Genetic instability and DNA amplification in Streptomyces lividans 66

Abstract

Streptomyces lividans 66 exhibits genetic instability, involving sequential loss of resistance to chloramphenicol (Cams) and subsequent mutation of argG. Associated with this instability is the amplification of a 5.7-kilobase (kb) amplified DNA sequence (ADS). We have characterized a second, independent pathway of genetic instability, involving sequential loss of resistance to tetracycline (Tet') followed by mutation in nitrogen assimilation (Ntr). We detected DNA amplification in many of these mutant strains, as well as other reiterations coresident with the 5.7-kb ADS in Cam' Arg mutants. However, in contrast to the 5.7-kb ADS, none of the novel elements were observed to amplify at high frequency. The mutation of argG is due to a deletion, one endpoint of which is defined by the 5.7-kb ADS. This amplification derives from a structure, the tandemly duplicated amplifiable unit of DNA (AUD), present in the wild-type genome. We found that progenitor strains containing just a single-copy AUD failed to reproducibly generate amplification of this element in Cam' argG mutants, and DNA deletion endpoints proximal to the element were found to be unspecific. These results suggest that a duplicated AUD structure is required for high-frequency amplification and that this reiteration can subsequently buffer the extent of deletion formation in the relevant chromosomal region.

Characterization of a Streptomyces-lividans-type site-specific DNA modification system in the avermectin-producer Streptomyces avermitilis permits investigation of two novel giant linear plasmids, pSA1 and pSA2

“…Streptomyces strains and plasmids are listed in Table 1. Cultures were grown on GHM agar (glucose, 4 g 1-' ; yeast extract, 4 g 1-'; malt extract, 10 g I-' , CaCO,, 2 g 1-I; agar 15 g 1-' , pH 7.2) and, for liquid cultivation, in tryptic soy medium (Dyson & Schrempf, 1987). Plasmid-curing was achieved by nitrosoguanadine mutagenesis (Hopwood et al, 1985) using conditions permitting 10 ?h survival.…”

Soil To Genomics: The Streptomyces Chromosome

“…Robinson et al (97) described variants carrying a 6-kb sequence tandemly amplified ∼300 times, and many other reports followed, some very detailed. Amplifiable units of DNA (AUDs) may generate variants by unequal crossing over between two pre-existing copies (2), and ampli- DNA segment (27). All these gross structural changes were found in laboratory-grown cultures, so it is an open question how far they are relevant in natural habitats.…”

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

“…The association between deletion and genetic instability was frequently reported in Streptomyces species (2,5,6). In S. glaucescens, for example, the double mutant phenotype Str-Mel-was ascribed to deletions whose sizes were estimated to range from 270 to over 800 kb (2).…”

“…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.…”