May 24, 1982 - Streptomyces glaucescens strain GLAO (= ETH 22794) produces hydroxystreptomycin and ..... Tyrosinase inheritance in Streptomyces scabies.
Journal of General Microbiology (1983), 129, 5 19-527.
Printed in Great Britain
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Chromosomal Instability in Streptomyces glaucescens: Mapping of Streptomycin-sensitive Mutants By R E T O C R A M E R I , TOBIAS K I E S E R , t H I D E 0 ONO,: JESUS S A N C H E Z ยง A N D R A L F H U T T E R * Mikrobiologisches Institut, Eidgeniissische Technische Hochschule, ETH-Zentrum, CH-8092 Zurich, Switzerland (Received 24 May 1982; revised 30 August 1982) Streptomyces glaucescens strain GLAO (= ETH 22794) produces hydroxystreptomycin and has a high natural resistance to hydroxystreptomycin, dihydrostreptomycin and streptomycin. The wild-type strain gives rise spontaneously to streptomycin-sensitive (StrS-) variants at a frequency of 0.2 to 1-4%. These mutants lack streptomycin phosphotransferase activity responsible for the wild-type resistance to streptomycin group antibiotics and are unable to produce detectable amounts of hydroxystreptomycin. Mapping experiments showed that the strS marker lies between the chromosomal markers lys-2 and ura-3 on the linkage map of S. glaucescens. The molecular basis for instability of this marker is as yet unknown.
INTRODUCTION
Genetic instability is a widespread phenomenon among streptomycetes. The most thoroughly investigated unstable characters are natural resistance to antibiotics (Freeman et al., 1977 ; Freeman & Hopwood, 1978; Sermonti et al., 1977, 1978,1980; Fedorenko & Danilenko, 1980; Kirby & Lewis, 1981), aerial mycelium formation (Pogell, 1979; Redshaw et al., 1979), production of pigments (Gregory & Huang, 1964a, b; Pogell, 1979; Hutter et al., 1981; Schrempf, 1981) and the production of antibiotics (Shaw & Piwowarski, 1977; Kirby, 1978; Schrempf, 1981). An extensive genetic analysis is available only for chloramphenicol resistance in Streptomyces coelicolor A3(2) (Freeman et al., 1977; Sermonti' et al., 1977, 1978, 1980) and for erythromycin resistance in the same strain (Puzynina et al., 1979). Chloramphenicol sensitivity was found to be reversible and no constant map position for the resistance gene could be established (Freeman et al., 1977). According to Sermonti et al. (1980) two chromosomal map positions q a y be deduced from crossing data and the authors therefore deduced that a jumping gene might be responsible for chloramphenicol resistance in this strain. However, the interpretation of the data may be questioned (Lanfaloni et al., 1980). In the case of erythromycin resistance the marker was found to segregate chromosomally and the authors postulated, that erythromycin sensitivity is caused by transposition of genetic material (Puzynina et al., 1979). In both cases, chloramphenicol and erythromycin resistance, no data about the resistance mechanism were published. Similarly, Streptomyces glaucescens possesses a natural resistance towards streptomycin group aminoglycoside antibiotics, but the resistance is highly unstable. Streptomycin-sensitive variants arise with high frequency spontaneously after induction by curing agents or after other Present address: John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K.
2 Present address : Takeda Chemical Ind., Central Research Division, Jusohonmachi 2, Yodogawa-ku,
Osaka 532, Japan. Present address : Departemento Interfacultativo de Microbiologia, Universidad de Oviedo, Oviedo, Spain. 0022-1287/83/0001-0592$02.00 0 1983 SGM
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R . CRAMERI A N D OTHERS
Table 1. Streptomyces glaucescens strains Strain
Genetic markers*
Status of streptomycin resistance?
GLAO GLA15 GLA20 GLA21 GLA23 GLA24 GLA31 GLA46 GLA58 GLA62 GLA65 GLA71 GLA72 GLA75 GLA84 GLA 128 GLA205 GLA207 GLA212 GLAS 12 GLAS 16 GLA528 GLA541 GLA543 GLA544 GLA547 GLA565 GLA567 GLA572 GLA877
W ild-type ura-3 met-2 gua-4 lys-1 hom-1 lys-1 lys-2 ura-5 thr-1 lys-2 lys-2 thr-1 nic-2 lys-2 ura-3 ura-5 met-2 pro-Z ade-1 lys-2 ade-1 leu-2 ura-2 ade-1 lys-2 pro-I rif-565 met-8 Prototroph Prototroph Prototroph his-2 nic-2 ura-3 lys-3 leu-2 ura-3 nic-2 lys-2 ade-1 nic-2 ura-2 nic-2 rif-547 met-8 rif-565 met-8 rif-567 lys-2 thi-3 rif-572 rif-567 pro- I ade-I
strS+ strS+ strS+ strS+ strS+ strS-24 strS+ strS-24 strS+ strS+ strS+ strS+ strS+ strS+ strS+ strS-I28 strS-205 strS-207 strS-212 strS+ strS+ strS+ strS-541 strS-543 strS-544 strS+ strS+ strS+ strS+ strS+
Mutagenic treatment3
-
NTG (GLA31)
-
EB (GLA565) CS (GLAO) A 0 (GLAO) EB (GLAO) -
EB (GLA516) Spontaneous (GLA72) EB (GLA58)
* Marker designations are according to Demerec et al. (1966) (see also Baumann et al., 1974 and Hutter et al., 1981). t Two loci determining the degree of streptomycin resistance are distinguished (Hutter et al., 1981). The wildtype strain GLAO strA+ strS+ can grow on agar media containing up to 10 mg streptomycin 1-'. Strains carrying the mutant allele strA-4 (strS+ or strS) exhibit a high streptomycin resistance (growth on media with 2 100 mg streptomycin l-'), while strA+ strS strains are highly streptomycin sensitive (minimal inhibitory concentration 0.2 mg streptomycin 1-l). All strains used had the genotype strA+. 3 NTG, N-methyl-N'-nitro-N-nitrosoguanidine; EB, ethidium bromide ;CS, cold storage ;AO, acridine orange. The strain numbers in parentheses designate the direct parent of the corresponding mutant strain. -, Designates mutational and recombinational derivatives from the stock culture collection. mutagenic treatments (Freeman & Hopwood, 1978; Suter et al., 1978; Hutter et al., 1981). The natural streptomycin resistance observed in the wild-type strain is due to the presence of a streptomycin phosphotransferase. Sensitive mutant strains lack this enzyme. Simultaneously they lose the capacity to produce detectable amounts of hydroxystreptomycin,indicating either the participation of the phosphotransferase both in antibiotic production and antibiotic resistance or the presence of multiple gene defects in strS mutant strains (On0 et al., 1983). In this paper we report on the genetic analysis of the streptomycin-sensitive mutants. A preliminary report of the data was given in Hutter et al. (1981). METHODS Strains. All mutant strains used are derivatives of Streptomyces glaucescens strain GLAO (corresponding to strain ETH 22794, Baumann et al., 1974; Hutter et al., 1981) and are listed in Table 1 . The linkage map of the markers used is given in Fig. 1 .
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Mapping of streptomycin-sensitive mutants
Table 2. Isolation of streptomycin-sensitive colonies fiom the wild-type and from mutant strains of Streptomyces glaucescens Strain
No. of colonies tested*
* % StrS isolates
Total
(a) Spontaneously occurring StrS mutants
GLAO GLA15 GLA20 GLA21 GLA23 GLA62 GLA65 GLA71 GLA72 GLA75 GLA84 GLA512 GLA516 GLA528 GLA547 GLA567 GLA572
1856 757 218 280 134 700 700 2514 822 558 650 337 1800 170 880 1654 1356
Strain
No. of colonies tested?
8 5 0 0 1 0 0 0 3 8 0 0 0 0 2 0 0
0.43 0.66 -
0.75 -
0.36 1.43 -
0.23 -
* % StrS isolates
Total
-
(b) StrS mutants after growth on media containing ethidium bromide GLAO 9090 6819 75-01 GLA65 956 653 68.30 GLA516 1247 1156 92-70
Strain GLAO GLA65 GLA516
Strain
No. of colonies tested
* %
Auxotrophic mu tan ts Total
(c) StrS mutants after UV irradiation 9658 64 0.66 8899 58 0.65 9223 49 0-53 No. of colonies tested
* %
Auxotrophic mutants Total
StrS isolates
Total
%
17 27 3
0.18 0.30 0.03
* % StrS isolates
Total
(d) StrS mutants after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine 3856 34 0.88 11 0.29 GLAO 37 0.70 23 0.44 GLA65 5260 0.50 35 6941 59 0.85 GLA516
* No spontaneous auxotrophic mutants could be detected (spontaneous mutant
frequency
