Hypervariability, a new phenomenon of genetic instability, related to DNA amplification in Streptomyces ambofaciens

Abstract

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 ...

Genetic instability and associated genome plasticity in Streptomyces ambofaciens: pulsed-field gel electrophoresis evidence for large DNA alterations in a limited genomic region

“…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).…”

“…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).…”

“…BamHI restriction fragments used as probes in hybridization experiments have been cloned: S1 (SlL and S1R correspond to the endpoints of the AUD joined in the reiteration fragment); S2, which corresponds to the left flanking sequence of the AUD; and IHS (internal homologous sequence), which represents a sequence, included in the 12-kb BamHI fragment, homologous to SiR (4). independent genetic instability events; 14 were derived from the WT strain by the basic genetic instability and exhibited a stable pigment-defective mutant phenotype, and 12 were derived from hypervariability and exhibited various mutant phenotypes (12).…”

“…All of the amplified BamHI fragments generated from ADS120 were present in ADS90 (12), revealing overlapping stretches. These facts suggested that the fragment created by the reiteration of AUD120 was comigrating with one BamHI fragment of ADS90.…”

Organization of the Bacterial Chromosome

“…Often accompanying the amplification are deletions of adjacent DNA, sometimes large tracts of DNA that contain known genes and confer mutant phenotypes (for reviews, see references 27, 61, and 159). The genetic instability that is characteristic of most Streptomyces species gives rise to spontaneous mutants at rates as high as 1%, rising to 10% and more after such treatments as exposure to UV light or growth with ethidium bromide (159,201). The generation of a tract of highly amplified sequences is proposed to be equivalent to attaining a hypervariable state, one that generates further mutants at unusually high frequencies, such as deletions of sequences adjacent to the amplified tract (201).…”