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

Abstract

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)

A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome

“…For example, those in S. glaucescens can be 800 kb or more long (11), and in S. ambofaciens, PFGE analysis has revealed deletions of up to 2 Mb of contiguous DNA (57). In S. lividans and S. coelicolor, frequent deletions at least 40 kb long include the argG locus and a determinant of chloramphenicol resistance (8,29); these genes were located, by recombination analysis, near to 3 o'clock in S. lividans (8).…”

Occurrence of deletions, associated with genetic instability in Streptomyces ambofaciens, is independent of the linearity of the chromosomal DNA

“…This phenomenon was shown to be closely related to chromosomal rearrangements such as large-scale deletions, up to 2 Mb, and DNA sequence amplifications in a particular chromosomal area called the unstable region (1,3,8,9,15,20,23,25).…”

An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes

“…NSA205, a mutant derived from RP181110, has an 89 kb amplification (ADS204 in the AUD90 locus and is Spi- (Dary etal., 1992). S. ambofaciens NSA97H, in which the AUD90 locus is totally deleted, is a spontaneous mutant of the DSM 40697 strain (Leblond et al, 1991). Escbericbia coli SURE (Stratagene) was the host for cloning experiments.…”