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Thymine-requiring mutants of the insect pathogen Bacillus thuringiensis
JrWANAL OF IZVEHTEBRATE PATHOLOGY 16, 321-324 (19701
Thymine-Requiring
Mutants
of the Insect
Pathogen
Bacillus
dzudngiensk
H. DE BARJAC Service des Vaccins
Bactiriens,
Institut
Received December
Pasteur,
Paris,
France
8, 1969
By action of aminopterin with thymine in the medium, auxotrophic thymine-requiring mutants of Bacillus thuringiensis var. thuringiensis have been obtained. The study of one of these mutants (AM 28 t- ) shows that its optimal growth requires thymine or thymidine at a concentration of 20 ug/ml. Thymine starvation produces very long, filamentous cells with noticeable lysis and no further DNA-synthesis. Mixtures of other ribosides, such as adenosine + guanosine or uridine + cytidine, and of vitamins, such as folic acid or thiamine, cannot be substituted for thymine. By incubation at 30°C without thymine, this mutant progressively loses its viability, which decreases by about 1 X 103 cells within 8 hr. A minimal dose of 2 &ml thymine can afford total survival of cells. The prototrophic, wild-type strain is fully inhibited by aminopterin at 2 X 10-4 M, but its thymine-requiring mutant grows well with aminopterin at 5 X or 7 X lo-* M. The results of this study show many similarities with the phenomenon of thymineless death which have been described in mutants of Escherichia coli and of B&i&s megaterium.
method of Okada, as modified by Wachsman et al. ( 1964), was applied to these Thymineless mutants of Escherichia CO& mutants with the following changes. and Salmonella typhimurium have been The basal medium for growing B. isolated by Okada et al. (1960, 1962) by thuringiensis contained: P0.1H2K, 6.8 g; action of aminopterin. Stacey and Simson S04Mg.7Hs0, 0.123 g; S04Mn*4Hz0, (1965) obtained the same results with E. 0.002 g; S01Zn*7H20, 0.014 g; (SOa):$Fez, coli by using such diaminopyrimidines as 0.02 g; C1&a*4Hz0, 0.183 g; casamino The acids ( Difco), 10 g; glucose, 5 g; and dispyrimethamine and trimethoprim. method of Okada with aminopterin has tilled water, 1 liter. The pH was adjusted been also successfully applied to Bacillus to 7.4. For selecting thymineless mutants, megaterium (Wachsman et al. 1964) and this medium was supplemented by 200 to Bacillus subtilis (Farmer and Rothman, lig/rnl of aminopterin and 200 rig/ml of 1965). Reported herein is the effect of ami- thymine. All the cultures were made at nopterin on B. thuringiensis. 30°C with shaking. After incubation for 4 days, the cultures were diluted and plated on basal agar plates (basal medium with MATERIALS, METHODS, AND DISCUSSION 1.5% D&o agar) supplemented either Two spontaneous streptomycin-resistant with 20 yg/ml of thymidine or 20 ug/ml of mutants were previously isolated from thymine or with 20 ug/ml of thymine plus Bacillus thuringiensis var. thuringiensis 20 pg/ml of aminopterin. After incubation serotype 1: “Sm R 50,” which is resistant for 30 hr, the largest colonies were picked, to 50 pg/ml of streptomycin, and “Sm R reinoculated on plates containing the same 200,” which is resistant to 200 uglml. The medium as the original plate, and repliINTRODUCTION
321 Copyright
0
1971
by
Academic
Press, Inc.
322
DE
BARJAC
cated on plates of basal medium with agar. The thymine- or th!,midine-requiring colonies were repickcd and controlled by their ability of growing in liquid basal medium with or without 20 pg/ml of thymine. Almost all the largest colonies reinoculated on plates of basal agar medium -Ithymidine and on plates of basal agar medium + thymine + aminopterin gave negative replica on the basal agar medium. When controlled on liquid basal medium with or without thymine, a high percentage of these colonies appeared to be thyminerequiring. Some tests on this thymine requirement in B. thuringiensis were made on one mutant obtained from the isolate “Sm R 50,” namely, AM 28 t-.
1 0
and Thymine
Requirement
Exponentially growing cells of AM 28 tin basal medium + 20 pg/ml of thymine were centrifuged, washed twice with saline, and inoculated in liquid basal medium with or without different doses of thymine or thymidine. The subsequent growth, illustrated in Fig. 1, depends on the concentration of thymine or thymidine; 20 ug/ml of thymine or thymidine support full growth of AM 28, which then gives normal cultures with production of spores and crystals. Under these conditions, the thermostable toxin is also produced. Indeed, the autoclaved filtrates of these cultures have the same toxicity on Ephestia kuhniella as the autoclaved filtrates of cultures in basal medium of the mutant Sm R 50. Thymineless
Death
Washed cells of AM 28 t- in the exponential phase were inoculated at a final density of 6.2 X lo6 in liquid basal medium alone and in liquid basal medium supplemented with 2, 0.2, or 0.02 vg/ml of thymine. After incubation at 30°C samples were taken at every 2 hr, and counts of
I 8
I 12 HOURS
I
I
I
16
20
24
Growth of AM 28 t- mutant of Batiks var. thtcringiensis in basal medium (7); in basal medium + thymine, 20 ug/ml ( I), 2 ug/ml ( 2), and 0.2 pg/ml ( 3) ; in basal medium + thymidine: 20 ug/ml (4), 2 kg/ml (5), and 0.2 ug/ml (6). After 8 hr incubation: atypical bacteria with filamentous giant cells and lysis, except in cultures Nos. 1 and 4. After 24 hr incubation: presence of spores and crystals only in cultures Nos. 1 and 4. FIG.
Growth
I 4
1.
thuringiensis
viable cells were made by spreading dilutions on the surface of plates containing basal agar medium with 20 yg/ml of thymine. The results, given in Fig. 2, show that the total survival of the thymineless mutant needs 2 pg/ml of thymine. By thymine-starvation, the viability decreasesprogressively because the DNA synthesis is stopped. Tests of Growth on Other Substrates instead of Thymine Exponentially growing cells of AM 28 twere centrifuged, washed twice, and inoculated in liquid basal medium and in this same medium supplemented either by 20 pg/ml of thymine or by different ribosides or vitamins. The optical densities after incubation (Fig. 3) indicate that these differ-
THYMINE-REQUIRING
VIABLE
MUTANTS
OF
&X&s
323
thWif@msis
ent substrates cannot be used Instead of thymine.
CELLS/ml
Stability By plating washed cells of AM 28 t- in the exponential phase on plates of basal medium agar and plates of this medium + 20 pg/ml of thymine, it was found that 7.5 cells reverse to prototrophy in a population of 32.4 X 10F bacteria.
Resistance to Aminopterin
I
I
2
4 HOURS
I
I
6
8
Tests of comparative growth of AM 2.5t and of Sm R 50 in liquid basal medium + 20 yg/ml of thymine supplemented or not with different doses of aminopterin arc\ illustrated in Fig. 4. The strain Sm R 50 is
FIG. 2. Thymine-starvation of AM 28 t- mntant of Bacillus thuringiensis var. thuringiensis in basal medium (4) and in basal medium + thymine: 2 pg/ml ( 1 ), 0.2 pg/ml (2), and 0.02
ug/ml
(3).
I 0 1
I
0
4
I
8 HOURS
12
FIG. 3. Tests of growth of AM 28 t- mutant of Bacillus thuringiensis var. thuringiensis in basal medium ( 1) and in basal medium supplemented by 20 pg/ml of thymine (2)) 20 pg/ml of adenosine + 20 pg/ml of guanosine ( 3)) 20 pg/ml of cytidine + 20 pg/ml of uridine (4), 5 pg/ml of fohc acid (5), and 5 pg/ml of thiamine (6).
I 4
HOURS’
I
I 12
Frc. -L Comparative resistance to aminopterin of the wild strain and of the thymineless mutant of Bacillus thuringiensis var. thuringiensis. Wild strain: 1 = basal medium + thymine ( 20 pg/ml ): 2 = basal medium + thymine (20 wg/ml) + aminopterin ( 0.5 X IO-4 IU); 3 = basal medium + thymine (20 pg/ml) + aminopterin ( 1 X lo---’ hf); 4 = basal medium + thymine (20 pg/ml ) + aminopterin (2 X or 4 X 10-h hr). AM 28 t-: 5 = basal medium + thymine (20 &ml) + alnin:@erin (0.5 X “p to 5 X 10-4 hx 1.
DE BARJAC
fully inhibited by aminopterin at 2 X 10e4 M but the growth of the thymineless mutant is normal with aminopterin at 5 X 10m4 M. Even with aminopterin at 7 X 10e4 M, the growth of this mutant is noticeable.
AND RYAN, F. J. of thymineless 188, 340-341.
OKADA, T., HOMMA, J., AND SONOHARA, H. 1962. Improved method for obtaining thymineless mutants of E. coli and S. typhimurium. J.
Bacterial.
84, 602-603.
STACEY, K. A., AND SIMSON, E. 1965. Improved method for the isolation of thymine-requiring mutants of E. coli. J. Bacterial., 90, 554-555.
REFERENCES FARMER, J. L., AND ROTHMAN, formable thymine-requiring subtilis. J. Bacterial. 89,
OKADA, T., YANAGISAWA, K., 1960. Elective production mutants. Nature (London),
F. 1965. Transmutant of Bacillus
262-263.
1964. WACHSMAN, J. T., KEWP, S., AND How, L. Thymineless death in Bacillus megaterium. J. Bacterial. 87, 1079-1086.
Thymine-Requiring
Mutants
of the Insect
Pathogen
Bacillus
dzudngiensk
H. DE BARJAC Service des Vaccins
Bactiriens,
Institut
Received December
Pasteur,
Paris,
France
8, 1969
By action of aminopterin with thymine in the medium, auxotrophic thymine-requiring mutants of Bacillus thuringiensis var. thuringiensis have been obtained. The study of one of these mutants (AM 28 t- ) shows that its optimal growth requires thymine or thymidine at a concentration of 20 ug/ml. Thymine starvation produces very long, filamentous cells with noticeable lysis and no further DNA-synthesis. Mixtures of other ribosides, such as adenosine + guanosine or uridine + cytidine, and of vitamins, such as folic acid or thiamine, cannot be substituted for thymine. By incubation at 30°C without thymine, this mutant progressively loses its viability, which decreases by about 1 X 103 cells within 8 hr. A minimal dose of 2 &ml thymine can afford total survival of cells. The prototrophic, wild-type strain is fully inhibited by aminopterin at 2 X 10-4 M, but its thymine-requiring mutant grows well with aminopterin at 5 X or 7 X lo-* M. The results of this study show many similarities with the phenomenon of thymineless death which have been described in mutants of Escherichia coli and of B&i&s megaterium.
method of Okada, as modified by Wachsman et al. ( 1964), was applied to these Thymineless mutants of Escherichia CO& mutants with the following changes. and Salmonella typhimurium have been The basal medium for growing B. isolated by Okada et al. (1960, 1962) by thuringiensis contained: P0.1H2K, 6.8 g; action of aminopterin. Stacey and Simson S04Mg.7Hs0, 0.123 g; S04Mn*4Hz0, (1965) obtained the same results with E. 0.002 g; S01Zn*7H20, 0.014 g; (SOa):$Fez, coli by using such diaminopyrimidines as 0.02 g; C1&a*4Hz0, 0.183 g; casamino The acids ( Difco), 10 g; glucose, 5 g; and dispyrimethamine and trimethoprim. method of Okada with aminopterin has tilled water, 1 liter. The pH was adjusted been also successfully applied to Bacillus to 7.4. For selecting thymineless mutants, megaterium (Wachsman et al. 1964) and this medium was supplemented by 200 to Bacillus subtilis (Farmer and Rothman, lig/rnl of aminopterin and 200 rig/ml of 1965). Reported herein is the effect of ami- thymine. All the cultures were made at nopterin on B. thuringiensis. 30°C with shaking. After incubation for 4 days, the cultures were diluted and plated on basal agar plates (basal medium with MATERIALS, METHODS, AND DISCUSSION 1.5% D&o agar) supplemented either Two spontaneous streptomycin-resistant with 20 yg/ml of thymidine or 20 ug/ml of mutants were previously isolated from thymine or with 20 ug/ml of thymine plus Bacillus thuringiensis var. thuringiensis 20 pg/ml of aminopterin. After incubation serotype 1: “Sm R 50,” which is resistant for 30 hr, the largest colonies were picked, to 50 pg/ml of streptomycin, and “Sm R reinoculated on plates containing the same 200,” which is resistant to 200 uglml. The medium as the original plate, and repliINTRODUCTION
321 Copyright
0
1971
by
Academic
Press, Inc.
322
DE
BARJAC
cated on plates of basal medium with agar. The thymine- or th!,midine-requiring colonies were repickcd and controlled by their ability of growing in liquid basal medium with or without 20 pg/ml of thymine. Almost all the largest colonies reinoculated on plates of basal agar medium -Ithymidine and on plates of basal agar medium + thymine + aminopterin gave negative replica on the basal agar medium. When controlled on liquid basal medium with or without thymine, a high percentage of these colonies appeared to be thyminerequiring. Some tests on this thymine requirement in B. thuringiensis were made on one mutant obtained from the isolate “Sm R 50,” namely, AM 28 t-.
1 0
and Thymine
Requirement
Exponentially growing cells of AM 28 tin basal medium + 20 pg/ml of thymine were centrifuged, washed twice with saline, and inoculated in liquid basal medium with or without different doses of thymine or thymidine. The subsequent growth, illustrated in Fig. 1, depends on the concentration of thymine or thymidine; 20 ug/ml of thymine or thymidine support full growth of AM 28, which then gives normal cultures with production of spores and crystals. Under these conditions, the thermostable toxin is also produced. Indeed, the autoclaved filtrates of these cultures have the same toxicity on Ephestia kuhniella as the autoclaved filtrates of cultures in basal medium of the mutant Sm R 50. Thymineless
Death
Washed cells of AM 28 t- in the exponential phase were inoculated at a final density of 6.2 X lo6 in liquid basal medium alone and in liquid basal medium supplemented with 2, 0.2, or 0.02 vg/ml of thymine. After incubation at 30°C samples were taken at every 2 hr, and counts of
I 8
I 12 HOURS
I
I
I
16
20
24
Growth of AM 28 t- mutant of Batiks var. thtcringiensis in basal medium (7); in basal medium + thymine, 20 ug/ml ( I), 2 ug/ml ( 2), and 0.2 pg/ml ( 3) ; in basal medium + thymidine: 20 ug/ml (4), 2 kg/ml (5), and 0.2 ug/ml (6). After 8 hr incubation: atypical bacteria with filamentous giant cells and lysis, except in cultures Nos. 1 and 4. After 24 hr incubation: presence of spores and crystals only in cultures Nos. 1 and 4. FIG.
Growth
I 4
1.
thuringiensis
viable cells were made by spreading dilutions on the surface of plates containing basal agar medium with 20 yg/ml of thymine. The results, given in Fig. 2, show that the total survival of the thymineless mutant needs 2 pg/ml of thymine. By thymine-starvation, the viability decreasesprogressively because the DNA synthesis is stopped. Tests of Growth on Other Substrates instead of Thymine Exponentially growing cells of AM 28 twere centrifuged, washed twice, and inoculated in liquid basal medium and in this same medium supplemented either by 20 pg/ml of thymine or by different ribosides or vitamins. The optical densities after incubation (Fig. 3) indicate that these differ-
THYMINE-REQUIRING
VIABLE
MUTANTS
OF
&X&s
323
thWif@msis
ent substrates cannot be used Instead of thymine.
CELLS/ml
Stability By plating washed cells of AM 28 t- in the exponential phase on plates of basal medium agar and plates of this medium + 20 pg/ml of thymine, it was found that 7.5 cells reverse to prototrophy in a population of 32.4 X 10F bacteria.
Resistance to Aminopterin
I
I
2
4 HOURS
I
I
6
8
Tests of comparative growth of AM 2.5t and of Sm R 50 in liquid basal medium + 20 yg/ml of thymine supplemented or not with different doses of aminopterin arc\ illustrated in Fig. 4. The strain Sm R 50 is
FIG. 2. Thymine-starvation of AM 28 t- mntant of Bacillus thuringiensis var. thuringiensis in basal medium (4) and in basal medium + thymine: 2 pg/ml ( 1 ), 0.2 pg/ml (2), and 0.02
ug/ml
(3).
I 0 1
I
0
4
I
8 HOURS
12
FIG. 3. Tests of growth of AM 28 t- mutant of Bacillus thuringiensis var. thuringiensis in basal medium ( 1) and in basal medium supplemented by 20 pg/ml of thymine (2)) 20 pg/ml of adenosine + 20 pg/ml of guanosine ( 3)) 20 pg/ml of cytidine + 20 pg/ml of uridine (4), 5 pg/ml of fohc acid (5), and 5 pg/ml of thiamine (6).
I 4
HOURS’
I
I 12
Frc. -L Comparative resistance to aminopterin of the wild strain and of the thymineless mutant of Bacillus thuringiensis var. thuringiensis. Wild strain: 1 = basal medium + thymine ( 20 pg/ml ): 2 = basal medium + thymine (20 wg/ml) + aminopterin ( 0.5 X IO-4 IU); 3 = basal medium + thymine (20 pg/ml) + aminopterin ( 1 X lo---’ hf); 4 = basal medium + thymine (20 pg/ml ) + aminopterin (2 X or 4 X 10-h hr). AM 28 t-: 5 = basal medium + thymine (20 &ml) + alnin:@erin (0.5 X “p to 5 X 10-4 hx 1.
DE BARJAC
fully inhibited by aminopterin at 2 X 10e4 M but the growth of the thymineless mutant is normal with aminopterin at 5 X 10m4 M. Even with aminopterin at 7 X 10e4 M, the growth of this mutant is noticeable.
AND RYAN, F. J. of thymineless 188, 340-341.
OKADA, T., HOMMA, J., AND SONOHARA, H. 1962. Improved method for obtaining thymineless mutants of E. coli and S. typhimurium. J.
Bacterial.
84, 602-603.
STACEY, K. A., AND SIMSON, E. 1965. Improved method for the isolation of thymine-requiring mutants of E. coli. J. Bacterial., 90, 554-555.
REFERENCES FARMER, J. L., AND ROTHMAN, formable thymine-requiring subtilis. J. Bacterial. 89,
OKADA, T., YANAGISAWA, K., 1960. Elective production mutants. Nature (London),
F. 1965. Transmutant of Bacillus
262-263.
1964. WACHSMAN, J. T., KEWP, S., AND How, L. Thymineless death in Bacillus megaterium. J. Bacterial. 87, 1079-1086.