Nonsense mutants defining seven new genes of the lipid-containing bacteriophage PR4

VIROLOGY

177, 1 l-22

Nonsense

(1990)

Mutants

Defining Seven New Genes of the Lipid-Containing

Bacteriophage

PR4

THOMAS VANDEN BOOM* AND JOHN E. CRONAN, JR.*+’ Departments

of *Microbiology

and tBiochemistry,

Received October

University of Illinois, Urbana, Illinois 6 180 1

12. 1989; accepted March 7, 1990

Thirty-eight new nonsense mutants of the lipid-containing bacteriophage PR4 were isolated. These mutants define seven new viral genes, including the gene encoding the terminal genome protein and an accessory lytic factor. The defective gene products produced in uv-irradiated cells infected with representative mutants from each of the new genetic groups were identified using sodium dodecyl sulfate-polyactylamide gel electrophoresis (SDS-PAGE). Extracts of uv-irradiated cells infected with nonsense mutants that produce a defective major capsid protein, P2, also lacked two lower molecular weight proteins. The synthesis of all three protein species was recovered in cells infected with one-step revertants of two independent major capsid protein mutants, suggesting the possibility of post-translational processing or overlapping genes. The time course of protein synthesis in wild-type PR4-infected cells was examined using SDS-PAGE. These analyses revealed at least 34 proteins produced following phage PR4 infection that were not 0 1SSOAcademic Press. Inc. present in uninfected COfItrOl CUkUreS.

Muller and Cronan, 1983). However, this enrichment is not essential for phage assembly or infectivity (Vanden Boom and Cronan, 1988). The PR4 genome consists of a linear DNA molecule of about 15,000 base pairs (bp) covalently attached at the 5’ termini to a protein (Davis et a/., 1982; Davis and Cronan, 1985; Bamford and Mindich, 1984; Vanden Boom, 1989). Davis and Cronan (1983) have previously reported the isolation of 33 amber mutants of PR4. These mutants defined 12 complementation groups and permitted the identification of 7 virion and 3 nonvirion proteins (Davis and Cronan, 1983). We have undertaken a more extensive genetic analysis of bacteriophage PR4. In this report, we describe the isolation and preliminary characterization of nonsense mutants defining seven new genetic groups. In addition, we have examined the time course of protein synthesis in wild-type PR4-infected cells and have identified at least 34 proteins produced following PR4 infection that were not detected in uninfected cells.

INTRODUCTION Bacteriophage PR4 is one of a group of lipid-containing viruses which can infect Escherichia co/i and Salmonella typhimurium. These phages have been developed as model experimental systems to study membrane assembly and protein-lipid interactions (Davis and Cronan, 1985; Mindich and Bamford, 1988). Phages PR4 and PRDl are the best-characterized members of this group (Mindich and Bamford, 1988). PR4 and PRDl are known to be closely related, but nonidentical, originally isolated from Australia and the United States, respectively. Structural or morphogenic features determined for one virus are most likely relevant to the other. However, differences exist between phages PR4 and PRDl in both restriction fragment patterns and in the profile of phage-encoded proteins in SDS-PAGE gels (Mindich and Bamford, 1988). Sensitivityto these phages is conferred by a resident plasmid of the C, N, P, or W incompatibility groups (Bradley and Rutherford, 1975; Stanisich, 1976; White and Dunn, 1978). The PR4 virion consists of an icosahedral protein capsid which surrounds a membrane vesicle-enclosed double-stranded DNA genome (Lundstram et a/., 1979; Davis and Cronan, 1985). The viral membrane is composed of roughly 60% phospholipid and an assortment of at least 12 phage-encoded proteins (Davis et al., 1982; Davis and Cronan, 1985). The phage phospholipids are enriched in phosphatidylglycerol relative to the E. co/i or S. typhimurium host (Davis et al., 1982;

MATERIALS Media, reagents,

AND METHODS

and buffers

Rich broth (RB), Luria broth (LB), antibiotic medium 5, RB top agar, and SM buffer have been described previously (Davis et a/., 1982; Maniatis et a/., 1982). For labeling with a mixture of [35S]methionine and [35S]cysteine (Tran35S-label, ICN Biomedicals, Inc.) cells were grown in Mops-M medium which consisted of Mops (4-morpholinepropanesulfonic acid)-buffered synthetic medium (Neidhardt et al., 1974) supplemented with 4 g/liter glucose, 1 mg/liter thiamine, 2 mg/liter biotin, the

’ To whom requests for reprints should be addressed. 11

0042.6822/90

$3.00

Copynght 0 1990 by Academic Press. Inc. All rights of reproduction in any form resewed

12

VANDEN

BOOM AND CRONAN

amino acid mixture (below) diluted 50-fold, and 200 mg/liter each of leucine, isoleucine, tryptophan, and tyrosine. Tran35S-label is produced from a cellular hydrolysate of E. co/i and contains 70% L[35S]methionine (1 194 Ci/mmol) and 15% L[35S]cysteine. The amino acid mixture contained 10 mg/ml of the L-form of each of the following: alanine, aspartic acid, glutamic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine. Mops phage buffer contained 20 mM Mops, pH 7.2; 100 mM NaCI, and I mM MgC12. For convenience, a 1O-fold concentrate of this buffer was routinely made and subsequently diluted as needed. Kanamycin sulfate and tetracycline hydrochloride were used at final concentrations (per liter) of 50 and 10 mg, respectively. Bacterial and phage strains The bacterial strains used in this work are listed in Table 1. The IncP plasmid RPl derivative, pLM2, was transferred by plate spot matings (Miller, 1972) using strain MS1 550(pLM2) as the donor. Plvir-mediated transductions were performed as described by Miller (1972). Plasmid RPl confers resistance to kanamycin sulfate (100 pg/ml), sodium ampicillin (200 pg/ml), and tetracycline hydrochloride (10 pg/ml), and sensitivity to phage PR4 (White and Dunn, 1978). The piasmid RPl derivative, pLM2, contains amber mutations in the tet and b/a genes (Mindich et a/., 1976). JKl is an rpsL derivative of W31 10. JKl was transduced to tetracyclineresistance with Pl vir grown on LCD43 to give TVB56. A tetracycline-sensitive derivative of TVB56 was selected on fusaric acid medium plates (Malay and Nunn, 1981) and subsequently made kanamycin-resistant by conjugation with MS1550(pLM2) to give TVB58. TVB58 was scored for recA by testing the sensitivity of this strain to ultraviolet light (Miller, 1972). Wild-type bacteriophage PR4 was the gift of D. E. Bradley (1974; Bradley and Rutherford, 1975). Phage PR4 mutant strains 9 through 102 were described previously (Davis and Cronan, 1983). Bacteriophage

PR4 nomenclature

Individual phage mutants are identified by a threeletter designation preceding the unique isolation number (e.g., Nam9). The uppercase letter denotes the mutagen employed in the isolation of the mutant strain (N, IV-methyl-N’-nitro-Nnitrosoguanidine; H, hydroxylamine). This letter is followed by a two letter abbreviation of the mutant class designation (am, amber; ts, temperature-sensitive). Several mutant isolates contained identifiable secondary mutations which were subsequently removed from the mutant phage strain either through a backcross with wild-type phage or

through reversion. Such phage strains retain the original isolate designation and are followed by an additional uppercase letter to distinguish them from the original isolate (e.g., NamSA). Revertants derived from mutant bacteriophage strains are designated by an uppercase R preceding the relevant mutant designation and are assigned an additional revertant number (e.g., RNamS-1 is revertant number one derived from phage mutant Nam9). The bacterial strain used for the propagation of a bacteriophage strain may be included in parentheses following the bacteriophage strain [e.g., Nam9 (TVB500)]. PR4 protein nomenclature The protein designations of Davis and Cronan (1983) have been retained in this study (Fig. 1). Previously unidentified phage-specified proteins have been designated P15 through P28 as shown in Fig. 1. It should be noted that protein P5A retains the original letter designation of Davis and Cronan (1983) denoting a nonvirion protein, although this species is now known to be a virion component, the terminal genome protein (Davis and Cronan, 1985). Protein P5A was not previously identified as a component of purified virions since this species is complexed to the viral DNA and does not migrate into conventional SDS-PAGE gels. Bacteriophage

techniques

Techniques for the plaque purification of phage PR4 strains, production and storage of PR4 lysates, titration of phage PR4 stocks, and nitrosoguanidine mutagenesis have been described previously (Davis et al., 1982; Vanden Boom and Cronan, 1988; Vanden Boom, 1989). Hydroxylamine mutagenesis was done essentially as described by Davis eta/. (1980) (Vanden Boom, 1989). Treatment of phage preparations with 1.1 or 1.2 IVI NH,OH resulted in 98.0 or 99.6% killing after 24 hr, respectively. Appropriate dilutions of these mixtures giving about 200 PFU/plate were plated to screen for mutant bacteriophages. Mutagenesis with hydroxylamine gave mutants numbered 200-269 whereas nitrosoguanidine mutagenesis yielded mutants numbered 500-l 394. Isolation and scoring of phage with amber mutations Mixed indicator cek. The isolation of amber mutations was facilitated by use of the mixed indicator method, essentially as described by Howe et al. (1979) for phage Mu. The Su- and Su+ strains off. coliand S. typhimurium used in this work are given in Table 1. The ratio of Su+ to Su- cells was varied from 1 to 3 in different platings.

PHAGE PR4 NONSENSE

Stab tests. Plaques suspected of containing mutant phage were picked with a toothpick and stabbed into lawns containing about 1 X 10’ cells of a Su+ or Suindicator strain. Phage that grew poorly on the Su- indicator lawn were plaque purified from the permissive Su+ lawn and again examined using the stab test. High titer lysates of candidate phage mutants that survived this screening procedure were used in subsequent experiments. Cross-species

experiments

The broad host range of phage PR4 permitted the use of both E. co/i and S. typhimurium bacterial strains in this study (Table 1). Phage grown on E. co/i strains show only a minor decrease in plating efficiency on S. typhimurium strains and vice versa (Bamford et a/., 1981). Consequently, wild-type or mutant phage lysates propagated on one species were used in qualitative spot-test or stab-test experiments using indicator bacteria of the other species. However, for quantitative liquid complementation assays (see below) amber phage mutant lysates were always prepared using the appropriate permissive Su+ strains within the same species in order to avoid any minor effects due to host restriction. This procedure most often involved growing an amber mutant isolated using E. co/i strain TD6 (supE44) on the S. typhimurium strain TVB502 (supE20). Complementation

tests

Qualitative spot tests. Spot complementation tests with reference mutations in the 12 previously identified complementation groups were performed essentially as described earlier (Davis and Cronan, 1983). During the latter stages of this work, a collection of recombinant plasmids containing phage PR4 DNA fragments became available (Vanden Boom and Cronan, 1990). Consequently, in some cases a new mutation was first localized to a particular segment of the phage genome through marker rescue by a recombinant plasmid containing a phage PR4 DNA fragment. Spot complementation tests were then performed using a subset of the reference mutations given in Table 2 previously localized to that region. The recombinant plasmids used in this study are given in Table 1. Quantitative complementation assays. Liquid complementation tests were performed in certain cases in order to confirm the placement of a mutation in a particular complementation group (Davis and Cronan, 1983). TVB54 and TVB500 were used as the Su- host strains for the E. co/i and S. typhimurium-propagated phage mutants, respectively.

13

MUTANTS

Radioactive

labeling

A modification (Vanden Boom, 1989) of the procedure for uv irradiation, infection, and radiolabeling of MS1 550(pLM2) cells, described earlier (Davis and Cronan, 1983), was employed in this study. 35S-labeled virus particles were prepared from a loo-ml lysate of phage PR4. Strain TD6 was grown in MOPS-M medium at 37” with shaking. At 2.5 X 10’ cells/ml the culture was infected with PR4 at a m.o.i. - 10. At 20 and 30 min postinfection, 0.2 mCi Tran35S-label was added to the infected culture. Incubation was continued with shaking at 37” until lysis (-1.5 hr). The 35S-labeled phage were harvested and purified as described previously (Davis et al., 1982; Vanden Boom and Cronan, 1988). Gel electrophoresis Two discontinuous SDS-PAGE systems (Laemmli, 1970) were used in this study to resolve proteins synthesized in phage-infected uv-irradiated cells (Figs. 1 and 5). The resolution of proteins Pl, Pl A, P3, P4, and P5 was best achieved in a 16% acrylamide gel whereas the resolution of lower molecular weight PR4 proteins was improved in a glycerol-containing 15% acrylamide gel (Giulian et al., 1985). 14C-labeled protein molecular weight standards were obtained from Bethesda Research Laboratories. SDS-PAGE gels were fixed in 10% TCA for 15 min, rinsed thoroughly with distilled water, and then treated with Enlightning (New England Nuclear) for 30 min. The gels were then dried in a Hoeffer gel dryer under vacuum at 80” for 1 hr. Autoradiography was performed using preflashed (background fog absorbance of 0.15 at 540 nm) Kodak XAR film (Laskey and Mills, 1975). Exposure times varied from 2 days to 2 weeks at -70”. RESULTS AND DISCUSSION Time course of wild-type

PR4 protein synthesis

Strain MS1 550(pLM2) was uv-irradiated, infected with wild-type PR4, and labeled using 5-min pulses from 0 to 60 min postinfection as described in the legend to Fig. 1. Using the two SDS-PAGE systems shown in Fig. 5, we have identified at least 34 proteins produced following PR4 infection that could not be detected in uninfected cells. This represents improved resolution over the 13-l 7% gradient polyacrylamide gels described previously (Davis and Cronan, 1983). The time course of synthesis of individual phage proteins provides a useful signature for the identification of defective phage proteins in extracts of amber mutantinfected Su- cells (Fig. 1). It seems likely that phage PR4 encodes additional, as yet unidentified, gene prod-

14

VANDEN

BOOM AND CRONAN

P2

4B P15 P16 P17

~ P6A EA p7

P16 P19 P20 P21

-P22 -

P23

E PlO PlOA -

P24 PllA PllBP25

~

P26 P12A

Pl3

P27 P26

~%l

PULSE ( min ) FIG. 1. Time course of phage PR4 protein synthesis in uv-irradiated cells. Strain MS1 550 (pLM2) was grown to 50 Klett units in MOPS-M medium at 37”. The culture was irradiated as described under Materials and Methods. The irradiated culture was aerated at 37’ for 5 min and then infected with wild-type PR4 at a m.o.i. - 10. Aliquots (0.1 ml) were immediately dispensed into foil-covered 13 X loo-mm screw cap tubes. At the indicated pulse times, 38 j&i of Trar?S-label was added to a portion of the infected culture. For the O-to 5-min pulse, the Tran35S-label was dispensed into the appropriate tube prior to the addition of the infected culture. At the end of the 5-min radiolabel pulse, the samples were quick-frozen in a dry-iceeethanol bath. Two unrnfected portions of the culture were similarly labeled in parallel with the 0- to 5-min and 60- to 65.min phageinfected cultures. Samples (5-l 0 ~1) of the labeled cultures were mixed with an equal volume of 2X sample buffer (0.125 h/lTrisHCI, pH 6-8; 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, and 0.0025% (w/v) bromphenol blue) and loaded onto a 20-cmlong, 0.75.mm-thick, 15% glycerol-containing acrylamide gel (Giulian et a/., 1985). The separating gel contained 15% (w/v) acrylamide (200:1, acrylamide: bis-acrylamide), 10% (v/v) glycerol, 0.75 M Tris, pH 9.3; 0.1% (w/v) SDS, 0.028% (w/v) ammonium persulfate, and 0.14% (v/v) TEMED. The stacking gel contained 10% (w/v) acrylamide (2 1: 1, acrylamide: bis-acrylamide), 10% (v/v) glycerol, 0.03% (w/v) ammonium persulfate, and 0.24% (v/v) TEMED. The “C-protein molecular weight standards were obtained from Bethesda Research Laboratories and are as follows (in daltons): ovalbumin, 43,000; carbonic anhydrase, 29,000; P-lactoglobulin, 18,400; lysozyme, 14,300; bovine trypsin inhibitor, 6200; and insulin (A and B chain), 3000. Electrophoresis was carried out at 13. to 16.mA constant current. Autoradiography was performed as described under Materials and Methods. The first column of protein designations in the right margin is from Davis and Cronan (1983). Previously unidentified proteins have been consecutively numbered from 15 through 28, as shown. Abbreviations used: MW, molecularweight standards: UN, uninfected.

ucts since no defective gene products in phage Nam 14 (group 4)) and Nam71 (group 12)-infected cells (Davis and Cronan, 1983) could be detected using the two SDS-PAGE systems described in this report (data not shown).

Isolation

of amber mutants

In order to optimize recovery of wide variety of PR4 amber mutant strains, two mutagenesis procedures were employed. Moreover, a variety of Su+ strains (Ta-

PHAGE PR4 NONSENSE

15

MUTANTS

TABLE 1 BACTERIALSTRAINSAND PLASMIDSUSED IN THIS WORK Escherichia

TV854 TvB58

Source/Derivation

Genotype

Strain JKl TD6 LCD43

co/i strains’

F-thyA deoC2 IN(rrnD-rrnE) rpsL F-thi-1 thr-1 leuB6lacYl fhuA27 supE44 X-(RPl) F -A(argFlac)U 169 araD 139 thi non gyrA rpsL 150/metE70 A(recA-srl) TnlO::srl F-JKl(pLM2) F-JKl A(recA-sr/)(pLM2) Salmonella typhimurium

A(uvrB-bio) sup” leuA4 74(Am) hisC527(Am) leuA4 74(Am) hisC527(Am) leuA4 74(Am) hisC527(Am) leuA4 74(Am) hisC527(Am) leuA4 14(Am) hisC527(Am) DB7136 (pLM2) DB7154 (pLM2) DB7155 (pLM2) DB7156 (pLM2) DB7157 (pLM2)

This study This study

strainsb

L. Mindich (Youderian and Susskind, 1980; Mindich eta/., 1976) SGSC” SGSC SGSC SGSC SGSC This study This study This study This study This study

leuA4 14(Am) hisC527(Am) M51550 (pLM2) DB7136 DB7154 DB7155 DB7156 DB7157 TV8500 TV8501 TV8502 TV8503 TV8504

(Davis eta/., 1982)

Source/Derivation

Genotype

Strain

J. Konisky Laboratory collection L. DeVeaux

supD 10 supE20 supf30 supl60

Plasmids Plasmids pLM2 pcTV400 pCTV204

Description

Source

ampR(Am) tetR(Am) derivative of RPl W/l-D fragment of PR4 genome in pUC19 Hphl-B fragment of PR4 genome in pUC19

L. Mindich (Mindich eta/., 1976) Vanden Boom and Cronan (1990) Vanden Boom and Cronan (1990)

‘Allele numbers are those of the E. co/i Genetic Stock Center (Yale University, New Haven, CT). b Allele numbers are those of the S. tvDhimurium Genetic Stock Center (University of Calgary, Calgary, Alberta, Canada). ’ S. typhimurium Genetic Stock Center

bles 1 and 2) were used in order to isolate amber alleles which might require a specific suppressor (i.e., insertion of a specific amino acid) to restore function. Phage PR4 Ham mutants were isolated from PR4 lysates mutagenized in vitro with hydroxylamine, a chemical mutagen which causes unidirectional transition mutations (Tessman et a/., 1964). The Ham mutants probably represent independent isolates since mutagenesis of phage particles precludes the problem of siblings among recovered phage mutants. Cross-contamination of plaques being screened for amber mutations was minimized by control of plaque density (-200 PFWplate) and length of incubation of experimental plates. Stab tests were performed, as described under Materials and Methods, between 8 and 12 hr after plat-

ing of mutagenized phage. The Nam mutants were isolated from lysates of nitrosoguanidine-treated infected cells (Vanden Boom, 1989). At least 95% of the Nam phage strains isolated in this study represent independent isolates based on (i) the mutagenized lysate of origin, (ii) their behavior in complementation tests, and (iii) the pattern of proteins synthesized in mutant-infected cells. The mutants in groups 17, 18 and 19 were leaky for growth on Su- strains. However, these phage strains grew better on the Su+ host and were tentatively classified as amber mutants. The reversion frequency for each amber mutant phage strain was determined using the appropriate Su- and Su+ host strains (see Table 2 footnote). Mutant PR4 strains were obtained in this study having reversion frequencies that

16

VANDEN

BOOM AND CRONAN TABLE 2

SUMMARYOF BACTERIOPHAGEPR4 AMBER REFERENCEMUTANTS Groupa

Alleles

1

10

2

9

3

5

4 5 6

1 1 4

7

4 2

8 9 10

1 10

11

10

12 13 14 15

1 1 1 3

16

3

17 18

1 2

19 ?

1 1

Reference mutant(s)

Permissive strainb

Nam65 Ham237 Ham201 Nam1394 Nam68 Nam588 Nam14 Naml6 Nam72 Nam1358 Nam15 Nam41 Nam6 1 Ham207 Nam84 Naml142 Naml148 Nam40 Ham239 Nam7 1 Naml407 Ham269 Ham208 Ham224 NamlllO Naml139 Naml235A NamlOO9 Naml380A Nam1268 Naml031

TD6 TD6 TD6 TV8501 TD6 TVB503 TD6 TD6 TD6 TVB504 TD6 TD6 TD6 TD6 TD6 TVB503 TV8503 TD6 TD6 TD6 TVB501 TD6 TD6 TD6 TV8503 TVB503 TV8503 TD6 TVB501 TVB503 TD6

Reversion frequency’ 1.1 x 1o-5 1.6x 1O-5 2.6 X 1O-5 3.5 x 1o-6 5.8 X 1O-7 4.1 x 1o-6 4.9 x 1o-4 1.0x 1o-7 2.9 x 1o-6 4.5 x 1o-4 1.0x 10-S 7.7 x 1 o-’ 8.4 x 1 O-4 4.4 x 1 om7 7.0 x 1 o-5 2.5 x 1O-4 2.7 X 1 O-4 1.3x 1o-5 1.6x 1O-6 1.0x 1o-4 1.2 x 1o-6 6.1 x lo-’ 9.0 x 1o-6 2.2 x 1o-6 2.0 x 1o-5 4.5 x 1o-6 0.96 0.55 0.88 1.03 4.4 x 1om4

a Groups 1 through 12 have been described previously (Davis and Cronan, 1983). b The permissive strain given is the original Su+ strain used in the isolation of the mutant phage strain. c Reversion frequency is defined as the ratio of plaque titer on the nonpermissive Su- strain to the permissive d For phage PR4 protein designations see Fig. 1 and Davis and Cronan (1983).

varied from 1 .O (Nam1268) to 6.1 X 10e7 (Ham269). Although numerous amber mutants defective for protein PlA were isolated in this study only two mutants defective for protein Pl were recovered. This was unexpected since the genes encoding these two proteins are of similar size. Conversely, a surprisingly large number of mutants defective for protein P9A were recovered. The basis for this apparent bias in the mutagenesis and/or mutant recovery procedures used in this study is not known. We cannot reliably assign molecular weights to the cluster of PR4 proteins migrating faster than the 6.2-KDa protein standard (Fig. 1) since deviations from linearity of the log molecular weight versus Rr relationship have been noted for very low molecularweight proteins (Neville, 1971). However, these phage proteins probably range from 4-6 KDa. The predicted coding requirement necessary for such proteins

Defective

protein(s)d

PlA P2, P16. P18 P6A ? P4, PlO P13 P6 Pl P3 P7 P9A ? P4, Pl 1A P23 P24? P5A P4, P21 P5 P8A ?

Su’ strain given.

is only loo-140 bp, a target size that likely accounts for our failure to isolate mutants defective in these proteins. Assignment

of mutants to genetic groups

In most cases, amber mutants were tentatively assigned to a complementation group on the basis of spot complementation tests, as described under Materials and Methods. For convenience, dilutions of reference amber mutants were spotted onto lawns infected with a new amber mutant isolate. Mutant phage strains which complemented reference mutants in all 12 previously defined complementation groups were subsequently tested with each other (both as the phage in the lawn and as the phage spotted on the overlay). In order to verify assignments of certain amber mutants

PHAGE PR4 NONSENSE

to complementation groups, selected mutants were tested using the quantitative liquid complementation assay. In no case was an assignment based solely on the results of a qualitative spot complementation test. However, certain phage PR4 amber mutants were assigned to genetic groups strictly on the basis of SDSPAGE analysis of the proteins made in Su- uv-irradiated cells. These mutants either behave anomalously in liquid complementation tests (group 2) or are leaky under the experimental conditions employed in this study (groups 17-l 9). The number of alleles assigned to the previously described genetic groups 1 through 12 (Davis and Cronan, 1983) are given in Table 2. Majorcapsidprotein mutants (group 2). Five new amber mutants were isolated which affect the synthesis of the major capsid protein, P2 (Ham201, Ham218, Nam595, Nam1394, and Nam1402). Assignment to group 2 was based solely on SDS-PAGE analysis of the proteins made in mutant-infected Su cells since these phage strains behave anomalously in quantitative liquid complementation assays (Davis and Cronan, 1983). Putative amber fragments of P2 were visible in Su- host cells infected with mutants Ham201, Ham21 8, Nam595, and Nam1394. In addition to the major capsid protein P2, two previously unidentified minor phage proteins (P16 and P18) were also missing in Su- cells infected with any of the group 2 mutants. In experiments with three independent group 2 mutants, putative amber fragments were visible for both P2 and P16 (Fig. 2). One-step revertants of Ham201 and Nam1394 were obtained that recovered all three protein species. The basis of this observation is not known. However, the presence of putative P2 and P16 amber fragments in Su- cells infected with several group 2 mutants argues against polarity as the cause of the missing protein P16 in these cells. Several alternative explanations seem plausible: (i) the lower molecular weight virion proteins might represent processed products derived from protein P2; (ii) the three gene products might be derived from overlapping genes; or (iii) the three gene products might be derived from an alternatively translated or processed common mRNA transcript. Several examples of proteolytic cleavage of structural proteins during phage head assembly have been reported (for reviews, see Casjens and Hendrix, 1988; Black and Showe, 1983; Black, 1989). Interestingly, in phage T4 a phage-encoded protease, gp21, removes 65 amino acids from the N-terminal end of the coat protein gp23 (Parker et a/., 1984). Finally, it should be noted that the minor P2-related protein species identified in SDS-PAGE gels might be due to a host protease present in extracts of phage-infected cells (Maurizi eta/., 1985; Richardson eta/., 1988). Although possible, we believe this is unlikely since protein P16

MUTANTS

17

FIG. 2. Pattern of protern synthesis in group 2 mutant-infected cells. Radiolabeled extracts of mutant-infected MS1 550 (pLM2) cells were prepared as described under Materials and Methods. Samples were analyzed in a 16% acrylamide gel. The separating gel contained 16% (w/v) acrylamide (29: 1, acrylamide:bisacrylamide). 0.375 n/r Tns-HCI, pH 8.8; 0.1% (w/v) SDS, 0.1% (w/v) ammonium persulfate, and 0.033% (v/v) TEMED. The stacking gel contained 5.3% (w/v) acrylamide (29:1, acrylamide:bis-acrylamide), 0.125 AJTris-HCI, pH 6.8; 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate. and 0.05% (v/v) TEMED. The autoradiogram was overexposed to clearly show minor phage-encoded proteins. Arrows indicate the position of wildtype or putative amber fragments of proteins P2 and P16. Extracts of group 2 mutant-infected cells were also missing protein P18 (see legend to Fig. 1). This protein specres was not resolved in the gel shown. Abbreviations used: UN, uninfected; WT, wild type.

has been tentatively identified as a component of purified virions. Group 13. Mutant Nam 1407 was originally assigned to a new complementation group on the basis of spot complementation tests with the reference mutants given in Table 2. SDS-PAGE analysis identified two proteins missing in uv-irradiated Su- cells infected with Naml407, P4, and Pl 1A (Fig. 5). A one-step revertant, RNam 1407-l) was obtained that was able to grow on

18

VANDEN

BOOM AND CRONAN

the Su- strain TVB500. Interestingly, Su- cells infected with RNaml407-1 recovered synthesis of both P4 and Pl 1A (data not shown). Group 14. The Ham269 mutation was first localized to the /-/,&II-B fragment of the PR4 genome through marker rescue by a recombinant plasmid containing the /-@l-B fragment. Quantitative liquid complementation tests were performed with Group 8 mutant Nam61, the sole mutant previously localized to this region of the phage genome (Vanden Boom and Cronan, 1990). Mixed infection of Su- strain TVB54 with Nam61 and Ham269 resulted in a burst of 29 phage per infected cell whereas control infections with either Nam61 or Ham269 and group 2 mutant Nam9 yielded about 10 phage per infected cell. The bursts for the Nam61 and Ham269 self-test controls were 0.065 and 0.003 phage per infected cell, respectively. The assignment of Ham269 to a new genetic group was confirmed by examination of the proteins produced in Sucells infected with phage strain Ham269 (Fig. 5). Extracts of group 14 mutant Ham269-infected cells lacked a previously unidentified phage protein (P23) made late in infection (Figs. 1 and 5). Group 15. Mutants Ham208, Ham21 2, and Ham224 were assigned to a new group on the basis of their common defective lysis phenotype. All three members of this group failed to lyse strain TVB54 (Su-) in liquid culture. However, in contrast to group 11 endolysindefective mutants (Davis and Cronan, 1983), group 15 mutant-infected Su- cells could be artificially lysed by addition of l/l 00 volume of chloroform to the infected culture (Fig. 3). Complementation between mutant phage in group 15 and the group 11 reference mutant Nam70 was examined by following the turbidity of a culture of strain TVB54 (Su-) co-infected with phage Nam70 and a member of group 15. A representative experiment is shown in Fig. 3. Phage strains Nam70 and Ham208 appear to complement each other but mixed infections do not produce the wild-type pattern of lysis. Interestingly, phage Ham208-infected TVB54 (Su-) cells examined by phase contrast microscopy at 3-4 hr postinfection have a filamentous phenotype (Fig. 4). When Ham208-infected cells were artificially lysed by chloroform at 4 hr postinfection the resulting lysates typically yielded from 1000 to 1400 phage per infected cell. The variability in phage yield was most likely due to the sensitivity of phage PR4 to chloroform. The chloroform was merely removed through dilution in these experiments. The group 15 mutants define an accessory lytic factor possibly analogous to that encoded by the X S gene (Garret et al., 1981) or the T4 t gene (Josslin, 1970) suggesting that bacteriophage PR4 employs a multicomponent lysis system similar to that of the lambdoid and T-even phages (Garret et a/.,

100 90

2

20

101

wild type

’

I

I

I

I

I

I

I

I

I

I

0

20

40

60

80

100

120

140

160

180

200

220

MINUTES AFTER INFECTION

FIG. 3. Pattern of lysis of group 1 1 and group 15 mutant-infected cells. Sum strain TVB54 was grown in RB at 37” to a cell density of 2.5 X 10’ cells/ml. Wild type or mutant phage PR4 was added at a m.0.i. - 10. Aeration of the cultures was continued at 37”. The turbidity of the infected cultures was followed using a Klett-Summerson calorimeter with a green filter (1 Klett unit equals approximately 5 X 1 O6cells/ml). Complementation between group 1 1 mutant Nam70 and group 15 mutant Ham208 was assayed by following the turbidity of a culture of strain TV854 coinfected with each mutant phage strain at a m.0.i. - 7.5 per mutant (total m.o.i. - 15). The pattern of lysis of strain TV854 by wild-type phage PR4 is provided for comparison. Arrows indicate the time of addition of l/100 volume chloroform to the infected cultures.

1981; Renell and Poteete, 1985). The defective gene product in group 15 mutant-infected cells has been tentatively identified as protein P24 (data not shown). However, the presence of a host protein which is produced in uv-irradiated cells in the vicinity of protein P24 in the SDS-PAGE systems employed in this study precluded a definitive assignment. Group 15 mutants should prove useful in overproducing certain phage strains or in producing extracts from mutant-infected cells for in vitro complementation studies. The PR4 group 15 mutants appear similar to phage PRDl group A mutants (Mindich et al., 1982a). Since information is lacking regarding the lysis profile and complementation pattern of PRDl group A mutants, a comprehensive comparison with PR4 group 15 mutants is not possible. However, PRDl group A-infected cells could also be lysed by addition of chloroform and electron micrographs of thin sections of group A-infected cells showed them to be packed with phage particles (Min-

PHAGE PR4 NONSENSE

19

MUTANTS

I

10 pm

I

10 pm

FIG. 4. Light microscopy of group 15 mutant Ham208-Infected cells. Strain TV654 was grown in RB at 37” and infected with mutant Ham208 as described in the legend to Fig. 3. Slides were prepared as described by Pfennig and Wagener (1986). (A and 6) uninfected cells; (C and D) strain TV854 cells at 4 hr oostinfectron with mutant Ham208.

dich et a/., 198213) consistent with the yield from PR4 group 15 mutant-infected ceils noted above. Group 16. Mutant phage belonging to groups 1 or 16 were first identified through marker rescue by recombinant plasmid pCTV400. This clone contains the 2850bp /‘WI-D fragment of the PR4 genome and was known to rescue group 1 mutants (Vanden Boom and Cronan, 1990) which are defective for the phage PR4 DNA polymerase (Mindich and Bamford, 1988; Davis and Cronan, 1983). In spot complementation tests none of these mutants appeared to complement each other or the four known mutants in Group 1. However, liquid complementation assays indicated that these mutations comprise two distinct complementation groups, the previously identified group 1 and the new group 16 (data not shown). Group 16 mutants give low, but significant, burst sizes in mixed infection with group 1 mutants. Examination by SDS-PAGE of the proteins made

in Su- uv-irradiated cells infected with representative group 1 or group 16 mutants verified that these phage comprise two distinct genetic groups (data not shown). Extracts of Su- cells infected with group 16 mutants lacked a protein corresponding in apparent molecular weight to the terminal genome protein (P5A) (Davis and Cronan, 1983; Davis and Cronan, 1985). The observed low level of complementation between mutants in groups 1 and 16 might be due to the polar effect of a nonsense mutation on a promoter-distal gene (Newton et al., 1965). Group 17. Extracts of Su- cells infected with mutant Nam1235 lacked a previously unidentified phage protein produced late in infection. This protein species migrated between proteins P7 and P8A in the two SDSPAGE systems used in this study and was designated protein P2 1. Unfortunately, phage Nam1235 contained secondary mutations affecting proteins Pl and P4. A

20

VANDEN

BOOM AND CRONAN

* P5

* P21

PllAw

13 14 group

A

17 18 group

6

19 ? group

C

FIG. 5. (A) Pattern of proteins synthesized in group 13 and group 14 mutant-infected cells. Radiolabeled extracts of mutant-infected cells were prepared as described under Materials and Methods. Samples were analyzed in a 15% glycerol-containing acrylamide gel as described in the legend to Fig. 1. (B) Pattern of proteins synthesized in group 17 and group 18 mutant-infected cells. Radiolabeled extracts of mutant-infected cells were prepared as described under Materials and Methods. Samples were analyzed in a 15% glycerol-containing acrylamide gel as described in the legend to Fig. 1. (C) Pattern of proteins synthesized in group 19 mutant Nam 1268 and mutant NamlO31 -infected cells. Radiolabeled extracts of mutant-infected cells were prepared as described under Materials and Methods and analyzed in a 16% acrylamide gel as described in the legend to Fig. 2. Protein molecular weight standards are identical to those shown in Fig. 1. Abbreviations used: UN, uninfected; WT, wild type; MW. molecular weight standards.

derivative of Naml235, Naml235A, was obtained that lacked only proteins P4 and P21 (Fig. 5). Mutant Naml235A grew on Su- strain TVB500. Thus, we conclude that proteins P4 and P21 are both dispensible under the experimental conditions used in this study. Group 18. Two mutants, Nam 1009 and Naml380A, were assigned to group 18. The original isolate of Nam1380 contained a secondary mutation which affected P2 synthesis. Nam1380 was cleared of this mutation to yield Naml380A. This phage strain had

no other detectable secondary mutations. Mutants NamlOO9 and Naml380A both showed leaky growth on Su- strains and could not be characterized genetically in complementation tests. Analysis by SDSPAGE of the proteins synthesized in Su- uv-irradiated ceils infected with NamlOO9 or Naml380A indicated that these phage failed to synthesize virion protein P5 (Fig. 5). This was the sole basis for their assignment to group 18. We were particularly interested in obtaining mutants defective for protein P5 since this is an integral

PHAGE PR4 NONSENSE

component of the phage membrane. Protein P5 is thought to be closely associated with viral membrane phospholipids since (i) this protein migrates anomalously in SDS-PAGE gels, characteristic of very hydrophobic integral membrane proteins (Davis et al., 1982); and (ii) treatment of empty (DNA-less) phage particles with phospholipase liberates protein P5 and -90% of the phage phospholipids (Davis et al., 1982). The characteiization of the P5-defective virus particles made in group 18 mutant-infected Su- cells should aid in the elucidation of the role of this membrane component. Group 19. The single member of group 19, phage Naml268, was defective for the nonvirion protein P8A produced early in phage infection (Figs. 1 and 5). Mutant Nam1268 was leaky for growth in Su- strain TVB500 and could not be characterized by genetic complementation tests. The identification of the defective protein produced in Su- uv-irradiated cells infected with phage Nam 1268 was the basis for assignment to group 19. Mutant Naml268-infected cells also produced a protein P5 of altered electrophoretic mobility. A putative amber fragment which migrated just Jelow the position of the wild-type P8A protein in SDS-PAGE gels was readily seen in Nam 1268-infected Su- cells (Fig. 5). Thus, the amber mutation seemed to be located in the region encoding the COOH segment of the protein. The leakiness of mutant Nam 1268 suggests that either protein P8A is not essential for phage growth or that the amber fragment retains partial P8A activity. Phage Nam1268 plated on the Su+ strain TVB503 as the indicator bacteria produced a novel “bull’s-eye” plaque morphology which was readily distinguished from wild-type plaques and may be useful as a genetic marker. Mutant /Vam 1037. One potentially useful amber mutant which could not be reliably assigned to any of the above genetic groups should be noted. In mutant Nam 1031 -infected Su- uv-irradiated cells, the early phage protein P8A appeared to be overexpressed with a concomitant decrease in late phage protein synthesis (Fig. 5). This phage strain may be defective for nonvirion protein P12A. However, the pleiotropic phenotype of this mutant precluded a definitive assignment. CONCLUSION We describe the isolation and preliminary characterization of 38 new amber mutants of bacteriophage PR4. These new mutants define seven additional PR4 genes. The current collection of PR4 nonsense mutants have been assigned to 19 genetic groups on the basis of complementation assays and the pattern of proteins made in mutant-infected Su- cells (Table 2). The refined SDS-PAGE analysis of phage-specified proteins presented in this study reveals a complexity

21

MUTANTS

that was originally not fully appreciated for phage PR4 (Davis et al., 1982; Mindich and Bamford, 1988). The analysis of group 2 amber mutants suggests that at least one segment of the PR4 genome codes for more than one phage gene product. Thus, the potential coding capacity of the 15,000-bp PR4 genome may have been underestimated. The present collection of phage PR4 amber mutants should facilitate a genetic approach to understanding membrane assembly in bacteriophage PR4. ACKNOWLEDGMENTS We thank Heejoon Myung for helpful discussions and K. Sanderson for providing bacterial strains. We also thank Jane Matheus for technical assistance. This work was supported by Public Health Service Grant GM 26156. This work is in partial fulfillment of the requirements for the doctoral degree for T.V.B. in the Department of Microbiology, University of Illinois.

REFERENCES BAMFORD, D. H., and MINDICH, L. (1984). Characterization of the DNA-protein complex at the termini of the bacteriophage PRDl genome. J. Viral. 50, 309-315. BAMFORD, D. H., ROUHIAINEN, L., TAKKINEN, K., and SBDERLUND, H. (1981). Comparison of the lipid-containing bacteriophages PRDl, PR3, PR4, PR5 and L17. J. Gen. Virol. 57,365-373. BLACK, L., and SHOWE. M. (1983). Morphogenesis of the T4 head. ln “Bacteriophage T4” (C. Mathews, E. Kutter. G. Mosig, and P. Berget, Eds.), pp. 2 19-245. ASM Publications, Washington. BLACK, L. W. (1989). DNA packaging in dsDNA bacteriophages. Annu. Rev. Microbial. 43,267-292. BRADLEY, D. E. (1974). Adsorption of bacteriophages specific for Pseudomonas aeruginosa R factors RPl and R1822. Biochem. Biophys. Res. Commun. 57,892-900. 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. Canad. J. Microbial. 21, 152-l 63. CASJENS,S., and HENDRIX, R. (1988). Control mechanisms in dsDNA bacteriophage assembly. In “The Bacteriophages” (R. Calendar, Ed.), Vol. 1, pp. 15-9 1. Plenum, New York. DAVIS, R. W., BOTSTEIN, D., and ROTH, J. R. (Eds.) (1980). “A Manual for Genetic Engineering: Advanced Bacterial Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. DAVIS, T. N., and CRONAN, J. E., JR. (1983). Nonsense mutants of the lipid-containing bacteriophage PR4. Virology 126, 600-613. DAVIS, T. N., and CRONAN, J. E., JR. (1985). An alkyl imidate labeling study of the organization of phospholiprds and proteins in the lipidcontaining bacteriophage PR4. /. Biol. Chem. 260, 663-67 1. DAVIS, T. N.. MULLER, E. D., and CRONAN, J. E., JR. (1982). The virion of the lipid-containing bacteriophage PR4. Virology 120, 287-306. GARRET. J., FUSSELMAN, R., HISE, J., CHIOU, L., SMITH-GRILLO, D., SCHULZ, J., and YOUNG, R. (1981). Cell lysis by induction of cloned lambda lysis genes. MO/. Gen. Gene?. 182, 326-33 1. GIULIAN, G. G., SHANAHAN. M. F., GRAHAM, J. M., and Moss, R. L. (1985). Resolution of low molecular weight polypeptides in a nonurea sodium dodecyl sulfate slab polyacrylamide gel system. Fed. Proc. 44, 686. HOWE, M. H., O’DAY, K. J., and SCHULTZ, D. W. (1979). Isolation of mutations defining five new cistrons essential for development of bacteriophage Mu. Virology93, 303-319.

22

VANDEN

BOOM AND CRONAN

JOSSLIN,R. (1970). The lysis mechanism of phage T4: Mutants affecting lysis. Virology 40, 719-726. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14C in polyacrylamide gels byfluorography. fur. /. Biochem. 56,335-341. LUNDSTROM, K. H., BAMFORD, D. H.. PALVA, E. ,T.. and LOUNATMAA, K. (1979). Lipid-containing bacteriophage PR4: Structure and life cycle. 1. Gen. Viral. 43, 583-592. MAURIZI, M. R., TRISLER, P., and GOT~ESMAN. S. (1985). Insertional mutagenesis of the /on gene in Escherichia co/i:/on is dispensible. 1. Bacreriol. 164, 1124-l 135. MALOY, S. R., and W. D. NUNN (1981). Selection for loss of tetracycline resistance by Escherichia co/i. J. Bacterial. 14, 1 1 1O-l 1 12. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982) “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MILLER, 1. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MINDICH, L., and BAMFORD. D. (1988). Lipid-containing bacteriophages. In “The Bacteriophages” (R. Calendar, Ed.), Vol. 2, pp. 475-520. Plenum, New York. MINDICH, L., BAMFORD, D., GOLDTHWAITE,C., LAERTY, M., and MACKENZIE,G. (1982a). Isolation of nonsense mutants of lipid-containing bacteriophage PRDl .J. Viral. 44, 1013-l 020. MINDICH, L., BAMFORD, D., MCGRAW, T., and MACKENZIE, G. (1982b). Assembly of bacteriophage PRDl: Particle formation with wildtype and mutant viruses. 1. Viral. 44, 1021-l 030. MINDICH, L., COHEN, J., and WEISBURD, M. (1976). Isolation of nonsense suppressor mutants in Pseudomonas. /. Bacterial. 126, 177-182. MULLER, E. D., and CRONAN, J. E. JR. (1983). The lipid-containing bacteriophage PR4: Effects of altered lipid composition on the virion. /. Mol. Biol. 165, 109-l 24. NEIDHARDT. F. C., BLOCH. P. L., and SMITH, D. F. (1974). Culture medium for enterobacteria. J. Bacterial. 119, 736-747.

NEVILLE, D. M. (1971). Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. J. Biol. Chem. 246,6328-6334. NEWTON,A., BECKWITH,J. R.. ZIPSER,D., and BRENNER,S. (1965). Nonsense mutations and polarity in the lac operon of E. co/i. /. Mol. Biol. 14, 290-295. PARKER,M., CHRISTENSEN,A.. BOOSMAN, A., STACKARD,J.. YOUNG, E., and DOERMANN, G. (1984). Nucleotide sequence of bacteriophage T4 gene 23 and the amino acid sequence of its product. /. Mol. Biol. 180, 399-416. PFENNIG,N., and WAGENER,S. (1986). An improved method of preparing wet mounts for photomicrographs of microorganisms. /. Microbiol. Methods 4, 303-306. RENELL, D., and POTEETE,A. R. (1985). Phage P22 lysis genes: Nucleotide sequences and functional relationships with T4 and X genes. Virology 143, 280-289. RICHARDSON,D. L., JR., AOYAMA, A., and HAYASHI, M. (1988). Proteolysis of bacteriophage 0X174 prohead protein gpB by a protease located in the fscherichia co/i outer membrane. J. Bacterial. 170, 5564-557 1. STANISICH,V. A. (1976). Isolation and characterization of plasmids in Pseudomonas aeruginosa. Bull. inst. Pasteur 74,285-294. TESSMAN, I., PODDAR, R. K., and KUMAR, S. (1964). Identification of the altered bases in mutated single-stranded DNA. 1. Mol. Biol. 9, 353-363. VANDEN BOOM, T. (1989). Genetic and Biochemical Studies of the Lipid-Containing Bacteriophage PR4, Ph.D. thesis, University of IIlinois. VANDEN BOOM, T., and CRONAN, J. E., JR. (1988). Enrichment of the bacteriophage PR4 membrane in phosphatidylglycerol is not essential for phage assembly and infectivity. J. Bacferiol. 170, 28662869. VANDEN BOOM, T., and CRONAN, J. E.. JR. (1990). A physical-genetic map of the lipid-containing bacteriophage PR4. Virology 177, 2332. WHITE, G. P., and DUNN, N. W. (1978). Compatibility and sex specific phage plating characteristics of the TOL and NAH catabolic plasmids. Gener. Res. 32,207-213. YOUDERIAN,P., and SUSSKIND,M. M. (1980). Identification of the products of bacteriophage P22 genes, including a new late gene. Virology 107,258-269.