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Chapter 32 : The Tn-family of Replicative Transposons

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Abstract:

The ampicillin-resistance transposon Tn is the archetype (“Tn” being synonymous with “Tn” or “Tn”; ( )) of a large and widespread family of transposons with representatives in nearly all bacterial phyla including proteobacteria, firmicutes, and cyanobacteria. Family members are modular platforms allowing assembly, diversification, and redistribution of an ever-growing arsenal of antimicrobial resistance genes, thereby contributing along with other mobile genetic elements, to the emergence of multi-drug resistances at a rate that challenges the development of new treatments ( ). They are also prevalent in horizontal transfer of large catabolic operons, allowing bacteria to metabolize various families of compounds, including industrial xenobiotic pollutants ( ).

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014

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Mobile Genetic Elements
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Figures

Image of Figure 1
Figure 1

Overview of the replicative transposition cycle of Tn-family transposons. Intermolecular transposition (curved arrow) from a donor (purple) to a target DNA molecule (blue) generates a cointegrate in which both molecules are fused together by directly repeated copies of the transposon. This step requires the transposase and the host replication machinery. The cointegrate is resolved by resolvase-mediated site-specific recombination (double arrow) between the duplicated copies of the transposon resolution site (boxed cross). Bracketed triangles are the terminal inverted repeats (IRs) of the transposon. The transposase and resolvase genes are represented by a purple and a white arrow, respectively. Small triangles show the short (usually 5-bp) direct repeats (DRs) that are generated upon insertion into the target. The red stippled circle indicates that a molecule that contains a copy of the transposon is immunized against further insertions due to target immunity. doi:10.1128/microbiolspec.MDNA3-0060-2014.f1

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 2
Figure 2

The modular structure of Tn-family transposons and derived elements. (A) Tn-family transposons are constituted by the association of three classes of functional modules. The transposition module comprises the transposase gene (, purple arrow) and its associated ∼38-bp inverted repeats (IRs, bracketed triangles). The cointegrate resolution module is made of a gene encoding a site-specific recombinase from the serine- (green) or tyrosine- (magenta) family and their cognate recombination site (boxed cross). Resolution modules working with a tyrosine recombinase of the TnpS/OrfI subgroup (pale magenta) contain an additional gene coding for an accessory recombination protein (TnpT/OrfQ). “Long ” (pale green) refers to a resolvase gene that encodes a C-terminally extended member of the serine recombinase family. Passenger genes comprise a variety of phenotypic determinants and transposons that are specific to each element (gray arrows). (B) Autonomous transposons are characterized by a fully functional transposition module to mediate the mobility of the element . The simplest elements (ISs) are solely constituted by the minimal transposition module. Most characterized transposons have a typical unitary structure in which the transposase gene and its associated modules are flanked by the IR ends. Unitary elements can associate to form composite transposons containing a pair of full-length elements flanking a specific genomic segment; or pseudo-composite transposons carrying an autonomous element on one side and an isolated IR end on the other side. (C) Nonautonomous elements are Tn-family derivatives whose mobility requires a functional transposase to be provided in by a related transposon. Miniature Inverted-repeats Transposable Elements (MITEs) are solely constituted by a pair of IR ends flanking a short DNA segment that sometime contains a cointegrate resolution site. Mobile Insertion Cassettes (MICs) are nonautonomous unitary elements carrying one or more passenger genes between the ends. MITEs can also associate with passenger genes and isolated ends to form composite and pseudo-composite mobilized structures. See the text for details and the indicated examples. doi:10.1128/microbiolspec.MDNA3-0060-2014.f2

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 3
Figure 3

Phylogenetic tree of the Tn-family transposase proteins. The different clusters and subgroups identified within the family are boxed with different colors as indicated. Transposons that contain a cointegrate resolution module working with a tyrosine recombinase are underlined. Transposons that encode a “long”, C-terminally extended resolvase of the serine-recombinase family are marked with an asterisk. The length of the branches is proportional to the average number of substitutions per residue. doi:10.1128/microbiolspec.MDNA3-0060-2014.f3

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 4
Figure 4

Phylogenetic tree of transposon-encoded and plasmid-encoded resolvases of the S-recombinase family. Transposons belonging to different subgroups are boxed with different colors as in Fig. 3 . Transposons encoding a “long”, C-terminally extended resolvase are marked by an asterisk. Plasmids are highlighted in blue with the name of the corresponding recombinase (when assigned) in brackets. “Mu-like” transposons are highlighted in magenta. The length of the branches is proportional to the average number of substitutions per residue. doi:10.1128/microbiolspec.MDNA3-0060-2014.f4

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 5
Figure 5

Phylogenetic tree of transposon-encoded and plasmid-encoded resolvases of the Y-recombinase family. Transposons belonging to different subgroups are boxed with different colors as in Fig. 3 . Plasmids are highlighted in blue with the name of the corresponding recombinase (when assigned) in brackets. Representative and well-characterized XerC and D recombinases are from (Ec), (Bs) and (Vc). The length of the branches is proportional to the average number of substitutions per residue. doi:10.1128/microbiolspec.MDNA3-0060-2014.f5

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 6
Figure 6

Cointegrate resolution modules working with a resolvase of the S-recombinase family. (A) Serine-resolvases are typically small proteins containing an N-terminal catalytic domain (CD) linked to a short helix-turn-helix (HTH) C-terminal DNA-binding domain (DBD). Resolvases of the “long” resolvase subgroup have a ∼100-amino acid extension (white cylinder) at the C-terminus. Position of important catalytic residues (248) is indicated, with the active site serine (circled) highlighted in magenta. (B) Organization of the resolution sites . Open arrows are the 12-bp resolvase binding motifs. Shaded arrows are for motifs with a poorer match to the consensus. Coordinates (in bp) of the first position of each motif are indicated above the recombination sites. Boxed triangles show the position of the putative Hbsu binding sites in the site of Tn and Tn. Organization of Tn, Tn and IS res sites is as proposed by Rowland ( ). Adapted from figure 6, p. 152 of reference ( ). doi:10.1128/microbiolspec.MDNA3-0060-2014.f6

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 7
Figure 7

Cointegrate resolution modules working with a resolvase of the Y-recombinase family. (A) Typical two-domain structure of a tyrosine recombinase. The catalytic domain is in the C-terminal part of the protein. Positions of the conserved catalytic residues (RKHR/Y) are indicated, with the active site tyrosine (circled) highlighted in magenta. Both the N- and C-terminal domains contain the DNA-binding determinants of the protein (DBD). (B) Organization of the transposon recombination sites. Open arrows indicate the recombinase binding motifs. Shaded arrows in the site of Tn are the putative DNA recognition motifs for the auxiliary protein TnpT. Numbers indicate the length and the spacing between each motif (in bp) in the recombination site. Brackets in the site show the extent of the functional recombination site as determined by deletion analysis ( ). doi:10.1128/microbiolspec.MDNA3-0060-2014.f7

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 8
Figure 8

Model for copy-in replicative transposition. The diagram illustrates the case of intermolecular movement of a transposon (double black line delineated by brackets) between a donor molecule (purple) and a target molecule (blue). (A) Transposition starts when the transposase introduces specific single-strand nicks at both 3′ ends of the element, releasing 3′ OH groups in the donor (red triangles). (B) The transposon 3′ OH ends are then used as a nucleophile to attack separate phosphodiester bonds in both target DNA strands. (C) The reaction generates a strand transfer product (often called a “Shapiro intermediate”) in which the transposon is linked to the target DNA through its 3′ ends, and to the donor DNA through its 5′ ends. The target phosphates (black dots) are usually staggered by five base pairs, which leaves 5-bp single-strand gaps at the junctions between the transposon and the target DNA in the strand transfer intermediate. (D) Replication initiates at the 3′ OH end(s) released by cleavage of the target to synthesize the complementary strands of the transposon (red line) and form the cointegrate. DNA synthesis also repairs the single-stranded gaps at the ends of the transposon generating directly repeated (DR) 5-bp target duplications that flank both copies of the element in the final cointegrate. doi:10.1128/microbiolspec.MDNA3-0060-2014.f8

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 9
Figure 9

The Tn-family transposase protein. (A) Structural organization of the transposase. The protein is depicted with three main domains based on limited proteolysis (vertical arrows) of the Tn transposase (987 amino acids) ( ). The N-terminal DNA-binding domain (DBD) has a predicted bipartite structure analogous to that of the two-helix-turn-helix (HTH) DNA-binding domain of CENP-B. The C-terminal catalytic domain contains the predicted RNase H fold region (shaded in blue). Vertical bars correspond to highly conserved (>90%) amino acid residues in a representative subset of 21 transposases of the family, with the 15 perfectly conserved residues highlighted in red. Residues of the DD-E catalytic triad are indicated. Characterized mutations that selectively affect target immunity (T+/I– mutations) are reported below the diagram. (B) Structural models for the CENP-B-like DNA-binding domain (left panels) and the RNase H fold catalytic core (right panels). The actual structures of the CENP-B–DNA co-complex ( ) and the HIV integrase RNase H fold ( ) are shown on the top, and the predicted models derived for the Tn transposase are shown below. Nonstructured regions are represented by dashed lines. Positions of secondary structures and other critical structural elements are indicated and highlighted in different colors (see text for details). Nonstructured regions are represented by dashed lines. In the RNase H fold, the DD-E catalytic residues are shown in a stick configuration colored in red. The predicted location of the 90-amino acid insert in the putative RNase H fold of TnpA is indicated by a rectangle. doi:10.1128/microbiolspec.MDNA3-0060-2014.f9

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 10
Figure 10

The Tn-family transposons ends. The 38-bp terminal inverted repeat (IR) sequences of 21 representative transposons of the Tn family are aligned, and perfectly or highly (>75%) conserved positions are boxed in red and yellow, respectively. The resulting consensus sequence is shown below the alignment, and a cartoon showing the orientation of the IR sequence (purple triangle) with respect to the inside (in) and outside (out) regions of the transposon is reported on the top. Position of the transposase 3′-end cleavage site is indicated by an arrow. Conserved regions corresponding to the external cleavage domain of the IR (Box A) and the internal transposase recognition domain (Box B) are indicated with brackets. The transposase recognition sequence is further subdivided into two conserved motifs (Box B 1 and Box B 2). doi:10.1128/microbiolspec.MDNA3-0060-2014.f10

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Image of Figure 11
Figure 11

Mechanism of cointegrate resolution by resolvases of the S-recombinase family. (A) The rotational strand exchange reaction catalyzed by S-recombinases. Representation of the recombination complex is inspired from the structure of the synapse showing the activated γδ resolvase tetramer bound to paired core sites I of ( ). The recombination sites are aligned in parallel. Blue arrows represent the 12-bp resolvase recognition motifs. The partner resolvase dimers are colored in pale and dark green with their catalytic domain (CD) lying at the inside of the synapse and their DNA-binding domain (DBD) at the outside. The four recombinase molecules have cleaved the DNA, generating double-strand breaks with phosphoseryl DNA–protein bonds at the 5′ ends (shown as yellow dots linked to red connectors) and free OH groups at the 3′ ends of the breaks (half-arrows). DNA strands are exchanged by 180° rotation of one pair of partner subunits with respect to the other around a flat hydrophobic interface within the tetramer. For the rejoining reaction, each free 3′ OH end attacks the phosphoseryl bond of the opposite DNA strand. (B) Topological selectivity in resolvase-mediated cointegrate resolution. Binding of resolvase dimers (green spheres) to sites I, II and III of the partner sites results in the formation of a synaptosome in which the two sites are inter-wrapped, trapping three negative crosses from the initial DNA substrate. This complex only readily forms if the starting sites are in a head-to-tail configuration on a supercoiled DNA molecule. Strand exchange by right-handed 180° rotation as in (A) generates a two-node catenane product. doi:10.1128/microbiolspec.MDNA3-0060-2014.f11

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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Figure 12

Mechanism of cointegrate resolution by the TnpI recombinase of Tn. (A) Ordered DNA strand exchange catalyzed by TnpI at the IR1–IR2 core site of the IRS. The TnpI tetramer bound to synapsed IR1–IR2 core sites is drawn according to the structure of the related Cre recombinase complexes (232). Only the C-terminal catalytic domain of the protein is shown for clarity. Each recombinase subunit is connected to its neighbors though a cyclic network of allosteric interactions that dictates its activation state during the consecutive steps of recombination. The recombination sites are brought together in an antiparallel configuration exposing one specific pair of DNA strands at the center of the synapse. In this configuration, the IR1-bound TnpI subunits (magenta) are activated to catalyze the first strand exchange and generate the Holliday junction (HJ) intermediate. The complex then isomerizes, which deactivates the IR1-bound TnpI subunits and activates the IR2-bound subunits (pink) for catalyzing the second strand exchange. For each strand exchange, the recombinase catalytic tyrosine (curved arrow) attacks the adjacent phosphate (yellow circle) to form a 3′ phosphotyrosyl protein–DNA bond, which is in turn attacked by the 5′ OH end (half-arrow) of the partner DNA strand. (B) Possible model for the topological organization of the TnpI/IRS recombination complex. TnpI binding to the DR1–DR2 accessory motifs of directly repeated IRSs generate a synaptic complex in which three DNA crosses are trapped. Proper antiparallel pairing of the IR1–IR2 core sites introduce a positive twist in the DNA so that strand exchange as in (A) generates a two-node catenane product. doi:10.1128/microbiolspec.MDNA3-0060-2014.f12

Citation: Nicolas E, Lambin M, Dandoy D, Galloy C, Nguyen N, Oger C, Hallet B. 2015. The Tn-family of Replicative Transposons, p 693-726. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0060-2014
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