Mutational cloning in Streptomyces and the isolation of antibiotic production genes

Abstract

An attachment site-deleted derivative, phi C31KC400, of the Streptomyces temperate phage phi C31 was used to clone fragments of the genetic determinants (mmy) for the biosynthesis of an antibiotic, methylenomycin A. The vector carries a cloned viomycin resistance gene (vph), and can transduce a recipient to viomycin resistance when DNA sequences are common to the phage and the recipient: the phage integrates into the recipient's genome through a Campbell type of recombination at the site of the homology. For the cloning of mmy DNA, the homology was provided by the in vitro insertion into the vector of DNA from a methylenomycin A-producing streptomycete. Clones carrying mmy DNA could integrate into a methylenomycin-producing recipient's mmy genes, sometimes disrupting their expression: thus a search of viomycin-resistant transductants for methylenomycin non-producing derivatives identified lysogens which spontaneously released phi C31 phages carrying mmy DNA. Some of these lysogens participated in methylenomycin co-synthesis with previously isolated mmy mutants. At least 7 kb of mmy DNA was identified among the clones. Screening for mmy non-producers was simplified by exploiting the presence of the mmy genes on the (albeit unisolable) plasmid, SCP1. In the course of the experiment, SCP1, a low copy number plasmid in its primary host S. coelicolor A3(2), was shown to have a copy number of about 30 in the single S. parvulus SCP1+ transconjugant strain tested, and a molecular size probably greater than 200 kb.