Insertional mutagenesis of <italic>Streptomyces</italic> plasmid SCP2*

SCP2* (31.4kb) is a spontaneous high fertility variant of the low copy number and broad host-range Streptomyces plasmid SCP2. Amongst other applications, derivatives of SCP2* had previously been used as vectors for the cloning of Streptomyces gene clusters specifying antibiotic biosynthesis. SCP2* is 100% self-transmissible and has the ability to form 'pocks' which are typical for, and evidently unique to conjugative Streptomyces plasmids. A detailed physical map of SCP2* had been constructed and the transfer and fertility functions, along with sufficient functions for pock formation, had been located to a c.9kb region of SCP2* using deletion derivatives. The aim of this investigation was to identify individual transfer and pock formation genes by the in vitro insertion of ΩSPT, a specifically constructed resistance cassette, into plJ903 (25.8kb), a bifunctional derivative which retains all the known phenotypic properties of SCP2*. As a new construct, the establishment of ΩSPT's structural and functional integrity and suitability for widespread application presented further investigative aims. Firstly, the ends of ΩSPT were sequenced to confirm the presence and structural integrity of the T7 and SP6 promoter sequences which form an important part of the cassette. Their functional integrity was demonstrated by in vitro transcription using the corresponding RNA polymerases. It was also demonstrated that it is possible to obtain sequence information from regions of pIJ903 flanking the ΩSPT insertion site using the commercially available T7 and SP6 primers, plasmid templates isolated by small-scale procedures from E.coli or Streptomyces lividans, and a doublestrand sequencing procedure. ΩSPT was inserted into pIJ903 linearised with Sau3AI to yield 104 putative recombinants (p903Ωs) in S.lividans TK64. Seven pock deficient and seventeen novel small pock mutants were found, the remainder exhibited pocks indistinguishable in size from pIJ903. Three of the small pock and all of the pock deficient inserts mapped were located within or very close to the transfer/pock region. The ΩSPT inserts of the remaining small pock mutants were located external to this, many being clustered within a region which had been previously deleted without affecting pock formation, and were interspersed with inserts which did not affect pock formation. Amongst further p903Ω constructs isolated from E.coli and then transformed into S.lividans, a further two pock deficient mutants were found. These possessed inserts within the transfer/pock region and could not be recovered by replica plating after mixing with plasmid-free recipients. ΩSPT was found to be structurally stable in E.coli JM107 but unstable in S.lividans TK64. This deletional instability was insertion site dependent and probably occurred through illegitimate recombination between the ΩSPT inserts and the pBR327 region of pIJ903. Alterations interpretable as the tandem duplication of extensive lengths of pIJ903 DNA were also observed. This too appeared to be ΩSPT insertion site dependent and included the duplication of sequence within the transfer/pock region of pIJ903 in normal pock formers