VIROLOGY 26, 6‘&6‘i9 (1965)

Effect of Acridines and Temperature on a Strain of Bacillus

megaterium Lysogenic for Phage a’

R. RITA ARDITTI AND ANNA COPPO

International Laboratory of Genetics and Biophysics, Naples, Italy

Accepted January 11, 196’5

The unstable lysogenic strain B. megaterium (0~) exhibits the phenomenon of curing at different rates depending on the temperature. The frequency of lysis and the burst size also depend on the temperature. Lysogenic cultures show a higher rate of curing when grown in the presence of acridines than in their absence. To explain these facts, the favored hypothesis is that phage LY is a genetic element not integrated with the genome of the-host cell.

INTRODUCTION

Temperate phage, after infection of the bacterial cell, can either undergo reduction to the prophage state or enter into the vege- tative state, leading to lysis of the bacterium and the release of new phage particles.

In the first case a stable association is established between the bacteriophage and the bacterium, and the lysogenic system so formed behaves as a single reproductive unit.

In some cases, however, the lysogenic condition can be lost spontaneously. This phenomenon, called “curing,” has been shown to occur in B. megaterium (Clarke, 1952), in Corynebacterium diphtheriae (An- derson and Cowles, 1958), and in Salmonella typhi (Felix and Anderson, 1951). Recently it has been studied in the lysogenic system formed by B. megaterium and temperate phage a (Donini and Guerrini, 1962). These authors have followed the dynamics of curing for a large number of generations and have established the rate of curing per cell division. At 35°C the curing rate is 0.02- 0.03, while at 18” it is about ten times lower: 0.003-0.004. They have shown curing to be a continuous process which takes place during bacterial multiplication and is influenced by temperature.

1 This work has been supported by EURATOM- C.N.R.C.N.E.N. Contract no. 012-61-12 BIAI.

One of the hypotheses put forward to explain curing in this system was that phage (Y behaved in a manner similar to that of a cytoplasmic particle with a high degree of independence from the genetic structure of its host. In this case, curing could be as- cribed to the statistical distribution of the particles in the progeny. A similar situation is that existing in stable lysogenic systems in the stage that follows infection and pre- cedes definite lysogenization. There is a transitory unstable condition, a so-called “preprophage” state, which has been de- scribed by Lieb (1953) and by Luria (1959). However, in our case no stable lysogenic condition is attained. Curing could also be attributed to lack of homology between phage CY and the host chromosome.

Phage (Y has some peculiar properties that could very well be the basis for the dissimi- larity of the genetic structures of the phage and of the host. In fact the densities of phage (Y DNA and its host DNA are different whereas in a classic lysogenic system, such as X and E. coli, bot’h DNA’s have the same densities (Schildkraut et al., 1962). Native phage cr DNA in the CsCl density gradient has a density of 1.705 g/cm3 while B. mega- terium DNA has a density of 1.695 g/cm3 (Graziosi, personal communication). Fur- thermore, heat denaturation of phage (Y DNA at 100” followed by fast cooling causes

643

644 ARDITTI ANI) COPPO

two components of different densities to appear in CsCl densitly gradient’ cent,rifuga- tion. It is assurned t,hat t,he t’wo bands ob- served correspond to the two strands of the DNA, differing in nucleotide composition (Aurisicchio et al., 1961). B. megate&um D?;A, however, does not show t,his behavior after denaturat’ion and fast cooling (Aurisic- chio, personal communication). In this paper new evidence favoring the cytoplasmatic hypot’hesis is presenbed. Acridines have been used to test their effect on the lysogenic system. Differential production of free phage by lysogenic cells at different t>empera- tures has been observed, giving further sup- port to this hypothesis.

MATERIALS AND METHODS

Phage and bacteria. The following strains were used:

Temperate phage (Y and its host, Paris strain of B. megaterium. For a detailed description of the phage and bacteria, see Donini and Guerrini (1962).

The lysogenic strain formed by temperate phage CY and its host strain: B. megaterium (4.

Lysogeny in t’he case of B. megaterium (CY) has been inferred from the following observations (Donini and Guerrini, 1962; Donini, personal communication) : (1) The cells were still lysogenic after four consecu- tive transfers in brot’h plus antiphage serum. (2) xv0 loss of lysogenicity was observed when spores obtained from a lysogenic cul- ture were heated for 10 minutes at 80” (t.his treatment eliminates vegetative cells and free phage). (3) The strain could be induced wit’h UV, X-rays, and mitomycin.

B. megateriuna (a) grown in a broth cul- ture liberates phage into the medium. Phage CII adsorbs very poorly in standard liquid media; this fact made it easy to avoid re- infection during our experiments.

A&dine solutions. Two acridine dyes, acriflavine (AC) (Calbiochem, mixture of 2,8-diamino-lo-methyl acridinium chloride and 2,8-diaminoacridine) and acridine or- ange (AO) (Gurr’s, 2, g-bisdiamethyl- aminoacridine) were used. Stock solutions containing 1 and 10 mg/ml of the dyes in

broth were sterilized and st>ored in the dark al 4” for periods up to 3 months.

Media. All media had the following con-

position per liter: KaCl, 5 g, Costa&lo peptone, 10 g; and Costant,ino meat extract, 10 g. In addition, the bottom layer agar contained per liter 15 g of Costantino agar, and the overlay agar, 10 g of the Costantino agar.

Spore suspensions. Stocks of lysogenic spores were prepared by growing a lysogenic culture to lo* cells/ml. Cells were centri- fuged, resuspended in saline solution, and kept, at 37”. After 24 hours prospores could be seen in most] of the cells; after 48 hours spores were formed. The suspension was again centrifuged, concentrated to the de- sired tit,er, heated at 80” for 10 minutes to eliminate free phage and vegetative cells, and kept’ at -20” in 50 %;’ glycerol.

Acridine method. A standard inoculum of lysogenic spores (lo6 spores per milliliter) was made in brot)h containing per milliliter A0 at, 1 pg, 10 pg, and 20 pg; or AC at 0.75 pg and 1 pg. Cultures were grown to about lo* cells per milliliter by aeration at 37°C for 5-6 hours. The suspensions were appropri- ately diluted, plated, and teshed as described below for t’he number of lysogenic and cured cells.

Control cultures. Whenever an experiment was made with acridines, a similar inoculum was made in normal broth to check the rate of spontaneous curing under similar condi- tions.

Assay for lysogenic and cured colonies. In order to distinguish between lysogenic and cured colonies, the replica plating tech- nique wit’h velvet, was used. Cells were plated on petri dishes, each of which had a cardboard cover tjo diminish the humidity and allow uniform growth of the colonies. Plates were incubated overnight at 30” and then were replicated on plates with t*he a-sensitive indicator. After 4-5 hours the distinction between lysogenic and cured colonies was already clear cut. Lysogenic colonies had a transparent halo due to the lysis of the sensitive strain by the phage particles liberated during growth. Cured colonies lacked such a halo.

Determination of the frequency of lysis and

EFFECT OF ACRIDINES ON R. MEGATERIUM (a) 645

TABLE 1

EFFECT OF ACRIDINE DYES ox CURING OF B. ~~g~~~r~u?~ (m) AT 37°C~

Treated Control Expt. No.

DF Dose Cured/lysogenic %

Wml) colonies Cured Cured/lysogenic %

colonies Cured

I AC 0.75 935/1884 49.6 72412024 35.7 II AC 1 1143/1576 72.5 2771779 35.5

III A0 1 650/1542 42.1 948/2380 39.8 IV A0 10 581,‘846 68.6 948/2380 39.8 V A0 20 738/892 82.7 646/1657 38.9

a For each experiment, a value is given for a cont.rol culture grown under similar conditions but with no acridine. AC, acriflavine; AO, acridine orange. Aerated cultures containing different doses of the dyes were grown to about W/ml and tested for lysogenic and cured colonies, using the replica plating technique (see Materials and Methods).

the burst size. To determine the frequency of lysis of lysogenic cells, the following procedure was followed: The spores or vegetative cells from a culture in exponential phase were distributed in about, a hundred tubes, 5-10 per tube, and the free phage was eliminated by centrifugation. Ten tubes were plated with and 10 tubes without the sensitive strain at, zero time in order to count infective centers and the total num- ber of cells. The tubes were t,hen incubated at 22” or 37” to allow for a few bacterial multiplications to take place, and t,hen 10 tubes were plated for bacterial count,s and the rest with sensit,ive sl-rain. It, was t,hen possible to calculate t,he frequency of lysis (per cell division) from the number of plates that did not show any burst (Bertani, 1951). Application of the Poisson distribution gave m (mean number of bursts for each tube). The rat’io between nt and the number of divisions that had taken place gave the num- ber of bursts per cell division. The mean burst, size was obtained from the relation between the total number of plaques (minus infect,ive centers) and the tot,al number of bursts.

RESULTS

Efect of the A&dine Treatment

Table 1 shows that after growth in the presence of acridines at 37’, the number of cured colonies was significantly greater than in controls (see also Donini and Guerrini, 1962).

0.7

0.6

--I 0.5 +A u I

B 0.4 -I

0.3

of A0

0.2

Without A0

I ! / i 0 t 2 3

TIME OF SAMPLING IN “OURS

FIG. 1. C = cured cells; L = lysogenic cells; Temper&me, 37°C. Logarithms to the base 10 have been used.

The effect of acridine orange as a function of time was followed. Samples were taken each hour for 4 hours and the number of lysogenic and cured colonies was determined for each sample. The results are shown in Fig. 1.

From the resuIts of these experiments it is possible to calculate the rat.e of curing per cell per division, p. The equation used is that proposed by Shapiro (1946) for the accumulation of mutants in a population of wild-type cells. The equation can be sim- plified in the case of curing because one can safely assume t,hat the growth rat,e of the two kinds of cells is the same (see later).

646 ARDITTI ANI) COPPO

TABLE 2

EFFECT OF ACRIDINE DYES ON THE R.ITE OF CTXIXG OF R. megateriunl (a) AT 37°C”

Treated Control Expt. No.

Dye Dose bdml) P 6 x- P 6 iv

I AC 0.75 0.07 22.32 8.OG 0.03 21.78 8.26 II AC 1 0.18 22.32 8.06 0.03 19.86 9.06

III A0 1 0.04 19.08 9.43 0.03 18.00 9.9G IV A0 10 0.13 19.44 9.25 0.03 18.06 9.9G V A0 20 0.28 24.42 7.37 0.03 19.20 9.37

VI A0 20 0.21 25.20 7.14 0.03 17.40 10.34

D AC, acriflavine; AO, acridine orange; p, rate of curing; 6, division time, in minutes; N, number of duplications that have taken place. See legend of Table 1. Same cultures as Table 1.

This way of representing our data is valid only if we assume that only one particle (integrated or not) is present per cell. It is of course possible Do formulate models which do not have formal analogy with the spon- taneous mutation process. The equation has already been used by Donini and Guer- rini and can be presented in the following form :

Ct -- z-z e Pkt

Lt ( >

2+1 -1 0

in which Ct and Lt are the number of cured and lysogenic cells respectively at time t; Co and Lo are the number of cured and lyso- genie cells respectively at time 0; k is the growth rate, t is the time in hours; p is the rate of curing per cell division.

Table 2 gives the values of p for experi- ments done with acridines and with control cultures. The value of p for t’he control cultures is 0.03 as expected for a temperature of 37”. It is seen that the rate of curing in- creases with concentration of the dye. For AC, it becomes 0.07 and 0.18. For AO, it reaches the value of 0.28 in a particular experiment, using a dose of 20 kg/ml. Higher doses of AC and A0 are inhibitory for growth of the cultures.

The increase in the number of cured cells after growth in the presence of acridines might be attributed, in principle at least, to a selective effect that could favor the cured cells already present in the population. To test this possibility, reconstruction experi- ments were carried out with the sensitive and the lysogenic strains grown in t’he presence

of acridines. No difference in the growth rate was observed.

Production of Phage by Lysogenic Cultures

In the course of the experiments on curing, the production of free phage by t’he lysogenic cultures was followed. A new fact came out of these observations: the production of free phage by the lysogenic cultures was dependent on temperature. The production of phage was followed, in part’icular, at 22” and 37” (see Figs. 2 and 3).

It is seen, for instance, that when there are about lo6 cells per milliliter, the free phage in the supernatant is about 3 X 107/ ml if the culture had been grown at 22”; but the free phage is only 105/ml if growth had been at 37”. Reconstruction experi- ments have shown that LX is not, inactivated at 37”.

These differences in the product’ion of free phage by lysogenic cultures could be due either to a different frequency of lysis or to a difference in the burst size.

Frequency of Lysis and Burst Size

The frequency of lysis of the lysogenic cells was studied under two different con- ditions: (A) cells obtained by germination of lysogenic spores; (B) cells obtained from a lysogenic culture in exponential phase. Table 3 shows that the results obtained under both conditions were almost the same, and in each case the frequency of lysis was higher at the lower temperature.

The burst size was about 600 under both conditions, except for when the cells were

EFFECT OF ACRIDINES ON B. MEGATERIUM (a) 647

obtained from a lysogenic culture in exponential phase at 22”. In that case the burst size was about 1500.

The increase in the production of free

lo9 PHAGE Ci -i; 108 &

E 107 _

g 106 - 5 105 E ,TP

0

= loL BACTERIA

is 103- 22’C

g 102-

10’ I 1 I 5 1 123456789

TIME IN HOURS

FIG. 2. Production of phage during growth of B. megaterium (a) at 22°C. The cultures were in- oculated with lysogenic spores. The free phage was measured by titration of the supernatant of twice- centrifuged samples. A small crystal of thymol was added to kill residual bacteria and prevent contamination.

BACTERIA

TIME IN HOURS

FIG. 3. Production of phage during growth of B. megateri?Lrn (01) at 37°C. See for details the legend of Fig. 2.

phage at 22’ is therefore due to both a higher burst size and a higher frequency of lysis, since condition (B) was the usual one used in our experiments.

Experiments at Higher Temperatures

When lysogenic cultures were grown at 39”, 40”, and 42”, we observed that the rate of curing increased and t’he titer attained by free phage decreased in relation to the temperature. The results are summarized in Table 4. Controls showed no inactivation of phage at the temperature used.

DISCUSSION

The above data show that the relation of phage Q to its host is peculiar. No stable lysogenic strain has ever been isolated; the unstable lysogenic strain shows t’he phenom- enon of curing at different rat’es depending on the temperature; the rates of spontaneous curing vary from 0.003 at 18” to 0.217 at 42”.

The production of free phage in the lyso- genie cultures shows an inverse relation to the curing rate: it diminishes with the in- crease of temperature. In two particular cases, at 22” and at 37”, this difference in the production of free phage has been shown to arise from a difference in t’he frequency of lysis and in burst size.

The instability of a! in the intracellular stage can be attributed either to a high frequency of spontaneous detachment from the bacterial chromosome (prophage hy- pothesis) or to a behavior similar to that of an element not integrabed with the genome

TABLE 3

FREQUENCY OF LYSIS (PER CELL DIVISION) AND MEAN BURST SIZE OF B. megaterium (a) AT 22°C AND 37°C”

Expt. no. Temperature Number of Zero event9 Number of divi- Bursts per cell Mean burst (“Cl tubes sions per tube division size

I 22 78 17 50 l/33 600 II 22 50 6 231 l/110 1400

III 22 50 4 399 l/158 1600 IV 37 100 85 228 l/1400 690 V 37 78 75 26 l/660 620

0 Experiments I and V were done with cells obtained by germination of spores; experiments II, III, and IV with cells obtained from a lysogenic culture in exponential phase.

b Zero events = the number of plates that do not show any burst. These data refer only to lysogenic cells; correction has been made for cured ones.

648 ARDITTI AKD COPPO

TABLE 4 EXPERIMENTS AT HIGHER TEMPERATURES

Temperature e-2

Free phage when cells are 10B/ml

39 0.05 9 x 105 40 0.08 1 x 105 42 0.21 6 x 104

aF = rate of curing. Broth cultures were inoculated with Iysogenic spores of B. megaterium (a). The aerated cultures were grown up until about 108 cells/ml at different temperatures; then the cells were plated and the resulting colonies were tested for lysogenicit,~ {see Materiafs and Met~iods) .

of the host cell; curing should be the con- sequence of the loss of such an element. One could also imagine a situation where several copies of the phage genome are present in the cell cytopiasm. Curing in this last case could be attributed merely t,o a statistical distribution of the phage genomes among the progeny cells. It, is obvious that, if so, the mathematical representation adopted by us would be of a very dubious validity.

The fact that lysogenic cultures grown in the presence of acridines show a higher rate of curing suggests that the “nonintegration” hypothesis is the more plausible. In other systems acridines are known t,o affect, cyto- plasmic particles: respiratory factors in yeast (Ephrussi, 1953) and the F fact’or in $7. coli (Hirota, 1960) as examples. When the F factor is bound to the chromosome in Hfr strains, acridines are not effective in eliminating it. Recently, in the E. co& K12- Pl dE system (Luria et al., 1960), it, was found that out of eleven strains of E. coti carrying 1’1 dl phages that were tested, one strain, 30S0 (PI dl 13-4), could be cured of the prophage by A0 treatments (Amati, 1965). There are indications that’ Pl dZ 13-4 may have an extrachromosomal location (Boice and Luria, 1963).

The results obtained with Iysogenic cul- tures at different tem~ratures can be ~terpre~ in the following way: in some way the replicative system of phage DNA is favored at lower temperatures resulting in a higher frequency of lysis than at higher temperatures.

In other words, the two phenomena, wing and production of free phage would be cor- related and be due basically to the same mechanism. At higher temperatures the replication of phage (Y USA would not keep up in pace w&h replication of bacterial DNA, resulting in greater curing. At, t,he same time these would be a decrease in the production of free phage.

At lower temperatures, phage and bac- terial DNA’s would replicate at the same rate, resulting in less curing, and t’he pro- duction of free phage would be enhanced.

If we regard a as an element not ~tegrated into the genome of its host, the problem arises as to the number of copies of such a particle that could be present in the host cell. Further work is necessary to elucidate t,his very important aspect of the relation- ships between (Y genome and its host.

Professor F. Graziosi’s guidance and interest throughout this investigation are gratefully ac- knowledged. Drs. P. Donini and M. Terzi are thanked for many suggestions and helpful dis- cussions.

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