Bacteriophage P4: a satellite virus depending on a helper such as prophage P2

VII~OLOGY

61, 327-344 (1973)

Bacteriophage

P4: a Satellite

Virus

as Prophage ERICH Depurtmerd

W. SIX”

AXI)

of !Vicrobioloy~,

CAROL The UGversity

Depending

on a Helper

Such

P21v2 A. CONNELLY of Iowa,

KLUG

Iowa Cilly, Iowa 52240

Accepted Oclober 25, 19Y2 Bacteriophage P4 was isolated from cultures of Escherichia coli strain K-235, and two spontaneous mutants, P4 imp and P4 virl, were obtained from I’4 wild type. P4 was found to be a satellite virus, depending on a helper genome for the completion of its lytic life cycle. Genomes of phage P2 and of Pa-related phages can serve as helpers. They are able to assist PI either if present in the host as a prophage or if introduced by coinfection. P4 does not depend on a helper for lysogenizing its host. A specific P4 prophage site was recognized on the host chromosome. 1’4 depends on its helper for the expression of genes with late functions. This is indicated by the finding that the Pi virion reflects in certain of its properties, for example, host range and neutralizability, the genotype of its most recent helper. P4 production in cells lysogenic for a helper and infected with P4 proceeds without induction of the helper prophage and without lifting the (helper-specific) immunity of such cells. It is proposed that P4 produces a transactivating factor that triggers the expression of the helper genes that have to fulfill the late functions needed by P4. Some other findings concerning the propagation of P4 and properties of P4 lysogens are also reported. INTllODUCTION

Bacteriophage 1’4 was discovered (Six, 1963) dm~ examining the phages spontanccoli strain ously produced by lkherich,ia K235, reported to be lysogenic for a 1’21 Symbols P4 and 1’2, used without further specification of the genotype, denote not on!y the respect,ive wild types, but also mutant genotypes derived from wild-type 1~4 and 1’2, respectively. Wherever necessary, genot,ypes are specified, P4+ amd P2+ referring to the wild types of P4 and P2, respectively. The last host in which phage P4 was propagated may be indicated by an added symbol denoting the host; e.g., P4.C(P2), refers to 1’4 (wild type or mutant) grown in strain C lysogenic for P2 (wild type or mutant). 2 SlIpported by Grant No. AI-04043 from the Nationa,] institutes of Health, Unit,ed States Public IIenlt,h Service, and also aided in part by n grant from the Swedish Medical Research Council (Visiting Scientist Fellowship, Project No. K70-10I(-3280). 3 Recipient of the fellowship from the Swedish Medical Research Cou~~~il.

rclatcd phagc: PI< (,Jesaitis and Hutton, 1963). Culturos of K-235 were found to produce in addition to PII another tempcrate phage, designated P4. It became clear immediately that P4 can form plaques only on lawns of bacteria lysogenic for either P2 or certain related phages such as PM, but not on lawns of the corresponding nonlysogenie strains. We report hcrc on some basic asp&s of the propagation of P4, and in particular \VC,show the following: (i) P4 is genetically defective and depends on a helper gcnomc such as that of phage P2 for gene products needed to produce progeny phage, but (ii) 1’4 dots not depend on a helper for lysogenizing its host. (iii) Cells lysogcnic for helper and infected with P4 will product essentially only P4 but no helper type phage, whereas (iv) nonlysogenie cells infected with 1’4 and simultancously with a helper type phage will produce bot’h kinds of phagc. The implications of these and some related 327

Copyright All riphts

@ 1973 by Academic Press, of reprodluction in any furm

Inc. reserved

32s

SIX

AND

findings will hc discussed. Brief accounts of the original observations concerning P4 were previously published by Six (1963), and Six and Connclly (1966). Further studies of 1’4 have since beon puhlishcd by Inman et al. (1971), Lindqvist and Six (1971), and Six and Lindqvist (197 1). MATEItIALS

AND

MHTHODS

Media. L broth contains per liter of water: 10 g tryptonc (Difco) or t,rypticase (BBL), 5 g yeast extract (Difco), 10 g NaCl, and 1 g glucose. I, agar and LO agar contain L broth plus 1% Difco-agar. I, agar also contains 2.5 X 10e3 M CaC12. The agar medium used in bacterial crosses to select for recombinants, as described by Bertani and Six (1958), contains per liter of water: 7 g KZ2HP04, 2 g KHzP04, 1 g (NH&SO+ 1g r>-asparagine, 1 g glucose, 0.5 g r\‘a-citrat,e. 5Hz0, 0.1 g ;\IgSOb, and 14 g Difco-agar. After autoclaving streptomycin sulfate is added to a final concentration of 250 mg/l. Depending on the strains used and the sclection desired, this medium rnaJ; be further supplemented with the appropnatc L-amino acid(s) or xanthinc or both, at final concentrations of 20 mg/l for each. Bacterial strains. &cherichia coli strain I<-235 (F&d&&4, 1950) is the original source of phagcs PK and P4. Other E. coli strains used, all dcrivativcs of strain C, are listed in Table 1. As indicators for plaque assays were used t’hc streptomycin-resistant strains C-S, C-1055, their lysogcnic derivatives, especially C-290, and also the following streptomycin-resistant derivatives of X. dysenteriae Sh : the nonlysogenic strain Sh-16, Sh-201 = Sh-lG(PK), and Sh-203 = ShlB(PK Hy dis). Only strains lysogcnic for P2 or a related phage could be used as indicators for P4. Phage strains. 1’4 phages include wild type, as produced by I<-235, and two spontaneous mutants obtained from the wild type: the temperate P4 imp, originally recognizcd as forming slightly larger plaques than those of P4+, and the immunityinsensitive P4 via. I’2 and its relatives are reviewed by Bertani and Bcrtani (1971). Besides wildtype P2 the following phages that arc homo-

KLUG

immune with P2 wcrc used: the> cxtrcmely CaZ+ dependent 1’2 rd 1 and I’2 rd 1 Cai, a Ca%ndependent derivative of P2 rd 1, possibly a 1’2 rd l+ revertant,, (SW Tublc 7), and PK (Jesaitis and Hutt’on, 1963). Other P2-related phages include I’2 Hy’dis (Cohen, 1959) and it’s following homoimmunc rclatives: I’2 Hy’dis vir14 (Cohen, 1959), 1’2 Hy*dis (Six, 1961), and PK Hy dis, presumably a recombinant between 1’1~ and P2 Hyldis showing the host range and Ca*+ independence of PK. Phaye stock preparation. Stocks were obtained from phage-infected cultures of the appropriate host in tither L broth or A medium (Bcrtani and Bcrtani, 1970). Details of the proccdurcs employed varied. The most commonly used hosts for P4 were C-77, C-235, and C-295, for other phages tither C-la or C-2. WC found it preferable to add to the cultures after lysis EDTA at’ a final concentration of 0.01 X rnt’lier than KP (= potassium phosphate; XC Bcrtani and Bertani, 1970), since apparently the KP addition, but not the addition of 15DTA may lead to a rapid decay of the> titer of P4 lysates. After lysis cell debris were rcmoved by 101~ speed centrifugation, then the phages were pelleted by centrifugation (usually for 2 hr at 54,000 g), finally t.he pellets were resuspcndcd, usually in a MgCl2 solution (0.075 M). All P4 st’ocks contained also a small number of helper type phage. This admixture can be considered insignificant for our studies, since t’he plaquc-forming unit(s) (PFU) ratio of helper-type phage to I’4 was found to range from lop4 to lo-“. Rntiphage seTa. Prior to their immunization, rabbits were tested for the presence in their serum of neut’ralizing antibodies to the phage to be used as antigen. n’onc were found. Each animal then received a subcutaneous injection of betbvccn 8 X 10’” and 4 X 10” PFU5 of phage suspended in 0.075 M MgClz and emulsified with an equal volume of Freund’s complete adjuvant (Difco). About 10 n-ceks later, n-hen the peak of the primary immune response had been reached, the animals received intravenously a single booster dose of phage suspension (1 to 7 X 10” PFUjanimal). Four to .5 days later the sera were collected

__ Collection number

Genetic structure

Mating type*

C-la c-2 C-8 c: -27 C-G6 c-77 c-03 c -208 c-212 C-218 c-231 C-233 C-235 C-236 c-237 C-243 c-244 C-245 C-246 C-249 C-284

++ + i+ -Iit + + iii-t-

c-290 C-295 C-330 c-331 c-417

+ + -

C-429 C-43G c-443 c-445

-

See C-1055 See C-8 arg 2’1, try sir arg 7’1, lry nrg ?'I, lry m-g Tl, tru arg 2’1, try

C-1055

+

his xan thr leu str ~2


urg Tl, try str (P2 rd lc)l uru w+)IrI

(defective P2) (P2 rd l)r (1’2 Hy*dis)l (P2 Hyldis)l

See C-8 See C-8

Wf)I

See C-8 See C-8 sir s lr P2

(1’2 rd 1 Ca’) @WI Wf)I (P4+) (P2 Hyldis)I (P4+) (P2 rd 1)1 (P4+) (P4$) (P2 rd 111 P4+1 U’WI (P4+) 0’2 rd 1 Ca’) (P4+) W’K)

w+)I (pa+) @‘2+h(I’4+) (P2+)1 (P4 imp)

See C-1055

met purJ thr his pro ilvz

--

-~

Origin or reference Prophages”

Mutations*

arg 7'1, lr!J sir

~__~

str sir str sir

d

Sasaki and Bertani (1965) Bertani and Six (1958) Bertani and Six (1958) Bertani and Six (1958) Bertani and Six (1958) Six (1966) Obtained from 11. E. Bertani Six (1961) From C-2, by lysogenizat,ion Six (1966) Six (19G6) From C-2, by lgsogenization From C-2, by lysogenization From C-231, by lysogenization From C-218, by lysogenization From C-208, by lysogenization From a cross C-66 X C-236 From a cross C-208 X C-236 From a cross C-235 X C-236 From C-233, by lysogenization By spontaneous mutation of a C-8 derivative to PB-resistance and subsequent lysogenization From C-1055 by lysogenization From C-la by lysogenization From C-290 by lysogenization From C-231 by lysogenization Wiman et al., 1970 Wiman et al., 1970 Wiman el al., 1970 Wiman et al., 1970 from C-8 by UV-induced mut,ation; B. Kelly (personal communirat,ion) Wiman et cl., 1970 ____..-__

u +: F+; -: F-. ISFor explanation of symbols and further details, see Wiman et al. (1970). c Roman numeral subscripts indicate P2 (or PK) prophage location, if determined. The locations for all P-1+ prophages listed were found to be identical (see results). The site occupied by P4 imp (in C-331) has not, yet been determined. d This strain is nonlysogenic, but a derivative lysogenic for P4+ was obtained and used in bacterial crosses (see Table 11). The site occupied by P4+ was found to be the same as that occupied in the other P4+ lysogens listed.

by cardiac puncture. Scra with neutralization constants 117 of about 500 to 1000 min-’ could be obtained. The I’4 suspension used ns antigScn contained no more than 2 X

10B5 PI< per 1’4, according to plaque assays. One-step growth and single-burst experiments. All cultures were gro1v-n in and di-

luted into L brot’h. The incubation

tempera-

330

SIX AND KLUG

ture for all experiments was 37”. The usual procedures were as follows: Host bacteria in the exponential growth phase were suspended in broth containing 2.5 or 5 X 1O-3 M CaC12, aiming at a cell titer of 2 to 3 X 108/m1, and were assayed for colony formers. CaClz could be omitted for infections with PK or P4-C(PK). After addition of phage, the culture was incubated for a period of 5-10 minutes. Then the extent of phage adsorption was determined by assaying the culture for st.reptomycin-resistant plaque formers (adding about 0.5 mg streptomycin to each assay plate), provided the host cells were streptomycin-sensitive. The infected culture was diluted into broth containing either anti-P2 or anti-PK serum at a lc value of 1-2 min-’ and was incubated for 5-10 min to neutralize most of the unadsorbed phages. Then the culture was further diluted and assayed for phage-yielding cells. The yielder frequency is the number of yielders expressed as the percentage of infected cells, the latter as estimated from the multiplicity of infection. In some experiments treatment of the infected culture with antiphage serum was omitted. Determination of phage production in the infected cultures followed standard procedures. For one-step growth experiments the average burst size given, unless stated otherwise, is the ratio of the number of phages produced to the number of infected cells; for single-burst experiments it is the mean of the individual burst sizes found. The indicator used for single-burst assays was either Sh-203 or C-1055(P2 Hy’dis) to allow plaque formation by both: P4 and either P2 or PK. The phages could be distinguished by their plaque types. In some cases the plaque type classification was checked (and confirmed) by testing the phages contained in the plaques against the appropriate selective indicators. Tests for P4 prophuge. The simplest test for the presence of P4 prophage relies on the inhibit’ion of the growth of P2 Hy’dis vir14 ;n cells lysogenic for P4. L broth cultures of isolates to be tested are grown to a ccl1 titer of about 108/ml and spotted on L agar plat,es with an overlay containing about lo7 1’2 Hy’dis vir 14.After incubation of the

plates overnight, P4 lysogens can be identified by confluent bacterial growth on the spots, usually with nibbled edges, whereas spots of isolates not carrying P4 show very little bacterial growth, usually just a few colonies. This test allows detection of P4 prophage in cells not carrying a helper and also in cells carrying either P2 or PK. For cells lysogenic for a helper, the presence of a P4 prophage can also be detected by testing cultures of such cells for the types of spontaneously produced phagc. Bacterial crosses. The procedures employed were essentially the same as described by Bertani and Six (1958) with the following minor modifications. In crosses where only one or two closely linked markers were selected from the donor, the number of donor cells in the mixture with recipient cells was reduced aiming at donor:recipient ratios of about’ 1: 10 or 1: 100 (rather than 1: 1). Recombinant colonies were picked into L broth containing streptomycin (200 mg/l), incubated, and then streaked either on LO agar plates or, if appropriate, on plates with the above described synthetic agar medium, not containing any supplements. One colony from each streak was tested for P4 lysogeny. ILESULTS

Isolation of Pd and Its Dependence on a Lysoqenic Host Cultures of K-235, grown in L broth to a cell titer of about 108/ml, contain per milliliter between lo4 and lo5 free phages that form plaques with a nonlysogenic indicator such as C-8 or Sh-16. These plaque formers can bc identified as phage PK. PK is homoimmune with P2 (Jesaitis and Hutton, 1963), hence it cannot form plaques on a lawn of cells Iysogcnic for P2. However, if a C or Sh strain lysogenic for either P2 or PK is chosen as indicator for the phages produced by K-235, plaques are obtained, corresponding to a PFU titer about one-fourth OJthat found with a nonlysogenic indicator) These plaques are rather small (with a diameter of about 1 mm vs 2-3 mm for PK. and quite turbid. They indicate t’he presence of another phage produced by K23.5, phage P4.

HELPER-DEPENDENT

SATELLITE

From such playucs I’4 phagc can be isolated and then propagated lytically in an appropriate lysogenic host (see Materials and Methods). Strains lysogenic for P4 can also easily be obtained from the plaques. Such P4 lysogens still carry the prophage (= “helper”) originally present in the indicator strain (e.g., P2 or PK) and (in culture) they produce both P4 and helper-type phage. That I’4 is unable to form plaques on lawns of nonlysogenic cells, became evident from an examination of plaques produced on such lawns by phages from either I-235 itself or from C-236, a strain lysogenic for 1’2 and for P4 (obtained from K-235). All of the tot.al of 833 plaques examined contained phagc able to lyse a nonlysogenic indicator, but only two of them also produced (I’4 like) plaques with a lysogenic indicat’or. Hence 831 of the 833 plaques were caused by eit,her PK or P2 (depending on the source of the phages). The two exceptional plaques were shown to contain a mixture of PK and P4. Presumably they resulted from the accident’al deposition of a P4 phage within a PK plaque arca. The failure to detect any “pure” P4 plaques puts t’he plating efficiency of P4 for nonlysogenic indicators at < 1 %,4 and, as shown below, t,he cficicncy can actually be regarded as nil. P4, as produced by K-235, defines the wild type> of this phagc. From the wild type \ve obtained t,wo spontaneous mutants, I’4 imp and I’4 virl, which like the wild-type phage can form plaques only wit’h indicators lysogcnic for either I’2 or a related phage. The inability of 1’4 (wild type and mutants) to form plaques on lawns of nonlysogcnic cells is demonstrated by the assays of P4 lysates obtained from P4 infected cells lysogenie for P2 or PK (see Materials and Methods). The PFU titer of such lysates, as dctcrmined with a nonlysogenic indicator, al\~a?;s is much lower (by a factor ranging bct\veen lo4 and 10fi) than the titer (of P4) determined with a lysogenic indicator. The plaques appearing on nonlysogenic lawns resemble helper-type plaques and arc in4 Since Iho proportion of P4 among the phages produced by K-235 and C-23F is about one-fifth; compare Table 12.

BACTERIOPHAGE

P4

331

deed caused by helper-type phages contained in the P4 lysatcs. This can be shown by assaying the P4 lysatc with an indicator lysogenic for a defective P2 (e.g., strain C-93). No plaques at all are observed in such assays. The helper-type phages present in the lysate are blocked by the immunity of the indicator and 1’4 cannot grow, presumably because of thn defect of the prophagc carried by the indicator. If the P4 lysatcs examined contain any phage able to grow in C-93, then its frequency must bc less than 1OF per 1’4 (as assayed with an indicator lysogenic for wild-type Pa). From the evidence presented in the following sections it will become apparent that P4 is restricted to forming plaques on lawns of lysogenic indicators because it is genetically defective and depends on a helper such as a P2 prophagc to produce progeny phage. il~ultiplication of P/, in Lysogenic Hosts Infected with P4 Since bacteria lysogenic for P2 (or a related phage) appear to be the natural hosts for P4, the multiplication of P4 in such hosts \vas studied in “one-step growth” experiments. The results of one such experiment’ (Fig. 1) show that after a latent period of about 60 min E. coli C cells lysogenic for PK and infected with one P4 wild-type phage \vill rclcasc on the average about 300 new I’4. In a similar experiment, using a host lysogcnic for I’2 Hy’dis, the average burst size was about 100. Similar results were obtained for the P4 mutants, I’4 imp and P4 virl (Table 2). In all these expcriments a latent period of about 60 min was observed. Under comparable conditions P2 and I’K (infecting nonlysogenic cells) have a latent period of only about 30 min. In those cxperimcnts in which most cells wcrc infcctcd (multiplicity of infection = 3 or more) the ratio of the number of hclpcr type phage (I’2 wild type or 1’2 Hyj’dis) to the number of P4 virl produced was also determined. It ranged from less than lo-” to about 1 X 10-4, similar to the helper: 1’4 ratios for P4 lysatcs. Since the P4 burst size is about 100, it follows that most of the infected culls must rclcasc: only 1’4.

332

SIX

AND

KLUG

Multiplication Phaye

of P/t and Coinfectinq Helper

In view of the virtual absence of helper type among the phages liberated bv P4 infected lysogenic cells, it became of”interest to find out whether lysogenic cells, infected with P4 and simultaneously with a phage related to the helper prophage, would produce only P4 or also the coinfecting phage. In the first series of such experiments, the coinfecting phage chosen (PK or P2) was sensitive to the immunity of the lysogenic host. As shown in Table 4, such mixedly infected cells produced a normal yield of P4, but released only very little phagc of the coinfect’ing (and prophage) type, usually less than OIIC per cell. (An average yield above one, about 1.5, was found only in one experiment in which the multiplicity of the coinfecting phage was rather high.) The 0 20 40 60 80 100 120 small number of coinfecting type phages TIME (min) could result from a repackaging of some of the coinfccting phage genomes, in the abFIG. 1. One-step growth experiment for wildsence of any multiplication of the coinfecting type P4 with C(PK) as host. At time = 0, a phagc. culture of C-235 (titer: 2.9 X 108/ml) was inQuite different results were obtained when fected with P4, from a Sh(PK)(P4) culture, at a multiplicity of about 3 X 1O-3 and incubated at the coinfecting phage (P2 vin) was hetero37”; 5 min later the culture was diluted 1:400 and immune wit,h the prophage (P2 Hy*dis) in 1:2000. At that time 96% of the P4 input had been the host (Table 5). In this case the mixedly adsorbed. The diluted cultures were kept at 37” infected cells produced, on the average, P4 and assayed for plaque formers at the times and coinfecting P2 in about equal amounts. shown. The PFU titers indicated refer to the unThe total number of phages (P4 and P2) diluted adsorption mixture and are corrected for produced per mixedly infected cell apunadsorbed phage. proached the average burst size of cells infectcd with either phage alone. In each of This conclusion was confirmed by “single the experiments summarized in Table 5, burst” experiments (Table 3). In experimore than 80 % of the mixedly infected cells ments a-c the multiplicity of infection was were shown to be P4 yielders, and also 80 % very low, hence the number of uninfected or more of the mixedly infected cells proved host cells in the burst tubes was correspondto be P2 yielders. Hence most of the cells ingly large (about 200). The large number must have produced both types of phage, of uninfected cells accounts for the tubes in indicating that P4 and P2 can multiply which helper type phage was produced. It together. can also account for the one tube in which This point was further studied in singleboth P4 and helper-type phage was released. burst experiments with nonlysogenic cells Of the remaining total of 79 tubes with P4, mixedly infected with P4 and either PK or 77 contained only P4, two tubes each con- P2 (Table 6). These experiments also protained also a single helper-type plaque vided evidence that a coinfecting phagc can former. Hence, in these 79 tubes, 2 helperserve as helper for P4 in a nonlysogenic type plaques were observed for about 9000 host Of the total of 94 tubes in which phage P4 plaques, in good agreement with the was produced, 21 contained only 1’4. Fifty helper: 1’4 ratios found for P4 lysatcs. nine tubes (or 68 %) contained a mixt,urc of

HELPER-DEPENDENT

SATELLITE TABLE

ONE-STEP

-__~

P4 type

GROWTH

___

Yielder frequenc9 vi)

d

INFECTING

LYSOGENIC

HOSTS

Average burst size per yielderbt e

Helper/P4ratio*, f

2


ND0

(107-258)

ND

(P2)

5

(0.1

ND

132 (199-227)

ND

C(P2)

13

imp

co74

virl

c

vii-1

virl

333

I’4

2

FOR P4 imp AND P4 virl

EXPERIMENTS

Number Multi$icity of experiments infection68 c

Host5

BACTERIOPHAGE

C (P2Hy*dis)

-

(37ll)

(SFlO4)

137 (74-248)

((lo-6

2 x 10-s to 1 x W-4)

(3-i)

(72?08 )

117 (73-148)

(<10-S

2 x 10-s to 1 x 10-4)

9

a All C(P2) hosts carried wild-type P2; for twelve experiments C(P2) = C-295. C(P2Hy*dis) = c-212. b The average of the values from individual experiments is listed first; their range is given in parentheses. c Corrected for unadsorbed phage; for 10 experiments adsorption was not determined, for these experimenm it was assumed that adsorption was 9870 of the phage input, equal to the average of the adsorption values found for the other experiments included in this table. d Percentage of infected cells producing P4 as determined by plaque assay before lysis. 6 P4 titer after lysis: titer of P4 yielders. f Titer of helper type phage P4 titer; determined after lysis. g ND: not determined. TABLE SINGLE

EXFk

BURST

ANALYSIS

3

OF LYSOGENIC

I I-

I

INFECTED

WITH

P4 Uninfected culture

Infected culture

Host

P4 type

CELLS

-

.I.

-7

Multiplicit of infectia

of tubes with YI Number bursts 7

Average burst size@ P4

IIelper

~f%l: with bursts (helper)

60 60 80

98 222 50

184 180 44

6 6 3

120 119

103 78

-

-

Pure P4

Mixed (P4 and helper)

PUR helper

2 x lo-3 2 x 10-a 1 X lo-$

12 19d 12’

0 1e 0

4 1 6

I

7 29

0 0

0 0

No

-

+” +”

T; c

l t”

d e

+ virl

-

C-235 = C(PK) C-235 = C(PK) C-233 = C(P2 rd I Cai) c-77 = c(P2+) c-295 = C(P2$)

8:k

60 60 40

-

a IV = number of tubes examined. h Sizes of individual bursts range from about fit to about 3 X t,he average values. c Instead of a P4 lysate, the supernatant of a culture of strain Sh lysogenic for P4 and PK was used as a source of P4, after sterilization with chloroform. ‘1 Including one tube with 423 P4 and 1 PK. e Expected number of tubes in which coincidentally a pure P4 burst and a pure helper burst occurred is 0.7. f Including one tube with 36 P4 and 1 P2. 0 ND not determined.

334

SIX

INFECTION

KLUG

TABLE

4

OF P2 LYSOGENS WITH P4 AND A P2 IMMUNITY-SENSITIVE (ONE-STEP GROWTH EXPERIMENTS)

P4

Coinfecting

Tee

m.0.i.”

+ virl viri virl

4 2.3 7 10

0 Multiplicity

AND

phage

Host

m.o.i.a

‘Me

4 4 16 16.5

PK P2virl P2virl P2viri

C-27 c-295 c-295 c-295

= = = =

TABLE

infection P4 P2 -~~~~~__ 5 3 3 9 7

7 6 5 11 8

PHAGE

P4 yielder frequency (%)

P4

Coinfecting phage

Coinfecting phage plus pro)hage type

67 85 89 96

60 517 140 190

0.19 0.39 1.4 0.3

0.23 0.40 1.6 0.4

C(P2 rd 1 c) C(P2f) c(P2+) c(P2+)

Average burst sizes

of infection. 5

MIXED INFECTION OF C-212 = C(P2 Hy* dis) WITH P4 viri AND P2 viri (ONE-STEP GROWTH EXPERIMENTS) Multi$icity

COINFECTING

Yielder frequency (%I

Average burst size

P4

P4

P2

P4

P2

31 39 65 106 59

29 39 38 22 36

73 93 108 124 118

91 ND* ND* 151 164

82 86 100 111 89

P2 80 86 98 82 89

Average burst size of controlsa

a Infection with only one phage type; P4 values from those experiments listed in Table 2 that were performed together with the corresponding mixed infection experiments listed in this table. * Not determined.

P4 and either PK or P2. This percentage is larger than could be expected on the basis of a coincidental occurrence within the same tube of two yielders, one producing only P4, the other only PK or P2. Although, in principle, some of “mixed yields” observed could have been produced by cell “doublets,” one member of the doublet yielding only P4, the other only helper type phage, the majority of the tubes containing phage mixtures must have been produced by single bursts yielding both types of phage. This conclusion is supported by the finding that the average numbers of phage of either type

found for the tubes containing phage mixtures are lower than the corresponding numbers for tubes containing only one type of phage. (Compare with similar data in Table 5.) For experiments (b), (c), and (d) (Table 6) the percentage of “mixed” yielders was also estimated from the number of cells that produced plaques with (i) a common indicator for P4 and PK or P2, such as C(P2 Hy’dis), (ii) a specific P4 indicator, e.g., C(P2), and (iii) a specific PK or P2 indicator, namely a nonlysogenic strain. For experiment (b) the estimate agrees well with the single-burst data, whereas for experiments (c) and (d) the estimates are even higher (82 and 94%, respectively) than the single burst data indicate. In these last two experiments, involving cells infected with wild-type P2 and P4 via, the mixed yielders could also be recognized on nonlysogenic lawns, since, unlike cells yielding only P2, they produced plaques with clear centers containing P4 virr as well as P2. We conclude that in mixed infection, P2 (and PK) can multiply together with P4 within the same cell, but only if the host has no P2 immunity. The immunity of a P2 lysogen for a superinfecting homoimmune phage is apparently not lifted by simultaneous infection with P4. Helper-Determined Vision

Properties

of

the

PJ

Further insight into the nature of the helper-dependent propagation of P4 was

HELPER-DEPENDENT

SATELLITE TABLE

SINGLE-BURST

EXperiment

-

P4

Helper

:

INFECTED WITH P4 AND

-

Host

Nb

Number of tubes with bursts

-

__

+

6

+ virl virl

5.5 2 5

m.o.La

-

~

PK PK P2+ P2+

14 9 3.4 6

c-2 c-2 C-la C-la

335

P4

6

ANALYSIS OF NONLYSOGENIC CELLS MIXEDLY HELPER PHAGE

Type

:

BACTERIOPHAGE

60 80 140 160

Average burst sizes for tubes with

1Pure Mixed Pure P4 (P4 and helper helper)

P4 only

_.___

__-

1 6 13 1

10 16 21 12

1 4 5 4

192 258 119 52

Mixed Bursts P4

Helper ~-

76 206 55 32

104 64 17 36

Helper only

343 270 66 / 77

___~ a Multiplicity of infection. b Number of tubes examined.

gained t’hrough experiments that revealed that certain properties of the P4 virion depend on t’he genotype of the helper used. Such properties of the P4 virion were found to reflect the corresponding properties of the phage whose genome was employed as helper for the propagation of P4. One aspect in which helper t’ype phages differ is their ability to form plaques at differcnt Ca*+ concentrations in the agar medium. Hence the Ca2+ requirements were compared for P4 and helper type phage produced by cultures of C strains lysogenic for both: P4+ and for one of several helper type phages knon-n to differ in their Ca*+ rcquircment’s. Cultures of such double lysogens WYC grown in L broth t’o a titer between 2 and 10 X 10’ cells/ml, t,rcat’ed with chloroform, and centrifuged. The supernatants \vere then assayed for plaque formers with the> appropriate indicators on 1, agar plates (with CaClT) and also on LO agar plated (no CaClz added). The result’s for somo reprcsent,ativc strains (Table 7) shorn that omission of CaClz from tho agar reducw thr plaqw-forming ability of P4 to a similar extent as that of the particular hclpcr type: phngc produced by the same double lysogc>n. Henw the CaZ+ requirement of 1’4 phagc appears to bc determined by it#s helper. In similar experiments we compared the heat stability of 1’4 and helper-type phages wleascd from different double lysogens (Table 8). Comparing the phages produced

by C(PK) (P4) and by C(P2) (P4) we found that PK is more stable than P2 and that P4 reflects the heat stability of its helper. However, P4 from C(P2 Hy’dis)(P4) did not share the extremely low stability of P2 Hy’dis, but showed about the same stability as P4 from C(P2)(P4). An interpretation of this difference in the heat stability of P4 and P2 Hyldis will be given in the Discussion. P2 and PK differ also in t’heir host range and in their neutralizability by anti-P2 and anti-PK sera (Jcsaitis and Hutton, 1963). Hence we test,ed next if P4 expresses the genotype of its helper with respect to its host range. A P2-resistant., but PK-sensitivc mutant of strain C was lysogenized with PK. The resulting strain (=C-2S4) was used as indicator for P4 with a PK-like host range in the experiment summarized in Table 9. P4 virl from a stock propagated in a PK lysogen showed a plating efficiency of about 50 % in plaque assays with C-B4 as indicator, as compared to assays \j-it’h a standard (= P2-sensit’ive) indicator. This P4. C (1%) was then grown in cells lysogenic for P2. Assays of the phage progeny indicated that after only one cycle of growth in a P2 lysogen P4 had lost its ability to ovcrcome 1’2 resist,ance, i.e., to form plaques on a C-2S4 lawn. In a subsequent, second growth cycle this P4.C(1’2) was propagated in a PK lysogen. As a result of its passage through this host, P4 regained its ability to form plaques on C-2S4 lawns to the extent

336

SIX TABLE

AND

7

PLAQUE-FORMING EFFICIENCY IN THE ARSENCE OF CaClz FOR P4 AND FORHELPKRTYPEPHAGE PRODUCED BY DOUBLY LYSOGENIC DONORS Ca2+ independence6

Phage donor” experiments

Strain

K-235 C-236 C-246d C-245” C-2498

PK

p2+ PK P2 rd 1 P2 rd 1 Cal

3 9 4 5 5

Helpertype phw

0.82 0.32 0.80 Of 0.80

P4/ helper type”

P4

0.78 0.95 0.32 1.02 0.87 1.09 0.OOt 0.86 1.07

a All donors carried wild-type P4 as prophage in addition to the helper. * Number of plaques on LO agar (no CaC12): number of plaques on L agar (with CaClt); the values given are the geometric means of the ratios determined in the individual experiments. The nonlysogenic strain Sh-16 was used as indicator for helper-type phage, Sh-201 = Sh-16(PK) as indicator for P4. Hence, regardless of the donor, P4 phages assayed depended during plaque formation on the same helper: PK. Differences in the helper-determined CaZi requirements of P4 from different donors, therefore, affect only the first infection of a Sh-201 cell on the assay plates. This circumstance appears especially important for P4 from C-245. c Ratio of the Ca*+ independence values for P4 and for the helper type phage. On the average these ratios for a given donor were found to vary by about 15% from experiment to experiment. d A recombinant between strains C-236 and C-235. e A recombinant between strains C-236 and C-208. f That is, <1 X 10m5on the basis of the pooled data. Similar experiments for P2 rd 1 from C-208 indicated a value <3 X 1OW. Q Obtained from C-233 by lysogenization with P4 that was propagated with wild-type P2 as helper and was obtained originally from C-236.

shown by the original P4. C(PK) stock. Hence P4 appears to reflect the host range characteristic for its most recent helper. Phages P4eC(P2) and P4.C(PK) as well as P2, and PK wert also compared with regard to their neutralizability by anti-P2 and anti-PK serum with the following rosuit’s (Fig. 2A and B, respectively). As expected, the helper type phages P2 and PK

KLUG

were found to be antigenically different, though related. The same was observed for phages P4.C(P2) and P4sC(PK). Their neutralization rates resembled those for P2 and PK, respectively. The P4.C(P2) stock used had been obtained from P4. C(PK) by a one cycle growth passage through a C(P2+) host. Hence a single cycle of propagation sufficed to change the neutralizability of P4 from “PK like” to “P2 like”. Similar experiments (not shown) indicated that one growth cycle of P4. C(P2) in C(PK) would lead to the reverse change in the neutralizability of P4. These findings suggest that the antigens involved in the neutralization of P4 by anti-P2 and anti-PK sera are identical to those of either P2 or PK, depending on whether P4 was propagated in either a P2 or a PK lysogen. Above results do not exclude the possibility that the P4 virion may also possess P4 specific, i.e., helper-independent, antigens of a comparable nature. Such antigens, if they exist, might be the target of neutralizing antibodies present only in anti-P4 sera, but missing from anti-P2 and anti-PK sera. In an attempt to detect any such P4 specific antigens, the rates of neutralization by anti-P4. C(PK) serum were compared for the two types of P4 and for PK and P2 (see Fig. 3). The rates for P4*C(PK) and P4eC(P2) were found to resemble closely those for PK and P2, respectively, i.e., the results obtained with anti-P4.C(PK) serum TABLE

8

HEAT STABILITY OF P4 AND HELPER TYPE PHAGES Phage donor

C&W (P4-t) C(P2f) (P4+) C(P2 Hyr dis) (P4+)

Number of experiments

4 5 4

Fraction of phages surviving after 10 min at 56” C” Helper me

P4

0.24 0.08 0.003

0.35 0.10 0.09

a Phages were suspended in L broth supplemented with an additional 1% NaCl and 1OV :lf MgS04. The values given are geometric means of the values found in the different experiments.

HELPEIL-DEPENDE?;T

SATELLITK TABLE

HBLPEK-DEPENDENT Growth cycle

Host

BACTERIOPHAGE

337

P4

9

HOST RANGE MODIFICATION Multiplicity infection

of

OF P4 virla

Percent adsorption

P4 yield

-

Burst sizeb -0 1 2

C-235 = C(PK) C-208 = C(P2 rd 1) C-235 = C(PK)

-

-

0.1 4 x 10-s

-

99.3 99.2

53 250

EOP on C-284” 0.49 Od 0.52

a Phages from a P4 virl stock propagated in C-235 (= growth cycle 0) were used to infect C-208 cells at, a cell density of 3 X 108/ml. After an adsorption period of 8 min, most of the remaining unadsorbed phages were removed by pelleting the cells in the centrifuge and then resuspending them in fresh L broth at a cell density of about 3 X 10S/ml. This cell suspension was incubated at 37”; 100 minutes after phage addition, cells and debris were removed from the culture by filtration and the filtrate was assayed for P4 produced in the first growth cycle. To initiate the second growth cycle, C-235 cells were suspended in the filtrate at a cell density 01 about 4 X lO*/ml after addition of CaClz (final concentration 5 X 10d3 Rf) to facilitate adsorption. After an 8-min adsorption period, unadsorbed phages were neutralized with anti-P2 serum (see Materials and Methods). The culture was then diluted into L broth, thereby reducing the anti-P2 serum concentration to a negligible value (i.e., to a final k value of about lV/min) and incubated at 37”. Ninety minutes after the cells were added to the filtrate, the P4 produced in the second cycle was assayed. b Assayed with a P2-sensitive P4 indicator. c Number of plaques on C-284: number of plaques on C-290; C-284 is a Pa-resistant strain, lysogenic for PK, C-29fJ is a P2-sensitive strain, lysogenic for wild-type P2. d That is,
1.0

.

. 02IP‘ll o- 03IPa

0

0.1 f

0.01

t--&-l

0

IO 20 30

40 50

\

t IAIPKI I 0 IP4l

B

~~ L

I

I

I

I

I

I

0

IO

20

30

40

50

60

TIME (min) FIG. 2. Neutralization of P4 and of helper type phages by antihelper sera. Anti-P2 serum (Fig. 2A) and anti-PK serum (Fig. 2B), respectively, were diluted 1:2400 into L broth with 2.5 X 10-a M CaClz. At time 0 either phages P2 + (-0-), PK (-A-), P4.C(P2+) (--a---), or P4. C(PK) (--A-) were added to the diluted sera at final titers of approximately 1 X lo7 PFU/ml. The phage-serum mixtures were then incubated at 37” and assayed for plaque formers at various times. The fraction of surviving plaque formers is plotted as a function of the time of incubation with the serum. From these data the following neutralization constants are obtained for the undiluted sera with respect to the homologous phage: L = 640 min-’ for anti-P2 serum (vs P2) and k = 720 min-1 for anti-PK serum (vs PK). The k values of the sera with respect to the different phages tested are shown in t,he graphs, expressed as fract.ions of the k values for the homologous phage.

338

SIX AND KLUG ,004 (PKI .Ol (P41

P4 virion are under the control of the particular helper genome present during P4 multiplication strongly suggests that gene products obtained by P4 from the helper are incorporated into the P4 virion and thereby account for the helper-determined P4 properties observed. of Nonlysogenic Cells with P4 and the Establishment of a P4 Prophage in the Absence of a Helper

Infection

P4 adsorbs to nonlysogenic host cells as readily as to cells lysogenic for a helper. Its adsorption rate appears not to differ from 39 (PKI that of helper-type phages; hence under standard conditions P4 adsorption is usually better than 90 %. \ I I I I I Nonlysogenic cells are not lysed following IO 20 30 40 50 60 0 infection with P4, and most or all the inTIME (mid fected cells survive as colony formers. In FIG. 3. Neutralization of P4 and of helper type two experiments in which either C-la or phage by anti-P4.C(PK) serum. Procedures C-2 cells were infected with wild-type P4 at followed and presentation of the results are the a multiplicity of 4, the titer of colony same as for Fig. 2, except that the anti-P4 serum formers, assayed after a 10 min adsorption was used at a final concentration of 1:4000. The period, was 108 % and 95 %, respect’ively, of data for P4.C(PK) correspond to a neutralization the titer prior to infection. The correspondconstant k = 1030 min-’ for the undiluted serum ing values for contro1 cultures of uninfected with respect to the homologous phage. The figure cells similarly incubated for 10 min prior t.o also contains data for anti-P4.C(PK) serum prethe assay were 139% and 133 Yc, respecadsorbed with PK. Anti-P4.C(PK) serum, 9 ml tively. This difference in the titer between diluted lOO-fold, was mixed with 1 ml of a PK suspension containing approximately 3 X 10r2 infected and uninfected cultures could rePFU and kept at 0” for 48 hr. The preadsorbed sult from a delay in the division of the inserum was then further diluted and at time 0 was fected cells rather than from the killing of mixed with either P4eC(PK) (--b) or PK some of these cells. (---cl-). The mixtures were incubated at 37”, A majority (about 70 %) of nonlysogenic and the survival of plaque formers was determined cells infected wit,h wild-type P4 was found as a function of incubation time. The k values to yield P4 lysogens (Table 10). Infection of the untreated and of the preadsorbed serum with a single P4+ phage appeared to suffice with respect to the different phages tested are for lysogenization, and the fraction of in shown on the graph, expressed as fractions of the fected cells yielding P4 lysogens show-cd no k value of the untreated serum with respect to dependence on the multiplicity of the infccP4.C(PK). tion. These results indicate that P4 needs no helper for infecting a suitable host or for mere quite similar to those found for anti-PK lysogenizing it. Properties of P4 lysogens serum (Fig. 2B). Furthermore, preadsorpare described below. tion of the anti-P4eC(PK) serum with PK not only abolished the ability of the serum Location of Prophage P4 to neutralize PK, but also reduced i6s neuThe location of prophage P4 was studied tralization rate constant for P4sC(PK) by crosses involving Ff and I?about 99 % (see also Fig. 3). Hence these in bacterial preliminary tests failed to establish the strains. These crosses showed that P4 brexistence of P4 specific antigens. longs to the linkage group of the bacterial The finding that several properties of the chromosome. The sites occupied by each of

1 \

HELPER-DEPENDENT

SATELLITE

BACTERIOPHAGE

P4

339

or C-244. For 7 of these 9 P4 lysogens (identified in Table 1) lysogenization had occurred in the absence of a helper prophage, the remaining two (= C-243 and C-330) had been obtained from P2 lysogens. For each Host Experi- MultipliFrequency of LysoP4 lysogen between 60 and 200 recombinants C (P4) (P) c genization ment city of frequency infectionb were tested, and all were found to carry P4, WY (Ml indicating that the P4 sites of all 9 strains are allelic with the reference site. Similar tests C-la a o/100 0 for further P4 lysogens obtained by lysogeni0.83 6/100 a 0.074 zation from different, helper carrying strains, 1.22e 0.25 27/100 a and crossed wit’h C-243, gave the same rc1.W 0.74 G2/100 sults. Hence in all 15 P4 prophage establish0.52 ; 0.83 35/120 ments that were compared, the same site on 0.82 2.5 75/100 0.80 94/ 120 it 4.1 the bacterial chromosome appears to be 0.83 a. 7.4 83/lao occupied, and the same site was chosen 0.71 c-2 Cf 2.5 05/99 whether the P4 prophage was established in 105/119 0.88 d 4.2 the absence or in the presence of a helper. 0.39 5.2 54/139 d In crosses between P4 lysogens and non0.80 d 96/120 8.3 lysogens the linkage of the P4 site with bac0.74 d 11 87/118 terial markers, used for the selection of a Phage-cell mixtures were incubated for 10 recombinants, was studied (Table 11). The results indicate that the P4 site is located min (except for experiment c) at 37” to allow adsorption, then assayed for unadsorbed phage between the pm3 and pro markers, i.e., and plated for colonies after the appropriate within a 16-min segment of the map for C dilution. Colonies were isolated and streaked on (Wiman et al., 1970), presumably near the agar plates. From each streak one colony thr and leu markers. Attempts to cotranswas test,ed against P2 Hy’dis vir14. Isolates duce the P4 site with either the pur3 or thr resist,ant t,o this phage were scored as P4 lysogens marker failed (M. G. Sunshine, personal (see Materials and Methods). communication). For a given selection, the b Corrected for unadsorbed phage. transfer frequencies for the P4 site tended to c F = number of P4 lysogens found: number be somewhat higher when the donor rather of colonies tested. than the recipient carried P4 (Table 11). But d L = F/(1 - eCm). this difference does not affect the conclusion e Values >l indicate an underestimate of concerning the location of the P4 site. multiplicity of infection. J An adsorption period of 6 min was followed It should be noted that the P4 site appears by a 6.min phage neutralization. not to coincide with any of the three mapped P2 sites (Kelly, 1963; Wiman et al, 1970), namely the preferred site, I (near his), the 15 independently established P4 prophages were compared with each other. The P4 site most frequently chosen secondary site, II occupied in strain C-236 [= FL arg fry str (near ilv), and another secondary site: III (PJ),(P4)] was chosen as reference site. A (near try). Coincidence of the P4 site with second strain carrying P4 at the reference any of the further secondary P2 sites IVsite, C-244 = F+ (P4), was obtained from VIII (Six, 1966) can also be excluded, for site C-236 as a recombinant between C-236 and IV because of its close linkage to site I (Six, C-66. Eighty recombinants (= arg+ try+ 1966), for the other sites on the basis of addistr) from a cross C-244 X C-236 were all tional bacterial crosses between P2 lysogens found to carry P4, as expected. The I’4 sit’es and P4 lysogens (Six, unpublished). We conof 9 further P4 lysogens of independent ori- clude that P4 possrsscs its own, hclpcrgin were compared with the reference site in independent systr>m for prophage integracrosses of these lysogens :vith either C-236 tion. TABLE

10

FREQUENCY OF LYSOGENIZATION WITH WILDTYPE P4 FOR NONLYSOGENIC Escherichia coli 0

340

SIX AND KLUG TABLE LINKAGE

OF THE P4 SITE WITH BACTERIAL

Recipient type

Selection

Marker

Position (min)d

11

MARKERS USED FOR SELECTION IN CROSSEP

A P4 in Recipient6 iv”

Transfer frequency’ for P4 site

B P4 in Donorc .-___ iV8 Transfer frequencyi for P4 site 7

xan+

0

c-1055

200

7 (3; 11)

arg+

6

ilv2+ pun+ thr+

31h 37 41

led pro+

41 54

C-436 C-8 c-445 c-445 c-417 c-429 c-1055 c-1055 c-443

100 99 loo 200 100 197 200 200

313 1 4 14.5 53 59 61 (53; 69) 59.5 (59; 60)

try+

73.5 91

C-436 c-445 C-8 C-436 c-1055 c-1055 C-8 C-231”

100 100 100 100 100 280 180 80

his+ thr+leu+ arg+try+

47 61 10 2 4 68 (59; 72.5; 73) 14 14 (12; 17.5) 10

100

21s

100

28

200 99 100 300

70 910 808 50

99 180 160 75

::“5 (69; 69p) 16 14 11

5 In all crosses counter selection was for streptomycin resistance (position of str: 14 min from zan). The P4 lysogens used either carry wild-type P4 at the reference site (C-236 and C-244) or were shown in allelism tests (see text) to carry wild-type P4 at an identical site. Except for C-231 and C-236 none of the strains included is lysogenic for P2 (or another helper). The data for double marker selection are shown since the crosses to test for allelism of the P4 sites (see text) involved either one of the single marker selections included in this table or, in the majority of crosses, selection for either thr+Zeu+ or arg+try+.

derivative of the recipient type listed. a Donor : C-2; recipient: a P4-carrying c Donor : C-244, except for two crosses with C-2(P4) as donor (thr+ selection selection) d Clockwise

pro+

; recipient: strain listed. distance from the zan. marker,

according

with

to the E. coli map of Wiman

recipient

C-429;

et al. (1970), with

a total map length of 101 min.

e Total number of recombinants

tested in all crosses of this type.

J Percentage of recombinants receiving the P4 site from the donor; if more than one cross of a given type was performed, the average frequency is given first; the values obtained in the individual experiments are given in parentheses. The presence of P4 prophage was determined by testing the recombinants for resistance to P2 Hyldis vir14 (see Materials and Methods). 0 Values obtained for the same C-244 X C-1055 mating mixture, on plates with different supplements for the different selections. h Position of the iZvI mutation. Since the rep locus is closely linked to iZv2 as well as to other ila markers (Calendar et al. 1970), ilvz should map near ilv~. i The P4-carrying derivative is C-236.

Phage Production. by P4 Lysogens and Their Superinfection Immunity Cultures of cells lysogenic only for P4 do not produce any phage spontaneously. But following superinfection with helper phage P2 such cells will produce some P4, on the

average between 0.5 and 1 P4 per cell, as well as a normal yield of P2 (100 or more per cell). Cultures of cells lysogenic for a helper type phage in addition to P4 produce b0t.h phage t ypes spontaneously (Table 12). K-235, the the original donor of wild-type P4 and of PI<, and C-236 = C(P:!+)(P4+) both produce

HELPER-DEPENDENT

SATELLITE TABLE

SPONTANEOUS

PRODUCTION

BACTERIOPHAGE

P4

341

12

OF P4 AND HELPER-TYPE

PHAGE

BY CULTURES

OF, LYSOGENIC

STRAINS”

Donor Strain

K-235

Experiment

Prophages

Colony Phages formers (X 10-7) Helper P4 :x 10-y we x 10-4

(PK) (P4+)

b

C-236

: p.r. (15)b

C-231 c-331

w+) (Pa+) (P4 imp)

Titer ratios

Titers (per ml)

d d

former (X 109

14

5

11

3.4 4.3 4.8

6.4 4.8 1.4

5.4 2.0 ~___

1800

1.6 3.9 1.7

3.0 4.3 0.5

2.8 3.0

1.5 0.61

540

-

P4/Helper type phage

p4/ colony

pklg:, colony former (X 109 --

4.7 9.0 3.6

--

Helper

.I-

0.23 0.24c (O.ll-0.29)d

0.03 0.17" (0.09-0.27)d 89

a L broth cultures of the lysogenic strains were grown to reach a cell titer of about 3 to 10 X lO’/ml. Then the cultures were assayed for colony formers and immediately thereafter were sterilized by the addition of chloroform and kept at room temperature. About 20 min after chloroform addition, cells were removed by centrifugation, and the culture fluids were assayedfor phage. h Pooled results from several experiments in which the titer of colony formers was not determined. The number of experiments for which results were pooled is given in parentheses. 0 Average value for pooled data.

d Range of pooled data. more helper type phages than P4, the level of P2 production by C-236 being similar to that of its precursor, C-231 = C(P2+). By contrast, C-331 like C-236 derived from C-231, hut carrying P4 imp instead of P4+, produces much more P4 than any P4 urild-type carrying lysogen. Other strains lysogenic for P4 imp and P2 showed a similarly high level of spontaneous P4 production. Attempts to

induce P4 prophages with ultraviolet light failed. P4 (+ or imp) carrying cells appear to be immune to superinfectmg P4+ and P4 imp, as shown by the inability of these phages to produce plaques on lawns of cells lysogenic not only for P2 but also for P4. In addition it was found that a C(P2) (P4+) culture did did not lyse after infection with m
By contrast, P4 virl does form plaques on lawns of C(P2) (P4+). The plaques are not as large, but are produced with the same efficiency as with an indicator not carrying P4. In one-step growth experiments the multiplication of P4 virl in cells of C-235 = C(PK) was compared with t)hat in cells of a derivative of C-235, carrying P4+ in addition to PK. Lysis of the latter host was found to be delayed by about 30-40 min. This delay presumably accounts for the P4 yields from this host being about three times as high (average burst size: 650 in the first, 9.50 in a second experiment) as the yields from the host not lysogenic for P4t. No wild-type P4 was observed among the P4 produced by the P4+ carrying’ host. The findings described in t’his section indicate that I’4 lysogens are not unusual wit’h regard to the two main properties of lysogenie cells, phage production and immunity,

342

SIX

AND

except, of course, for the dependence of P4 on a helper for the production of phage. Other Properties of P4 Lysogens Two further properties of P4 lysogens shall be mentioned. Both have in common that the P4 prophage interferes with another genetic element, and in both instances the effect of P4 does not depend on the presence of a helper prophage. Prophage P4 interferes with the multiplication of phages P2 Hy’dis and P2 Hy’dis vir14, although P4 is heteroimmune with respect to these phages. The efficiency of plaque formation by P4+ with indicators lysogenic for P2 Hy’dis is good, but for P2 Hyldis and P2 Hyldis vir14 the efficiency of plaque formation with P4 lysogens is less than 1% of that found with an indicator not carrying P4. This property of P4 lysogens allowed us to design a simple spot test for recognizing P4 Iysogens, especially in the absence of a helper prophage (see Materials and Methods). The inhibition of P2 Hy’dis and P2 Hyldis vir14 by P4 prophage cannot be related directly to the immunity specificity of these phages since two other phages of the same immunity class, P2 Hy*dis and PK Hy dis, are not markedly affected in their plaque-forming efficiency by the presence of a P4 prophage in the indicator. The same was also observed for phages P2 and PK. A very unexpected property of P4 lysogens came to light in the course of the bacterial crosses described in a preceding section. The presence of a P4 prophage in F+ strains was found to lower their efficiency as donors, leading to a reduction in the number of recombinants obtained in the crosses by a factor ranging from about 2 to 20, depending on the experimental conditions. The presence of P4 in the recipient did not affect the number of recombinants obtained, whether the donor carried P4 or not. Hence, the reduction of fertility by P4 cannot be explained on the basis of zygotic induction (see Usher and Six, 1971, and in preparation for further studies concerning the fertility reduction by P4). DISCUSSION

Escherichia coli K-235 was first described by Fred&icq (1950) as a strain both lyso-

KLUG

genie and colicinogenic. Amano et al. (1958) found this strain to be lysogenic for two phages but did not investigate their nature. One of these phages, able to form plaques with a nonlysogenic indicator, was isolated by Jesaitis and Hutton (1963) and termed PK. The other phage mentioned by Amano et al. presumably is identical with P4 which we found to form plaques only with lysogenic indicators. Our studies show that plaque formation by P4 occurs only with lysogenic indicators because P4 is a helper-dependent or “satellite” virus. Unlike Salmonella phage G4 which can adsorb only to cells lysogenic for cl5 but presumably does not need this prophage as a helper (Uetake et al. 1958), P4 adsorbs well to nonlysogenic cells. Moreover, P4 must be able to infect such cells since it can lysogenize them. Two more specific conclusions emerge from the studies of the lysogenization by P4 of cells lacking a helper. First, P4 should have at its disposal its own integration mechanism, since it establishes a prophage at a P4-specific site on the host chromosome. Second, the P4 genome appears to be able to replicate without, a helper. This is suggested by the finding that the probability of a cell to become lysogenized with P4 is high and does not depend on the number of P4 infecting the cell. In the meantime direct evidence for P4 DNA synthesis in cells without helper has been obtained by Lindqvist and Six (1971). P4 does need its helper to complete the lytic growth cycle. The determination by the helper of several properties of the P4 virion indicates that P4 depends on the helper to provide proteins that become incorporated into the P4 virion. Since most or possibly all of the helper-determined P4 properties studied are associated with the phage’s ability to attach to a host cell (namely, Ca2+ dependence, host range, neutralizability) it appears that the helper has to contribute tail fiber, and possible other tail subunits for the assembly of the P4 virion. These conclusions are consistent with the finding of Inman et al. (1971) tha,t the tail of P4 is indistinguishable from that of a helper-type phage. Parenthetically it may be noted here, that P4, propagated in lysogenic hosts, may be

HELPER-DEPENDENT

SATELLITE

said to be subject to host-controlled modification, inasmuch as the host range, for instance, of the P4 virion reflects the genotype of the helper present in the previous P4 host. We realize, however, that’ the term host-controlled modification is now being used only in a restricted sense that would not allow its application in the case of P4. The failure of cells infected with P4 to lyse if no helper is present, suggests that P4 depends on its helper also for the gene(s) concerned with cell lysis. The observation that helper P2 Hy’dis does not impart on the P4 virion the heat-sensitivity of the P2 Hyldis virion presumably should not be considered evidence for (heat stable) P4 gene products being part of the P4 virion. For it appears likely that the unusually low heat stability of P2 Hy’dis (see Cohen, 1959) does not result from a heat-labile protein but rather is a consequence of the large size of the DNA of this phage and the resulting tight. packaging of the DNA in t’he phage head (Bertani and Bertani, 1971). Fur&er studies of the helper-dependence of P4 have since led to the conclusion that P4 depends on all known P2 (= helper) genes with a “late” function (Six and Lindqvist, 1970; Six, in preparation). As evident from our studies, gcnomes of phages other than 1’2 also can supply 1’4 with t,he needed gent product’s. In addition to the helpers we employed, several other helper type phages have been identified. All such phages are related to P2, but among them at lrast five different immunity classes have been recognized (Bertani and Bertani, 197 I ; Six, unpublished). The most intriguing aspect’ of the helperdcpcndencc of I’4 is the ability of P4 to obtain the necessary assistance from a helper prophagc (rather than only from a coinfccting helper tvpc phage). Cells lysogcnic for a helper and infected with P4 produce cssent’ially only I’4 but no helper t.ypc phage. Hence I’4 cithcr must be able to activate the helper prophagc gcn~s on which it depends without, inducing replication of the helper, or, altcrnativcly, 1’4 dots induct the helper prophagr: thereby triggering the activation of the entire helper genome, but then prevents maturation of hclpcr type phagc. The latter altcrnntivc> nppcxars unlik(~ly sinccl it was

BACTERIOPHAGE

P4

343

found that most nonlysogenic cells, simultaneously infected with P4 and helper type phage, will produce phages of both kinds. Further evidence in support of the former alternative, activation of helper prophage genes without induction of helper replication, was obtained from an analysis of the DNA synthesized in cells lysogenic for P2 following infection with P4 (Six and Lindqvist, 1971). Such cells were found t#o synthesize P4 DNA, but no P2 DNA synthesis (indicative of P2 prophage induction) could be detected. This result is consistent with our finding that cells lysogenic for I’2 and simult8aneously infected with P4 and P2 produce, in addition to P4, no more P2 than could be accounted for, in t’he absence of P2 DNA replication, by repackaging of the DNA of the infecting P2 phages. Hence the immunity of cells lysogenic for a helper such as P2 appears not to be lifted following infection with P4. We, therefore, assume that P4 produces (in the cell it infects) a “transactivating” factor. This “transactivator” effects the transcription of the helper prophage genes needed by P4. At present we can only speculate on the nature of this hypothetical transact’ivator. Conceivably it could be a factor determining the promoter specificity of t’he transcription enzyme, analogous to the u’ factor found in T4 infected cells (Burgess et al., 1969), or a new DNA-dependent RNA polymerase as in the case of T7-infected cells (Chamberlin and McGrat’h, 1970), or an antitermination factor as postulated for cells infected with X (Roberts, 1970). Whatever the exact funct’ion of the P4-transactivator may be, no serious “gene dosage” problem is posed by t8he ability of P4 to obtain gene products in sufficient quantities from helper prophages, as was already pointed out’ by Six and Lindqvist (1971). Prom the size of t’he I’4 DNA, as determined by Inman et al. (1971), it may be estimated that the P4 genomc could contain from about ten to fifteen genes. From the properties of P4 examined so far WCinfrr that among these P4 genes should be those responsible for the following functions : (i) initiation of the replication of the P4 genome as a helper-independent’ raplicon, (ii) t,ransactivation of hclpcr prophag(>s, (iii) intrgra-

344

SIX

AND

tion of a P4 prophage at a specific site on the host chromosome, (iv) repression of P4 in cells lysogenic for P4 (needed at least if the cell also contains a helper prophage), (v) interference by prophage P4 with the multiplication of P2 Hy’dis (and P2 Hy’dis vir14), and (vi) reduction of the fertility of male cells carrying P4 (see Usher and Six, 1971). Conceivably, fewer than six genes could be involved in the six P4 functions listed. ACKNOWLEDGMENTS We thank Dr. M. A. Jesaitis for providing us with strain K-235. One of us (E. W. S.) performed several of the experiments reported here in the laboratory of Dr. G. Bertani, Department of Microbial Genetics, Karolinska Institute, Stockholm. Sincere thanks are due to Dr. Bertani for his hospitality and for many valuable discussions. REFERENCES AMANO, T., GOEBEL, W. F., and SMIDTH, E. M. (1958). Colicine K. III. The immunological properties of a substance having colicine K activity. J. Exp. Med. 108,731-752. BERTANI, G., and SIX, E. (1958). Inheritance of prophage P2 in bacterial crosses. Virology 6, 357-381. BERTANI, L. E., and BERTANI, G. (1970). Preparation and characterization of temperate, noninducible bacteriophage P2 (host: Escherichia coli). J. Gen. Viral. 6, 201-212. BERTANI, L. E., and BERTANI, G. (1971). Genetics of P2 and related phages. Advan. Genet. 16, 199-237. BURGESS, R. R., TRAVERS, A. A., DUNN, J. J., and BAUTZ, E. F. K. (1969). Factor stimulating transcription by RNA polymerase. Nature (London) 221, 4346. CALENDAR, R., LINDQVIST, B., SIRONI, G., and CLARK, A. J. (1970). Characterization of REP mutants and their interaction with phage P2. Virology 40, 72-83. CHAMBERLIN, M., and MCGRATH, J. (1970). Characterization of a T7-specific RNA polymerase isolated from E. coli infected with T7 phage. Cold Spring Harbor Symp. Quant. Biol. 35, 259-262. COHEN, D. (1959). A variant of phage P2 originat-

KLUG ing in Escherichia coli, strain B. Virology 7, 112-126. FR~D~RICQ, P. (1950). Sur une souche d’Escherichia coli a la fois lysogene et antibiotique. Antonie van Leeuwenhoek J. Microbial. Serol. 16, 41-44. INMAN, R. B., SCHN~S, M., SIMON, L. D., SIX, E. W., and WALKER, D. H. JR. (1971). Some morphological properties of P4 bacteriophage and P4 DNA. Virology 44, 67-72. JESAITIS, M. A., and HUTTON, J. J. (1963). Properties of a bacteriophage derived from Escherichia coli K-235. J. Exp. Med. 117, 285302. KELLY, B. (1963). Localization of P2 prophage in two strains of Escherichia coli. Virology 19, 32-39. LINDQVIST, B. H., and SIX, E. W. (1971). Replication of bacteriophage P4 DNA in a nonlysogenic host. Virology 43, l-7. ROBERTS, J. W. (1970). The p factor: termination and anti-termination in Lambda. Cold Spring Harbor Symp. Quant. Biol. 35, 121-126. SASAICI, I., and BIZRTANI, G. (1965). Growth abnormalities in Hfr derivatives of Escherichia coli strain C. J. Gen. Microbial. 40, 365-376. SIX, E. (1961). Inheritance of prophage P2 in superinfection experiments. Virology 14, 220233. SIX, E. W. (1963). A defective phage depending on phage P2. Bacterial. Proc. 1963, 138. SIX, E. (1966). Specificity of P2 for prophage site I on the chromosome of Escherichia coli strain C. Virology 29, 106-125. SIX, E. W., and CONNELLY, C. (1966). Helperdependent multiplication of defective phage P4. Bacterial. Proc. 1966, 112. SIX, E. W., and LINDQVIST, B. (1970). Helperdependent reproduction of coliphage P4. Bacteriol. Proc. 1970, 202. SIX, E. W., and LINDQVIST, B. H. (1971). Multiplication of bacteriophage P4 in the absence of replication of the DNA of its helpers. Virology 43, 8-15. UETAKE, H., LURIA, S. E., and BURROUS, J. W. (1958). Conversion of somatic antigens in Salmonella by phage infection leading to lysis or lysogeny. Virology 5, 68-91. USHER, D. C., and SIX, E. W. (1971). Fertility reduction of Escherichia coli by P4 prophage. Bacterial. Proc. 1971, 180. WIMAN, M., BERTSNI, G., KELLY, B., and SASAKI, I. (1970). Genetic map of Escherichia coli strain C. Mol. Gen. Genet. 107, 1-31.