Excisive recombination of the SLP1 element in Streptomyces lividans is mediated by Int and enhanced by Xis

Abstract

The functions mediating site-specific recombination of the SLP1 element have been mapped to a 2.2-kb region that includes the site of integration (attP), a gene (int) that specifies a function both necessary and sufficient for integration of SLP1, and an open reading frame, orf61, suspected of encoding a protein, Xis, that shows limited similarity to the excisionases of other site-specific recombination systems. Here we describe experiments that investigate the respective roles of orf61 and int in the excision of SLP1. We constructed derivatives of the high-copy-number Streptomyces plasmid pIJ101 that express orf61, int, or both orf61 and int from transcriptional fusions to the Tn5 aph gene and tested the ability of these constructs to promote excision of an adventitious attP-containing plasmid that had been integrated site-specifically into the attB site of the Streptomyces lividans chromosome. Expression of the int gene product alone from an exogenous promoter was sufficient for excision of the integrated plasmid. This result indicates that the SLP1 int-encoded protein can carry out excisive, as well as integrative, recombination. The orf61 gene product, when expressed from an exogenous promoter, inhibited int-mediated integration at the chromosomal attB site. Moreover, under conditions in which excision and transfer normally occur, precise excision of SLP1 was enhanced by the orf61-encoded protein. On the basis of these findings, we here designate the orf61 gene as xis.