUTANTS OF PHAGE PR4

605

FIG. 1. Proteins synthesized in uv-irradiated cells. Strain MS1550.pLM2 was grown to 50 Klett units in LPM and uv irradiated at a dose of 2000-3000 e r g s / m m 2. The culture was aerated for 10 min and divided into portions each of which was infected (m.o.i. of 12-15) with a member of one of the complementation groups (1-12) or wild type phage (wt). One portion was left uninfected (un). Early proteins were labeled for 20 min starting 1 min after infection by adding 0.1 mCi/ml [~S]methionine (0.4 Ci/mg) to a portion (0.2 ml) of a culture. Late proteins were labeled for 30 min starting 40 rain after infection by adding 0.05 mCi/ml [~S]methionine (0.2 Ci/mg) to a portion (0.2 ml) of each culture. The labeling was terminated by the addition of methionine (0.5 mg/ml) and phenylmethylsulfonyl fluoride (1 mM) and the proteins were precipitated with 5% TCA and stored on ice until the end of the experiment. All samples were then pelleted in an Eppendorf centrifuge for 3 rain and washed once with 5% TCA followed by careful removal of the final drops of acid. Using this procedure, between 70% and 90% of the TCA precipitable counts were recovered. The pellets were resuspended in 1% SDS in 100 m M Tris-HC1 (pH 8.0), 0.006% bromophenol blue and stored at -70 ~ until electrophoresis on SDS-polyacrylamide gradient gels as described (Davis et at., 1982). The following mutants were used: group 1, 81; group 2, 76, 9; group 3, 46; group 4, 14; group 5, 16; group 6, 72; group 7, 41; group 8, 61; group 9, 84; group 10, 101; group 11, 40; group 12, 71. The minor band with molecular weight of 37,200 produced by group 2 mutants infecting uvirradiated cells coincided in size and amount with a host protein synthesized by uninfected cells. This gel does not clearly show this point because fewer counts of the mutant-76 extract were loaded than of the mutant-9 extract. As expected, this minor host protein was synthesized by all group 2 mutants (data not shown). Also not clearly seen in this reproduction, group 9 synthesized both protein P1 and protein P1A, whereas group 8 only synthesized protein P1A.

ing c a p a c i t y of the p h a g e genome, a s s u m ing no o v e r l a p p i n g genes.

Isolation and Characterization of Nonsense Mutants P h a g e stocks m u t a g e n i z e d w i t h N T G w e r e s c r e e n e d for p h a g e able to plaque on

a host c a r r y i n g an a m b e r s u p p r e s s o r (Su +) b u t unable to plaque on a h o s t lacking a s u p p r e s s o r (Su-). One h u n d r e d nonsense m u t a n t s were isolated f r o m 11,000 plaques. The m a j o r i t y of the m u t a n t s w e r e v e r y leaky and w e r e not f u r t h e r characterized. T h i r t y - t h r e e nonsense m u t a n t s t h a t plated >10a-fold m o r e efficiently on an Su + s t r a i n

606

DAVIS A N D CRONAN

t h a n on an S u - s t r a i n w e r e identified and characterized. The 33 m u t a n t s w e r e placed in 12 comp l e m e n t a t i o n groups. The p r o t e i n ( s ) affected by each g r o u p of n o n s e n s e m u t a n t s w a s identified in a u v - i r r a d i a t e d , S u - host (Fig. 1). In each case, a single s t e p r e v e r t a n t ( t h a t could grow on an S u - host) synthesized the protein(s), thus indicating t h a t the n o n s e n s e m u t a t i o n caused t h e loss of the protein(s). To d e t e r m i n e the function of the defective protein, one or two m e m bers of each c o m p l e m e n t a t i o n g r o u p were g r o w n on an S u - h o s t and the a m o u n t of D N A s y n t h e s i z e d (Fig. 2) as well as the c o m p o s i t i o n of a n y p a r t i c l e s a s s e m b l e d (Fig. 3) w a s d e t e r m i n e d . The ability of the p a r t i c l e s to a d s o r b to host cells w a s also e x a m i n e d (Table 2). Finally, each g r o u p w a s tested for its ability to lyse an Suhost.

A protein required for viral D N A synthesis. Nalidixic acid inhibits replication of the E. col• c h r o m o s o m e by i n a c t i v a t i n g

i

i

I

,,

I

i

i

i

4.0

80

uninfected

~

89

F-

c~ ~_j

40

so

4o so Time (n~in)

FIG. 2. DNA s y n t h e s i s by nonsense m u t a n t s . Strain T D l l 4 was g r o w n in LPC supplemented with thymine (2 mg/1) to 55 Klett units. Nalidixic acid (40 mg/1) was added to inhibit the h o s t D N A replication. A f t e r 10 min of incubation, the culture w a s divided. Each portion w a s infected (m.o.i. of 15) w i t h a m u t a n t phage of one of the complementation g r o u p s (1-12), or wild type phage (wt), or left uninfected. Then 8 m C i / l i t e r of [methyl-Nil]thymine was added. Samples (0.02 ml) were removed and pipetted onto filter disks soaked in t h y m i n e (2 m g / m l ) and 5% TCA. The filter disks were w a s h e d 3 times with cold 5% TCA (20 ml per filter disk per wash), w a s h e d once w i t h cold 95% ethanol, dried, and counted in Econofluor. The same m u t a n t s were used as in Fig. 1 with the following exceptions: group 1, 56; group 2, 76; g r o u p 6, 10; group 7, 15; and g r o u p 10, 60.

TABLE 2 ADSORPTION

OF PARTICLES

BY NONSENSE

PRODUCED

MUTANTS

Percentage particles adsorbed a Group

32pi

[3H]thymine

8 9 10 11 12 wt b

11 10 38

11 • 17 12 • 13 62• 1 56 • 9 67 • 1 53• 5

62 61

[14C]leucine 4 • 5 • 37• 20 • 41 • 34•

24 15 17 10 6 10

a Presented as m e a n • SD. b Wild type phage. Note. The particles assembled by the phage a m b e r m u t a n t s growing in s t r a i n TD17 (Su-) were labeled with 32/)i as described u n d e r Materials and Methods. Alternatively, the particles were labeled with [methyl3H]thymine and [14C]leucine by infecting strain TD130 (Su-, thy , and leu ) g r o w i n g in a modified LPM medium containing 20 m C i / l i t e r [Nil]thymine (~1000 m C i / m m o l ) and 1.1 m C i / l i t e r [14C(U)]leucine or [1'ac]leucine (9.6 m C i / m m o l ) . Lysates were cleared by centrifugation. Aliquots of the cleared lysates were mixed with 1 ml of exponentially g r o w i n g cells ( T D l l 8 ) carrying PLM2, the plasmid required for adsorption. As a control for nonspecific adsorption (which was minimal), lysates were also mixed with a culture of s t r a i n 7155 which does not carry PLM2. The m i x t u r e s were incubated for 25-30 min at 37 ~ and centrifuged to pellet the cells. The unadsorbed particles in the s u p e r n a t a n t s were immunoprecipitared with antibody to PR4 (6.3 t d / m l s u p e r n a t a n t ) as described under Materials and Methods. The immunoprecipitate w a s w a s h e d two or three times with 0.5 ml of 50 m M Tris-HC1 (pH 8.0) and counted. The fraction of total immunoprecipitable m a t e r i a l removed from the lysates by exposure to cells c a r r y i n g PLM2 but not removed by exposure to cells lacking PLM2 was taken as the fraction of particles adsorbed by the cells.

D N A g y r a s e (Gellert, 1981). However, nalidixic acid a p p a r e n t l y does not inhibit replication of the PR4 g e n o m e because infected cells ( t h y ) i n c o r p o r a t e d [methyl3H]thymine a t twice the r a t e seen in uninfected cells (Fig. 2). We thus were able to a s s a y PR4 D N A s y n t h e s i s with m i n i m a l b a c k g r o u n d f r o m h o s t s y n t h e s i s by addition of this inhibitor. Cells infected w i t h m u t a n t s in g r o u p 1 i n c o r p o r a t e d v i r t u a l l y no [3H]thymine, w h e r e a s cells infected with a m e m b e r of each o t h e r g r o u p incorpo-

NONSENSE MUTANTS OF PHAGE PR4 rated more [3H]thymine t ha n uninfected cells (Fig. 2). Upon infection of a uv-irradiated, Su- host, the m u t a n t s of group i did not synthesize the early protein P1A (Fig. 1) or any of the late proteins (in a few experiments, small amounts of the late proteins were synthesized). Single-step revertants able to grow on an Su- host synthesized all 20 proteins. The most straightforward explanation of these data is t h a t group 1 has a nonsense mutation in protein P1A which is required for viral DNA synthesis. The fact t hat none (or a small amount) of the late proteins were made may reflect that few DNA templates were available for phage mRNA synthesis. Alternatively, P1A may be required to activate the genes coding for the late proteins. Cells treated with nalidixic acid and infected with group 1 incorporated 90% less [3H]thymine than uninfected cells treated with nalidixic acid (Fig. 2). This was not due to degradation of the host DNA because host DNA labeled before infection was stable (data not shown). The inhibition of host DNA synthesis by group 1 did not require nalidixic acid because in the absence of the inhibitor, cells infected with group 1 incorporated 80% less [3H]thymine in 80 min than uninfected cells (data not shown). Thus, inhibition of host DNA synthesis may be a function of one or more of the three early proteins (P5A, PSA, and P l l B ) synthesized by the group 1 mutants.

Proteins required for particle assembly. Previously, we have shown t h a t two types of particles separable by velocity sedimentation are found in lysates of wild type PR4 (Davis et at, 1982). The faster sedimenting particles are infectious and contain phospholipid, protein, and DNA. The slower sedimenting particles contain phospholipid, all the virion proteins except P6, but lack DNA (Davis et al., 1982; also see Fig. 3). We assayed each of the m u t a n t groups for particle formation in an Suhost strain. Mutants of four complementation groups (groups 1 to 4) did not accumulate particles evident on sucrose velocity gradients (Fig. 3). As discussed above group 1 mutants are defective in phage DNA synthesis and synthesize only traces of the virion

607

proteins. Group 2 mutants lacked the major capsid protein, P2 (Fig. 1). The group 2 mutants also had decreased synthesis of P13 but since m u t a n t s t h a t synthesized P2 but not P13 accumulated particles (see below), the particle assembly defect of the group 2 m u t a n t s is attributed to the lack of P2. The defect in the group 3 nonsense mutants was the loss of protein P6A (Fig. 1), a protein not found in the mature virion. The protein was synthesized in large quantities in the infected cells and could be a core protein analogous to gp8 in phage P22 or gpNu3 of phage ~ (Murialdo and Becker, 1978). However, no evidence for a transient association of P6A with the particle is available. We have not yet identified a protein missing in Su- cells infected with the nonsense m u t a n t of group 4. The one member of this group had a protein P7 of altered mobility but this lesion was not a nonsense mutation (see above). The one nonsense m u t a n t of group 5 failed to synthesize two minor virion proteins P4 and P10, in an Su- host (Fig. 1). We have not isolated a m u t a n t deficient in the synthesis of only P4 or P10. In the absence of P4 and P10, a few particles containing virion proteins P2, P5, PT, P9, P l l , P12, P13, and P14 plus phospholipid were assembled. These particles sedimented in a broad peak at approximately the same position as the wild type particles that lack DNA (Fig. 3). Because the particles were heterogeneous, we believe t h a t they are products of an a b e r r a n t assembly process and thus P4 and P10 are required for proper assembly of the virion. Proteins involved in DNA packaging. The particles accumulated by mutants in groups 6-12 infecting an Su- host formed sharp, discrete peaks on sucrose gradients. These m ut ant s can be divided into those that assembled only particles lacking DNA and those t h a t accumulated particles containing DNA. Mutants of groups 6 and 7 accumulated particles having similar sedimentation properties to those of the DNA-less particles found in lysates from cells infected with wild type phage (Fig. 3). The particles found in group 6 lysates had a normal phospholipid composition and lacked virion proteins P5, P6, P8, and P13 as well

608

DAVIS AND CRONAN

4

E o F

x

E (J

b

v

x

o (.9

o9

15 I0 5

I0

20

Io 20 Fraction Number

I0 b

20

I0

2o

Fraction Number

FIG. 3. Velocity s e d i m e n t a t i o n of n o n s e n s e m u t a n t lysates. A: P r o t e i n - c o n t a i n i n g particles. A c u l t u r e of s t r a i n P A O I . p L M 2 w a s g r o w n a t 37 ~ to 60 K l e t t u n i t s in L P P a n d divided into p o r t i o n s each of w h i c h w a s infected a t a n m.o.i, of 15 w i t h a m e m b e r of one of t h e c o m p l e m e n t a t i o n g r o u p s (1-12) or wild type p h a g e (wt). One portion w a s left uninfected. A f t e r 30 m i n of a e r a t i o n a t 37 ~ 40 m C i / l i t e r [4,5-SH]leucine (20 m C i / m g ) w a s added. A f t e r 25 rain of labeling, t h e cells were lysed by 5 m M E D T A a n d 0.15 m g / m l e g g - w h i t e lysozyme. A f t e r 30 m i n at 25 ~ MgS04 (20 m M ) a n d a c r y s t a l of D N a s e were added a n d t h e l y s a t e s s t o r e d a t 10 ~ o v e r n i g h t . T h e l y s a t e s were s e d i m e n t e d t h r o u g h 5-20% (w/v) sucrose g r a d i e n t s in 50 m M T r i s - H C l (pH 8.0) c o n t a i n i n g 0 4 M NaC1 in a B e c k m a n SW41 r o t o r for 50 m i n at 76,400 g (25,000 r p m ) a n d 20 ~ F r a c t i o n s (11 drops) were collected. A 0.05-ml s a m p l e of each fraction w a s m i x e d w i t h w a t e r (0.2 ml) a n d P C S ( A m e r s h a m ) (3.8 ml) a n d counted. T h e s a m e m u t a n t s were used in Fig. 1 w i t h t h e following exceptions: g r o u p 1, 56; g r o u p 2, 76; g r o u p 10, 89; a n d g r o u p 11, 44. B: P h o s p h o l i p i d - c o n t a i n i n g particles. To avoid u s i n g E D T A w h i c h we f e a r e d m i g h t d i s r u p t p h o s p h o l i p i d vesicles if t h e y were produced, m u t a n t s in g r o u p s 1, 5, a n d 11, w h i c h could n o t lyse h o s t cells w e r e g r o w n on s t r a i n TD125 (Su-) w h i c h could be easily lysed by a f r e e z e - t h a w procedure. A c u l t u r e of s t r a i n TD125 w a s g r o w n in m e d i u m L P C (a g l u c o s e - c o n t a i n i n g m e d i u m ) to 80 K l e t t units, filtered, w a s h e d a n d diluted five-fold into L P F m e d i u m (which lacks glucose a n d t h u s induces t h e endolysins). T h e d i l u t e d c u l t u r e w a s g r o w n to 60 K l e t t u n i t s a n d divided into p o r t i o n s each of w h i c h w a s infected (m.o.i. of 15) w i t h a m e m b e r of g r o u p 1, 5, or 11, or wild type p h a g e (wt 2). T h e infected c u l t u r e s were labeled w i t h 20 m C i / liter of [2-SH]glycerol (10 C i / m m o l ) f r o m 45 m i n to 2 h r p o s t i n f e c t i o n a n d lysed by f r e e z i n g in liquid Nu a n d t h a w i n g at room t e m p e r a t u r e (wt 2 lysed w i t h o u t t h e f r e e z e - t h a w procedure). T h e m u t a n t s of all o t h e r g r o u p s lysed s t r a i n T D l l 6 (Su-). A c u l t u r e of s t r a i n T D l l 6 w a s g r o w n to 100 K l e t t u n i t s in L P F m e d i u m s u p p l e m e n t e d w i t h 0.2 m g / m l glycerol, filtered, w a s h e d , diluted fivefold a n d allowed to grow to 60 K l e t t u n i t s in L P F m e d i u m w i t h o u t glycerol before it w a s divided a n d infected (m.o.i. of 10). Infected c u l t u r e s were labeled w i t h 20 m C i / l i t e r [2-~H]glycerol (10 C i / m m o l ) f r o m 7 m i n p o s t i n f e c t i o n u n t i l lysis. All t h e l y s a t e s were cleared by c e n t r i f u g a t i o n (2 m i n , E p p e n d o r f ) followed by i m m u n o p r e c i p i t a t i o n w i t h anti-E, coli a n t i s e r u m (20 ~ l / m l cell s u p e r n a tant). The cleared l y s a t e s were loaded on 5-20% (w/v) s u c r o s e g r a d i e n t s f o r m e d in 50 m M s o d i u m

NONSENSE MUTANTS OF PHAGE PR4 as D N A . T h e s e m u t a n t s did n o t s y n t h e s i z e P13 b u t s y n t h e s i z e d P5 a n d P 6 (Fig. 1) (P6 h a d a n a l t e r e d m o b i l i t y ) . ( T h e s y n t h e s i s of P8 c o u l d n o t be a s c e r t a i n e d . ) 2 T h u s P13 ( a n d / o r P8) a r e r e q u i r e d f o r a s s e m b l y of p r o t e i n s P5 a n d P6, a n d D N A i n t o t h e v i r a l particle. The particles a s s e m b l e d in S u - s t r a i n s i n f e c t e d w i t h m u t a n t s of g r o u p 7 w e r e i n distinguishable from the DNA-less particles f o u n d i n l y s a t e s of t h e w i l d t y p e p h a g e a n d c o n t a i n e d all t h e v i r i o n p r o t e i n s e x c e p t P6. T h e s e m u t a n t s w e r e d e f e c t i v e i n p r o t e i n P 6 (Fig. 1). T h u s , P 6 is r e q u i r e d for p a c k a g i n g t h e v i r a l D N A . A l l t h e p a r ticles t h a t l a c k D N A , w h e t h e r p r o d u c e d b y m u t a n t s or w i l d t y p e PR4, also l a c k P6. S i m i l a r l y , all p a r t i c l e s l a c k i n g P 6 also lack D N A . T h u s , P 6 a p p e a r s to be d i r e c t l y i n volved in the D N A packaging, a n d the ins e r t i o n of P6 a n d p a c k a g i n g of D N A i n t o the virion seem interdependent.

Proteins involved in adsorption o f the virion to the host cell. N o n s e n s e m u t a n t s i n t h e g r o u p s 8, 9, 10, 11, a n d 12 a s s e m b l e d particles containing phospholipid, protein

2Mutants 10 and 72 of group 6 did not synthesize either P6 or P13. However, the band corresponding to P6A was characteristically broader in the mutant 10 and 72 infections than that seen in wild type infections (Fig. 1). Infection of an Su- strain with a revertant of mutant 72 gave P13 synthesis but no synthesis of P6 was seen and the abnormally broad P6A band remained. Infection of an Su§ strain with mutant 72 gave the same result. Thus, the mutants of group 6 appear to carry an amber mutation in the gene encoding P13. It also seems that either P6 is not required for phage growth or mutants 10 and 72 produce an altered P6 having an electrophoretic mobility similar to that of P6A. We favor the latter explanation because group 7 mutants have amber mutations in P6 and, hence, P6 seems essential for growth. It should be noted that P8 is obscured by P8A and thus it remains possible that in mutants 10 and 72, the gene encoding P8 could carry an amber mutation affecting P13 synthesis.

609

a n d D N A (Fig. 3). T h e p h o s p h o l i p i d c o m p o s i t i o n s of t h e p a r t i c l e s w e r e i d e n t i c a l to t h e p h o s p h o l i p i d c o m p o s i t i o n of w i l d t y p e phage (data not shown). Nonsense mut a n t s i n g r o u p s 8, 9, 10, a n d 11 w e r e def e c t i v e i n t h e v i r i o n p r o t e i n s P1, P3, a n d P 7 a n d t h e n o n v i r i o n p r o t e i n P9A, r e s p e c t i v e l y (Fig. 1). T h e p r o t e i n d e f e c t of p h a g e i n g r o u p 12 h a s n o t b e e n i d e n t i f i e d . T h e five p r o t e i n s r e p r e s e n t e d b y t h e s e g r o u p s a r e n o t e s s e n t i a l f o r p a r t i c l e a s s e m b l y or D N A packaging, b u t are involved in some other required viral function. The particles a s s e m b l e d in infections w i t h m u t a n t s of g r o u p 8, 9, 10, 11, o r 12 w e r e a s s a y e d f o r t h e a b i l i t y to a d s o r b to h o s t cells, T h e p a r t i c l e s p r o d u c e d b y g r o u p 10, 11, or 12 a d s o r b e d to h o s t cells, w h e r e a s t h e p a r t i c l e s p r o d u c e d b y g r o u p 8 or 9 w e r e d e f e c t i v e i n a d s o r p t i o n ( T a b l e 2). 3 P h a g e i n g r o u p 8 did n o t s y n t h e s i z e p r o t e i n P1 (Fig. 1) a n d , i n a n S u - h o s t , a s s e m b l e d p a r ticles l a c k i n g p r o t e i n P1. P h a g e i n g r o u p 9 did s y n t h e s i z e p r o t e i n P1 b u t d i d n o t s y n t h e s i z e P3 (Fig. 1); h o w e v e r , p a r t i c l e s a s s e m b l e d b y g r o u p 9 l a c k e d P1 as well as P3. T h u s , p r o t e i n P1 w a s r e q u i r e d f o r ads o r p t i o n of t h e v i r i o n to t h e h o s t cells a n d P3 w a s r e q u i r e d f o r a t t a c h m e n t of P1 to t h e v i r i o n . T h e r e a r e 9 copies of P1 p e r v i r i o n a n d 39 copies of P3 p e r v i r i o n ( D a v i s et al., 1982). T h e i r a r r a n g e m e n t i n t h e vir i o n is u n k n o w n . T a i l - l i k e s t r u c t u r e s app e a r o n s o m e of t h e v i r i o n s i n e l e c t r o n m i s The results were disappointingly variable, but in each set of trials (one using s2p-labeled particles and four using 3H- and 14C-labeledparticles) the particles produced by mutants in group 8 or 9 adsorbed less than either wild type phage or the particles produced by mutants in group 10, 11, or 12, usually >threefold less. This was true whether we assayed phospholipid and DNA (32p-labeled), DNA alone ([methyl3H]thymine labeled) or protein ([14C]leucine-labeled). Thus, we believe the particles produced by mutants in either group 8 or group 9 are defective in adsorption.

phosphate buffer (pH 8.0) containing 0.1 MNaCl. The gradients contained 0.4 ml of a 60% sucrose solution as a cushion. Sedimentation was performed at 76,400 g (25,000 rpm) for 1 hr at 20~ in an SW41 rotor. The gradients were fractionated and counted as described in A. Lysates of groups 1, 5, and 11, and wild type phage (wt 2) were prepared using strain TD125 as the host. Lysates of all other groups and wild type phage (wt 1) were prepared using strain TDll6 as the host. The same mutants were used as in Fig. 1 with the following exceptions: group 1, 56; group 2, 9; group 7, 15; and group 10, 60.

610

DAVIS AND CRONAN

c r o g r a p h s ( L i i n d s t r o m et al., 1979; M u l l e r , 1981; B r a d l e y a n d R u t h e r f o r d , 1975), b u t whether these structures represent fragile tails or pilus fragments (Coetzee and Bekk e r , 1979) is u n k n o w n .

TABLE 3 PHAGE LYTIC ENZYME ACTIVITY

Lysozyme activity

Phage/host

units mg protein

units ml extract

Mutant 40/Su Wild type/SuMutant 40/Su § Wild type/Su §

<0.25 20 6.5 17

<0.03 3.6 0.97 2.7

Identification of a phage lytic enzyme. R e p r e s e n t a t i v e s of e a c h c o m p l e m e n t a t i o n g r o u p w e r e t e s t e d f o r t h e a b i l i t y to l y s e a n Su h o s t . G r o u p s 1, 5 , a n d 11 w e r e d e f e c t i v e in l y s i s a n d c o u l d n o t be l y s e d w i t h chloroform. Upon lysozyme-EDTA treatm e n t , t h e g r o u p 11 m u t a n t s r e l e a s e d i n f e c t i o u s p a r t i c l e s , b u t t h o s e of g r o u p s 1 a n d 5 d i d not. P r o t e i n P 9 A w a s m i s s i n g f r o m g r o u p 11 i n f e c t i o n s of a n S u h o s t ( F i g . 1) a n d h e n c e , w a s t e n t a t i v e l y i d e n tified as a phage lytic enzyme. Extracts w e r e m a d e f r o m a n Su s t r a i n (TD17) i n f e c t e d w i t h w i l d t y p e p h a g e o r m u t a n t 40 of g r o u p 11. T h e s e e x t r a c t s w e r e a s s a y e d f o r c e l l u l a r l y t i c a c t i v i t y a s d e s c r i b e d in T a b l e 3. T h e e x t r a c t of c e l l s i n f e c t e d w i t h the wild type phage had considerable lytic a c t i v i t y w h e r e a s t h a t of m u t a n t 4 0 - i n f e c t e d c e l l s h a d no d e t e c t a b l e a c t i v i t y . H o w e v e r , u p o n g r o w t h on a s t r a i n c a r r y i n g supE, e x t r a c t s of m u t a n t 4 0 - i n f e c t e d c e l l s h a d a p p r o x i m a t e l y 35% of t h e l y t i c a c t i v i t y o f w i l d t y p e e x t r a c t s ( T a b l e 3), a l e v e l of e x p r e s s i o n c o n s i s t e n t w i t h t h e eff i c i e n c y of t h e s u p p r e s s o r ( G o r i n i , 1970). S i n c e t h e m u t a n t s of g r o u p 1 m a d e n o n e of t h e l a t e p r o t e i n s i n c l u d i n g P 9 A i t is n o t s u r p r i s i n g t h a t no l y s i s o c c u r r e d . T h e g r o u p 5 m u t a n t s d i d n o t l y s e t h e Su h o s t , although protein P9A was synthesized ( F i g . 1). T h e i n f e c t e d c e l l s a c c u m u l a t e d only a few noninfectious particles and could not be lysed with chloroform. Thus, this mutant does not represent a second lyric factor such as that encoded by the t gene of p h a g e T4 ( J o s s l i n , 1970) o r t h e S g e n e of p h a g e ~ ( H a r r i s et al., 1967). Other proteins. G r o u p 10 n o n s e n s e m u tants did not synthesize the virion protein P7 ( F i g . 1) a n d a s s e m b l e d n o n i n f e c t i o u s p a r t i c l e s l a c k i n g P7. T h e specific f u n c t i o n o f P7 is u n k n o w n . T h e m u t a n t in g r o u p 12 assembled noninfectious particles otherwise indistinguishable from the wild type virion. The defective protein has not been i d e n t i f i e d ; h o w e v e r , t h e g r o u p 12 m u t a n t d i d s y n t h e s i z e a p e p t i d e n o t p r e s e n t in w i l d type infections. This peptide may repre-

Note. The substrate of tyophilized cells was prepared as described (Tsugita et al., 1968). Just before assaying, the lyophilized cells were resuspended in 50 mM sodium phosphate buffer, pH 7.0, to an ODs~o of approximately 0.4. To prepare the extracts to be assayed, strain TD17 ( S u ) or strain TD6 (Su § was grown in M9C medium to 60 Klett units. The culture was divided in half. One half was infected with wild type PR4; the other half was infected with a member of group 11, mutant 40. After 90 min, the cells were passed through a French press at 700 lb/in. 2. The lysates were cleared in a Ti 70.1 rotor for 70 min at 109,000 g at 5 ~ This treatment removed greater than 99% of the infectious particles but less than 5% of the lytic activity. Dilutions of the cleared extract (0.1 ml) and substrate (0.6 ml) were mixed in a cuvette (1-cm path length) and the decrease in absorbance at 350 nm was followed. The enzyme activity was determined at several extract concentrations that gave proportional rates of absorbance decrease. A unit of PR4 lytic activity is defined as the rate of absorbance decrease given by 1 #g of egg-white lysozyme (Sigma) under the same conditions. One microgram of eggwhite lysozyme caused a decrease in absorbance of approximately 0.75 A/min. The extracts were dialyzed before determination of the protein concentration by a microbiuret procedure (Munkres and Richards, 1965). s e n t a n a m b e r f r a g m e n t of a p r o t e i n t h a t is o b s c u r e d b y o t h e r p r o t e i n s in o u r g e l system. CONCLUSION W e h a v e i s o l a t e d n o n s e n s e m u t a t i o n s in 12 p h a g e g e n e s r e p r e s e n t i n g m o r e t h a n 60% o f t h e c o d i n g c a p a c i t y of t h e P R 4 g e nome. We have identified proteins inv o l v e d in D N A s y n t h e s i s , p a r t i c l e a s s e m bly, D N A p a c k a g i n g , a d s o r p t i o n to t h e h o s t cell, a n d l y s i s of t h e h o s t cell. T h e r e s u l t s a r e s u m m a r i z e d in T a b l e 4. T h e f u n c t i o n s of s e v e r a l of t h e p h a g e p r o t e i n s r e m a i n

NONSENSE

MUTANTS

+

+

+

OF PHAGE

I

+

+

+

PR4

+

II

~o

+

L +

+

+ + + + r

I I + +

i

z~

611

+

+

i ~-}- +

+

+

+

§

+

+

+

+

-F +

-b

+

+

+

+

+

+

+

o

E

612

DAVIS AND CRONAN

unknown; however, because we chose only to study mutants that were not leaky, we would not have identified proteins not absolutely required for infection. We cannot yet describe an assembly pathway because we do not yet know which of the particles assembled by mutants represent true intermediates in the assembly pathway. Our data, however, are consistent with a model where PR4, like other double-stranded DNA phages, first assembles a prohead which is then filled with DNA. For PR4, the candidate for the prohead is the DNA-less particle found in lysates of group 7 mutants infecting an Suhost. The fact that such particles are also found in wild type lysates suggests t h a t they either are true intermediates or are derived from intermediates. Unlike proheads of other double-stranded DNA phages (Murialdo and Becker, 1978), however, PR4 DNA-less particles contain the adsorption protein (P1). The particles also possess a normal phospholipid composition. Our primary interest is in the assembly of the phospholipid-protein membrane of the phage. We have identified four proteins required for assembly of a particle containing phospholipid and protein. We have found one additional complementation group which is defective in this process but we have not yet identified the missing protein. We have not found mutants t h a t can assemble a protein coat without a membrane or a membrane vesicle without a protein coat even though such particles would have sedimented in the center of the sucrose gradients. It is possible t h a t such particles were unstable and disintegrated or they aggregated and pelleted. Excepting these possibilities, we have mutants in two classes, mutants t h a t assemble no particles and mutants t h a t assemble particles containing both the membrane and the protein coat, thus suggesting t h a t the protein coat and the membrane are assembled together. Our future studies will focus on these early steps in assembly. Mindich and co-workers (Mindich et al., 1982a, b) very recently reported the isolation and characterization of nonsense mutants of the lipid-containing phage

PRD1. Since, PR4 and PRD1 are closely related (but nonidentical)phages (Bamford et al., 1981), a comparison of our data with the PRD1 data is of interest. Many of the proteins of PR4 identified in our studies appear to have proteins analogous (in size, abundance, and function) to those of PRD1 (Mindich et aL, 1982a, b). The early proteins P1A of PR4 and P1 of PRD1 are required for phage DNA synthesis. The early proteins P5A, P8A, and P l l B of PR4 correspond in size and abundance to the PRD1 early proteins P8, P12, and P19, respectively; however, no mutants in these early proteins of PR4 have yet been isolated. Among the late proteins of the two phages, several provisional alignments seem warranted: PI(PR4) with P2(PRD1) (adsorption); P2(PR4) with P3(PRD1) (major capsid protein); P6(PR4) with P9(PRD1) (DNA packaging, a minor virion protein); and P6A(PR4) with P10 of PRD1 (abundant nonvirion protein required for particle assembly). These alignments must be tested by testing for complementation of PR4 mutants with PRD1 mutants. Finally Mindich et aL, (1982b) have shown that the assembly pathway of PRD1 first involves the formation of a particle containing phospholipid and all the proteins except possibly one (P9). The particles are then filled with DNA. This is completely consistent with our PR4 results. DNA-less particles of PR4 clearly lack P6. Further experiments are required to establish the exact relationship of PR4 and PRD1. ACKNOWLEDGMENTS We thank E. Muller for advice and encouragement and L. Mindich for strains and communication of unpublished results. This work was supported by NIH Grant GM26156. T.N.D. was a graduate fellow of the NSF. REFERENCES BAMFORD, D. n., ROUHIAINEN,L., TAKKINEN, K., and SODERLUND, H. (1981). Comparison of lipid-containing bacteriophages PRD1, PR3, PR4, PR5 and L17. J. Gen. ViroL 57, 365-373. BRADLEY, D. E., and RUTHERFORD, E. L. (1975). Basic characterization of a lipid-containing bacteriophage specific for plasmids of the P, N, and W compatibility groups. C a n a ~ J. MicrobioL 21, 152-163.

NONSENSE MUTANTS OF P H A G E PR4 BRADLEY, D. E., TAYLOR, D. E., and COHEN, D. R. (1980). Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12. J. BacterioL 143, 1466-1470. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M. W., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. BioL C h e ~ 252, 1102-1106. COETZEE, W. F., and BEKKER, P. J. (1979). Pilus-specific, lipid-containing bacteriophages PR4 and PR772: Comparison of physical characteristics of genomes. J. GeE. ViroL 45, 195-200. CRONAN,J. E., SILBERT,D. F., and WULFF,D. L. (1972). Mapping of the fabA locus for u n s a t u r a t e d fatty acid biosynthesis in Escherichia col4 J. BacterioL 112, 206-211. DAVIS, TIN., MULLER, E. D., and CRONAN, J. E., JR. (1982). The virion of the lipid-containing bacteriophage PR4. Virology 120, 287-306. GARDNER, J. F., SMITH, O. H., FREDERICKS, W. W., and McKINNEY, M. A. (1974). Secondary-site att a c h m e n t of coliphage lambda near the thr operon. J. Mol. Biol. 90, 613-631. GARRETT, J., FUSSELMAN,R., HISE, J., CHIOU,L., SMITHGRILLO, D., SCHULZ, J., and YOUNG, R. (1981). Cell lysis by induction of cloned lambda lysis genes. Mol. GeE. Genet. 182, 326-331. GELLERT, M. (1981). DNA Topoisomerases. Annu~ Rev. Biochem. 50, 879-910. GORINI, L. (1970). Informational suppression. Annw Rev. Genetics 4, 107-134. HARRIS, A. W., MOUNT, D. W. A., FUERST, C. R., and SIMINOVITCH,L. (1967). Mutations in bacteriophage lambda affecting host cell lysis. Virology 32, 553569. HOWE, M. M., O'DAY, K. J., and SCHULTZ,D. W. (1979). Isolation of mutations defining five new cistrons essential for development of bacteriophage MR. Virology 93, 303-319. JOSSLIN, R. (1970). The lysis mechanism of phage T4: Mutants affecting lysis. Virology 40, 719-726. LIVINGSTON, D. U. (1974). Immunoaffinity chromatography of proteins. In "Methods in Enzymology" (W. R. Jakoby and M. Wilchek, eds.), Vol. 34, pp. 723-731. Academic Press, New York. LUNDSTR(JM, S. H., BAMFORD, e . H., PALVA, E. T., and LOUNATMAA, K. (1979). Lipid-containing bacteriophage PR4: Structure and life cycle. J. GeE. ViroL 43, 583-592.

613

MILLER, J. H. (1972). "Experiments in Molecular Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. MINDICH, L., BAMFORD, D., GOLDWAITE, C., LAVERTY, M., and MACKENZIE, G. (1982a). Isolation of nonsense m u t a n t s of lipid-containing bacteriophage PRD1. J. ViroL 44, 1013-1020. MINDICH, L., BAMFORD, D., McGRAw, T., and MACKENZIE, G. (1982b). Assembly of bacteriophage PRDI: Particle formation with wild-type and m u t a n t viruses. J. Viro~ 44, 1021-1030. MINDICH, L., COHEN, Z., and WEISBURD, M. (1976). Isolation of nonsense suppressor m u t a n t s in Pseudomonas. J. Bacteriol. 126, 177-182. MULLER, E. D., and CRONAN, J. E., JR. (1983). Lipidcontaining bacteriophage PR4: Effects of altered lipid composition on the virion. J. MoL BioL In press. MULLER, E. G. (1981). "The Lipid-Containing Phage PR4." Ph.D. thesis. Yale University, New Haven, Connecticut. MUNKRES, K. D., and RICHARDS, F. M. (1965). The purification and properties of Neurospora malate dehydrogenase. Arch. Bioche~ra Biophys. 109, 466479. MURIALDO, H., and BECKER, A. (1978). Head morphogenesis of complex double-stranded deoxyribonucleic acid bacteriophages. Microbiol. Rev. 42, 529-576. OESCHGER, U. P., and WIPRUD, G. T. (1980). High efficiency temperature-sensitive amber suppressor strains of Escherichia coli K-12.: Construction and characterization of recombinant strains with suppressor-enhancing mutations. Molec. Ge~ Genet. 178, 293-299. TSUGITA, A., INOUYE, M., TERZAGHI, E., and STREIS[NGER, G. (1968). Purification of bacteriophage T4 lysozyme. J. BioL Che~r~ 243, 391-397. ULLMANN, A., and PERRIN, D. (1970). Complementation in fl-galactosidase. In "The Lactose Operon" (Beckwith, J. R., and Zipser, D., eds.), pp. 143-172. Cold Spring H a r b o r Laboratory, Cold Spring Harbor, New York. WHITE, G. P., and DUNN, U. W. (1978). Compatibility and sex specific phage plating characteristics of the TOL and NAH catabolic plasmids. Genet. Res. 32, 207-213. YOUDERIAN, P., and SUSSKIND, M. U. (1980). Identification of the products of bacteriophage P22 genes, including a new late gene. Virology 107, 258-269.