Satellite bacteriophage P4: Characterization of mutants in two essential genes

VIROLOGY 53, 24-39 (1973)

Satellite Bacteriophage P4 : Characterization of Mutants in Two Essential Genes WARREN GIBBS, RICHARD NEAL GOLDSTEIN, ROBERTA WIENER, BJORN LINDQVIST', AND RICHARD CALENDAR Department of Molecular Biology, University of California, Berkeley, California 94720 (Lad Department of Microbiology, University of Iowa Medical Center, Iowa City 52240 Accepted January 17, 1973 P4 is a helper-dependent bacteriophage which can use the late gene products of temperate phage P2 to encapsulate P4 DNA (Six, 1963 ; Six and Lindqvist, 1970 ; Gibbs, 1972 ; Six and Klug, 1973) . P4 phage heads (d = 450 A) contain only onethird the volume of P2 phage heads (d = 620 A ; Inman et al ., 1971), and heads of the P4 size are not detected during a normal P2 infection (D . Walker, personal communication) . Thus P4 directs the formation of 450 A phage heads . P4 causes P2 prophage late genes to be expressed in the presence of immunity (Six and King, 1973), and without excision or replication of the prophage genome (Six and Lindqvist, 1971) . In the absence of a helper, P4 DNA can replicate (Lindqvist and Six, 1971) . We have isolated and characterized mutants in two essential P4 genes . P4 gene A mutants are unable to synthesize P4 DNA, but they retain the ability to transactivate' P2 prophage genes under nonpermissive conditions . Thus the A gene product may participate directly in the process of P4 DNA replication . P2-lysogenie, nonpermissive cells infected with P4 gene A mutants synthesize empty phage heads, 80% of which are intermediate in size between P4 heads and P2 heads . Heads of this intermediate size are also formed in small quantity during a normal P2 infection . The inability of P4 gene A mutants to synthesize P4-size heads may he due to a lack of replicating DNA or to lack of a size-directing protein . IP4 gene B is defined by one temperature-sensitive mutation which is partially dominant to P4 wild type at 42° . At the nonpermissive temperature this mutant can synthesize P4 DNA and cause a P2 prophage to be transcribed, but cannot cause the formation of head-like particles . Unlike P4 wild type, this mutant kills nonlysogenic cells at 42°, and greatly depresses the synthesis of DNA, RNA, and protein . INTRODUCTION

duced in the resulting burst . However, only P4 is produced if the helper genome is present as a prophage (Six and Klug, 1973) . The P2 genome can provide helper functions without replicating its DNA and in the presence of P2 immunity (Six and Lindqvist, 1971 ; Six and Klug, 1973) . Furthermore, the P2 early genes, A and B, which arc required for P2 DNA replication and late gene expression, are not necessary for satellite phage P4 production (Six and Lindqvist, 1970 ; Six, in preparation) . In the absence

Satellite phage P4 requires a helper phage genome for lytic multiplication (Six, 1963) . Temperate coliphages related to P2 can provide helper function either as a prophage or as a coinfecting phage (Six and mug, 1973) . When P4 and P2 coinfect a nonlysogenic strain, both phages are pro' Department of Microbiology, University of Iowa Medical Center, Iowa City 52240. ' Thomas (1970) used the term transactivation to describe the induction of gene expression from a X prophage by a superinfecting heteroimmune phage .

of a helper, P4 DNA can replicate (Lindqvist and Six, 1971) and lysogenize by attaching 24

Copyright ~ 1973 by Academic Press, Inc . All rights of reproduction in any form reserved.



SATELLITE PHAGE MUTANTS to the host genome at a site near pro, where no P2 prophage has been found (Six and King, 1973) . Genetic studies using conditional-lethal mutants of bacteriophage P2 have shown that P4 requires all known P2 late genes : six head genes, cloven tail genes, and one gene involved in cell lysis (Six and Lindqvist, 1970 ; Six, in preparation ; Gibbs, 1972) . These findings strongly suggest that the P4 virion is composed mainly, if not entirely, of helper proteins . As a corollary, the morphology of P4 should be quite similar to that of P2 . This has been confirmed by electron microscopy (Lunan et al ., 1971) . P4 and P2 phage tails are of similar size and structure. The P2 and P4 heads are both isometric in shape, but the P4 head contains only one-third the volume contained by the P2 head . P4 must direct the formation of small phage heads composed of P2 proteins, since P2-infected cells contain no head particles as small as P4 heads (D . Walker, unpublished observations) . The DNA of P4 (molecular weight 6 .7 X 106 daltons) is only one-third the size of P2 DNA (molecular weight 2 .2 X 10' daltons ; Inman et al., 1971) . From the known molecular weight of P4 DNA it can be estimated that P4 codes for 10-15 genes . Two are probably required for lysogenization : a repressor and an integration gene . In addition, P4 should carry at least one essential gene for transactivation of helper phage functions ; P4 might also carry essential genes for DNA replication and for head size direction . We have attempted to define some of these P4 essential genes by the use of conditionallethal mutations . We report here the isolation and characterization of 14 P4 amber mutants and one P4 temperature-sensitive mutant, which fall into two different genes . MATERIALS AND METHODS Phage strains . The P2 strains used were the following : P2 vir, which does not express immunity (L . E . Bertani, 1957) ; P2 cox, and cox 4 which are excision-deficient (Lindahl and Sunshine, 1972) ; the P2 tail amber mutant, P2 vir, amD 6 (Lindahl, 1971) ; the head mutant, P2 vir, amM,2 (Sunshine et al ., 1971) ; and the hetero-

25

immune hybrid phage P2 Hy* dis (Six and Klug, 1973) . P4 vir, is a spontaneous clear plaque-type mutant described by Lindqvist and Six (1971) . Bacteria . The bacterial strains used in this study are listed in Table 1 . They are all derivatives of E . coli C (Bertani and Weigle, 1953), although the amber suppressors have been introduced by transduction from E . coli K strains (Sunshine et al ., 1971) . In most cases, the lysogenic strains carry a cox mutation in the P2 prophage, to reduce the level of P2 produced after infection with P4 from 10 -4 to 10-6 . Media and chemicals . The following media were used : LB, a rich broth, described by Bertani (1951) ; TPG-CAA (Lindqvist and Six, 1971) and A medium (Bertani and Bertani, 1970), which are minimal salts media supplemented with glucose and casein amino acids . P4 stocks were stored in P4. buffer, which contains 1 % ammonium acetate, 0 .05 M MgSO 4 , and 0.05 M Tris • Cl, pII 7 .2 . 'H-methylthymidine, 'H-uridine, and 14 C-leucine were obtained from Schwarz-Mann Bioresearch ; Mitomycin C from Sigma Chemical Company and NG (N-methyl-N'-nitrosoguanidine) from the Aldrich Chemical Company . Isolation of P4 mutants . The P4 mutants used in this study were isolated from four separate mutagenesis experiments . The general procedure for mutagenesis was based on Adelberg et al . (1965) : a P2-lysogenic strain grown in LB was infected with P4 Vir, at an m .o .i . of about 5 . The phage were allowed to adsorb ; then unadsorbed phage were removed either by washing or by neutralization with anti-P2-serum . The infected cells were allowed to grow for 10-20 min, at which time they were washed with Trismaleic buffer pH 6 .0 and resuspended in the same buffer supplemented with 100 µg,: ml of freshly prepared NG . The suspension was allowed to incubate for 30 mum at 30° with aeration ; then the infected cells were washed twice with Tris-maleic buffer and resuspended in LB . The mutagenized cells were allowed to grow at either 30 ° or 37 ° for about 140 min . Cell debris was removed by centrifugation and the mutagenized stock



26

GIBBS ET AL . TABLE

1

BACTERIAL STRAINS

Collection number

Pertinent genetic characters°

Origin or reference

HF4704

her thy

C-1a C-212 C-520 C-1055 C-1748 C-1749 C-1757 C-1758 C-1760 C-1766 C-1792 C-1794 C-1971 C-1994

prototrophic prototrophic (P2 Hy' dis) prototrophic xupD polyauxotrophic sty prototrophic (P2 cox,) polyauxotrophic sty (P2 cox t ) polyauxotrophic sty xupD polyauxotrophic str s-upD (P2 eel4) her thy (P2) prototrophic supD (P2 eox4) polyauxotrophic str supF polyauxotrophic str sup? (P2) her thy rite her thy rife (P2)

Lindqvist and Sinsheimer (1967a) Sasaki and Bertani (1965) Six and Klug (1973) Sunshine et at . (1971) Sironi (1969) From C-la by lysogenization From C-1055 by lysogenization Sunshine et at . (1971) From C-1757 by lysogenization From HF4704 by lysogenization From C-520 by lysogenization Sunshine et al . (1971) From C-1792 by lysogenization Ljungkvist (1973) Ljungkvist (1973)

- str = streptomycin-resistant ; supD and supF = amber suppressors D and F, according to Taylor's (1970) nomenclature ; her = host cell reactivation deficient ; thy = thymine requiring ; rife = rifamyein sensitive . was sterilized with chloroform . Individual phage plaques from the mutagenized stock were tested for potential mutant properties . Temperature sensitivity was tested by stabbing each plaque with a sterile toothpick onto two plates which were seeded with C-1749 . One plate was incubated at 33° and the other at 42° . Phage which failed to lyse the indicator lawn at 42° but did so at 33 ° were retested . After two singleplaque isolations a stock was grown starting with three plaques obtained from the last test . Stocks of the one is mutant isolated (P4 vir 1 tsBsr) contain is+ revertants at a frequency of about 10 4 . Most of the P4 vir 1 amber mutants were selected by plating the mutagenized phage on a mixed indicator lawn containing four parts of the suppressing indicator C-1758 (supD) or C-1794 (supF) and one part of the nonsuppressing indicator, C-1749 . Amber mutants gave a turbid plaque on such a mixed indicator (Parkinson, 1968) . Turbid plaques were tested by picking and stabbing into two different plates : one seeded with suppressing indicator and the other with nonsuppressing indicator . The plates were incubated at 37° and checked for clearing at the stabbed loci . After two single plaque isolations a

stock of each mutant was grown and checked for am.- revertants . Those stocks which contained less than 10 -' arn+ revertants were used in the studies reported here . Preparation of high titer stocks of P4 sagotarots . Plate stocks were grown according to the procedure of Walker and Anderson (1970) except that P4 vir 1 tsB g, was incubated at 32° . The lysates obtained usually contained 10 8 PFU/m1 and were purified only by removal of cell debris and agar . These lysates were then used as inoculum for liquid cultures . The P4 amber mutants were grown in LB, while P4 vir1 tsB e7 was grown in TPG-CAA ; 400 ml of medium was inoculated with the proper host and allowed to grow to a cell density of 2 X 10 7 /ml . The cells were infected with the P4 mutant at an m.o .i . between 0 .01 and 0 .05 . The infected cells were incubated at 37 ° for the amber mutants or at 32° for P4 air, tsB 87 . The optical density of the culture was monitored (600 ma), and when it stopped increasing 10 ml of 4 % EDTA, pH 7 .0 was added to block phage readsorption . The lysates were incubated for an additional 40-60 min, and the cell debris was removed by centrifugation . The phage were concentrated by making the solution 0.5 31 in



27

SATELLITE PHAGE MUTANTS NaCl and 10 % in polyethylene glycol 6000 (PEG) (Yamamoto et al ., 1970) . The solution was made 0 .08 11 in -\Ig 2 }, to stabilize the P4 (Lindqvist and Six, 1971) . Upon complete solubilization of the PEG, the phage were pelleted by centrifugation and resuspended in P4 buffer . This procedure usually resulted in phage stocks of 10 10-10" PFU,/ml . Electron microscopy. (A) . Sectioning . Preparation of thin sections is described in the proceeding publication (Lengyel et al ., 1973) . (B) . Negative staining . Phage preparations were negatively stained with either 2 phosphotungstate (pH 7 .0) or 2 % sodium silicatungstate (pH 7 .0) on collodian-carbon covered 400-mesh Cu grids (Ladd Co .) . One drop of cell lysate or purified phage suspension was placed on each grid for approximately 2 min, after which excess suspension was removed, leaving a thin film (Anderson, 1961) . One drop of negative stain was then added and immediately removed by touching the edge of the grid with filter paper . The grid was allowed to air dry . Polystyrene latex spheres of known diameter were sprayed onto the other side of the grid using a nebulizer ; these beads were used for astigmatic corrections, particle size determination and quantitization . (C) . Use of the ekzctron microscope . Cell lysates, purified particles, and thin sections were observed using a Siemans Elmiskop I electron microscope at 60 kV equipped with a 200 µm condenser aperture, a 50 µm objective aperture, and a pointed filament at magnifications between 20,000 and 50,000 . Micrographs were taken on Kodak Projector Slide Plates (contrast) and generally enlarged three times when printed . (D) . Isolation of

phage particles by high ,speed sedimentation . The appropriate bacterial host was grown in LB to a cell density of 2 X 10 2/ml and concentrated 20-fold by centrifugation, and resuspended in LB supplemented with 1 mM CaC12 . Phage were added to m .o .i . = 5, and adsorption was allowed to proceed for 10 min at 0 ° . Injection was allowed to occur at 37 ° for 10 min, after which the cells were centrifuged and washed twice with broth to remove input phage particles . The cells were resuspended in LB at the original cell density and incubated at 37° with aeration . Twenty

minutes after the lysis of infected cells, cell debris was removed by centrifugation at 10,0008 for 20 min . (All centrifugation were performed at 2 ° .) The supernatant was assayed for plaque-forming units, and then MgC12 was added to yield a final concentration of 0 .1 if and formaldehyde was added to 1 .85 % in order to cross-link (stabilize) the proteins of the phage particle . Lysates were then centrifuged at 13,000g for 40 to 65 hr . The supernatant was decanted and the pellet was covered with P4 buffer and allowed to resuspend for 24 hr at 2 ° . The pellets were then resuspended by repeated pipetting, after which insoluble material was removed by centrifugation at 6000g for 15 min . Aliquots of the supernatant were then placed on collodian-covered copper grids, negatively stained, and examined under the electron microscope . RESULTS

Cwnplementation analysis of P4 mutants . In order to determine the number of genes affected by the P4 conditional-lethal mutants, we have performed the complementation tests presented in Tables 2 and 3 . Surprisingly, all 14 amber mutants fall into a single complementation group . In the complementation experiments described in Table 2 we have used P4 vir, amA 1 as a representative mutant against which the other amber mutants were tested . Additional experiments testing P4 vir1 amA17, le And 26 against one another failed to reveal any complementation (data not shown) . The P4 temperature-sensitive mutant defines a second complementation group, since it can complement P4 amber mutants (Table 3) . In these experiments the yield of the temperature-sensitive mutant increases more than 1000-fold, while the yield of amber mutant increases 22- to 230-fold . The complementation is symmetric : both mutant types are represented in the burst in approximately equal numbers . However, the total burst never exceeds 22% of the P4 vir 1 control burst . This low yield of phage is due to the partial dominance of the temperature-sensitive mutation over its wild-type allele : as shown in Table 4, the burst of P4 vir, at 42 ° is reduced fivefold by coinfeclion with P4 vir 1 fa•B3r . The above complementation results



28

GIBBS

ET AL .

TABLE 2

TABLE 3

LIQUID COMPLEMENTATION STUDIES' : P4 AMSae MUTANTS AGAINST P4 m.r, arnA,

LIQUID COMPLEMENTATION STUDIES" : P4 AMDER MUTANTS AGAINST P1 inrl taB,7

Amber mutant

Percentage of control burst Amber mutant alone

amA,

0 .60

amA 6

0 .43

amA 7 amA 8 amA,

0 .30 0 .75

amA 1 , amA,, amA n amA,, amA,, amA,, 4mA 2 , amA,,

Mutant

with P4 trir,

amA, <0 .05 0 .31

0 .90 2 .10 0 .12 0 .004 0 .25

0 .15 0 .15 0 .29 0 .09 0 .92 0 .08 0 .01 0 .21

0 .11 0 .02 <0 .002 0 .63

0 .08 0 .02 0 .02 0 .40

° P2-lyaogenic strain 0-1748 was grown to a cells in LB and conconcentration of 2 X 10' ./ml centrated tenfold in LB supplemented with 5 =11 Ca°' . The P4 mutant to be tested was added at a m .o .i . = 10 along with 10 P4 vir, antA l per cell . The phage were allowed to absorb for 15 min at 37 ° without aeration . An aliquot was removed and the amount of unabsorbed phage was measured . Anti-P2 serum was added to k = 2 and the infected cells were allowed to shake for 15 min at 37 ° . [Anti-P2 serum inactivates P4 grown with a P2 helper (Six and Klug, 1973) .] The infected cells were then diluted 10,000- fold and incubated at 37° for 120 min . Aliquots were removed at time = 0 (after the 10,000-fold dilution) and at 120 min . The 0 time point was taken to insure that there was an increase of phage above background at 120 min . P4 amber mutants were assayed at 37° on C-1758, which is lysogenic for P2 and carries supD . The results are presented as the percentage of the burst produced when, in a control experiment, C-1748 is infected with P4 vir, . The average burst of P4 Dir t in C-1748 is 50-120 phage per cell .

define two essential genes of satellite phage P4 . The amber mutants are assigned to gene A, while the temperature-sensitive mutant defines gene B . Recombinati
Percentage of control burst Mutant alone For amber For P4 uir, mutant laB,,

tAB37

amA, amA, amA,7 am4, s

<0 .004' 0 .27 2 0 .03 2 0 .18 2 0 .04 2

3 .7' 6 .8' 5 .3 2 9 .4 2

4 .5 2 8 .3 2 9 .2 2 12 .1 2

"P2 lyaogenic strain 0-1748 was grown to a concentration of 2 X 102 cell/ml in LB and concentrated tenfold in LB supplemented with 5 m,11 Ca'* . The P4 Tin am mutant to be tested and P4 t4r, tsB, 7 were added at a m .o .i . of 10 each or 20 when the mutant was being tested alone . The phage were allowed to absorb for 15 min at 41° without aeration . An aliquot was removed and the amount of unabsorbed phage was measured . AntiP2 serum was added to k = 4-8 and the infected cells were allowed to incubate for 5 min at 42° . The infected cells were then diluted 10,000-fold into prewarmed LB and incubated at 42 ° for 60 min . Aliquots were removed at time = 0 (after the 10,000-fold dilution) and at 60 min . The 0 time point was taken to insure that there was an increase of phage above background at 60 min . The burst was analysed for the proportion of amber and temperature sensitive phage produced . Amber mutants were assayed at 42° on C-1758 while temperature sensitive mutants were assayed on C-1749 at 33° . The results are presented as the percentage of the burst produced by P4 vir, , which is 40-60 phage per cell at 42 ° . A superscript denotes the number of experiments on which an average value is based .

(data not shown) . In contrast, all the amber mutants recombine with the temperaturesensitive mutant at frequencies from 0 .3 to 1 .7% . These results are consistent with the complementation results .

DNA synthesis by P4 conditional-lethal mutants under non permissive conditions . In order to determine whether either class of P4 mutants was defective in phage DNA synthesis, we have utilized mitomycin C to suppress host DNA replication (Lindgvist and Sinshcimer, 1967b) . Nonlysogenic cells pretreated with mitomycin C were infected with the P4 mutant to he tested and incubated at either 33 °, 37 °, or 42° . DNA syn-

29

SATELLITE PHAGE MUTANTS TABLE 4

TABLE 5

DOMINANCE OF P4 vir, tsBa7 ovER P4 vsrP

Two-FACTOR CRossEs BETWEEN P4 CONDITIONALLETHAL MUTANTS'

Infecting phage

P4 vir, and P4 vir, amA, P4 vir, and P4 vir, amA, P4 vir, and P4 vir, tsB,, P4 vir, and P4 vir, tsB,,

Temperature C

Percentage of control burst For amber mutant

For tsar

For P4 tire

42

36 1

76'

42

28 1

621

32

23'

23'

42

3 .6'

8 .01

° The P2-lysogenic strain C-1748 was grown to a cell density of 2 X 10 1 in LB and concentrated tenfold in LB which was 5 ruff in Caa+ . The cells were infected with r4 vir, , P4 vir, amA,,,, or P4 vir, tsB,, at a m .o .i . = 20 in the case of infection with only one phage and an m .o .i- of 10 for each phage in the case of coinfection . The phage were allowed to absorb for 15 min at 42° or 32° at which time anti -P2 serum was added to a k -4 -8 . The cells were allowed to incubate for an additional 5 min . The infected cells were diluted 10,000fold into prewarmed LB and incubated for 60 min at 42 ° or for 120 min at 32 °. Progeny phage were assayed on either C-1749 at 32° in the case of P4 vir, and P4 vir, tsB,, or C-1758 at 37° in the case of P4 vir, and P4 stir, amA, . The percentage of each phage in the burst was determined by replica testing single plaques for the appropriate mutation . The results are presented as the percent burst of a P4 vir, control . The average burst of P4 vir, at 42° is about 40-60 phage per cell . A superscript denotes the number of experiments on which an average value is based . thesis was measured by the incorporation of 'H-thymidine into trichloroacetic acidinsoluble material . Figure 1A shows that the three amber mutants A l , AV, and A26 failed to synthesize any DNA above background levels in a nonpermissive host . Furthermore, amber mutants An, 14, 19 and 21 when tested in a similar way, also failed to synthesize DNA in a initomycin-treated host (data not shown) . In contrast, P4 vir, lsB,, was able to synthesize DNA at 42° (Fig . 1B) . Thus the gene A product is needed for phage DNA replication while the product of gene B is not . Transcription of P2 prophage after infection by P4 mutants . In order to determine

P4 mutant

amA, amA, aurA, amA, amA, amA, o amA, : amA 14 amA 17 amA,, amA„ mnA,, amA,, amA, tsB,,

Percentage am* ts- recombinants P4 vir, amA,

P4 vir, tsBe,

<0 .003 <0 .003 <0 .003 <0 .02 <0 .00003 <0 .00007 <0 .002 <0 .00004 <0 .0004 <0 .00006 <0 .00007 <0 .00004 <0 .001 <0 .0004

0 .97 1 .30 0 .71 0 .79 0 .65 0 .79 1 .10 1 .50 0 .84 0 .38 1 .10 1 .70 0 .82 0 .34 <0 .04

° A log phase culture of C-1766 grown on LB was concentrated to 5 X 101 cells/rd in LB supplemented with 5 mM Ca" . The cells were infected with an m .o .i . = 8 for each phage used in the cross . The phage were allowed to absorb for 12 min at 30' . The infected cells were diluted 1000-fold and incubated for 120 min at 30 ° in the case of crosses performed with P4 vir, tsBae and at 37° for crosses performed with P4 amber mutants . Total phage released was measured by plating on C-1758 at 33 ° . In experiments where amber mutants were crossed with P4 vir, tsB,r , the amp ts^" recombinants were scored on C-1749 at 42° . In the case where amber mutants were crossed with another amber mutant, am+ recombinants were scored on C-1749 at 37° . The figures represent percentage of recombination, which is twice the percent . of arm* ts+ phage in the burst . whether P4 gene A or B is needed to activate transcription of the P2 helper prophage, we have measured P2-specific RNA synthesized at late times after P4 mutant infection of a P2-lysogenic strain . The data presented in Table 6 show that both classes of P4 mutant are able to activate late P2 transcription under nonpermissive conditions . The P2 mRNA synthesized is qualitatively similar to that produced during a normal P2 infer tion, as determined by annealing to the separated strands of P2 DNA (Lindqvist and Bevre, 1972) . During a normal P2 infec-



30

GIBBS ET AL.

60

60

90

0 M . .utes

ma,

0 0 ofealon

60

90

120

Ft(; . 1 . Measurement of DNA synthesis in cells infected with P4 conditional-lethal mutants . We have used a modification of the Lindgvist and Sinsheimer (1967b) procedure . E . coli HF4704 (her) was grown in TPG-CAA medium supplemented with 10 pg/mI of thymidine to a concentration of I X 10 8 cells/ml . The cells were divided into the appropriate number of aliquots and mitomycin C was added at a con. The cells were allowed to incubate without aeration in the dark at 37° for 10 centration of 60 mg/ml min . The cells were washed once with 0 .01 M Tris • 1101 pH 7 .4, 0 .01 M EDTA pH 7 .4 and resuspended in an equal volume of TPG-CAA medium supplemented with 2 .5 pg/ml of thymidine . The phage were added at a m .o .i . equal to 16 and allowed to absorb for 15 min at 30° .'li-methyl-thymidine (sp . act . 14 .6 Ci/ mmole) was added to give a final concentration of 6 pCi/ml . The infected cells were then incubated at 37° or 42° . Aliquots of 0 .01 ml were removed at the times indicated and added to 1 ml of cold 10% trichloroacetic acid (TCA) . Salmon sperm DNA (0 .05 mg) was added as carrier, and the samples were placed on ice for 30 min . The samples were centrifuged for 10 min at 15,000g . The pellets were resuspended in 0 .5 ml 1 M NaOH and incubated overnight at 37° . In the morning, 0 .5 ml of 1 M HC1 was added to neutralize the NaOH and 1 ml of cold 20% TCA added . This solution was placed on ice for 20 min and the precipitates were collected by filtration on glass fiber filters (Whatman GF/C) dried, and counted in a Packard scintillation counter using a toluene-based scintillation fluid .

tion, 90-95 % of the RNA is specific for the heavy strand of P2 DNA, and a similar proportion of heavy strand RNA is obtained, regardless of which P4 mutant infects a P2-lysogenic strain (Table 6) . However, the amount of P2 transcription induced by the P4 mutants is decreased . Under nonpermissive conditions, the gene A mutant induces 15-20 % the amount of P2 mRNA which is synthesized under permissive conditions (Table 6) . This decrease in transcription might be due to a simple gene dosage effect, since mutants in P4 gene A cannot replicate their own DNA under nonpermissive conditions . The P4 temperaturesensitive mutant also shows reduced synthesis of P2 mRNA (7% of control), and this effect may be part of the general reduction of macromolecular synthesis observed in

cells infected with this mutant at 42° . We will return to this point in a later section . Complementation analysis of P2 prophage transactivation . Our analysis of P2 mRNA does not determine whether all the required P2 prophage genes are transactivated after infection with P4 mutants. However, it is possible to test which prophage genes are being transactivated using the heteroimmune hybrid pbage, P2 Hy*dis, as prophage helper for coinfecting P4 and P2 amber mutants (Six, 1971 ; Six, in preparation) . P2 Hy*dis phage can complement P2 amber mutants during coinfection ; however, P2 Hy*dis prophage cannot complement P2 amber mutants when they superinfect a P2 Hy*dis-lysogenic strain . Nevertheless, P4 causes P2 Hy*dis prophage to complement coinfecting P2 amber mutants to a



31

SATELLITE PHAGE MUTANTS TABLE 7

TABLE 6 Acriv .a'rioN OF P2 TRANSCRIPTION try P4 MUTANTS' Source

of RNA

P4 vir, 56-57 min : (42°) P4 vir, tsB,37 55-57 min nonpermissive : (42°) P4 vir, amA, 60-62 min permissive : P4 vir, amA, 60-62 min nonpermissive :

Input term X 10 - s

1 .68

Percentage rem hybridized with P2 strandst "heavy "light" h1 (I) 9 .8

0 .40

TRANSACTIVATION

By

P4

vir, amA,°

ratio (I/1 +

hi

P2 amber mutant

P4 transactivator

Progeny P2 per cell

Fraction P2 am+ in

progeny

0 .04

0 .34

3 .4

0 .21

0 .06

1 .48

18 .1

0 .866

0 .05

2 .63

1 .6

0 .16

0 .09

° E . coli TTF4704(P2)rife or C-1794 were grown at 37° in TPG-CAA (supplemented with 10 pg thymine,/ml when necessary) to a titer of about 5 X 10' cells/ml . Two 5 ml portions of HF4704(P2)rif° were infected at 42 ° (the nonpermissive temperature for P4 vir n tsB37) with P4 vir, and P4 vir, tsB3, , respectively ; 5 ml portions of C-1794 (permissive) and HF4704(P2)rif' (nonpermissive) cells were infected separately at 37° with P4 vir, amA, . The m .o .i . in the above infections ranged between 3 and 10 . Two minutes pulse labeling of the RNA was performed by administering 100 yCi 3 H-uridine (sp . act . 24 Ci/mmole) to the 5 Oil aliquots of the infected cells at the indicated times after addition of phage . P2 DNA strand separation, preparation of RNA extracts and liquid RNA-DNA hybridization were carried out as described by B6vre and Szybalski, (1971) and by Lindqvist and Bgvre (1972) . Linearity of hybridization is observed under these conditions . In the case of extracts from uninfected P2 lysogens about 0 .02% of the radioactivity hybridizes to either of the P2 strands .

limited degree . We have asked whether P4 virl amA, can cause P2 IIy*dis prophage to complement two P2 amber mutants : P2 amD 5 , a mutant deficient in tail synthesis, and P2 a nM32, a mutant deficient in phage head synthesis . P4 virr, amA 1 induces the heteroimmune prophage to complement both these P2 mutants, as measured by a large relative increase in P2 burst size (Table 7) . 2 3

OF PROPHAGE GENES

In fact, P4 amA mutants are superior to their

&f a3 Nl 3s Mac D8 1) 8 DR

none vir, amA, none vir, vir, amA,

0 .03° 1 .1 1 3 .0 1 <0 .1 2 0 .22 2 .1 8

01 ,20 0/75 1/75 0/100 0/75 0/66 1

° The P2 H7f*dis-lysogenie, sup strain C-212 was grown to 2 X 10 8 cells/ml in LB broth . The cells were concentrated 2 .5-fold by centrifugation and resuspended in LB broth supplemented with 0 .5 m,47 CaCI 1 . The P2 ,sir, amber mutant indicated and P4 vin or P4 vir, amA, phages were added at m .o .i . = 10 for each type . After 15 min of adsorption at 37°, the infected cells were centrifuged and washed to remove unadsorhed phage . The infected cells were diluted 10,000-fold in LB broth and aerated at 37 ° for 60 min . Progeny phage were assayed . When these cells are infected with P4 vir, alone, a burst of 30 ph age/cell results . P2 progeny phage were tested for the presence of the amber mutation by stabbing isolated plaques with a toothpick into 0 .05 nil of phage diluent, and replicating these phages with nails onto two plates, one seeded with C-1757 (sup*) and the other with C-1055 (sup - ) . Since very few P2 am* are found in the burst, recombination between P2 am and P2 21J*dis prophage cannot account for the apparant transactivation phenomenon . A superscript denotes the number of experiments on which an average value is based . From these data we conclude that P4 gene A is not needed for the transactivation of P2 head gene M or P2 tail gene D . Experiments similar to those reported in Table 7 were also performed using the P4 tsB mutant, but the results were inconclusive because this mutant inhibits P2 growth at 42° (unpublished results) . Electron microscopy of P4 mutant-infected cells . In order to determine whether either P4 mutant was able to assemble a phage amt parent in ability to transactivate, as meas-

ured by this test . This result is probably due to the fact that P4 amA mutants do not replicate their DNA and therefore cannot compete with P2 amber mutants for late gene products .

32

GIBBS ET AL .



SATELLITE PHAGE MUTANTS particle, we have examined in the electron microscope thin sections of P4-infected P2lysogenic cells . Escherichia coli (P2) sup infected with P4 vir, amA 1 is able to synthesize phage capsid structures which do riot appear to contain any DNA (Fig . 2C, D) . These P4 vir, amA,-infected cells contain about 9 empty and 0 .1 full phage heads per section ; as compared to these same cells infected with P4 vir,, which have about 2 .0 empty and 5 .0 full heads per section (Fig . 2A, B) . These results confirm that P4 vir1 amA 1 can cause transcription of at least some P2 late genes and demonstrate that the P2 mRNA is translated into proteins which can form head-like particles . Since the number of heads per cell (>_27) is greater than the input m.o .i . of 5, and since the input DNA does not replicate, it seems likely that these phage heads condense without the help of phage DNA . Thin sections of a P2-lysogenic strain infected with P4 vir, taB37 at 32° and 42° contain about 3 .5 and 0 .1 phage heads per section, respectively . P4 vir,-infected cells at 42° synthesized about 8 phage heads per section . Thus, although P4 vir, tsB37 is capable of inducing some P2 mRNA, it is defective either in translation of this messenger or in assembly of the P2 proteins into a phage particle . Size distribution of head-like Particles . Although P4 heads arc composed of P2 proteins (Six and Lindqvist, 1970 ; Gibbs, 1972), the P4-size head is only synthesized in the presence of the P4 genome (D . Walker, personal communication) . We have tested P4 vir, amA1 under nonpermissive conditions to 4 Sections are usually about 500-600AA in thickness . One bacterial cell, depending on the orientation of the cell when it is sectioned, yields three to six sections .

33

see whether it can cause this size-direction phenomenon . P2-lysogenic sup cells were infected with P4 vir, amA,, and after cell lysis, the phage heads were isolated by high-speed centrifugation and photographed (Fig . 3A) . The distribution of head diameters is shown in Fig . 4 . Eighty percent of the heads synthesized after infection with the P4 amber mutant have a diameter of 550 A, which is intermediate in size between P4 heads (d = 450 A) and P2 heads (d = 620 A) . The remaining 20 % are P4-size and P4 petit size (d = 400 A) . By comparison, P4 vir1-infected cells yield only P4 and P4 petit size heads (Fig. 4) . Thus P4 virl amA 1 has a severely reduced ability to cause condensation of P4-size head-like particles . The 550 A heads found after P4 amA mutant infection are also found during a P2 infection (Fig. 4, and D . Walker, personal communication) where they constitute 19-3 .5% of the total . Synthesis of phage tails in P2-lysogenic cells infected by P4 vir, amA1. Thirty-seven phage tails can be seen for every 100 heads in the pellet of particles purified from lysates of P4 vir 1 amnA1-infected cells . This result confirms the proposition that tail genes are being transcribed during infection of nonpermissive, P2-lysogenic cells by P4 an1A mutants. Host killing by P .4 vir, tsB, 7 . We have already noted the unusual ability of P4 vir, tsB37 to suppress the growth of P4 vir 1 at 42° (Table 4) . In addition, the tsB, 7 mutation inhibits the growth of nonlysogenic E . coli. Inhibition of host growth was measured by change in optical density and by colony-forming ability . Nonlysogenic cells were infected with P4 vir1 tsB37 at m .o .i . = 7 and were incubated at 32 ° for 60 min in order to allow expression of the P4 genome .

Fio . 2 . E . coil (P2) infected with P4 vir, and P4 vir, am-4 1 . The sup- , P2-lysogenic host C-1748 was infected with the phage indicated at m .o .i . = 5 as described in Materials and Methods . Five minutes before lysis, cells were pelleted, fixed, and then embedded and sectioned . At the time of lysis (60 min) a sample of bacterial cell debris was pelleted, fixed, and then embedded and sectioned as described in Materials and Methods . (a) Thin section of a cell infected with P4 vir, for 55 min . Intracellular full heads (FH) are seen . X 55,400 . (b) Thin section through bacterial membranes and debris after lysis of host cells by P4 vir, . Empty heads (EH) and full heads are observed . X 124,000 . (d) Thin section of cells infected for 55 min with P4 vir, n:mA, . Only empty heads are seen . X 85,400 . (c) Thin section through bacterial membranes and debris after lysis of the host by P4 vir, amA, . Only empty heads are observed . X 97,700 .

34

GIBBS ET AL,

Fio . 3 . (a) Negatively stained (2% PTA) heads isolated by high-speed sedimentation from lysates of E . coli (P2) sup (strain C-1748) infected with P4 vir, amA, . Cells were infected at m_o .i . = 5 in LB

at 37° as described in Materials and Methods . At 80 min-20 min after Iysis -particles were purified by high-speed sedimentation and negatively stained as described in Materials and Methods . Several sizes of empty heads (EH) are observed . X 152,200 . (b) Negatively stained heads isolated by high speed sedimentation from E . coil C-la (sup ) after lysis by P2 vir, am? 4 . Full heads (FH) and two sizes of empty heads (EH) arc seen . X 153,300. The infected cells were then shifted to 42° for 60 min and then shifted back to 32° . Immediately following the shift to 42°, the cells ceased to increase in optical density (Fig. 5) . Upon shifting back to 32 ° , the infected cells began again to increase in optical density . Thus the tsB 37 mutation affects bacterial growth in a temperaturedependent and reversible fashion . The number of bacterial survivors obtained from the above experiment 60 min after infection was dependent upon the

temperature at which they were plated . Colony-forming ability was 1000-fold reduced for P4 virr, tsB a 7-infected cells incubated at 42° when compared to these same cells incubated at 32° (Table 8) . P4 vir l infection has much less effect on the colonyforming ability of nonlysogenic cells (Table 8) . These results show that the tsB,, mutation causes nonlysogenic cells to be killed at 42 ° . The effect of P4 vir r, tsB 97 on host macromolecular synthesis . In order to understand



35

SATELLITE PHAGE MUTANTS P4 normol size (mostly full)

50

P4

40 30

P4 Petite Size (empty)

20 10

II

0

.

,l .

. (amp 1

50

P4 amA

40 30

u x I

(empty)

20 (empty) 10

16

0 Imost

0E t

amp

5

P2 amF

40

(amply) 3 2

'J11

~~~lll

u

P2 normal size (full and empty)

5

P2

4 3 1 2

1

P2 petite size Memory)

I I1I iI }

3 0

400

450

500

1 550

III 600

I

It , 650

700

Head Diameter, A

Fim . 4 . Histograms showing the size-distribution of heads isolated from P2 and P4 mutant-infected cells . The number of particles measured of a specific head diameter are plotted against that specific head diameter . The particles were prepared as described in Materials and Methods, with the exception of the P2 vir,, which was concentrated by polyethylene glycol precipitation instead of high-speed sedimentation . the effect of P4 vir, 1sB, on host metabolism we have monitored DNA, RNA, and protein synthesis during an infection of nonlysogenic cells at 42 ° (Fig . 6) . The amount of 3H-thymidine, 'H-uridine, and 14C-leucine incorporated into acid-insoluble material 40 min after infection is reduced to 20, 33, and 18%, respectively, when P4 vtir, tsB37-infected cells are compared to P4 virrinfected cells . This reduction of all macromolecular synthesis can be detected within 5 min after infection by P4 vir, tsBxr . The depression of mRNA translation may account for the absence of phage heads in P4 viii tsB37 -infected cells at 42° . DISCUSSION

We have isolated 14 amber mutants and one temperature sensitive mutant from sat-

ellite bacteriophage P4 . All 14 nonsense mutants fall into one complementation group (gene A), while the temperature-sensitive mutant defines a second gene, B . The A gene mutants do not define a series of noncomplementing genes, since they yield so few am+ recombinants after coinfection under permissive conditions . It is puzzling that all 14 amber mutants fall into the same gene, even though they were isolated from four independent mutagenesis experiments . This phenomenon cannot be due to the mutagenie action of nitrosoguanidine, since hydroxylamine mutagenesis gives similar results (B . Lindgvist and E . Six, unpublished data) . The reason for this effect is not understood at present . The P4 A mutants are unable to synthe-



36

GIBBS ET AL . Shift to 4I2

Shift to 32

°

°

C a

100

10 0

I

20

I

40

I

1

I

P

vir,

P4 vil,

/

I

120 40 60 80 100 Time after infection, minutes

3537

I

160

180

FIG . 5 . Growth of nonlysogenic cells infected with P4 vir, tsBar . The nonlysogenie strain C-la was grown to a cell density of 2 X 10a/ml in TPG-CAA medium and concentrated tenfold in TPG-CAA base which lacked amino acids and glucose . The concentrated cells were infected with P4 vir, or P4 vir, tsBB7 at an m .o .i . = 7-10 and allowed to absorb for 30 min at 32° . The infected cells were removed from the incomplete medium by centrifugation and diluted back to their original concentration with TPG-CAA medium . The cells were allowed to incubate with aeration for 60 min at 32° at which time they were shifted to 42° . The infected cells were allowed to incubate for an additional 60 min at which time they were returned to 32° . The growth of the cells was followed using a Klett spectrophotometer (600 mm) . TABLE 8 BACTERIAL SURVIVORS AFTER INFECTION WITH vir, AND P4 vir, tsBB,°

Infecting phage

None P4 vir, P4 virt tsBa7

P4

Number of colony formers X 10 -7 32°

42°

31 13 7

33 2 .0 0 .007

The nonlysogenic strain C-la was grown to a cell density of 2 x 10 8 in TPG-CAA medium and concentrated tenfold in TPG-CAA base which lacked amino acids and glucose . The concentrated cells were infected with P4 sir, or P4 zzr, feB,7 at an m .o .i . = 7-10 and allowed to absorb for 30 min at 32° . The infected cells were removed from the nonreplicating medium by centrifugationD and diluted back to their original concentration with TPG-CAA medium . The infected cells were allowed to incubate with aeration for 6o min at 32° at which time the survivors were plated at 32° or 42° .

strands of P2 DNA in a pattern which is characteristic of RNA obtained from a lytic P2 infection . The P4 amA mutants activate both the head and the tail regions of the P2 prophage, as measured by complementation analysis (Table 7) . P4 arm mutants cause E . coli (P2) sup cells to synthesize phage head- and tail-like particles, confirming the transactivation of P2 prophage head and tail genes (Figs . 2, 3) . From these results it is apparent that P4 DNA synthesis is not a prerequisite for transcription of P2 prophage . P4 amA mutants provide an opportunity to study phage head synthesis in the absence of DNA replication . Phage DNA appears to be unnecessary for condensation of P2 petit phage heads (d = 550 1), since these particles are formed in excess of phage DNA copies in P2-lysogenic sup cells infected with the nonreplicating mutant P4 virl amA, . Heads with 550 A diameter are not a unique product of P4 amA infection :

size P4 DNA (Fig. IA), but they can cause transcription of the P2 prophage at a re- they also constitute about 25% of the heads synthesized after P2 infection (Fig . duced level under nonpermissive conditions (Table 6) . The P2 mRNA produced by- 4) . When wild-type P4 infects E . cola (P2) 75 % of the heads formed are 450 A in dibridizcs to the "light" and the "heavy"



SATELLITE PHAGE MUTANTS

37

sH

HF4?04

uridine

6000

P4 .r.,

4000

2000

P4 0 0 0 10 20 30 40 0 Time after infection at 42, minutes

10

20

30

40

FIG. 6 . Measurement of macromolecular synthesis in P4 cer, taB,, infected cells . E . coil HF4701 was grown to a density of 2 X 10' cells/'ml in TPG-CAA medium which contained 100 pg/ml of each L-amino acid except for 5 pg/nil L-lcucine and was supplemented with 5 pg/nil of thymidine and uridine . The cells were centrifuged and concentrated tenfold in TPG-CAA base (without glucose or amino acids) . The cells were divided into three aliquots and two were infected with P4 vir, or P4 air, tsB,, at an m .o .i . = 10 . The phagc were allowed to absorb for 10 min at 42° . The cells were removed from the TPG-CAA base by centrifugation and resuspended in an equal volume of TPG-CAA medium (as above) . Each of the three aliquots were divided into three and each diluted 20-fold into a flask which contained TPGCAA supplemented with either 5 pCi/ml of [%H]methyl thymidino, 2 .5 pCi/ml of [ 1 H]uridine, or 0 .5 pCi/ml of [ 1 °CJlcucine . At the times indicated 20 ml were removed and added to 20 mm circles of Whatman 30 mm filter paper . The filters were added to 5% cold TCA, (10 nil/filter), batchwashed once with 5% TCA, washed twice with cold water, washed twice with hot water, and washed twice with 95% enthanol (Bollum, 1966) . The filters were dried and counted in toluene liquid scintillation fluid .

ameter while 25 % have a diameter of 400 A (Fig . 4) . The distribution of head sizes may be governed by the availability of replicating phage DNA . (This hypothesis assumes that DNA participates in the initial condensation of phage heads .) According to this model, 550 A heads would be made when no replicating phagc DNA is available, as during infection of E . coli (P2) sup- with a P4 aniA mutant . Replicating P2 DNA would cause 620 A heads to form, while replicating P4 DNA would cause 450 A heads to form . This model does not explain the condensation of 400 A heads . An alternative model assumes that P4 codes for a "size-directing protein ." According to this scheme, the size-directing protein is needed for condensation of 450 A and 400 A heads . P4 amA mutants arc said to make little or no size-directing protein, and thus they produce very few 450 A and 400 A heads . This model cannot be strictly correct,

since it predicts the presence of 620 A heads in E . coli (P2) sup- cells infected with P4 amnA mutants . In a separate communication (Barrett et at ., 1972) we report the discovery of a P4induced transcribing enzyme which can be assayed by its ability to synthesize polyriboguanylic acid upon a poly (dG) .poly (dC) template . The template specificity of this enzyme and the phenotype of the P4 amA mutants suggest that the enzyme activity is not involved in the transactivation of the P2 genome, but rather in the synthesis of P4 DNA . A conceivable role of this enzymatic activity may be to synthesize a small RNA primer which could serve as an initiator for P4 DNA replication (Brutlag et at ., 1971) . The P4 A gene may he the structural gene for this P4 transcribing enzyme . Alternatively, the gene A product may control the expression of other P4 genes, including the structural gene for the poly (dG)-poly (dC) transcribing enzyme .

38

C1BBS ET AL .

In contrast to the P4 amA mutants, the P4 tsR mutant is able to synthesize P4 DNA (Pig . 1) . This mutant induces the P2 prophage mRNA under nonpermissive conditions, although at reduced levels (about 7 %i Table 7) . Thin sections of cells infected with 1'4 tnr l tsB37 at 42 ° contain less than 0 .1 phage heads per section as compared with eight heads per section in cells infected with P4 vir, at 42° . Furthermore all macromolecular synthesis is drastically reduced during an infection by P4 vir, tsA37 at 42° . The inhibitory effects of this mutant are thought to be caused by the mutant gene 13 product, since the tsB37 mutation is partially dominant to the wild-type allele (Table 4) . The function and site of action of the stood .

B

gene product are not yet under-

ACKNOWLEDGMENTS We thank Erich Six for providing us with phage P4 before publication, communicating with us his unpublished results and for suggesting that P4tsB might affect nonlysogenic cells . We also thank Robley C . Williams for allowing us the use of his electron microscopy facilities . We are grateful to Alex Lumsden, whose steady hand and keen eye were essential for the isolation of the P4 mutants, as well as Margaret Marsh, who fixed and embedded the samples prior to thin-sectioningThis work was supported by U . S . Public Health Service Research Grants At 08722 and AT 01267 from the National Institute of Allergy and Infectious Diseases, by Training Grant GM 01389 from the National Institute of General Medical Sciences, by Cancer Contract 71-2173 from the National Cancer Institute Special Virus-Cancer Program, by Grant 507 from the California Division of the American Cancer Society, by Grant 277 from the Jane Coffin Childs Memorial Fund for Medical Research, and by Grant B72-99F3667-01 from the Swedish Medical and Natural Sciences Research Councils and the Swedish Cancer Society . R.K .G. is the recipient of an American Cancer Society Postdoctoral Fellowship (PF-663) . REFERENCES ADILBERG, E . A ., MANuEL, M ., and CHnN, G . C . (1965) . Optimal conditions for mutagenesis by N-methyl-N'-nitro-iV-nitrosoguanadine in Escherichia colt K 12 . Biochem . Biophys . Res . Comm . 18, 788-795 . ANnnnsoN, T . F . (1961) . On the fine structures of the temperate bacteriophages P1, P2 and P22 .

Proc . Eur . Regional Conf . o,, Electron Micros-

copy, Delft, 11, 1008-1011. BARRETT, K ., Glees, W ., and CALENDAR, R . (1972) . A transcribing activity induced by satellite phage P4 : (Helper dependent/RNA polymerase/DNA synthesis) . Proc . Nat. Acael. Sci . U . S. 68, 2986-2990 . BERTANI, G . (1951) . Studies on lysogenesis . 1 . The mode of phage liberation by lysogenie Escherichia colt . J . Bacteriol . 62, 293-300 . BEnmANI, G, and WEIGLE, J . J . (1953) . Host controlled variation in bacterial viruses . J . Bacterial . 65, 113-121 . BE RTANI, L . E . (1957) . The effect of the inhibition of protein synthesis on the establishment of lysogeny . Virology 4, 53-71 . BERTANI, L . E ., and BERTANI, G . (1970) . Preparation and characterization of temperate noninducible bacteriophage P2 (host : Escherichia coli) . J. Gen . Viral . 6, 201-212 . BOLLUM, F . J . (1966) . Filter paper disk techniques for assaying radioactive macromolecules . In "Proc . in Nucleic Acid Research" (G . C, Cantoni and D . R . Davies, eds .), pp . 296-300 . Harper & Row, New York . BpYRE, K ., AND SZYBALSKI, W . (1971) . In "Methods in Enzymology" (L . Grossman and K . Moldave, eds .), Vol . 21, pp . 350-351, Academic Press, New York and London . BRUTLAG, D ., SEES&MAN, R . anti KORNRERG, A . (1971) . A possible role for RNA polymerase in the initiation of M13 DNA synthesis . Proc . Nat . Acarl . Sci . U . S . 68, 2826-2829 . GIBes, W . (1972) . Studies on satellite bacteriophage P4 . Ph .D . Thesis, University of California, Berkeley . INMAN, R . B ., SCHNISS, M., SIMON, L . D ., Six, E . W ., and WALKER, D . H ., SR . (1971) . Some morphological properties of P4 bacteriophage and P4 DNA . Virology 44, 67-72. LENGYEL, J . A ., GOLDSTEIN, R . N ., SUNSHINE, M ., MARSH, M., and CALENDAR, R . (1973) . Bacteriophage P2 head morphogenesis : cleavage of the major capsid protein . Virology 53, 1-23 . LINDAHL, 0 . (1971) . On the control of transcription in bacteriophage P2 . Virology 46, 620-633 . LINUAHL, G ., and SUNSHINE, M. (1972) . Excisiondeficient mutants of bacteriophage P2 . Virology 49, 180-187 . LINmQVIST, B . H ., and SINSHmMER, R . L . (1967a) . Process of infection with bacteriophage X17 .1 . XIV : Studies on macromolecular synthesis (luring infection with a lysis-defective mutant . J . Mol . Biol . 28, 87-94 . LTNDQVIST, B . H . and SINSHEIMER, R . L . (1967b) . Process of infection with bacteriophage 4'X174 . IV : Bacteriophage DNA synthesis in abortive



SATELLITE PHAGE MUTANTS infections with a set of conditional lethal mutants . J . Mot . Biol . 30, 6940 . LINDQVIST, B ., and Six, E . (1971) . Replication of bacteriophage P4 DNA in a nonlysogenic host . Virology 43, 1-7 . LINDQVIST, B . H ., and B¢vRE, K . (1972) . Asymmetric transcription of the coliphage P2 genome during infection, Virology (in press) . LJUNGKVIST, E . (1973) . Interaction of phage P2 DNA with some fast-aedimenting host components during infection . Virology (in press) . PARKINSON, J . S . (1968) . Genetics of the left arm of the chromosome of bacteriophage lambda . Genetics 59, 311-325, SASAKI, I . and BERTANI, G . (1965) . Growth abnormalities in Hit derivatives of E . coli C . J . Gen . Ificrobiol . 40, 365-376 . SinoNI, G . (1969) . Mutants of Escherichia coli unable to be lysogenized by the temperate bacteriophage P2 . Virology 37, 163-176 . Six, E . W . (1963) . A defective phage depending on phage P2 . Bact . Proc . p . 138 . Six . E . W . (1971) . High degree of complementability by prophages : a special property of phage P4 . Ract . Proc. . p . 150 . Six, E . W ., and KLUG, C . A . (1973) . Bacteriophage P4 : a satellite virus depending on a helper such as prophage P2 . Virology 51, 327-344 .

39

Six, Ii . W ., and LINDQVIST, B . H . (1970). Helperdependent reproduction of coliphage P4 . Bact . Proc . p . 202 . Six, E . W ., and LINDQVIST, B . H . (1971) . Multiplication of bacteriophage P4 in the absence of replication of the DNA of its helper . Virology 43, 8-15. SUNSHINE, M . G ., THORN, M ., GIBBS, W ., CALENDAR, R ., and KELLY, B . (1971) . P2 phage amber mutants : characterization by use of a polarity suppressor. Virology 46, 691-702 . TAYLOR, A . L . (1970) . Current linkage map of Escherichia coli . Bacterial . Rev . 34, 155-175 . THOMAS, R . (1970) . Control of development in temperate bacteriophages . III . Which prophage genes are and which are not trees-aetivable in the presence of immunity. J . bfol . Biol. 49, 393-404 . WALKER, D . H ., JR ., and ANDERSON, T. F . (1970) . Morphological variants of coliphage P1 . J . Virol . 5, 765-782 . YAM,MOTO, K . R ., ALBERTS, B . M., BENZINGER, R ., LAWHORNE, L., and TREIBER, G . (1970) . Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification . Virology 40, 734-744 .