A stable temperature sensitive mutant of Streptomyces hygroscopicus JA6599 defective in both DNA and RNA syntheses is described. The mutant ts35 is characterized by an immediate stop of DNA synthesis and continued protein synthesis after transfer to restrictive temperature. The reinitiation of DNA synthesis begins immediately after a return to the permissive temperature. This kinetics of macromolecular synthesis at restrictive temperature appears to share similarities with a defect in the DNA elongation process, as described for Escherichia coli (CARL 1970, HANNA and.
The simultaneous stop of both DNA and RNA syntheses may be caused by an additional mutational event affecting also the RNA synthesis. The data were discussed with respect to similar results in E. coli.
Genetic studies in the primary metabolism of streptomycetes were carried out mainly with S. coelicolor A3(2) (for reviews see HOPWOOD 1967. Temperature sensitive mutants of this species were isolated and mapped , but knowledge about the biochemical properties of these mutants is rather limited and studies of macromolecule synthesis have not been carried out. BEHAL et al. (1979) studies DNA-and protein syntheses refering the secondary metabolism in S. aureofaciens. With respect to spore germination MIKULIK et al. (1 977), HARDISSON et al. (1980), GARCIA DIAZ et al. (1983), GUIJARRO et al. (1983) and SALAS et al. (1985) studied macromolecule synthesis in different species of streptomycetes.
In our paper the pattern of DNA-, RNA-and protein syntheses are described as occurs during growth of the heat sensitive mutant of S. hygroscopicus ts3.5 at permissive and restrictive temperatures and the results are discussed.
Methods
Bacterial strains: Streptomyces hygroscopicus NG14, a prototrophic derivative of the wild type IMET JA6599 was used for the isolation of the temperature sensitive mutant ts35.
Media: AL53 agar and the liquid complete medium AL53 were used for all experiments.
Isolation of the mutant: rs35 was isolated from S. hygroscopicus NG14 after combined UV-X-irradiation. Spores were irradiated by UV-(2 min with a high pressure mercury lamp, dose rate 1 J/mz) followed by Xirradiation (12 min, 50 KV, dose rate 16000 Rjmin). Then the spores were plated and incubated at 28 "C. Colonies were replica-plated and incubated, both at a temperature of 28 "C and of 37 "C. One colony which grew at 28 "C buth which was missing on the 37 "Cplate was selected and tested for stability after 4 subsequent sporulation steps. The standard growth temperature for S. hygroscopicus is 28 "C. The wild type grows well at 37 "C. This temperature was found to be nonpermissive for the mutant zs35. The mutant shows no additional nutritional requirement.
Chemicals: 3H-thymidine-6 (12-20 Ci/mM, 10 pCi/ml) and 'H-uridine-5 (29 Ci/mM, 2-8 pCi/ml), Institute of Research, Recovery and Application of Radioisotopes, Prague, CSSR.
Measurement of DNA and RNA syntheses: DNA and RNA synthesis in the wild type S. hygroscopicus (JA6599) and the mutant (ts35) were measured by the incorporation of 3H-thymidine (10 FCi/ml) and 3Huridine (2-8 FCi/ml) inturn into the acid unsoluble fraction. Two methods were used.