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To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase.

ϕC31, ϕBT1, R4, and TG1 are temperate bacteriophages with broad host specificity for species of the genus Streptomyces. They form lysogens by integrating site-specifically into diverse attB sites located within individual structural genes that map to the conserved core region of streptomycete linear chromosomes. The target genes containing the ϕC31, ϕBT1, R4, and TG1 attB sites encode a pirin-like protein, an integral membrane protein, an acyl-CoA synthetase, and an aminotransferase, respectively. These genes are highly conserved within the genus Streptomyces, and somewhat conserved within other actinomycetes. In each case, integration is mediated by a large serine recombinase that catalyzes unidirectional recombination between the bacteriophage attP and chromosomal attB sites. The unidirectional nature of the integration mechanism has been exploited in genetic engineering to produce stable recombinants of streptomycetes, other actinomycetes, eucaryotes, and archaea. The ϕC31 attachment/integration (Att/Int) system has been the most widely used, and it has been coupled with the ϕBT1 Att/Int system to facilitate combinatorial biosynthesis of novel lipopeptide antibiotics in Streptomyces fradiae.

The 113,463-bp nucleotide sequence of the linear plasmid pSLA2-M of Streptomyces rochei 7434AN4 was determined. pSLA2-M had a 69.7% overall GC content, 352-bp terminal inverted repeats with 91% (321/352) identity at both ends, and 121 open reading frames. The rightmost 14.6-kb sequence was almost (14,550/14,555) identical to that of the coexisting 211-kb linear plasmid pSLA2-L. Adjacent to this homologous region an 11.8-kb CRISPR cluster was identified, which is known to function against phage infection in prokaryotes. This cluster region as well as another one containing two large membrane protein genes (orf78 and orf79) were flanked by direct repeats of 194 and 566 bp respectively. Hence the insertion of circular DNAs containing each cluster by homologous recombination was suggested. In addition, the orf71 encoded a Ku70/Ku80-like protein, known to function in the repair of double-strand DNA breaks in eukaryotes, but disruption of it did not affect the radiation sensitivity of the mutant. A pair of replication initiation genes (orf1-orf2) were identified at the extreme left end. Thus, pSLA2-M proved to be a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L that might have been generated by multiple recombination events.

Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.

Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage (attP(TG1)) and bacterial (attB(TG1)) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis. The attB(TG1) was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N-succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis, however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attB(TG1) sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus. The gene encoding the putative TG1 integrase (int(TG1)) was located adjacent to the attP(TG1) site. TG1 integrase deduced from the int(TG1) gene was a protein of 619 amino acids having a high sequence similarity to phiC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific recombination systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli.

Gene targeting allows precise tailoring of the mouse genome such that desired modifications can be introduced under precise temporal and spatial control. This can be achieved through the use of site-specific recombinases, which mediate deletion or inversion of genomic DNA flanked by recombinase-specific recognition sites, coupled with gene targeting to introduce the recombinase recognition sites at the desired genomic locations within the mouse genome. The introduction of multiple modifications at the same locus often requires use of multiple recombination systems. The most commonly used recombination system is Cre/lox. We here evaluated in vivo the ability of PhiC31 phage integrase to induce a genomic deletion in mouse. We engineered a self-excision cassette, modeled after one previously designed for Cre, containing a positive selection marker and PhiC31 driven by a testis-specific promoter, all flanked by PhiC31 specific attP/B sites. We found in vivo PhiC31 mediated self-excision in 38% of transmitted alleles, although 18% of these showed evidence of imprecise deletion. Furthermore, in the 69% of un-recombined cassettes, sequence analysis revealed that PhiC31 mediated an intra-molecular deletion of the attB site preventing any subsequent recombination. This study demonstrates that PhiC31 can be used to automatically remove Neo, in the male chimera germline, although it is not as efficient or as accurate as Cre.

Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

Streptomyces PhiC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the PhiC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.

Bacteriophage C31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, C31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of C31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein-protein interactions.

We have established the integrase of the Streptomyces phage phiBT1 as a tool for eukaryotic genome manipulation. We show that the phiBT1 integrase promotes efficient reciprocal and conservative site-specific recombination in vertebrate cells and in Schizosaccharomyces pombe, thus establishing the utility of this protein for genome manipulation in a wide range of eukaryotes. We show that the phiBT1 integrase can be used in conjunction with Cre recombinase to promote the iterative integration of transgenic DNA. We describe five cycles of iterative integration of a candidate mouse centromeric sequence 80 kb in length into a human mini-chromosome within a human-Chinese hamster hybrid cell line. These results establish the generality of the iterative site-specific integration technique.

We have used the phiC31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than approximately 150 kb we have established a method that we call; 'Iterative Site Specific Integration' (ISSI). ISSI combines the activities of phiC31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.

We have investigated the ability of the integrase from the Streptomyces phiC31 'phage to either delete or invert 1 Mb of DNA around the centromere of the human Y chromosome in chicken DT40 hybrid somatic cells. Reciprocal and conservative site-specific recombination was observed in 54% of cells expressing the integrase. The sites failed to recombine in the remaining cells because the sites had been damaged. The sequences of the damaged sites indicated that the damage arose as a result of repair of recombination intermediates by host cell pathways. The liability of recombination intermediates to damage is consistent with what is known about the mechanism of serine recombinase reactions. The structures of the products of the chromosome rearrangements were consistent with the published sequence of the Y chromosome indicating that the assembly of the highly repeated region between the sites is accurate to a resolution of about 50 kb. Mini-chromosomes lacking a centromere were not recovered which also suggested that neo-centromere formation occurs infrequently in vertebrate somatic cells. No ectopic recombination was observed between a phiC31 integrase attB site and the chicken genome.

The Streptomyces phage phiC31 encodes an integrase belonging to the serine recombinase family of site-specific recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5' ends. Rotation of one pair of half sites by 180 degrees relative to the other pair occurs, to form the recombinant configuration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by phiC31 integrase when acting on its substrates attP and attB. We have identified intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identified. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we confirmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.

The integrase of the phage PhiC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of PhiC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved PhiC31-integrase as a new tool for genome engineering.

Aims: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil., Methods and Results: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency., Conclusions: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil., Significance and Impact of the Study: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.

The completed genome sequence of the temperate Streptomyces phage straight phiC31 is reported. straight phiC31 contains genes that are related by sequence similarities to several other dsDNA phages infecting many diverse bacterial hosts, including Escherichia, Arthrobacter, Mycobacterium, Rhodobacter, Staphylococcus, Bacillus, Streptococcus, Lactobacillus and Lactococcus. These observations provide further evidence that dsDNA phages from diverse bacterial hosts are related and have had access to a common genetic pool. Analysis of the late genes was particularly informative. The sequences of the head assembly proteins (portal, head protease and major capsid) were conserved between straight phiC31, coliphage HK97, staphylococcal phage straight phiPVL, two Rhodobacter capsulatus prophages and two Mycobacterium tuberculosis prophages. These phages and prophages (where non-defective) from evolutionarily diverse hosts are, therefore, likely to share a common head assembly mechanism i.e. that of HK97. The organisation of the tail genes in straight phiC31 is highly reminiscent of tail regions from other phage genomes. The unusual organisation of the putative lysis genes in straight phiC31 is discussed, and speculations are made as to the roles of some inessential early gene products. Similarities between certain phage gene products and eukaryotic dsDNA virus proteins were noted, in particular, the primase/helicases and the terminases (large subunits). Furthermore, the complete sequence clarifies the overall transcription map of the phage during lytic growth and the positions of elements involved in the maintenance of lysogeny.

Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage phiC31. Because expression of the two smaller isoforms, 54 and 42 kDa, is sufficient for superinfection immunity, the interaction between these isoforms was studied. The native 42 kDa repressor (Nat42) and an N-terminally 6x histidine-tagged 54 kDa isoform (His54) were shown by co-purification on a Ni-NTA column to interact in Streptomyces lividans . In vitro three repressor preparations, containing Nat42, His54 and the native 54 and 42 kDa isoforms expressed together (Nat54&42), were subjected to chemical crosslinking and gel filtration analysis. Homo- and hetero-tetramers were observed. Previous work showed that the smallest isoform bound to 17 bp operators containing aconservedinvertedrepeat (CIR) and that the CIRs were located at 16 loci throughout the phiC31 genome. One of the CIRs (CIR6) is believed to be critical for regulating the lytic pathway. The DNA binding activities of the three repressor preparations were studied using fragments containing CIRs (CIR3-CIR6) from the essential early region as templates for DNase I footprinting. Whereas Nat42 bound to CIR6, poorly to CIR5 but undetectably to CIR3 or CIR4, the Nat54&42 preparation could bind to all CIRs tested, albeit poorly to CIR3 and CIR4. The His54 isoform bound all CIRs tested. Isoforms expressed from the phiC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

Plasmid pTO1 containing the oriT fragment from RK2, the Escherichia coli replication function from pBR322, and a DNA fragment of actinophage phi C31 with the attachment site was transferred from E. coli S17-1 to strains of the genera Actinomadura, Arthrobacter, Micromonospora, Nocardia, Rhodococcus, and to 16 strains of the genus Streptomyces. The frequency of conjugant formation was 1 x 10(-3)-1 x 10(-5) depending on the strain. Hybridization experiments demonstrated that plasmid pTO1 integrates into chromosomes of a number of the recipient strains examined.

A cosmid, pR4C1, composed of the actinophage R4 cos sequence and Streptomyces plasmid pIJ365, was encapsidated in R4 phage particles in vivo. [T. Morino et al., Mol. Gen. Genet., 198, 228-233 (1985)] In this report a cosmid derivative, pR4C4, is shown to be also encapsidated by heterogeneous actinophages, SPA10 and SPA38, and transferred to Streptomyces lividans. Use of this transduction and conservation of the DNA packaging mechanism are discussed.

Previous work has identified three intergenic regions from the early region of actinophage øC31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated eta, etb and etc. Transcripts through eta-c would be expected to form stable RNA stem-loops but would lack poly-U tails. Eta-c contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by eta-c from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphII). In pUGT1 etb was at best a minor terminator in vivo whilst eta and etc exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing eta-c inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies eta > etc > etb. As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at etb was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of etc and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Eta was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of eta inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that eta-c are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.

The temperate actinophage RP3 integrates site-specifically into the chromosome of Streptomyces rimosus R6-554. The phage attachment site attP and the hybrid attachment sites of the integrated prophage--attL and attR--were cloned and sequenced. The 54nt core sequence, common to all RP3 related attachment sites, comprises the 3' terminal end of a putative tRNA(Arg)(AGG) gene. AttB bears the complete tRNA gene which is restored in attL after integration. A 7.5kb HindIII fragment, bearing attP, was used to construct an integrative plasmid to simulate the integration process in vivo and to localize the phage genes necessary for site specific integration. The int and xis genes were sequenced and compared to other recombination genes.