Search

The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein ( gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. [ABSTRACT FROM AUTHOR]

Genome engineering benefits from the availability of DNA modifying enzymes that have different target specificities and have optimized performance in different cell types. This variety of site-specific enzymes can be used to develop complex genome engineering applications at multiple loci. Although eight yeast site-specific tyrosine recombinases are known, only Flp is actively used in genome engineering. To expand the pool of the yeast site-specific tyrosine recombinases capable of mediating genome manipulations in mammalian cells, we engineered and analyzed variants of two tyrosine recombinases: R and TD. The activity of the evolved variants, unlike the activity of the native R and TD recombinases, is suitable for genome engineering in Escherichia coli and mammalian cells. Unexpectedly, we found that R recombinase benefits from the shortening of its C-terminus. We also found that the activity of wild-type R can be modulated by its non-consensus “head” sequence but this modulation became not apparent in the evolved R variants. The engineered recombinase variants were found to be active in all recombination reactions tested: excision, integration, and dual recombinase-mediated cassette exchange. The analysis of the latter reaction catalyzed by the R/TD recombinase pair shows that the condition supporting the most efficient replacement reaction favors efficient TD-mediated integration reaction while favoring efficient R-mediated integration and deletion reactions. [ABSTRACT FROM AUTHOR]

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence ( att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available. [ABSTRACT FROM AUTHOR]

The use of site-specific recombinases to manipulate eukaryotic genomes began nearly three decades ago. Although seemingly parallel efforts were being made in animal and plant systems, the motivation for its development in plants was unique to, at least at the time, crop bioengineering issues. The impetus behind site-specific deletion in plants was to remove antibiotic resistance genes used during transformation but unnecessary in commercial products. Site-specific integration in plants was more than academic curiosity of position effects on gene expression, but a necessary step towards developing the serial stacking of DNA to the same chromosome locus - to insure that bioengineered crops can be improved over time through transgene additions without inflating the number of segregating loci. This article is not a review of the literature on site-specific recombination, but a first person account of the series of events leading to the development of a gene stacking transformation system in plants. [ABSTRACT FROM AUTHOR]

Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of research has identified several DNA recombinases that carryout efficient cis and trans recombination between the recombination sites artificially introduced into the plant chromosome. The specificity and efficiency of recombinases make them extremely attractive for genome engineering. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes and have rarely been extended to more complex applications. The reversibility of recombination, a property of the tyrosine family of recombinases, does not lend itself to gene stacking approaches that involve rounds of transformation for integrating genes into the engineered sites. However, recent developments in the field of recombinases have overcome these challenges and paved the way for gene stacking. Some of the key advancements include the application of unidirectional recombination systems, modification of recombination sites and transgene site modifications to allow repeated site-specific integrations into the selected site. Gene stacking is relevant to agriculturally important crops, many of which are difficult to transform; therefore, development of high-efficiency gene stacking systems will be important for its application on agronomically important crops, and their elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking. [ABSTRACT FROM AUTHOR]

Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro., Methods and Major Results: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing., Conclusions and Implications: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Site-specific recombinases (integrases) can mediate the horizontal transfer of genomic islands. The ability to integrate large DNA sequences into target sites is very important for genetic engineering in prokaryotic and eukaryotic cells. Here, we characterized an unprecedented catalogue of 530 tyrosine-type integrases by examining genes potentially encoding tyrosine integrases in bacterial genomic islands. The phylogeny of putative tyrosine integrases revealed that these integrases form an evolutionary clade that is distinct from those already known and are affiliated with novel integrase groups. We systematically searched for candidate integrase genes, and their integration activities were validated in a bacterial model. We verified the integration functions of six representative novel integrases by using a two-plasmid integration system consisting of a donor plasmid carrying the integrase gene and attP site and a recipient plasmid harboring an attB site in recA -deficient Escherichia coli. Further quantitative reverse transcription-PCR (qRT-PCR) assays validated that the six selected integrases can be expressed with their native promoters in E. coli. The attP region reductions showed that the extent of attP sites of integrases is approximately 200 bp for integration capacity. In addition, mutational analysis showed that the conserved tyrosine at the C terminus is essential for catalysis, confirming that these candidate proteins belong to the tyrosine-type recombinase superfamily, i.e., tyrosine integrases. This study revealed that the novel integrases from bacterial genomic islands have site-specific recombination functions, which is of physiological significance for their genomic islands in bacterial chromosomes. More importantly, our discovery expands the toolbox for genetic engineering, especially for efficient integration activity. IMPORTANCE Site-specific recombinases or integrases have high specificity for DNA large fragment integration, which is urgently needed for gene editing. However, known integrases are not sufficient for meeting multiple integrations. In this work, we discovered an array of integrases through bioinformatics analysis in bacterial genomes. Phylogeny and functional assays revealed that these new integrases belong to tyrosine-type integrases and have the ability to conduct site-specific recombination. Moreover, attP region extent and catalysis site analysis were characterized. Our study provides the methodology for discovery of novel integrases and increases the capacity of weapon pool for genetic engineering in bacteria.

Escherichia coli is the most studied and well understood microorganism, but research in this system can still be limited by available genetic tools, including the ability to rapidly integrate multiple DNA constructs efficiently into the chromosome. Site-specific, large serine-recombinases can be useful tools, catalyzing a single, unidirectional recombination event between 2 specific DNA sequences, attB and attP, without requiring host proteins for functionality. Using these recombinases, we have developed a system to integrate up to 12 genetic constructs sequentially and stably into in the E. coli chromosome. A cassette of attB sites was inserted into the chromosome and the corresponding recombinases were cloned onto temperature sensitive plasmids to mediate recombination between a non-replicating, attP -containing "cargo" plasmid and the corresponding attB site on the chromosome. The efficiency of DNA insertion into the E. coli chromosome was approximately 10 7 CFU/μg DNA for six of the recombinases when the competent cells already contained the recombinase-expressing plasmid and approximately 10 5 CFU/μg DNA or higher when the recombinase-expressing plasmid and "cargo" plasmid were co-transformed. The "cargo" plasmid contains ΦC31 recombination sites flanking the antibiotic gene, allowing for resistance markers to be removed and reused following transient expression of the ΦC31 recombinase. As an example of the utility of this system, eight DNA methyltransferases from Clostridium clariflavum 4-2a were inserted into the E. coli chromosome to methylate plasmid DNA for evasion of the C. clariflavum restriction systems, enabling the first demonstration of transformation of this cellulose-degrading species. IMPORTANCE More rapid genetic tools can help accelerate strain engineering, even in advanced hosts like Escherichia coli. Here, we adapt a suite of site-specific recombinases to enable simple, rapid, and highly efficient site-specific integration of heterologous DNA into the chromosome. This utility of this system was demonstrated by sequential insertion of eight DNA methyltransferases into the E. coli chromosome, allowing plasmid DNA to be protected from restriction in Clostridium clariflavum and enabling genetic transformation of this organism. This integration system should also be highly portable into non-model organisms.

Transgenic approaches using I-SceI are powerful genome modification methods for creating heritable modifications in eukaryotic genomes. Such modifications are ideal for studying putative promoters and their temporal and spatial expression patterns in real time, in vivo. Central to this process is the initial engineering of a plasmid construct containing multiple DNA modules in a specific order prior to the integration into the target genome. One popular way of doing this is based upon the pGateway system, the modular form of which described in this chapter is known as pTransgenesis. We will initially describe the protocol of obtaining the plasmid construct containing the required sequence modules, and then the process of integrating the construct into the genome of a Xenopus embryo via co-injection with I-SceI and subsequent screening for transgenics., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The Cre/loxP system is a versatile and powerful tool that has been used to develop many kinds of genetically modified mice, such as conditional knockout mice and mutant protein-expressing mice through the excision of a STOP cassette. However, while numerous in vivo and in vitro applications of the Cre/loxP system have been reported, it remains difficult to target at one time more than one set of recognition sites in an identical single cell in mice using the Cre/loxP system. To overcome this barrier, we developed two novel site-specific recombination systems called VCre/VloxP and SCre/SloxP. These systems allow multiple independent site-specific recombination, for example, multiple targeted deletions in the same cell at different times. In this chapter, I describe the features of VCre/VloxP and SCre/SloxP, practical protocols and tips on how to use them in genomic engineering applications, potential problems in their use, and how problems can be identified and solved., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of the structure–function relationship of several previously identified non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harboring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision of the phage DNA. [ABSTRACT FROM AUTHOR]

Background: Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts. Recombinases such as Cre-lox, on the other hand, efficiently drive site-specific integrations in plants. However, the reversibility of Cre-lox recombination, due to the incorporation of two cis-positioned lox sites, presents a major bottleneck in its application in gene stacking. Here, we describe a strategy of resolving this bottleneck through excision of one of the cis-positioned lox, embedded in the marker gene, by nuclease activity. Methods: All transgenic lines were developed by particle bombardment of rice callus with plasmid constructs. Standard molecular approach was used for building the constructs. Transgene loci were analyzed by PCR, Southern hybridization, and DNA sequencing. Results: We developed a highly efficient gene stacking method by utilizing powerful recombinases such as Cre-lox and FLP-FRT, for site-specific gene integrations, and nucleases for marker gene excisions. We generated Cremediated site-specific integration locus in rice and showed excision of marker gene by I-SceI at ~20 % efficiency, seamlessly connecting genes in the locus. Next, we showed ZFN could be used for marker excision, and the locus can be targeted again by recombinases. Hence, we extended the power of recombinases to gene stacking application in plants. Finally, we show that heat-inducible I-SceI is also suitable for marker excision, and therefore could serve as an important tool in streamlining this gene stacking platform. Conclusions: A practical approach for gene stacking in plant cell was developed that allows targeted gene insertions through rounds of transformation, a method needed for introducing new traits into transgenic lines for their rapid deployment in the field. By using Cre-lox, a powerful site-specific recombination system, this method greatly improves gene stacking efficiency, and through the application of nucleases develops marker-free, seamless stack of genes at pre-determined chromosomal sites. [ABSTRACT FROM AUTHOR]

The precise elimination of selectable marker genes is highly desirable, when their function is no longer needed, because their presence raised worldwide public concerns against the release of genetically modified plants. This is the first report of simultaneous application of the minimal gene cassette and cold-inducible Cre/lox recombination system in wheat. The bar selection and cre-recombinase genes were eliminated from T and T transgenic lines with 44 and 51 % efficiency. This approach provides a new, reasonably effective technique to produce selection gene-free transgenic wheat lines either immediately after tissue culture, or from the subsequent transgenic generation. The advantage of this method is that it does not require any additional cold treatment during the plant regeneration/growing because the transgene elimination is ensured by the vernalisation. Application of this method prevents gene flow by pollen and seed, because the selection and recombinase genes are eliminated before pollen development, therefore reducing the risk of GM plants. [ABSTRACT FROM AUTHOR]

Genome engineering relies on DNA-modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ target-specific variants of the self-sufficient, versatile site-specific DNA recombinase Flp can be developed into promising alternatives. We demonstrate that the Flp variant evolved to recombine an FRT-like sequence, FL- IL10A, which is located upstream of the human interleukin-10 gene, and can target this sequence in the model setting of Chinese hamster ovary and human embryonic kidney 293 cells. This target-specific Flp variant is able to perform the integration reaction and, when paired with another recombinase, the dual recombinase-mediated cassette exchange reaction. The efficiency of the integration reaction in human cells can be enhanced by 'humanizing' the Flp variant gene and by adding the nuclear localization sequence to the recombinase. [ABSTRACT FROM AUTHOR]

Background and aims: Different varieties of Chinese Hamster Ovary (CHO) cells are being used frequently for producing most pharmaceutical products for many years. Permanent production in these host cells is a major challenge. The aim of this study was to integrate the vector permanently in the genome. In this study, the phiC31 site-specific recombination system was used for prolonged maintenance of the vector in the CHO cells. Methods: In this laboratory experimental study, a restriction enzyme cut site (PvuII) was selected as a cloning site in the pSVM vector using CLC software. Cutting with this enzyme led to create two blunt ends into the vector. Two specific primers were designed for isolation and amplification of attB region from pDrBB2 vector, using Oligo®5 software. Gel electrophoresis and PCR analysis were performed on the amplified fragment in order to confirmation of its structure. Results: The attB region was successfully integrated in to pSVM vector and was shown by gel electrophoresis. Colony PCR technique has revealed the correct structure of the reconstructed vectors. Conclusion: In this study, the phiC31 integrate catalyzes recombination between pseudo-attP sites (in CHO chromosomes) and attB site. A new construct has been made in this project containing attB site. This construct has to be introduced into CHO cells accompanied with pCMV-int vector (co-transfection) containing integrase. This integrase facilitates the incorporation of the pSVM vector into the chromosome, in order to get a permanent existence of the new construct into the CHO cells. [ABSTRACT FROM AUTHOR]

Recombinant baculoviruses have traditionally been generated by homologous recombination, in insect cells, of the virus DNA and a transfer vector. This method is, however, restricted to the small number of baculoviruses for which permissive cell lines are available. An alternative method, which avoids this limitation, is the bacmid system, in which a bacterial DNA cassette (BAC cassette) comprising a bacterial replication origin, an antibiotic resistance marker gene, and a transposon element, is inserted into a unique restriction endonuclease site of the virus genome and the recombinant DNA is produced in Escherichia coli K-12 strain. However, if the unique cleavage site is located within an open reading frame (ORF), the ORF will be disrupted by BAC cassette insertion. In this study, we propose a gene-repair BAC method which simultaneously restores the disrupted ORF and inserts a BAC cassette into the full genome of Adoxophyes honmai nucleopolyhedrovirus. [ABSTRACT FROM AUTHOR]

Phase variation of the UU172 phase-variable element of Ureaplasma parvum is governed by a DNA inversion event that takes place at short inverted repeats. The putative tyrosine recombinase XerC of Ureaplasma has been suggested as a mediator in the proposed site-specific recombination event. Here, we provide evidence that XerC mediates DNA inversion at the inverted repeats located on a synthetic locus that was introduced into the model organism Escherichia coli . Synthetic loci were created by exchanging the genes UU171 and UU172 with the two reporter genes gfp (green fluorescent protein) and mrfp1 (monomeric red fluorescent protein 1) either containing or missing the inverted repeats of the UU172 phase-variable element. E. coli was transformed with these loci and also co-transformed with the expression vector pBAD24 that contained the xer C gene behind the arabinose inducible pBAD promoter. Upon XerC expression, DNA inversion was observed only in the locus that contained the inverted repeat regions. We also demonstrate that XerC can process the recombination event with both an N-terminal maltose binding protein tag and a C-terminal 6×His tag in E. coli . A XerC mutant, where the proposed catalytic tyrosine residue 228 was exchanged with an alanine, did not process the recombination event. [ABSTRACT FROM AUTHOR]

Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes. [ABSTRACT FROM AUTHOR]

The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the ‘x’ number of transgenic loci into a single genome may seem trivial, (¼)x for a diploid species, but given the ‘y’ number of other nontransgenic traits that breeders also need to assemble into the same genome, the (¼)x+y probability for a ‘breeding stack’ could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific integration was mediated by the mycobacteriophage Bxb1 integrase-catalyzed recombination between attP and attB sites.Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of lox sites. Site-specific integration occurred in ~10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of ~3% of the transformed clones—a workable frequency for the development of commercial cultivars. Moreover, since neither the Bxb1–att nor the Cre–lox system is under patent, there is freedom to operate. [ABSTRACT FROM AUTHOR]

Efficient methods for DNA excision are needed for removing selectable marker genes from transgenic plants. The present work evaluated the enhanced FLP recombinase, FLPe, for excising FLP recombination target ( FRT)-flanked marker genes, and generating marker-free rice lines. Previously, the transient FLPe activity was found to be at least threefold higher on the transgene locus compared to that of FLPwt, the wild-type FLP recombinase. In this study, transgenic plants expressing FLPe were cross-pollinated with the plants harboring FRT site to analyze marker excision in F1 plants, and the transmission of marker-free locus to F2 progeny. The FLPe activity, expressed by the strong promoter (maize ubiquitin-1 gene), efficiently excised FRT-flanked marker gene in rice plants. However, marker excision in F2 progeny was tightly linked with the presence of FLPe gene, suggesting insufficient recombination in the gametophyte. The maize ubiquitin-1 promoter is reportedly active in gametophytic tissue and effective in meiotic transmission of the marker-free locus generated by Cre- lox recombination. Therefore, the observed lack of meiotic transmission in this study is possibly due to the limited efficiency of FLPe recombinase. While the reason for the FLPe inefficiency in the gametophyte is not clear, this work highlights the constraints of FLPe recombinase in generating stable marker-free plant lines through cross-pollination or gene induction methods. [ABSTRACT FROM AUTHOR]

The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp- zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the e gfp- zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp- zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals. [ABSTRACT FROM AUTHOR]

The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination. [ABSTRACT FROM AUTHOR]

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 and 8.2 × 10, 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. [ABSTRACT FROM AUTHOR]

Horizontal transfer of genomic islands (GEIs), that is, chromosomal regions encoding functions that can be advantageous for the host, plays a key role in bacterial evolution, but their mechanisms of transfer remained elusive for a long time. Recent data suggest that numerous GEIs belong to noncanonical classes of mobile genetic elements (MGEs) that can transfer by conjugation. Among them, the integrative and conjugative elements encode their own excision, conjugative transfer, and integration, whereas the integrative mobilizable elements are autonomous for excision and integration but require the conjugation machinery of helper elements to transfer. Others can self-transfer but require the recombination machinery of the recipient cell to integrate. All these MGEs evolve by acquisition, deletion, or exchange of modules, that is, groups of genes involved in the same function. Moreover, composite GEIs can result from the insertion of a MGE within another or from the site-specific integration of an incoming MGE into one of the recombination sites flanking a resident GEI (tandem accretion). Tandem accretion enables the cis-conjugative mobilization of highly degenerated and nonautonomous GEIs, the cis-mobilizable elements. All these mechanisms contribute to the plasticity and complex evolution of GEIs and explain the highly diverse tableau revealed by more and more genome comparisons. [ABSTRACT FROM AUTHOR]

Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore "linked in repulsion." Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner. [ABSTRACT FROM AUTHOR]

The complete sequencing and annotation of the genomes of industrially-important Bacillus species has enhanced our understanding of their properties, and allowed advances in genetic manipulations in other Bacillus species. Post-genomic studies require simple and highly efficient tools to enable genetic manipulation. Here, we summarize the recent progress in genetic engineering strategies for Bacillus species. We review the available genetic tools that have been developed in Bacillus species, as well as methods developed in other species that may also be applicable in Bacillus. Furthermore, we address the limitations and challenges of the existing methods, and discuss the future research prospects in developing novel and useful tools for genetic modification of Bacillus species. [ABSTRACT FROM AUTHOR]

Marker genes are essential for the selection and identification of rarely occurring transformation events generated in biotechnology. This includes plastid transformation, which requires that multiple copies of the modified chloroplast genome be present to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here, we demonstrate the precise excision of attP- and attB-flanked DNA from the plastid genome mediated by the large serine recombinase Bxb1. We transformed the tobacco plastid genome with the pTCH-PB vector containing a stuffer fragment of DNA flanked by directly oriented nonhomologous attP and attB recombinase recognition sites. In the absence of the Bxb1 recombinase, the transformed plastid genomes were stable and heritable. Nuclear-transformed transgenic tobacco plants expressing a plastid-targeted Bxb1 recombinase were crossed with transplastomic pTCH-PB plants, and the T1 hybrids exhibited efficient excision of the target sequence. The Bxb1- att system should prove to be a useful tool for site-specifically manipulating the plastid genome and generating marker-free transplastomic plants. [ABSTRACT FROM AUTHOR]

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Phage-encoded serine integrases are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (<50bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs. [Copyright &y& Elsevier]