Tn552 transposase purification and in vitro activities.

Rowland, Sally-J.; Sherratt, David J.; Stark, W. Marshall; Boocock, Martin R.

doi:10.1002/j.1460-2075.1995.tb06990.x

Cited by 23 publications

(19 citation statements)

References 62 publications

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“…The complete list of strains and plasmids used in this study is shown in Table 1. The expression vector pKETH-8c that carries the Tn552 transposase with an N-terminal His tag (59) was kindly provided by Nigel D. F. Grindley. The C. jejuni derivative of the Tn552 transposon (Tn552kan-Campy) has been described previously (7).…”

Section: Methodsmentioning

confidence: 99%

Identification of Campylobacter jejuni Genes Involved in Its Interaction with Epithelial Cells

2010

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Campylobacter jejuni is the leading cause of infectious gastroenteritis in industrialized nations. Its ability to enter and survive within nonphagocytic cells is thought to be very important for pathogenesis. However, little is known about the C. jejuni determinants that mediate these processes. Through an extensive transposon mutagenesis screen, we have identified several loci that are required for C. jejuni efficient entry and survival within epithelial cells. Among these loci, insertional mutations in aspA, aspB, and sodB resulted in drastic reduction in C. jejuni entry and/or survival within host cells and a severe defect in colonization in an animal model. The implications of these findings for the understanding of C. jejuni-host cell interactions are discussed.

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Section: Methodsmentioning

confidence: 99%

Identification of Campylobacter jejuni Genes Involved in Its Interaction with Epithelial Cells

2010

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“…Using the same combination of expression plasmid and host strain as Rowland et al (11), we explored a variety of growth and induction parameters, including culture medium, growth temperature, cell density at induction, length of induction, and concentration of inducer. We found that using a defined medium and growing the cultures at 30°C were To test for activity, we incubated our soluble transposase with a labeled, preprocessed (i.e., with exposed CA OH 3Ј ends) substrate, Tn552kan, which consists of a kanamycin-resistance gene flanked by the terminal 48 bp from the transposon's ends (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The Tn552 DNA substrate is also simpler. Each end of Tn552 contains two adjacent 23-bp transposase-binding sites and, as is the case with other elements of this superfamily, terminates with the dinucleotide -CA OH 3Ј (11). By contrast, the ends of Tn7 (and also of Mu) are complex, containing three or more transposase-binding sites spread irregularly over 90-100 bp (5).…”

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confidence: 99%

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Tn 552 transposase catalyzes concerted strand transfer in vitro

Leschziner

Griffin

Grindley

1998

Proc. Natl. Acad. Sci. U.S.A.

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The Tn552 transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer in vitro, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn552 termini. As is the case with transposition in vivo, the insertions recovered from the reaction in vitro are f lanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg 2؉ . Although it stimulates strand transfer overall, Mn 2؉ promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn552 transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.The transposon Tn552 is a 6.5-kb element isolated from Staphylococcus aureus responsible for resistance to ␤-lactam antibiotics (1, 2). It carries genes for three proteins likely to be involved in its transposition: a transposase (TnpA, previously called p480); a protein similar to the Mu B and Tn7 TnsC proteins (TnpB, previously called p271), which, by analogy, may enhance transposition activity and play a role in target selection; and a resolvase (3). The presence of these three genes suggests that Tn552 transposes by consecutive formation and resolution of a cointegrate between donor and target DNAs; however, this remains to be demonstrated.The Tn552 transposase belongs to the D,D(35)E superfamily of transposase proteins (3). Members of this superfamily include the integrase proteins of retroviruses and retrotransposons, the transposases of bacterial insertion sequences of the IS3 family, bacteriophage Mu A protein, and TnsB of Tn7. The defining characteristic of this family of phosphoryl-transfer proteins is a homologous catalytic core domain containing a triad of invariant acidic residues, two aspartates and a glutamate, which are essential for catalytic activity and give the superfamily its name (3, 4). Despite their homology, transposases of the D,D(35)E superfamily form a rather divergent and heterogeneous group. They range in size from the small retroviral integrases (about 290 residues) to Mu A (663 residues) and Tn7 TnsB (702 residues), and in many cases the region of homology does not appear to extend beyond the catalytic core (160-220 residues). Extensive biochemical studies of a few members of the family have provided a remarkably detailed picture of the transposition process including the substrates and proteins involved, the assembly of the transpositionally active protein-DNA complex, and the mechanism of strand transfer (5-7). However, complete understanding has been hampered by a lack of detailed structural information; despite substantial efforts, t...

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“…This transposon exhibits features that resemble those of Mu (12), such as the coding of a single-subunit transposase and the requirement of a single accessory protein for in vivo transposition (15). However, the ends of Tn552, consisting of only 48-bp terminal inverted repeats (10,16), are much simpler than those of Mu and Tn7. These are the only sequences required for transposition, which facilitates the engineering of Tn552 for different applications.…”

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In Vitro Transposition System for Efficient Generation of Random Mutants of Campylobacter jejuni

et al. 2001

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Campylobacter jejuni is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on the Staphylococcus aureus transposable element Tn552 that allows the efficient generation of insertion mutants of C. jejuni. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of C. jejuni. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.

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Tn552 transposase purification and in vitro activities.

Cited by 23 publications

References 62 publications

Identification of Campylobacter jejuni Genes Involved in Its Interaction with Epithelial Cells

Identification of Campylobacter jejuni Genes Involved in Its Interaction with Epithelial Cells

Tn 552 transposase catalyzes concerted strand transfer in vitro

In Vitro Transposition System for Efficient Generation of Random Mutants of Campylobacter jejuni

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