Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis

The insertion sequence IS6100 was shown to undergo intermolecular transposition from a temperature-sensitive delivery plasmid to the genome of the avermectin-producer Streptomyces avermitilis, creating cointegrates. Evidence from both Southern hybridization and the range of auxotrophic mutations present in a transposon library was consistent with random transposition. It was not possible to increase transposase expression by readthrough transcription from a copy of the tipA promoter located adjacent to the insertion sequence. This was in part due to the absence of a homologue of the Streptomyces lividans transcriptional activator TipAL in S. avermitilis. However, recombinant S. avermitilis strains carrying the S. lividans tip operon were also deficient for induction of the promoter. The frequency of reversion of different auxotrophic mutations by precise excision, involving recombination across 8 bp direct repeats, was shown to vary by at least five orders of magnitude. This dependence of recombination frequency on chromosomal location may contribute to the stability of repetitive modular type I polyketide biosynthetic genes.