Transposon-mediated site-specific recombination: A defined in vitro system

doi:10.1016/0092-8674(81)90178-1

Cell

Volume 25, Issue 3, September 1981, Pages 713-719

https://doi.org/10.1016/0092-8674(81)90178-1 Get rights and content

Abstract

Transposition of the insertion element γδ is thought to involve formation of intermediates in which the element is present at each junction between donor and target replicons. In vivo these cointegrate structures are rapidly converted to the end products of transposition by site-specific recombination at a defined sequence, res, that is present in each directly repeated γδ element. Resolvase, an element encoded protein of molecular weight 21,000 is required for cointegrate resolution. I have demonstrated site-specific recombination in vitro using purified resolvase and a cointegrate analog substrate. The required components of the system described here are resolvase, negatively supercoiled substrate DNA, buffer and Mg²⁺. Neither host-encoded products nor high energy cofactors appear to be required for resolution in vitro. Catenated, resolved molecules are the major products of the reaction. Elimination of Mg²⁺ from the reaction yields different product molecules. The in vitro system described here provides an opportunity for detailed study of the resolution reaction.

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Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site
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Cited by (154)

The antivirulence: From concept to applications (DOI:10.1016/j.antib.2008.12.002)
2009, Antibiotiques
Pendant des décennies, la recherche antibactérienne a été essentiellement focalisée sur le développement de molécules à activité bactériostatique et bactéricide. Cette stratégie basée sur la recherche de nouvelles cibles bactériennes n’a pour le moment pas amené chez les patients de nouveaux antibiotiques pouvant échapper au problème de résistance croisée.
Les connaissances fondamentales dans le domaine de la physiopathologie cellulaire et moléculaire de bon nombre de processus infectieux ont rapidement évolué. Parallèlement, des technologies permettant d’identifier de nombreux gènes et facteurs de virulence bactériens s’exprimant essentiellement chez l’hôte ont été mis au point. Ces éléments dans leur ensemble représentent le point de départ de la validation de cibles pour molécules antivirulence susceptibles d’empêcher certaines bactéries d’exprimer leur pathogénicité.
Quelques firmes pharmaceutiques et sociétés de biotechnologie développent actuellement des projets précliniques concernant des molécules dirigées contre différents aspects de la virulence bactérienne tels que : l’adhésion, le quorum sensing, la sécrétion de type III, la résistance aux défenses naturelles. Des premières validations du concept d’antivirulence ont été réalisées à l’aide de modèles d’infections expérimentales et des dossiers précliniques sont en phase de finalisation. Dans la mesure où l’on aborde la prévention et/ou le traitement de maladies infectieuses de manière innovante, il est nécessaire de mettre en place des protocoles d’essais cliniques adaptés, ainsi que des nouvelles méthodologies de diagnostic et de suivi microbiologiques.
Les prochaines années devraient voir arriver chez les patients les premiers représentants de cette nouvelle classe de molécules anti-infectieuses.
For decades, antibacterial research has been primarily focused on the development of molecules with bacteriostatic and bactericidal activity. For the moment, this strategy based on the search for new bacterial targets has not brought new antibiotics that can escape the cross-resistance problem among patients.
Fundamental knowledge in the field of cellular and molecular pathophysiology of a great number of infectious processes quickly evolved. In parallel, technologies allowing identifying many genes and virulence factors expressed primarily in the host were developed. These elements as a whole represent the starting point of targets validation for antivirulence molecules capable of preventing certain bacteria from expressing their pathogenicity.
Some pharmaceutical firms and biotechnology companies currently develop preclinical projects relating to molecules directed against various aspects of bacterial virulence such as: adhesion, quorum sensing, type III secretion, resistance to natural defenses. First validations of the antivirulence concept were carried out using models of experimental infections and preclinical files are in the phase of finalization. Insofar as one approaches the prevention and/or the treatment of infectious diseases in an innovating way, it is necessary to set up protocols of adapted clinical trials as well as new methodologies for microbiological diagnosis and follow-up of treatment.
In the coming years, the first representatives of this new class of anti-infective molecules should be available to patients.
The antivirulence: From concept to applications
2009, Antibiotiques
Pendant des décennies, la recherche antibactérienne a été essentiellement focalisée sur le développement de molécules à activité bactériostatique et bactéricide. Cette stratégie basée sur la recherche de nouvelles cibles bactériennes n’a pour le moment pas amené chez les patients de nouveaux antibiotiques pouvant échapper au problème de résistance croisée.
Les connaissances fondamentales dans le domaine de la physiopathologie cellulaire et moléculaire de bon nombre de processus infectieux ont rapidement évolué. Parallèlement, des technologies permettant d’identifier de nombreux gènes et facteurs de virulence bactériens s’exprimant essentiellement chez l’hôte ont été mis au point. Ces éléments dans leur ensemble représentent le point de départ de la validation de cibles pour molécules antivirulence susceptibles d’empêcher certaines bactéries d’exprimer leur pathogénicité.
Quelques firmes pharmaceutiques et sociétés de biotechnologie développent actuellement des projets précliniques concernant des molécules dirigées contre différents aspects de la virulence bactérienne tels que : l’adhésion, le quorum sensing, la sécrétion de type III, la résistance aux défenses naturelles. Des premières validations du concept d’antivirulence ont été réalisées à l’aide de modèles d’infections expérimentales et des dossiers précliniques sont en phase de finalisation. Dans la mesure où l’on aborde la prévention et/ou le traitement de maladies infectieuses de manière innovante, il est nécessaire de mettre en place des protocoles d’essais cliniques adaptés, ainsi que des nouvelles méthodologies de diagnostic et de suivi microbiologiques.
Les prochaines années devraient voir arriver chez les patients les premiers représentants de cette nouvelle classe de molécules anti-infectieuses.
For decades, antibacterial research has been primarily focused on the development of molecules with bacteriostatic and bactericidal activity. For the moment, this strategy based on the search for new bacterial targets has not brought new antibiotics that can escape the cross-resistance problem among patients.
Fundamental knowledge in the field of cellular and molecular pathophysiology of a great number of infectious processes quickly evolved. In parallel, technologies allowing identifying many genes and virulence factors expressed primarily in the host were developed. These elements as a whole represent the starting point of targets validation for antivirulence molecules capable of preventing certain bacteria from expressing their pathogenicity.
Some pharmaceutical firms and biotechnology companies currently develop preclinical projects relating to molecules directed against various aspects of bacterial virulence such as: adhesion, quorum sensing, type III secretion, resistance to natural defenses. First validations of the antivirulence concept were carried out using models of experimental infections and preclinical files are in the phase of finalization. Insofar as one approaches the prevention and/or the treatment of infectious diseases in an innovating way, it is necessary to set up protocols of adapted clinical trials as well as new methodologies for microbiological diagnosis and follow-up of treatment.
In the coming years, the first representatives of this new class of anti-infective molecules should be available to patients.
Production of unmarked mutations in mycobacteria using site-specific recombination
2003, FEMS Microbiology Letters
Gene disruption experiments play an important role in the functional characterization of genes in mycobacteria and rely mostly on the use of one or two antibiotic resistance markers. We have developed a system for mycobacteria which features both the advantages of the use of antibiotic resistance markers for gene disruption experiments and the ability to efficiently rescue the marker leaving an unmarked mutation on the chromosome. This new genetic tool relies on the transposon γδ site-specific recombination system. A res-ΩKm-res cassette was used to generate an insertional mutation by allelic exchange both in Mycobacterium smegmatis and Mycobacterium bovis BCG. Upon expression in the mutated strains of tnpR, the transposon γδ resolvase gene, res-ΩKm-res, was excised efficiently leaving behind a single res sequence at the mutated locus. A plasmid was engineered allowing expression of tnpR from an easily curable mycobacterial vector. This system will be useful for simple construction of unmarked mutations or repeated use of the same antibiotic marker to generate multiple mutants.
Interactions of protein complexes on supercoiled DNA: The mechanism of selective synapsis by Tn3 resolvase
2002, Journal of Molecular Biology
Citation Excerpt :
Thus, inversion reactions may give products which are unknotted, or knots of varying complexity, and resolution reactions may give unlinked DNA circles, or circles linked as catenanes of varying complexity (Figure 1(a)). Other “restrictive” recombinases (for example, the Tn3, γδ, and Tn21 resolvases, and the Hin and Gin DNA invertases) give a single major product; in the case of Tn3 resolvase, a catenane in which the two DNA circles are simply linked (Figure 1(a)).5,6 Permissive recombination systems do not distinguish between pairs of recombination sites in direct repeat (“head to tail”) or in inverted repeat, and can recombine sites on different DNA molecules.
“Looping” interactions of distant sites on DNA molecules, mediated by DNA-binding proteins, feature in many regulated genetic processes. We used plasmids containing up to six res recombination sites for Tn3 resolvase to analyse looping interactions (synapsis) in this system. We observed that in plasmids with four or more res sites, certain pairs of sites recombine faster than others. The relative rates of recombination depend on the number, relative orientation, and arrangement of the sites. To account for the differences in rate, we propose that pairing interactions between resolvase-bound res sites are in a state of rapid flux, leading to configurations in which the maximum number of sites within each supercoiled substrate molecule are synapsed in a topologically simple arrangement. Recombination rates reflect the steady state concentrations of these synapse configurations. Our results are at variance with models for selective synapsis that rely on ordered motions within supercoiled DNA, “slithering” or “tracking”, but are compatible with models that call for reversible synapsis of pairs of sites by random collision, followed by formation of an interwound productive synapse.
IVET and RIVET: Use of gene fusions to identify bacterial virulence factors specifically induced in host tissues
2000, Methods in Enzymology
IVET was designed to identify those bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes encode virulence factors, products specifically required for the infection process. The paradigm IVET system is based on complementation of an attenuating auxotrophic mutation by gene fusion and is designed to be of use in a wide variety of pathogenic organisms. In S. typhimurium, we have used this system successfully to identify a number of genes that are induced in a BALB/c mouse and that, when mutated, confer a virulence defect.
The RIVET system is based on recombinase gene fusions, which, on induction during infection, mediate a site-specific recombination, the product of which can be screened for after recovery of bacteria from host tissues. In V. cholerae, we have used this system successfully to identify genes that are induced transcriptionally during infection of the gastrointestinal tract of infant mice. RIVET is also uniquely designed for postidentification analysis of in vivo-induced genes: (1) it has been used to analyze the temporal and spatial patterns of virulence gene induction during infection and (2) it has been used to dissect the regulatory requirements of in vivo induction with respect to both bacterial regulatory factors and host-inducing environments.
The IVET system has several applications in the area of vaccine and antimicrobial drug development. This technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live-attenuated vaccines. In addition, the identification of promoters that are expressed optimally in animal tissues provides a means of establishing in vivo-regulated expression of heterologous antigens in live vaccines, an area that has been problematic previously. Finally, we expect that our methodology will uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.
The topological mechanism of phage λ integrase
1999, Journal of Molecular Biology
Bacteriophage λ integrase (Int) is a versatile site-specific recombinase. In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome. Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules. This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination. By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination. We find that, even in the absence of (−) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product. We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates. We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and −2, reflecting two different substrates with different topology but the same chirality. Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (−) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates. The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1 , show the same intrinsic chirality as λ Int.

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Abstract

References (27)

Use of the λ phage promoter P_L to promote gene expression in hybrid plasmid cloning vehicles

Meth. Enzymol.

The γδ sequence of F is an insertion sequence

J. Mol. Biol.

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3

Cell

Catenation and supercoiling in the products of bacteriophage λ integrative recombination in vitro

J. Mol. Biol.

Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site

Cell

A comprehensive molecular map of bacteriophage lambda

Gene

Dissection of the transposition process: a transposon-encoded site-specific recombination system

Mol. Gen. Genet.

Identification and characterization of a self-regulated repressor of translocation of the Tn3 element

Transposition protein of Tn3: identification and characterization of an essential repressor-controlled gene product

Nature

Excision of F plasmid sequences by recombination of directly repeated insertion sequence 2 elements: involvement of recA

Analysis of sequences transposed by complementation of two classes of transposition-deficient mutants of Tn3

J. Bacteriol.

Identification of the protein encoded by the transposable element Tn3 which is required for its transposition

Nature

Analysis of the structure and function of the kanamycin resistance transposon, Tn903

Cited by (154)

The antivirulence: From concept to applications (DOI:10.1016/j.antib.2008.12.002)

The antivirulence: From concept to applications

Production of unmarked mutations in mycobacteria using site-specific recombination

Interactions of protein complexes on supercoiled DNA: The mechanism of selective synapsis by Tn3 resolvase

IVET and RIVET: Use of gene fusions to identify bacterial virulence factors specifically induced in host tissues

The topological mechanism of phage λ integrase

Cell

ArticleTransposon-mediated site-specific recombination: A defined in vitro system

Abstract

Meth. Enzymol.

J. Mol. Biol.

Cell

J. Mol. Biol.

Cell

Gene

Dissection of the transposition process: a transposon-encoded site-specific recombination system

Mol. Gen. Genet.

Identification and characterization of a self-regulated repressor of translocation of the Tn3 element

Transposition protein of Tn3: identification and characterization of an essential repressor-controlled gene product

Nature

Excision of F plasmid sequences by recombination of directly repeated insertion sequence 2 elements: involvement of recA

Analysis of sequences transposed by complementation of two classes of transposition-deficient mutants of Tn3

J. Bacteriol.

Identification of the protein encoded by the transposable element Tn3 which is required for its transposition

Nature

Analysis of the structure and function of the kanamycin resistance transposon, Tn903

Article
Transposon-mediated site-specific recombination: A defined in vitro system