The Tn3-family of Replicative Transposons EMILIEN NICOLAS,1,3 MICHAEL LAMBIN,1,2 DAMIEN DANDOY,1,2 CHRISTINE GALLOY,1 NATHAN NGUYEN,1 CÉDRIC A. OGER,1 and BERNARD HALLET1 1Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348, Louvain-la-Neuve, Belgium; 2GSK Biologicals, B-1300, Wavre, Belgium; 3Laboratoire du Métabolisme de l’ARN FRS/FNRS-IBMM-CMMI- Université Libre de Bruxelles, B-6041 Charleroi-Gosselies, Belgium ABSTRACT Transposons of the Tn3 family form a widespread arsenal of antimicrobial resistance genes, thereby con- and remarkably homogeneous group of bacterial transposable tributing along with other mobile genetic elements, to elements in terms of transposition functions and an extremely the emergence of multi-drug resistances at a rate that versatile system for mediating gene reassortment and genomic challenges the development of new treatments (2–4). plasticity owing to their modular organization. They have made major contributions to antimicrobial drug resistance They are also prevalent in horizontal transfer of large dissemination or to endowing environmental bacteria with novel catabolic operons, allowing bacteria to metabolize var- catabolic capacities. Here, we discuss the dynamic aspects ious families of compounds, including industrial xeno- inherent to the diversity and mosaic structure of Tn3-family biotic pollutants (5, 6). transposons and their derivatives. We also provide an overview A distinguishing feature contributing to the prolifera- of current knowledge of the replicative transposition mechanism tion and evolutionary success of Tn3-family transposons of the family, emphasizing most recent work aimed at is their replicative mode of transposition (7, 8)(Fig. 1). understanding this mechanism at the biochemical level. “ ” “ ” Previous and recent data are put in perspective with those They transpose using a copy-in (or paste-and-copy ) obtained for other transposable elements to build up a tentative mechanism in which replication of the transposon occurs model linking the activities of the Tn3-family transposase protein during integration into the target. Intermolecular trans- with the cellular process of DNA replication, suggesting new position generates a cointegrate intermediate, in which lines for further investigation. Finally, we summarize our current the donor and target molecules are fused by directly re- view of the DNA site-specific recombination mechanisms peated transposon copies (Fig. 1). Cointegrate forma- responsible for converting replicative transposition tion requires both the transposon-encoded transposase intermediates into final products, comparing paradigm systems using a serine recombinase with more recently characterized (TnpA), to cut and rejoin the transposon ends with systems that use a tyrosine recombinase. the target DNA, and the cell replication machinery, to copy the complementary strands of the transposon. INTRODUCTION Received: 31 October 2014, Accepted: 3 November 2014, Published: 23 July 2015 The ampicillin-resistance transposon Tn3 is the arche- Editors: Mick Chandler, Université Paul Sabatier, Toulouse, France, type (“Tn3” being synonymous with “Tn1” or “Tn2”; and Nancy Craig, Johns Hopkins University, Baltimore, MD Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, (1)) of a large and widespread family of transposons Oger CA, Hallet B. 2014. The Tn3-family of replicative transposons. with representatives in nearly all bacterial phyla includ- Microbiol Spectrum 3(4):MDNA3-0060-2014. doi:10.1128 ing proteobacteria, firmicutes, and cyanobacteria. Fam- /microbiolspec.MDNA3-0060-2014. ily members are modular platforms allowing assembly, Correspondence: Bernard Hallet, [email protected] © 2015 American Society for Microbiology. All rights reserved. diversification, and redistribution of an ever-growing ASMscience.org/MicrobiolSpectrum 1 Downloaded from www.asmscience.org by IP: 200.145.191.169 On: Mon, 20 Feb 2017 14:06:40 Nicolas et al. to resolve the cointegrate intermediate by catalyzing a re- ciprocal recombination reaction between the newly syn- thesized copies of the element (Fig. 1). Recombination takes place at a specific site, the “resolution site” (res) or “internal recombination site” (IRS) located inside the transposon (7). The reaction completes the transpo- sition cycle, restoring the original donor molecule and producing a target molecule with a transposon copy (Fig. 1). The resolvase of most characterized Tn3-family transposons is a member of the serine recombinase (S- recombinase) family, but in a few cases it is a member of the tyrosine recombinase (Y-recombinase) family (7, 9). Although cointegrate resolution has been the subject of intense biochemical, topological, and structural stud- ies over the past decades, much less is known regarding the transposase-catalyzed reactions in both the transpo- sition and target immunity processes. After an overview of the biological diversity and significance of Tn3-family transposons, this chapter will provide an update of the mechanistic aspects of the transposition cycle with a special focus on the initial steps of the reaction. MODULAR AND DYNAMIC STRUCTURE OF TN3-FAMILY TRANSPOSONS Mobile genetic elements can be described as a juxtapo- FIGURE 1 Overview of the replicative transposition cycle of sition of functional modules, which together provide Tn3-family transposons. Intermolecular transposition (curved each element with its own specificities (10). Members of arrow) from a donor (purple) to a target DNA molecule (blue) the Tn3 transposon family include three types of module generates a cointegrate in which both molecules are fused (Fig. 2): the core transposition module, which is the mo- together by directly repeated copies of the transposon. This bility signature of the family; the cointegrate resolution step requires the transposase and the host replication ma- module, which optimizes the transposition pathway by chinery. The cointegrate is resolved by resolvase-mediated reducing the risk of generating aberrant replicon fu- site-specific recombination (double arrow) between the du- plicated copies of the transposon resolution site (boxed cross). sions and making the transposon less dependent on the Bracketed triangles are the terminal inverted repeats (IRs) of host recombination functions; and various sets of cargo the transposon. The transposase and resolvase genes are rep- genes and operons that were assimilated, presumably be- resented by a purple and a white arrow, respectively. Small cause they proved to be useful for their host under cer- triangles show the short (usually 5-bp) direct repeats (DRs) tain conditions. that are generated upon insertion into the target. The red stip- pled circle indicates that a molecule that contains a copy of The transposition module: the ID of the family the transposon is immunized against further insertions due to target immunity. doi:10.1128/microbiolspec.MDNA3-0060 Autonomous Tn3-family members carry a transposition -2014.f1 core module comprising the transposase gene (tnpA)and typical ∼38-bp inverted repeats (IRs) at the transposon The transposase is also involved in a process termed tar- ends (Fig. 2). TnpA proteins are unusually large (from get immunity whereby the transposon avoids inserting ∼950 to ∼1020 amino acids [aa]) compared to other more than once into the same DNA molecule (7). This transposases. A BLAST search performed with any Tn3- process is specific to each member of the family and is family transposase identifies only other members of the thought to act over large distances (up to several dozens family. However, phylogenetic analysis of 924 full-length of kilobases) within the genome. transposases from the Pfam database (http://pfam.xfam Tn3-family transposons generally encode a DNA site- .org/) reveals five protein clusters sharing less than ∼30% specific recombinase, or “resolvase”, whose function is sequence identity (Fig. 3). One large cluster comprising 2 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 200.145.191.169 On: Mon, 20 Feb 2017 14:06:40 The Tn3-family of Transposons 595 sequences can be further divided into three sub- family (Fig. 3), suggesting that these minimal structures groups exemplified by the Tn4430,Tn5393, and Tn21 have evolved separately. transposases (Fig. 3). An alignment of 21 active trans- posases from the different clusters and separate bacterial The cointegrate resolution modules phyla showed that only 15 residues, including the “DD- S-recombinases and Y-recombinases cleave and reseal E” catalytic triad, are perfectly conserved (11) (see also DNA molecules at specific sequences using a serine or The transposase section, below). a tyrosine nucleophile, respectively. Both recombinase Remarkably, this phylogeny does not faithfully su- families are involved in a broad range of biological pro- perimpose on that of the bacterial species from which cesses such as integration and excision of temperate bac- the transposons were originally isolated, underlining the teriophages, movement of different classes of mobile important level of horizontal transfer associated with genetic elements and control of gene expression through the family. In contrast, there is a good correlation be- programmed DNA rearrangements (17) (see also the tween the transposase phylogeny and the ∼38-bp ter- section on “Conservative site-specific recombination” of minal transposon IRs, suggesting that both transposition this volume). In all of these types of biological process, module partners