The course of phage ∅e infection in sporulating cells of Bacillus subtilis strain 3610

VIROLOGY

39, 265-276 (1969)

The Course

of Phage Bacillus

ABRAHAM Department of Biology,

4e Infection subtilis

Strain

L. SONENSHEIN Massachusetts

Institute

in Sporulating

AND

Cells

of

3610

DAVID

of Technology,

H. ROSCOE’

Cambridge,

Massachusetts

02199

Accepted June 3, 1969 Infection of Bacillus subtilis strain 3610 by the virulent phage +e at a critical stage during sporulation results in failure to produce phage and in incorporation of the phage genome within the developing spore. This phage “trapping” is maximal if infection occurs 5-6 hours after the end of logarithmic growth and is correlated with the failure of three phage-induced enzyme activities to appear at measurable levels. A nonsporulating mutant has been isolated which allows phage production in cells infected at a time when cells of the wild-type bacterium produce no phage. This mutant is blocked before the prespore stage but produces an antibiotic factor and a protease charact,eristically formed in the early steps of the sporulation process. INTRODUCTION

Some phage-bacterium relationships cannot be classified as either productive infection or lysogeny. Examples of this are the persistent infection of Escherichia coli by filamentous male-specific phages (HoffmanBerling and Maze, 1964) or of Shigella dysenteriae by phage T7 (Li et al., 1961). Such pseudolysogenic relationships (Lwoff, 1953) have been studied in some detail in Bacillus subtilis (Thorne, 1962; Takahashi, 1964; Bott and Strauss, 1965; Kawakama and Landman, 1968). This paper describes the interaction between a spore-forming bacterium and a phage whose genome becomes incorporated within the developing spore if infection takes place at a critical time during sporulation. Bacillus subtilis 3610 is an abundant sporeformer on whose growing cells phage 4e multiplies as a virulent phage (Roscoe and Tucker, 1966). When the bacteria are placed under conditions leading to sporulation they reach a state after which they no longer support phage multiplication. At this stage of sporulation the phage genome can enter the

developing spore but gives rise to no new phage. If the infected sporulating cells are allowed to complete the spore-forming process the resulting free spores act as infective centers. The observation of trapping of phage genomes inside spores is not new. Takahashi (1964) extracted DNA from spores of B. subtilis SB19 carrying phage PBS1 and demonstrated the presence of phage DNA in a CsCl density gradient. Yehle and Doi (1967) isolated two phages, one of which could grow on sporulating cells of B. subtilis W23, the other not. They correlated the ability of a phage to grow on sporulating cells with the abilities to repress synthesis of host messenger RNA and withstand repression of phage mRNA synthesis by the host. By studying a phage-bacterium relationship in the sporulating process, as described in the present paper, we hope to clarify the mechanisms by which the sporulating bacterium prevents phage growth and, by extension, to explore the control mechanisms in the switchover from vegetative growth to sporulation.

1 Present address: Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, W. C. 2, England.

Bacteria and phage. Bacillus subtilis 3610 (ATCC 6051), a Marburg strain, and its

MATERIALS

265

AND

METHODS

266

SONENSIIEIN

hydroxymet,hyluracil(HMU)-containing phage, de, have been described previously (Roscoe and Tucker, 1966). High-titer phage stocks were prepared as follows. An overnight culture of B. subtibis 3610 in LB broth was diluted 1: 20 into fresh broth and shaken at 37” for 30 min. Phage de was added t,o approximately lo7 plaque-forming units (PFU) per milliliter, and the culture n-as allowed to shake at 37” until lysis was complete (4-6 hours). After storage overnight at 4”, the lysate was treated with two cycles of low and high speed centrifugation. The final low speed supernatant was stored over a crystal of thymol at 4”. Typical titers of such lysates are 2 to 5 X 1O1lPFU/ml. Staphylococcus uureus strain NRRL was from our laboratory collection. Media. LB broth, LB agar, and top agar (Hall, 1967) were used for growing and assaying bacteria and phage. T2 buffer contained per liter: 5.7 g of NazHP04.7Hz0, 1.5 g of KHZPOI, 4.0 g of NaCl, and 5.0 g K&Oh (sterilized together); plus 0.25 g of MgSO4. 7H,O; 0.011 g of CaClZ, and 10 ppm gelatin added separately. Phage stocks were maintained and diluted in T2-buffer. Two modifications of basal medium 121 (Hall, 1967) were used: (1) medium 121A, supplemented with 0.8% glucose, 0.01% casamino acids, and lop3 M K2HPOI; (2) medium 121B, containing 0.2 % glucose, 0.2% casamino acids; 2.5 X 1 OV3M K2HP04, 10e4 M &InC&, 3.4 X 10m3M sodium citrate, and 1.5 X 1O-4 M FeC13. Spore suspensions used as indicator for phage platings were grown in potato extract medium (Spizizen, 1958). Bacterial stocks were maintained on slants of Potato Dextrose Agar Special (Difco). Plating conclitions. Unless otherwise stated, bacteria and phage were plated on LB agar. For colony counts, aliquots of bacterial suspensions were either spread directly or embedded in 2.5 ml of top agar. For plaqueforming assays, phage or infected bacteria were mixed with lo8 spores of 3610 in 2.5 ml of top agar. All plates were incubated at 30”. Measurements on bacterial cultures. A Klett-Summerson calorimeter was used to measure turbidity at 540 rnp. Total cell numbers were estimated in a Petroff-

AND

ROSCOE

Hnusser counting ch:amber. The appearance of refractile presporcs w-as judged by phasecontrast microscopy. Heat resistance was determined from ability to withst,and incubnt,ion at SO” for 10 min without loss of viability. Octanol resistance was estimated from survival in 0.2% octanol for 5 min at 37”. For chloroform treatment, 2 drops of CHC13 were added to 0.5 ml cells; the mixt’ure w-as shaken vigorously for 15 set; an aliquot taken as soon as the chloroform settled out was diluted and plated. Dipicolinic acid was assayed according to Janssen et al. (1958). Phuge physiology. For single-step growth experiments cells were infected at a multiplicity of 0.1 and shaken at 37” for 10 min. The adsorption mixture was diluted into phage antiserum of sufficient activity to inactive 99.9% of any remaining free phage within 5 min at 37”. After dilution at least lOOO-fold, an aliquot was plated for assay of infective centers. The last dilution was shaken at 37”, and samples were removed at intervals up to 90 min to determine the average burst size. Incorporation of 14C-leucine. 14C-leucine (2.6 #&‘~mole) was added to samples of cultures in medium 121B to a final concentration of 0.02 rCi/ml (1 pg/ml). Aliquots of 0.2 ml were removed in duplicate to 2 ml ice-cold 5% trichloroacetic acid (TCA) containing 50 pg/ml 1-leucine to minimize nonspecific adsorption of free 14C-leucine. After overnight storage at 4”, the precipitates were collected by centrifugation, resuspended in 0.75 ml 1 IV nTaOH, and kept for 20 min at room temperature. Then 3 ml of 10% TCA was added and the suspension n-as heated to 95” for 30 min and then cooled. The precipitates were collected on membrane filters presoaked in 5% TCA containing 50 pg/ml 1-leucine and were washed thoroughly with the same solution. Filters \+-ere dried on planchets and counted in a Nuclear-Chicago gas flow system. Total protein was determined after precipitation in 5%’ TCA. The precipitates were dissolved in 1 N SaOH by heating to 100” for 10 min, diluted to 0.1 N NaOH, and assayed for protein according to Lowry et al. (1951). Preparation of bacterial extracts. B. subtilis 3610 grown in medium 121B was infected

SPORULATION

AND

with phage 4e at a multiplicity of 10. After 25 min at 37”, the cells were harvested, washed with 0.05 M Tris-HCI buffer, pH 8.5, and resuspended in 5 ml Tris buffer (0.1 M, pH 8.5) for every 100 ml of initial culture. An uninfected control culture was treated in the same manner. Cells were disrupted by sonication (MSE sonic power unit) for 8 min in the cold in 1-min pulses with 0.5-min intervals. Debris was removed by spinning at 12,000 g for 5 min. The supernatant (“crude extract”) was assayed immediately for deoxycytidylate deaminase and stored at 4”. For dUlKP hydroxymethylase and thymidylate synthetase assays, the extracts were centrifuged at 100,000 g for 60 min and the supernatants were stored at 4” until assayed. dTTPase was assayed on the crude extract. Enzyme assays. (1) Deoxycytidylate deaminase was assayed as described by Roscoe and Tucker (1966) except that the pH was 8.5 instead of 7.5 and the final volume was 1.0 ml; (2) the dTTPase assay has been described (Roscoe, 1969); (3) thymidylate synthetase was assayed by the spectrophotometric assay of Wahba and Friedkin (1962); (4) deoxyuridylate hydroxymethylase was assayed as described by Roscoe and Tucker (1966) except that the appropriate ultraviolet adsorbing spots were cut from the chromatogram and counted in 5 ml of toluene-POPOP-PPO or toluene-Omnifluor (New England Nuclear) scintillator in a Nuclear-Chicago Mark I liquid scintillation system. Protein concentrations were estimated by the method of Lowry et al. (1951). Chemicals. N-Methyl-N’-nitro-N-nitrosoguanidine was from Aldrich Chemical Company; HCHO-14C was obtained from Volk Radiochemical Company; 1-leucine-14C was from New England Nuclear Corporation; dTTP-3H was from Schwarz BioResearch, Incorporated; dCMP, dUMP, dTMP, dTTP and THFA were from Sigma Chemical Company; and Renografin 76 was from E. R. Squibb and Sons. RESULTS

Trapping of Phase When a complex medium favorable to sporulation is inoculated with B. subtilis 3610

PHAGE

267

DEVELOPMENT

and phage +e, a large fraction of the resulting spores behave as infective centers even after treatment with antiphage serum, chloroform, octanol, and incubation at 80” for 10 min (Table 1). To examine this phenomenon under reproducible conditions suitable for biochemical and radiochemical experiments, cell populations partially synchronized with respect to the sporulation process were obtained as follows. Twenty-five-milliliter cultures of B. subtilis 3610 in medium 121A, started from slants and incubated overnight at 37”, were harvested, washed in medium 121, and resuspended in 5 ml of medium 121; 1.2 ml was used to inoculate 30 ml of medium 121B. This culture was shaken at 30 or 37” until the early stationary phase was reached (4-6 hours at 37”, 12 hours at 30”). The cells were harvested and resuspended in 6 ml of medium 121. Thirty milliliters of fresh medium 121B was inoculated with 1.2 ml of this suspension. At 37” this culture entered the logarithmic phase of growth within 15-30 minutes. The synchronization achieved is evidenced by the fact that 90 % of the culture enters the prespore stage within a l-hour interval (see Fig. 4b). By contrast, a culture kept in medium 121B without transfer would enter the prespore stage over a 4-hour period. As shown in Fig. 4, growth of the synchronized population reaches a maximum after 4 hours at 37”; there follows a period of cell lysis. Of the cells that do not lyse (50% or more of the peak number) at least 95% become spores after 24 hours of incubation TABLE PRODUCTION Total spores per ml 2.8 x

107

1

OF SPORES THAT CENTERS~

ARE INFECTIVE

Colony-forming units per ml

Plaque-forming units per ml

5.8 x

2.0 x

106(21%)

lO’(717,)

a Potato extract (25 ml) was inoculated simultaneously with a loopful of Bacillus subtilis 3610 and a loopful of phage +e stock lysate and shaken at 37” for 4 days. The resulting spores were washed three t,imes with distilled water, treated with antiphage serum, chloroform, octanol, and antiphage serum again and heated to 80” for 10 min before plating.

26s

SOTENSHEIN

at 37”. In log phase, phage infect’ion is productive, yielding an average burst of (jOGSO new phages per infected bacterium. The trapping of phage in spores is easily demonstrated in cells grown in defined medium under the standard conditions. Figure 1 shows the results of a typical experiment. Aliquots of 2.5 ml of the culture Tverc removed at various times and mixed wit,h phage at a multiplicity of 10. Aft’er 10 minutes at 37”, the mixture received 0.25 ml of antiphage serum suitably diluted in “depleted medium,” that is, in supernatant of a sample of the same culture centrifuged at the time of infection. The infected cells were incubated at 37” in the presence of antiserum until sporulation was complete; this takes about 24 hours after inoculation of the culture in medium 121B. An uninfected control sample was treated with antiserum in the same way. The spores were harvested, washed, diluted, and plated with or without pretreatment with antiserum; there were no free phage. As seen in Fig. 1 the optimum time of infection for trapping of phage is 5-6 hours after the end of logarithmic growth (7.5-8.5 hours after inoculation). The peak of phage trapping is fairly sharp (10 % of the maximum value is found if infection occurs 2 hours earlier or later than the optimal time). Although SO% or more of the spores recovered from cells infected at 8 hours carry the phage, the total number of spores recovered is only 30-50 % of that in uninfected cultures. This is probably related to the observation, described in more detail below, that the peak time of phage trapping occurs between two periods during which phage infection results in cell lysis. Thus the 5070% loss of spores (and hence phage-carrying spores) observed after infection at S hours probably reflects incomplete synchrony. In taking the number of infective centers produced to be indicative of the number of carrier spores in the population, the assumption is made that, when a carrier spore is plated, it germinates and gives rise to a burst of phage, which produces a plaque on indicator cells. Studies of phage development during germination will be presented in a future publication.

AND ROSCOE

:-.-,-GM-i

sporesfrom uninfected cells

IO9

107.~ infective

centers

Time of infection in hours

1. Trapping of phage genomes as infective center-producing spores. (0) Total spores produced per milliliter by infected cells as counted in Petroff-Hausser chamber. (0) Colony-forming units per milliliter among the infected spores. (A) Plaque-forming units per milliliter among the infectedspores. (A) Total spores and colony-forming units per milliliter produced by uninfected spores. FIG.

The phage trapping described above suggests that at some stage during sporulation the 4e genome can enter potential spores, but normal phage development is prevented. A cell infected in this way is able to compIete the sporulation process. Further experiments have provided support for this hypothesis. Growth of Phage To analyze the relation between the trapping of phage into spores and its ability to grow productively, the average phage burst size was determined in single-step growth experiments for cultures infected at various stages of growth and sporulation (Fig. 2). The phage yield was highest in mid-log phase and then fell off, reaching approximately 1 at S-10 hours. Single-burst experiments, shown in Table 2, indicated that at late times (S-10 hours) all infected cells

SPORULATION r

I

BND

PHAGE

-71

.-*1 ./

-/ a-.4 20 I

40

60 -.

--

100

00

0

269

results are compatible with the assumption that at late times only about 1 infected cell in 10 produces phage. This is not due to lack of phage adsorption (see Fig. 5), but may be related to the fragility exhibited even by uninfected cells at this stage if they are transferred to solid media. This is discussed more fully below. The average phage yields shown in Fig. 2 and Table 2 were calculated as the ratios of phage present at 90 min to the infective centers at 15 min. Since in the S- to lo-hour period infective centers are underestimated by a factor of 5-10, the true burst sizes (per infected cell) are lower by a similar factor.

120

Time (min ) after infection I 1 i 00

k 0’

DEVELOPMENT

Time (hours)

FIG. 2. Phage growth in single infection. (a) Single-step growth for cells infected in log phase (0) and while sporulating (9-10 hours of growth) (0). (b) Average burst size for infection at various times during growth and sporulation. (a) Average burst size, (0) turbidity at 540 rnp.

produced little or no phage, so that the low yields could not be ascribed to a minority of cells producing normal-size bursts. Cells infected at late times, as they approach sporulation, not only gave smaller burst sizes but produced fewer infective centers. For example, only lo-20% of cells that received phage when infected at 9.5 hours in a standard culture appeared as infective centers. Similarly, the single-burst

Phage-SpeciJic Proteins Production of new phage in sporulating cells might be blocked at any one of several levels. Phage DNA must enter the cells since it becomes incorporated into spores. It is not certain that the DNA which enters sporulating cells can be replicated or transcribed to generate phage-specific proteins. To obtain information on these points the synthesis of phage-specific proteins was measured in infected sporulating cells. Infection of growing bacteria with 4e induces the production of at least four proteins whose functions relate to nucleic acid metabolism (Roscoe and Tucker, 1966; Roscoe, 1969). Three new enzymatic activities are detected: deoxycytidylate deaminase, deoxyuridylate hydroxymethylase, and thymidine triphosphate nucleotidohydrolase (dTTPase). The fourth protein is an inhibitor of the bacterial thymidylate synthetase. These activities were assayed after infection of cells in log phase, in stationary phase, and in sporulating phase, as shown in Table 3. The pattern of burst size reduction and phage trapping was correlated with reduced ability to induce the three enzymatic activities. The inhibition of thymidylate synthetase, however, does not follow the same pattern: there was full inhibition in infected sporulating cells. The meaning of this observation is unclear since the assays in Table 3 show only the presence of inhibitor, not its amount, and since it is not known whether the inhibitor is coded for by the phage genome.

270

SONENSHEIS

AND ROSCOE

TABLE SINULI+BERHT

2

EX~KIXIMEXTS”

Expt. No.

Infection time (hoursj

Average burst size

12(== average number of infected cells per tube)

0

1

1 1 1 2 2 3

1.25 4.5 9.0 9.0 9.0 10.0

51.4 13.3 5.9 5.7 5.7 0.9

1.2 2.0 10.0 15.0 1.5 15.0

10 14 21 5 41 8

Number 0 0 3 1 1 16

Expected*

Phages per tube 2-10

11-100

>lOrl

0

>o

16 3 0 0 0 0

15 7 0 0 11 0

33 41 48 50 39 46

of tubes 19 3 25 6 15 9 1G 28 2 6 1 21

a Aliquots of a culture were infected at the indicated times and treated with antiserum as in singlestep growth experiments (see Materials and Methods). After 5 min, the infected cells were diluted and distributed so that an average of 12infected cells were delivered to each of 46-50 tubes. After 75 min at 37”, 2.5 ml top agar containing lo* spores of 3610 were added to each tube and the mixture was poured over an LB agar plate. Plates were incubated at 30”. The average burst size was determined in the standard way. b From Poisson’s equation, assuming that each infected cell produces phage. TABLE ASSAYS OF PHAGE-INDUCED

Extract Log-phase Log-phase Stationary Stationary Sporulating Sporulating

3

PROTEINS IN CELLS INFECTED DURING GROWTH AND SPORULATION~

infected uninfected infected uninfected infected uninfected

dCMP deaminase (units/mg protein)

dUMP HMase (units/mg protein)

dTTPase (units/mg protein)

dTMP synthetase (units/mg protein)

1.40 0.01 0.28 0.07 0.14 0.15

0.121 0.006 0.017 0.002 0.018 0.012

39.3 1.4 11.2 0.6 1.8 1.4

0.0011 0.0172 0.0020 0.0193 0.0019 0.0201

0 Preparation of extracts and enzyme assays are described in Materials and Methods. Log-phase cells were infected after l-l.5 hours of growth, stationary cells after 4.5-5.5 hours, and sporulating cells after 8-10 hours.

It is clear that the phage-directed enzymes are not, made at the normal levels. Whether or not small amounts of enzyme are present, sufficient for replication of phage DP\IA at least to a limited

extent,

remains

undecided.

In order to test whether the failure to produce measurable amounts of phagedirected enzymes was due to a general deficiency in protein synthesis in sporulating cells, we compared the ability of infected and uninfected sporulating cells to incorporate leucineJ4C into protein with that of log phase cells (Fig. 3). Sporulating cells incorporated leucineJ4C at about two-thirds the rate of log phase cells. We conclude that the deficiency in phage-directed protein syn-

thesis in sporulating cells is due to a specific inhibition, probably related to the failure of sporulating cells to make most, of the logphase enzymes. Efects of Phage Infection on the Properties of Sporulating Cultures Total cell numbers, colony-forming ability, and resistance to potentially lethal agents were measured in uninfected sporulating cultures (Fig. 4). In addition, some effects of infection with phage +e were examined (Fig. 5). The relevant findings, shown in Fig. 4, are as follows: (1) after active growth ceases, about half the culture lyses; 95% of the remaining cells will give spores; (2) the

SPORULATION

1

I

271

AND PHAGE DEVELOPMENT

I

I

. /

tion. Adsorption of phage and production of infective centers at low multiplicity are shown in Fig. 5b. Adsorption was normal throughout. Until the twelfth hour, the ability to produce infective centers was roughly correlated with the ability of the cells to produce colonies. Later colony-forming ability recovered but infective center formation remained low. This suggested that the failure of sporulating cells to produce phage even in single-burst experiments might simply be due to the sensitivity of these cells to transfer to fresh media. To check this possibility single-burst experiments similar to those of Table 2 were repeated using depleted medium from sporulating cultures for all dilutions, so that the cells were never exposed to fresh medium

IO 20 30 Time (min.) after addition of ‘4C- leucine

FIG. 3. Incorporation of W-leucine. Experimental details are given in Materials and Methods. (0) Uninfected log-phase cells; (0) uninfected sporulating cells; (A) infected log-phase cells; (a) infected sporulating cells.

ability of the surviving cells to produce colonies on nutrient agar plates continues to decrease even after lysis stops and then increases again after the first appearance of prespores; (3) the appearance of prespores precedes that of resistance to heat and octanol (and also the appearance of resistance to chloroform and the production of dipicolinic acid; not shown in Fig. 4). The transient loss of colony-forming ability (“viability”) during the 7- to 12-hour period must be due to the transfer to the plat’ing medium since the cells, if left in liquid medium, would proceed to form spores. It is probably due to the shift in environmental conditions, including exposure to fresh medium. Colony counts were not higher, however, if cells were diluted in de&e&d instead’of fresh medium, or if the dilution was made at 4’, 25”, or 37”, or if 1 ,. 1 1. *, plating was Dy spreaamg or in a solt-agar layer. Figure 5a shows the adsorption of phage, production of infective of cells by phage infection

centers, and killing at high multiplicity

at various times during growth and sporula-

/f 100 i : , , : I O- 2 4

0 p , , , Tfime(ho;rsj10 12

I

IO

f z g o) kg 0, i g g; 2 a s z oo, z i E E Time

of incubation,

hours

FIG. 4. Parameters of cell growth and sporula-

tion. (a) (0) Turbidity at 540 rnp; (0) colonyforming units per milliliter; (A) total cells per ml. CJJ) (0) fraction

Fraction of cells that are prespores; of cells resistant to octanol; (A)

tion of cells resistant to heat.

(0) frac-

SONENSHEIN

0

j--m-

I -
I 12 for

ANIl

,

(b) adsorption

0

I I I I 2 4 6 8 IO Time (hours) when sample removed for infection

Fro. 5. Parameters of phage infection. (a) At high multiplicity of phage infection (ca. 10 phages per cell): (0) fraction of input phage adsorbed; (0) fraction of cells that become infective centers; (A) fraction of uninfected cells that can form colonies;: (A) 4fraction of colony-forming cells that survive infection. (b) At low multiplicity of phage infection (ca. 0.1 phages per cell): (0) fraction of input phage adsorbed; (0) fraction of input phage that yield infective centers.

until the time of plating. Average burst sizes, infective center production, and singlebursts were the same as in the previous experiments. Phage Infection of a Nonsporulatirq

Mutant

In an attmept to link exclusion of phage growth with processes specific to sporulation, bacterial mutants were sought which would simultaneously lose the ability to form spores and to exclude phage. Log-phase cells of B.

ROSCOE

subtiZis ?A10 were treat,ed with nitrosoguanidine (Adelberg et al., 1965), filtered, and resuspended in medium 12lB. After 23 hours at X0, a l..?ml aliquot was layered on a 30-ml gradient of Renografin 76 (Tamir and Gilvarg, 1966) constructed so that the spores would be pelleted and vegetative cells would float near the top of the tube. Centrifugatiori was at 20,000 rpm for 20 min in a Spinco SW 25 rotor in a Spinco Model L centrifuge. A thin band near the top of the tube was removed with a syringe and filtered on a Xlillipore membrane, which was then washed alternately with medium 121 and 0.2 ill MgCh. The cells from the membrane were resuspended in 1.5 ml of medium 121 and spread on potato dextrose agar plates, on which sporulating colonies are brown. Colonies that remained white after 36 hours at 30” were picked, purified, and tested for spore-forming ability, production of the sporulation-specific antibiotic active against Staphylococcus aureus (Balassa et al., 1963), and production of extracellular protease (Michel et al., 1968a). Seventeen mutants that failed to produce prespores, as judged by phase contrast microscopy, were tested for their ability to support phage growth after 8-10 hours of growth in the standard culture method. Eight mutants allowed more phage growth than wild type, with burst sizes ranging from 3 to 20. Mutant spr8 was studied in some detail. The burst size of 4e on spr8 at various times is shown in Fig. 6. Since 90-100% of the infected cells of spr8 produce infective centers, phage production per infected cell at the 8- to lo-hour stage is more than 100 times that of wild type. This confirms that exclusion of phage growth in sporulating cells is related to the sporulation process. Preliminary experiments with extracts of phageinfected mutant cells indicated that dTTPase was induced in a culture infected at the ninth hour. Unlike wild-type cells, the mutant spr8 did not become fragile to plating in the first 12 hours of growth (cells at later times have not been tested). Also, at high multiplicity of infection virtually all the infected cells became infective centers. This strongly sug-

SPORULATION

AND PHAGE

A20

I

0

2

4 6 8 IO Time of infection, hours

12

FIG. 6. Burst size of qie on nonsporulating mutant. (a) Turbidity (at 546 nq) of mutant spr8; (0) turbidity of parent strain 3610; (I(r) burst size of +e on spr8 at the indicated times of growth; (A) burst size of +e on 3610 after indicated times of growth.

DEVELOPMENT

273

ably because the few uninfected cells have a chance to multiply before sporulating. Since antiserum is present from 10 min after infection, there is no secondary infection of originally uninfected cells. The second period covers the 4- to 7-hour interval, during which the cells are in stationary phase. In this period, infected cells (1.5 to 2.5 X log/ml) yield 1.0 to 1.5 X lo* spores per milliliter, some of which carry phage, especially if infected after 6 hours; infection leads to cell death with low production of phage (see Figs. 2 and 5), and the cells that survive infection have little chance of growing due to depletion of the medium. During the third period, 7-9.5 hours, infection results in considerable trapping of phage, with about 50% recovery of spores compared to uninfected cells. The loss of spores probably reflects the asynchrony of the sporulation process. Between 9.5 and 12.5 hours (period 4; prespore stage), infection results in substantial loss of spores without either trapping of phage or production of new phage. It is possible that phage infection during this

gests that the sensitivity of sporulating wildtype cells to plating, as well as the decreased production of infective centers by phageinfected wild-type cells, are phenomena specifically related to sporulation. .-.-.

4-.*-

.-a--.

Effect of Phage Infection on Spore Formation Spore production by infected and uninfected cells is shown in Fig. 7, which is based on the same series of experiments shown in Fig. 1. The course of spore production in infected standard cultures, as illustrated in Fig. 7, is quite reproducible from experiment to experiment. Virtually all the spores produced by uninfected cells yield colonies when plated on nutrient agar. Formation of spores by infected cells is greatly dependent on the time of infection. The data of Fig. 7 can be interpreted in terms of five time periods. In the first period, lasting 4 hours in the standard culture, the cells are growing rapidly and are presumably highly sensitive to phage infection. A culture infected in this period yields some spores (mostly without phage), prob-

-I

QJ

-I-2+3+-4+5-

I

I

2

4

I

I

1

I

I

I

6 8 IO I2 I4 I6 Time of infection (hours)

I

I8

I

FIG. 7. Recovery of spores from cells infected at various times during growth and sporulation. The experiment is the standard trapping experiment and is identical to that shown in Fig. 1. (0) Recovered spores per milliliter from uninfected cells; (0) recovered spores per milliliter from infected cells (m.o.i. about 10).

274

SONENSHEIN

period initiates phage functions that result in cell lysis, but it seems more likely that cells at the prespore stage are very sensitive to external factors, such as attachment of phage, which might damage the sporangium. In period 5 (12.5-24 hours), infection leads to little loss in spore production; the spores are virtually all capable of forming colonies, and few, if any, carry phage. Heat Sensitivity of Phage-Infected Spores Yehle and Doi (1967) reported that spores of B. subtilis W23 carrying phage fl3 were heat-sensitive, showing a 20% decrease in the infective center titer after 10 min at 80”, whereas colony formation by either infected or uninfected spore preparations decreased only 34 %. We found a similar situation in the B. subtilis-phage 4e system (Fig. S); the infective center titer drops to about 30% in

(b)

uninfected

infected

spores (cfu 1

spores (PfU 1

P P s

/ 1

IO6I-

infected *

*

I

IO" L-_. 0

-I-. IO

spores (dfu,,

I.-.-I

Time of incubation

30 20 at 65 or 80°C

FIG. 8. Heat sensitivity of infected and uninfected spores. Spores were heated to (a) 65” or (b) 80" for the indicated period of time before dilution and plating. (0) Colony-forming units of uninfected spores; (a) plaque-forming unit.s of infected spores; (A) colony-forming units among infected spores.

ANU the

ROSCOIC first

10 mire at SO”, arid

to 3%

after

30

min. Uninfected spores survive for 20 min at 80” and lose about 50 % viability after 30 min. Colony formation by an infected spore preparation is, if anything, stimulated by incubation at SO”. Infective center production by infected spores and colony formation by uninfected spores are hardly affected by incubation at 65”. DISCUSSION

We have described a system for the study of bacterial sporulation through the use of a phage which grows normally on log-phase cells but fails to infect sporulating cells productively. This system may provide information about the interplay between the control mechanisms of phage development and those of bacterial sporulation. Having confirmed that spores of B. subtilis can carry phage genomes, we established the existence of a critical period in the sporulation process during which this trapping phenomenon can occur. The trapping of phage was correlated with loss of ability of the phage to yield normal bursts and to induce detectable levels of phage-induced enzymes. A fourth phage-induced protein, the inhibitor of bacterial thymidylate synthetase, was still made after infection of sporulating cells. Whether this is indicative of a separation of phage functions or only of different sensitivity of their detection remains to be seen. In agreement with these enzyme studies, preliminary experiments using polyacrylamide gel electrophoresis indicate the absence of phage-specific proteins. However, the possibility that very small amounts of phage enzymes are induced, sufficient for some replication of phage DNA, is not excluded. The question of whether phage DSA is replicated after infection leading to phage trapping is currently under investigation. Phage-induced enzymes are not detectable in sporulating cells infected at a time when total protein synthesis is operating at a reasonable rate. This implies that phage proteins are specifically excluded. Whether phage expression is excluded by a mechanism which also represses vegetative functions in sporulating cells is one of the questions we aim to answer in future work.

SPORULATION

AND PHAGE

The study of infection at various times showed that phage trapping into spores occurs at a time when sporulating cells have not yet acquired the refractile polar body characteristic of prespores. Furthermore, the phage trapping phase precedes resistance to heat, octanol, and chloroform in the sequence of events in sporulation. In that sense, trapping is a relatively early event. It occurs at a time when the cells are sensitive to plating, producing colonies at low efficiency. The use of a nonsporulating mutant blocked before the production of prespores enabled us to link exclusion of phage growth with sporulation. This mutant supports substantial phage growth after many hours in stationary phase. This is correlated with the absence of other sporulation properties such as the sensitivity of cells to plating in nutrient agar. In addition, cultures of spr8 show less than 1% resistance to heat, chloroform, and octanol after 24 hours at 37”. Yet, the bacterial mutant produces both the antibiotic and the protease characteristic of sporulation. Asporogenic mutants deficient in these proteins are blocked at a very early step in sporulation (Balassa et al., 1963; Michel et al., 1968b). Thus exclusion of phage, as an event in the sporulation process, occurs after the earliest known functions but before the prespore stage. This agrees with our observation that trapping of phage is maximal 5-6 hours after the end of logarithmic growth and 1.5-2.5 hours before prespores appear. A reasonable prediction is that mutants which cannot produce either antibiotic or protease should be able to support phage growth whereas mutants that form prespores should exclude phage growth. The characterization of additional mutants should enable us to pinpoint the position of phage exclusion in the sequence of events of sporulation. We have found that at the 9.5- to 12.5hour stage prespores are very sensitive to phage infection. Even though no new phage are produced, up to 90% of the would-be spores are destroyed. This may be due to effects of the phage which have little to do with phage function, such as interference with spore coat assembly by the attachment

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of phage particles to the sporangial membrane. Experiments are in progress to determine whether phage functions are necessary for prespore killing. Note added in proof: Recent experiments (R. M. Losick and A. L. Sonenshein, to be published) indicate that during sporulation, the RNA polymerase of B. subtilis 3610 undergoes a change in template specificity that prevents transcription of @e DNA in an in vitro system.

ACKNOWLEDGMENTS We thank Dr. S. E. Luria for helpful guidance in this project and for assistance in preparing the manuscript. We also thank Dr. C. P. Georgopoulos for useful suggestions. The research reported here was supported by grants from the National Science Foundation (GB-5304X) and the National Institutes of Health (AI 03038) to Dr. S. E. Luria; one of the authors (ALS) is a predoctoral fellow of the National Institute of General Medical Sciences (5Fl-GM-34,47103). REFERENCES ADELBERG, E. A., MANDEL,

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in bact.eriophage-infected Bacillus subtilis. Virology 29, 157-166. SPIZIZEN, J. (1958). Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S. 44, 1072-1078. TAKAHASHI, I. (1964). Incorporation of bacteriophage genome by spores of Bacillus subtilis. J. Bacterial. 87, 1499-1502. TAMIR, H., and GILVARG, C. (1966). Density gradient centrifugation for the separation of sporulating forms of bacteria. J. Biol. Chem. 241, 1085-1090. THORNE, C. B. (1962). Transduction in Bacillus subtilis. J. Bacterial. 83, 106-111. WAHBA, A. J., and FRIEDKIN, H. (1962). The enzymatic synthesis of thymidylate. I. Early steps in the purification of thymidylate synthetase of Escherichia coli. J. Biol. Chem. 237, 3794-3801. YEHLE, C. O., and DOI, R. H. (1967). Differential expression of bacteriophage genomes in vegetative and sporulating cells of Bacillus subtilis. J. Viral. 1, 935-947.