The genome of the broad host range Streptomyces temperate phage, C31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase͞invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP͞B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB͞attB, attP͞attP, attL͞R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL͞R recombination) requires an additional phage-or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in C31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases͞invertases.In site-specific recombination a recombinase interacts with a specific site in the DNA, brings the sites together in a synapse, and then catalyzes strand exchange so that the DNA is cleaved and religated to opposite partners (1, 2). The reaction can result in integration, inversion, or resolution͞excision depending on the position and orientation of the recombination sites, their interactions with recombinase, and the presence or absence of accessory factors or sites. Site-specific recombinases in bacteria fall into one of two very distinct families, the integrase-like enzymes and the resolvase͞invertases, on the basis of amino acid sequence similarities and their different mechanisms of catalysis (1-3). Recombination by members of the integrase family (e.g., integrase, P1 Cre-loxP) is well understood and involves the formation and resolution of a Holliday junction intermediate during which the DNA is transiently attached to the enzyme through a phosphotyrosine linkage (4-6). The resolvase͞invertase family of enzymes (e.g., Tn3 or ␥␦ resolvases, Mu Gin invertase) act via a concerted, four-strand staggered break and rejoining mechanism during which a phosphoserine linkage is formed between the enzyme and the DNA (2, 7). The crystal structure of ␥␦ resolvase bound to a cleavage site reveals a unique arrangement of the catalytic and DNA-binding domains in that they bind to different faces of the helix (8). Although two models have been proposed (9-11), the structure of the synapse and the changes in the conformation of...
Summary Most site‐specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases or λ integrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N‐terminal catalytic domain and a C‐terminal DNA binding domain. Phage‐encoded serine recombinases (e.g. φC31 integrase) cause integration and excision with strictly controlled directionality, and have an N‐terminal catalytic domain but much longer C‐terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N‐terminal to the catalytic domain (e.g. IS607 transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607 transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.
Despite extensive similarities between the genomes of the Streptomyces temperate phages C31 and BT1, the attP-int loci are poorly conserved. Here we demonstrate that BT1 integrates into a different attachment site than C31. BT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein.Integration vectors based on BT1 integrase were shown to have a broad host range and are fully compatible with those based on the C31 attP-int locus.The attP-int locus from C31 has been heavily exploited in the construction of versatile, low-copy-number, and convenient vectors for use in a broad range of Streptomyces species (5, 10). Despite their wide use and clear advantages, it has been reported that integration of these vectors into the C31 attB site can cause detrimental effects on antibiotic production in some strains (2). C31 integrates intragenically into SCO3798, a highly conserved gene in prokaryotes and eukaryotes but is not essential for the growth of Streptomyces coelicolor in the laboratory (8). Although some phages can regenerate a functional gene after insertion (7), there is no evidence that this is the case with C31. Furthermore, a vector, pSET152 containing the C31 attP-int locus, introduced by conjugation from Escherichia coli can integrate into secondary or pseudo-attB sites in both S. coelicolor and Streptomyces lividans (8). The reported reductions in antibiotic synthesis could be caused by insertional mutagenesis into SCO3798 or by integration into one of the pseudo-attB sites or some other factor. Another potential problem with integrating vectors could be the absence of an efficiently recognized attB site in some streptomycete strains. Indeed, Saccharopolyspora erythraea appears to lack a C31 attB site (P. Leadlay, personal communication). For these reasons and as many workers would like to use two compatible integrating vectors in the same organism, we have investigated the integration site of the Streptomyces phage BT1, a homoimmune relative of C31. We demonstrate that BT1 does indeed integrate into a different attB site in S. coelicolor, and we have constructed novel integrating vectors derived from the BT1 attP-int locus.The organization of the BT1 genome is highly similar to that of C31, and the majority of gene products are closely related (9). There is evidence, however, of mosaicism between the two genomes where DNA has been inserted and/or deleted in one genome but not in the other, and there are sudden transitions in the level of sequence similarity (9). One of the most noticeable differences is the relatively poor sequence similarity of int and the three genes upstream, genes 26 to 28. BT1 integrase and gp26 to gp28 exhibit 26% and 10 to 18% identity to their C31 homologues, respectively. Despite this poor similarity, BT1 integrase is clearly a member of the large serine recombinase family, as it contains conserved motifs present in other members of this group (12). Furthermore, no significant similarity could be detected between the C31 attP site and any BT1 sequence....
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