Integrase-mediated Recombination Research Articles

Cassette recombination dynamics within chromosomal integrons are regulated by toxin-antitoxin systems.

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here, we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

A novel approach for Escherichia coli genome editing combining in vivo cloning and targeted long-length chromosomal insertion

Despite the abundance of genetic manipulation approaches, particularly for Escherichia coli, new techniques and increased flexibility in the application of existing techniques are required to address novel aims. The most widely used approaches for chromosome editing are based on bacteriophage site-specific and λRed/RecET-mediated homologous recombination. In the present study, these techniques were combined to develop a novel approach for in vivo cloning and targeted long-length chromosomal insertion. This approach permits direct λRed-mediated cloning of DNA fragment with lengths of 10kb or greater from the E. coli chromosome into the plasmid vector pGL2, which carries the ori of pSC101, the ϕ80-attP site of ϕ80 phage, and an excisable CmR marker bracketed by λ-attL/attR sites. In pGL2-based recombinant plasmids, the origin of replication can be eliminated in vitro via hydrolysis by SceI endonuclease and recircularization by DNA ligase. The resulting ori-less circular recombinant DNA can be used for targeted insertion of the cloned sequence into the chromosome at a selected site via ϕ80 phage-specific integrase-mediated recombination using the Dual-In/Out approach (Minaeva et al., 2008). At the final stage of chromosomal editing, the CmR-marker can be excised from the chromosome due to expression of the λint/xis genes. Notably, the desired fragment can be inserted as multiple copies in the chromosome by combining insertions at different sites in one strain using the P1 general transduction technique (Moore, 2011). The developed approach is useful for the construction of plasmidless, markerless recombinant strains for fundamental and industrial purposes.

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system.

Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines.

The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ΦC31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ΦC31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to carry out transient transfection of interested DNA fragments into the eukaryotic cells for various purposes.

Using phage integrases in a site-specific dual integrase cassette exchange strategy.

ΦC31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ΦC31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites. Previously, we have used these integrases sequentially to integrate plasmid DNA into the genome. This approach relied on placing a landing pad attP for Bxb1 integrase in the genome by using phiC31 integrase-mediated recombination at a genomic pseudo attP site. In this chapter, we present a protocol for using these integrases simultaneously to facilitate cassette exchange at a predefined location. This approach permits greater control and accuracy over integration. We also present a general method for using polymerase chain reaction assays to verify that the desired cassette exchange occurred successfully.

ΦC31 Integrase Mediates Efficient Site-Specific Integration in Sheep Fibroblasts

ΦC31 integrase, a site-specific recombinase, can effectively mediate the integration of foreign genes bearing an attB sequence into pseudo attP sites of genomes in human, mouse, and Drosophila cells. In this study, we measured ΦC31 integrase-mediated homologous recombination between attB and pseudo attP sites in sheep cells. The integration efficiency of the EGFP expression cassette with the attB sequence increased at least 2-fold in sheep fibroblasts. Three pseudo-attP sites were identified in the sheep genome, located in the intergenic regions on chromosomes 4, 13, and 7 respectively. Moreover, the transgene that was integrated at the three pseudo attP sites exhibited high levels of expression. Our study indicates that the ΦC31 integrase system provides an efficient integration tool for genetic engineering of the sheep genome.

Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems

FK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strain Streptomyces tsukubaensis by the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact that S. tsukubaensis is amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.

Toward Improvement of Erythromycin A Production in an IndustrialSaccharopolyspora erythraea Strain via Facilitation of Genetic Manipulation with an Artificial attB Site for Specific Recombination

Large-scale production of erythromycin A (Er-A) relies on the organism Saccharopolyspora erythraea, in which lack of a typical attB site largely impedes the application of phage ΦC31 integrase-mediated recombination into site-specific engineering. We herein report construction of an artificial attB site in an industrial S. erythraea strain, HL3168 E3, in an effort to break the bottleneck previously encountered during genetic manipulation mainly from homologous or unpredictable nonspecific integration. Replacement of a cryptic gene, nrps1-1, with a cassette containing eight attB DNA sequences did not affect the high Er-producing ability, setting the stage for precisely engineering the industrial Er-producing strain for foreign DNA introduction with a reliable conjugation frequency. Transfer of either exogenous or endogenous genes of importance to Er-A biosynthesis, including the S-adenosylmethionine synthetase gene for positive regulation, vhb for increasing the oxygen supply, and two tailoring genes, eryK and eryG, for optimizing the biotransformation at the late stage, was achieved by taking advantage of this facility, allowing systematic improvement of Er-A production as well as elimination of the by-products Er-B and Er-C in fermentation. The strategy developed here can generally be applicable to other strains that lack the attB site.

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken β actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.

Sp100 interacts with phage ΦC31 integrase to inhibit its recombination activity.

Phage ΦC31 integrase is a potential vector for the insertion of therapeutic genes into specific sites in the human genome. To understand the mechanism involved in ΦC31 integrase-mediated recombination, it is important to understand the interaction between the integrase and cellular proteins. Using a yeast two-hybrid system with pLexA-ΦC31 integrase as bait, we screened a pB42AD human fetal brain cDNA library for potential interacting cellular proteins. From the 10⁶ independent clones that were screened, 11 potential interacting clones were isolated, of which one encoded C-terminal fragment of Sp100. The interaction between Sp100 and ΦC31 integrase was further confirmed by yeast mating and co-immunoprecipitation assays. The hybridization between a ΦC31 integrase peptide array and an HEK293 cell extract revealed that residues 81RILN84 in the N-terminus of ΦC31 integrase are responsible for the interaction with Sp100. Knocking down endogenous Sp100 with Sp100-specific siRNA increased ΦC31 integrase-mediated recombination but did not impact reporter gene expression. Therefore, endogenous Sp100 may interact with ΦC31 integrase and inhibit the efficiency of ΦC31 integrase-mediated recombination.

Critical Amino Acid Residues Within the ɸC31 Integrase DNA-Binding Domain Affect Recombination Activities in Mammalian Cells

The bacteriophage-derived ϕC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells. Its integration reaction is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild-type attachment site attP or pseudo-attP attachment sites (attP') present in the mammalian genome. In the present study we aimed at increasing the safety and efficiency of ϕC31 integrase-mediated recombination by mutating the DNA-binding domain located at the C terminus. Using an alanine mutagenesis approach, we generated 22 ϕC31 point mutants that were screened for activities in mammalian cells. Intramolecular excision assays based on recombination between attB and attP revealed five mutants with 2-fold enhanced excision activity. Importantly, we also identified mutants showing enhanced recombination activities between attB and three previously described attP' sites detected in the mammalian genome, indicating that there may be enhanced specificity for these hot spots. Several mutants showed, in mammalian cells, integration activities that increased in a cell line-dependent manner. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies up to 5.5-fold. We also identified three ϕC31 integrase mutants that were recombination defective in all applied assays, suggesting that these amino acid residues are essential for the functionality of ϕC31 integrase in mammalian cells. In summary, we identified critical amino acid residues within the ϕC31 DNA-binding domain. With respect to site-directed recombination and genome engineering these findings have important implications for improved ϕC31 protein design.

The synthetic integron: an in vivo genetic shuffling device

As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with ‘regulatory’ elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.

Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion

The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH2-THF) in the malaria parasite Plasmodium falciparum. One carbon (C1) donor units are necessary for amino acid and nucleotide biosynthesis, and for the initiation of mitochondrial and plastid translation. In other organisms, GCV activity is closely coordinated with the activity of serine hydroxymethyltransferase (SHMT) enzymes. P. falciparum contains cytosolic and mitochondrial SHMT isoforms, and thus, the subcellular location of the GCV is an important indicator of its role in malaria metabolism. To determine the subcellular localization of the GCV, we used a modified version of the published method for mycobacteriophage integrase-mediated recombination in P. falciparum to generate cell lines containing one of the component proteins of the GCV, the H-protein, fused to GFP. Here, we demonstrate that this modification results in rapid generation of chromosomally integrated transgenic parasites, and we show that the H-protein localizes to the mitochondrion.

An Antimicrobial Peptide That Targets DNA Repair Intermediates In Vitro Inhibits Salmonella Growth within Murine Macrophages

The hexapeptide WRWYCR was previously identified on the basis of its ability to inhibit bacteriophage lambda integrase-mediated recombination by trapping and preventing resolution of the Holliday junction intermediate. This peptide inhibits several unrelated DNA repair enzymes that bind to and process Holliday junctions and branched DNA substrates. WRWYCR and its d stereoisomer, wrwycr, are bactericidal against both Gram-positive and Gram-negative bacteria, causing the accumulation of DNA breaks, chromosome segregation defects, and the filamentation of cells. DNA repair is a novel target of antibiotics. In the present study, we examined the ability of the peptides to inhibit the growth of Salmonella in mammalian cells. J774A.1 macrophage-like cells and murine peritoneal macrophages were infected with Salmonella enterica serovar Typhimurium and grown in the presence or absence of peptide. We found that peptide wrwycr reduced the number of Salmonella cells recovered after 24 h growth in J774A.1 cells by 100 to 1,000 times, depending on the multiplicity of infection. The peptide also inhibited Salmonella growth in peritoneal macrophages, and although higher doses were required, these were not toxic to the host cells. The apparent lower level of potency of the peptide paralleled the lower level of replication of Salmonella and the lower level of permeation of the peptide in the peritoneal macrophages than in the J774.1 cells. Treatment with peptide wrwycr elicited the SOS response in a significant fraction of the intracellular bacteria, as would be expected if the mechanism of bacterial killing was the same in pure culture and in host cells. These results represent a proof of principle of the antimicrobial activities of compounds that target DNA repair.

The relaxed requirements of the integron cleavage site allow predictable changes in integron target specificity

Integrons are able to incorporate exogenous genes embedded in mobile cassettes, by a site-specific recombination mechanism. Gene cassettes are collected at the attI site, via an integrase mediated recombination between the cassette recombination site, attC, and the attI site. Interestingly, only three nucleotides are conserved between attC and attI. Here, we have determined the requirements of these in recombination, using the recombination machinery from the paradigmatic class 1 integron. We found that, strikingly, the only requirement is to have identical first nucleotide in the two partner sites, but not the nature of this nucleotide. Furthermore, we showed that the reaction is close to wild-type efficiency when one of the nucleotides in the second or third position is mutated in either the attC or the attI1 site, while identical mutations can have drastic effects when both sites are mutated, resulting in a dramatic decrease of recombination frequency compared to that of the wild-type sites. Finally, we tested the functional role of the amino acids predicted from structural data to interact with the cleavage site. We found that, if the recombination site triplets are tolerant to mutation, the amino acids interacting with them are extremely constrained.

Intracellular Steady-State Concentration of Integron Recombination Products Varies with Integrase Level and Growth Phase

IntI1 mediates the recombination of antibiotic-resistant gene cassettes between different integrons in the same cell, facilitating the persistence and dissemination of these genes. Historically, integrase activity has been measured by conjugating recombinant products from donor cells overexpressing integrase and quantifying them in recipient cells. Here we report the first measurements of the steady-state intracellular abundance of integrase-mediated recombination products in strains expressing natural or high IntI1 levels. Recombination products in both high and natural integrase strains increased markedly through late log phase and continued to rise in stationary phase in the high integrase strain, but declined in the natural expression strain. Simple acquisition of gene cassettes was seen only in strains expressing high integrase; in strains with natural integrase levels, only cointegrates between the two integron-bearing plasmids were detectable at all growth stages. Unexpectedly, more attI×attI than attC×attI recombination products were seen in log phase for both strains; however, in stationary phase, the high integrase strain increased attC recombination, consistent with earlier observations of integrase crossover site preferences. Thus, direct quantification of the steady-state concentration of recombination products reveals that the integrase's intracellular concentration affects the amount and type of recombination events in a growth-phase-dependent manner.

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.

DNA repair, a novel antibacterial target: Holliday junction‐trapping peptides induce DNA damage and chromosome segregation defects

Holliday junction intermediates arise in several central pathways of DNA repair, replication fork restart, and site-specific recombination catalysed by tyrosine recombinases. Previously identified hexapeptide inhibitors of phage lambda integrase-mediated recombination block the resolution of Holliday junction intermediates in vitro and thereby inhibit recombination, but have no DNA cleavage activity themselves. The most potent peptides are specific for the branched DNA structure itself, as opposed to the integrase complex. Based on this activity, the peptides inhibit several unrelated Holliday junction-processing enzymes in vitro, including the RecG helicase and RuvABC junction resolvase complex. We have found that some of these hexapeptides are potent bactericidal antimicrobials, effective against both Gm+ and Gm- bacteria. Using epifluorescence microscopy and flow cytometry, we have characterized extensively the physiology of bacterial cells treated with these peptides. The hexapeptides cause DNA segregation abnormalities, filamentation and DNA damage. Damage caused by the peptides induces the SOS response, and is synergistic with damage caused by UV and mitomycin C. Our results are consistent with the model that the hexapeptides affect DNA targets that arise during recombination-dependent repair. We propose that the peptides trap intermediates in the repair of collapsed replication forks, preventing repair and resulting in bacterial death. Inhibition of DNA repair constitutes a novel target of antibiotic therapy. The peptides affect targets that arise in multiple pathways, and as expected, are quite resistant to the development of spontaneous antibiotic resistance.