Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.     

Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: the Structure of Lactococcal Phage TP901-1

Tailed phages are genome delivery machines exhibiting unequaled efficiency acquired over more than 3 billion years of evolution. Siphophages from the P335 and 936 families infect the Gram-positive bacterium Lactococcus lactis using receptor-binding proteins anchored to the host adsorption apparatus (baseplate). Crystallographic and electron microscopy (EM) studies have shed light on the distinct adsorption strategies used by phages of these two families, suggesting that they might also rely on different infection mechanisms. Here, we report electron microscopy reconstructions of the whole phage TP901-1 (P335 species) and propose a composite EM model of this gigantic molecular machine. Our results suggest conservation of structural proteins among tailed phages and add to the growing body of evidence pointing to a common evolutionary origin for these virions. Finally, we propose that host adsorption apparatus architectures have evolved in correlation with the nature of the receptors used during infection.     

Key players in the genetic switch of bacteriophage TP901-1

After infection of a sensitive host temperate phages may enter either a lytic or a lysogenic pathway leading to new phage assembly or silencing as a prophage, respectively. The decision about which pathway to enter is centered in the genetic switch of the phage. In this work, we explore the bistable genetic switch of bacteriophage TP901-1 through experiments and statistical mechanical modeling. We examine the activity of the lysogenic promoter Pr at different concentrations of the phage repressor, CI, and compare the effect of CI on Pr in the presence or absence of the phage-encoded MOR protein expressed from the lytic promoter Pl. We find that the presence of large amounts of MOR prevents repression of the Pr promoter, verifying that MOR works as an antirepressor. We compare our experimental data with simulations based on previous mathematical formulations of this switch. Good agreement between data and simulations verify the model of CI repression of Pr. By including MOR in the simulations, we are able to discard a model that assumes that CI and MOR do not interact before binding together at the DNA to repress Pr. The second model of Pr repression assumes the formation of a CI:MOR complex in the cytoplasm. We suggest that a CI:MOR complex may exist in different forms that either prevent or invoke Pr repression, introducing a new twist on mixed feedback systems.     

Structure and molecular assignment of lactococcal phage TP901-1 baseplate

P335 lactococcal phages infect the gram(+) bacterium Lactococcus lactis using a large multiprotein complex located at the distal part of the tail and termed baseplate (BP). The BP harbors the receptor-binding proteins (RBPs), which allow the specific recognition of saccharidic receptors localized on the host cell surface. We report here the electron microscopic structure of the phage TP901-1 wild-type BP as well as those of two mutants bppL (-) and bppU(-), lacking BppL (the RBPs) or both peripheral BP components (BppL and BppU), respectively. We also achieved an electron microscopic reconstruction of a partial BP complex, formed by BppU and BppL. This complex exhibits a tripod shape and is composed of nine BppLs and three BppUs. These structures, combined with light-scattering measurements, led us to propose that the TP901-1 BP harbors six tripods at its periphery, located around the central tube formed by ORF46 (Dit) hexamers, at its proximal end, and a ORF47 (Tal) trimer at its distal extremity. A total of 54 BppLs (18 RBPs) are thus available to mediate host anchoring with a large apparent avidity. TP901-1 BP exhibits an infection-ready conformation and differs strikingly from the lactococcal phage p2 BP, bearing only 6 RBPs, and which needs a conformational change to reach its activated state. The comparison of several Siphoviridae structures uncovers a close organization of their central BP core whereas striking differences occur at the periphery, leading to diverse mechanisms of host recognition.     

Analysis of the Collar-Whisker Structure of Temperate Lactococcal Bacteriophage TP901-1

Proteins homologous to the protein NPS (neck passage structure) are widespread among lactococcal phages. We investigated the hypothesis that NPS is involved in the infection of phage TP901-1 by analysis of an NPS⁻ mutant. NPS was determined to form a collar-whisker complex but was shown to be nonessential for infection, phage assembly, and stability.     

Host genetic requirements for DNA release of lactococcal phage TP901-1

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.     

Analysis of the collar-whisker structure of temperate lactococcal bacteriophage TP901-1

Proteins homologous to the protein NPS (neck passage structure) are widespread among lactococcal phages. We investigated the hypothesis that NPS is involved in the infection of phage TP901-1 by analysis of an NPS- mutant. NPS was determined to form a collar-whisker complex but was shown to be nonessential for infection, phage assembly, and stability.     

Crystal Structure and Function of a DARPin Neutralizing Inhibitor of Lactococcal Phage TP901-1: COMPARISON OF DARPin AND CAMELID VHH BINDING MODE*

Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppU·BppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 Å resolution. The DARPin·RBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry for the production of an array of fermented milk products. Several industrial strains are sensitive to various distinct bacteriophages, mostly belonging to the Siphoviridae family. The lactococcal phage population is divided in at least 10 genetically distinct groups, of which the 936, c2, and P335 groups are prominent (1, 2). These L. lactis-infecting phages are considerably problematic in causing milk fermentation failures and resulting in decreased yields as well as low quality products (3). Preventing these infections has proven to be difficult because of lactococcal phage ubiquity, biodiversity, and genomic plasticity (4).Phage infection is initiated by binding of the phage receptor-binding protein (RBP),⁵ located within the baseplate at the distal part of the tail, to its receptor on the host cell surface (5). We have previously solved the crystal structures of the three RBPs of the lactococcal phages p2 (936) (6), bIL170 (936) (7), TP901-1 (P335) (8), and their chimera (9) as well as characterized their saccharide binding sites (10). The RBPs of these phages have a similar homotrimeric architecture related by a 3-fold axis. They comprise three domains: the N terminus shoulder domain, the interlaced β-prism neck domain, and the jellyroll head domain at the C terminus. The head domain has a saccharide binding site likely involved in host recognition. The lactococcal phage TP901-1 contains a double-disk-shaped baseplate at the tip of its tail which is made of a lower baseplate protein (BppL) and an upper baseplate protein (BppU) (11).One strategy to minimize bacteriophage infections is to competitively block phage adsorption by adding a protein that specifically binds to the phage RBP. A neutralizing llama VHH domain recognizing the head domain of the phage p2 RBP has been used to block L. lactis phage infection in milk fermentation (12). Lactococcal phages could readily escape neutralization by generating mutations interfering with VHH binding over the large interaction surface while keeping the central polysaccharide receptor binding pocket intact (10). Designed ankyrin repeat proteins (DARPins) may be another tool to neutralize viral infection, as they display distinct characteristics from VHHs and contain the required properties in terms of stability and facility of expression (13).Ankyrin repeat proteins are found in virtually all phyla and mediate specific protein-protein interactions in all cell compartments (14). The ankyrin elementary module is composed of 33 amino acids structured as a β-turn followed by two antiparallel α-helices and a loop connected to the β-turn of the next repeat. The repeats are stacked in a rigid manner. In creating a DARPin library, residues in each repeat were subdivided in two groups; (i) randomized residues constituting potential target interaction points and (ii) framework residues, important for maintaining the ankyrin fold (13). Libraries with varying repeat numbers were assembled and named according to the constituent repeat number; N2C and N3C libraries were used in this study, with two and three internal repeats inserted between the N and C capping repeats, respectively. DARPins are a powerful alternative to the use of antibodies, notably because of their very high expression rates in Escherichia coli, their high stability paired with high affinity, and successful reports of their use in co-crystallization (15–19). Their architecture results in a very rigid structure that facilitates multiple crystal contacts and may promote crystal formation of the protein of interest by providing additional surfaces for such crystal contacts.We report here the selection and analysis of DARPin binders directed against a macromolecular multiprotein ensemble, the TP901-1 baseplate BppU·BppL protein complex. Ribosome display selection, ELISA screening, and surface plasmon resonance (SPR) measurements allowed us to isolate and characterize three N2C DARPins that recognized the RBP (BppL of the BppU·BppL complex) with high specificity and affinity. Further studies showed that the three DARPins bound to a unique area of the RBP at the tip of the head domain. QELS, MALS, UV, and refractometry coupled online with a size exclusion chromatography (SEC) column allowed us to monitor complex formation in solution as well as to estimate DARPin binding stoichiometry. Crystals of one of these selected DARPins in complex with the RBP were obtained, and the x-ray structure was solved at 2.1 Å resolution. This constitutes the first structure of a DARPin complex originating from the N2C library and the highest resolution for a DARPin complex structure reported to date. Finally, phage adsorption inhibition experiments demonstrated that the three N2C DARPins strongly inhibited L. lactis infection by TP901-1. We describe the DARPin·RBP interface and compare it to other DARPin interfaces. We also compare it to the p2 RBP·VHH5 complex, a previously selected llama VHH domain inhibiting p2 phage adsorption (12), to highlight the different binding mode of these two types of binders.     

Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated into attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.     

Structural characterization and assembly of the distal tail structure of the temperate lactococcal bacteriophage TP901-1

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate Lactococcus lactis phage TP901-1 of the Siphoviridae family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes dit (orf46), tal(TP901-1) (orf47), bppU (orf48), bppL (orf49), and orf50. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal(TP901-1) were shown to be structural tail proteins essential for tail formation, and Tal(TP901-1) was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.     

Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB(+) lines with a dual-plasmid system, expressing a transgene on an attP-containing plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB x attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.     

Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells

Aims:  To investigate if the site-specific tyrosine integrase (Int) from phage P2 has features that would make it interesting for use of gene transfer into eukaryotic cells. These include the possibility of promoting recombination with a nonphage sequence, abolishing the requirement for the bacterial DNA-binding and -bending protein integration host factor (IHF), and localization to the nucleus of eukaryotic cells.
Methods and Results:  We show that the Int protein catalyzes site-specific recombination using a human sequence in Escherichia coli and in vitro although not as efficiently as with the wild-type bacterial sequence, and that insertion of high mobility group recognition boxes in the phage attachment site substrate abolish the requirement of IHF and allows efficient recombination in vitro in a eukaryotic cell extract. Furthermore, we show by fluorescence that the Int protein contains a functional intrinsic nuclear localization signal, localizing it to the nucleus in both HeLa and 293 cells.
Conclusions:  We conclude that P2 Int may be a potential tool for site-specific integration of genes into the human chromosome.
Significance and Impact of the Study:  The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes.     

Cleavage properties of an archaeal site-specific recombinase, the SSV1 integrase

SSV1 is a virus infecting the extremely thermophilic archaeon Sulfolobus shibatae. The viral-encoded integrase is responsible for site-specific integration of SSV1 into its host genome. The recombinant enzyme was expressed in Escherichia coli, purified to homogeneity, and its biochemical properties investigated in vitro. We show that the SSV1 integrase belongs to the tyrosine recombinases family and that Tyr(314) is involved in the formation of a 3'-phosphotyrosine intermediate. The integrase cleaves both strands of a synthetic substrate in a temperature-dependent reaction, the cleavage efficiency increasing with temperature. A discontinuity was observed in the Arrhenius plot above 50 degrees C, suggesting that a conformational transition may occur in the integrase at this temperature. Analysis of cleavage time course suggested that noncovalent binding of the integrase to its substrate is rate-limiting in the cleavage reaction. The cleavage positions were localized on each side of the anticodon loop of the tRNA gene where SSV1 integration takes place. Finally, the SSV1 integrase is able to cut substrates harboring mismatches in the binding site. For the cleavage step, the chemical nature of the base in position -1 of cleavage seems to be more important than its pairing to the opposite strand.     

Characterization of site-specific recombination by the integrase MJ1 from enterococcal bacteriophage phiFC1

Bacteriophage phiFC1 integrase (MJ1) was previously shown to perform a site-specific recombination between a phage attachment site (attP) and a host attachment site (attB) in its host, Enterococcus faecalis, and also in a non-host bacterium, Escherichia coli. Here, we investigated biochemical features of MJ1 integrase. First, MJ1 integrase could perform in vitro recombination between attP and attB in the absence of additional factors. Second, MJ1 integrase interacted with att sites. Electrophoretic mobility shift assays and DNase I footprinting revealed that MJ1 integrase could efficiently bind to all the att sites and that MJ1 integrase recognized relatively short sequences (approximately 50 bp) containing an overlapping region within attB and attP. These results demonstrate that MJ1 integrase indeed catalyzes an integrative recombination between attP and attB, the mechanism of which might be simple and unidirectional, as found in serine integrases.     

PhiC31 integrase induces efficient site-specific excision in zebrafish

Site-specific recombinases catalyze recombination between specific targeting sites to delete, insert, invert, or exchange DNA with high fidelity. In addition to the widely used Cre and Flp recombinases, the phiC31 integrase system from Streptomyces phage may also be used for these genetic manipulations in eukaryotic cells. Unlike Cre and Flp, phiC31 recognizes two heterotypic sites, attB and attP, for recombination. While the phiC31 system has been recently applied in mouse and human cell lines and in Drosophila, it has not been demonstrated whether it can also catalyze efficient DNA recombination in zebrafish. Here we show that phiC31 integrase efficiently induces site-specific deletion of episomal targets as well as chromosomal targets in zebrafish embryos. Thus, the phiC31 system can be used in zebrafish for genetic manipulations, expanding the repertoire of available tools for genetic manipulation in this vertebrate model.