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Journal of Bacteriology, October 2002, p. 5746-5752, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5746-5752.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Streptomyces Genome Contains Multiple Pseudo-attB Sites for the {phi}C31-Encoded Site-Specific Recombination System

Patricia Combes,{dagger} Rob Till, Sally Bee,{ddagger} and Margaret C. M. Smith*

Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom

Received 30 April 2002/ Accepted 22 July 2002

The integrase from the Streptomyces phage {phi}C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of {lambda} or its relatives. Moreover, {phi}C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. {phi}C31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site ({Delta}attB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB+ and {Delta}attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.

* Corresponding author. Mailing address: Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom. Phone: 44 115 9709778. Fax: 44 115 9709906. E-mail: Maggie.smith{at}nottingham.ac.uk.

{dagger} Present address: INSERM U384, Faculté de Médecine, 63000 Clermont-Ferrand, France.

{ddagger} Present address: Qiagen GmbH, 40724 Hilden, Germany.

Journal of Bacteriology, October 2002, p. 5746-5752, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5746-5752.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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