The form of the DNA substrate required for excisive recombination of bacteriophage lambda.

We have studied the excision reaction of bacteriophage lambda, both in vivo and in vitro, using as a substrate a λatt²(L × R) phage carrying both the right and left-hand prophage attachment sites. Int and Xis are provided by induction of the heat-inducible defective prophage, λc1857 ΔH1. After a brief induction (5 min) of these cells, excisive recombination is blocked in the presence of the DNA gyrase inhibitor, coumermycin. However, after a longer induction (greater than 30 min) excisive recombination occurs efficiently under conditions where λ integrative recombination is inhibited by coumermycin. In such extensively induced coumermycin-treated cells, infecting λatt²(L × R) DNA is not supercoiled, and recombinants are found among the relaxed covalently closed circular DNA.In vitro, starting with a hydrogen-bonded λatt² DNA substrate, excision is insensitive to high concentrations of coumermycin and novobiocin. To study the DNA substrate requirements for excisive recombination in more detail, we have developed a restriction fragment assay for excisive recombination. With this assay, we demonstrate that supercoiled, hydrogen-bonded, and linear λatt² DNA molecules are all efficient substrates in the in vitro excision reaction. Spermidine is required but ATP and Mg²⁺ are not. We conclude that supercoiling is not an absolute requirement for site-specific recombination of λ.     

Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803

The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10^?5 and 8.2 × 10^?5, respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.     

Am E. coli gene product required for λ site-specific recombination

We report characteristics of himA mutations of E. coli, selected for their inability to support the site-specific recombination reaction involved in the formation of lysogens by bacteriophage λ. The himA allele lies at minute 38 on the chromosome. Three noncomplementing and closely linked mutations define the himA locus; one is a nonsense mutation which shows that the gene product is a protein. HimA mutations reduce both λ integrative and excisive site-specific recombination. Since dominance tests demonstrate that himA mutations are recessive, it is probable that the himA protein is either a necessary component for site-specific recombination or, alternatively, regulates the expression of such a function. HimA mutations exhibit pleiotropic effects. They reduce integration of phages that have different attachment specificities from λ and inhibit the growth of phage mu. In addition, himA mutations reduce precise excision of integrated phage mu as well as Tn elements. This pleiotropy suggests that the role of himA protein is nonspecific. Since all of the processes affected by himA mutations ultimately rely on protein-DNA interactions, we suggest that himA protein may act in an auxillary manner to facilitate these interactions.     

Site-specific recombination Xis-independent excisive recombination of bacteriophage lambda

The integration of bacteriophage lambda into the Escherichia coli chromosome depends on the phage-encoded Int protein; prophage excision depends on Int and a second phage function, Xis. Limited excisive recombination has been observed in vivo with certain xis mutants, suggesting that Int may be able to carry out excision without Xis.We report here that purified Int protein carries out lambda site-specific excisive recombination in vitro in the absence of Xis. This reaction requires host factors derived from a non-lysogenic E. coli strain and is influenced strongly by ionic strength. Excision in the absence of Xis occurs slowly at low salt concentrations (40 mm-NaCl) and very little excision occurs at high salt concentrations (100 mm-NaCl). In the presence of Xis, excisive recombination proceeds rapidly at both low and high ionic strengths. These observations are consistent with previous experiments that suggested the partial dispensability of Xis for excision.     

int-h: An int mutation of phage lambda that enhances site-specific recombination

The mutation int-h3 maps in the int gene of coliphage λ and results in the synthesis of an integrase with enhanced activity, which is manifested by an ability to support λ site-specific recombination relatively efficiently under conditions where the wild-type integrase functions inefficiently. The level of site-specific recombination seen in the presence of the int⁺ integrase in himA^? hosts is greatly reduced, as measured by lysogen formation, intramolecular site-specific integration and excision, and excision of a cryptic λ prophage. In contrast, the int-h3 integrase shows relatively high levels of activities under these conditions. Int-h3 is also more active in other host mutants (himB and hip) that reduce λ site-specific recombination. In the absence of the normal attB site, the frequency of lysogen formation (at secondary sites) by λ int⁺ is reduced 200 fold. Although λ int-h3 will integrate preferentially at the attB site if it is present, the mutant phage forms lysogens at a high frequency in attB-deleted hosts. λ int-h3 requires himA function for integration at secondary sites. The fact that the int-h3 integrase uses the same att sites as well as the same host functions as the int⁺ integrase suggests that the mutation results in a quantitative rather than a qualitative change in integrase activity; that is, the int-h3 integrase is more active. The mutant integrase supports site-specific recombination with att sites that carry the att24 mutation. We propose that the int-h3 integrase is endowed with an enhanced ability to recognize att sequences, including some that are not effectively recognized by wild-type integrase.     

The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3′ end of a putative proline tRNA gene

Bacteriophage 16-3 inserts its genome into the chromosome of Rhizobium meliloti strain 41 (Rm41) by site-specific recombination. The DNA regions around the bacterial attachment site (attB) and one of the hybrid attachment sites bordering the integrated prophage (attL) were cloned and their nucleotide sequences determined. We demonstrated that the 51 by region, where the phage and bacterial DNA sequences are identical, is active as a target site for phage integration. Furthermore it overlaps the 3′ end of a putative proline tRNA gene. This gene shows 79% similartiy to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.     

Plastid marker gene excision by the phiC31 phage site-specific recombinase

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

Initiation of synthesis of the structural proteins of Semliki Forest virus.

Insertion of phage λ DNA into the normal attachment site of the DNA of the host Escherichia coli has been studied by ultracentrifugation analysis of the conversion of covalent circles of F′450 (F′gal att^λ bio) to F′450(λ) circles. We have found that integration proceeds at the normal rate if, in addition to the int gene product and a proper combination of phage and bacterial attachment sites, a large pool of λ DNA and some activity of the excision gene xis are present. In addition, turnoff of both phage DNA synthesis and xis gene activity are required.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F₁, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.     

Hotspots for generalized recombination in the Escherichia coli chromosome.

A naturally occurring hotspot for Rec recombination of Escherichia coli was located in the biotin operon. The phenotypes of the bio hotspot as observed in λbio transducing phage were identical to those of Chi mutations in phage λ. In addition to recA⁺ function, the site-specific stimulation of recombination required recB⁺ function. The stimulation took place when the hotspot was present in only one parent of the cross and even when present opposite a region of heterology.The demonstration of a Chi element in E. coli provoked us to measure the density of Chi elements on the chromosome. E. coli DNA sampled in λ transducing phage (either obtained by induction of secondary site lysogens or made in vitro from EcoRI cleavage fragments) showed one hotspot per 5 to 15 × 10³ bases. The high density and the fact that Chi stimulation of recombination can span the inter-Chi distance suggest that Chi might be important in Rec recombination in the absence of λ.     

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host''s chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.     

Attachment site mutants of bacteriophage lambda

Insertion and excision of the chromosome of phage λ occurs by recombination at special regions of the phage and bacterial chromosomes known as attachment sites (alt's). We have isolated att mutants which display reduced recombination frequencies. The mutations are cis-dominant, trans-recessive, and can be crossed into a phage, bacterial or prophage att. These results suggest that the att's, although different over-all, include the same DNA sequence as part of their structure, and that the mutations reside in these sequences. Crosses between mutant and wild-type att's occasionally yield heterozygotes. This result suggests that recombination of the att's generates complementary single-strands via staggered nicks in these common sequences. Recombinant att's are then formed by the interannealing of single-strands of different att's followed by ligation.     

Analysis of the Site-Specific Integration System of the <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> Phage μ1/6

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3′ end of a putative tRNA^Thr gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.     

Attachment site recognition and regulation of directionality by the serine integrases

Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between ‘attachment sites’ in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function.     

Directional control of site-specific recombination by bacteriophage lambda. Evidence that a binding site for Int protein far from the crossover point is required for integrative but not excisive recombination

Phage lambda controls its integration and excision by differential catalysis of the forward and reverse reactions. The lambda Int protein is required for both directions, but Xis for excision only. To investigate the substrate requirements for directional control, we have characterized two mutations of the phage attachment site that are defective in integrative but not excisive recombination. Both of these mutations produce the same base change in the P'3 binding site for Int protein 79 base-pairs from the center of the crossover region for site-specific recombination. We infer that differential utilization of this distant binding site is crucial for directional control of recombination.