Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.     

Mechanism of transposition of bacteriophage Mu: structure of a transposition intermediate

Mu transposition works efficiently in vitro and generates both cointegrate and simple insert products. We have examined the reaction products obtained under modified in vitro reaction conditions that do not permit efficient initiation of DNA replication. The major product is precisely the intermediate structure predicted from one of the current models of DNA transposition. Both cointegrates and simple inserts can be made in vitro using this intermediate as the DNA substrate, demonstrating that it is indeed a true transposition intermediate. The requirements for efficient formation of the intermediate include the Mu A protein, the Mu B protein, an unknown number of E. coli host proteins, ATP, and divalent cation. Only E. coli host proteins are required for conversion of the intermediate to cointegrate or simple insert products. Structures resulting from DNA strand transfer at only one end of the transposon are not observed, suggesting that the strand transfers at each end of the transposon are tightly coupled.     

Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10(4) to 10(6) CFU/microg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.     

Differential role of the Mu B protein in phage Mu integration vs. replication: mechanistic insights into two transposition pathways

The Mu B protein is an ATP-dependent DNA-binding protein and an allosteric activator of the Mu transposase. As a result of these activities, Mu B is instrumental in efficient transposition and target-site choice. We analysed in vivo the role of Mu B in the two different recombination reactions performed by phage Mu: non-replicative transposition, the pathway used during integration, and replicative transposition, the pathway used during lytic growth. Utilizing a sensitive PCR-based assay for Mu transposition, we found that Mu B is not required for integration, but enhances the rate and extent of the process. Furthermore, three different mutant versions of Mu B, Mu BC99Y, Mu BK106A, and Mu B1-294, stimulate integration to a similar level as the wild-type protein. In contrast, these mutant proteins fail to support Mu growth. This deficiency is attributable to a defect in formation of an essential intermediate for replicative transposition. Biochemical analysis of the Mu B mutant proteins reveals common features: the mutants retain the ability to stimulate transposase, but are defective in DNA binding and target DNA delivery. These data indicate that activation of transposase by Mu B is sufficient for robust non-replicative transposition. Efficient replicative transposition, however, demands that the Mu B protein not only activate transposase, but also bind and deliver the target DNA.     

Conformational isomerization in phage Mu transpososome assembly: effects of the transpositional enhancer and of MuB.

Initiation of phage Mu DNA transposition requires assembly of higher order protein-DNA complexes called Mu transpososomes containing the two Mu DNA ends and MuA transposase tetramer. Mu transpososome assembly is highly regulated and involves multiple DNA sites for transposase binding, including a transpositional enhancer called the internal activation sequence (IAS). In addition, a number of protein cofactors participate, including the target DNA activator MuB ATPase. We investigated the impact of the assembly cofactors on the kinetics of transpososome assembly with the aim of deciphering the reaction steps that are influenced by the cofactors. The transpositional enhancer IAS appears to have little impact on the initial pairing of the two Mu end segments bound by MuA. Instead, it accelerates the post-synaptic conformational step(s) that converts the reversible complex to the stable transpososome. The transpososome assembly stimulation by MuB does not require its stable DNA binding activity, which appears critical for directing transposition to sites distant from the donor transposon.     

Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA

Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported. Recently, this system has been used by Craigie and Mizuuchi [Cell 41 (1985) 867-876] to identify and biochemically characterize intermediates in the transposition process. We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods. Partial denaturation mapping has shown the intermediates to be theta-shaped structures in which the phi X174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome. Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition [Proc. Natl. Acad. Sci. USA 76 (1979) 1933-1937].     

DNA intermediates in transposition of phage Mu

Transposable genetic elements can insert into DNA sites that have no homology to themselves. Evidence that there is a physical linkage between a transposable element and its target DNA sequence during transposition comes from studies on bacteriophage Mu DNA transposition in which plasmids containing Mu DNA have been shown to attach to host DNA. We report the isolation of key structures, seen after induction of Mu DNA replication, after cloning lac operator into Mu DNA and using the lac repressor-operator interaction to trap Mu DNA on nitrocellulose filters. We have localized Mu sequences within these structures in the electron microscope by visualizing the lac operator-repressor interaction after binding with ferritin-conjugated antibody. This analysis shows that key structures contain replicating Mu DNA linked to non-Mu DNA and that replication can begin at either end of Mu.     

Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains.

Two mutations within the transposase (the A protein) gene of phage Mu with distinct effects on DNA transposition have been studied. The first mutation maps to the central domain (domain II) of A, a protein consisting of three major structural domains. The variant protein is normal in synapsis and cleavage of Mu ends but is temperature-sensitive in the strand transfer reaction, joining the Mu ends to target DNA. The second mutation is a deletion at the C terminus (within domain III); on the basis of genetic studies, the mutant protein is predicted to have lost the ability to interact with the Mu B protein. The B protein, in conjunction with A, promotes efficient intermolecular transposition, while inhibiting intramolecular transposition. We show that the purified mutant protein is proficient in intramolecular, but not intermolecular transposition in vitro. The interactions between A and B proteins have been followed by a proteolysis assay. The chymotrypsin sensitivity of the interdomainal Phe221-Ser222 peptide bond within the bidomainally organized B protein is exquisitely modulated by ATP, DNA and A protein. The sensitive or "open" state of this bond in native B protein becomes partially "open" upon binding of ATP by B, attains a "closed" or resistant configuration upon binding of DNA in presence of ATP, and is rendered "open" again upon addition of the A protein. In this test for the interaction of A protein with B protein-DNA complex, the domain II mutant behaves like wild-type A protein. However, the domain III mutant fails to restore chymotrypsin susceptibility of the Phe221-Ser222 bond.     

Altering the DNA-binding specificity of Mu transposase in vitro.

We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.     

A truncated form of the bacteriophage Mu B protein promotes conservative integration, but not replicative transposition, of Mu DNA

The phage-encoded proteins required for conservative integration of infecting bacteriophage Mu DNA were investigated. Our findings show that functional gpA, an essential component of the phage transposition system, is required for integration. The Mu B protein, which greatly enhances replicative transposition of Mu DNA, is also required. Furthermore, a truncated form of gpB lacking 18 amino acids from the carboxy terminus is blocked in replicative transposition, but not conservative integration. Our results point to a more prominent role for gpB than simply a replication enhancer in Mu DNA transposition. The ability of a truncated form of B to function in conservative integration, but not replicative transposition, also suggests a key role for the carboxy-terminal domain of the protein in the replicative reaction. The existence of a shortened form of gpB, which uncouples conservative integration from replicative transposition, should be invaluable for future dissection of Mu DNA transposition.     

Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes

Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K. (1987) Cell 51, 493-501). Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins. Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU. The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes. Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B. However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl. Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product. Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.     

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.     

Mini-Mu mediates deletion-inversions in vivo by intra-transposon transposition

We have shown that a mini-Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion-inversion product, which has previously been observed in vitro, is formed by non-replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini-Mu can undergo non-replicative reactions in vivo and that these generate a 5 bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.     

Mu DNA replication in vitro: Criteria for initiation

Summary An in vitro system for investigating Mu replication and transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates.Two conditions reported to specifically decrease Mu DNA replication in vivo (Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.     

转座子相关技术在微生物功能基因组研究中的应用

转座子是DNA插入因子的一种,是指能在基因组间或组内跳跃的DNA片段。转座子作为插入突变剂或分子标签已被广泛地应用于基因的分离和克隆,且因其独特的性质已成为发现新基因和基因功能分析的有效工具。这使得转座子无论是在单基因水平还是全基因组水平,都成为细菌、酵母和其他微生物研究的有力工具。简单而有效的体外转座反应可以对一些以往难以进行分析的顽固微生物进行转座诱变分析。而建立在转座子基础上的信号标签诱变技术和遗传足迹法的应用则发现了一些新的病原微生物毒力因子,从而可以更好地对这些病原微生物的致病机理进行阐述。这些再次说明转座子是微生物功能基因组研究中的有力工具。本文综述了转座子及其衍生载体介导的一些技术,并讨论其在微生物功能基因组研究中的应用。     

Studies on a "jumping gene machine": higher-order nucleoprotein complexes in Mu DNA transposition.

Studies in my lab have focused on DNA transposition in the bacterial virus, Mu. In vitro studies have shown that Mu DNA transposition is a three-step process involving DNA breakage, strand transfer and DNA replication. In the first step, a nick is introduced at each end of the transposon. The liberated 3'-OH groups subsequently attack a target DNA molecule resulting in strand transfer. The transposon DNA, now covalently linked to the target, is finally replicated to generate the transposition end-product, referred to as a cointegrate. The DNA cleavage and strand transfer reactions are mediated by a "jumping gene machine" or transpososomes, which we discovered in 1987. They are assembled by bringing together three different DNA regions via a process involving multiple protein-DNA and protein-protein interactions. The action of four different proteins is required in addition to protein-induced DNA bending or wrapping to overcome the intrinsic stiffness of DNA, which would ordinarily prohibit the assembly of such a structure. Transpososome assembly is a gradual process involving multiple steps with an inherent flexibility whereby alternate pathways can be used in the assembly process, biasing the reaction towards completion under different conditions.     

Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu

An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed. Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes. Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells. Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10⁴ to 10⁶ CFU/μg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica. Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria. An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied. This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.