Margaret C. M. Smith

Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31.

The genome of the Streptomyces temperate phage φC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, φC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non‐permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non‐permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.

Bacteriophages with tails: chasing their origins and evolution.

Comparative genomic analysis of the tailed bacteriophages shows that they are genetically mosaic with respect to each other, implying that horizontal exchange of sequences is an important component of their evolution. Horizontal exchange occurs intensively among closely related phages but also at reduced frequency across the entire population of tailed phages. It results in exchange of homologous functions, exchange of analogous but non-homologous functions as with the prophage integrases, and introduction of novel functions into the genome as with the morons. Extrapolation of these processes back in evolutionary time leads to a speculative model for the origins and early evolution of phages.

A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination

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.

Switching the polarity of a bacteriophage integration system

During lysogenic growth many temperate bacteriophage genomes are integrated into the hosts chromosome and efficient integration and excision are therefore an essential part of the phage life cycle. The Streptomyces phage φC31 encodes an integrase related to the resolvase/invertases and is evolutionarily and mechanistically distinct from the integrase of phage λ. We show that during φC31 integration the polarity of the recombination sites, attB and attP, is dependent on the sequences of the two base pairs (bp) where crossover occurs. A loss or switch in polarity of the recombination sites can occur by mutation of this dinucleotide, leading to incorrectly joined products. The properties of the mutant sites implies that φC31 integrase interacts symmetrically with the substrates, which during synapsis can align apparently freely in either of two alternative forms that lead to correct or incorrect joining of products. Analysis of the topologies of the reaction products provided evidence that integrase can synapse and activate strand exchange even when recombinant products cannot form due to mismatches at the crossover site. The topologies of the recombination products are complex and indicative of multiple pathways to product formation. The efficiency of integration of a φC31 derivative, KC859, into an attB site with switched polarity was assayed in vivo and shown to be no different from integration into a wild‐type attB. Thus neither the host nor KC859 express a factor that influences the alignment of the recombination sites at synapsis.

Sequences in attB that affect the ability of ϕC31 integrase to synapse and to activate DNA cleavage

Phage integrases are required for recombination of the phage genome with the host chromosome either to establish or exit from the lysogenic state. ϕC31 integrase is a member of the serine recombinase family of site-specific recombinases. In the absence of any accessory factors integrase is unidirectional, catalysing the integration reaction between the phage and host attachment sites, attP × attB to generate the hybrid sites, attL and attR. The basis for this directionality is due to selective synapsis of attP and attB sites. Here we show that mutations in attB can block the integration reaction at different stages. Mutations at positions distal to the crossover site inhibit recombination by destabilizing the synapse with attP without significantly affecting DNA-binding affinity. These data are consistent with the proposal that integrase adopts a specific conformation on binding to attB that permits synapsis with attP. Other attB mutants with changes close to the crossover site are able to form a stable synapse but cleavage of the substrates is prevented. These mutants indicate that there is a post-synaptic DNA recognition event that results in activation of DNA cleavage.

Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage ϕBT1

We have established the integrase of the Streptomyces phage ϕBT1 as a tool for eukaryotic genome manipulation. We show that the ϕBT1 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 ϕBT1 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.

Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage φC31

Mutants of Streptomyces coelicolor A3(2) J1929 (ΔpglY) were isolated that were resistant to the Streptomyces temperate phage φC31. These strains could be transfected with φC31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate‐d‐mannose:protein O‐d‐mannosyltransferases. Concanavalin A (ConA) inhibited φC31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, α‐d‐methyl‐pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to φC31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, φDT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the φC31 receptor is a cell wall glycoprotein, and that the φC31h mutation compensates for the lack of glycosylation of the receptor.

Control of lytic development in the Streptomyces temperate phage phi C31.

The repressor gene. c, is required for maintenance of lysogeny in the Streptomyces phage φC31. The c gene expresses three in‐frame N‐terminally different protein isoforms at least one of which is thought to bind to a 17bp highly conserved inverted repeat (CIR) sequence found at 18 (or more) loci throughout the φC31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in φC31. To the right of CIR6, according to the standard map of φC31, an ‘immediate‐early’ promoter. ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin‐resistance gene. aphll, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a φC31 lysogen of S. coelicolor. Operator‐constitutive (Oc) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature‐sensitive lysogen of S. coelicolor localized a mRNA 5′ endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, φC31 vir1, capable of forming plaques on an S. coelicolorφC31 lysogen, φC31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7kbp to the right of CIR6) and in the essential early region between CIR6 and the ‐10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in φC31 vir1. Band‐shift assays and DNase I footprinting experiments using purified 42kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high‐affinity binding to CIR6 was abolished with the Oc mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor–CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.

3 Exploitation of Bacteriophages and their Components

Publisher Summary This chapter discusses the exploitation of bacteriophages for genetic analysis and in vitro recombinant DNA techniques in diverse bacterial species. Bacteriophages are obligate intracellular parasites and are uniquely adapted to growth in their hosts. They will inevitably, therefore, encode genetic elements that are easily accessible as tools for the genetic analysis of their hosts. The numbers of papers relating to phage research would indicate that the interest in phage has not peaked. Indeed the numbers of papers reporting new integrases are increasing at an apparently exponential rate. As microbiology moves into detailed analysis of diverse species, examining their evolution, ecology and physiology, phages are both integral to these processes (e.g. the movement of pathogenicity islands in the evolution of harmful bacteria) and easily exploitable. Phages are the most numerous and most diverged entities on this planet. This must be a good place to look for new genetic tools.

Chromosome engineering in DT40 cells and mammalian centromere function

Chromosome engineering is the term given to procedures which modify the long range structure of a chromosome by homologous and site specific recombination or by telomere directed chromosome breakage. DT40 cells are uniquely powerful for chromosome engineering because mammalian chromosomes may be moved into them, efficiently modified and then moved back into a mammalian cell lines (Dieken et al., 1996). The high rate of sequence targeting seen in DT40 cells carrying human chromosomes is necessary but not sufficient for chromosome engineering. The ability to either delete or introduce long tracts of DNA subsequent to a sequence targeting reaction depends upon the use of site specific recombinases. We have made important progress in the development of this technology in the past few years and much of this review will be used to describe this work.