The Streptomyces genome contains multiple pseudo-attB sites for the fC31-encoded site-specific recombination system

P. Combes, R. Till, S. Bee, Margaret Caroline MacHin Smith

Research output: Contribution to journalArticle

95 Citations (Scopus)

Abstract

The integrase from the Streptomyces phage phiC31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, phiC31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 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 57T present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site (DeltaattB), 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 DeltaattB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.

Original languageEnglish
Pages (from-to)5746-5752
Number of pages6
JournalJournal of Bacteriology
Volume184
DOIs
Publication statusPublished - 2002

Keywords

  • ESCHERICHIA-COLI
  • PHAGE PHI-C31
  • IN-VITRO
  • INTEGRATION
  • GENE
  • DNA
  • BACTERIOPHAGE-PHI-C31
  • REPLACEMENT
  • CELLS

Cite this

The Streptomyces genome contains multiple pseudo-attB sites for the fC31-encoded site-specific recombination system. / Combes, P.; Till, R.; Bee, S.; Smith, Margaret Caroline MacHin.

In: Journal of Bacteriology, Vol. 184, 2002, p. 5746-5752.

Research output: Contribution to journalArticle

Combes, P. ; Till, R. ; Bee, S. ; Smith, Margaret Caroline MacHin. / The Streptomyces genome contains multiple pseudo-attB sites for the fC31-encoded site-specific recombination system. In: Journal of Bacteriology. 2002 ; Vol. 184. pp. 5746-5752.
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AB - The integrase from the Streptomyces phage phiC31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, phiC31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 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 57T present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site (DeltaattB), 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 DeltaattB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.

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