DNA rearrangements associated with instability of an arginine gene in Streptomyces coelicolor A3(2)
✍ Scribed by Fiona Flett; Jacqueline Platt; Dr. J. Cullum
- Publisher
- John Wiley and Sons
- Year
- 1987
- Tongue
- English
- Weight
- 584 KB
- Volume
- 27
- Category
- Article
- ISSN
- 0233-111X
No coin nor oath required. For personal study only.
✦ Synopsis
Streptomyces coelicdor
A3( 2 )
gives rise t o spontaneous chloramphenicol sensitive mutants a t a frequency of about 0.3% per spore. These mutants are often genetically unstable and give rise to arginine auxotrophs ( h g -) a t frequencies of 1-7% per spore. These Arg-mutants usually lack the enzyme argininosuccinate synthetase (one exception was found that lacked ornithine carbamoyltransferase) and were shown to have deleted the corresponding argG gene by hybridisation analysis using a cloned 8. cattleya argG gene. The Arg-strains also showed a variety of different DNA amplification and deletion events in a region homologous to an amplified DNA sequence found in spontaneous A g -mutants in S. lividans 66.
Many Streptomyces species are genetically unstable and spontaneous mutants can occur at B frequency above 0.1% per spore in properties such as pigment production (SCHREMPF 1983), antibiotic resistance (FREEMAN and HOPWOOD 1978, FEDERENKO and DANILENKO 1980), sporulation and arginine biosynthesis (REDSHAW et al. 1979, NAKANO and OGAWARA 1980). Genetic studies suggested that genetic instability in Streptomyces cannot be explained by plasmid loss but might be due to DNA rearrangements (FREEMAN et al. 1977). Recently deletions (SCHREMPF 1983, HINTERMA" et al. 1984) and amplifications (ROBINSON et al. 1981, ONO et al. 1982, FISHMAN and HERSHBERGER 1983) have been associated with instability.
Streptomyces coelicolor A3(2) was chosen for these studies on instability since it is genetically tractable and has been used as a model organism in Streptomyces Genetics for many years (HOPWOOD et at. 1973). FREEMAN et al. (1977) first discovered the unstable chloramphenicol resistance in S. coelicolor A3(2) and showed that it was not possible to define a unique chromosomal map location for the CmlR/CmlS character. Furthermore, work of SERMONTI et al. (1978) indicated that some Cmls mutants of S. coelicolor A3(2) gave rise to arginine auxotrophs at high frequency (> 5% per spore).
ISHIHARA et al. (1985) showed that CmlSArg-mutants of S. coelicolor A3(2) had deleted the argG gene (the structural gene for argininosuccinate synthetase). We tested out Arg-mutants for argininosuccinate synthetase activity and for the presence of the argG gene. ALTENBUCHNER and CULLUM (1984, 1985) showed that, in the closely relaed species S. lividans 66, CmlSArg-mutants had high copy number amplification of a 5.7 kb DNA sequence. We used the S. lividans amplified sequence as a hybridisation probe to look for DNA rearrangements in S. coelicolor Arg-mutants.
Methods
Strains, media and culture conditions: The strains (Table 1) were derivatives of S. coelicolor A3(2). Plasmid pJOE756 is described in ALTENBUCHNER and CULLUM (1985) and plasmid pTGlll carries the argff gene of 8. eattleya on a 1.5 kb BamHI fragment cloned into the BamHI site of pBR322 1'