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 or16 and int in the excision of SLP1. We constructed derivatives of the high-copy-number Streptomyces plasmid pU101 that express ori6, int, or both orf6 and int from transcriptional fusions to the TnS 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 atiB 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 orf6l gene product, when expressed from an exogenous promoter, inhibited int-mediated integration at the chromosomal atB site. Moreover, under conditions in which excision and transfer normally occur, precise excision of SLP1 was enhanced by the or61-encoded protein. On the basis of these findings, we here designate the or61 gene as xis.SLP1, a 17.2-kbp DNA segment indigenous to the chromosome of Streptomyces coelicolor, is the prototype of a family of transmissible chromosomally integrating plasmidogenic genetic elements of the actinomyces (reviewed in references 1, 2, and 23). 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. The product of this open reading frame was shown to be both necessary and sufficient for integration of SLP1 in S. lividans and accordingly has been designated as the Int protein. The second open reading frame, orf6l, which lies immediately 5' to int, corresponds to a 61-aminoacid basic peptide showing limited similarity to the excisionase (xis) gene products of other site-specific recombination systems.In other recombination systems that encode proteins resembling the int and orf6l gene products, integration requires, in addition to host factors, only integrase, whereas excision ordinarily requires both integrase and an excisionase protein (1,2,14,19). We report here the results of studies that analyze the respective roles of the SLP1 int and orf6l genes in excisive recombination. We find that, under conditions in which the integrase is expressed under the contr...

Actinomycete integrative and conjugative pMEA-like elements of Amycolatopsis and Saccharopolyspora decoded

“…An excision-ase homologue appears to be absent on pSE222. Conceivably, its integrase can carry out both integration and excision, as shown for the integrase of SLP1 (Brasch and Cohen, 1993). The integrases of SLP1 and pSE222 only show 23% (84/352) identity to each other.…”

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

“…Additional high-molecular-weight bands in lanes 1 and 5 result from incomplete digestion. functional analysis of these genes as well as similarity of the derived amino acid sequences to those of known recombinases, we believe that the putative 455-amino-acid product of the int gene is an integrase while the product of the orf6l gene, a putative 61-amino-acid peptide, is an excisionase (9).…”

“…A summary of this region is presented in Fig. 3a [2] and extending to Scal [9]. orf6l initiates at nt 132 and terminates at nt 315. orf455 (also designated int) initiates at nt 378 and terminates at nt 1743.…”

“…The nucleotide sequence (Fig. 3b) of a 2.2-kb region of SLP1 DNA beginning just upstream of the BstBI site [2] and extending to the ScaI site [9] was determined. Two likely ORFs provisionally designated orf6l and orf455 were identified with the Codon Preference program of the University of Wisconsin Genetics Computer Group software package.…”

“…To confirm that the product of the int gene is responsible for the ability of the 2-kb HindIII [3]-ScaI [9] fragment to promote integration and to support the assignment of ATG-378 as the start of this gene, we constructed a derivative of pSUM154 that contains a nonsense codon at a position corresponding to amino acid 4 of the putative Int protein. This was accomplished by inserting a 14-bp linker that contains nonsense codons in each of the three reading frames (Materials and Methods) at the Spll site (position 388, Fig.…”