Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7

Mitra, Rupak; McKenzie, Gregory J.; Liang, Yi; Lee, Cherline A; Craig, Nancy L.

doi:10.1186/1759-8753-1-18

Cited by 57 publications

(52 citation statements)

References 38 publications

(64 reference statements)

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“…The canonical Tn7 element and especially the transposition pathway that directs the element into the attTn7 site located downstream of the conserved glmS gene have been studied extensively. The Tn7 TnsD(TniQ) protein is a sequence-specific DNA-binding protein that recognizes a highly conserved 36-bp sequence in the downstream region of the glmS gene-coding sequence (40,56). Transposition events promoted by TnsABC+D are directed into a position 23 bp downstream of the region bound by TnsD.…”

Section: Chromosomal Insertions Of the I-f Crispr-cas-associated Elemmentioning

confidence: 99%

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Peters

Makarova

Shmakov

et al. 2017

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

214

246

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A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNAguided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.CRISPR-Cas systems | Tn7 transposon | transposition strategy | crRNA guide | target-site selection

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Section: Chromosomal Insertions Of the I-f Crispr-cas-associated Elemmentioning

confidence: 99%

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Peters

Makarova

Shmakov

et al. 2017

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

214

246

View full text Add to dashboard Cite

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“…All of these Tns proteins, as well as the DNA substrates for transposition (i.e., the Tn7 ends and the attTn7 target DNA), must assemble into the elaborate transpososome in which transposition actually occurs (11,18). The targeting protein TnsD binds to a specific sequence in attTn7 (11,19). Subsequent TnsC recruitment involves both TnsC-DNA interactions that depend on TnsDinduced distortions in attTn7 (20,21) and likely TnsC-TnsD interactions that we previously detected using yeast two-hybrid assays (19).…”

mentioning

confidence: 99%

The Tn7 transposition regulator TnsC interacts with the transposase subunit TnsB and target selector TnsD

Choi

Spencer

Craig

2014

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

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The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB-TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB-TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD.photocrosslinking | protein-protein interaction | transpososome | transposable element

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“…Tn7 transposition is ideal because it is directional (14), is highly specific for attTn7 sites (15), and results in single-copy insertion in most bacterial species (13). For Salmonella and E. coli, the single attTn7 site lies near the conserved glmS gene (16). Modifications have been made in an attempt to improve the flexibility of the Tn7 system, such as through the generation of new helper plasmids (17), helper plasmid and delivery plasmid hybrids (18), arabinoseinducible gene expression (19), and luciferase integration (20,21).…”

mentioning

confidence: 99%

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

Shivak

MacKenzie

Watson

et al. 2016

Appl Environ Microbiol

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Our goal was to develop a robust tagging method that can be used to track bacterial strains in vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies in Salmonella and Escherichia coli and Tn7 transposition. We generated kanamycin-and chloramphenicolresistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of 70 -dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7 system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10-to 30-fold boost in transposase gene expression and transposition efficiencies of 10 ؊8 to 10 ؊10 in Salmonella enterica serovar Typhimurium and E. coli APEC O1, whereas the existing Tn7 system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, and in vivo animal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within the Enterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics. IMPORTANCEThis article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructs in vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system in Salmonella and E. coli, and we predict that it will be widely applicable to other bacterial strains within the Enterobacteriaceae. This technology will allow for improved in vivo analysis of bacterial pathogens.T he ability to generate recombinant strains of Escherichia coli and Salmonella enterica has facilitated insight into their ecology and pathogenic mechanisms. In recent years, strain collections consisting of single-gene knockouts of the entire genomes of E. coli K-12 (1) and S. enterica serovar Typhimurium 14028 (2) have been assembled. These collections can be used for finely tuned analysis of gene function and host-pathogen interactions, as well as for strain fitness and competition experiments (3). There are also a wide array of reporter systems available to analyze gene expression in detail, for the ordering of hierarchical gene circuits (4), a deeper understanding of metabolism (5), or the development of biosensors (6). The use of these systems, coupled with next-generation sequencing approaches that have facilitated functio...

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Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7

Cited by 57 publications

References 38 publications

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Recruitment of CRISPR-Cas systems by Tn7-like transposons

The Tn7 transposition regulator TnsC interacts with the transposase subunit TnsB and target selector TnsD

A Modular, Tn 7 -Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

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