Analysis of recombination occurring at SLP1 att sites

Abstract

SLPlINt is a conjugative Streptomyces coelicolor genetic element that can transfer to Streptomyces lividans and integrate site specifically into the genome of the new bacterial host. Recombination of SLP1 previously has been shown to occur within nearly identical 112-base-pair act sequences on the plasmid and host chromosome. We report here that both integrative recombination and intermolecular transfer of SLPlINt require no more than a 48-base-pair segment of the att sequence and that SLP1 transfer occurs by a conservative rather than a replicative mechanism. The functions responsible for the excision of the element as a discrete DNA segment are induced during the conjugal transfer of SLP1.The SLP1 genetic element, which exists in nature as an integrated sequence (i.e., SLPlint) in the Streptomyces coelicolor chromosome, is capable of transfer into Streptomyces lividans and integration into a specific site on the S. lividans genome (1, 27). SLP1-mediated recombination events associated with transfer and integration occur at the attachment sites attP of SLP1 and attB of the host chromosome and at attL and attR, the recombination loci that flank the integrated element. The att sites are nearly identical 112-basepair (bp) sequences; a 1-bp difference occurring at position 95 yields a BclI site in attB and attR that is absent from attP and attL (Fig. 1). We previously have proposed (27,28) that SLPlint is transferred between species as a transiently existing, physically autonomous 17-kilobase (kb) plasmid that integrates into the attB site of the recipient upon entering the cell. Autonomously replicating SLP1-derived extrachromosomal elements (1, 27, 28) can result from deletion of segments of the SLP1 sequence that are required either for integration or for maintenance of SLP1 in the integrated state.Examination of the DNA sequences of the four SLP1 att sites reveals two regions of dyad symmetry (i.e., inverted repeats) (29) (Fig. 1). In several other systems, such inverted repeat sequences serve as loci for site-specific recombination (3,9,12,13,17,18). Both inverted repeats in the SLP1 att segments lie in the region where SLP1-mediated recombination events are known to occur (29). We report here the results of insertion and deletion analyses that identify regions of the att sites essential for SLP1-mediated recombination and localize more precisely the actual site of recombination. We also provide direct evidence that intermolecular transfer of SLPlint involves conservative excision of the 17-kb SLP1 sequence as a discrete DNA segment and show that the functions responsible for such excisive recombination are regulated rather than constitutively expressed. MATERIALS AND METHODSBacterial strains and plasmids. The plasmids pACYC177 (5), pUC18, and pUC19 (37) were used as vectors for cloning in Escherichia coli, and the pIJlOl-derived replicons pIJ364 (22) and pIJ702 (19) were used in S. lividans. E. coli MC1061 (4), S. lividans TK64 (16), and S. lividans 1326 (15) were * Corresponding author. used as cloning hosts...

Genetics ofStreptomyces rimosus, the Oxytetracycline Producer

“…Sedlmeier and collaborators (204) proposed that the lack of tRNA genes with rRNA genes might be characteristic for all actinomycetes. Based on the first reports that initiator tRNAs from Streptomyces griseus and S. rimosus lack CCA ends (74, 128) and on later publications indicating that potential tRNA Pro , tRNA Thr , and tRNA Tyr genes associated with site-specific recombination in Streptomyces also lack CCA termini (132,143,197), Plohl and Gamulin (185) predicted that most, if not all, tRNA genes in S. rimosus do not encode a CCA terminus. At present, this prediction is still valid and in agreement with current analyses of the tRNA genes based on genome sequencing data.…”

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

“…Similar experiments reported previously (18) failed to detect these free circular molecules in nonmating cells, leading to the conclusion that excision is induced by mating. While we found a 2-to 10-fold increase in the abundance of autonomous xis+ SLP1 molecules during mating, the presence of precisely excised SLP1 plasmids in nonmating cells indicates that mating is not required for excision.…”

“…It is now clear that because the attB site lies within the 3' end of a gene encoding a tRNAT3" essential for viability of S. lividans (29), at least part of the 112-bp homology is required to regenerate this gene upon integration. We have shown previously that no more than 48 bp of the 112-bp attB-attP homology is required for a sequence to function as an attB site (18). However, during construction of a substrate for excision assays, we found that, while the Int-expressing plasmid pSUM307 can promote integration of a plasmid that contains a 485-bp segment of SLP1 DNA flanking attP (pSUM317, Fig.…”

Organization of the Bacterial Chromosome

“…For example, a cross in which S. coelicolor A3(2) served as the donor and S. lividans 66 functioned as the recipient revealed the presence of a 17-kb plasmidogenic locus in the former (258). Upon transfer, the locus could exist in the recipient in three forms: transiently as a 17-kb plasmid containing all the genetic content present in the original locus, integrated at a specific site in the S. lividans chromosome (203,259,260), or as one of several smaller plasmids, 11 to 14.5 kb in size (153). In the smaller plasmids, genetic information for integration and maintenance was deleted.…”