We have developed a simple system to clone indigenous Rhizobium plasmids into E. coli. The strategy consists of three matings: the first is to insert Tn5 in the plasmid to be cloned, the second incorporates the integrative vector into the inserted Tn5 in the native Rhizobium plasmid, and the last mating transfers the target plasmid directly into E. coli. This mating-based system was successfull...

We examined insertion site profiles generated from three studies of Tc1/mariner elements: Tc1 (genomic copies and experimentally induced insertions (van Luenen and Through this comparison we found that bendability, protein-induced deformability and A-philicity are the most significant for determining insertion site preference. We further examined Tn5 and Ty1 insertion sites and found that Tn5 s...

Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and re...

Tn5 transposase (Tnp) is a 53.3-kDa protein that is encoded by and facilitates movement of transposon Tn5. Tnp monomers contain a single active site that is responsible for catalyzing a series of four DNA breaking/joining reactions at one transposon end. Based on primary sequence homology and protein structural information, we designed and constructed a series of plasmids that encode for Tnps c...

Synaptic complexes in prokaryotic transposons occur when transposase monomers bind to each of two specific end-binding sequences and then associate to bring the proteins and the two ends of the transposon together. It is within this complex of proteins and DNA that identical catalytic reactions are carried out by transposase on each of the ends of the transposon. In this study, we perform in vi...

Transposable elements induce spontaneous mutations, promote genome rearrangements, regulate gene expression, and participate in the horizontal spread of genes encoding traits such as antibiotic resistance among bacterial genera too distantly related to undergo homologous recombination. Here we review the bacterial transposon Tn5 and focus on those aspects of its functional organization and tran...

Pseudomonas sp. MIS38 produces an effective biosurfactant named arthrofactin, which is a cyclic lipopeptide synthesized by a mega complex composed of three nonribosomal peptide synthetases. In order to gain insight into the control mechanism of arthrofactin production, a Tn5 mutant library was constructed and screened for arthrofactin-deficient mutants. Along with a number of mutations that occ...

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon's site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host...

We report here a novel system for the conjugal transfer of bacterial chromosomes which utilizes the transfer origin (oriT) of plasmid RK2 cloned into transposon Tn5. Tn5 with oriT was inserted by transposition into the chromosomes of Escherichia coli and Rhizobium meliloti. The oriT sequence then served as the origin of high-frequency chromosome transfer when a helper RK2 plasmid was present in...