Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans

Abstract

SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage A integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf6M) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.Streptomyces are gram-positive filamentous soil bacteria notable for their complex morphology as well as the production of a variety of antibiotics and other secondary metabolites, many of which have medical or agricultural importance. Numerous extrachromosomal elements have been identified for these organisms, including a novel family of chromosomally integrated self-transmissible plasmidogenic genetic elements (reviewed in reference 35). SLP1, indigenous to the chromosome of Streptomyces coelicolor (6), was the first of this group to be identified. A model in which the integrated SLP1 element (i.e., SLPlr') can excise from the S. coelicolor chromosome during conjugation with a strain lacking SLP1, transfer to the recipient strain as a transiently existing circular molecule, and either integrate into the recipient chromosome at a site identical to that occupied in the donor strain or undergo deletion of sequences that suppress autonomous replication and consequently be maintained as an autonomously replicating plasmid (33) has been proposed.The recombination reactions mediated by SLP1 are sitespecific and involve 112-bp regions of homology on SLP1 (attP) and the host chromosome (attB) that have only a single-base-pair difference (34). The integration event generates sites at the left (attL) and right (attR) of the integrated element that retain the homologies between attP and attB. Characterization of these sequences (29) integrants. A derivative of SLP1 containing an insertion at a KpnI site 1.3 kb from attP ( Fig. 1), within a locus designated intB, fails to integrate. In addition, integration of SLP1-derived plasmids deleted for a second locus (i.e., intA, Fig. 1) situated at some distance from attP and intB was not observed. This defect could be c...

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

“…pSUM352 was constructed similarly: pSUM49 is pBR322 (6) Figure 1 summarizes the genetic organization of the segment of SLP1 involved in site-specific recombination. We have shown that the int gene encodes a product necessary for integration and have speculated that the open reading frame orf6l encodes a protein implicated in excision (7). To test directly the roles of orf6l and the int gene in SLP1 excision, we expressed Int and Orf6l both separately and concurrently in S. lividans and monitored excision of a nonreplicating hygromycin resistance (Hygr) plasmid that had been integrated site-specifically at the SLP1 attB site.…”

“…lividans as well as the conditions used for Southern blot analysis have been described elsewhere (7). The ( Fig.…”

“…In the accompanying report (7), we demonstrate that a 2.2-kb segment of SLP1 contains all of the functions required for site-specific integration of SLP1 into the Streptomyces lividans chromosome. Nucleotide sequence analysis of this DNA revealed two open reading frames, one of which encodes a putative 50.5-kDa protein having substantial similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage X integrase.…”

Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector

“…For many int genes described, the ORF is immediately preceded by or overlaps an ORF which is defined in most instances as, or speculated to be, the excisionase (xis) gene. Up to now, no ORF sharing similarity with the described Streptomyces excisionase proteins (Boccard et al, 1989;Brasch et al, 1993;Brown et al, 1990Brown et al, , 1994Gabriel et al, 1995) could be detected in the sequence surrounding VWB int.…”

“…(including two plasmids of Saccharopolyspora erythraea, previously classified as Streptomyces erythraeus) most often integrate site-specifically into the host chromosome (Bar-Nir et al, 1992;Boccard et al, 1988;Brasch et al, 1993;Brown et al, 1990Brown et al, , 1994Gabriel et al, 1995;Kendall & Cullum, 1986;Shirai et al, 1991;Sosio et al, 1989). In general, such site-specific integration is catalysed by a sitespecific recombinase.…”