VIROLOGY 93, 357-368 (1979)

On the Trapping of Phage Genomes in Spores of Bacillus subtilis 168

Reciprocal Exclusion of Phages $29 and +e during Outgrowth of Spores

FELIPE MORENO’

Institut de Microbiologic, Universitd Paris-Sud, Orsay, France, and Bioquimica Experimental, Clinica Puerta de Hierro, Facultad de Medicina, Universidad Authwma, Madrid, Spain

Accepted October 24, 1978

The trapping of phages 429 and 4e by spores of Bacillus subtilis 163 has been studied. The optimal time and the efficiency of trapping were different for each phage. The ef- ficiency of 429 was maximum (0.01-0.02) when infection occurred at stage I of sporulation; that of +e (0.5-l) 1.5 to 2 hr later. A single phage genome was sufficient to infect a spore and there is neither cooperation nor competition between genomes in the trapping event. Viral DNA synthesis is not required for trapping. A single spore may trap two viral genomes, either from the same phage or from the two unrelated phages, 429 and +e. During outgrowth, spores carrying both genomes, 429 and +e, produced particles of one or the other phage, but never of both. Normally 429 multiplied but +e developed when expression of 429 was impaired by a lethal mutation in cistron 2. However in doubly infected exponentially growing cells 4e dominated and prevented reproduction of 429.

INTRODUCTION

Several workers have noted that phages, which productively infect vegetative cells of Bacillus subtilis, will not produce infec- tive progeny when they infect sporulating cells. If infection occurs at a critical time during sporulation the phage genome may be incorporated into the developing spore (Yehle and Doi, 1967; Sonenshein and Ros- toe, 1969; Ito et al., 19’73; Kawamura and Ito, 1974, 1977; Osburne and Sonenshein, 1976). This incorporation has been named phage trapping and has been correlated with the failure to induce certain phage- specific enzymes related to nucleic acid me- tabolism (Sonenshein and Roscoe, 1969). Carrier spores act as infectious centers when allowed to germinate in fresh me- dium.

This work characterizes some of the pa- rameters required for trapping of phages $29 and $e into developing spores of Bacil-

1 Present address: Unite de Genetique Moleculaire, Departement de Biologie Moleculaire, Institut Pas- teur, 25 Rue du Docteur Roux, Paris 75015, France.

lus subtilis 168 and demonstrates that a single spore may trap two viral genomes from either the same phage or two unre- lated phages. In the latter case, progeny from only one phage will eventually be pro- duced when spore dormancy is relieved.

MATERIAL AND METHODS

Bacteria and phqes. Bacillus subtilis 168 spa+ su- was the host to study phage trapping. Bacillus subtilis spoOA-3NA (Michel and Cami, 1969) and Bacillus sub- tilis MO-99 spoOA-3NA (met- thr-)+ SU+~ (Moreno et al., 1974) were used to prepare stocks and to count the plaque-forming units (PFU).

The phages used were: wild type (wt) 429, its mutants ts 2 (98), sus 2 (513), sus 9 (422), and sus 13 (53), and wt +e (this one obtained from A. L. Sonenshein). Ge- netic nomenclature of 429 mutants is that of Mellado et al. (1976).

Media. The nutrient broth of Schaeffer et al. (1963) was used to obtain sporulation and to study phage growth; with agar added it was used to determine the number of

357 0042-6822/79/040357-12$02.00/O Copyright 0 19’79 by Academic Press, Inc. All rights of reproduction in any form reserved.

358 FELIPE MORENO

12345678 TIME (hours)

FIG. 1. Trapping efficiencies of 429 and +e. Aliquots of a culture of Bacillus subtilis 168 incubated at 42” were infected (m.o.i. = 0.05) with either 429 ts 2 (98) or wild type +e at different times. Viral adsorption was measured 10 min after infection. At t,,, spores were prepared and plated at 30” as indicated under Materials and Methods. Trapping efficiency is the ratio of infected spores/adsorbed phages. 0 - 0, Ab- sorbancy; 0 - 0, trapping efficiency of 429; H - n , trapping efficiency of I&; A - A, chloro- form-resistant CFU in the uninfected culture.

colony-forming units (CFU). Liquid and solid LB medium supplemented with Mg2+ and glucose (Moreno et al., 1974) were used to grow indicator strains, to prepare phages, and to titer PFU.

Phage buffers. $29 buffer was as de- scribed by Mendez et al. (19’71), but with MgS04 replacing MgCl,. 4e buffer con- tained 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 mM MgSO*, 0.05 mM Ca(NO&, and 0.1 mg of gelatin per ml.

Phage stocks. They were usually made as described by Moreno et al. (1974) and con- centrated by high-speed centrifugation in a

three-layered gradient of CsCl, as de- scribed by Ito et al. (1973).

Phage specific antisera. Anti-429 serum (K = 330) was a gift of M. Salas. Two batches of anti-+e serum were used; one (K = 360) was a gift of C. Orrego; the other (K = 1000) was prepared following standard techniques. Enough serum was used to neutralize at least 99.9% of the phage in 5 min.

Bacterial cultures and sporulation pa- rameters. Growth and sporulation of strain 168 was achieved at 42” and its course fol- lowed as indicated by Moreno (1977). Spor- ulation was fairly synchronized: the number of chloroform- and heat-resistant CFU dou- bled in about 10 min (Fig. 1, see also Moreno, 1977). At t5.5, 5.5 hr after the end of exponential growth (to), the chloroform resistance reached its maximum level: 70 to 80% of the CFU were resistant to it. The same occurred with the heat resistance at ts.

Phage trapping. Culture samples were infected at various times and at indicated multiplicities, and incubation was continued under the same conditions. At lo-15 min after phage infection, aliquots were re- moved to measure unadsorbed phage, and then anti-phage serum was added in all cul- tures, either infected or noninfected. When sporulation was completed cultures usually received 50 @g/ml of lysozyme, 10 pg/ml of DNase, and 5 pug/ml of RNase and incu- bation was continued for 20 min at 37“. One- milliliter samples were then heated for 10 min at 80”. When necessary, these spore preparations were washed with sterile water and treated again with anti-phage serum.

Finally spores were plated alone to count the noninfected (CFU), and with an indi- cator strain to count the infected (PFU). It was assumed that a spore counted as CFU could not be PFU and viceversa. Trap- ping frequency was defined as the ratio in- fected spores/(infected spores + uninfected spores) and trapping efficiency as the ratio infected spores/adsorbed phage (10 min af- ter infection). Adsorption phage was deter- mined by plating after killing of infectious centers with chloroform.

Mutant ts 2 (98) was used in most experi- ments to study $29 trapping; it was pre-

TRAPPING AND EXCLUSION OF PHAGES +29 AND c$e 359

viously demonstrated that this mutant is trapped as efficiently as is wt $29. The use of the mutant, under restrictive conditions (42”), and addition of anti-429 serum en- sured that the cells infected at the desired time were never super-infected.

RESULTS

Sporulation and Phage Trapping as a Function of Infection Time

When sporulating B. subtilis cells are in- fected with $e or 429 at certain critical times, sporulation proceeds and phage DNA is incorporated into spores (Sonen- shein and Roscoe, 1969; Ito et al., 1973; Kawamura and Ito, 1974). Since these au- thors used strains and/or sporulation condi- tions different from those used here, it seemed necessary to establish some param- eters of the infection of B. subtilis by 429 and +e under our conditions.

B. subtilis 168 spa+ cells were infected at 42” with wt 429 or wt 4e, at a multi- plicity of infection of 10, at different times during growth and sporulation. Exponen- tially growing bacteria support phage multi- plication and lyse (Moreno, 1977).

During the sporulation process the fraction of cells which, despite infection by 429, com- plete sporulation remains constant regard- less of the infection time. This fraction (80 to 100%) approaches that obtained in unin- fected cultures. However, when sporu- lating cells were infected by 4e the yield of spores was much smaller (lo-25%), not because sporulation was directly inhibited, but because phage infection killed the cells as indicated by clearing of the cultures.

Maximum trapping frequency (20%) was obtained for 429 when infection occurred just after the end of exponential growth, between to and tl (stage I of sporulation). Maximum trapping frequency for +e (90- 100%) was found over a period extending from tl to t3. These differences between phages (trapping frequency and optimum time) could not be attributed to different degrees of viral adsorption, which was al- ways 60-75% for $29 and 90-95% for +e. These results are consistent with those of Kawamura and Ito (1974) on 429 trapping but do not agree with those of Sonenshein

and Roscoe (1969) who found t,-t6 to be the optimum period for trapping 4e. This discrepancy may be due to differences in strains and/or culture conditions.

Since the maximum trapping for 429 is five times lower than for +e, study of phage trapping in infections at a low m.o.i. became interesting. When this was done (Fig. l), it was found that optimum trapping times were the same as for high m.o.i. infections. On the other hand, while up to 40% of the +e adsorbed were trapped in the spores, only 1% of the $29 were trapped. In another experiment values of 70 and 2%, respec- tively, were obtained. Consequently, the probability for a spore to trap a $29 genome is a function of the m.o.i.

Trapping as a Function of m.o.i.

In order to investigate if more than one (629 genome was necessary to yield an in- fected spore, the frequency of trapping was determined at various m.o.i. At low multi- plicities, in a log-log representation, the adjusted curve of Fig. 2 is a straight line with a slope of 1, indicating that a single infection is sufficient to ensure trapping.

Moreover, simultaneous infection with one 429 ts 2 (98) and an excess (20) of either $29 sus 2 (513) or $29 sus 9 (422) did not change the trapping frequency of the ge- nome ts 2 (98) found in single infection.

These results suggest that any 429 ge- nome injected into a cell competent for trap- ping has a certain probability of being trapped, independent of the presence of other genomes. This conclusion is also sup- ported by our study of double trapping (see below). Since the trapping efficiency, just after to, is 0.01-0.02, it can be predicted that in a bacterial population infected with an average multiplicity of 100, most of the spores produced will be infected by a least one phage genome. In fact, with multiplici- ties of 50 to 100, we found that 70 to 90% of the spores were carrying (629.

Double Trapping

Analysis of Takahashi (1964) of the DNA content of spores of B. subtilis SB19 in- fected by the phage PBS1 showed that 11% of the total DNA was phage DNA, suggest-

360 FELIPE MORENO

I I 1 I 1

0.01 0.1 1.0 IO lot

MULTIPLICITY OF INFECTION (h2883mutant)

FIG. 2. Trapping as a function of the m.o.i. Experiment A. Culture samples of Bacillus subtilis 168 at to were infected with 429 ts 2 (98) at increasing multiplicities. Ten hours later, spores were prepared and plated at 30” on Bacillus subtilis MO-99. 0 -0, Infected spores. Experiment B. Culture samples of Bacillus subtilis 168 (t,,,) were infected with 429 ts 2 (98) at increasing multi- plicities and with either 429 sus 2 (98) or 429 sus 9 (422) at a m.o.i. of 20. Ten hours later, the spores were prepared and plated at 30” on spo OA3NA su- indicator bacteria. Spores infected by ts 2 (98): 0 - 0, infection with ts 2 (98) alone; 0 - - - 0, double infection with phages ts 2 (98) and sus 2 (513); 0 - 0, double infection with phages ts 2 (98) and sus 9 (422).

ing that one spore might contain several copies of the phage genome.

In order to check this, double infection experiments were carried out with the mu- tants ts 2 (98) and sus 9 (422) of 429. The presence of the ts genome in the spore was detected by plating on the strain spo OA- 3NA (su-), and incubating at 30”. Under these conditions, spores infected by the sus mutant alone did not produce plaques. The presence of the sus genome was detected by plating on MO-99 SU+~ and incubating at 42” [ts 2 (98) alone does not develop un- der these conditions]. The simultaneous presence of sus 9 (422) and ts 2 (98) genomes in the spore was detected by plating on spo OA-3NA and incubating at 42”; only the

spores infected by both mutants will pro- duce plaques under these conditions. The total number of infected spores was deter- mined by plating on MO-99 at 30”. It should be pointed out, however, that not all the sus 9 (422) genomes trapped could be counted. When infected with a sus mutant, 168 su- cells produce plaques on MO-99 SU+~ less efficiently than when infected with the wt phage. Thus the number of sus 9 (422) ge- nomes trapped was underestimated by a factor of two (data not shown). Correcting for this, Table 1 shows that ts 2 (98) and sus 9 (422) are trapped in single infections with similar frequencies (g-13%). These frequencies are not changed in mixed infec- tions (12%). The number of plaque-forming

TRAPPING AND EXCLUSION OF PHAGES 429 AND 4e

TABLE 1

TRAPPING OF Two PHAGE GENOMES IN THE SAME SPORES

361

Infecting phages

ts 2 (98) SW 9 (422) ts 2 (98) sus 9 (422) No phage

PFU/ml

30” 42” CFU/ml (x 108) on MO-99 on 3NA on MO-99 on 3NA

3.8 5.9 x 107 5.8 x 10’ <lo2 <lo* 4.2 1.2 x 107 <lo* 1.1 x 10’ <lo2

3.8 6.6 x 10’ 6.0 x 10’ 1.7 x 10’ 6.6 x 106

5.5 -

a Bacillus subtilis 168 were infected around to of sporulation (m.o.i. = 20). Eighteen hours later spores were prepared and plated. The number of infectious centers (PFU/ml) was determined on the strains MO-99 (su+) and spo OA-3NA (SK) at 30 and 42”. The number of noninfected spores (CFU/ml) was determined at 42”.

spores at 42” on spo OA-3NA is 1.3% of the total. A spore can therefore trap ts 2 (98) and sus 9 (422) genomes simultaneously and the two trapping events are independent (the calculated frequency of double trap- ping, 1.45%, is quite close to the experi- mental value, 1.3%).

These conclusions were confirmed when some of the plaques obtained by plating spores on MO-99 at 30” were studied. Thus, among the plaques with phage able to grow on spo OA-3NA at 30”, 16% also contained phage which grow on MO-99 at 42”, a result that is again close to the expected one. Moreover analysis of the phage content of individual plaques shows that we are deal- ing with spores infected by the two mutant genomes and not by a wild-type phage ge- nome produced by recombination before or during the trapping event. Indeed the analysis of 20 of the plaques obtained by direct plating on spo OA-3NA at 42” showed that all of them contained sus+ ts and sus ts+ phages; 15 also contained wild-type re- combinants, and 8 at least double mutant recombinants.

Duration of Bacterial Capacity to Trup 429 Genomes

The optimal time for trapping 429 occurs during stage I of sporulation (Fig. 1). In order to determine more precisely the dura- tion of this competent period, several cul- tures were simultaneously infected with one

$29 mutant at &,, and at various times around t,, with another mutant. When spor- ulation was completed the fraction of doubly infected spores was determined in all cul- tures. Since phage adsorption, cell survival, and sporulation are not significantly af- fected by these super-infections, we con- clude from the data (Fig. 3) that the trap- ping capacity is lost exponentially over the course of time. This loss can be expressed by the equation N/N,, = e+, in which N, is the number of spores trapping the first genome, N the number of cells trapping the two genomes, t the time of super-infection, and A a constant. If the time is expressed in minutes, the value of A is 0.05 min-‘; at any time, each cell “able to trap” has one chance in 20 of losing its “competence” in the next minute, which means that a cell is capable of trapping during an average period of 20 min.

Infection of Both Phages 4.29 and 4e

In order to detect spores possibly infected with two unrelated viral genomes, samples of a culture were infected with 429 and 4e. It was hoped that mere examination of the plaques might suffice to detect the presence of mixedly infected spores (429 plaques are large and surrounded by a characteristic halo, while 4e plaques are much smaller and have no halo; the hypothetical “mixed” plaques might be intermediate in appear- ance). The results of such an experiment

362 FELIPE MORENO

TIME (MINUTES)

FIG. 3. Duration of “competence” for trapping of $29. A culture of 168 in late exponential phase was distributed in several flasks and incubation continued. Each particular culture was infected with 429 ts 2 (98) (m.o.i. = 10) at about t, of sporulation (time 0 min of experiment) and with 429 sus 13 (53) (m.o.i. = 10) at the times indicated. Anti-$29 serum was added at 15 min of superinfection. At t,,, spores were prepared and plated on spoOA-3NA at both 30 and 42“ to de- termine the number of spores infected by ts 2 (98) alone and by both ts 2 (98) and sus 13 (531, respec- tively. Control spores [infected by either sus 13 (53) or ts 2 (98) alone] did not produce any plaques at 30 or 42”, respectively.

(Table 2) indicate that (a) the effects of each infection on the survival and sporulation are additive in the double infection; (b) infection by $e does not discriminate between cells

which trap and cells which do not trap 429; and (c) none of the plaques produced could be recognized as “mixed.” However, these results do not rule out the existence of doubly infected spores. Indeed, as shown in Fig. 4, $29 develops earlier than f$e in the singly infected germinating spores. The rise period of $e begins only after 429 has reached its plateau. Thus, spores carrying both phages might not produce +e particles, or would produce so few that their plaques would still be of the (629 type.

Trapping of Both C$N and $29 ts 2 (98) in the Same Spore

The presence of $e in the doubly infected spores could be unmasked by using the mu- tant ts 2 (98) instead of wild type 429. This mutant cannot direct synthesis of viral DNA at 42” (Talavera et al., 1972) and lysis of the host is delayed (Talaveraet al., 1971).

Table 3 represents the results of such a mixed infection. The number of plaques at 42” was 10 times higher than that of 4e- type plaques at 30”, indicating that most +e-containing spores were not detected at 30”. That the plaques formed at 42” con- tained only $e particles was verified by neu- tralizing with anti-+e serum. About 90% of the spore population therefore contained $e; since the same percentage of spores con- tained 429, ts 2 (98), the majority of the spores surviving double infection contained genomes of both phages.

TABLE 2

SPORESOF CULTURES~NFECTEDBY~~~AND~~~

Infecting phages m.0.i.

CFU per ml

CFU per ml

WY

PFU per ml

429 type

PFU per ml

9e type

Total of spores

(%Y

Trapping frequency

(%)

429 10

4e 7

429 10

4e 7 None -

5.3 x 10’ 39 1.3 x 10’ - 49 20 3.8 x lo6 2.8 - 4.6 x 10’ 37 92

2.0 x 106 1.5 4.3 x 106 1.9 x 10’ 19 92’

1.35 x 108 100 - - 100 -

a Samples of a Bacillus subtilis 168 culture incubated at 42” were infected with 429 at about &, and with #e 2 hr later. At tzo spores were prepared and plated on MO-99 at 30” to count PFU. To count CFU incubation was carried out at 37”.

b Taking as 100% the number of CFU per ml in the noninfected culture. c Eighteen percent of these infective centers had the appearance of the 429 plaques and 82% that of the

$e plaques.

TRAPPING AND EXCLUSION OF PHAGES 429 AND +e 363

, I I 1 I 1 1 I

20 40 20 20 loo I20 I40 160 TIME (minutrs)

FIG. 4. One-step growth experiments with spores (+e), spores @29), and spores [$29 ts 2 (98)]. Spores prepared as indicated in Table 3 were incubated (about 1OVml) in fresh Schaeffer’s medium to allow germination and outgrowth. At 30 min, the cultures were diluted 500-fold in the same medium and incubation was continued. Temperature shift was achieved by diluting the cultures at 42” lo-fold in fresh medium prequilibrated at 31”. The PFU were measured by plating on strain MO-99 at 30”. 0 - 0, spores (&29) incubated at 42”; 0 - 0, spores @e), incubated at 42”; A- A, spores [@9 ts 2 (98)] incubated for 70 min at 42” and then at 31”; A - A, spores (4e) incubated for 70 min at 42” and then at 31”.

This conclusion was verified by studying a few of them had +e particles. Upon germi- the viral progeny of individual spores, in nation at 42”, however, all of the phage- single-burst experiments (Table 4). When containing tubes (50% of the total) produced germination was carried out at 31”, 54% of +e progeny. This indicates that almost all the tubes contained viral progeny but only the spores had incorporated +e, but that at

364 FELIPE MORENO

TABLE 3

TRAPPING OF 429 ts 2 (98) AND WT +e GENOMES IN THE SAME SPORE”

Incubation of plates at

Infecting phages

429 ts 2 (98)

g9 ts 2 (98) and $e

None

30

CFU per ml

PFU per ml

429 type 4e type

4.5 x 101 1.9 x 108 - 1.2 x 10’ - 1.8 x 10s 1.0 x 106 4.6 x 10’ 4.5 x 106

7.8 x 108 - -

42”

CFU per ml

PFU per ml

6.8 x 10’ 1.0 x 107 1.8 x 106

8.3 x lOa

None 1.6 x 10s 4.4 x 107

cbe-type plaques

-

u Samples of a Bacillus subtilis 168 culture incubated at 42” was infected either with 429 ts 2 (98) (m.o.i. = 75) at to-t,, with $e (m.o.i. = 15) 105 min later, or with both phages. Each antiphage serum was added 15 min after corresponding infection. Ten hours later, the cultures were centrifuged, and the pellets were resuspended in 1 ml of buffer (5 mkf Tris-HCl, 10 mM Mg2+, pH 7.1); after addition of lysozyme and anti-+e serum, suspen- sions were incubated 15 min at 37”, and received 2 ml of sterile water. Spores collected by centrifugation were resuspended in 1 ml of sterile water and heated for 10 min at 80”. Anti-429 and anti-de sera were added to the nutrient agar on which CFU were determined.

31” only a minority of such spores produced $e during germination.

The 12 petri dishes with $29 and +e prog- eny (Expt A) could have originated from spores in which development of 429 was delayed and that of +e advanced. If that were the case, the frequency of mixed prog- eny should be increased by modifying the incubation conditions in such a way that the rise periods of both phages overlap. This overlapping could be performed as indicated in Fig. 4. Although substantially different

from the ones obtained in experiments A and B, the distribution of types of progeny in experiment C of Table 4 shows that the frequency of mixed progeny (5%) is not sig- nificatively different from the one obtained in experiment A (8%). Indeed, these frequen- cies were close to the expected value (6%) for tubes that received at least two infected spores, one of which containing only $e.

Therefore it can be concluded that: (a) outgrowing spores of Bacillus subtilis 168 infected with the two genomes produce only

TABLE 4

SINGLE-BURST EXPERIMENTS WITH SPORES FROM A CULTURE MIXEDLY INFECTED WITH +e AND 429 tS 2 (98)”

Expt

Temperature of

incubation (“)

No plaques

Petri dish with Total

429 +e petri plaques plaques 429 and $e plaques dishes

A 31 66 56 10 12 144 B 42 72 0 72 0 144 C 42-31 48 14 33 5 100

o A spore suspension prepared as indicated in Table 3 was diluted to contain 2.5 infected spore per ml; 0.2-ml aliquots of this dilution were distributed in tubes and incubated for 4 hr, without shaking. When tempera- ture was shifted from 42 to 31” during incubation, the shift occurred at 70 min. Indicator bacteria and soft agar were added, and the whole volume was poured on plates. Petri dishes were incubated at 30”.

TRAPPING AND EXCLUSION OF PHAGES 429 AND 4e 365

TABLE 5

DOUBLE INFECTIONOFEXPONENTIALLYGROWING Bacillus subtilis 168 BY~~~AND$~~

Infecting phage Type of plaques Phage input Adsorption (%) PFU/ml (12 min) PFU/ml(90 min) Surviving CFU/ml

(10 min)

429 429

2.2 x 109 36

6.3 x 10’ 1.4 x 10’0

3.0 x 10’

4e 4e

2.8 x lo9 47

9.0 x 10’ 2.0 x 10’0

5.0 x 106

429 2.2 x 109

36 3.1 x 10’ 6.6 x 108

429 and de +e

2.8 x lo9 48

8.0 x 10’ 1.1 x 10’0

3.0 x 106

429 and 4eb 5.0 x 109

- 8.4 x 10’ 1.1 x 10’0

D Samples of a culture of Bacillus subtilis 163 were infected with 429 only, 4e only, or with both phages, at a m.o.i. of 10 and incubated at 42”. Adsorption was allowed for 5 min. Antiphage sera were added for 5 min; then cultures were diluted into fresh medium and incubated until 90 min when CHCl, was added. To determine the number of PFU contributed by each phage, plating was made in the presence of appropriate antiphage serum. Both sera were added to nutrient agar on which CFU were counted. Enough serum was used to avoid ambiguous results.

b Without antiphage sera.

one type of phage, either 429 ts 2 (98) or +e, but never both; (b) the incubation tem- perature determines which of the phages will be produced, presumably by controlling the extent of expression of $29 ts 2 (98).

Mixed Infection with $29 and r$e during Exponential Growth

It was of interest to know whether ex- clusion of one of the phages also occurred within exponentially growing cells. As it is not possible to study single bursts in this case because of the chains formed by the cells, the ratio between the two phages in the output has been determined. As shown in Table 5, $e dominates 429: the 429 prog- eny in mixed infection was about 5% of that released in single infection, while the cor- responding figure for $e was 55%. In an- other experiment, +e progenies from single and double infection were equally numerous. It should also be noted that lysis of mixedly in- fected cultures occurred at 50 min as with cultures infected only by +e, while lysis of cultures infected by 429 may require up to 80 min.

A comparison of the number of infectious centers at 12 min suggests that dominance is only partial. +29 PFU at that time is about 50% of that produced by singly in- fected cells, even though almost all cells were infected by +e (only 3-5% of CFU survive its infection, and the total number

of PFU at 12 min in mixed infections is close to the number corresponding to single be infection). The dominance of +e over 429 seems thus to be expressed at two levels: first, by preventing any production of $29 progeny in a fraction of the cells and sec- ond, by depressing its yield in the rest. This result is especially interesting since the la- tent and rise periods of the two phages are identical (Fig. 5). Incidentally one can ob- serve (Table 5, last line) that a high pro- portion of cells survive infection by 429. This fact has been consistently observed, particularly when infection was done in Schaeffer sporulation medium, as was the case here.

DISCUSSION

Trapping of 429 and $M Phages

The results presented and those of Sonen- shein and Roscoe (1969) and of Kawamura and Ito (1974) establish the existence of critical times, after cessation of exponential growth, for the trapping of phages +e and 429. Combined with those of Ito et al. (1973) they show that each phage has its own criti- cal time. 415, 429, M2 (Ito, personal com- munication), and be are trapped by Bacillus subtilis 168 in that order. Maximum effi- ciency for 429 occurs 0.5 hr after the end of exponential growth, and that for +e 1.5 hr later (Fig. 1).

Two other differences have been found

366 FELIPE MORENO

LIB I III II II

102030405060x)80 TIME (min)

FIG. 5. One-step growth of 429 and +e phages in Bacillus subtilis 168 during exponential growth. An aliquot of the bacterial culture of the experiment of Table 5 was infected with wt 429 and wt +e (m.o.i. for 0.05 for each) and incubated at 42”. At 5 min anti- phage sera were added and at 10 min the culture was diluted first l/2000 and then 20-fold further into fresh medium. The number of PFU of each phage was de- termined at different times on those diluted cultures. Enough of one anti-phage serum was added to the plating medium to neutralize this phage quantitatively and to study growth of the other phage. 0 - 0, 929; 0 - 0, 4e. Thirty percent of the 429 input and 75% of the $e input was adsorbed at 5 min; 25% of the 429 adsorbed and 63% of 4e adsorbed produced infectious centers.

between the trapping of 429 and $e. The first is the duration of the period during which phages can be trapped by the sporu- lating cells as illustrated in Fig. 1. Ninety minutes after the peak of maximum effi- ciency for $e trapping the cells have re- tained 4/4 of their maximum ability to trap. During the same time interval after the peak for 429 trapping, the efficiency de- creases by a factor 100 to 200. In fact, re- sults in Fig. 3 indicate that the average time during which a cell is capable of trapping $29 is only 20 min. The second difference resides in the actual value of the trapping efficiency. Under optimal conditions 40 to

100% of the +e adsorbed are recovered bio- logically active in spores, the corresponding value for 429 being only l-2%. This poor efficiency is not modified by increasing the m.o.i. (Fig. 2). In other words there is nei- ther competition nor cooperation between genomes in the trapping phenomenon. That is confirmed by results of double trapping, which has been shown to occur by two in- dependent events.

The different trapping efficiencies for the two phages could be due to a defective in- jection of DNA by phage 429. It is also possible that 429 DNA is normally injected but it is more sensitive to some mechanism of exclusion which would take place from the beginning of sporulation. Although our experiments do not allow a choice to be made between these possible mechanisms, results from one-step growth curves (Moreno, 1977) with sporulating cells tend to favor the second.

Identical trapping values were found whether the infecting phage is able to syn- thesize its DNA or not. This indicates that synthesis of viral DNA is not required for trapping to occur. Moreover, this suggests that, in fact, such a synthesis does not take place during trapping. Alternatively, if neo- synthesized 429 genomes appeared, they would not be trapped. Similar conclusions were reported by Sonenshein (1970) by using conditional lethal +e mutants and by measuring viral DNA synthesis after infec- tion under trapping conditions. The small quantity of synthesis of 429 DNA detected by Kawamura and Ito (1974) under these conditions was very probably carried out by cells which had not yet reached compe- tence for 429 trapping.

Phage trapping has been visualized “as simply mechanical penetration of an un- replicated phage DNA molecule into the de- veloping spore primordium” (Sonenshein, 1970). While this hypothesis cannot actually be set aside, the fact that phages such as 429 and +e are trapped at different times suggests that the viral genome could play an active role in its own trapping. Kawamura and Ito (1974) have reported that phage- specific messenger RNA is still synthesized when phage is trapped. This RNA could di- rect the synthesis of a viral protein(s) which

TRAPPING AND EXCLUSION OF PHAGES 429 AND $e 367

might protect the phage genome from the exclusion mechanism mentioned above. In order to test this hypothesis, the trapping of sus and ts 429 mutants, affected in 16 (2 through 17, Mellado et al., 1976) of the phage cistrons was studied. All the mutants were trapped with nearly equal efficiency as was the wt phage (data not shown) and no phage function required for trapping could thus be detected. Since about 20 pro- teins are coded by the 429 genome (Car- rascosa et al., 1973; Hawley et al., 1973; Pene et al., 1973; McGuire et al., 1974) a few more functions still remain to be tested. The possibility that the product(s) postu- lated are necessary for trapping, but not for viral development, should not be over- looked.

ReciprocuZ ExcZusion between @i!9 and +e Phuges

In order to demonstrate the simultaneous trapping of two unrelated phages in a single spore, Bacillus subtilis cells were mixedly infected, at appropriate times with $29 ts 2 (98) and wt +e. Among the formed spores, 90% contained ts 2 (98), 90% contained $e, and 80% contained both genomes. This illus- trates that in the trapping process, no inter- ference occurs between the two phages.

In contrast, during germination and out- growth, there is a complete reciprocal ex- clusion. A spore infected with $29 ts 2 (98) and 4e produces either +e or $29, but never both, depending on the incubation condi- tions. This may easily be accounted for. At 30”, 429 completes its own development and host lysis occurs before 4e morphopoiesis. At 42”, $e is produced only because mutant ts 2 (98) cannot synthesize its own DNA. However, total reciprocal exclusion occurs even under conditions where development of the two phages is synchronized. There- fore the decision to produce one or the other viral particle in mixed infection occurs at the individual cell level. When viral expres- sion of both phages starts simultaneously cells will produce +e, progeny 429 being mainly produced in cells in which expression of +e is delayed relative to expression of 429. The same seems to occur in mixed in- fected growing cells. In this case, however,

the burst-size in cells producing 429 is dra- matically reduced relative to single infec- tion; this was not the case when the host was outgrowing cells (data not shown).

Since su- cells infected by sus mutants in cistron 2 produce a protein pattern simi- lar to that of wt 429 (Anderson and Reilly, 1974; McGuire et al., 1974; Carrascosa et al., 1976), it follows that the 429 specific macromolecules, probably synthesized at 42” in doubly infected cells before the 4e genome is expressed, do not disturb devel- opment of the latter phage. By using other $29 mutants it should be possible to deter- mine which step in $29 development is re- sponsible for the exclusion of +e.

Vegetative cells infected from without re- lease progeny with similar rates after 40 min, whether $29 or +e was the infecting phage. In outgrowing carrier spores, 4e progeny is released with a 20-min delay relative to $29. In both hosts, 25-30 min elapse between the start of 429 DNA syn- thesis (results not shown) and the liberation of 429 progeny, suggesting that the viral development program, once started, is the same in both kinds of cells. Therefore one or more factors of the host, required for (inhibiting) +e multiplication, would appear (disappear) at 35-40 min of spore germina- tion. This host factor(s) should not play a role in the growth of $29.

ACKNOWLEDGMENTS

I am very grateful to P. Schaeffer for his advise in the preparation of the manuscript. I thank E. Eisenstadt who participated on some preliminary ex- periments, and A. L. Sonenshein, M. Salas, and H. A. Eisen for critically reading the manuscript.

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