JOURNAL OF BACTERIOLOGY, Sept. 1978, p. 1149-1150 0021-9193/78/0135-1149$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 135, No. 3
Printed in U.S.A.
Kasugamycin-Resistant Mutants of Bacillus subtilis AKIRA TOMINAGAt AND YASUO KOBAYASHI* Department of Biology, Faculty of Science, Hiroshima University, Hiroshima 730, Japan Received for publication 15 June 1978
Kasugamycin-resistant mutants of Bacillus subtilis were isolated and classified into two groups, one of which had resistance to kasugamycin in in vitro protein synthesis and mapped in the ribosomal region. The other group had no resistance to kasugamycin in in.vitro protein synthesis and had weak cross-resistance to gentamicin and kanamycin. Neither group could sporulate in the presence of kasugamycin. Kasugamycin (Ksg) is used to prevent rice blast caused by Piricularia oryzae (13). It also inhibits the growth of gram-negative bacteria such as Escherichia coli (2). Three Ksg-resistant (Ksgr) loci are known in E. coli, ksgA, ksgB, and ksgC, whose resistance is attributed to undermethylation of 16S rRNA, alteration of permeability, and alteration of ribosomal protein S2, respectively (3, 11, 14). So far, no Ksg-resistant mutant of Bacillus subtilis, one of the typical gram-positive bacteria, has been reported. In the present paper, the isolation and some characteristics of Ksg' mutants of B. subtilis are described. Ksgr mutants of B. subtilis 168 thy trp were obtained spontaneously at a frequency of 4 x 10-9 to 7 x 10-9. Thirty-three mutants were isolated after 5 days of incubation at 370C on Schaeffer sporulation plates (10) containing 3 to 5 mg of Ksg per ml. They consist of two groups, one of which has low-level resistance to Ksg and weak cross-resistance to low concentration of gentamicin and kanamycin and the other of which has high-level resistance to Ksg and no cross-resistance (Table 1). In in vitro protein synthesis with polyuridylic acid as messenger, the former is as sensitive as the parental strain, whereas the latter is resistant to Ksg (Fig. 1). Although more detailed analysis is required, these results suggest that Ksg5O2 may be a permeability mutant and Ksg618 and Ksg619 may be ribosome mutants (see below). The chromosomal locations of ksg loci were defined by transduction. This was carried out by the method of Takahashi (12), using PBS1. The low-level-resistant allele cotransduced 2% with leuA, 7% with argA, and 8% with aroG. These results suggest that the ksg-502 locus lies between aroG and sacQ, since the linkage between aroG and sacQ is 3% (8). High-level-resistant alleles have a close linkage with the cysA
marker. To correctly position the loci, threefactor transduction crosses were carried out (Table 2). Results showed that the loci lie at the ribosome region, where many ribosome mutations exist. The map order deduced from Table 2 is cysA-ery-ksg. The reciprocal data from direct selection of KsgW recombinants were unreliable because of unexplained difficulties with the selection method and the interdependence among ribosomal elements. A similar phenomenon has been reported in E. coli; i.e., the addition of a Ksg-resistant mutation to an erythromycinresistant strain frequently suppresses the expression of the erythromycin-resistant phenotype (9). This may result in the overproduction of "111"-type recombinants in Table 2. In any case, this result strongly suggests the possibility that Ksg618 and Ksg619 are ribosome mutants. According to our preliminary experiments, the high-level resistance could be attributed to the alteration of rRNA, since alteration of ribosomal proteins was not found in Ksg618 and Ksg619 by two-dimensional gel electrophoresis. It has been reported by several investigators that the sporulation of B. subtilis may be controlled at the level of translation (1, 5, 7). Several ribosome-directed, antibiotic-resistant mutants are defective in sporulation. We found that the sporulation of Ksg5O2 was normal, but that of Ksg618 and Ksg619 was a little lower than that of the parental strain (Table 1). This may be due to a defect in the protein-synthesizing system in these mutants. The sporulation ability of the mutants was reduced greatly in the presence of Ksg. The parental strain also shows a similar character at a lower concentration of Ksg. This result is of particular interest in regard to a regulatory mechanism of sporulation, since Ksg is known to exert a differential inhibition on the initiation of protein synthesis (6), and the result suggests the possibility that Ksg discriminates sporulation-specific initiation signals. This possibility will be clarified by further experiments.
t Preent address: Institute for Cancer Research, Osaka University Medical School, Fukushima-ku, Osaka 553, Japan. 1149
1150
J. BACTERIOL.
NOTES TABLE 1. Characteristics of Ksg-resistant mutants of B. subtilis
Group
Growth rate with Ksg (3
No. of mutants (typical strain)
MICa (mg/ml)
Cross-resistance SM, GM,toKMb
10
GM (0.05)
mg/mi) 1 (Ksg5O2)
Spores/ml after 24-h incubationc Without Ksg With Ksg (3 mg/mil)
(Mt/mi)
Low
2.9 x
10"
1.3 x 106
KM (0.25) 32 (Ksg618, Ksg619)
II
Normal
40
1.0 x 1071.5 x W 3.3 x 106
None
1 Parental strain a MIC, Minimum inhibitory concentration. b SM,
c
1.0 x 103-4.7 X 105
6.6 x 106 (100 jug/ml) 1.1 x 106 (500 ug/ml)
Streptomycin; GM, gentamicin; KM, kanamycin.
Survivors after heating at 800C for 10 min.
TABLE 2. Mapping order from three-factor transduction crosseea Se- Recombinant class" No. of Donor lected recomstrain markers yA ery ksg binants 1 1 107 Ksg6l8 Cys+ 1 OO
6060
1
V
Kag619 Cys+
40
168 thy trp .L
ksg
20
502
0-0
X
X
ksg 619
2
3
4
FIG. 1. Inhibition by Ksg of [1C]phenyla1anine incorporation directed by polyuridylic acid. Preparation of the S-30 fraction and the in vitro protein synthesis were carried out by the method of Kimura et al. (4). We are indebted to M. Matsuoka, Hokko Chemicals Co., Ltd., for the supply of kasugamycin.
2.
3. 4.
5.
1
1
0
103
1 1
0
1 i0
14 27
i0
cysA ery ksg
cysA ery ksg
5
Kosugamycin(mg/mi)
1.
12 24 83
0
aRecipient strain: B. subtilis 168 cysA14 er/ IeuA8. b1 and 0 refer to donor and recipient phenotypes, respectively.
keg 618
0
1
1 00 1 1 1
Possble order
LITERATURE CITED Bott, K., S. Graham, and G. Chambliss. 1973. Translational control and its relevance to sporulation. Regulation de la sporulation microbienne. Colloq. Int. C.N.R.S. 227:96-102. Hamada, M.,T. Hashimoto, T. Takahashi, S. Yokoyama, KL Miyake, T. Takeuchi, Y. Okami, and H. Umezawa. 1965. Antimicrobial activity of kasugamycin. J. Antibiot. Ser. A 18:104-106. Helser, T. L., J. E. Davies, and J. E. Dahlberg. 1972. Mechamism of kasugamycin resistance in Escherichia coli. Nature (London) New Biol. 235:6-9. Kimura, A., A. Muto, and S. Osawa. 1974. Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis. Mol. Gen. Genet. 130:203-214. Kobaynahi, Y., and T. Domoto. 1977. Role of ribosomes in bacterial sporulation, p. 115-131. In T. Ishikawa, Y. Maruyama, and H. Matsumiya (ed.), Growth and differentiation of microorganisms. University of Tokyo
Press, Tokyo. 6. Kozac, M., and D. Nathans. 1972. Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics. J. Mol. Biol. 70:41-65. 7. Leighton, T. 1974. Sporulation-specific translational dis-
crimination in Bacillus subtilis. J. Mol. Biol.
86:855-863. 8. Lepesant-Kejzlarova, J., J.-A. Lepesant, J. Walle, A. Billault, and R. Dedonder. 1975. Revision of the
linkage map of Bacillus subtilis 168: indications for circularity of the chromosome. J. Bacteriol. 121:823-834.
9.
10.
11. 12.
13. 14.
Saltzman, L A., M. Brown, and D. Apirion. 1974. Functional interdependence among ribosomal elements as revealed by genetic analysis. Mol. Gen. Genet. 133:201-207. Schaeffer, P., H. Ionesco, A. Ryter, and G. Balassa. 1963. La sporulation de BaciUus subtilis: etude genetique et physiologique. Regulation chez les micro-organisms. Colloq. Int. C.N.R.S. 124:553-563. Sparling, P. F., Y. Ikeya, and D. Elliot. 1973. Two genetic loci for resistance to kasugamycin in Escherichia coli. J. Bacteriol. 113:704-710. Takakash, L. 1961. Genetic transduction in Bacillus subtili.s. Biochem. Biophys. Res. Commun. 5:171-175. Umezawa, H., Y. Okami, T. Hashimoto, Y. Suhara, M. Hamada, and T. Takeuchi. 1965. A new antibiotic, kasugamycin. J. Antibiot. Ser. A 18:101-103. Yo.hkarwa, M., A. Okuyama,and N. Tanaka. 1975. A third kasugamycin resistance locus, ksgC, affecting nbosomal protein S2 in Escherichia coli K-12. J. Bacteriol. 122:796-797.
Vol. 135, No. 3
Printed in U.S.A.
Kasugamycin-Resistant Mutants of Bacillus subtilis AKIRA TOMINAGAt AND YASUO KOBAYASHI* Department of Biology, Faculty of Science, Hiroshima University, Hiroshima 730, Japan Received for publication 15 June 1978
Kasugamycin-resistant mutants of Bacillus subtilis were isolated and classified into two groups, one of which had resistance to kasugamycin in in vitro protein synthesis and mapped in the ribosomal region. The other group had no resistance to kasugamycin in in.vitro protein synthesis and had weak cross-resistance to gentamicin and kanamycin. Neither group could sporulate in the presence of kasugamycin. Kasugamycin (Ksg) is used to prevent rice blast caused by Piricularia oryzae (13). It also inhibits the growth of gram-negative bacteria such as Escherichia coli (2). Three Ksg-resistant (Ksgr) loci are known in E. coli, ksgA, ksgB, and ksgC, whose resistance is attributed to undermethylation of 16S rRNA, alteration of permeability, and alteration of ribosomal protein S2, respectively (3, 11, 14). So far, no Ksg-resistant mutant of Bacillus subtilis, one of the typical gram-positive bacteria, has been reported. In the present paper, the isolation and some characteristics of Ksg' mutants of B. subtilis are described. Ksgr mutants of B. subtilis 168 thy trp were obtained spontaneously at a frequency of 4 x 10-9 to 7 x 10-9. Thirty-three mutants were isolated after 5 days of incubation at 370C on Schaeffer sporulation plates (10) containing 3 to 5 mg of Ksg per ml. They consist of two groups, one of which has low-level resistance to Ksg and weak cross-resistance to low concentration of gentamicin and kanamycin and the other of which has high-level resistance to Ksg and no cross-resistance (Table 1). In in vitro protein synthesis with polyuridylic acid as messenger, the former is as sensitive as the parental strain, whereas the latter is resistant to Ksg (Fig. 1). Although more detailed analysis is required, these results suggest that Ksg5O2 may be a permeability mutant and Ksg618 and Ksg619 may be ribosome mutants (see below). The chromosomal locations of ksg loci were defined by transduction. This was carried out by the method of Takahashi (12), using PBS1. The low-level-resistant allele cotransduced 2% with leuA, 7% with argA, and 8% with aroG. These results suggest that the ksg-502 locus lies between aroG and sacQ, since the linkage between aroG and sacQ is 3% (8). High-level-resistant alleles have a close linkage with the cysA
marker. To correctly position the loci, threefactor transduction crosses were carried out (Table 2). Results showed that the loci lie at the ribosome region, where many ribosome mutations exist. The map order deduced from Table 2 is cysA-ery-ksg. The reciprocal data from direct selection of KsgW recombinants were unreliable because of unexplained difficulties with the selection method and the interdependence among ribosomal elements. A similar phenomenon has been reported in E. coli; i.e., the addition of a Ksg-resistant mutation to an erythromycinresistant strain frequently suppresses the expression of the erythromycin-resistant phenotype (9). This may result in the overproduction of "111"-type recombinants in Table 2. In any case, this result strongly suggests the possibility that Ksg618 and Ksg619 are ribosome mutants. According to our preliminary experiments, the high-level resistance could be attributed to the alteration of rRNA, since alteration of ribosomal proteins was not found in Ksg618 and Ksg619 by two-dimensional gel electrophoresis. It has been reported by several investigators that the sporulation of B. subtilis may be controlled at the level of translation (1, 5, 7). Several ribosome-directed, antibiotic-resistant mutants are defective in sporulation. We found that the sporulation of Ksg5O2 was normal, but that of Ksg618 and Ksg619 was a little lower than that of the parental strain (Table 1). This may be due to a defect in the protein-synthesizing system in these mutants. The sporulation ability of the mutants was reduced greatly in the presence of Ksg. The parental strain also shows a similar character at a lower concentration of Ksg. This result is of particular interest in regard to a regulatory mechanism of sporulation, since Ksg is known to exert a differential inhibition on the initiation of protein synthesis (6), and the result suggests the possibility that Ksg discriminates sporulation-specific initiation signals. This possibility will be clarified by further experiments.
t Preent address: Institute for Cancer Research, Osaka University Medical School, Fukushima-ku, Osaka 553, Japan. 1149
1150
J. BACTERIOL.
NOTES TABLE 1. Characteristics of Ksg-resistant mutants of B. subtilis
Group
Growth rate with Ksg (3
No. of mutants (typical strain)
MICa (mg/ml)
Cross-resistance SM, GM,toKMb
10
GM (0.05)
mg/mi) 1 (Ksg5O2)
Spores/ml after 24-h incubationc Without Ksg With Ksg (3 mg/mil)
(Mt/mi)
Low
2.9 x
10"
1.3 x 106
KM (0.25) 32 (Ksg618, Ksg619)
II
Normal
40
1.0 x 1071.5 x W 3.3 x 106
None
1 Parental strain a MIC, Minimum inhibitory concentration. b SM,
c
1.0 x 103-4.7 X 105
6.6 x 106 (100 jug/ml) 1.1 x 106 (500 ug/ml)
Streptomycin; GM, gentamicin; KM, kanamycin.
Survivors after heating at 800C for 10 min.
TABLE 2. Mapping order from three-factor transduction crosseea Se- Recombinant class" No. of Donor lected recomstrain markers yA ery ksg binants 1 1 107 Ksg6l8 Cys+ 1 OO
6060
1
V
Kag619 Cys+
40
168 thy trp .L
ksg
20
502
0-0
X
X
ksg 619
2
3
4
FIG. 1. Inhibition by Ksg of [1C]phenyla1anine incorporation directed by polyuridylic acid. Preparation of the S-30 fraction and the in vitro protein synthesis were carried out by the method of Kimura et al. (4). We are indebted to M. Matsuoka, Hokko Chemicals Co., Ltd., for the supply of kasugamycin.
2.
3. 4.
5.
1
1
0
103
1 1
0
1 i0
14 27
i0
cysA ery ksg
cysA ery ksg
5
Kosugamycin(mg/mi)
1.
12 24 83
0
aRecipient strain: B. subtilis 168 cysA14 er/ IeuA8. b1 and 0 refer to donor and recipient phenotypes, respectively.
keg 618
0
1
1 00 1 1 1
Possble order
LITERATURE CITED Bott, K., S. Graham, and G. Chambliss. 1973. Translational control and its relevance to sporulation. Regulation de la sporulation microbienne. Colloq. Int. C.N.R.S. 227:96-102. Hamada, M.,T. Hashimoto, T. Takahashi, S. Yokoyama, KL Miyake, T. Takeuchi, Y. Okami, and H. Umezawa. 1965. Antimicrobial activity of kasugamycin. J. Antibiot. Ser. A 18:104-106. Helser, T. L., J. E. Davies, and J. E. Dahlberg. 1972. Mechamism of kasugamycin resistance in Escherichia coli. Nature (London) New Biol. 235:6-9. Kimura, A., A. Muto, and S. Osawa. 1974. Control of stable RNA synthesis in a temperature-sensitive mutant of elongation factor G of Bacillus subtilis. Mol. Gen. Genet. 130:203-214. Kobaynahi, Y., and T. Domoto. 1977. Role of ribosomes in bacterial sporulation, p. 115-131. In T. Ishikawa, Y. Maruyama, and H. Matsumiya (ed.), Growth and differentiation of microorganisms. University of Tokyo
Press, Tokyo. 6. Kozac, M., and D. Nathans. 1972. Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics. J. Mol. Biol. 70:41-65. 7. Leighton, T. 1974. Sporulation-specific translational dis-
crimination in Bacillus subtilis. J. Mol. Biol.
86:855-863. 8. Lepesant-Kejzlarova, J., J.-A. Lepesant, J. Walle, A. Billault, and R. Dedonder. 1975. Revision of the
linkage map of Bacillus subtilis 168: indications for circularity of the chromosome. J. Bacteriol. 121:823-834.
9.
10.
11. 12.
13. 14.
Saltzman, L A., M. Brown, and D. Apirion. 1974. Functional interdependence among ribosomal elements as revealed by genetic analysis. Mol. Gen. Genet. 133:201-207. Schaeffer, P., H. Ionesco, A. Ryter, and G. Balassa. 1963. La sporulation de BaciUus subtilis: etude genetique et physiologique. Regulation chez les micro-organisms. Colloq. Int. C.N.R.S. 124:553-563. Sparling, P. F., Y. Ikeya, and D. Elliot. 1973. Two genetic loci for resistance to kasugamycin in Escherichia coli. J. Bacteriol. 113:704-710. Takakash, L. 1961. Genetic transduction in Bacillus subtili.s. Biochem. Biophys. Res. Commun. 5:171-175. Umezawa, H., Y. Okami, T. Hashimoto, Y. Suhara, M. Hamada, and T. Takeuchi. 1965. A new antibiotic, kasugamycin. J. Antibiot. Ser. A 18:101-103. Yo.hkarwa, M., A. Okuyama,and N. Tanaka. 1975. A third kasugamycin resistance locus, ksgC, affecting nbosomal protein S2 in Escherichia coli K-12. J. Bacteriol. 122:796-797.
