The wild-type strain Streptomyces ambofaciens DSM 40697 exhibits a high degree of genetic instability. Pigment-defective (26). Genomic instabilities are associated with these phenotypic instabilities. Thus, molecular analysis of mutant progeny often reveals genomic rearrangements such as large deletions including genes directly involved in the following phenomena: chloramphenicol sensitivity in S. coelicolor A3(2) and S. lividans 66 (1, 7), streptomycin sensitivity in S. glaucescens (11), melanin formation in S. reticuli (25), S. glaucescens (12), and several Streptomyces species (26), and A-factor biosynthesis in S. bikiniensis (14). However, in some cases of reversible phenotypes, it was suggested that the genes involved were affected by transposable elements (8,9).Highly amplified DNA sequences (ADS) have been detected within the DNA isolated from variants of many Streptomyces species arising either spontaneously (1,3,25) or during vegetative growth in the presence of ethidium bromide (10,19,24,25), formation and regeneration of protoplasts (6), or interspecific protoplast fusion (23). In some cases, ADS were associated with particular phenotypes, such as sequential loss of resistance to chloramphenicol and arginine synthesis (2) and loss of resistance to tetracyclihe and nitrogen assimilation (5). Some others were selected on the basis of high-level antibiotic resistance (7,15,17,21) Table 1. Most of the media used in the culture conditions were described previously (13). S. ambofaciens strains were grown at 37°C on plates of HickeyTresner (HT) medium (22) for maintenance, sporulation, and mutant isolation. Auxotrophic mutants were detected by replica-plating the WT cultures on minimal medium and further characterized on minimal medium supplemented with combinations of growth requirements. For large-scale isolations of genomic DNA, the S. ambofaciens cultures were grown aerobically at 37°C for 48 h in YEME liquid medium supplemented with glycine (0.25%). Small-scale isolations of genomic DNA were performed with mycelium grown at 37°C in HT liquid medium for 24 h.DNA extraction and restriction endonuclease analyses. The myceliumn was harvested by centrifugation, resuspended in TE buffer (10 mM Tris hydrochloride, 1 mM EDTA, pH 8.0) and mixed with lysozyme (Boehringer Mannheim) (2 mg/ml). Protoplasts were formed by incubation of the mixture at 30'C for 30 min and lysed with sodium dodecyl sulfate (1%) in the presence of proteinase K (50 ,ug/ml). DNA was purified by two phenol-chloroform extractions followed by one chloroform extraction. The aqueous phase was treated with RNase A (Sigma) (5 mg/ml) for 1 h at 37°C. DNA was precipitated with sodium acetate (0.3 M, pH 5.2) and isopropanol at -20°C. After centrifugation, the pellet was vacuum dried and dissolved in TE buffer. For the small-scale isolations, DNA was purified by the Geneclean process (Bio 101 Inc.). For the large-scale isolations, the DNA was purified by equilibrium density centrifugation in CsCl-ethidium bromide gradients, using a vTi 65.2 rotor ...

Using pulsed-field gel electrophoresis (PFGE) analysis, the amplifiable units of DNA (AUD) loci AUD6 and AUD90 of Streptomyces ambofaciens DSM40697 could be mapped in the wild-type genome within two adjacent AseI restriction fragments estimated to be about 75 and 850 kb. In addition, the genetic instability and formation of very large deletions were strictly correlated. Their sizes were estimated to range from 250 to more than 2,000 kb. These deletions affected the DNA region overlapping both amplifiable loci. PFGE also allowed us to localize the amplified DNA sequences and to establish their structure: amplification takes place at the AUD locus as a tandem array of the wild-type AUD sequence.The Streptomyces genome exhibits a high degree of genome plasticity, consisting of large deletions and amplified DNA sequences (ADS) associated with instability of many characteristics (1,11,13). In Streptomyces ambofaciens DSM40697 (9), two levels of genetic instability were characterized: (i) a basic genetic instability similar to that reported for other Streptomyces spp. and (ii) hypervariability, closely related to the first phenomenon, generating a phenotypic variability from mutant clones (12). At least two DNA regions undergo amplification in close association with hypervariability (3, 12). In addition, an amplifiable locus was recently characterized as a rearrangement hotspot, mainly for deletions (4). An analogous situation implying successive mutational events associated with changes in DNA structure is well documented in Streptomyces lividans (5). Using pulsed-field gel electrophoresis (PFGE), we investigated the localization of the amplifiable units of DNA (AUD) on large restriction fragments generated from the wild-type (WT) genome and the size of the deletions associated with genetic instability. We then studied the structure and the physical localization of the ADS in the mutant genome.Physical relationship between the AUD regions on the WT genome. Forty-eight ADSs, whose sizes ranged from 5.2 to 105 kb, generated through independent genetic instability events were considered. Three probes were used in hybridization experiments: S1, consisting of the cloned extremities of ADS6 (Fig. 1); S120, consisting of a 1.95-kb BamHI fragment of ADS120 (12) (Fig. 1). The relative intensity of the signals could be explained by the fact that the 75-kb fragment contained two homologous stretches (i.e., the right side of the AUD and the internal homologous sequence). On the other hand, the 850-kb fragment contained only the left side of AUD6. Both the S120 and S55 (from an ADS belonging to a region other than AUD90 or AUD6) probes revealed the 850-kb fragment. Faint signals could be detected that corresponded to almost all restriction fragments generated by AseI, particularly the 200-kb doublet (14)

Many Streptomyces species exhibit a very high degree of genetic instability which is usually manifested as genomic rearrangements such as large deletions. In Streptomyces ambofaciens DSM40697, two levels of genetic instability were previously described: (i) a basic genetic instability similar to that reported for other strains, and (ii) hypervariability, a phenomenon that we believe to be a new aspect of instability closely associated with DNA amplification. A large DNA region undergoes deletions, amplifications and large genomic changes strictly associated with both aspects of genetic instability. The genetic and molecular analyses of the different aspects of genetic instability allow us to propose that they result from a cascade of molecular events and to investigate the relationships between genetic instability phenomena and genome fluidity.

Four highly amplified DNA sequences (ADS) ranging from 5.8 to 24.8 kb were found in spontaneous mutant strains of Streptomyces ambofaciens DSM 40697. Restriction patterns of total DNA were hybridized with purified ADS6 (24.8 kb) as a probe to detect the amplifiable regions in the wild-type (WT) genome. The results suggested that the amplifiable unit of DNA (AUD) was present as a single copy in the WT genome. Moreover, similarities suggested by the restriction maps of three of the ADS were confirmed by hybridization experiments. The fourth ADS did not hybridize with the three others. Therefore, two families of DNA sequences are potentially amplifiable in the S. ambofaciens genome.

Genetic instability in Strepfomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.