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Direct induction of prophage lambda by spheroplast formation and indirect induction by spheroplasts
J. Mol. Biol. (1967) 29, 527-530
Direct Induction of Prophage Lambda by Spheroplast Formation and Indirect Induction by Spheroplasts In our previous work on direct and indirect induction of prophage lambda by 5-fluorouracil (Zgaga & Novak, 1967) we were able to conclude that indirect 5-fluorouracil induction depends neither upon the transfer of chromosomal DNA, nor upon the transfer of the sex factor F, but that it results from cell-wall contact between 5-fluorouracil-treated non-lysogenic and untreated lysogenic bacteria. The treatment was carried out with 100 pg 5-fluorouracil/ml., which interfered with cell-wall synthesis (Tomasz & Borek, 1959,196O). During further studies of prophage induction, we observed that direct induction of prophage lambda in Escherichia wli K12 can be obtained by treatment with lysozyme and EDTA which leads to spheroplast formation. Indirect induction is achieved when spheroplasts from either male or female non-lysogenic bacteria are mixed with untreated lysogenic bacteria of the opposite sex. Thus, this type of indirect induction presents the same main features as that described in our earlier paper (Zgaga & Novak, 1967). In the present work we used the following strains: W677: F-thr-leu-thi-hr non-lysogenic, called F-(h)-, and W677(X), called F-(h) +, both from Dr E. Wollman; W1817(h): F+thr-leu-thi-h=(h), called F+(h)+ from Dr E. Borek; K12SX from Dr E. Calef, called F+(h) - ; HfrC(h)Xmet - called Hfr(h)-, and HfrChRmet - called Hfr(h)-, both from Dr Wollman; and K12SXS, called K12S, from Dr R. Devoret. MA isotonic liquid medium plus 10% Tryptone broth and 1% nutrient brot,h, supplemented with amino acids and vitamin for auxotrophs (Zgaga & Novak, 1967) was used for bacterial growth. For experiments of direct induction, the female or male lysogenic culture in the logarithmic phase of growth was washed twice with MA medium by centrifugation at 3500 rev./min for ten minutes and divided into two parts. One part (control) was resuspended in SP hypertonic medium (Ryter & Jacob, 1966) supplemented as the MA medium, and the second part (treated) in the same medium plus lysozyme and EDTA (Ryter & Jacob, 1966), and incubated at 37°C in a very thin layer without bubbling. According to Ryter & Jacob, spheroplast formation in this medium is obtained after two hours. Microscope examination proved that, under our conditions, after three hours of incubation at 37”C, about 80% of treated bacteria were transformed into spheroplasts. The kinetics of direct induction of female and male lysogenic strains by spheroplast formation in the hypertonic medium is presented in Fig. 1. For phage assay, TP agar and top TP agar (Zgaga & MiletiE, 1965) were used. For experiments on the indirect induction of lysogenic bacteria by spheroplasts from non-lysogenic cells, we could not apply the treatment described for direct induction, because of the permanent presence of lysozyme and EDTA in the medium. For indirect induction, spheroplasts from non-lysogenic male or female cells were made also by lysozyme and EDTA but in a different way (Zgaga, 1967). These spheroplasts were washed twice with hypertonic SP medium and resuspended in 537
DJ.
528
NOVAK,
S. TKALAC
I
0
60
120
180
240
60
(a)
FIQ. 1. Direct
induction
of prophage
I
AND
I
V. ZGAGA
I
I20 180 240 Tome (b)
I
I
I
0
60
I20
lambda by spheroplast
I
I80
240
Cc)
formation.
Release of infective X particles from the control and treated lysogenic cultures. (a) F - (X) + . (b) F+(X)+. (c) Hfr(X)+. In the logarithmic phase of growth the lysogenic culture is washed twice with MA medium and divided into two parts. One part, the control (-o-m--), is resuspended in SP hypertonic in the same medium plus 20 pg lysozyme/ml. and medium, and the second part (-O--O-) 0.004 g EDTA/l. (in Fig. 1, 0 mm). The cultures were incubated at 37’C without bubbling. At different time intervals from the beginning of the treatment, we determined the number of free h phages, after subjection to chloroform for 30 mm, with K12S as sensitive strain.
fresh SP medium. Examination under a microscope showed that most spheroplasts lysed, although held in the hypertonic medium, after about 120 minutes. At the same time that spheroplasts from non-lysogenic bacteria were washed, female or male lysogenic cells and non-lysogenic untreated cells were also washed. Spheroplasts and untreated bacteria were resuspended in a ratio 1 male : 5 females in SP hypertonic medium, and a very thin layer of bacterial suspension was incubated at 37°C without bubbling. Figure 2 represents results of experiments in which F+ or Hfr non-lysogenic cells were transformed into spheroplasts and mixed with F- untreated lysogenic bacteria. In Fig. 3 the results of experiments in which F- non-lysogenic cells were transformed into spheroplasts and mixed with untreated lysogenic F+ or Hfr cells are shown. As previously described (Zgaga & Novak, 1967), in this type of experiment the Hfr bacteria used did not bring about zygotic induction after mating with F-(h)bacteria because of the very distant position of the prophage. In experiments on the indirect induction by spheroplasts, we had three types of control experiments. In the f&t control, indicated in Figs 2 and 3, results from the mating between untreated lysogenic and untreated non-lysogenic cells of the opposite sex are shown. The second control experiment was needed to verify the possibility that very small amounts of lysozyme and EDTA, which might have been present in the medium even after two washings of the spheroplasts, could achieve the induction
LETTERS
TO THE
EDITOR
529
Time (mm)
(a) Fra.
2. Indirect
(b)
induction
by spheroplasts.
Release of infective h particles after mating of female lysogenic with the control bacteria and spheroplasts from non-lysogenic male cells. (a) F+(X)- x F-(A)+. (b) Hfr(X)- x F-(h)+. In the logarithmic phase of growth, spheroplasts from non-lysogenic male bacteria were made by lysozyme and EDTA (as indicated in the text). Spheroplasts were washed twice with SP medium and resuspended in fresh SP medium. At the same time, female lysogenic and male non-lysogenic untreated bacteria were washed and resuspended into SP medium. Mixing in the ratio 1 male : 5 females was done (in Fig. 2, 0 min), and a very thin layer of bacterial suspension was incubated at 37°C without bubbling. Mating: control male bacteria x lysogenic female (-e--e-); spheroplasts from male bacteria x lysogenio female (--O-O-). Determination of the number of free h phages was done as in Fig. 1.
of the prophage in lysogenic cells. Therefore we investigated the inducing effect of 240 minutes incubation of lysogenic cells in a liquid medium obtained in the following way: (1) after two washings of spheroplasts with SP medium, resuspension in fresh SP medium and centrifugation; (2) after incubation of washed spheroplasts in hypertonic medium for (a) 30 minutes, (b) 60 minutes, (c) 90 minutes and centrifugation. The results of these experiments were totally negative. Suspension of lysogenic bacteria to which media described under (1) or (2) were added in the ratio described above, 1: 5 or 5 : 1, depending on whether male or female bacteria had been incubated in it, behaved, with respect to spontaneous induction, like the control lysogenic culture. This indicates that, at least up to 90 minutes after mixing, the observed increase in the number of free lambda phages is not the consequence of the possible release of lysozyme or EDTA from spheroplasts. In the third control experiment, we mixed non-lysogenic spheroplasts with untreated lysogenic bacteria of the same sex. The results of these experiments were also negative. This supports the view that the observed phenomenon was in fact cross-induction, effected by specific contact. From the data presented above we conclude that spheroplast formation by lysozyme and EDTA induces the development of the prophage h in the lysogenic bacteria
DJ.
530
NOVAK,
S. TKALAC
I
0
I
I
/
I
I
I
I
60
120
180
240
AND /
I 0 60 TrmeCmin)
(8)
V. ZGAGA I
I
/
I
I
I
120
180
240
_
(b)
FIG. 3. Indirect
induction
by spheroplasts.
Release of infective h particles after mating of male lysogenic with the control bacteria and spheroplasts from non-lysogenic female cells. (a) F-(h)- xF’+(A)+. (b) F-(A)- xHfr(X)+. The treatment is the same as that described in Fig. 2, with the exception that non-lysogenic treated bacteria are F-, and lysogenic untreated F+ or Hfr. Mating: control female x lysogenic male (-@-a-); spheroplasts from female bacteria x lysogenic male (--O-O-).
used. In the case of indirect induction, male or female non-lysogenic spheroplasts are equally potent in provoking the development of X phages in lysogenic bacteria of the opposite sex. We thank Miss M. Taiber discussion and for critically
for able technical assistance, reading the manuscript.
31 July
SLAVKO TKALAC VERA ZUAGA
1967 REFERENCES
Ryter, Tomasz, Tomasz, Zgaga, Zgaga, Zgaga,
for helpful
D JURDJANOVAK
Laboratory of Cellular Radiobiology Institut “Rudjer Boikovib” Zagreb, Yugloslavia Received
and Dr M. DrakuliC
A. & Jacob, F. (1966). Ann. Inet. Pasteur, 110, 53. A & Borek, E. (1959). Proc. Nat. Acad. Sci., Wash. 45, 929. A. & Borek, E. (1960). Proc. Nut. Acad. Sci., Wash. 46, 324. V. (1967). Virology, 31, 559. V. & Miletio, B. (1965). Virology, 27, 205. V. & Novak, DJ. (1967). J. Mol. Biol. 29, 125.
Direct Induction of Prophage Lambda by Spheroplast Formation and Indirect Induction by Spheroplasts In our previous work on direct and indirect induction of prophage lambda by 5-fluorouracil (Zgaga & Novak, 1967) we were able to conclude that indirect 5-fluorouracil induction depends neither upon the transfer of chromosomal DNA, nor upon the transfer of the sex factor F, but that it results from cell-wall contact between 5-fluorouracil-treated non-lysogenic and untreated lysogenic bacteria. The treatment was carried out with 100 pg 5-fluorouracil/ml., which interfered with cell-wall synthesis (Tomasz & Borek, 1959,196O). During further studies of prophage induction, we observed that direct induction of prophage lambda in Escherichia wli K12 can be obtained by treatment with lysozyme and EDTA which leads to spheroplast formation. Indirect induction is achieved when spheroplasts from either male or female non-lysogenic bacteria are mixed with untreated lysogenic bacteria of the opposite sex. Thus, this type of indirect induction presents the same main features as that described in our earlier paper (Zgaga & Novak, 1967). In the present work we used the following strains: W677: F-thr-leu-thi-hr non-lysogenic, called F-(h)-, and W677(X), called F-(h) +, both from Dr E. Wollman; W1817(h): F+thr-leu-thi-h=(h), called F+(h)+ from Dr E. Borek; K12SX from Dr E. Calef, called F+(h) - ; HfrC(h)Xmet - called Hfr(h)-, and HfrChRmet - called Hfr(h)-, both from Dr Wollman; and K12SXS, called K12S, from Dr R. Devoret. MA isotonic liquid medium plus 10% Tryptone broth and 1% nutrient brot,h, supplemented with amino acids and vitamin for auxotrophs (Zgaga & Novak, 1967) was used for bacterial growth. For experiments of direct induction, the female or male lysogenic culture in the logarithmic phase of growth was washed twice with MA medium by centrifugation at 3500 rev./min for ten minutes and divided into two parts. One part (control) was resuspended in SP hypertonic medium (Ryter & Jacob, 1966) supplemented as the MA medium, and the second part (treated) in the same medium plus lysozyme and EDTA (Ryter & Jacob, 1966), and incubated at 37°C in a very thin layer without bubbling. According to Ryter & Jacob, spheroplast formation in this medium is obtained after two hours. Microscope examination proved that, under our conditions, after three hours of incubation at 37”C, about 80% of treated bacteria were transformed into spheroplasts. The kinetics of direct induction of female and male lysogenic strains by spheroplast formation in the hypertonic medium is presented in Fig. 1. For phage assay, TP agar and top TP agar (Zgaga & MiletiE, 1965) were used. For experiments on the indirect induction of lysogenic bacteria by spheroplasts from non-lysogenic cells, we could not apply the treatment described for direct induction, because of the permanent presence of lysozyme and EDTA in the medium. For indirect induction, spheroplasts from non-lysogenic male or female cells were made also by lysozyme and EDTA but in a different way (Zgaga, 1967). These spheroplasts were washed twice with hypertonic SP medium and resuspended in 537
DJ.
528
NOVAK,
S. TKALAC
I
0
60
120
180
240
60
(a)
FIQ. 1. Direct
induction
of prophage
I
AND
I
V. ZGAGA
I
I20 180 240 Tome (b)
I
I
I
0
60
I20
lambda by spheroplast
I
I80
240
Cc)
formation.
Release of infective X particles from the control and treated lysogenic cultures. (a) F - (X) + . (b) F+(X)+. (c) Hfr(X)+. In the logarithmic phase of growth the lysogenic culture is washed twice with MA medium and divided into two parts. One part, the control (-o-m--), is resuspended in SP hypertonic in the same medium plus 20 pg lysozyme/ml. and medium, and the second part (-O--O-) 0.004 g EDTA/l. (in Fig. 1, 0 mm). The cultures were incubated at 37’C without bubbling. At different time intervals from the beginning of the treatment, we determined the number of free h phages, after subjection to chloroform for 30 mm, with K12S as sensitive strain.
fresh SP medium. Examination under a microscope showed that most spheroplasts lysed, although held in the hypertonic medium, after about 120 minutes. At the same time that spheroplasts from non-lysogenic bacteria were washed, female or male lysogenic cells and non-lysogenic untreated cells were also washed. Spheroplasts and untreated bacteria were resuspended in a ratio 1 male : 5 females in SP hypertonic medium, and a very thin layer of bacterial suspension was incubated at 37°C without bubbling. Figure 2 represents results of experiments in which F+ or Hfr non-lysogenic cells were transformed into spheroplasts and mixed with F- untreated lysogenic bacteria. In Fig. 3 the results of experiments in which F- non-lysogenic cells were transformed into spheroplasts and mixed with untreated lysogenic F+ or Hfr cells are shown. As previously described (Zgaga & Novak, 1967), in this type of experiment the Hfr bacteria used did not bring about zygotic induction after mating with F-(h)bacteria because of the very distant position of the prophage. In experiments on the indirect induction by spheroplasts, we had three types of control experiments. In the f&t control, indicated in Figs 2 and 3, results from the mating between untreated lysogenic and untreated non-lysogenic cells of the opposite sex are shown. The second control experiment was needed to verify the possibility that very small amounts of lysozyme and EDTA, which might have been present in the medium even after two washings of the spheroplasts, could achieve the induction
LETTERS
TO THE
EDITOR
529
Time (mm)
(a) Fra.
2. Indirect
(b)
induction
by spheroplasts.
Release of infective h particles after mating of female lysogenic with the control bacteria and spheroplasts from non-lysogenic male cells. (a) F+(X)- x F-(A)+. (b) Hfr(X)- x F-(h)+. In the logarithmic phase of growth, spheroplasts from non-lysogenic male bacteria were made by lysozyme and EDTA (as indicated in the text). Spheroplasts were washed twice with SP medium and resuspended in fresh SP medium. At the same time, female lysogenic and male non-lysogenic untreated bacteria were washed and resuspended into SP medium. Mixing in the ratio 1 male : 5 females was done (in Fig. 2, 0 min), and a very thin layer of bacterial suspension was incubated at 37°C without bubbling. Mating: control male bacteria x lysogenic female (-e--e-); spheroplasts from male bacteria x lysogenio female (--O-O-). Determination of the number of free h phages was done as in Fig. 1.
of the prophage in lysogenic cells. Therefore we investigated the inducing effect of 240 minutes incubation of lysogenic cells in a liquid medium obtained in the following way: (1) after two washings of spheroplasts with SP medium, resuspension in fresh SP medium and centrifugation; (2) after incubation of washed spheroplasts in hypertonic medium for (a) 30 minutes, (b) 60 minutes, (c) 90 minutes and centrifugation. The results of these experiments were totally negative. Suspension of lysogenic bacteria to which media described under (1) or (2) were added in the ratio described above, 1: 5 or 5 : 1, depending on whether male or female bacteria had been incubated in it, behaved, with respect to spontaneous induction, like the control lysogenic culture. This indicates that, at least up to 90 minutes after mixing, the observed increase in the number of free lambda phages is not the consequence of the possible release of lysozyme or EDTA from spheroplasts. In the third control experiment, we mixed non-lysogenic spheroplasts with untreated lysogenic bacteria of the same sex. The results of these experiments were also negative. This supports the view that the observed phenomenon was in fact cross-induction, effected by specific contact. From the data presented above we conclude that spheroplast formation by lysozyme and EDTA induces the development of the prophage h in the lysogenic bacteria
DJ.
530
NOVAK,
S. TKALAC
I
0
I
I
/
I
I
I
I
60
120
180
240
AND /
I 0 60 TrmeCmin)
(8)
V. ZGAGA I
I
/
I
I
I
120
180
240
_
(b)
FIG. 3. Indirect
induction
by spheroplasts.
Release of infective h particles after mating of male lysogenic with the control bacteria and spheroplasts from non-lysogenic female cells. (a) F-(h)- xF’+(A)+. (b) F-(A)- xHfr(X)+. The treatment is the same as that described in Fig. 2, with the exception that non-lysogenic treated bacteria are F-, and lysogenic untreated F+ or Hfr. Mating: control female x lysogenic male (-@-a-); spheroplasts from female bacteria x lysogenic male (--O-O-).
used. In the case of indirect induction, male or female non-lysogenic spheroplasts are equally potent in provoking the development of X phages in lysogenic bacteria of the opposite sex. We thank Miss M. Taiber discussion and for critically
for able technical assistance, reading the manuscript.
31 July
SLAVKO TKALAC VERA ZUAGA
1967 REFERENCES
Ryter, Tomasz, Tomasz, Zgaga, Zgaga, Zgaga,
for helpful
D JURDJANOVAK
Laboratory of Cellular Radiobiology Institut “Rudjer Boikovib” Zagreb, Yugloslavia Received
and Dr M. DrakuliC
A. & Jacob, F. (1966). Ann. Inet. Pasteur, 110, 53. A & Borek, E. (1959). Proc. Nat. Acad. Sci., Wash. 45, 929. A. & Borek, E. (1960). Proc. Nut. Acad. Sci., Wash. 46, 324. V. (1967). Virology, 31, 559. V. & Miletio, B. (1965). Virology, 27, 205. V. & Novak, DJ. (1967). J. Mol. Biol. 29, 125.