Transposons families/Tn3 family

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Historical

Members of the Tn3 family were among the earliest transposons to be identified. In fact, the word “transposon” was used for the first time in 1974 by Hedges and Jacob in a seminal article in which they showed that ampicillin resistance could be transmitted between a number of different plasmids <ref name=":0"><pubmed>4609125</pubmed></ref>:

“We designate DNA sequences with transposition potential as transposons (units of transposition) and the transposon marked by the ampicillin resistance gene(s) as transposon A “.

TnA, later called Tn1, was isolated from the plasmid RP4 <ref name=":0" /> while the closely related TnB and TnC (later called [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2-KT002541 Tn''2''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] respectively) were isolated from plasmids RSF1010 <nowiki><ref name=":0" /> and R1 <nowiki><ref>Meynell E, Data N. Mutant Drug Resistant Factors of High transmissibility. Nature. 1967;214:885–887.</ref><ref name=":1"><pubmed>1093180</pubmed></ref>. Tn3 proved to be inserted into another, larger Tn3 family transposon, Tn4 <ref name=":1" />''. A number of early studies using electron microscope DNA [[wikipedia:Heteroduplex|heteroduplex analysis]] (e.g. <nowiki><ref name=":10"><pubmed>796463</pubmed></ref><ref name=":4"><pubmed>606841</pubmed></ref><ref name=":93"><pubmed>PMC232870</pubmed></ref> Fig. Tn3.1) demonstrated that movement of ampicillin resistance was accompanied by insertion of a DNA segment of about 4-5 kilobases (kb).

[[File:Fig.Tn3.1.png|center|thumb|640x640px|Fig. Tn3.1. Early Electron microscope heteroduplex study. One of many studies at this time. This example is from Rubens et al. 1976 <ref name=":93" />. Examples of plasmid RSF1010 with [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1''] ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''A'']) insertions. '''Left'''. EcoRI linearized [[wikipedia:Heteroduplex|heteroduplex]] between two RSF1010 plasmids with different Tn1 insertions at the same position but in opposite orientations, showing 2 single strand [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1''] loops (SS [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''A'']). '''Right'''. Two RSF1010 plasmids with different [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1''] insertions at 150 bp distant, showing 2 single strands Tn''1'' loops. A scale bar of 1 kilobase is shown. Note the short stem, which could represent the ~40bp IRs.|alt=]] The DNA sequence of the 4957 base pair (bp) Tn''3'' was obtained in 1979 <nowiki><ref name=":2"><pubmed>391406</pubmed></ref> and shown to be bordered by two inverted repeat sequences of 38 bp and included 2 genes in addition to the ampicillin resistance (beta-lactamase, bla) gene: a transposase gene, tnpA, and a gene involved in regulating tnpA and its own expression, tnpR (R for repressor). TnpR was subsequently shown to be a site-specific recombinase intimately involved in the transposition pathway <ref><pubmed>PMC319721</pubmed></ref> which acts on a specific site, IRS (Internal Resolution Site) (Fig. Tn3.2 i). In its absence, donor and target replicons form a stable cointegrate with two directly repeated Tn3 copies <ref name=":2" />. It was suggested that this type of structure was an intermediate in Tn''3'' transposition and that the IRS site was required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn''3'' <nowiki><ref name=":7"><pubmed>PMC218601</pubmed></ref> according to the Shapiro cointegrate model of replicative transposition (Fig. Tn3.2 ii; <ref name=":94"><pubmed>PMC383507</pubmed></ref> Fig. 2.7 Early models).

Indeed, Tn3 was shown to be instrumental in permitting transfer of a non-transmissible plasmid by a co-resident conjugative plasmid <ref name=":3"><pubmed>322280</pubmed></ref> resulting in fusion of the two plasmids which were separated at their junctions by two directly repeated Tn copies <ref name=":3" /><nowiki><ref name=":41"><pubmed>PMC294055</pubmed></ref><ref name=":42"><pubmed>340918</pubmed></ref><ref name=":5"><pubmed>745233</pubmed></ref>. [[File:Fig.Tn3.2.png.png|center|thumb|640x640px|Fig. Tn3.2. Organization and transposition mechanism of TnA. i) Organization. Early information describing the structure of the 4957 base pair Tn1 (now called Tn1 and similar to Tn2 and Tn3). The figure shows the length (in amino acids, aa) and relative position and orientation of the transposase, tnpA, repressor (resolvase), tnpR and Beta-lactamase passenger gene, blaTEM. The genes are indicated by horizontal unfilled arrows. The long terminal inverted repeats (IRL for left, IRR for right) are shown as black triangles and their sequence is written below included in a blue oval. The TnpR binding site, res, is indicated as a black box <ref name=":2" />. '''ii) The transposition cycle.''' The pathway involves replicative transposition resulting in fusion of the donor replicon (thick black circle) carrying the transposon (unfilled box) whose orientation is indicated by an arrow., and a target replicon (thin black circle). Replicon fusion requires TnpA and produces a cointegrated in which both donor and target replicons are fused and separated at each junction by a copy of the transposon in direct repeat. In a second step, TnpR binds to each ''res'' site and catalyses inter-res recombination, to regenerate the donor replicon and a target replicon which carries a single copy of the transposon <nowiki><ref name=":94" /><nowiki><ref><pubmed>6297786</pubmed></ref>. ]] A related TE γδ, or Tn1000, was identified as part of the plasmid F and appeared as an insertion loop in heteroduplex analysis <ref name=":5" /><nowiki><ref name=":6"><pubmed>6271456</pubmed></ref>. It was also implicated in the integration of the F plasmid into the Escherichia coli host chromosome <ref name=":6" /> and deletion of chromosomal DNA in F’ plasmids <nowiki><ref><pubmed>PMC408333</pubmed></ref><ref><pubmed>PMC246114</pubmed></ref> derived from F-excision with flanking chromosomal DNA <ref>Deonier RC, Yun K, Kuppermann M. Gamma delta-mediated deletions of chromosomal segments on F- prime plasmids. MolGenGenet. 1983;190:42–50. </ref>. It generates 5bp direct target repeats (DR) on insertion <ref><pubmed>PMC413041</pubmed></ref> and carries similar ends to those of Tn3 and to IS101, a small 200bp sequence carried by the pSC101 plasmid <ref><pubmed>2993789</pubmed></ref><ref><pubmed>PMC294604</pubmed></ref>.

Many other related transposons have since been identified with a highly diverse range of passenger genes (see <ref name=":8"><pubmed>26350313</pubmed></ref> and Fig. Tn3.4B). The tetracycline resistance transposon, Tn1721 from plasmid pRSD1 <ref name=":11"><pubmed>377024</pubmed></ref> and the multi-resistance transposons, Tn4 from R6-5 and Tn21, a component of the 25 kb resistance determinant (r-det) of the plasmid NR1 (R100) <ref name=":4" /> are three of many early examples. ===General Organization=== [[File:Fig.Tn3.3.png|left|thumb|520x520px|'''Fig. Tn3.3.''' '''Tn3 family: diverse and variable.''' A number of examples of Tn''3'' family transposons are shown to illustrate the extent of diversity of the family. The Tn are indicated by pale-yellow horizontal boxes. Open reading frames are shown as horizontal arrows with the arrowheads indicating the direction of expression: purple, transposition-associated genes; blue, integron integrase; red, antibiotic resistance genes; bright yellow, plant virulence genes; chrome, heavy metal resistance genes; orange, toxin/antitoxin genes; pale salmon, other passenger genes. The inverted terminal repeats are shown as grey arrows. The names of each transposon are shown on the left and their length in base pairs on the right. Accession numbers: [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1071 IS''1071''] ([https://www.ncbi.nlm.nih.gov/nuccore/M65135.1/ M65135]); [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] ([https://www.ncbi.nlm.nih.gov/nuccore/V00613 V00613]); Tn''4330'' ([https://www.ncbi.nlm.nih.gov/nuccore/X07651.1 X07651.1]); [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXax1-AE008925 Tn''Xax1''] ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXax1-AE008925 Tn''7206'']) ([https://www.ncbi.nlm.nih.gov/nuccore/AE008925 AE008925]); [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc4-CP009039 Tn''Xc4''] ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc4-CP009039 Tn''7210'']) ([https://www.ncbi.nlm.nih.gov/nuccore/CP009039 CP009039]); [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], ([https://www.ncbi.nlm.nih.gov/nuccore/AF071413 AF071413]); Tn''4651''.]] Members of the Tn''3'' transposon family form a tightly knit group with related transposases and DNA sequences at their ends. The basic Tn''3'' family transposition module is composed of '''transposase''' and '''resolvase''' genes and two ends with related terminal inverted repeat DNA sequences, the IRs, of 38-40bp or sometimes even longer ([[:File:Fig.Tn3.2.png.png|Fig. 3.2 '''i''']]) <nowiki><ref name=":20"><pubmed>PMC4337579</pubmed></ref>.

There is a large (~1000 aa) DDE transposase, TnpA, significantly longer than the DDE transposases normally associated with Insertion Sequences (IS) (see <ref><pubmed>PMC98933</pubmed></ref>). TnpA catalyzes the DNA cleavage and strand transfer reactions necessary for formation of a cointegrate transposition intermediate during replicative transposition.

A second feature of members of this transposon family is that they carry short (~100-150bp) DNA segments, res (for resolution) or rst (for resolution site tnpS tnpT – see below; <ref name=":9"><pubmed>22878084</pubmed></ref>) at which site-specific recombination between each of the two Tn copies occurs to “resolve” the cointegrate into individual copies of the transposon donor and the target molecules each containing a single transposon copy (Fig. Tn3.2 ii)(see <ref name=":8" />). This highly efficient recombination system is assured by a transposon-specified sequence-specific recombinase enzyme: '''the resolvase'''. There are at present three known major resolvase types: TnpR (which includes two subgroups, '''long''' and '''short''' with and without a [[wikipedia:C-terminus|C-terminal]] extension; [[Transposons families/Tn3 family#Resolution|Resolution]]), TnpI, and TnpS+TnpT, distinguished, among other things, by the catalytic nucleophile involved in DNA phosphate bond cleavage and rejoining during recombination: TnpR, a [[wikipedia:Site-specific_recombination|classic serine (S)-site-specific recombinase]] (e.g. <nowiki><ref name=":60"><pubmed>2555940</pubmed></ref><ref name=":61"><pubmed>2548736</pubmed></ref>); TnpI, a tyrosine (Y) recombinase similar to phage integrases <ref name=":43"><pubmed>PMC458404</pubmed></ref> (see <ref name=":8" />); and a heteromeric resolvase combining a [[wikipedia:Site-specific_recombination|tyrosine recombinase]], TnpS, and a divergently expressed helper protein, TnpT, with no apparent homology to other proteins <nowiki><ref name=":9" /><nowiki><ref name=":76"><pubmed>PMC135285</pubmed></ref>.

The resolvase genes can be either co-linear, generally upstream of tnpA or divergent. In the former case the res site lies upstream of tnpR and in the latter case, between the divergent tnpR and tnpA genes. For relatives encoding TnpS and TnpT, the corresponding genes are divergent and the res (rst) site lies between tnpS and tnpT.

Examples of these architectures are shown in Fig. Tn3.3. Each res includes a number of short DNA sub-sequences which are recognized and bound by the cognate resolvases. These are different for different resolvase systems. But where analyzed, res sites also include promoters which drive both transposase and resolvase expression. Indeed, TnpR from Tn3 was originally named for its ability to repress transposase expression by binding to these sites <ref name=":2" /><nowiki><ref name=":7" /> (see later: [[Transposons families/Tn3 family#Resolution|Tn''3'' family resolution systems]]). ====Diversity: TnpA Tree==== The complexity of these Tn resides in the diversity of other mobile elements incorporated into their structures (such as [[General Information/What Is an IS?|IS]] and [[wikipedia:Integron|integrons]] as well as other Tn''3'' family members – see <nowiki><ref name=":8" /> - and other '''passenger genes'''). The most notorious of these genes are those for '''antibiotic''' and '''heavy metal resistance''' although other genes involved in organic catabolite degradation and virulence functions for both animals and plants ([[:File:Fig.Tn3.3.png|Fig. Tn3.3]]) also form part of the Tn''3'' family arsenal of passenger genes. The diversity of Tn''3'' family members was investigated using a library of carefully annotated examples in the [https://tncentral.ncc.unesp.br/ISfinder/index.php ISfinder database] <nowiki><ref><pubmed>PMC1347377</pubmed></ref>, those listed in Nicolas et al. <ref name=":8" />, those resulting from a search of [https://www.ncbi.nlm.nih.gov/ NCBI] for previously annotated Tn''3'' family members (March 2018) and those obtained using a script, Tn''3''_TA_finder, which can be searched for ''tnpA'' and ''tnpR'', genes located in proximity to each other (Tn''3''finder, https://tncentral.proteininformationresource.org/TnFinder.html; Tn''3''_TA_finder, https://github.com/danillo-alvarenga/tn3-ta_finder) in complete bacterial genomes in the RefSeq database at [https://www.ncbi.nlm.nih.gov/ NCBI]. This yielded 190 Tn''3'' family transposons for which relatively complete sequence data (transposase, resolvase, and generally both IRs) were available. Full annotations can be found at '''[https://tncentral.proteininformationresource.org/index.html TnCentral]''' (https://tncentral.proteininformationresource.org/index.html). A tree based on the transposases of these transposons is shown in [[:File:Fig.Tn3.4.png|Fig. Tn3.4A]] <nowiki><ref name=":30"><pubmed>PMC7157771</pubmed></ref>.[[File:Fig.Tn3.4.png|center|thumb|780x780px|Fig. Tn3.4A. A phylogenetic tree of 190 Tn3-family members based on their TnpA sequences. Lima Mendez et al. <ref name=":30" /> Tn''3'' family members were extracted from the ISfinder database, which served to generate the subgroups defined in Nicolas et al. <nowiki><ref name=":8" />. Many others were drawn from the literature and have been given official names (Tn followed by digits, e.g. Tn''1234'': https://transposon.lstmed.ac.uk/tn-registry) while others were identified using Tn''3''_finder software ([https://tncentral.ncc.unesp.br/ TnCentral]: https://tncentral.ncc.unesp.br/TnFinder.html) and given temporary names (these have now been registered at the Transposon registry and have been assigned Tn numbers which can be found as synonyms in [https://tncentral.ncc.unesp.br/ TnCentral]). Each is associated with its GenBank accession number; the GenBank file contains either the extracted transposon or the DNA sequence from which it was extracted (e.g., DNA fragment, plasmid or chromosome). Numbers above the lines of each clade indicate the maximum likelihood bootstrap values. The sub-groups adhere closely to those defined by Nicolas et al. <nowiki><ref name=":8" /> with some minor variations resulting from the significantly larger Tn sample. The majority of members carry ''tnpR'', '''serine resolvases''' (purple circles) although one group in the Tn''3'' clade encodes “long’ serine '''resolvases'''. Those that include ''tnpI'' or ''tnpT''/''tnpS'' are indicated by salmon and pink circles, respectively. The '''TA''' gene pairs are indicated by coloured squares. Note that [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501.5''] carries a mutation which truncates its toxin gene, leaving the antitoxin intact. The outer squares represent the toxin and the inner squares, the antitoxin. The five toxin types are: [http://pfam.xfam.org/family/PF13876 Gp49] ([https://pfam.xfam.org/family/PF05973 PF05973]), purple; [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR002850/ PIN_3] ([https://pfam.xfam.org/family/PF13470 PF13470]), dark green; [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR002850/ PIN] ([https://pfam.xfam.org/family/PF01850 PF01850]), bright blue; [https://www.uniprot.org/uniprot/P0C077 ParE] ([https://pfam.xfam.org/family/PF05016 PF05016]), yellow; and [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR008201/ HEPN], black. The antitoxins are: [https://pfam.xfam.org/family/PF13744 HTH_37] ([https://pfam.xfam.org/family/PF13744 PF13744]), orange; [https://www.ebi.ac.uk/interpro/entry/pfam/PF09957/ RHH_6] ([https://pfam.xfam.org/family/PF16762 PF16762]), blue; [https://www.uniprot.org/uniprot/Q06253][[Phd]]/[https://www.uniprot.org/uniprot/P69346 YefM] ([https://pfam.xfam.org/family/PF02604 PF02604]), magenta; RelB/[https://www.uniprot.org/uniprot/P22995 ParD]/CcdA/DinJ, dark grey; AbrB/[https://www.uniprot.org/uniprot/P0AE72 MazE], light grey; [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR008201/ MNT], bright green. The corresponding Tn names and accession numbers are highlighted in bold type for clarity. Note that the branches have been extended for clarity.|alt=]]The tree defines 7 deeply branching clades which supports the divisions proposed by Nicolas et al., <nowiki><ref name=":8" />. They were named after a representative Tn from each clade: [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']; Tn''4651''; [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3000-AF174129 Tn''3000'']; [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071'']; [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21'']; [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn163-L14931 Tn''163'']; and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430'']. As can be seen from [[:File:Fig.Tn3.4.png|Fig. Tn3.4A]], the vast majority of Tn''3'' family members encode a ''tnpR''/''res'' resolution system and encode a TnpR without the [[wikipedia:C-terminus|C-terminal]] extension (shown by blue circles) and a small group which encodes a TnpR derivative with the [[wikipedia:C-terminus|C-terminal]] extension ([[:File:Fig.Tn3.4.png|Fig. Tn3.4A]]). However, a significant sub-group of the Tn''4651'' clade encodes the ''tnpS''/''tnpT''/''rst'' resolution system (pink circles) while the ''tnpI''/''irs'' is represented in only three cases. An overview, extracted from [https://tncentral.proteininformationresource.org/index.html TnCentral], of the diversity and distribution of different passenger genes within the Tn''3'' family and their presence in different bacterial hosts is shown in Fig. Tn3.4B. [[File:Fig.Tn3.4B.png|center|thumb|820x820px|'''Fig. Tn3.4B. Distribution of Different Passenger Genes within the Tn''3'' Family.''' A key to the types of passenger genes is shown on the left of the figure. This figure was kindly provided by [https://uclouvain.be/en/directories/nicolas.aryanpour Nicolas Aryanpour] based on information extracted and collated from the [https://tncentral.ncc.unesp.br TnCentral] database by [https://scholar.google.com/citations?user=2DgPsLAAAAAJ&hl=en Gipsi Lima-Mendez].|alt=]] ====Tn3 family complementation groups==== Early studies on the relationship between different Tn''3'' family members revealed that they could be divided into different functional groups by genetic complementation of their ''tnpA'' and ''tnpR'' genes <nowiki><ref name=":62"><pubmed>6312271</pubmed></ref><ref name=":12"><pubmed>6294711</pubmed></ref>.

Transposition-deficient tnpA mutants of Tn1721 (Tn21 clade; Fig. Tn3.4A) and the mercury resistance transposon Tn501 <ref><pubmed>1660177</pubmed></ref><ref name=":36"><pubmed>3007931</pubmed></ref><ref><pubmed>6327411</pubmed></ref><ref><pubmed>PMC321896</pubmed></ref> (close to Tn1721 in the Tn21 clade;) could be complemented in trans by co-resident wild type copies of either Tn21, Tn501, or Tn1721, while transposition of a Tn21 tnpA mutant could only be restored by Tn21. Moreover, Tn3 was unable to complement either Tn21, Tn501, or Tn1721, and vice versa <ref name=":12" />. Similarly, a [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] ''tnpR'' mutant could be complemented by [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] or [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''], but not by [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']. Moreover, mutations in the Tn''2603'' ''tnpA'' and ''tnpR'' genes could be complemented by mercury resistance transposons Tn''2613'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] (although [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] was much less efficient in complementation than Tn''2613'') but not by gamma delta, Tn''2601'' or Tn''2602'' (both of which resemble the Tn''3'' group – see [[:File:Fig.Tn3.7A.PNG|Fig. Tn3.7 '''A''']]) <nowiki><ref name=":13"><pubmed>6314094</pubmed></ref>. In this context, it is perhaps useful to note the Tn501 and Tn1721 are located at some distance from Tn21 in the tnpA phylogenetic tree. This reinforced the idea, based principally on the direction of transcription of their tnpA and tnpR genes, that the Tn3 family could be divided into 2 major groups: Tn3 and Tn21 <ref>Heffron F. Tn3' ' and its relatives. In: James A. S, editor. Mobile' ' Genetic Elements. New York.: Academic Press,; 1983. p. 223–260. </ref>.

Tn3 and Tn21 groups

Grinsted et al. <ref name=":14"><pubmed>1963947</pubmed></ref> identified at least five Tn3 family subgroups which correspond to those shown in Fig. Tn3.4A. In addition to the Tn3 and Tn21 subgroups, the others included Tn2501 (Tn163 subgroup), Tn917/Tn551 (Tn4430 subgroup) and Tn4556 (Tn3000 subgroup). Tn917 and Tn551 are quasi-identical and Tn4430 was included in a separate subgroup because it encodes a resI/tnpI resolution system.

These divisions were based on the observations that: transposition proteins within each group were at least 70% similar or identical whereas this value was only about 30% between groups and that the IR sequences were less than 26/38 identical. The authors propose a model for the evolution of the Tn3 family transposition modules (Fig. Tn3.5) in which two ancestral modules were assembled: the first included a tnpR gene (which they suggest was flanked by an invertible DNA segment incorporating the res site) and a tnpA gene. This subsequently gave rise to each of the Tn3 subgroups by tnpR/res inversion and sequence divergence. For Tn such as Tn4430, the assembly involved tnpI/res and tnpA components. The tnpS/tnpT/rsc resolution system was not included since it had not been identified at that date but could easily be incorporated into this scheme. To our knowledge, the proposed ancestral components in this scheme have not yet been identified. [[File:Fig.Tn3.5.png|thumb|640x640px|Fig. Tn3.5. Hypothetical pathways Tn3 family evolution (adapted from <ref name=":14" />). The transposons are shown as yellow filled boxes, resolvase (R) and transposase (A) genes as horizontal purple arrows. Res sites are shown as green boxes. The figure proposes that present day Tn''3'' family members arose from three ancestors: one in which the ''tnpR'' gene lies downstream from ''tnpA'' with ''res'' between them (left: Tn''4556''), a second which encodes a ''tnpI'' resolvase and an alternative ''res'' site (right: Tn''4330'') and one which is a precursor to the majority of family members (middle). This is proposed to include ''tnpR'' and ''tnpA'' genes expressed in the same direction and two sequences, one at the left end and another between ''tnpR'' and ''tnpA'' which gave rise to ''res''. Inversion of tnpR generated one Tn''3'' class which, on acquisition of the ''[[wikipedia:Beta-lactamase|bla]]'' ([[wikipedia:Penicillin|penicillin resistance]]) passenger gene, became [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'' itself], while in the other branch, the internal res sequences were lost to generate the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] class and acquisition of an erm ([[wikipedia:Erythromycin|erythromycin resistance]]) passenger gene gave rise to Tn''917''/[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn551-Y13600.1 Tn''551'']. The branches also correspond to the sequence relationships of ''tnpR'' and ''tnpA'' in these groups.|alt=|center]]The diversification of different [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] clade members was also examined <nowiki><ref name=":14" /> (Fig. Tn3.6) and forms two subclades. One includes [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], Tn''2613'' (whose sequence is not available but which may be identical to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5060-AJ551280.1 Tn''5060'']-[https://www.ncbi.nlm.nih.gov/nuccore/AJ551280.1/ AJ551280.1]) and Tn''3926'' (with only a partial sequence available but which complements a ''tnpA''-defective [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] but not [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''] or [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] mutants <nowiki><ref><pubmed>3005130</pubmed></ref>). The other includes Tn501, Tn1722, Tn1721 and Tn4653. Tn501 and Tn1721 are located in a sub-clade distinct from Tn501 and Tn5060 (Fig. Tn3.4A). In this scheme, mercury resistance was proposed to have been acquired twice independently in each subclade, early in the Tn21 subclade lineage and later in the line leading to Tn501. The ancestor of Tn21 had acquired an integron platform transported by a Tn402 family transposon and Tn1721 was derived from Tn1722 by acquisition of a tet resistance gene. [[File:Fig.Tn3.6.png|thumb|640x640px|Fig. Tn3.6. Proposed Evolution of elements of the Tn21 subgroup (adapted from <ref name=":14" />). This is based on genetic complementation and sequence similarities. The ancestral Tn of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] group was proposed to have acquired a [http://parts.igem.org/Part:BBa_K1420000 mercury] (''[http://parts.igem.org/Part:BBa_K1420000 mer]'') resistance operon early in the pathway ('''left''') with the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] lineage acquiring sulfonamide resistance by insertion of an [[wikipedia:Integron|integron]] platform. In the right-hand pathway, acquisition of a [http://parts.igem.org/Part:BBa_K1420000 mercury] (''[http://parts.igem.org/Part:BBa_K1420000 mer]'') resistance operon was proposed to give rise to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''], while [[wikipedia:Tetracycline|tetracycline]] was proposed to have been acquired leading to formation of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''] and the ability to degrade [[wikipedia:Toluene|toluene]] (''xyl'') was acquired to generate Tn''4651'' (see [[:File:Fig.Tn3.3.png|Fig. Tn3.3]]).|alt=|center]] =====The Tn''21'' Clade===== The [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] is a large group with 49 members at present in TnCentral (most of these are shown in [[:File:Fig.Tn3.7A.PNG|Fig. Tn3.7 '''A''']]). Like the entire Tn''3'' family, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] clade members possess highly conserved IRL and IRR ([[:File:Fig.Tn3.7B.PNG|Fig. Tn3.7 '''B''']], '''[[:File:Fig.Tn3.7C.png|C]]''' and '''[[:File:Fig.Tn3.7D.PNG|D]]''').[[File:Fig.Tn3.7A.PNG|thumb|640x640px|'''Fig. Tn3.7A'''. The relationship between the transposases of different members of the Tn''21'' clade expanded from [[:File:Fig.Tn3.4.png|Fig.Tn3.4A]]. Each transposon is shown together with its Genbank accession number. The small purple circles indicate that all members encode a ''tnpR'' resolution system. One member, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnSod9-NC_004349 Tn''Sod9''], encodes a [[wikipedia:Toxin-antitoxin_system|toxin/antitoxin gene pair]] (black and green squares) and is shown in bold type. Those Tn encircled by blue boxes are treated in detail in the text. The lozenges on the outer circle indicate the presence and type of passenger gene carried by the corresponding Tn. '''''mer''''': [http://parts.igem.org/Part:BBa_K1420000 mercury resistance operon]; '''''ars''''': [[wikipedia:Arsenic|arsenic resistance]]; '''''chr''''': [[wikipedia:Chromate_and_dichromate|chromate resistance]]; '''red''': [[wikipedia:Integron|integron]] plataform; '''pale-yellow''': other passenger genes; '''pale-yellow/red''': other passenger genes + non integron; '''white''': no passenger genes; '''black line''': not in [https://tncentral.ncc.unesp.br/images/transparent_dot.png TnCentral].|alt=|center]] Many clade members encode ''tnpR'' with a ''res'' site immediately upstream and, in a majority (but not all), ''tnpA'' is located downstream and in the same orientation. The ''res'' sites of this class ([[:File:Fig.Tn3.7E.PNG|Fig. Tn3.7 '''E''']]) show a high degree of identity ([[:File:Fig.Tn3.7F.PNG|Fig. Tn3.7 '''F''']]). However other ''tnpR''/''tnpA'' configurations also occur ([[:File:Fig.Tn3.3.png|Fig. Tn3.3]]; [[:File:Fig.Tn3.7E.PNG|Fig. Tn3.7 '''E''']]) and their ''res'' sites (see below: [[Transposons families/Tn3 family#The Tn1721.2C Tn21 and Tn501 res.|The Tn''1721'', Tn''21'' and Tn''501'' ''res'']]) show relatively good conservation ([[:File:Fig.Tn3.7F.PNG|Fig. Tn3.7 '''F''']]) <gallery mode="slideshow" caption="'''Fig. Tn3.7.''' Panels B, C and D (use the arrows to scroll the figures)."> File:Fig.Tn3.7B.PNG|'''Fig. Tn3.7B.''' The Ends of Members of the Tn''21'' Clade. Left (IRL) and right IRR inverted terminal repeats are shown in [https://weblogo.berkeley.edu/logo.cgi WebLogo] format. Three well conserved regions are interested in horizontal double-headed arrows. File:Fig.Tn3.7C.png|'''Tn3.7C and D.''' Alignments of IRL and IRR of Tn''21'' family members. Transposon names are shown on the left. Those with non-conforming names have all now been attributed names in The [https://www.lstmed.ac.uk/technical-services/the-transposon-registry Transposon Registry]. File:Fig.Tn3.7D.PNG|'''Tn3.7C and D.''' Alignments of IRL and IRR of Tn''21'' family members. Transposon names are shown on the left. Those with non-conforming names have all now been attributed names in The [https://www.lstmed.ac.uk/technical-services/the-transposon-registry Transposon Registry]. </gallery> [[File:Fig.Tn3.7E.PNG|center|thumb|640x640px|'''Tn3.7E.''' Tn''21'' clade ''tnpR''/''tnpA'' configurations. The right-hand column shows members with the “classical” Tn''21'' configuration in which the passenger genes are located upstream from ''tnpR'' and the ''res'' site; the next column indicates those members with the same ''tnpR''/''tnpA'' configuration but in which the ''res'' site has been split by insertion of a Tn''401''-like [[wikipedia:Integron|integron]] structure; the third column includes those clade members with divergent ''tnpR''/''tnpA'' and a ''res'' site between the two genes; the fourth column lists members with the divergent ''tnpR''/''tnpA'' but in which passenger genes have been inserted in between. Where appropriate, registered transposon names are shown in brackets]] [[File:Fig.Tn3.7F.PNG|center|thumb|640x640px|'''Fig. Tn3.7F.''' ''res'' site alignment of Tn''21'' clade members with co-linear ''tnpR''/''tnpA'' genes. The Tn names are shown to the left of the figure. Sequence conservation is shown by the depth of the blue background. Sites I, II and III are indicated.]] ======Derivatives with a simple mercury operon.====== In general, passenger genes in this clade are located upstream of ''tnpR'' and the ''res'' site ([[:File:Fig.Tn3.7G.PNG|Figs. Tn3.7 '''G-N''']]). Ten clade members carry only genes for resistance to mercury salts. Two of these, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5060-AJ551280.1 Tn''5060''] ([https://www.ncbi.nlm.nih.gov/nuccore/AJ551280.1 AJ551280.1]) ([[:File:Fig.Tn3.7G.PNG|Tn3.7 '''G''']]), the proposed ancestor of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] [[wikipedia:Integron|integron]] group ([[:File:Fig.Tn3.7I.PNG|Tn3.7 '''I''']]) <nowiki><ref name=":15"><pubmed>14553919</pubmed></ref>, and Tn20 (AF457211.1) are nearly identical except for a few SNP and a deletion of a few base pairs in ufrM (Tn20).[[File:Fig.Tn3.7G.PNG|center|thumb|640x640px|Fig. Tn3.7G. Integron platform insertion sites. The top of the figure shows a probable generic ancestor of Tn21 clade Transposons which carry Tn402-based class I integrons. Horizontal filled arrows represent the open reading frames: chrome color, mercury resistance genes (merR, merT,merP,merC,merA,merD,merE); pink, reading frame of unknown function (urfM); purple, transposition- related genes tnpR (resolvase) and tnpA (transposase). The resolution site, res, is shown in green. The points of integration (into an ancestor, such as Tn5060 or Tn20) are shown by vertical red and blue arrows. Integration into urfM (blue) gave rise to Tn21 and its related transposons (blue box, left). Those which for which the DNA sequence is available (blue) share an identical insertion site together with a 5 base pair flanking target duplication expected to be generated by Tn402 family integration. Those for which no sequence is available also appear to carry the inserted integron in an identical position as judged by restriction mapping. Integration into the res site of an ancestor such as Tn1696.1 or Tn5036 (right) gave rise to Tn1696 and Tn6005 (red box, right). The transposons in the middle column are referred to in the text.]]These are quite different in sequence both in the mer operon and in tnpR/tnpA segments from the other transposons of similar organization. Tn1696.1 (CP047309) and Tn5036 (Y09025) differ by only a few SNPs while Tn4378 (CP000355), Tn6203 (CP065412) and Tn6346 (KM659090) are also quite different from the these. Tn4378 and Tn6203 show many sequence differences along their entire length as does TnAs2 (JN106175.1) while clearly, Tn6346 shares identity with Tn4378 over the entire length of the mer operon up to res but shows variability in the tnpR/tnpA region.

This clearly indicated that there has been an exchange by inter res recombination between two different transposons (Fig. Tn3.7H). A similar recombination has occurred with Tn501. In addition, Tn4380 appears to have been derived from Tn6346 by deletion of the entire res site. Thus Tn4378, Tn6436 (Tn4380) and Tn501 share highly related mer operons but vary in the sequences of tnpR and tnpA.

Fig. Tn3.7H. Relationship between mercury resistant transposons without integron insertions and of similar organization. i) The features are identical to those in Fig. Tn3.7G. Low resolution alignments against Tn4378 are shown as horizontal red lines above. While TnAs2 (Tn7144; JN106175.1) and Tn6203(CP065412) show significant divergence along their entire length, Tn6346 (KM659090), Tn4380 (CP000354), and Tn501 (Z00027) all share their mercury resistance genes with those of Tn4378. However, Tn6346 (KM659090), Tn4380 (CP000354) exhibit significant divergence in tnpR and tnpA and Tn501 shows even a higher level of divergence. The changes in levels of identity occur in or close to the res site. The Tn is indicated by a pale-yellow horizontal box. Open reading frames are shown as horizontal arrows with the arrowheads indicating the direction of expression: purple, transposition-associated genes; chrome, mercury resistance genes; pale salmon, other passenger genes. The inverted terminal repeats are shown as grey arrows. ii) The DNA sequences in the res site region showing the three subsites, resI, resII and resIII of Tn6346 and Tn4378 and Tn501. The non-identical bases are shown in red, conserved bases in two of the three sequences are in bold and underlined and the probable recombination point ATA is shown in red/bold.


Derivatives with class I integrons: 2 events leading to multiple antibiotic resistance

At least 22 Tn21 clade members carry class I integrons (Fig. Tn3.7 A and Fig. Tn3.7 I) although the DNA sequence of some of these is not available. These are transmitted by Tn402 derivative transposons which exhibit pronounced target specificity (more details at: Tn402 family) and show a preference for insertion into or close to Tn3 family res sites or into plasmid res sites. A major pathway for the acquisition of passenger genes was the initial integration of a Tn402-like transposon which carried a class I integron platform. The integron insertions have occurred at one of two positions in the Tn5060 /Tn20 related examples (Fig. Tn3.7 G). In one group, which all encode an identical mer operon, insertion occurred in a precise position in a gene of unknown function, ufrM (see: The Tn21 Lineage) (Fig. Tn3.7 I). Since these occur at the same nucleotide, it seems possible that all diverged from a single insertion event.

Fig. Tn3.7I. Integron insertion into ufrM. An alignment of various Tn21 group transposons (red horizontal lines) against Tn5060 (AJ551280.1; features identical to those described in Fig. Tn3.7G) show that insertion of the integron platform (small red triangles) occurs at the same position in ufrM. A number of small sequence differences with Tn5060 are shared with Tn20, suggesting that Tn20 was perhaps the ancestor of this group. Tn21 (AF071413); Tn2411 (FN554766); Tn2424 (UGCJ01000005); TnAs3 (Tn7145; CP000645.1); Tn5086 (CP054343); Tn4 (KY749247.1); Tn21.1 (MH257753); Tn21.2 (MH626558); Tn20 (AF457211.1). The Tn is indicated by a pale-yellow horizontal box. Open reading frames are shown as horizontal arrows with the arrowheads indicating the direction of expression: purple, transposition-associated genes; chrome, Mercury resistance genes; pale salmon, other passenger genes. The inverted terminal repeats are shown as grey arrows.

In the others, the res site itself has been targeted: at two slightly different positions both in the Tn1696 (Fig. Tn3.7 J) (also carrying a mer operon) and Tn1721 (with an mcp gene) groups (Fig. Tn3.7 K) while a third example can be observed in Tn5045.1 carrying the tao gene cluster (Fig. Tn3.7 L). The fact that integrons In2 and In4 are located in different sequence environments in two distinct mercury resistance transposons, Tn21 and Tn1696 has previously been noted <ref><pubmed>PMC90453</pubmed></ref>. Thus, although widespread in nature, class 1 integrons appear to have inserted in only six target sequences in the entire Tn21 clade in TnCentral. The significant variability therefore arises principally by acquisition and loss of integron cassettes and by frequent various degrees of loss by deletion/inactivation (see: Tn21 lineage) of the Tn401 transposition genes tniA,B,Q and its resolvase tniR (see: Tn402 family).

Fig. Tn3.7J. Tn1696 and relatives. The transposon features are identical to those in previous figures. The Tn is indicated by a pale-yellow horizontal box. Open reading frames are shown as horizontal arrows with the arrowheads indicating the direction of expression: purple, transposition-associated genes; chrome, Mercury resistance genes; pale salmon, other passenger genes. The inverted terminal repeats are shown as grey arrows. The presumed ancestor, Tn16961.1 (CP047309), is shown below and alignments of Tn5036 (Y09025) a similar transposon without the integron insertion), Tn1696 (U12338.3) and Tn6005 (EU591509.1; both with integron insertions; small vertical triangles) are shown as horizontal red lines above. The top of the figure shows the sequence of the Tn1696.1 res site, indicating the position of integron Insertions (vertical blue arrows). The target sequences which are the flanking DRs are shown in red.
Fig. Tn3.7K. Tn1721 and relatives. The bottom panel shows the tetracycline resistance transposon Tn1721 (X61367.1) in which the passenger genes are located both upstream and downstream of tnpR and tnpA. The Tn are indicated by pale-yellow horizontal boxes. Open reading frames are shown as horizontal arrows with the arrowheads indicating the direction of expression: purple, transposition-associated genes; red, antibiotic resistance genes; pale salmon, other passenger genes. The inverted terminal repeats are shown as grey arrows. A potential ancestor, TnpCTXM9 (Tn7181; probably identical to Tn1722; CP031724) is shown above. It carries the upstream passenger gene, but not the downstream tet genes. Alignments of Tn1721.1 (HQ730118.1) and TnCfrpOZ172 (Tn7154; CP016763.1) both carrying integron insertions (small vertical triangles) are shown as red lines above. The DNA sequence of the res site is shown with the positions of resI, resII and resIII and the points of integration. The target sequences which are the flanking DRs are shown in red.
Fig. Tn3.7L. Tn5045.1 and relatives. An integron insertion into Tn5045.1 (NC_008357.1) generated Tn5045 (FN821089.1). The DNA sequence of the Tn5045.1 res site is shown with the positions of resI, resII and resIII and the point of integration. The target sequences, which are the flanking DRs, are shown in red.
Derivatives with upstream passenger genes: colistin resistance.

Of the four colistin resistant examples (Fig. Tn3.7 M): TnSen1.1 [Tn7191] and TnSen1.2 [Tn7192] are nearly identical except that TnSen1.2 carries an ISPa96 insertion; both TnEc026 [Tn7159] and TnMCR5ECO26H11 [Tn7163] are identical but TnEcO26 has two right ends.

Moreover, while the left segment of all 4 are closely related, there appears to have been a recombination event in the region of the res site two right ends and TnSen11.2/TnSen1.2 and TnEcO26/ TnMCR5ECO26H11 carry divergent tnpR and tnpA.

Fig. Tn3.7M. Tn21 Colistin resistance. Top panel: the colistin (mcr5) resistance transposon TnSen1.1 (Tn7191; KY807921) in which the passenger genes are located upstream of tnpR and tnpA. Alignments of TnSen1.2 (Tn7192; CP028162), TnMCR5ECO26H11 (Tn7163; BEPM01000040) and TnEc026 (Tn7159; BDIH01000107.1) are shown as red lines above. Apart from three small sequence differences, the major difference with TnSen1.1 is that TnSen1.2 carries an insertion of ISPa96 within the transporter gene(s) at its left end (red vertical triangle). TnMCR5ECO26H11 and TnEc026 are identical except that TnEc026 includes an extension at its right end which duplicates an IRR sequence. Bottom panel: The DNA sequence of the res site is shown below with the proposed positions of resI, resII and resIII and the points of recombination. The non-identical bases are shown in red, conserved bases are in bold and underlined and the probable recombination point ATA is shown in red/bold.
Derivatives with upstream passenger genes: other passengers.

There are a number of other Tn21 clade members with different upstream passenger genes. Analysis of these reveals that, although there has been some diversification of the tnpR and tnpA genes (Fig. Tn3.7 N), there is a clear breakpoint in identity which occurs at the res site. Sequence analysis (Fig. Tn3.7 N) indicates that the break in identity occurs at the potential AT recombination dinucleotide (see: Resolution topic below) strongly suggesting that acquisition of various passenger genes frequently occurs by modular exchange via inter-res recombination.

Fig. Tn3.7N. Tn21 other passenger genes. Top panel: TnPa40 carrying chromate resistance genes (Tn7173; CP003149) is shown together with alignments of TnPa40.1 (Tn7174; CP020704.1; carrying a miscellaneous set of passenger genes and an insertion of IS1411), Tn5045.1 (Tn1013; NC_008357.1; carrying a tao operon) and Tn4656 (NC_008275.1; carrying mcp genes). Bottom panel: The DNA sequence of the res site is shown below with the proposed positions of resI, resII and resIII and the points of recombination.
Derivatives with divergent tnpR and tnpA

There are a number of Tn21 clade members in which the tnpR and tnpA genes are expressed divergently. Several of these (e.g. Tn4659, TnAcsp1 [Tn7133], TnEc1 [Tn7158] and TnSba14 [Tn7190]) (Fig. Tn3.7 O) do not encode passenger genes and are not closely related, while others encode heavy metal resistance operons located between tnpR and tnpA (e.g. TnLfArs [Tn7162], TnOtChr [Tn7169]) while TnPa38 [Tn7172] encodes genes of unknown function and TnSod9 [Tn7199] is the only example in the Tn21 clade to encode a Toxin/Antitoxin gene pair. These are not closely related.

Fig. Tn3.7O. Tn21 clade Members with Divergent tnpR and tnpA. The Tn are indicated by pale-yellow horizontal boxes. Open reading frames are shown as horizontal arrows with the arrowheads indicating the direction of expression: purple, transposition-associated genes; chrome, heavy metal resistance genes; pale salmon, other passenger genes; bordeaux, hypothetical. The inverted terminal repeats are shown as grey arrows. TnLfArs (Tn7162, DQ057986.1) ; TnOtChr (Tn7169, EF469735.1); TnPa38 (Tn7172, CP003149).
The Tn21 Lineage.

The Tn21 lineage is an example of the plasticity of Tn3 family transposons. Tn21 was originally identified in the multiple antibiotic resistance plasmid NR1/R100 <ref><pubmed>13727669</pubmed></ref>, as part of the IS1-flanked r-determinant <ref name=":10" /> and its component antibiotic resistance genes were first mapped by restriction enzyme digestion and cloning <nowiki><ref><pubmed>340913</pubmed></ref>. The Tn21 group of transposons appear to be very successful as judged by their distribution.

Fig. Tn3.7P. Early Restriction site maps of related antibiotic resistance Tn21 clade transposons. Redrawn from <ref name=":17"><pubmed>6298184</pubmed></ref>. Transposons are indicated as horizontal yellow boxes. Retriction sites: EcoRI (down-arrow); HindIII (up-arrow); BamHI (down circle-arrow)); PstI (up circle-arrow). Passenger genes: mer: mercury; Su: sulphonamide; Sm: streptomycin; bla: beta-lactamase. There is no available sequence for many of these transposons: Tn2613, Tn2608, Tn21 (AF071413), Tn2603, Tn2607, Tn2601, Tn4 (KY749247.1), Tn3 (V00613).

This is arguably the result of acquisition of an integron platform permitting incorporation of various resistance genes as integron cassettes <ref name=":14" /><nowiki><ref name=":16"><pubmed>PMC103744</pubmed></ref> (Fig. Tn3.7 A and Fig. Tn3.7 G). Tanaka and collaborators proposed in the early 1980s that Tn21-like transposons which carry a variety of antibiotic resistance genes are related and evolved from an ancestor carrying a mercury resistance operon <ref name=":17" /> ([[:File:Fig.Tn3.5.png|Fig. Tn3.5]]; F[[:File:Fig.Tn3.7P.PNG|ig. Tn3.7 '''P''']]). [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] itself is a complex collection of intercalated TE and a comprehensive and detailed scheme for its formation has been proposed <nowiki><ref name=":14" /><nowiki><ref name=":17" /><nowiki><ref name=":16" /> (see [[:File:Fig.Tn3.6.png|Fig. Tn3.6]].; [[:File:Fig.Tn3.7P.PNG|Fig. Tn3.7 '''P''']]). Unfortunately, although the DNA sequences of some of the component transposons are now available (e.g. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4-KY749247.1 Tn''4'']'','' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411'']), many are not and comparison was based on physical and functional maps (restriction, genetic features) <nowiki><ref name=":13" /><nowiki><ref name=":17" /><nowiki><ref><pubmed>PMC217832</pubmed></ref><ref name=":18"><pubmed>2991421</pubmed></ref>.

This scheme was later expanded with the addition of more up-to-date information to include a number of potential Tn21 descendants (see <ref name=":16" />) ([[:File:Fig.Tn3.7Q.PNG|Fig. Tn3.7 '''Q''']]). It was proposed that a [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] precursor ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21'']) acquired an [[wikipedia:Integron|integron]] platform such as is found in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4-KY749247.1 Tn''4''] (for convenience, called [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn''4''] here) which then received an insertion of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1353-AF071413 IS''1353''] into a resident [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] copy to generate [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In2-AF071413 In2] found in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] <nowiki><ref name=":17" />. [[File:Fig.Tn3.7Q.PNG|thumb|640x640px|'''Fig. Tn3.7Q.''' The proposed lineage of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] [[wikipedia:Integron|integron]]-carrying group of transposons. Redrawn and amended from <nowiki><ref name=":16" /><nowiki><ref name=":18" />. The steps leading to each transposon (within a red box) are shown in blue boxes. Filled red boxes show transposons for which the DNA sequence is available. The sequences of Tn''2608'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5086-CP054343 Tn''5086''] were reconstructed from known sequence elements. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411''] ([https://www.ncbi.nlm.nih.gov/nuccore/FN554766 FN554766]); [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4-KY749247.1 Tn''4''] ([https://www.ncbi.nlm.nih.gov/nuccore/KY749247.1 KY749247.1]); [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] ([https://www.ncbi.nlm.nih.gov/nuccore/AF071413 AF071413]); [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2424-UGCJ01000005 Tn''2424''] ([https://www.ncbi.nlm.nih.gov/nuccore/UGCJ01000005 UGCJ01000005]).|alt=|center]]Although the Tn''21'' group ancestor prior to acquisition of the [http://parts.igem.org/Part:BBa_K1420000 mercury resistance genes] is at present unknown, the later identification of a [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] transposon, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5060-AJ551280.1 Tn''5060''] ([https://www.ncbi.nlm.nih.gov/nuccore/AJ551280.1 AJ551280.1]), isolated from the Siberian permafrost <nowiki><ref name=":15" /> ([[:File:Fig.Tn3.7R.PNG|Fig. Tn3.7 '''R''']]) provided a possible candidate for the hypothetical Tn''21'' precursor, Tn''21°''. [[File:Fig.Tn3.7R.PNG|center|thumb|640x640px|'''Fig. Tn3.7R.''' Proposed lineage of the Tn''21'' [[wikipedia:Integron|integron]]-carrying group of transposons. Redrawn from <nowiki><ref name=":16" />. Integration of a type 1 [[wikipedia:Integron|Integron]] was proposed to occur into an ancestral transposon Tn''21D'' ('''Top''') into the ''ufrM'' gene of unknown function, giving rise to a characteristic five base pair DR (ATGGA) and to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411''] ([https://www.ncbi.nlm.nih.gov/nuccore/FN554766 FN554766]) ('''Middle'''). Insertion of a copy of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1353-AF071413 IS''1353''] into the resident [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1536 IS''1536''] then gave rise to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] ('''Bottom''').]]Other examples of this Tn such as [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn20-AF457211.1 Tn''20''] ([https://www.ncbi.nlm.nih.gov/nuccore/AF457211 AF457211]) ([[:File:Fig.Tn3.7I.PNG|Fig. Tn3.7 '''I''']]) can be identified which share a number snips with other members of the group compared to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5060-AJ551280.1 Tn''5060''] <nowiki><ref name=":37"><pubmed>PMC149298</pubmed></ref> and therefore is perhaps a better candidate as an ancestor.

An alternate view of the path from Tn5060 to Tn21 is that evolution of the integron platform occurred “in situ” by the gradual loss/accumulation of component TE. In this scheme (Fig. Tn3.7 S and Fig. Tn3.7 P), a first step would be insertion into the ufrM (unknown function) gene of a Tn402 family transposon to provide the integron platform (Fig. Tn3.7 S).

Fig. Tn3.7S. Modified proposed lineage of the Tn21 integron-carrying group of transposons. The identification of Tn5060 (Top) and Tn20 (Fig. Tn3.7G) revealed possible Tn21 ancestors. The figure shows (Bottom) insertion of a simple active copy of a Tn402 derivative, the vector of type 1 integrons, carrying a complete set of transposition genes at position 4626 – 4633 to generate the 5 bp DR ATGGA.

Although it has been shown that transposition of defective Tn402 transposons (e.g. In0 and In2) can be complemented by a related, wildtype copy <ref><pubmed>PMC2838034</pubmed></ref>, it seems simpler to hypothesize that an initial insertion involved a Tn402 derivative with a complete functional set of Tn402 transposition genes. We have chosen a simple integron platform, In_Tn1721.1 from Tn1721.1 (HQ730118.1), for convenience.

This carries tniA,B,Q, the resolvase tniR together with the Tn402 res site, both ends (IRt and IRi), the integron integrase int and a common qac gene cassette. Insertion into the Tn5060 urfM gene generates a 5 bp DR (Fig. Tn3.7 S) and leads to the formation of tnpM from the 3’ end of ufrM (serendipitously generating an ATG initiation codon) <ref name=":16" /><nowiki><ref name=":63"><pubmed>2992807</pubmed></ref>. TnpM has been suggested to be a transposition regulatory gene (but see Resolution below). Subsequent steps in the Tn21 lineage (Fig. Tn3.7 T) would then involve modification of the integron platform by acquisition of the typical GNAT (previously known as orf5) and sul genes, decay of the Tn402 transposition genes and insertion, first of IS1326 (resulting in In0) followed by acquisition of the aadA integron cassette (generating In_Tn4) and, finally, insertion of IS1353 into IS1326 (IS1326::IS1353) between IRL and the start of the istA gene presumably not affecting IS1326 transposition functions (generating In2).

Fig. Tn3.7T. A plausible lineage of the Tn21-related integrons. The insertion point of all transposons of the Tn21 group is identical, suggesting that insertion occurred only once and subsequence variation to occurred from this common ancestor. The figure shows a plausible pathway for the changes in the integron structure leading to that found in Tn21. The relevant transposons are indicated at the left of the figure. The ancestral integron structure chosen as the simplest known example of a functional Tn401 derivative transposon is that found at present in Tn1721.1 (In_Tn1721.1; HQ730118.1) (first panel) although Tn1721.1 cannot be a direct source since the integron is inserted into another target in this transposon (Fig. Tn3.7K). In_Tn1721.1 could then lead to In0 (U49101) (second panel) by acquisition of two integron cassettes, loss of the Tn402 tniQ and tniR genes and part of tniB and insertion of IS1356. Acquisition of an additional integron cassette would then generate In_Tn4 (KY749247.1) (third panel) found in Tn4 and Tn2411 (FN554766) and insertion of IS1356 would generate In2 (AF071413) in Tn21 (fourth panel).

Due to their conservation in a large number of class I integron platforms, the DNA region including the sul, qac and GNAT family (previously called orf5) genes has been called the 3’CS (conserved segment) while that including the attI site and intI gene has been called the 5’CS <ref><pubmed>2560119</pubmed></ref> (however, using a more extended data set it was noted that, while the 5’CS was highly conserved across a number of integrons, the 3’CS proved to be somewhat more variable <ref><pubmed>PMC196970</pubmed></ref>).

Tn2411 is not only the precursor of Tn21. It was proposed to give rise to additional transposons (Fig. Tn3.7 Q)<ref name=":16" />: to Tn''4'' by insertion of a Tn''3'' transposon copy into the ''[https://www.uniprot.org/uniprot/P04131 merP]'' gene ([[:File:Fig.Tn3.7U.PNG|Fig. Tn3.7 '''U''']]); to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5086-CP054343 Tn''5086''] <nowiki><ref name=":19"><pubmed>PMC203974</pubmed></ref> by deletion of the In_Tn4 IS1326 copy to generate Tn2608 <ref name=":16" /> and replacement of the [https://www.uniprot.org/uniprot/P0AG05 ''aadA''] cassette and acquisition of ''dfrA7'' ([[:File:Fig.Tn3.7V.PNG|Fig. Tn3.7 '''V''']]); and to Tn''2410'' by replacement of the [https://www.uniprot.org/uniprot/P0AG05 ''aadA''] cassette by an [[wikipedia:Oxacillin|''oxa'' cassette]] <nowiki><ref name=":18" />. [[File:Fig.Tn3.7U.PNG|center|thumb|640x640px|'''Fig. Tn3.7U.''' Relationship between [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4-KY749247.1 Tn''4''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5060-AJ551280.1 Tn''5060'']. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411''] carries a copy of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn''4''] ('''right''') while [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4-KY749247.1 Tn''4''] also includes a copy of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] inserted into ''[https://www.uniprot.org/uniprot/P04131 merP].'']][[File:Fig.Tn3.7V.PNG|center|thumb|640x640px|'''Fig. Tn3.7V.''' Relationship between [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411''], Tn''2608'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5086-CP054343 Tn''5086'']. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411''] ('''Top panel''') carries a copy of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn''4''] which includes a copy of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] while this insertion is absent in In_Tn''2608'' (which carries the same [[wikipedia:Integron|integron]] cassette as [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn''4'']) in Tn''2608'' ([https://www.ncbi.nlm.nih.gov/nuccore/FN554766 FN554766]) ('''Second panel''') and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In22-CP054343 In''22''] in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5086-CP054343 Tn''5086''] ([https://www.ncbi.nlm.nih.gov/nuccore/CP054343 CP054343]) ('''Third panel'''). Moreover, in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In22-CP054343 In''22''], the ''aad'' [[wikipedia:Integron|integron]] cassette of In_Tn''2608'' has been exchanged for a ''dfr'' cassette. It seems probable, from the DNA sequence ('''Fourth panel''') that In_Tn''2608'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In22-CP054343 In''22''] were derived by deletion from a structure similar to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn''4''] because neither carry an [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] copy although they both retain the tip of the IRL (4 bp for In_Tn''2608'' and 3bp for [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In22-CP054343 In''22'']) at one end and are missing 5bp of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn''4''] DNA flanking the right [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] end. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] IR are shown in red and contained within a blue horizontal arrow. Identical nucleotides are underlined.]]The complete DNA sequences of many of these Tn are not available but [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5086-CP054343 Tn''5086''] or Tn''2608'' could be reconstructed from [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] using the limited sequence data in refeference <nowiki><ref name=":19" />. Moreover, using the reconstructed [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5086-CP054343 Tn''5086''] sequence in a BLAST search revealed an identical sequence in the [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA624897 ''E. coli'' SCU-164 chromosome] ([https://www.ncbi.nlm.nih.gov/nuccore/CP054343 CP054343]) and a nearly identical copy, in which the IRL had been interrupted by an insertion of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS4321-U67194 IS''4321''], in [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA624897 ''E. coli'' plasmid pSCU-397-2] ([https://www.ncbi.nlm.nih.gov/nuccore/CP054830 CP054830]) in addition to many closely related copies. This analysis suggests that deletion of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] had occurred by nearly-precise excision <nowiki><ref><pubmed>6260376</pubmed></ref> since the deletion junction observed in Tn5086 <ref name=":19" /> is not the original sequence identified in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411'']. Indeed, the DNA sequences of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2411-FN554766 Tn''2411'']'','' Tn''2608'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5086-CP054343 Tn''5086''], ([[:File:Fig.Tn3.7V.PNG|Fig. Tn3.7 '''V''']]) suggest that In_Tn2608 and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In22-CP054343 In22] were derived by deletion from a structure similar to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn4] because neither carry an [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] copy although they both retain the tip of the IRL (4 bp for In_Tn2608 and 3bp for [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In22-CP054343 In22]) at one end and are missing 5bp of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=In_Tn4-KY749247.1 In_Tn4] DNA flanking the right [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] end. [[File:Fig.Tn3.7W.PNG|center|thumb|640x640px|'''Fig. Tn3.7W.''' Relationship between [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] and Tn''1831''. Tn''1831'' was generated from [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] by deletion mediated by [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326_IS1353-AF071413 IS''1326''::IS''1353'']. Since no sequence is available for Tn''1831'', its restriction map ('''bottom''') <nowiki><ref name=":18" /> was mapped against the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] restriction map derived from its DNA sequence (top). The segment of DNA missing in Tn''1831'' compared to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] is shown in red. ]] [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] was also proposed to give rise to a number of different transposons <nowiki><ref name=":16" /><nowiki><ref name=":18" />: to Tn''1831'' by [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326'']-mediated deletion ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326-KY749247.1 IS''1326''] in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS1326_IS1353-AF071413 IS''1326''::IS''1353''] is almost certainly functional) rightwards towards or past the IRt end of the [[wikipedia:Integron|integron]] while retaining the IS ([[:File:Fig.Tn3.7Q.PNG|Fig. Tn3.7Q]] and [[:File:Fig.Tn3.7W.PNG|Fig. Tn3.7 '''W''']]); to Tn''2607'' by insertion of Tn''2601'' (probably similar to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']) into the ''mer'' genes; to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2424-UGCJ01000005 Tn''2424''] by insertion of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS161 IS''161''] to first generate Tn''2425'' and subsequent acquisition of two [[wikipedia:Integron|integron]] cassettes ''[https://www.uniprot.org/uniprot/P10051 aacA1]'' and ''[https://www.uniprot.org/uniprot/P26838 catB2]'' ([[:File:Fig.Tn3.7Q.PNG|Fig. Tn3.7Q]] and [[:File:Fig.Tn3.7X.PNG|Fig. Tn3.7 '''X''']]); and to Tn''2603'' by insertion of an [[wikipedia:Oxacillin|''oxaA1'' cassette]]. [[File:Fig.Tn3.7X.PNG|center|thumb|640x640px|'''Fig. Tn3.7X.''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] to Tn''2425'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2424-UGCJ01000005 Tn''2424'']. The figure shows how [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2424-UGCJ01000005 Tn''2424''] is thought to be generated from [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] (Top) by insertion of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS161 IS''161''] to first generate Tn''2425'' ('''Bottom''') and subsequent acquisition of two [[wikipedia:Integron|integron]] cassettes ''[https://www.uniprot.org/uniprot/P10051 aacA1]'' and ''[https://www.uniprot.org/uniprot/P26838 catB2]'' to generate Tn''2424'' ('''Middle'''). Since no sequence is available for Tn''2424'' or Tn''2425'', their restriction maps <nowiki><ref name=":18" /> were mapped against the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] restriction map ('''Top''') derived from its DNA sequence. The relevant restriction fragments are shown as blue lines.]] <br /> =====Tn''1721'' and (tandem) amplification of the ''tet'' genes===== [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''] ([[:File:Fig.Tn3.7K.PNG|Fig. Tn3.7 '''K''']]) carries resistance to [[wikipedia:Tetracycline|tetracycline]] (''[[wikipedia:Tetracycline|tet]]''), is present on plasmid pRSD1 and is capable of undergoing amplification to generate tandem repeats <nowiki><ref><pubmed>377009</pubmed></ref>. It was isolated by transposition to a lambda phage followed by a further transposition event onto plasmid R388 <ref name=":11" /> where it retained the ability to amplify <nowiki><ref name=":11" /> . Amplification was identified using restriction enzyme mapping ([[:File:Fig.Tn3.7Y.PNG|Fig. Tn3.7 '''Y''']]) which showed a duplication of an Eco''R''I fragment and presumably occurs via replication slippage or unequal crossing over during replication between the full ''tnpA'' gene and the 5’-end ''tnpA'' segment at the right end of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721'']. Indeed, amplification was shown to depend on the host [[wikipedia:RecA|''recA'' gene]] <nowiki><ref><pubmed>PMC216121</pubmed></ref>.

[[File:Fig.Tn3.7Y.PNG|center|thumb|640x640px|Fig. Tn3.7Y. Tn1721. 1 amplification of tet. Tet module duplication following acquisition by Tn1722 of the tet gene module (Fig. Tn3.7K) <ref name=":11" />.]] =====The Tn''163'' Clade===== There are 39 members of this clade (May 2021). Two ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnSku1-CP002358.1 TnSku1] [[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnSku1-CP002358.1 Tn''7197'']] ([https://www.ncbi.nlm.nih.gov/nuccore/CP002358.1 CP002358.1]) and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnAmu1-AP012041 TnAmu_p] ([https://www.ncbi.nlm.nih.gov/nuccore/NC_015188.1 NC_015188.1]) have acquired [[wikipedia:Toxin-antitoxin_system|toxin/antitoxin gene pairs]] and most members ([[:File:Fig.Tn3.8A.PNG|Fig. Tn3.8 '''A''']]; [[:File:Fig.Tn3.8B.png|Fig. Tn3.8 '''B''']]) encode divergent ''tnpR'' and ''tnpA'' genes. There are a number of members without passenger genes as in the Tn''21'' clade (e.g. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6137-AB610648.1 Tn''6137''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnMex22-CP001511 Tn''Mex22''][ [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnMex22-CP001511 Tn7165]], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnMex38-CP001513 Tn''Mex38''] [[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnMex38-CP001513 Tn7166]], Tn''Che1'', [[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnChe1-CP000391 Tn7155]], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnAmu1-AP012041 Tn''Amu1''] [[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnAmu1-AP012041 Tn7138] ], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnAli20-FQ311873.1 Tn''Ali20''] [[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnAli20-FQ311873.1 Tn7136]], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6122-JN127372.1 Tn''6122'']'','' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3434-AY232820 Tn''3434'']).[[File:Fig.Tn3.8A.PNG|thumb|640x640px|'''Fig. Tn3.8A.''' The Tn''163'' Clade. The relationship between the transposases of different members of the Tn''163'' clade expanded from [[:File:Fig.Tn3.4.png|Fig.Tn3.4]]. Each transposon is shown together with its Genbank accession number. The small purple circles indicate that all members encode a ''tnpR'' resolution system. Those carrying a [[wikipedia:Toxin-antitoxin_system|toxin/antitoxin gene pair]] (Bordeaux, orange and turquoise squares) are shown in bold type. Those Tn encircled by blue boxes are treated in detail in the text. The losenges on the outer circle indicate the presence and type of passenger gene carried by the corresponding Tn: '''''mer''''': [http://parts.igem.org/Part:BBa_K1420000 mercury resistance operon]; '''''ars''''': [[wikipedia:Arsenic|arsenic resistance]]; '''red:''' antibiotic resistance; '''pale-yellow''': other passenger genes; '''purple''': hypothetical; '''white:''' no passenger genes; '''orange''': toxin/antitoxin; '''black-line''': not in [https://tncentral.ncc.unesp.br/ TnCentral].|alt=|center]]One small related group ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6137-AB610648.1 Tn''6137''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6136-AB610647.1 Tn''6136''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6134-AB610645.1 Tn''6134''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6138-AB610649.1 Tn''6138'']) ([[:File:Fig.Tn3.8B.png|Fig. Tn3.8 '''B''']]) all identified within the [[wikipedia:Hexachlorocyclohexane|hexachlorocyclohexane-degrading]] bacterium [[wikipedia:Sphingobium_japonicum|''Sphingobium'' ''japonicum'']] UT26 genome <nowiki><ref><pubmed>22142724</pubmed></ref> show evidence at the DNA sequence level of several recombination events including acquisition of an sdr passenger gene and exchange of tnpR and tnpA by exchange at a location at which res should occur (Fig. Tn3.8 C).

Fig. Tn3.8B. The Tn163 Clade: Organisation of TnpA, TnpR and res. The configuration of the various genes is shown above each column, with the direction of expression shown by arrow heads. Where appropriate, registered transposon names are shown in brackets. Note that several Tn5393 derivatives are not included, since they have undergone extensive insertion and rearrangement.

Alignment against Tn6136 (Fig. Tn3.8 Ci) shows that Tn6137 carries the left half while Tn6134 carries the right section while Tn6137 carries the right while Tn6134 carries the left segments of Tn6138 (excluding the passenger gene insertion). Although the res sites have yet to be defined in detail, comparisons clearly show sequence divergence in this region (Fig. Tn3.8 Cii). Both Tn6134 and Tn6138 carry the same passenger gene (Fig. Tn3.8 Ciii) whose insertion has occurred proximal to IRL (Fig. Tn3.8 Civ).

Fig. Tn3.8C. Acquisition of passenger genes and inter-res site exchange in Tn6134 and relatives. i) The map of Tn6134 (AB610645.1) is shown with alignments of Tn6136 (AB610647.1), Tn6137 (AB610648.1) and Tn6138 (AB610649.1) above showing that Tn6134 and Tn6138 carry a sdr passenger gene while Tn6136 and Tn6137 do not. ii) The DNA sequences in the passenger gene region show that Tn6136 and Tn6137 have identical IRL sequences (blue filled box) while Tn6134 and Tn6138 (unfilled box) are also identical but have a number of SNPs compared to Tn6136 and Tn6137. Both passenger gene insertions are identical, with a 39 bp non-coding region downstream (shown in red) of the sdr gene (shown as a pink horizontal arrow). All four transposons have quasi identical sequences upstream of sdr but those of Tn6134/Tn6138 (red filled box) have a number of SNPs compared to Tn6136/Tn6137 (blue filled box).
Fig. Tn3.8C (continuation). Acquisition of passenger genes and inter-res site exchange in Tn6134 and relatives. iii) This shows an alignment which emphasizes the probable recombination at the res site. iv) DNA sequence around the probable res site (although res site sub-sites have not been identified). To the left Tn6134/Tn6138 (red filled box) are identical as are Tn6136/Tn6137 (blue filled box) but differ from Tn6134/Tn6138 by a number of SNPs. To the right, the Tn6134 and Tn6136 sequences are identical (blue filled box) as are those of Tn6137 and Tn6138 (red filled box) but differ from Tn6134 and Tn6136 by a number of SNPs. This indicates that recombination has occurred somewhere within the 29 identical base pairs in the open box.

The ancestor of another group of related transposons, the Tn5393 group (Fig. Tn3.8 D), appears to be Tn5393c (AY342395.1; Pseudomonas syringae pv. syringae plasmid pPSR1) which underwent an insertion of Tn5501.6 to generate Tn5393.1 (MF487840.1; Pseudomonas aeruginosa PA34), of IS1133 to generate Tn5393 (M95402; Erwinia amylovora plasmid pEa34) (Fig. Tn3.8 E) and of a complex set of mobile elements to generate Tn5393.4 (AJ627643; Alcaligenes faecalis).

Fig. Tn3.8D. The Tn5393 group: Organisation of TnpA, TnpR and res. Different members of this group are shown within red boxes on a pale red background. Insertions, deletions and rearrangements leading from one to the other are shown in blue boxes.

Tn5393 also gave rise to a number of other derivatives: Insertion of Tn3 into its transposase gene generated Tn5393.7 (LT827129; Escherichia coli strain K12 J53); insertion of Tn10 into IS1133 to generate Tn5393.2 (CP030921; Escherichia coli KL53 plasmid pKL53-M) (Fig. Tn3.8 F) followed by insertion of IS903 to generate Tn5393.11 (CP000602; Yersinia ruckeri YR71 plasmid pYR1); insertion of Tn10 in res to generate Tn5393.8 (CP002090; Salmonella enterica subsp. enterica plasmid pCS0010A).

Fig. Tn3.8E. Detailed map of the relationship between Tn5393c, Tn5393 and Tn5393.1. Target Tn5393c sequences of the insertion of Tn5501.6 to generate Tn5393.1 and of IS1133 to generate Tn5393 are shown in red.

There are also 4 examples carrying derivatives of Tn5 inserted into tnpA. They have an identical 3’ junction. In Tn5393.12 (KM409652; Escherichia coli REL5382 plasmid pB15), carries a complete Tn5. A second, Tn5393.13 (AB366441; Salmonella enterica subsp. enterica serovar Dublin plasmid pMAK2) is derived from Tn5393.12 by insertion of Tn2 into the IS1133 copy. In Tn5393.3 (LT985287; Escherichia coli strain RPC3 plasmid: RCS69_pI) the Tn5 insertion is a partial head-to-head Tn5 dimer, and in the other, Tn5393.10 (CP019905; Escherichia coli MDR_56 plasmid unnamed 6), insertion(s) and deletion(s) have occurred leaving only a partial Tn5 sequence. Finally, Tn5393 also gave rise to Tn5393.9 (KU987453; Klebsiella pneumoniae 05K0261 plasmid F5111) by multiple insertion including a type II intron, IS5708, ISCR1, ISEc28, ISEc29 and Tn2. A number of intermediate structures have yet to be identified but can probably be found in the large number of Tn5393 derivatives in the public databases. This group of Tn163 clade members have undergone a large number of modifications and constitute a broad network of related elements.

Fig. Tn3.8F. Detailed map of the relationship between Tn5393, Tn5393.7 and Tn5393.8. Target Tn5393 sequences of the insertion of Tn10 to generate Tn5393.8 and of Tn3 to generate Tn5393.7 are shown in red.
The Tn4430 Clade

At present (May 2021) this clade is composed of only 11 examples (Fig. Tn3.9 A). One example, Tn4430 (X07651.1), encodes a tnpI gene and a res site with its associated organization but no passenger genes. The others encode a tnpR gene (Fig. Tn3.9 B). There are two small groups: Tn1546 which carry vancomycin resistance genes, and Tn6332 which carry mercury resistance genes. [[File:Fig.Tn3.9A.png|center|thumb|580x580px|Fig. Tn3.9A. The Tn4430 Clade. The relationship between the transposases of different members of the Tn4430 clade expanded from Fig.Tn3.4. Each transposon is shown together with its Genbank accession number. The small purple circles indicate that all members encode a tnpR resolution system. The lozenges on the outer circle indicate the presence and type of passenger gene carried by the corresponding Tn: mer: mercury resistance operon; ars: arsenic resistance; red: antibiotic resistance; pale-yellow: other passenger genes; purple: hypothetical; white: no passenger genes; black-line: not in TnCentral.|alt=]]

ig. Tn3.9B. The Tn4430 Clade: Organisation of TnpA, TnpR and res. The configuration of the various genes is shown above each column, with the direction of expression shown by arrow heads. Where appropriate, registered transposon names are shown in brackets.
The Tn1564 Vancomycin Resistance Group

Resistance to Vancomycin in Enterococci appeared in 1988 <ref><pubmed>2891921</pubmed></ref>, was shown to be transmissible <ref><pubmed>2968517</pubmed></ref><ref><pubmed>PMC171412</pubmed></ref> and carried by a transposon, Tn1546 (M97297.1) <ref name=":21"><pubmed>PMC196104</pubmed></ref>. The relationship within the Tn1546 vancomycin resistant transposons is relatively simple and the result of insertions/deletions mediated by several different insertion sequences: Tn1546.2 (AB247327) is derived from Tn1546 <ref name=":21" /><nowiki><ref name=":22"><pubmed>PMC89148</pubmed></ref> by insertion of IS1216E between vanYA and vanXA and Tn1546.1_p (KR349520.1) appears to be derived from Tn1546.2 by insertion of IS1251 between vanHA and vanSA and a neighboring deletion to the right of IS1216E bringing vanYA and vanXA closer to each other. Other examples identified in surveys of vancomycin-resistant Enterococci from human and other animal sources also include insertions of ISEf1, IS1542 and IS19 <ref name=":23"><pubmed>PMC404624</pubmed></ref>, in addition to a number of other IS1216 insertions (often in multiple copies and accompanied by neighboring deletions) <ref name=":22" /><nowiki><ref name=":24"><pubmed>PMC2258525</pubmed></ref>. A number of these insertion/deletion derivatives have been identified from several sources and different geographical locations <ref name=":22" /><nowiki><ref name=":23" /><nowiki><ref name=":24" /><nowiki><ref><pubmed>PMC162963</pubmed></ref> (Fig. Tn3.9 C).

[[File:Tn3.9C.png|center|thumb|640x640px|Fig. Tn3.9C. Examples of Tn1546 derivatives showing the position of various insertions. A map of Tn1546 without insertions is shown below, with the vancomycin resistance genes in red. Data from <ref name=":21" /><nowiki><ref name=":23" /><nowiki><ref name=":24" />.]] ======The Mercury Resistance Group====== Within the [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] group ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6294-LC015492.1 Tn6294]-[https://www.ncbi.nlm.nih.gov/nuccore/LC015492.1 LC015492.1], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5084-AB066362.1 Tn5084]-[https://www.ncbi.nlm.nih.gov/nuccore/AB066362.1 AB066362.1], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6332-LC155216.1 Tn6332]-[https://www.ncbi.nlm.nih.gov/nuccore/LC155216.1 LC155216.1] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6332-LC155216.1 TnMERI1]-[https://www.ncbi.nlm.nih.gov/nuccore/LC152290 LC152290] – note that we have reconstituted the left end by comparison with [https://www.ncbi.nlm.nih.gov/nuccore/Y08064 Y08064]; [[:File:Tn3.9D.png|Fig. Tn3.9 '''D''']]), the [http://parts.igem.org/Part:BBa_K1420000 mercury resistance genes] are expressed to the left while TnpR and TnpA are expressed to the right. All four carry additional copies of ''[https://www.uniprot.org/uniprot/P16172 merB]'' and ''[https://www.uniprot.org/uniprot/P0A183 merR]''. Huang et al <nowiki><ref name=":103"><pubmed>10548738</pubmed></ref> have shown that expression of the mercury resistance genes of TnMERI1 is driven by three promoters (Fig. Tn3.9 E). Comparison with Tn6294 suggests that the mercury gene set has been exchanged by recombination at the level of the res site (Fig. Tn3.9 D). The sequences of two closely related members of the same group, Tn5083 and Tn5085, are incomplete <ref><pubmed>26802071</pubmed></ref>.

Fig. Tn3.9D. The Tn4430 Clade: Tn6332 group i) Alignment against Tn6294 to illustrate probable inter transposon recombination at the res site. ii) Alignment against Tn6332.

[[File:Tn3.9E.png|center|thumb|640x640px|Fig. Tn3.9E. Tn6332 group Expression of Mercury Resistance. A map of TnMERI1_p showing the position of three operator/promoter sequences identified by <ref name=":103" />. These are shown as blue vertical lines, with the horizontal lines indicating the direction of expression. Although this transposon is only partial because it lacks at least part of the left end, it is similar to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn6332-LC155216.1 Tn''6332'']. ]] =====The Tn''3'' Clade===== This clade includes the classical [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1''], ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2-KT002541 2]'' and ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 3]'' (see [[Transposons families/Tn3 family#Historical|Historical]]) as well as [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000'']. There are 29 examples of the Tn''3'' clade (of which 26 can be found in [https://tncentral.ncc.unesp.br/index.html TnCentral]) ([[:File:Fig.Tn3.10A.PNG|Fig. Tn3.10 '''A''']]) which fall into two subgroups. The majority have divergently expressed ''tnpR'' and ''tnpA'' and most carry passenger genes ([[:File:Fig.Tn3.10B.PNG|Fig. Tn3.10 '''B''']]). The ''res'' sites of each sub-group show significant similarity ([[:File:Fig.Tn3.10C.PNG|Fig. Tn3.10 '''C''']]). A number carry [[wikipedia:Toxin-antitoxin_system|toxin-antitoxin genes]] ('''TA''') generally located between the divergent ''tnpR'' and ''tnpA''. These are of two types ([[:File:Fig.Tn3.10A.PNG|Fig. Tn3.10 A]]) and appear to be specific for each subgroup. Passenger genes can be located upstream of downstream of the ''tnpR''/''tnpA'' transposition module ([[:File:Fig.Tn3.10B.PNG|Fig. Tn3.10 '''B''']]). All except two encode ''tnpR'' type resolvases. The two which do not, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnBth4-CP010092.1 Tn''Bth4''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''], also encode a TA module. [[File:Fig.Tn3.10A.PNG|center|thumb|640x640px|'''Fig. Tn3.10A.''' The Tn''3'' Clade. The relationship between the transposases of different members of the Tn''3'' clade expanded from [[:File:Fig.Tn3.4.png|Fig.Tn3.4]]. Each transposon is shown together with its Genbank accession number. The small purple circles indicate that all members encode a ''tnpR'' resolution system. Members with [[wikipedia:Toxin-antitoxin_system|toxin/antitoxin gene pairs]] are shown in bold type. The lozenges on the outer circle indicate the presence and type of passenger gene carried by the corresponding Tn: '''''mer''''': [http://parts.igem.org/Part:BBa_K1420000 mercury resistance operon]; '''small-red''': intron-associated antibiotic resistance; '''large-red''': non-intron associated antibiotic resistance; pale-yellow: other passenger genes; '''yellow''': plant pathogenicity genes; '''orange''': toxin/antitoxinf gene pairs; '''white''':no passenger genes|alt=]] [[File:Fig.Tn3.10B.PNG|center|thumb|640x640px|'''Fig. Tn3.10B.''' The Tn''3'' Clade: Organisation of TnpA, TnpR and ''res''. The configuration of the various genes is shown above each column, with the direction of expression shown by arrow heads. Where appropriate, registered transposon names are shown in brackets.]] [[File:Fig.Tn3.10C.PNG|center|thumb|640x640px|'''Fig. Tn3.10C.''' Res site alignment of Tn''3'' clade members with divergent ''tnpR''/''tnpA'' genes. The Tn names are shown to the left of the figure. Sequence conservation is shown by the depth of the blue background. Sites I, II and III are indicated. '''Top''': Tn''3''-like without passenger genes between ''tnpR'' and ''tnpA''. '''Bottom''': [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc4-CP009039 Tn''Xc4'']-like with TA passengers between ''tnpR'' and ''tnpA'']] ======Importance of IS''Ecp1'' in ''bla'' CTX-M-expression====== There are examples of members of the Tn''3'' clade which carry insertions of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISEcp1 IS''Ecp1'']-like sequences (see: [[IS Families/IS1380 family|IS''1380'' family]]) closely upstream of a [[wikipedia:Beta-lactamase|''bla''-CTX-M gene]]. Indeed, upstream insertion of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISEcp1 IS''Ecp1''] derivatives have been identified associated with a number of different [[wikipedia:Beta-lactamase|''bla''-CTX-M]] variants in both Tn''3'' and other groups <nowiki><ref><pubmed>11470367</pubmed></ref><ref><pubmed>12007800</pubmed></ref><ref><pubmed>PMC139670</pubmed></ref><ref><pubmed>PMC127467</pubmed></ref><ref><pubmed>PMC127047</pubmed></ref><ref name=":25"><pubmed>15135523</pubmed></ref>. In some examples, this is limited to an isolated right end <ref name=":25" /> which is responsible for expression of the [[wikipedia:Beta-lactamase|''bla''-CTX-M]] gene by providing a mobile promoter <nowiki><ref><pubmed>PMC182628</pubmed></ref>.

The Tn3 group

Tn3, Tn1, Tn1MER, Tn2, Tn2.1 and Tn3.1. all carry a probable internal IR upstream of the bla gene (Fig. Tn3.10 D) which acts as a hotspot for IS231A insertion and was initially observed in the bla gene of plasmid pBR322 <ref name=":31"><pubmed>7830551</pubmed></ref>. Tn2 and Tn2.1 are identical except for the ISEcp1 insertion which also carries an internal IS1 insertion (Fig. Tn3.10 E). Note that an ISEcp1 promoter drives bla CTX-M-expression. There are a number of closely related derivatives (e.g. Tn6339-MF344565) in which the IS1 copy appears to have been involved in small rearrangements of the ISEcp1 copy while maintaining the ISEcp1 promoter. Three examples carry a number of integron cassettes without either the integrase gene, the Tn402 ends or the Tn402 transposition genes that are often associated with integrons in the Tn21 clade.[[File:Fig.Tn3.10D.PNG|center|thumb|640x640px|Fig. Tn3.10D. Tn3 showing the potential internal IR. (Top). Potential internal IR sequence. (Bottom left) Map of Tn3. (Bottom right) plasmid pBR322. The position of the potential internal IR, a hotspot for Tn4430 insertion, is shown as a blue arrow. From <ref name=":31" />.]] [[File:Fig.Tn3.10E.PNG|center|thumb|640x640px|'''Fig. Tn3.10E.''' Tn''3'' group alignment against [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']. This shows the variation in ''tnpR'', the ''res'' site and the 5’ end of the ''tnpA'' gene]]Inspection of the alignment ([[:File:Fig.Tn3.10E.PNG|Fig. Tn3.10 '''E''']]) shows that apart from insertion of different mobile elements, the major sequence variations occur in the region of the ''res'' sites, the 5’ ends of ''tnpA'' and ''tnpR'' as had been previously noted for [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1''], ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2-KT002541 2]'' and ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 3]'' <nowiki><ref><pubmed>PMC549287</pubmed></ref> (for res, see Fig. Tn3.10 C) and an evolutionary pathway involving a combination of homologous and resolvase-mediated recombination has been proposed.

This can be detected by the distribution of SNPs on each side of the res site (e.g. Tn1331 and Tn1332). In this respect, the integron carrying Tn6238 is more similar to Tn3 while Tn1MER, Tn1331, and Tn1332 are more similar to Tn1 and Tn2.1 resembles Tn2.

The Xanthomonas group

This group except for TnPsy39 (Tn7187), all members of this group in the tree carry the same TA pair and the passenger genes are located to the right of the transposition module. The Xanthomonas transposon cluster (Fig. Tn3.10 F) are closely related and differ essentially by insertion of ISXac1 and ISXac5 (Fig. Tn3.10 G) as well as deletions (in particular of the res site in TnXc4.2 [Tn7212]). TnXc4.1 [Tn7211], although having an organisation identical to that of TnXc4 [Tn7210] has undergone significant sequence divergence along its entire length. TnThsp9 [Tn7202] also shows sequence variation within the region carrying transposition and TA functions (but includes mercury genes instead of plant pathogenicity functions while TnPsy39 [Tn7187] only exhibits similarity in the TnpA gene.[[File:Fig.Tn3.10F.PNG|center|thumb|640x640px|Fig. Tn3.10F. Variation in the Xanthomonas Tn3 clade transposons. An alignment against TnXc4 (Tn7210). ]]

Fig. Tn3.10G. Relationship between Xanthomonas Tn3 derivative transposons. The figure shows that sequential IS insertions are involved in the decay of the TnXc4 (Tn7210) secC gene.

All members of the second cluster, which encode for the same TA gene pair as the Tn3 group (Fig. Tn3.33A), also carry mercury resistance genes although these have undergone some rearrangements and sequence divergence (Fig. Tn3.10 H) and are also divergent from those present in TnThsp9 (Tn7202).

The Tn3000 Clade

This clade is composed of nearly 30 members (25 in TnCentral) all of which encode TnpR resolvases and carry tnpR-related res sites. Most also encode TA gene pairs and these are of three types (Fig. Tn3.11 A).

Fig. Tn3.11A. The Tn3000 Clade. The relationship between the transposases of different members of the Tn3000 clade expanded from Fig.Tn3.4. Each transposon is shown together with its Genbank accession number. The small purple circles indicate that all members encode a tnpR resolution system. Members with toxin/antitoxin gene pairs are shown in bold type. The lozenges on the outer circle indicate the presence and type of passenger gene carried by the corresponding Tn: red: non-intron associated antibiotic resistance; pale-yellow:other passenger genes; yellow: plant pathogenicity genes; orange: toxin/antitoxin gene pairs; white: no passenger genes; purple: hypothetical.
Fig. Tn3.11B. The Tn3000 Clade: Organisation of TnpA, TnpR and res. The configuration of the various genes is shown above each column, with the direction of expression shown by arrow heads. Where appropriate, registered transposon names are shown in brackets.
The Tn5501 cluster.

There are a number of Tn5501 examples (Fig. Tn3.11 B). All have their passenger genes located upstream of the transposition module and all except TnPysy42 [Tn7188] and Tn5501.12 encode the same parE/parD TA genes (Fig. Tn3.11 A). Tn5501.12 appears to have acquired different TA genes (HTH_37, GP49) by recombination at the res site (Fig. Tn3.11 C).

The relationship between members of the cluster is shown in Fig. Tn3.11 C. Most have retained the same transposition and TA modules but vary in the type of passenger genes they carry. They all carry deletions with respect to Tn5051.3. For 8 of these, the right junctions of the deletions are close but not identical (Fig. Tn3.11 Di and Dii). All leave the TA module intact. In only one example, the toxin gene has undergone deletion leaving the antitoxin intact (Fig. Tn3.11 Diii). The left junction is less clear and difficult to interpret. A number of Tn5501 derivatives are related by IS insertions and deletion (Fig. Tn3.11 E).

Fig. Tn3.11C. The Tn5501 cluster, showing the acquisition of different set of passenger genes, including toxin-antitoxin gene pairs.

Finally, a small group of Tns which, like Tn5501.12, all carry the HTH_37/GP49 TA pair is shown in Fig. Tn3.11 F. It appears that there has been an exchange between a Tn5501.5-like transposon and a derivative of Tn4662a (lacking the ISAs20 insertion) by recombination at the res site to generate Tn5501.12.

Fig. Tn3.11D. The Tn5501 cluster: right deletion junction The DNA sequence of the deletion junctions compared to Tn5501.3. i) Junction of Tn5501 ii) Tn5501.7, Tn5501.9, Tn5501.6, Tn5501.10, Tn5501.1, Tn5501.8, Tn5501.4, Tn5501.11. iii) toxin deletion endpoint in Tn5501.5.
Fig. Tn3.11E. The Tn5501 derivatives with IS insertions.
Fig. Tn3.11F. The Tn4662 cluster.
Clinical Importance of Tn4401

In the past decades, carbapenemase-producing Enterobacteriaceae (CPE) have appeared that are resistant to most or all clinically available antibiotics, including carbapenems, which are often considered antibiotics of last resort <ref><pubmed>23887414</pubmed></ref>. The 10kb transposon, Tn4401 has been instrumental in the spread of the carbapenem resistance gene blaKPC. It was described in 2008 in a number of clinical isolates of Klebsiella pneumoniae and Pseudomonas aeruginosa from the United States, Colombia and Greece <ref><pubmed>PMC2292522</pubmed></ref><ref><pubmed>PMC2681555</pubmed></ref>.

Members of this small group have divergently expressed tnpR and tnpA genes located towards the left end and blaKPC towards the right end downstream from tnpA (Fig. Tn3.11 B) flanked by two different insertion sequences, ISKpn6 and ISKpn7 (Fig. Tn3.11 G). The ISKpn7 insertion had occurred within an additional Tn4401 IR. It was further observed that there were two “isoforms” of Tn4401: Tn4401a and Tn4401b. Tn4401a, isolated in the United States and Greece carried a 100bp deletion upstream of the bla gene compared to Tn4401b from Colombia. The Tn4401 backbone appears to have undergone a number of recombination events. A third derivative, Tn4401c <ref><pubmed>PMC3294963</pubmed></ref>, was found to carry a deletion of about 200 bp upstream of bla while in a fourth, Tn4401d <ref><pubmed>PMC3294926</pubmed></ref>, the ISKpn7 copy along with flanking DNA has undergone deletion to leave a 3’ segment of blaKPC and a 5’ segment of tnpA and therefore would not be capable of autonomous transposition.

Fig. Tn3.11G. Tn4401 an Important Vector in the Spread of blaKCP (Top) A map of Tn4401 indicating the large deletion carried by Tn4401d. The region representing the DNA sequences below is circled. (Bottom) Nucleotide sequences located upstream of blaKCP show the location of three potential promoters. The transcription start sites (+1), the ribosome binding site (RBS), and the translation start site (ATG) are also indicated. The IRR end of ISKpn7 is shown in yellow, and the disrupted Tn4401 IR is shown in pink. ISKpn7 insertion creates a hybrid promoter using a -35 box in ISKpn7 and a -10 sequence in the disrupted Tn4401 IR.

Furthermore, analysis of a number of clinical isolates from different regions of the United States which exhibited various levels of carbapenem resistance, revealed deletions of different extent in the region upstream of blaKPC <ref><pubmed>PMC2944623</pubmed></ref>. Closer analysis using RACE (Rapid amplification of cDNA ends) to locate transcriptional start points revealed 3 (possibly 4) promoters, one of which had been generated from the -35 element located in the IR of the inserted ISKpn7 (as is characteristic for a member of the IS21 family (see IS21 chapter; formation of hybrid promoters figure IS21.1).

The Tn4651 Clade
Fig. Tn3.12A. The Tn4651 Clade. The relationship between the transposases of different members of the Tn4651 clade expanded from Fig.Tn3.4. Each transposon is shown together with its Genbank accession number. The small purple circles indicate that all members encode a tnpR resolution system. The lozenges on the outer circle indicate the presence and type of passenger gene carried by the corresponding Tn.

The Tn4651 mix of radically different structures

This Tn3 family clade (Fig. Tn3.12 A) contains members with very diverse structures (Fig. Tn3.12 B). They fall into three major clusters. Two encode the tnpT/S/rst while the third encodes the tnpR/res system.

Fig. Tn3.12B. The Tn4651 Clade: Organisation of TnpA, TnpR and res. The configuration of the various genes is shown above each column, with the direction of expression shown by arrow heads. Where appropriate, registered transposon names are shown in brackets.
The tnpT/S/rst clusters

In the first tnpT/S/rst cluster, mostly from the plant pathogen Xanthomonas (Fig. Tn3.12 C), TnXax1.1 [Tn7207] appears to have undergone res-recombination in which the upstream passenger genes and tnpT have been exchanged. TnpT is significantly different from the other four. TnXax1.3 [Tn7209] differs from the others (TnXax1 [Tn7206]; TnXax1.2 [Tn7208]; TnXax1.3 [Tn7209] in the 3’ region of tnpA and there is some variation in tnpS and tnpT.

Fig. Tn3.12C. The Tn4651 Clade: Organisation of TnpA, TnpR, and res of the TnXax1 group. Alignment of different TnXax1 derivatives against TnXax1.3. The blue boxes indicate significant sequence variation between TnXax1.3 and the other derivatives.

TnXax1 derivatives <ref name=":20" /> are generally vehicles for pathogenicity genes such as [[wikipedia:Transcription_activator-like_effector|Transcriptional Activator Like Effectors]] ([[wikipedia:Transcription_activator-like_effector|TALE genes]]), lytic transglycosilases (''mtlB2'') and genes (''xop'') involved in [[wikipedia:Type_three_secretion_system|type III secretion system]] ([[wikipedia:Type_three_secretion_system|TTSS]]) translocation of effector proteins into host plant cells <nowiki><ref><pubmed>19400638</pubmed></ref> (Fig. Tn3.12 C). TnXax1 derivatives can include IR which are significantly longer (72/92 bp) than the 38-40bp characteristic of the Tn3 family (Fig. Tn3.12 D) although the functional significance of this has not been investigated. The IR also terminate in a GAGGG pentanucleotide. The left end of group members is quite variable (Fig. Tn3.12 E) while their right ends appear more homogeneous (Fig. Tn3.12 F).

Fig. Tn3.12D. i) Genetic organization of the canonical TnXax1 from X. citri strain 306 plasmid pXAC64. Genes are indicated by colored boxes, with the direction of transcription shown by the arrow heads. Transposition-related genes are shown in purple, passenger genes in yellow. The presumed resolution site is located between the tnpT and tnpS genes, as observed in the transposable element Tn4651. This includes two palindromes: IR1 and IR2 are probably part of the core site at which recombination occurs, recognized by the TnpS recombinase whereas IRa and IRb are potential binding sites for TnpT. The terminal inverted repeats (IRL and IRR) are shown as grey triangles. The convention used for orientation of the transposon is that the transposase (TnpA) is transcribed from left to right. ii) Sequence of the long IRs; iii) IRs identified from other Tn3-like Transposable Elements. All include the GAGGG tips, sharing sequence similarities to TnXax1 IRs. These are: TnPa43 from Pseudomonas aeruginosa; TnStma1 from Stenotrophomonas maltophilia strain D457; TnTin1 from Thiomonas intermedia strain K12 and TnXca1 from X. campestris pv. vesicatoria plasmid pXCV183.
Fig. Tn3.12E. Genetic organization of the Left end of the TnXax1 related structures found in other Xanthomonas species. Abbreviations of the TnXax1 related structures: (1) X. fuscans subsp. aurantifolii: XauB ctg 621_0 (2) X. axonopodis strain 29-1, chromosome copy: XccA-29-1 (3) X. citri subsp. citri strain Aw 12879 chromosome copy: XccA(w) (4) TnXax1 (5) X. axonopodis strain 29-1, chromosome copy: XccA-29-1 (7) plasmid copy: XccA-29-1 pXac64 (8) X. campestris pv. vesicatoria 85-10, chromosomal copy: Xcv B (9) X. axonopodis pv. citrumelo F1, chromosomal copy: Xac F1 A (10) X. axonopodis pv. citrumelo F1, chromosomal copy Xac F1 B (11) X. campestris pv. vesicatoria 85-10, chromosomal copy: XcvA (12) X. arboricola pv. pruni plasmid pXap41: Xap pXap41 (13) X. fuscans subsp. aurantifolii: XauC ctg 1147_0.
Fig. Tn3.12F. Genetic organization of the Right end of the TnXax1 related structures found in other Xanthomonas species. Abbreviations as for Fig. Tn3.12E.

The second tnpT/S/rst cluster is characterized by Tn4651, a toluene-catabolic transposon identified in from Pseudomonas putida plasmid pWW0 <ref name=":26"><pubmed>2830457</pubmed></ref>. In addition to the tnpS/T resolution system, it encodes an additional small transposition-related gene, tnpC which impacts cointegrate formation.

Using, Tn4652, a Tn4651 deletion derivative lacking the toluene-catabolic genes <ref name=":26" />, TnpC was shown to regulate TnpA expression post-transcriptionally <nowiki><ref><pubmed>PMC103765</pubmed></ref>. Moreover, the host protein IHF binds to sites in both Tn4652 ends (Fig. Tn3.12 G) <ref name=":50"><pubmed>PMC107244</pubmed></ref><ref name=":27"><pubmed>15009901</pubmed></ref>. These overlap the region protected by TnpA binding <ref name=":27" /> and binding positively regulates both ''tnpA'' transcription and TnpA binding to the terminal IRs. Indeed, transposase binding to the IRs ''in vitro'' was shown to occur only after binding of IHF <nowiki><ref name=":27" />. TnpA protects an extensive region encompassing the IRs and 8-9 bp of flanking DNA ([[:File:Fig.Tn3.12G.PNG|Fig. Tn3.12 '''G''']]). [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4652-AF151431.1 Tn''4652''] transposition appears to be elevated in stationary phase, involves the stationary phase sigma factor, sigma S <nowiki><ref><pubmed>PMC95431</pubmed></ref>, and is limited by the levels of IHF <ref name=":27" /> whose level is increased in stationary phase. Another DNA chaperone host factor, FIS, has a negative effect on transposition, apparently by competing for [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF binding]] <nowiki><ref name=":27" /><nowiki><ref><pubmed>19332822</pubmed></ref>. [[File:Fig.Tn3.12G.PNG|center|thumb|640x640px|Fig. Tn3.12G. Integration host Factor (IHF) and the Ends of Tn4652 (Top) Map of Tn4652. (Bottom) Left and right Tn4652 ends. The position of the IHF binding sites at each end of Tn4652 is shown in red. The bracketed regions indicate the extent of IHF protection. The IR are indicated within the blue boxes <ref name=":27" />.]][[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF and FIS]] have been implicated in other transposition systems such as [https://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=IS10L-AF162223 IS''10''] (see: [[IS Families/IS4 and related families|IS''4'' family]]). Moreover, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] (Tn''3'' clade) carries an [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF binding site]] proximal to each IR which acts copoperatively to increase TnpA binding and immunity <nowiki><ref name=":44"><pubmed>PMC213150</pubmed></ref><ref name=":45"><pubmed>PMC457184</pubmed></ref>. One additional interface with host physiology is the observation that the CorR/CorS two component system regulates transposition positively <ref><pubmed>15491368</pubmed></ref>.

Other members of the cluster include: Pseudomonas sp. mercury resistance transposon Tn5041 <ref name=":28"><pubmed>9274008</pubmed></ref><ref name=":77"><nowiki><pubmed>12427948</pubmed</ref></nowiki> ; Tn4676, a long (72,752bp) and complex Pseudomonas resinovorans carbazole-catabolic transposon from plasmid pCAR1 <ref name=":78"><pubmed>12547188</pubmed></ref><ref><pubmed>15856217</pubmed></ref>; and Tn4661, a Pseudomonas aeruginosa cryptic transposon <ref name=":9" />. All include ''tnpA'', ''tnpC'' and the ''tnpS''/''T'' resolution system. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''] transposition has also been addressed experimentally<nowiki><ref name=":28" /><nowiki><ref name=":29"><pubmed>10822806</pubmed></ref> and was observed to be host-dependent <ref name=":29" />: it occurred in the original [[wikipedia:Pseudomonas|''Pseudomonas sp.'']] KHP41 host but not in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 ''P. aeruginosa'' PAO-R] or in [[wikipedia:Escherichia_coli|''Escherichia coli'' K12]]. Interestingly, transposition in these strains was found to be complemented by the Tn''4651'' transposase gene (''tnpA'') and the region which determines this host dependence was mapped to a 5’ ''tnpA'' gene segment by construction of hybrid [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041'']-Tn''4651'' ''tnpA'' genes. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''] apparently acquired its [http://parts.igem.org/Part:BBa_K1420000 ''mer'' operon] from a derivative of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] or [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] <nowiki><ref name=":29" />. It is reported to be preceded by a 24 bp element with 75% sequence similarity to the outermost part of IRs typical for [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21'']-like transposons. =====The Tn''1071'' Clade===== ======The Tn''1071'' group.====== Members of this small group are often associated with [[wikipedia:Xenobiotic|xenobiotic]] catabolism and other “exotic” functions ([[:File:Fig.Tn3.13A.png|Fig. Tn3.13 '''A''']]). [[File:Fig.Tn3.13A.png|thumb|580x580px|'''Fig. Tn3.13A.''' The Tn''1071'' Clade.|alt=|center]] [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] itself ([[:File:Fig.Tn3.13B.png|Fig. Tn3.13 '''Bi''']]), the founding member, was identified as part of a [[Transposons families/compound transposons|compound transposon]], Tn''5271'', in [[wikipedia:Comamonas_testosteroni|''Comamonas'' ''testosteroni'']] where it flanks a [[wikipedia:4-Chlorobenzoic_acid|chlorobenzoate catabolic operon]] in <nowiki><ref><pubmed>PMC52498</pubmed></ref>. It is unusual since it carries only tnpA and not tnpR, has unusually long (110bp) IR (Fig. Tn3.13 Bii) and was first described as IS1071. Two other members of this small group, IS882 from Ralstonia eutropha H16 megaplasmid pHG1 encoding key enzymes for H2-based lithoautotrophy and anaerobiosis <ref><pubmed>12948488</pubmed></ref> and ISBusp1 (aka ISBmu13; NC_007509.1) from the Burkholderia multivorans ATCC 17616 genome <ref><pubmed>PMC1082539</pubmed></ref>, were also originally identified as IS. Their structure fits the definition of an IS since they all contain a single transposase open reading frame located between two IR.

Fig. Tn3.13B. Tn1071 organisation. (Top panel) The structure of Tn1071 (M65135) showing the single tnpA reading frame and the long terminal IRs. (Bottom panel) Sequence of the long IRs. Boxed sequence indicate the length of normal Tn3 family IRs. Bold and underlined bases indicate identity, red bases indicate non identity.

A limited functional analysis of Tn1071 transposition is available <ref><pubmed>PMC1352228</pubmed></ref>. It was only able to transpose at high frequencies in two environmental β-proteobacteria Comamonas testosteroni and Delftia acidovorans but not in Agrobacterium tumefaciens (α-proteobacteria) or Escherichia coli, Pseudomonas alcaligenes and Pseudomonas putida (all γ-proteobacteria). These studies showed that Tn1071 generates cointegrates as a final transposition product (since it has no resolution functions), produces 5bp DR on insertion and requires the entire 110bp IRs for activity. This is therefore in contrast to many other Tn3 family members which only require the 38 bp IR.

The absence of a resolution system implies that, like IS26(see: IS6 family) , Tn1071 probably forms “pseudo-compound transposons” <ref><pubmed>32871211</pubmed></ref><ref>Galas DJ, Chandler M. Bacterial Insertion Sequences. In: Berg DE, Howe MM, editors. Mob DNA. Washington, D.C.: American Society for Microbiology; 1989. p. 109–162. </ref><ref><pubmed>PMC7986276</pubmed></ref>. In these structures the flanking Tn1071 copies must be in direct orientation as a consequence of the homologous recombination event required to resolve the cointegrates structure. Transposition is initiated by one of the flanking IS to generate a cointegrate structure with three Tn1071 copies (similar to those generated by the IS6 family of insertion sequences; Fig. IS6.8 B). “Resolution” resulting in transfer of the transposon passenger gene requires recombination between the “new” IS copy and the copy which was not involved in generating the cointegrate.

The implications of this model, as for IS6 family members, are that the transposon passenger gene(s) are simply transferred from donor to target molecules in the “resolution” event and are therefore lost from the donor “transposon” leaving a single Tn1071 copy in the donor plasmid. However, it is possible that both Tn1071 copies are used in transposition in which case the cointegrates would be expected to contain two directly repeated copies of the entire transposons at the donor/target junctions.

A significant number of Tn1071-associated xenobiotic-degrading genes on many catabolic plasmids have been documented by population-based PCR <ref name=":32"><pubmed>23802695</pubmed></ref><ref><pubmed>30207068</pubmed></ref><ref name=":33"><pubmed>PMC201273</pubmed></ref> and genetic studies <ref><pubmed>11495991</pubmed></ref><ref><pubmed>PMC111268</pubmed></ref>.

Tn5271 itself is widely distributed in bacteria isolated from a large ground water bioremediation site <ref name=":33" /> and plasmid derivatives carrying the transposon together with a third [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] copy in an inverted orientation were also identified. The interstitial DNA segment between the old and new copy in these derivatives was also inverted as expected from intra-molecular transposition events <nowiki><ref name=":33" /> ([[:File:Fig.Tn3.16A.png|Fig. Tn3.16 '''A''']]). A number of additional potential compound transposons have been identified although these may be inactive: a >28kb transposon, Tn''5330'' ([https://www.ncbi.nlm.nih.gov/nuccore/AF029344 AF029344]), from ''[[wikipedia:Delftia|Delftia acidicorans]]'' <nowiki><ref><pubmed>12949179</pubmed></ref> carries the entire 2,4-dichlorophenoxyacetic acid degradation pathway and, although the sequence data for the flanking Tn1071 copies is not complete, both carry inactivating insertions of IS1471; a similar ~48 kb transposon (NC_005793) with 5bp flanking DR from Achromobacter xylosoxidans plasmid, pEST4011, also carries identical IS1471 inactivating insertions in each flanking Tn1071 copy <ref name=":102"><pubmed>PMC523222</pubmed></ref> and a 7kb internal tandem duplication compared to the Delftia acidovorans transposon.

When analyzed in more detail, these genes are sometimes flanked by Tn1071 copies in direct repeat as in the original Tn5271 but are found in more complex Tn1071-based structures.

TnHad2 <ref name=":34"><pubmed>PMC127583</pubmed></ref> (Fig. Tn3.13 Ci), for example, from a Delftia acidovorans haloacetate-catabolic plasmid, pUO1, carries a nested copy of a potential Tn1071-based compound transposon, TnHad1 which does not carry flanking DR. TnHad1 is inserted into a larger structure, TnHad2 with flanking 5bp DR, typical Tn3 family ends related to those of Tn21 but no apparent dedicated transposase except that of the Tn1071 copies.

The authors state that TnHad2 was unable to transpose as judged by a “mating out” assay using the plasmid R388 as a target. However, The TnHad2 Tn21-like IRs were found to be active in transposition if supplied with Tn21 but not with Tn1722 transposition functions <ref name=":34" />. Tn''Had2'' also appeared to carry a functional ''res'' site. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] also flanks [[wikipedia:Atrazine|atrazine degrading genes]] in plasmid ''[[wikipedia:Pseudomonas|Pseudomonas]]'' pADP-1 ([https://www.ncbi.nlm.nih.gov/nuccore/U66917 U66917]) <nowiki><ref name=":102" /> in a structure with three directly repeated [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] copies intercalated with three copies of an [[IS Families/IS91-ISCR families|IS''91'' family member]], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISPps1 IS''Pps1'']''.'' These are apparently generated by duplication events since regions with identical sequence stretch from the ''oriIS'' end of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISPps1 IS''Pps1''] through [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] and terminate just before the [[wikipedia:Atrazine|''atz'' genes]] ([[:File:Fig.Tn3.13C.png|Fig. Tn3.13 '''Cii''']]). The repeated regions also includes the DR sequences at each [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] except for that at the far right. [[File:Fig.Tn3.13C.png|center|thumb|620x620px|'''Fig. Tn3.13C.''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] Involvement in Compound transposons. '''i)''' Tn''Had1'', a composite transposon inserted into Tn''Had2'' <nowiki><ref name=":34" />. '''ii)''' Amplified [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] copies flanking [[wikipedia:Atrazine|atrazine]] degrading genes ]] There are a number examples of other structures with multiple [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1071-M65135 Tn''1071''] copies and in a large proportion of these cases, the multiple copies occur in direct repeat. They are associated with plasmids which degrade the [[wikipedia:Linuron|phenylurea herbicide linuron]] e.g. pBPS33-2 ([https://www.ncbi.nlm.nih.gov/nuccore/CP044551 CP044551]) <nowiki><ref><pubmed>PMC7039861</pubmed></ref> and have been isolated from a variety of bacteria with the capacity to degrade a wide range of chlorinated aromatics and pesticides <ref name=":32" /> or [[wikipedia:P-Toluenesulfonic_acid|p-toluene sulfonate]] ([[wikipedia:P-Toluenesulfonic_acid|PTSA]]) where they flank the [[wikipedia:P-Toluenesulfonic_acid|PTSA]] genes in plasmid pTSA ([https://www.ncbi.nlm.nih.gov/nuccore/AH010657 AH010657]) <nowiki><ref><pubmed>PMC168534</pubmed></ref>.

MITES, MICs and TALES

Many TE families also include non-autonomous transposable derivatives with no transposition related genes. These are simple and composed of two correctly oriented ends with or without an intervening passenger gene and are called MICs (Minimal Insertion Cassette) and MITEs (Miniature inverted-repeat transposable elements) respectively. For Tn3, related MITEs are known as TIMEs (Tn3-Derived Inverted-Repeat Miniature Elements) <ref><pubmed>PMC4133298</pubmed></ref><ref name=":79"><pubmed>PMC4588545</pubmed></ref>. [[File:Fig.Tn3.14A.png|center|thumb|640x640px|Fig. Tn3.14A. Map of Xanthomonas plasmid pXAC64 This identifies a number of Tn3 family transposable elements including TnXc4 (Tn7210) and TnXax1 (Tn7206) and a TAL-carrying MITE. Our standard labelling convention and colour coding is used. ]]


Studies have shown that Xanthomonas genomes are often havens for MICs carrying genes involved in pathogenicity towards their host plants <ref name=":20" />. A number of Tn''3'' family structures were identified in a conjugative plasmid, pXac64 ([https://www.ncbi.nlm.nih.gov/nuccore/CP024030 CP024030]), of the principal pathogen of citrus trees, ''[[wikipedia:Xanthomonas_citri|Xanthomonas citri]]'', an important economic problem (e.g., reference <nowiki><ref><pubmed>PMC3751643</pubmed></ref>) (Fig. Tn3.14 A). The plasmid includes two Tn3 family transposons, TnXc4 (Tn7210) and TnXac1.4 (Tn7206) and a MIC (MIC XAC64.T1; 3948bp) which carries a TAL effector gene (Transcriptional Activator Like effector). Other TAL effector-carrying MICs can be identified in other Xanthomonas plasmids such as pXac33 (CP008996) <ref><pubmed>PMC4579464</pubmed></ref> (two TAL-carrying MIC: MIC XAC33.T1, 3739bp, and MIC XAC33.T2, 3538bp; and the Tn3 family transposon TnXc5) and from the Xanthomonas fuscans plasmid pplc XAF (FO681497)<ref name=":20" /> (a single MIC, MIC XAF.T1 ,3768bp, and a 10kb MIC with a number of virulence genes. Some MICs, e.g. MIC XAC33.T1 ([[:File:Fig.Tn3.14B.png|Fig. Tn3.14 '''B''']] right), are flanked by 5bp DR, a hallmark of Tn''3'' family transposition. A global analysis of [[wikipedia:Transcription_activator-like_effector|TAL effector genes]] in ([[:File:Fig.Tn3.14C.png|Fig. Tn3.14 '''C''']]) within the [[wikipedia:Xanthomonas_citri|''Xanthomonas'']] genus (available in 2014) identified a large number which were flanked by Tn''3''-like IR although a some carried a single identifiable IR while others failed to exhibit clear IRs <nowiki><ref name=":20" />. [[File:Fig.Tn3.14B.png|center|thumb|640x640px|'''Fig. Tn3.14B.''' Plasmids pXAC33 and plc XAF This figure shows additional examples of both transposons and MICs.]] [[File:Fig.Tn3.14C.png|center|thumb|720x720px|'''Fig. Tn3.14C.''' Molecular Phylogenetic analysis by Maximum Likelihood method of the [[wikipedia:Transcription_activator-like_effector|TALEs]] genes found in completely sequenced ''[[wikipedia:Xanthomonas|Xanthomonas]]'' genomes. Each [[wikipedia:Transcription_activator-like_effector|TALEs]] gene is shown with its respective genomic coordinates (for [[wikipedia:Transcription_activator-like_effector|TALEs]] extracted from complete genomes) or [[wikipedia:Transcription_activator-like_effector|TALEs]] gene Genbank Accession Number inside the brackets. Red diamonds indicate [[wikipedia:Transcription_activator-like_effector|TALEs]] genes associated with MIC structures, yellow triangles [[wikipedia:Transcription_activator-like_effector|TALEs]] genes associated with a solo IR flanking one of their extremities, blue circles genomes which carry [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXax1-AE008925 Tn''Xax1''] or related structures and asterisks indicate MICs with the direct TACTC(G) target repeat . The blue arrow indicates the [[wikipedia:Transcription_activator-like_effector|TALEs]] gene from [[wikipedia:Xanthomonas|''X. fuscans'' subsp. ''fuscans'']] strain 4834-R plasmid pla and plc. [[wikipedia:Transcription_activator-like_effector|TALEs]] from different Xo pathovars are highlighted as follows: XooP, blue; XooK, green; XooM, purple; and XocB, pink. TA [[wikipedia:Transcription_activator-like_effector|TALEs]]LEs from ''[[wikipedia:Xanthomonas|X. citri]]'', ''[[wikipedia:Xanthomonas|X. axonopodi]]'', ''[[wikipedia:Xanthomonas|X. vesicatoria]]'' and ''[[wikipedia:Xanthomonas|X. campestris]]'' are highlighted in gray <nowiki><ref name=":20" />.|alt=]] <gallery mode="slideshow" caption="'''Fig. Tn3.14.''' Panels D1,2 and 3 (use the arrows to scroll the figures)."> File:Fig.Tn3.14D1.png|'''Fig. Tn3.14D1.''' List of MICs present in the Genomes of ''[https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 X. oryzae PXO99A]'', [https://www.ncbi.nlm.nih.gov/nuccore/NC_007705.1 MAFF] and [https://www.ncbi.nlm.nih.gov/nuccore/AE013598.1 KACC] For each strain, the columns show the relative positions of each MIC along the respective genome from the point of origin together with the type of MIC. Those carrying genes other than [[wikipedia:Transcription_activator-like_effector|TAL]] effectors are marked as such, with their passenger gene noted. The Tale containing MICs are marked MIC P.T, MIC P.M and MIC P.K for [https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 PXO99A], [https://www.ncbi.nlm.nih.gov/nuccore/NC_007705.1 MAFF] and [https://www.ncbi.nlm.nih.gov/nuccore/AE013598.1 KACC] respectively. The coloured boxes indicate MICs that are identical from one strain to another. File:Fig.Tn3.14D2.png|'''Fig. Tn3.14D2 (continuation).''' List of MICs present in the Genomes of ''[https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 X. oryzae PXO99A]'', [https://www.ncbi.nlm.nih.gov/nuccore/NC_007705.1 MAFF] and [https://www.ncbi.nlm.nih.gov/nuccore/AE013598.1 KACC] For each strain, the columns show the relative positions of each MIC along the respective genome from the point of origin together with the type of MIC. Those carrying genes other than [[wikipedia:Transcription_activator-like_effector|TAL]] effectors are marked as such, with their passenger gene noted. The Tale containing MICs are marked MIC P.T, MIC P.M and MIC P.K for [https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 PXO99A], [https://www.ncbi.nlm.nih.gov/nuccore/NC_007705.1 MAFF] and [https://www.ncbi.nlm.nih.gov/nuccore/AE013598.1 KACC] respectively. The coloured boxes indicate MICs that are identical from one strain to another. File:Fig.Tn3.14D3.png|'''Fig. Tn3.14D3 (continuation).''' List of MICs present in the Genomes of ''[https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 X. oryzae PXO99A]'', [https://www.ncbi.nlm.nih.gov/nuccore/NC_007705.1 MAFF] and [https://www.ncbi.nlm.nih.gov/nuccore/AE013598.1 KACC] For each strain, the columns show the relative positions of each MIC along the respective genome from the point of origin together with the type of MIC. Those carrying genes other than [[wikipedia:Transcription_activator-like_effector|TAL]] effectors are marked as such, with their passenger gene noted. The Tale containing MICs are marked MIC P.T, MIC P.M and MIC P.K for [https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 PXO99A], [https://www.ncbi.nlm.nih.gov/nuccore/NC_007705.1 MAFF] and [https://www.ncbi.nlm.nih.gov/nuccore/AE013598.1 KACC] respectively. The coloured boxes indicate MICs that are identical from one strain to another. </gallery> [[File:Fig.Tn3.14E.png|center|thumb|820x820px|'''Fig. Tn3.14E.''' MIC clusters in [https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 PX099] Panels '''i)''' and '''ii)''' show two tandem repeats at different genome positions which share one identical MIC. These have presumably arisen by transposition and diversification of the second MIC which is missing the right-hand IR. In panels '''v)''' and '''vi)''' we have not been able to identify IRs. Note that in '''i)''', '''ii)''', '''iii)''', '''iv)''', '''vii)''' and '''viii)''' the interstitial DNA sequence between each MIC is identical.|alt=]] Inspection showed that the chromosome of ''[[wikipedia:Xanthomonas_citri|Xanthomonas citri]]'' strains do not carry identifiable [[wikipedia:Transcription_activator-like_effector|TAL]]-carrying MICs but those of ''[[wikipedia:Xanthomonas_oryzae|X. oryzae]]'' carry relatively high numbers <nowiki><ref name=":20" />. A smaller number of MICs carrying other pathogenicity-related genes are also observed (e.g. [[wikipedia:Type_three_secretion_system|Type III ''Xop'' genes]]) It is notable that the majority of the [[wikipedia:Transcription_activator-like_effector|TAL]]-associated MICs occur as two or more tandem copies. These are listed for three example genomes, [https://www.ncbi.nlm.nih.gov/nuccore/CP000967.2 ''X. oryzae'' PXO99A], [https://www.ncbi.nlm.nih.gov/nuccore/NC_007705.1 MAFF] and [https://www.ncbi.nlm.nih.gov/nuccore/AE013598.1 KACC] in [[:File:Fig.Tn3.14D1.png|Fig. Tn3.14 '''D.1-3''']]. [[File:Fig.Tn3.14F.png|center|thumb|640x640px|'''Fig. Tn3.14F.''' A Model for generating variability in the number of [[wikipedia:Transcription_activator-like_effector|TALE]] genes repeats: Expansion and Contraction by Replication Slippage Replication forks traversing a MIC are shown. The [[wikipedia:Transcription_activator-like_effector|TAL]] repeat sequences are shown in different shades of green, the IRs in either red or green boxes. These structures could arise from normal replication or during the Tn''3''-family transposition process. The intermediate structure shows leading strand synthesis ('''bottom branch of the fork''') and lagging strand synthesis ('''top branch of the fork'''); replication fork slippage on the leading strand template ('''left panel''') results in removal of repeat sequences, while slippage on the nascent strand ('''right panel''') results in expansion of repeat sequences. These are resolved in a second round of replication.]] Those where no IRs could be detected at either end are shown simply as open reading frames. In each case, the DNA segment between tandemly repeated MICs is identical ([[:File:Fig.Tn3.14E.png|Fig. Tn3.14 '''E''']]), suggesting that the tandem [[wikipedia:Protein_dimer|dimer]]<nowiki/>s and multimers arose by amplification possibly via replication slippage and unequal crossing over <nowiki><ref name=":20" /> ([[:File:Fig.Tn3.14F.png|Fig. Tn3.14 '''F''']]). Another characteristic is that they are often flanked by transposase genes raising the possibility that their appearance at different chromosome locations (“radiation”) has occurred by transposition of a single ancestral MIC. This might have been mediated either by flanking transposable elements or by complementation from a Tn''3'' family transposase. In many cases, one of the terminal MICs is truncated and does not exhibit an IR and could often be attributed to insertion of an IS. [[File:Fig.Tn3.14G.png|center|thumb|640x640px|'''Fig. Tn3.14G.''' MIC Duplication and Diversification The figure shows an alignment of the MIC clusters i) and ii) from [[:File:Fig.Tn3.14E.png|Fig. Tn3.14E]] clearly indicating the occurrence of a deletion in '''i)''' compared to '''ii)''' or an insertion of repeats in '''ii)''' compared to '''i)'''.]] It is clear that this “radiation” of [[wikipedia:Transcription_activator-like_effector|TAL]]-associated MICs does not only occur by transposition. In one case ([[:File:Fig.Tn3.14E.png|Fig. Tn3.14 '''Ei''']], '''Eii''' and [[:File:Fig.Tn3.14G.png|Fig. Tn3.14 '''G''']]) an entire DNA segment containing a tandem MIC [[wikipedia:Protein_dimer|dimer]] (MIC P.T11-M<nowiki/>IC P.T13) appears to have been translocated together with surrounding genomic sequences with MIC P.T13 undergoing deletion to generate MIC P.T11-MIC P.T12. [[File:Fig.Tn3.14H.png|center|thumb|640x640px|'''Fig. Tn3.14H.''' [[wikipedia:Transcription_activator-like_effector|TALE protein]] Architecture. ('''Top''') Structure of a 34 amino acid repeat. ('''Bottom''') Organisation of a [[wikipedia:Transcription_activator-like_effector|TAL]] protein showing the N- and C-terminal domains separated by a series of peptide repeats of different colours, each with its recognition dipeptide. Below is shown the DNA base recognition code. From Amanda Zucoloto Group: iGEM13_Calgary (2013-09-17).]] This variability in MIC sequence can be observed within the longer arrays (e.g. [[:File:Fig.Tn3.14E.png|Fig. Tn3.14 '''E''' '''viii''']]) suggesting that diversification follows amplification. This is due to changes in the [[wikipedia:Transcription_activator-like_effector|TAL genes]]. [[wikipedia:Transcription_activator-like_effector|TAL proteins]] are composed of conserved [[wikipedia:N-terminus|N-terminal]] and [[wikipedia:C-terminus|C-terminal]] regions separated by a variable number of 34 amino acid repeats (Fig. Tn3.14H) which can number between 1.5 and 35.5 tandem copies. Each repeat includes a pair of adjacent amino acids capable of recognizing a single base in a DNA sequence ([[:File:Fig.Tn3.14H.png|Fig. Tn3.14 '''H''']]; [[:File:Fig.Tn3.14I.png|Fig. Tn3.14 '''I''']]) e.g. <nowiki><ref name=":20" /><nowiki><ref><pubmed>19933107</pubmed></ref><ref><pubmed>22781676</pubmed></ref>. A tandem array of repeats therefore enables the TAL protein to recognize specific sequences within the target plant genome. This is illustrated by the TAL effector carried by MIC P.T14 (Fig. Tn3.14 J) which includes 19.5 such repeats. The TAL effectors encoded by other members of this cluster (Fig. Tn3.14 Eviii), MIC P.T15, MIC P.T16, MIC P.T17, MIC P.T18, which have presumably all arisen by amplification of a single ancestral MIC, each carry a different number of repeats and vary in their sequence recognition properties.

  • Fig. Tn3.14. Panels I and K (use the arrows to scroll the figures).

  • Fig. Tn3.14I. TALE Protein Specificity. Left column show the recognition dipeptide. The right column shows the nucleotide which is recognised by the amino acid pair. Data from <ref><pubmed>22223738</pubmed></ref><ref><pubmed>21106758</pubmed></ref><ref><pubmed>22828628</pubmed></ref>.

  • Fig. Tn3.14J. Amino acid Sequence of the TAL expressed from MIC P.T14 This shows the 19.5 34 amino acid repeats together with the recognition dipeptides (boxed).

  • Fig. Tn3.14Ki. Comparison of the MIC P.T14 Clusters in PXO99A and MAFF Strains The Sequence of the 34 amino acid repeats of each TAL protein in consecutive MICs is shown in equivalent positions in the clusters. Recognition dipeptides are boxed. Right: PXO99A, left, MAFF. i) The recognition sequences are presented between the two panels and show some variation; ii) both PXO99A and MAFF include a single repeat with an insertion while MAFF also has a repeat which is deleted for a single amino acid in the recognition dipeptide; iii) PXO99A MIC P.T16 carries a repeat with a single amino acid removed, while both MIC P.T17 are identical; iv) shows the final MIC in the PXO99A cluster with no equivalent in the MAFF cluster.

  • Fig. Tn3.14Kii. Comparison of the MIC P.T14 Clusters in PXO99A and MAFF Strains The Sequence of the 34 amino acid repeats of each TAL protein in consecutive MICs is shown in equivalent positions in the clusters. Recognition dipeptides are boxed. Right: PXO99A, left, MAFF. i) The recognition sequences are presented between the two panels and show some variation; ii) both PXO99A and MAFF include a single repeat with an insertion while MAFF also has a repeat which is deleted for a single amino acid in the recognition dipeptide; iii) PXO99A MIC P.T16 carries a repeat with a single amino acid removed, while both MIC P.T17 are identical; iv) shows the final MIC in the PXO99A cluster with no equivalent in the MAFF cluster.

  • Fig. Tn3.14Kiii. Comparison of the MIC P.T14 Clusters in PXO99A and MAFF Strains The Sequence of the 34 amino acid repeats of each TAL protein in consecutive MICs is shown in equivalent positions in the clusters. Recognition dipeptides are boxed. Right: PXO99A, left, MAFF. i) The recognition sequences are presented between the two panels and show some variation; ii) both PXO99A and MAFF include a single repeat with an insertion while MAFF also has a repeat which is deleted for a single amino acid in the recognition dipeptide; iii) PXO99A MIC P.T16 carries a repeat with a single amino acid removed, while both MIC P.T17 are identical; iv) shows the final MIC in the PXO99A cluster with no equivalent in the MAFF cluster.

  • Fig. Tn3.14Kiv. Comparison of the MIC P.T14 Clusters in PXO99A and MAFF Strains The Sequence of the 34 amino acid repeats of each TAL protein in consecutive MICs is shown in equivalent positions in the clusters. Recognition dipeptides are boxed. Right: PXO99A, left, MAFF. i) The recognition sequences are presented between the two panels and show some variation; ii) both PXO99A and MAFF include a single repeat with an insertion while MAFF also has a repeat which is deleted for a single amino acid in the recognition dipeptide; iii) PXO99A MIC P.T16 carries a repeat with a single amino acid removed, while both MIC P.T17 are identical; iv) shows the final MIC in the PXO99A cluster with no equivalent in the MAFF cluster.

It is interesting to note that while the amino acid repeats are always maintained in phase, certain TAL effectors have undergone removal of a single amino acid while another has acquired a short insertion. These changes might be expected to influence the capacity of the proteins to recognise their cognitive DNA sequence.

Diversification can also be observed between clusters in related X. oryzae strains such as MAFF and KACC.

Strain PXO99A and MAFF share the cluster MIC P.T15, MIC P.T16, MIC P.T17 (Fig. Tn3.14 Ki; Fig. Tn3.14 L). Both clusters have identical genomic environments (with some sequence variation) and the inter MIC sequences are identical. Not only has there been a large deletion of MIC P.T18 in the MAFF cluster, but sequence variations are apparent along the entire cluster length both within and between the clusters potentially modifying the DNA sequence recognition properties. Strain MAFF and KACC also share a cluster (MIC M.T2 and MIC M.T3).

Further analyses and experimental approaches are necessary to fully understand the role of MICs in the dispersal and diversification of these important instruments of Xanthomonad virulence, the TAL effectors.

Fig. Tn3.14L. Alignment of the MIC P.T14 Clusters in PXO99A and MAFF Strains. The figure shows an alignment of the MAFF (top) and PXO99A (bottom) MIC clusters, indicating a deletion in MAFF in MIC P.T18 or an insertion in PXO99A of MIC P.T18.

Acquisition of Passenger Genes.

Tn3-family transposons carry large and diverse and diverse sets of passenger genes (e.g. Fig. Tn3.3). These have been acquired by a number of different processes.

Tn402 and integron platforms.

One major source of antibiotic passenger genes has been by ancestral insertions of Tn402 derivatives which have often “decayed” to lose their transposition properties but have retained their abilities to acquire (and lose) integron gene cassettes (Fig. Tn3.7 G; Fig. Tn3.7 I; Fig. Tn3.7 J; Fig. Tn3.7 R; Fig. Tn3.7 S; Fig. Tn3.7 U; Fig. Tn3.7 V; Fig. Tn3.18 C).

Additional TE

A second pathway to acquisition is by insertion of additional transposable elements with, or without rearrangement (Fig. Tn3.7 U; Fig. Tn3.8 E; Fig. Tn3.8 F). It is also interesting to note that there are a number of cases in which additional IR appear within certain structures (e.g. Fig. Tn3.10 D; Fig. Tn3.11 G) such as Tn3 <ref name=":31" /> and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] <nowiki><ref name=":35"><pubmed>6098802</pubmed></ref> raising the possibility that these have been involved in generating the host transposon.

Recombination at res.

A third major pathway to passenger gene acquisition is by inter-transposon exchange via res sites (see: Resolution). This was first suggested to explain the formation of Tn501, by exchange of a transposition module with a Tn1721-related transposon <ref name=":35" />. It was later observed by Kholodii and coworkers <nowiki><ref><pubmed>11376944</pubmed></ref><ref name=":38"><pubmed>9159519</pubmed></ref> and called “shuffling”, by Yano et al., <ref><pubmed>17408691</pubmed></ref> and by others <ref name=":30" />. As judged by the analyses included here, this seems to be a recurring type of event and can be found in members of most clades ('''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21'']:''' [[:File:Fig.Tn3.7H.PNG|Fig. Tn3.7 '''H''']]; [[:File:Fig.Tn3.7M.PNG|Fig. Tn3.7 '''M''']]; [[:File:Fig.Tn3.7N.PNG|Fig. Tn3.7 '''N''']]; [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn163-L14931 '''Tn''163''''']''':''' [[:File:Fig.Tn3.8C.png|Fig. Tn3.8 '''Ci''']] and [[:File:Fig.Tn3.8C2.png|Fig. Tn3.8 '''Cii''']]; '''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430'']:''' [[:File:Tn3.9D.png|Fig. Tn3.9 '''D''']]; '''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3000-AF174129 Tn''3000'':]''' [[:File:Fig.Tn3.11C.PNG|Fig. Tn3.11 '''C''']]; [[:File:Fig.Tn3.11F.PNG|Fig. Tn3.11 '''F''']]; and '''Tn''4561'':''' [[:File:Fig.Tn3.12C.PNG|Fig. Tn3.12 '''C''']]). This type of behavior can also lead to “suicide” of a transposon in which the transposition module is removed by res recombination with a site outside the transposon <nowiki><ref><pubmed>8387603</pubmed></ref>.

Mercury Resistance: a Major Passenger Gene Group

The Mercury Operon and the Tn3 family
Fig. Tn3.15A. The mercury resistance operon. A typical mercury operon redrawn from Liebert et al 1997. Each gene is represented by a yellow horizontal arrow indicating the direction of expression. merO is the divergent operator site to which the merR regulator binds. The mercury transport genes, merC and merF, shown bracketed by dotted lines, are not always present and only one or the other is ever included in a given operon. The organo-mercurial lyase, merB, is not always present. Both merC and merT encode transmembrane segments (grey boxes).

Not surprisingly, bacteria carrying mer operons are particularly abundant in areas with increased mercury concentrations such as mercury mines and contaminated soil or water <ref>Jobling MG, Peters SE, Ritchie DA. Plasmid-borne mercury resistance in aquatic bacteria. FEMS Microbiol Lett. 1988;49:31–37.</ref><ref><pubmed>6394954</pubmed></ref><ref><pubmed>PMC183436</pubmed></ref> and it was suggested that mercury resistance is an ancient system as reflected by its wide geographical, environment and species range and it has been speculated that it evolved as a response to increased levels of mercury in natural environments resulting, for example, from volcanic activity <ref><pubmed>9167257</pubmed></ref>.

It is certainly present in the Murray collection <ref><pubmed>6316165</pubmed></ref>, a collection of Enterobacteriaceae isolated in the pre-antibiotic era, as part of transposons Tn5073 and Tn5074 which show high similarity to present day examples such as Tn5036 and Tn1696 (Tn3 family members of the Tn21 clade) and Tn5053 (a Tn402 family member of the Tn5053 clade) and Tn5075 respectively <ref name=":37" />. Although Tn''3'' family members carry a large variety of passenger genes, [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] is found repeatedly within the family and is thought to be one of the first sets of passenger genes to have been acquired (Fig. Tn3.6) and appears in precursors of the major groups of antibiotic resistance carrying Tn''3'' family members ([[:File:Fig.Tn3.7G.PNG|Fig. Tn3.7 '''G''']]). [http://parts.igem.org/Part:BBa_K1420000 Mercury resistance operons] were proposed to have been acquired at least twice <nowiki><ref name=":14" />([[:File:Fig.Tn3.6.png|Fig. Tn3.6]]): once by an ancestor of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] and once by an ancestor of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501'']. Their acquisition presumably predates the acquisition of antibiotic resistance [[wikipedia:Integron|integron]] platforms since a number of [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] Tn''3'' family transposons have been identified and, in at least two cases, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1696-U12338.3 Tn''1696''] (whose ''mer'' genes appear to fall largely into different groups; Fig. Tn3Bi-vii), clear precursors devoid of [[wikipedia:Integron|integrons]] ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5060-AJ551280.1 Tn''5060''] <nowiki><ref name=":15" /> and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn20-AF457211.1 Tn''20''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1696.1-CP047309 Tn''1696.1''] respectively) have been identified. [http://parts.igem.org/Part:BBa_K1420000 Mercury resistance genes] are found in a number of Tn''3'' family clades ([[:File:Fig.Tn3.15B.PNG|Fig. Tn3.15 '''B''']] and [[:File:Fig.Tn3.15B.PNG|Fig. Tn3.15 '''Bi-vii''']]). These include [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn163-L14931 Tn1''63''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] and Tn''4651''. Those associated with the Tn''21'' clade occur upstream of, and are generally expressed towards, ''tnpR'' ([[:File:Fig.Tn3.7G.PNG|Fig. Tn3.7 '''G''']]); those of the Tn''3'' clade are located downstream of ''tnpA'' ([[:File:Fig.Tn3.15Ci.png|Fig. Tn3.15 '''C''']]) and in those carrying the tnpS/T genes, they are between the transposase module and the ''tnpS''/''tnpT'' module ([[:File:Fig.Tn3.15D.png|Fig. Tn3.15 '''D''']]). [[File:Fig.Tn3.15B.PNG|thumb|640x640px|'''Fig. Tn3.15B.''' Table showing the distribution of various ''mer'' resistance gens in Tn''3'' family transposons. The ''mer'' genes '''R''' through '''E''' are indicated at the top. The various Tn''3'' family transposons are cited in the left-hand column. Each line is coloured according to the Tn3 family clade. Transposons highlighted in yellow have ''mer'' gene duplications.|alt=|center]]<gallery mode="slideshow" caption="'''Fig. Tn3.15.''' Panels C ''mer'' phylogenetic trees (use the arrows to scroll the figures)."> File:Fig.Tn3.15Ci.png|'''Fig. Tn3.15Ci.''' MerR phylogenetic tree. File:Fig.Tn3.15Ciii.png|'''Fig. Tn3.15Ciii.''' MerT phylogenetic tree. File:Fig.Tn3.15Civ.png|'''Fig. Tn3.15Civ.''' MerP phylogenetic tree. File:Fig.Tn3.15Cv.png|'''Fig. Tn3.15Cv.''' MerC phylogenetic tree. File:Fig.Tn3.15Cvi.png|'''Fig. Tn3.15Cvi.''' MerA phylogenetic tree. File:Fig.Tn3.15Cvii.png|'''Fig. Tn3.15Cvii.''' MerD phylogenetic tree. File:Fig.Tn3.15Cviii.png|'''Fig. Tn3.15Cviii.''' MerE phylogenetic tree. </gallery>A survey of 29 functional [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] transposons isolated from Gram negative bacteria in environmental isolates revealed that the most widespread of transposons belong to two types: transposons of the Tn''21'' clade of the Tn''3'' family and relatives of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5053-L40585.1 Tn''5053''], a member of the [[Transposons families/Tn402 family|Tn''402'' family]] <nowiki><ref name=":38" /><nowiki><ref><pubmed>11763242</pubmed></ref>. In addition, Yurieva et al <ref name=":38" /> identified a third group, related to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''], a member of the Tn''4651'' clade They also identify “mosaic” [http://parts.igem.org/Part:BBa_K1420000 ''mer'' operons]which, they suggest, are generated by homologous recombination between short DNA sequences. While [https://www.uniprot.org/uniprot/P0A183 MerR] appears to be very similar between different [http://parts.igem.org/Part:BBa_K1420000 ''mer'' operons], while [https://www.uniprot.org/uniprot/P00392 MerA] showed a higher degree of mosaicism as did [https://www.uniprot.org/uniprot/P04140 MerT] and [https://www.uniprot.org/uniprot/P04131 MerP] to some extent <nowiki><ref name=":38" />. =====The Mercury Operon: Organization, Regulation, and Resistance Mechanism===== The mechanism underlying [[wikipedia:Mercury_(element)|mercury]] resistance has been extensively reviewed a number of times <nowiki><ref name=":16" /> . Briefly, [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] in gram-negative bacteria results in the release of gaseous mercury Hg<sub>0</sub>. Mercury salts (HgII) are captured by the periplasmic [https://www.uniprot.org/uniprot/P04131 MerP], transferred across the periplasm to the inner membrane proteins [https://www.uniprot.org/uniprot/P22905 MerC] or [https://www.uniprot.org/uniprot/P04140 MerT] and then across the cytoplasmic membrane to the mercuric reductase, [https://www.uniprot.org/uniprot/P00392 MerA] which converts it to the volatile Hg<sub>0</sub>. The operon is regulated by two genes, ''[https://www.uniprot.org/uniprot/P0A183 merR]'' and ''[https://www.uniprot.org/uniprot/P0A2Q6 merD]'' ([[:File:Fig.Tn3.15A.PNG|Fig. Tn3.15 '''A''']]). The order of these genes is generally ''[https://www.uniprot.org/uniprot/P04140 merT]'', ''[https://www.uniprot.org/uniprot/P04131 merP]'', [https://www.uniprot.org/uniprot/P22905 ''merC''], [https://www.uniprot.org/uniprot/P00392 merA], ''[https://www.uniprot.org/uniprot/P0A2Q6 merD]'' and ''[https://www.uniprot.org/uniprot/P06690 merE]''. ''[https://www.uniprot.org/uniprot/P0A183 merR]'' is located upstream and is transcribed in the opposite direction with overlapping promoters. Binding of [https://www.uniprot.org/uniprot/P0A183 MerR] represses expression of the operon and of itself. Interaction with Hg(II) releases [https://www.uniprot.org/uniprot/P0A183 MerR] repression of the ''mer'' structural genes permitting their expression without significantly impacting on its autorepression <nowiki><ref><pubmed>PMC210155</pubmed></ref> and its interaction with RNA polymerase creates a pre-transcription initiation complex <ref><pubmed>10079080</pubmed></ref>.

The product of the secondary regulator gene, merD <ref name=":36" />, appears to play a role in down-regulating the [http://parts.igem.org/Part:BBa_K1420000 ''mer'' operon] <nowiki><ref name=":39"><pubmed>1917975</pubmed></ref>. It binds weakly but specifically to the merOP region and DNase I footprinting identified a common operator binding sequence for both MerR and MerD <ref name=":39" />. The genes essential for [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] were identified as ''[https://www.uniprot.org/uniprot/P0A183 merR]'', ''[https://www.uniprot.org/uniprot/P04140 merT]'', ''[https://www.uniprot.org/uniprot/P04131 merP]'' and ''[https://www.uniprot.org/uniprot/P00392 merA]'' <nowiki><ref><pubmed>PMC168397</pubmed></ref>. An additional mercury ion transmembrane transporter gene, merE (UniProtKB - D4N5J4) involved in the accumulation of methyl-mercury <ref name=":16" /><nowiki><ref><pubmed>19265693</pubmed></ref> is often present. Not all mercury operons include merC and some have a gene, merF <ref name=":40"><pubmed>8063107</pubmed></ref>, an alternative mercury ion transmembrane transporter (UniProtKB - Q1H9Y3). Some also include a mercury lyase gene, merB, involved in resistance to organo-mercury <ref><pubmed>PMC304818</pubmed></ref><ref><pubmed>3542022</pubmed></ref>.

The Mercury Operon: Diversity in various Tn3 family clades.

The mer carrying Tn3 family members (Fig. Tn3.15 B) all lack merF. Most examples carry a full mer gene complement although a small group (Tn501, Tn511, Tn1412, Tn4378 and Tn4380) lack the merC gene and only 3 (Tn5084, Tn6294, Tn6332), all members of the Tn4430 clade) carry a merB gene and have a duplicated or partially duplicated mer operon.

Phylogenetic trees generated for MerR (Fig. Tn3.15 Ci and Cii), MerT (Fig. Tn3.15 Ciii), MerP (Fig. Tn3.15 Civ), MerA (Fig. Tn3.15 Cvi), MerD (Fig. Tn3.15 Cvii) and MerE (Fig. Tn3.15 Cviii) reveal that, in general, Tn501-related mer genes group separately from those of Tn21 relatives. This provides some support for the hypothesis that the mer operon had been acquired at least twice. These groups are separated by mer genes from Tn402 family relatives.

Within the Tn21 clade, all members carry the mer operon upstream of tnpR with the direction of transcription to the right (Fig. Tn3.15 D top). merR, on the other hand, is transcribed in the opposite direction and terminates with a TAG codon within the IRL sequence (Fig. Tn3.15 D bottom) with one exception, Tn6023.

Fig. Tn3.15D. MerR and the Tn21 clade. The top of the figure shows a generic Tn21 clade transposon with a typical mer operon. merR is shown at the left. An alignment of the DNA sequences of members of the Tn21 clade is shown underneath indicating the transposon name and accession number, IRL (enclosed in a blue filled box emphasized with a blue arrow), and the merR termination codon, TAG (enclosed in the red filled box). Note that in the case of Tn6203, there is an upstream termination codon. The termination codon is located within the IR.

On the other hand, for the few members of the Tn3 clade, the mer genes are located downstream of tnpA and are transcribed to the left (Fig. Tn3.15 E top) except for merR which is transcribed towards and terminates some distance from IRR (Fig. Tn3.15 E bottom), while for the unique Tn4651 member, the mer operons is located between tnpA and tnpS/T (Fig. Tn3.15 F).

Fig. Tn3.15E. MerR and the mer operon Tn3 clade. The top of the figure shows a generic Tn3 clade transposon with a typical mer operon. Note that here, the mer operon is not complete and the mercury genes are located downstream. merR is shown at the right. An alignment of the DNA sequences of members of the Tn3 clade are shown underneath to indicating the transposon name and accession number, IRR (enclosed in a blue filled box emphasised with a blue arrow.
Fig. Tn3.15F. MerR and mer operon in a tnpS/tnpT-carrying Tn3 family members A map of Tn5041 (X98999.3) is shown. Note that the mercury operon is located between the tnpA and tnpS/tnpT genes.
The Mercury Operon: Tn21 in mer acquisition by Tn402?

It is worth noting that members of the Tn402 family Tn5053 mercury resistance subgroup carry a single copy of a sequence closely related to Tn21 IRL (Fig. Tn3.15 G top) located in a similar position with respect to the mercury operon as the resident IRL in the Tn21 group (Fig. Tn3.15 D and Fig. Tn3.15 G top and middle) (see <ref name=":40" />). There is some variability in the 10 [[wikipedia:C-terminus|C-terminal]] amino acid tail of the neighboring [https://www.uniprot.org/uniprot/P0A183 MerR] protein ([[:File:Fig.Tn3.15G.png|Fig. Tn3.15 '''G''']] bottom) although the major part of [https://www.uniprot.org/uniprot/P0A183 MerR] amino acid sequence is highly conserved. This raises the possibility that the [http://parts.igem.org/Part:BBa_K1420000 mercury resistance genes] carried by the [[Transposons families/Tn402 family|Tn''402'' family elements]] was derived from an ancestral Tn''21'' group transposon. [[File:Fig.Tn3.15G.png|center|thumb|640x640px|'''Fig. Tn3.15G.''' Tn''3'' family Mercury resistance and its relationship with the [[Transposons families/Tn402 family|Tn''402'' family]]. Members of the [[Transposons families/Tn402 family|Tn''402'' family]] in the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5053-L40585.1 Tn''5053''] mercury resistance subgroup carry a single copy of a sequence closely related to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] IRL (Top panel) located in a similar position with respect to the mercury operon as the resident IRL in the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] group ('''Middle panel'''). There is some variability in the 10 C-terminal amino acid tail of the neighboring [https://www.uniprot.org/uniprot/P0A183 MerR] protein ('''Bottom panel''') although the major part of [https://www.uniprot.org/uniprot/P0A183 MerR] amino acid sequence is highly conserved.]] <br /> ===Transposition Mechanism Overview=== ====Early Studies==== In early studies of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1''] and ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn2-KT002541 2]'') <nowiki><ref name=":3" /><nowiki><ref name=":41" /><nowiki><ref name=":42" /><nowiki><ref name=":5" />, Tn''4651'' <nowiki><ref name=":26" /> and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] <nowiki><ref name=":43" /><nowiki><ref name=":80"><pubmed>16629665</pubmed></ref> it was clearly demonstrated that Tn3 family transposition occurs in a two-step process involving a replicative step in which the transposon first couples the donor and target replicons by single strand transfer to create a forked allowing replication to generate a fully double stranded cointegrates structure followed by a site-specific recombination step, resolution, catalyzed by a dedicated enzyme, the resolvase (Fig. Tn3.2). While the resolution step for a number of Tn3 family members has been studied in exquisite detail (see Resolution below), study of the initial strand transfer and replication steps have proved problematic.

Fig. Tn3.16A. The Consequences of Replicative Transposition. i) intermolecular transposition. a) donor and target molecules; b) cleavage at both terminal inverted repeats (TIRs) of the Insertion Sequence (IS; yellow double-headed arrow) results in nicks on both strands generating 3’-OH groups (grey circles) that attack the target site (red arrow); c) DNA replication generates a cointegrates containing a duplication of the IS and the target site; d) this can be subsequently resolved into a plasmid identical to the original donor plasmid and a modified target plasmid carrying an IS copy flanked by target site duplications arranged as direct repeats(DRs). ii) intramolecular transposition. a) molecule showing target sequence and two genes, a and b; b) the 3’-OH groups generated by cleavage at both TIRs can either attack the target site on the same (cis) or c) the opposite (trans) strand; d) When in cis, DNA between the IS and target site becomes circularized and contains one IS copy and target site; e) In trans, DNA between IS and the target site is instead inverted ("a b" becomes "b a"), bracketed by the original IS and a new copy in an inverted orientation. The target site is also duplicated but in inverted orientation and each TSD is associated with one IS copy. Black arrows indicate potential DRs from previous transposition events; different numbers represent different sequences. Oval and square represent the origins of replication on the donor and target molecules respectively. The red arrow marked “0” represents the target sequence which becomes the newly duplicated sequence following transposition. Black arrows marked “1” and “2” represent the original DR sequences. Figure from <ref><pubmed>PMC5142620</pubmed></ref>.

The consequences of these pathways are shown in greater detail in Fig. Tn3.16A. This underlines why not all Tn3 family transposition events yield transposons flanked by 5bp direct repeats. Fig. Tn3.16 Ai shows intermolecular transposition generating a cointegrates which, following resolution yields donor and target each with a single copy of the transposon in flanked by two DR copies. In intramolecular transposition, one pathway leads to a deletion while the other to an inversion. In neither case is the transposon flanked by direct target repeats. Tn3-mediated inversions and deletions of this type have been described a number of times with Tn3, Tn1, Tn2660 and Tn1721 <ref name=":4" /><nowiki><ref name=":51"><pubmed>385227</pubmed></ref><ref><pubmed>PMC294047</pubmed></ref><ref><pubmed>6294460</pubmed></ref><ref><pubmed>6329911</pubmed></ref><ref><pubmed>6308391</pubmed></ref><ref name=":52"><pubmed>6090862</pubmed></ref>.

Early studies also demonstrated that, like a number of transposons, the transposition frequency of Tn3 family transposons appears to decrease exponentially with increasing length <ref name=":13" /> ([[:File:Fig.Tn3.16B.png|Fig. Tn3.16B]]). Tanaka and colleagues investigated Tn''2603'' and various derivatives ranging in length from approximately 5kb to 22.5kb from a number of different donor plasmids to both R386 and R388 target plasmids and noted a steep exponential reduction in transposition frequencies of over 1000-fold with increasing length. This observation would be more robust if transposition frequencies had been measured from the same donor plasmid and the transposons all had identical genetic contexts. [[File:Fig.Tn3.16B.png|thumb|580x580px|'''Fig. Tn3.16B.''' Transposition frequencies (relative) of Tn''2603''-related Tn''3'' family transposons of different lengths. Two target plasmids were used : R386 (blue squares) and R388 (blue circles). The donors were '''A:''' pMK1::Tn''2613''#1; '''B:''' pTY61 (Tn''2603''del-61); '''C:''' ColE1::Tn''2608''#1; '''D:''' pMK1::[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21'']#1; '''E:''' pMK1::Tn''2603''#1; '''F:''' pSC101::[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4-KY749247.1 Tn''4''].|alt=|center]] ====Replicative transposition==== One of the major problems in studying transposition of Tn''3'' members is that their transposases, TnpA, are long (~1000 amino acids) and difficult to solubilize. ====Interaction of transposase and transposon ends==== The [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] transposase, TnpA<sub>Tn''3''</sub>, was first purified in 1981 and shown to bind DNA in a salt resistant way <nowiki><ref><pubmed>6262296</pubmed></ref> and one of the first attempts to investigate TnpATn3 activity in vitro <ref><pubmed>2986006</pubmed></ref> concluded that addition of ATP was necessary to obtain TnpATn3 binding to the Tn3 ends. However, in a subsequent article this was shown to be erroneous and probably due to a pH effect of the added ATP solution <ref><pubmed>PMC299513</pubmed></ref>. Purified TnpATn3 was observed to bind specifically to both IRTn3 and protect a sub-terminal DNA region within the IR (Fig. Tn3.16 Ci) in a heparin resistant manner a measure of its strong and highly sequence-specific DNA binding activity while another study using a different TnpATn3 purification scheme and DNA binding conditions <ref><pubmed>2843651</pubmed></ref> showed a much less sequence-specific protection which included the entire IRTn3 and a significant region of flanking DNA. Further functional analysis of the Tn3 ends <ref><pubmed>2155858</pubmed></ref> demonstrated that mutations in the first 10 IRTn3 base pairs (domain A) did not influence TnpATn3 binding while mutations in the 13-38 base pair region (domain B) inhibited binding (Fig. Tn3.16 Ci), behavior confirmed in a second study <ref name=":53"><pubmed>1849179</pubmed></ref>.

This is a similar functional architecture to the ends of other transposable elements (see: General Information/IS Organization/Terminal Inverted Repeats). In addition, the effects of mutations in the Tn3 ends on transposition in vivo <ref><pubmed>2162965</pubmed></ref> indicated that mutations in the TnpATn3 binding site have a stronger effect when present at both transposon ends than when located at only one end.

Similar binding studies have been undertaken for Tn1000 (γΔ) (Fig. Tn3.16 Cii). Protection against DNAse is more extensive than for Tn3 although this depends critically on the binding and digestion conditions <ref name=":45" />. The protection pattern is broadly similar with the tip of the terminal IR<sub>Tn''1000''</sub> remaining unprotected and protection extended to the inner end of the IR. Some weak protection occurred on the DNA region flanking the IR tip. In addition, however, the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] ends include a binding site for the host DNA architectural protein, [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF]], and both proteins were found to bind cooperatively <nowiki><ref name=":45" />. However, [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF]] appeared to downregulate [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] transposition <nowiki><ref name=":44" />. The juxtaposition of [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF]] sites and transposon ends has been observed in several other TE (see <nowiki><ref><pubmed>7744253</pubmed></ref><ref><pubmed>PMC553657</pubmed></ref><ref><pubmed>2821273</pubmed></ref><ref name=":112"><pubmed>9630232</pubmed></ref>).

Binding studies have also been carried out with the transposase of Tn4430, TnpATn4430, a Tn3 derivative which encodes a TnpI resolvase <ref name=":46"><pubmed>PMC5293067</pubmed></ref>. Here, it was necessary to use a mutant transposase (Fig. Tn3.16 Ei) which had been selected for a reduction in its transposition immunity (see Transposition immunity below) and which concomitantly showed an increase in transposition activity. Similar protection patterns (Fig. Tn3.16 Eii) were observed as with TnpATn3 and TnpATn1000: transposase binding protects the distal IRTn44300 internal region. The IR was divided into three regions (A, B1 and B2) based on sequence conservation, which largely correspond to the A and B regions of IRTn3 (Fig. Tn3.16 Ci).

Fig. Tn3.16C. Organisation of Tn3 and Tn1000 IRs. A) Nucleotide sequence of Tn3 terminal IRs. The IRL and IRR, which are perfect inverted repeats, are boxed and flanking DNA is shown as XXXXXXX. The horizontal striped boxes marked A and B indicate the two functional IR domains. The horizontal green lines represent the extent of protection by transposase binding to Dnase I digestion (redrawn from Ichikawa et al., 1987). B) Nucleotide sequence of Tn1000 terminal IRs. IRL (g) and IRR (D) sequences are boxed. The internal abutting IHF binding site is shown in red. The horizontal green and blue lines represent the extent of protection by transposase anf IHF binding respectively to Dnase I digestion (redrawn from Wiater and Grindley, 1988).

TnpA functional domains

The TnpATn3 is 1004 amino acid residues long. Like many other transposases, it carries a DDE catalytic motif (General Information/Reaction mechanisms). Characterization of a series of fusions of TnpATn3 segments to β-galactosidase <ref name=":104"><pubmed>8382339</pubmed></ref><ref name=":47"><pubmed>8080658</pubmed></ref> (Fig. Tn3.16 Di) revealed that the N-terminal segment (residues 1-242) exhibited sequence-specific binding to the 38 base pair IR and that this region could be dissected into two sub-regions, amino acids 1-86 and 87-242, which showed non-specific DNA binding activity, implying that both were involved in sequence-specific end binding. The large central region also included two regions with non-specific DNA binding properties while the C-terminal region encodes the DDE catalytic site.

Fig. Tn3.16D. Tn3 and Tn1000 Transposases. i) A map of the 1004 amino acid residues long TnpATn3. The length of each of the three domains is shown above in amino acids while the regions with DNA binding functions are shown as orange boxes with the amino acid residues within; ii) N-terminal sequences of Tn3 (1-242) and Tn1000 (1-243). * and black residues indicate identical amino acids, red residues indicate differences. The boxed region is responsible for the different DNA sequence-specific binding activities of the two proteins. iii) A dot-plot of the two proteins showing their shared C-terminal end sequence and the more variable N-terminal regions. Data from <ref name=":104" /><nowiki><ref name=":47" /><nowiki><ref><pubmed>1652540</pubmed></ref>.

The region of TnpA involved in DNA sequence recognition for binding to the transposon IRs was further investigated using a series of hybrid TnpA genes carrying the N-terminal IR-binding region constructed between TnpATn3 and TnpATn1000 <ref name=":47" />. TnpA<sub>Tn''3''</sub> and TnpA<sub>Tn''1000''</sub> were found to share over 64% identity ([[:File:Fig.Tn3.16D.png|Fig. Tn3.16 '''Dii''']]). This enabled the definition of a region of TnpA which permits distinction between binding to an IR<sub>Tn''3''</sub> and an IR<sub>Tn''1000''</sub> <nowiki><ref name=":47" /> ([[:File:Fig.Tn3.16D.png|Fig. Tn3.16 '''Dii''']]). A dotplot comparison of tnpA<sub>Tn''3''</sub> and tnpA<sub>Tn''1000''</sub> nucleotide sequences indicated that the 3’ ends of both genes were conserved whereas the 5’ ends showed some variation ([[:File:Fig.Tn3.16D.png|Fig. Tn3.16 '''Diii''']]) <nowiki><ref name=":47" />. A functional map of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] transposase, TnpA<sub>Tn4430</sub>, was obtained by partial proteolysis with trypsin and chymotrypsin ([[:File:Fig.Tn3.16D.png|Fig. Tn3.16 '''Di''']]) <nowiki><ref name=":48"><pubmed>22624153</pubmed></ref>. This treatment indicated that, like TnpATn3, TnpATn4430 has three major domains: an N-terminal domain (amino acids 1-152) similar to a CENP-B DNA binding domain <ref><pubmed>9097724</pubmed></ref>; a central region (amino acids 153-682); and a C-terminal domain (amino acids 683-980) with an RNase H fold-like domain including the catalytic DDE triad. Like other members of the family, the distance between the second D and E residues is somewhat longer than in typical DDE transposases and has been called an insertion domain and is likely composed of alpha-helical structures <ref name=":49"><pubmed>PMC3107681</pubmed></ref>. The presence of insertion domains between the D and E residues observed in other transposases does not disturb the catalytic RNAse fold <ref name=":49" /> and, in both cases studied in detail <nowiki><ref name=":54"><pubmed>10884228</pubmed></ref><ref><pubmed>PMC7780238</pubmed></ref>, performs crucial functions in the transposition chemistry specific for each element.

Cleavage and Strand transfer.

In spite of the extensive DNA binding studies, the biochemistry of Tn3 family transposition has proved refractory to detailed analysis. A single study with Tn3 <ref><pubmed>2172235</pubmed></ref> in vitro used a cell extract with high TnpA levels, a donor minimal plasmid replicon containing a mini transposon with Tn3 ends and a target molecule composed of concatemeric phage lambda DNA. Following the reaction, the phage DNA was packaged in an in vitro system and used to infect suitable recipient cells. The process yielded cells which appeared to carry large plasmids consistent with the formation of cointegrates. However, these were not physically characterized and the approach does not seem to have been developed further. Additionally, sequence-specific 3’ cleavage at the ends of a plasmid carried mini Tn3 derivative was observed with a cell-free extract containing TnpATn3 in a reaction which required Mg2+ and was stimulated by a host factor determined to be acyl carrier protein (ACP) <ref><pubmed>9077464</pubmed></ref>. A similar observation had been made for the Tn7 transposition reaction<ref name=":112" />. In a more recent a study using the mutant TnpA<sub>Tn''4430''</sub> <nowiki><ref name=":46" /> an ''in vitro'' system including both strand cleavage and strand transfer was developed. The mutant TnpA<sub>Tn''4430''</sub> carried 3 mutations ([[:File:Fig.Tn3.16E.png|Fig. Tn3.16 '''Ei''']]) selected for a reduced level of transposition immunity <nowiki><ref name=":48" /> but exhibiting a hyper transposition efficiency <nowiki><ref name=":46" />. It was shown, using a gel shift assay and differentially fluorescently labeled IR, that this TnpA derivative formed two types of complex which appeared to be '''S'''ingle '''E'''nd and '''P'''aired '''E'''nd (SEC and PEC) species containing one or two IR<sub>Tn''4430''</sub> molecules bridged by the transposase.[[File:Fig.Tn3.16E.png|thumb|640x640px|'''Fig. Tn3.16E.''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] Transposase. '''i)''' A map of the 980 amino acid residues long TnpA[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430'']. The length of each of the three domains identified by partial trypsin and chymotrypsin digestion is shown above in amino acids. Mutations are shown as orange vertical lines marked with their positions in black below. Those mutations conferring hyper-transposition and reduced immunity are indicated by red vertical arrows below with the mutant residues marked. '''ii)''' DNA footprint analysis. SEC and PEC protection against DNAse on both strands (green horizontal lines). Small red dots in flanking DNA indicate additional protection of flanking DNA in the PEC. [(OP)2-Cu+] DNA enhanced cleavage in the PEC is indicated by orange triangles and in the SEC by purple triangles. '''iii)''' Consequences of strand transfer into a circular target. Left: a single strand transfer event; right a concerted strand transfer reaction which linearises the target DNA. Data from <nowiki><ref name=":46" /><nowiki><ref name=":48" />.|alt=|center]]Footprinting both types of complex revealed an identical pattern of DNase protection ([[:File:Fig.Tn3.16E.png|Fig. Tn3.16 '''Eii''']]) except for some additional weak protection of flanking DNA in the PEC. When probed with the [[wikipedia:Phenanthroline|1,10-phenanthrolinecopper]] [(OP)2-Cu+] nuclease, the PEC showed significantly enhanced cleavage at the IR tip and in the DNA flank, particularly on the lower strand indicating a change of DNA conformation ([[:File:Fig.Tn3.16E.png|Fig. Tn3.16 '''Eii''']]). Correct single strand cleavage at the 3’ end of the IR tip was observed in typical cleavage conditions as well as some double strand cleavage (3’ and 5’). This was examined using both wildtype TnpA<sub>Tn''4430''</sub> and mutant derivatives with different transposition activities. The unexpected 5’ cleavage increase with increasing TnpA<sub>Tn''4430''</sub> activity and when Mn<sup>2+</sup> was used instead of Mg<sup>2+</sup> indicating that this is an aberrant activity. Furthermore, precleaved IR substrates were able to form a more stable PEC as observed in other ''in vitro'' transposition systems such as those of transposon [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn10-AF162223 Tn''10''] and [[wikipedia:Bacteriophage_Mu|bacteriophage Mu]]. The system was also shown to support strand transfer of a precleaved IR into a supercoiled target plasmid. Integration of both single and to a lower extent concerted integration of two IR was observed ([[:File:Fig.Tn3.16E.png|Fig. Tn3.16 '''Eiii''']]). Initial data have also suggested that TnpA<sub>Tn''4430''</sub> binds preferential to DNA structures which resemble replication forks ''in vitro'' (cited in <nowiki><ref name=":8" />) <nowiki><ref>Oger C. End-synapsis and target integration during replicative transpositon of the Tn3-family transposon Tn4430 [Doctoral dissertation]. Université de Louvain la Neuve; 2018.</ref> and insertion appears to be influenced by replication of the target molecule in vivo (cited in <ref name=":8" />). Some initial evidence was also presented suggesting that the PEC was composed of a pair of IRs and a single TnpA<sub>Tn''4430''</sub> molecule. This has proved to be a misinterpretation of the data. In all other transposition systems, PEC complexes include two (or more) transposase molecules (e.g.<nowiki><ref name=":54" /><nowiki><ref><pubmed>PMC3977044</pubmed></ref><ref><pubmed>PMC7292550</pubmed></ref>). Recent data both from Atomic Force Microscopy (AFM) and Cryoelectron microscopy demonstrates that the TnpATn4430 is indeed a dimer (B. Hallet personal communication; <ref name=":55">Shkumatov AV, Aryanpour N, Oger CA, Goossens G, Hallet BF, Efremov RG. Metamorphism of catalytic domain controls transposition in Tn3 family transposases. BioRxiv. 2022 Feb 24.</ref><ref>Hallet B. Tn4430 AFM.</ref>).

Mechanism in the Light of Structure

A 3.6 Å average resolution cryoelectron microscopy structure has demonstrated that TnpATn4430 is indeed dimeric and has provided some insight into how it might function in transposition <ref name=":55" />. Moreover, using the hyperactive immunity deficient TnpA mutant it was possible to resolve a structure for the PEC which was composed of the transposase [[wikipedia:Protein_dimer|dimer]] and two double strand [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] ends. The structural model permitted a refinement of the TnpA<sub>Tn''4430''</sub> functional modules obtained from partial proteolysis and footprinting ([[:File:Fig.Tn3.16E.png|Fig. Tn3.16 '''Ei''']] and '''[[:File:Fig.Tn3.16E.png|Eii]]'''). Four [[wikipedia:DNA-binding_domain|DNA binding domains]] were identified (DBD1-4; [[:File:Fig.Tn3.16F.png|Fig. Tn3.16 '''F''']] top). DBD1,2 and 4 bind the IR in a sequence-specific manner. The first ([[wikipedia:N-terminus|N-terminal]] proximal) DBD1 establishes both base and phosphate contacts largely with the internal region of the IR previously defined as B2 while DBD2 and DBD4 interactions are located towards the external end of B2 and into A. DBD3 interacts principally with the DNA flank in a non-sequence-specific manner ([[:File:Fig.Tn3.16F.png|Fig. Tn3.16 '''F''']] bottom). There are also phosphate contacts across the IR/flank junction by residues in the catalytic [[wikipedia:Ribonuclease_H|RNH domain]]. When bound, the flank is bent from the IR axis, an observation which was expected from the enhanced [(OP)2-Cu+] cleavage sites in this region. Note the similarities with the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']/[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] transposase organization ([[:File:Fig.Tn3.16D.png|Fig. Tn3.16 '''Di''']]). [[File:Fig.Tn3.16F.png|thumb|640x640px|'''Fig. Tn3.16F.''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] Transposase Functional Modules '''i)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] Transposase organization from <nowiki><ref name=":48" /> ([[:File:Fig.Tn3.16D.png|Fig. Tn3.16D]]); '''ii)''' refined functional module organization. The 10 functional modules are '''DBD1''' and '''DBD2''' separated by an alpha-helical arm domain; a dimerization domain, '''DD'''; '''DBD3''' and '''DBD4''' separated by a linker domain, '''LN'''; the catalytic domain containing the [[wikipedia:Ribonuclease_H|RNaseH fold]], '''RNH''', interrupted by an insertion domain called a scaffold domain, '''SFD'''; and a c-terminal domain, '''CT'''. '''iii)''' a combination of the footprinting data of <nowiki><ref name=":46" />, and structural data from Shkumatov et al 2022. '''SEC''' and '''PEC''' protection against DNAse on both strands (green horizontal lines). Small red dots in flanking DNA indicate additional protection of flanking DNA in the '''PEC'''. [(OP)2-Cu+] DNA enhanced cleavage in the '''PEC''' is indicated by orange triangles and in the '''SEC''' by purple triangles. Phosphate and base contacts are indicated by orange letters in the sequence which are boxed with the appropriate color corresponding to the interacting protein domain. The IR and the DNA flank are boxed in yellow and pale yellow respectively to correspond to the color in the structural figure.|alt=|center]]The apo-protein appears relatively compact ([[:File:Fig.Tn3.16G.png|Fig. Tn3.16 '''Gi''']]). The [[wikipedia:Protein_dimer|dimer]] is held together at the bottom by the '''DD''' domains and at the top by the [[wikipedia:C-terminus|C-terminal]] domain which docks onto the surface of the adjacent monomer. The CT interaction appears to be further stabilized by [[wikipedia:DNA-binding_domain|DNA binding]] ([[:File:Fig.Tn3.16G.png|Fig. Tn3.16 '''Gi''']]). The authors point out that this is an unusual [[wikipedia:Protein_dimer|dimer]] interface. IR binding is accompanied by large conformational change ([[:File:Fig.Tn3.16G.png|Fig. Tn3.16 '''Gi''']]). In this pre-cleavage complex, the protein “arms” align the 4 [[wikipedia:DNA-binding_domain|DBD]] along IR, bend the DNA at Site A ([[:File:Fig.Tn3.16F.png|Fig. Tn3.16 '''Fiii''']]) which moves the flank with respect to the IR tip and places the [[wikipedia:Scissile_bond|scissile phosphate bond]] at the catalytic site of the opposite monomer both '''LN''' and '''RNH''' residues are involved. [[File:Fig.Tn3.16G.png|thumb|640x640px|'''Fig. Tn3.16G.''' A sketch of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] Cryo-em Structure. '''i)''' The dimeric apo-protein (green and purple); '''ii)''' PEC structure. The bound IRs (yellow) and flank (pale yellow) are shown; '''iii)''' 90-degree rotation of '''i)''' (view from the top); '''iv)''' 90 degree rotation of '''ii)''' (view from the '''top'''). The dimerization domain ('''DD'''; '''bottom''') is ringed in red as is the '''CT''' (top) domain. |alt=|center]] Like other transposition systems cleavage appears to be “in trans” (see: [[General Information/Transposase expression and activity#Cleavage in Trans: A Committed Complex|Cleavage in Trans: A Committed Complex]]), a constraint which ensures that the transpososome complex has been assembled before cleavages occur and prevents adventitious initiation of transposition. The two [[wikipedia:Scissile_bond|scissile phosphate bonds]] are correctly positioned to generate the expected 5bp DR. The '''S911''' mutation which leads to hyper transposition and decreased immunity (T<sup>+</sup>/I<sup>-</sup>) would appear to assist the apo-PEC transition, as indeed would the other T<sup>+</sup>/I<sup>-</sup> mutations. Another consequence of the transition is that, while the [[wikipedia:Ribonuclease_H|RNaseH fold]] is poorly defined in the apo-protein, it becomes more easily recognizable in the rearranged PEC. However, in this conformation only '''E881''' ([[:File:Fig.Tn3.16E.png|Fig. Tn3.16 '''Ei''']]) is stably positioned while the other two members of the triad '''D679''' and '''D751''' are mobile. The authors suggest that this is part of a regulatory process, protein metamorphism, and that additional factor(s) are involved in stabilising the catalytic pocket. It seems possibly that this may be regulated by correct docking of the target DNA. Which, they propose, could enter by opening of the '''DD''' interaction domains, a suggestion from studies with a branched DNA substrate ([[:File:Fig.Tn3.16H.png|Fig. Tn3.16 '''Hi''']] and [[:File:Fig.Tn3.16H.png|Fig. Tn3.16 '''Hiii''']]) representing a strand transfer product. The low-resolution structure suggests that the target segment of the branched molecule is located at the base ([[:File:Fig.Tn3.16H.png|Fig. Tn3.16 '''Hii''']]). [[File:Fig.Tn3.16H.png|thumb|640x640px|'''Fig. Tn3.16H.''' Proposed Target Engagement. The figure shows how a branched DNA molecule appears to dock with the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] transposase. '''i)''' and '''iii)''' representation of the branched molecule used in the study: These include the IR (yellow box) attached by a single DNA strand to the double-strand flank (pale yellow box) and by the other strand to a double-strand target (pink/red box). '''ii)''' representation of the cryo-em structure showing the pathway of the branched molecule with the « exit » position of the target and flank. '''iv)''' a reminder of the uncleaved target molecule.|alt=|center]]These are proposed to be the position at which the target ([[:File:Fig.Tn3.16H.png|Fig. Tn3.16 '''Hiv''']]) may dock. This led to a model of stepwise transpososome assembly in which the apo-protein first engages a target molecule which opens a “cavity” between the two protomers and subsequently allows engagement of the IR. ====Tn''3'' Transposition immunity, a poorly understood phenomenon.==== In some of the earliest studies on [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 TnA] ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1'']) <nowiki><ref><pubmed>PMC234940</pubmed></ref> it was observed that transposition into a plasmid already carrying a TnA copy was severely inhibited, a phenomenon known as Transposition Immunity. The effect, identified by transposition of TnA from the E. coli chromosome to plasmid R388 or a derivative already carrying TnA was pronounced (a 105 fold reduction in the immune target).

Two other Tn3 family transposons, Tn501 and Tn1721, also exhibited this inhibition phenomenon (cited as personal communication in <ref name=":56"><pubmed>6278249</pubmed></ref>). However, other studies have identified plasmids having received two copies of TnA but these probably occurred at the same time rather than consecutively <ref><pubmed>672895</pubmed></ref><ref><pubmed>6244100</pubmed></ref>.

Transposition Immunity is a poorly understood phenomenon and some of the early studies gave a number of conflicting results. Immunity has since been observed for bacteriophage Mu and for transposon Tn7 (e.g. <ref><pubmed>6317201</pubmed></ref><ref><pubmed>PMC282071</pubmed></ref><ref><pubmed>2544738</pubmed></ref>) where it involves proteins with ATPase activity, MuB <ref><pubmed>PMC3703974</pubmed></ref><ref><pubmed>PMC1170876</pubmed></ref> and TnsC <ref><pubmed>PMC312388</pubmed></ref><ref><pubmed>26104363</pubmed></ref> respectively. However, Tn3 and its relatives do not encode this type of protein and only a single large transposase with no demonstrated ATPase activity is involved in transposition. It is therefore possible that immunity here is mechanistically distinct from that of both phage Mu and Tn7.

Immunity Requires a Transposon End

Further analyses of TnA <ref name=":56" /> demonstrated that between 290 bp and 470 base pairs at the right end ([[:File:Fig.Tn3.16I.png|Fig. Tn3.16 '''Ii''']]) were sufficient to confer immunity <nowiki><ref name=":56" />. These measurements were made either by accumulation of transposition events in bacteria grown on agar “slopes” or transpositions from the chromosome into a plasmid target in stationary phase cell <nowiki><ref name=":56" />. While plasmids carrying the right end showed immunity, those carrying the left end showed no immunity or only “partial-immunity”. [[File:Fig.Tn3.16I.png|center|thumb|640x640px|'''Fig. Tn3.16I.''' Immunity determinant in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000'']. '''i)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] Map ('''top''') showing the region involved in immunity as a red dotted line and ('''bottom''') IRR sequence. Differences with the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] IRL are shown in red <nowiki><ref name=":56" />. '''ii)''' Tn''3'' IRR-IRL junction and deletion derivatives showing the level of immunity ('''right column''') <nowiki><ref name=":58" />. Differences with [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] IR are shown in red.|alt=]]Unfortunately, the quantitative effects are not clear from this publication. However, the conclusions are generally supported by another study which uses a different assay system involving a temperature sensitive replication mutant of plasmid pSC101 carrying a [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] derivative in which ''tnpR'' was inactivated by linker insertion. In this system <nowiki><ref name=":51" /><nowiki><ref name=":64"><pubmed>6271635</pubmed></ref>, cointegrates are not resolved and were isolated by “rescue” of the temperature sensitive donor plasmid by a coresident target plasmid following a shift to high temperature <ref name=":57"><pubmed>PMC390066</pubmed></ref>. Here, plasmids carrying restriction fragments containing one or other Tn3 ends conferred immunity; inclusion of both ends did not enhance immunity; and immunity was observed regardless of the orientation of the 38 bp IR end. Intriguingly, the distribution of insertions into immune and non-immune targets appeared to be different <ref name=":57" />. However, the study also indicated in some cases that the orientation of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] DNA fragment in the target affected the immunity level. This could be the result of directional processes such as replication or transcription through the region. Furthermore, it was observed that deletions within the IRs which eliminated transposition, also eliminated immunity ([[:File:Fig.Tn3.16I.png|Fig. Tn3.16 '''Iii''']]) <nowiki><ref name=":58"><pubmed>3009272</pubmed></ref>. However, studies comparing TnpATn3 binding and immunity <ref name=":53" /> suggested that some mutants which do not affect transposase binding capacity do impact on transposition immunity. Moreover, a study which implicated TnpR<sub>Tn''3''</sub> in immunity <nowiki><ref><pubmed>6278250</pubmed></ref> was not supported by subsequent studies <ref name=":58" />. A finer scale analysis of the extent of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] IR sequence required for immunity was obtained by sequential deletion analysis of one IR <nowiki><ref name=":44" /> ([[:File:Fig.Tn3.16Iiii.png|Fig. Tn3.16 '''Iiii''']]). While a number of the deletions resulted in retention of certain internal IR nucleotides, a clear pattern is that the distal end of the IR segment rather than the tip of the IR is important (sequences in Box B; Fig. Tn3.16Ci). This is also largely in agreement with the results from Huang et al.<nowiki><ref name=":58" />. [[File:Fig.Tn3.16Iiii.png|center|thumb|640x640px|'''Fig. Tn3.16Iiii.''' Immunity determinant in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000'']. '''iii)''' Effect of [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF]] on the immunity of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000'']. The left column shows the relative transposition immunity levels in wild-type and [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF]]-cells <nowiki><ref name=":44" /> . '''iv)''' Effect of [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF]] on immunity of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000'']. The left column shows the relative transposition immunity levels in wild type and [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF]]-cells<nowiki><ref name=":44" />.|alt=]] Interestingly, Bishop and [https://www.bioch.ox.ac.uk/article/2021-genetics-society-medal-for-professor-david-sherratt Sherratt] <nowiki><ref name=":52" />, using a plasmid system which allows identification of both inter- and intra-molecular [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1-NC_008357 Tn''1''] transposition Inversions and deletions were found to occur at frequencies similar to insertion suggesting that insertion into its own vector plasmid is not significantly subject to immunity. However, when [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] sequences, such as those present in pBR322, were also present in the transposon donor plasmid, inversions and deletions occurred at significantly lower frequencies. For [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''], it was observed that 200 base pairs of the IRL (Gamma end) or 400 base pairs of the IRR (delta end) showed immunity to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] insertion <nowiki><ref name=":59"><pubmed>PMC213758</pubmed></ref> while no other segment of Tn1000 conferred immunity. This was further refined to the terminal 38-base-pairs of IRR which were sufficient to confer immunity, whereas the 38-bp sequence of IRL conferred only moderate immunity (note that we use the standard nomenclature for IRL and IRR: viz IRR is defined as the IR towards which the transposase is expressed. This is the opposite of the nomenclature originally used for Tn1000). The IR sequence of both ends is identical for the first 35 base pairs and it was observed that this common sequence alone was not able to confer immunity <ref name=":59" />. Like [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4652-AF151431.1 Tn''4652''] ([[:File:Fig.Tn3.12G.PNG|Fig. Tn3.12 '''G''']]) <nowiki><ref name=":50" /><nowiki><ref name=":27" /> in which [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF binding]] to sites located close to the ends positively regulates TnpA binding <nowiki><ref name=":27" /> to the terminal IRs, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] also carries IHF sites proximal to the IRs. A more detailed analysis of the related [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] IRR <nowiki><ref name=":44" /><nowiki><ref><pubmed>PMC213151</pubmed></ref> using a mating-out assay <ref><pubmed>6281440</pubmed></ref> to measure transposition frequencies, showed that while the 38 base pair end was capable of conferring immunity on a target replicon, the neighboring IHF site (which is not present in TnA/Tn1,Tn2,Tn3) conferred a significantly higher level of immunity in the presence of IHF (Fig. Tn3.16Iiv) while removal of the terminal 2 GC base pairs at the tip had no real effect. IHF has been shown to bind cooperatively with TnpATn1000 <ref name=":45" />. This result strongly suggested that it is the [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF-enhanced binding]] strength TnpA<sub>Tn''1000''</sub> which determines the level of immunity <nowiki><ref name=":44" />. The available data is relatively old and restricted by the experimental approaches available at that time. Since every assay system is different, it is not possible to directly compare results. However, in spite of the apparently conflicting detailed data, it appears likely that TnpA<sub>Tn''3''</sub> and TnpA<sub>Tn''1000''</sub> binding to an IR in the immune target is necessary for immunity. =====Immunity in Tn''4430''===== More recent studies on immunity of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] <nowiki><ref><pubmed>PMC2916420</pubmed></ref> have involved isolation of TnpATn4430 mutants which escape immunity <ref name=":48" />. The mutants were screened for both transposition and loss of immunity (T<sup>+</sup>/I<sup>-</sup>) using a papillation test. Surprisingly, these were not localized to a specific region of the protein but occurred over its entire length ([[:File:Fig.Tn3.16E.png|Fig. Tn3.16 '''Ei''']]). The frequency of transposition into the permissive (non-immune) target of most mutants was similar to that of wild-type TnpA<sub>Tn''4430''</sub>. However, immune-deficient mutations in the [[wikipedia:N-terminus|N-terminal]] region appeared to have a slightly increased transposition frequency whereas those clustering in the C-terminus exhibited a slightly decreased transposition frequency. Based on the cryo-em structure, these T<sup>+</sup>/I<sup>-</sup> mutants are expected to positively affect the apo-PEC transition <nowiki><ref name=":55" />. Although some data suggested that immunity could be observed in a relatively crude cell-free system <nowiki><ref><pubmed>9077463</pubmed></ref>, the establishment of a more defined and robust in vitro transposition system <ref name=":46" /> might permit further experimental investigation into the molecular basis of Tn''3'' family transposition immunity. ====On Ended Transposition.==== Early in the study of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''], it was observed that, In the presence of the cognate transposase, plasmids containing a single inverted repeat (IR) can fuse efficiently with other plasmids <nowiki><ref name=":98"><pubmed>6092854</pubmed></ref><ref name=":99"><pubmed>6092853</pubmed></ref> in a reaction that requires neither the resolution system nor a functional host recA gene.

Insertion occurred at different sites in the target plasmid and the products contained a complete copy of the IR-carrying donor plasmid often with a duplication of various lengths of donor DNA. The sequence across the junction showed that the segment of donor DNA started precisely at the IR at one end, was variable at the other and the insertion was generally flanked by a 5bp DR generated in the target plasmid <ref name=":100"><pubmed>PMC341238</pubmed></ref>. Some recombinants were observed to contain only short segments of the donor plasmid <ref name=":101"><pubmed>PMC210914</pubmed></ref>.

Models involving asymmetric (rolling circle or processive) replicative transposition or simple insertion have been proposed for this type of transposition and it seems possible that this in some way results from insertion into an extant replication fork in the target DNA.

Resolution

The serine recombinases.

Efficient resolvase-catalyzed recombination between two directly repeated res sites is instrumental in completing transposition by physically separating donor and target molecules. This was first recognized in studies on complementation of transposition deficient Tn1 and Tn3 mutants where mutation of tnpR resulted in accumulation of cointegrates <ref name=":2" /><nowiki><ref name=":7" /><nowiki><ref name=":64" /><nowiki><ref><pubmed>392228</pubmed></ref> (Fig. Tn3.2 ii). It therefore showed that TnpR functions not only as a repressor of TnpA and TnpR expression by binding to the res site and blocking the promoters <ref name=":65"><pubmed>6313485</pubmed></ref> for both genes (see Fig. Tn3.17 Ci), but that it has an active function in the transposition process.

A number of resolvase enzymes have since been recognized (for a comprehensive review see <ref name=":8" />([[:File:Fig.Tn3.17A.png|Fig. Tn3.17 '''Ai-iv''']]). [[File:Fig.Tn3.17A.png|thumb|640x640px|'''Fig.Tn3.17A.''' Tn''3'' family ''res'' sites. Transposons are shown as pale yellow boxes ending in arrowheads. The transposon length in base pairs is indicated. Terminal inverted repeats (IRs) are indicated by gray arrowheads (IRL and IRR, respectively, labeled by convention with respect to the direction of ''tnpA'' transcription from left to right). Recombination sites (''res'', irs, and rst) are shown in green, transposition genes in purple, passenger genes in red (antibiotic resistance genes), orange-yellow (heavy metal resistance genes), and bright yellow (plant pathogenicity genes). '''(i)''' Tn''3''. Accession number [https://www.ncbi.nlm.nih.gov/nuccore/V00613 V00613] ([[Transposons families/Tn3 family#The Tn3 Clade|Tn''3'' clade]]). Carries the [[wikipedia:Beta-lactamase|''bla''TEM-1a beta-lactamase]] gene and divergent serine recombinase/resolvase (''tnpR'') and transposase (''tnpA'') genes. The recombination site, ''res'', composed of three subsequences, I, II, and III, is located between ''tnpR'' and ''tnpA'', with site III proximal to ''tnpR''. Recombination occurs within site I. '''(ii)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501'']. Accession number [https://www.ncbi.nlm.nih.gov/nuccore/Z00027 Z00027] ([[Transposons families/Tn3 family#The Tn21 Clade|Tn''21'' clade]]). Carries an operon containing [http://parts.igem.org/Part:BBa_K1420000 mercury resistance genes] (''mer'') and colinear serine recombinase/resolvase (''tnpR'') and transposase (''tnpA'') genes. The res site is located upstream of ''tnpR''. It has a similar organization as that of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] with site III proximal to ''tnpR''. Recombination occurs within site I. '''(iii)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430'']. Accession number [https://www.ncbi.nlm.nih.gov/nuccore/X07651.1 X07651.1] ([[Transposons families/Tn3 family#The Tn4430 Clade|Tn''4430'' clade]]). Carries no known passenger genes. Tyrosine recombinase/resolvase (''tnpI'') and transposase (''tnpA'') genes are colinear, and the recombination site, ''irs'', is located upstream of and proximal to the resolvase gene with four subsites: inverted repeats IR1 and IR2 and direct repeats DR1 and DR2. Recombination occurs at the recombination core site IR1-IR2. '''(iv)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXax1-AE008925 Tn''Xax1'']. Accession number [https://www.ncbi.nlm.nih.gov/nuccore/AE008925 AE008925] ([[Transposons families/Tn3 family#The Tn4651 Clade|Tn''4651'' clade]]). Carries two passenger genes involved in plant pathogenicity located at the left (''xopE'') and right (''mlt'') ends of the transposon. The resolvase has two components: a tyrosine recombinase (''tnpT'') and a helper protein (''tnpS'') expressed divergently. The ''res'' site, ''rst'', is located between ''tnpT'' and ''tnpS'' and is composed of two pairs of inverted repeats, IR1 and IR2 and IRa and IRb. Recombination occurs at the IR1-IR2 inverted repeat. From <nowiki><ref name=":30" />.|alt=|center]] The majority so far identified appear to be recombinases which use a serine residue as the nucleophile during recombination ([[:File:Fig.Tn3.17A.png|Fig. Tn3.17 '''Ai''']] and ii). These [[wikipedia:Site-specific_recombination|serine recombinases]] can be divided into two major groups (Fig. Tn3. 17A): the “classical” recombinases, TnpR encoded by [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] and their relatives (~185 aa); and “long” [[wikipedia:Site-specific_recombination|serine recombinases]] <nowiki><ref name=":8" /><nowiki><ref name=":91"><pubmed>11994145</pubmed></ref> (~300aa) (Fig. Tn3.17 B) (see <ref name=":8" /><nowiki><ref><pubmed>PMC3775659</pubmed></ref><ref name=":92"><pubmed>PMC4384473</pubmed></ref>. In both types, the catalytic center is located at the N-terminal end in a large catalytic domain which is followed by a smaller helix-turn-helix DNA binding domain. In the case of the “long” recombinases, there is a C-terminal extension compared to the “classic” resolvases. These fall largely within a small subclade in the Tn3 subgroup which includes Tn5044, the Xanthomonas transposons TnXc4 and TnXc5 and Tn1412 (Fig. Tn3.4A). It is worth noting that all members of this Tn group also encode a toxin/antitoxin system located between the divergent tnpA and tnpR genes (Fig. Tn3.4).

Fig. Tn3.17B. Organisation of Serine resolvases. The short (top) and long (bottom) serine resolvases are shown. Both contain the catalytic site near their N-terminal ends. The active site serine is indicated in red. The catalytic domain is followed by a short helix which acts as an oligomerization helix and a helix-turn-helix DNA binding domain. In the case of the long serine resolvase derivatives, a C-terminal extension of approximately 100 amino acids is present <ref name=":8" /><nowiki><ref><pubmed>26104558</pubmed></ref>.
Studies with Tn1000 (γδ) and Tn3 res.

Early studies using the resolvase of Tn1000 (aka γδ) in vitro demonstrated that the enzyme could introduce double strand breaks in a res site and, in the absence of the divalent cation Mg2+, formed covalent TnpR-DNA intermediates <ref name=":66"><pubmed>6269756</pubmed></ref>.

Cleavage occurred at a crossover point in a palindromic sequence to generate a cleavage product with a free 3’OH group and a 2 base 3’ overhang <ref name=":66" /> ([[:File:Fig.Tn3.17C.png|Fig. Tn3.17 '''Ci''']]). Furthermore, formation of a free 3’OH implied that the covalent protein-DNA linkage occurred at the 5’ end and was cleavage more efficient if the substrate carried 2 directly repeated ''res'' copies. This led to the hypothesis that although TnpR acts as a repressor at ''res'', binding simultaneously to two ''res'' copies in some way changes the protein conformation allowing recombination to proceed <nowiki><ref name=":66" />. It was further shown using DNase and footprinting that ''res''<sub>Tn''3''</sub> and ''res''<sub>Tn''1000''</sub> carry three TnpR binding sites <nowiki><ref name=":67"><pubmed>6290077</pubmed></ref>, I, II and III (where sites II and III, known as accessory sites, are closely spaced and site I known as the core site, is very slightly distanced) (Fig. Tn3.17 Ci) <ref name=":67" /> and that the recombination point (the dinucleotide '''AT''') <nowiki><ref name=":68"><pubmed>PMC319581</pubmed></ref> is included within site I. Each site shows some degree of two-fold symmetry <ref name=":67" /><nowiki><ref name=":69"><pubmed>PMC553467</pubmed></ref> (Fig. Tn3.17 Ci). The resTn3 has an identical organization <ref name=":70"><pubmed>PMC555234</pubmed></ref> and almost identical sequence to that of Tn1000 and the Tn3 and Tn1000 TnpR products are interchangeable <ref name=":70" />. These similarities were exploited to determine the crossover point using [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] TnpR-mediated resolution between ''res''<sub>Tn''3''</sub> and ''res''<sub>Tn''1000''</sub> carried by a single plasmid <nowiki><ref name=":68" />. [[File:Fig.Tn3.17C.png|center|thumb|640x640px|'''Fig. Tn3.17C.''' The Sequence and organization of ''res'' sites used by short serine resolvases. Short sequences required for resolution of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] ('''i''') and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''] ('''ii''') are shown. The regions of resolvase binding defined by footprinting are shown as horizontal green lines. Sites I (core site) and sites II and III (accessory sites) are indicated and the generally accepted functional limits are boxed in blue were determined. The conserved inverted repeats defining “half-sites” are shown as horizontal blue arrows for [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000'']. -35 and -10 promoter elements are shown in red. And the direction of expression of the flanking genes is indicated and represented by thick horizontal blue arrows. The TA dinucleotide at which recombination occurs is shown in red and the cleavage is indicated at the bottom of the panel ('''i'''). Data for [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] <nowiki><ref name=":65" /><nowiki><ref name=":70" /><nowiki><ref name=":71" /><nowiki><ref name=":67" />; for [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721'']<nowiki><ref name=":97"><pubmed>2998784</pubmed></ref>.|alt=]]

Tn3 res, tnpR and tnpA gene expression.

In both Tn3 and Tn1000, tnpA and tnpR are divergent and the res site is located in the intergenic space with subsite III proximal to tnpR (Fig. Tn3.17 Ci). Promoters for both tnpA and tnpR, were located by footprinting of RNA polymerase and lie within res <ref name=":65" /><nowiki><ref><pubmed>6094833</pubmed></ref> (Fig. Tn3.17 Ci). The -35 promoter elements of both gene are only 10 bp distant from each other and the -10 element of tnpA is located within site I straddling the point of recombination crossover (Fig. Tn3.17 Ci). The transcription start point for both genes has been mapped. Clearly, tnpA and tnpR expression would be regulated by TnpR binding.

Variant res sites with this configuration have been observed in which the center of sites I and II are separated by 4, 5, 6 and seven helical turns (see <ref name=":8" />). =====The Mechanics of Resolution.===== TnpR binding to ''res'' generates a highly compact protein-DNA complex as judged by [[wikipedia:Electron_microscope|electron microscopy]] <nowiki><ref><pubmed>PMC457183</pubmed></ref>. This was explained by the observation that TnpR binding to res-containing linear DNA fragments results in significant bending of the DNA although it was noted that the complex contains a single DNA molecule under the conditions use rather than two res sites <ref name=":71"><pubmed>2850169</pubmed></ref>.

Gentle proteolysis of purified Tn1000 TnpR was observed to generate two fragments: a large N-terminal fragment which includes the catalytic center and a smaller C-terminal fragment which binds to each of the three res sites <ref name=":72"><pubmed>PMC345424</pubmed></ref>(Fig. Tn3.17 B). Unlike full length TnpR which binds the res sub-sites with equal affinity, the C-terminal fragment binds to each of the half-sites but with different, weaker, affinities suggesting that the N-terminal part of TnpR is involved in protein-protein interactions within the TnpR-res complex <ref name=":72" />. Footprinting of small fragment binding indicated that the protection was centered on the 9bp half-sites ([[:File:Fig.Tn3.17C.png|Fig. Tn3.17 '''Ci''']]). Further studies using saturated mutagenesis of a halfsite from subsite I and chemical probing identified how the protein contacts DNA in both the major and minor grooves <nowiki><ref><pubmed>PMC551726</pubmed></ref>.

A model of the overall architecture of single TnpR-res complexes was proposed <ref name=":71" /> based on results using a number of footprinting agents to reveal sensitive sites on the DNA and permutation experiments to identify DNA curvature <nowiki><ref name=":81"><pubmed>PMC306530</pubmed></ref> in which each subsite binds a TnpR dimer (with one monomer recognizing each partial diad symmetry element called “half-sites”) <ref name=":67" /><nowiki><ref name=":69" /> and introduces an “intra-site” bend in the DNA at each site while at another level, protein-protein interactions introduce inter-site bends ([[:File:Fig.Tn3.17D.png|Fig. Tn3.17 '''D''']]). Experimentally, this conformation requires all 3 sites and a correct spacing between sites I and II. [[File:Fig.Tn3.17D.png|thumb|640x640px|'''Fig. Tn3.17D.''' Model for the structure of a single resolvase-bound ''res'' site. The larger N-terminal Catalytic domain has been eliminated for clarity. The DNA binding domain is indicated as blue circle. The DNA is shown as black parallel lines. The resolvase-induced bends Predicted by circular permutation experiments are indicated, together with the large kink at the recombination site I. Half sites containing individual repeated sequences are shown as orange arrows.|alt=|center]]''In vitro'' resolution systems have been developed and require supercoiled DNA (see <nowiki><ref><pubmed>7821562</pubmed></ref> together with a divalent cation, Mg2+ <ref name=":61" /><nowiki><ref name=":66" /><nowiki><ref name=":70" /><nowiki><ref name=":73"><pubmed>6301692</pubmed></ref> although later studies showed that neither the Tn3/Tn1000 <ref name=":61" /> nor [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] resolvases show an absolute requirement for Mg<sup>2+</sup> ions <nowiki><ref name=":108"><pubmed>3020509</pubmed></ref>. However, in their absence and in low ionic strength, the reaction can be very slow but high activity can be restored by increasing Na+ concentration or adding multivalent amines such as spermidine <ref name=":61" />. It has been suggested that Mg<sup>2+</sup> ions may enhance resolvase activity, but are not directly involved in the catalytic process and probably affect recombination by altering the DNA conformation <nowiki><ref name=":108" />. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] resolvase relaxes its DNA substrate even in the absence of Mg2+, and also in ionic conditions that inhibit recombination. A number of laboratories have contributed to an understanding of how the complex [[wikipedia:Site-specific_recombination|site-specific resolution recombination]] reaction takes place. These studies have used extremely clever techniques to understand the mechanics of this process including topology, mutagenesis and structural biology. ''In vitro'' resolution requires a supercoiled DNA substrate carrying two directly repeated ''res'' sites and results in a simple concatenated recombination product with a specific change in linking number (the number of time one DNA strand crosses another) ([[:File:Fig.Tn3.17E.png|Fig. Tn3.17 '''E''']]) <nowiki><ref name=":61" /><nowiki><ref name=":66" /><nowiki><ref name=":70" /><nowiki><ref name=":73" /> indicating that the synaptic complex must have a very precise type of protein-DNA architecture. The ''in vitro'' reaction is very inefficient when the ''res'' sites are in an inverted orientation raising the question of how the two ''res'' sites are aligned for recombination (for review see <nowiki><ref name=":60" />). Random collision between ''res'' sites on a supercoiled molecule was ruled out since this would generate a complex concatenated product with a variable number of supercoils trapped between the recombined product ([[:File:Fig.Tn3.17E.png|Fig. Tn3.17 '''Eii''']]). Alignment of the two ''res'' sites was first proposed to occur when TnpR recognizes one site and tracks along the DNA molecule until encountering the second site. However, present evidence, in particular the observation that ''res'' site recombination can occur intermolecularly, suggests that this is not the case. In particular the observation that ''res'' site recombination can occur intermolecularly. A second hypothesis was that ''res'' sites meet via “slithering” i.e. continuous one-dimensional diffusion of supercoils in plectonemically ([[:File:Fig.Tn3.17E.png|Fig. Tn3.17 '''Eii''']]) wound DNA molecules (for review see <nowiki><ref name=":60" />).[[File:Fig.Tn3.17E.png|thumb|640x640px|'''Fig. Tn3.17E.''' The serine recombinase resolution mechanism. '''A)''' The main product of the resolution reaction. The [[wikipedia:Cointegrate|cointegrate]] ('''left''') gives rise to a recombination product, a linked catenane. The entire res site is shown as a single filled arrow and the [[wikipedia:Cointegrate|cointegrate]] as a circle with two replicons in blue and orange. Recombination results in two interlinked molecules. '''B)''' Recombination following random collision of two ''res'' sites. This would trap a variable number of negative supercoils depending on the position of the two ''res'' sites relative to the supercoils. '''C)''' In the two ''res'' sites, shown as three filled arrows ('''Ci''') are synapsed to bring the crossover sites I into apposition ('''Cii''') while trapping three supercoils. Resolvase tetramers at each site are shown as filled circles. Strand exchange between both sites I ('''Ciii''') gives rise to the simple catenane ('''Civ''') with two crossovers.|alt=|center]]Intensive studies using both [[wikipedia:Gel_electrophoresis|gel electrophoresis]] and [[wikipedia:Electron_microscope|electron microscopy]] to visualize TnpR recombination activities <nowiki><ref><pubmed>PMC397197</pubmed></ref><ref><pubmed>2990045</pubmed></ref> led to a model in which the two res sites Fig. Tn3.17 Eiii) sites are constrained in a configuration which entraps 3 supercoils (Fig. Tn3.17 Eiiib) and which takes into account the observation that Tn3 resolution (Fig. Tn3.17 Eiiic) removes four negative supercoils on recombination (Fig. Tn3.17 Eiiid) <ref name=":61" />. The resulting energy change probably drives the reaction server an "architectural" function allowing the recombining site to finalize the recombination event (Fig. Tn3.17F). This occurs by simple rotation at site I <nowiki><ref name=":60" /> on the flat hydrophobic surface between subunits in the resolvase tetramer after simultaneous cleavage of all four strands in the synaptic complex ([[:File:Fig.Tn3.17F.png|Fig. Tn3.17 '''F''']] and [[:File:Fig.Tn3.17G.png|Fig. Tn3.17 '''G''']]). The TnpR monomers remain attached to the 5’ ends ([[:File:Fig.Tn3.17F.png|Fig. Tn3.17 '''F''']] left) and the serine-DNA bond is then broken by attack by the 3’OH of the recombining site ([[:File:Fig.Tn3.17F.png|Fig. Tn3.17 '''F''']] right) to complete recombination. More than a single round of recombination can occur and this results in the generation of knots of increasing complexity with increasing numbers of recombination events (not shown) <nowiki><ref><pubmed>1655422</pubmed><br /></ref>.

Fig. Tn3.17F. Mechanism of cointegrate resolution by Serine-recombinases by rotational strand exchange (Tn3 and Tn1000). The two res sites at which strand crossover occurs, sites I, are shown as filled arrows interrupted at the point of recombination by the TA dinucleotide (in red) at which cleavage occurs. The repeated sequences to which resolvase binds are shown as blue arrows within the res sites. The recombination sites are aligned in parallel and their strand polarities are shown. The colors are as for those in Fig. Tn3.17E. The resolvase tetramer formed of two dimers is shown in dark and light green, in which the monomer DNA binding (B) and catalytic (C) domains are separated by a linker. The 5’ phospho-serine bond at the recombination point is indicated by a small blue circle and the free 3’OH as a thin blue arrowhead. The resolvase dimers at the left and right part of the site I interact through a flat hydrophobic surface. Note that all 4 strands are interrupted simultaneously. Left: In the pre-strand exchange complex, the catalytic domains are shown on the inside of the synapse and the DNA-binding domain on the outside. Right: Strand exchange occurs by a 180° rotation of one partner dimer pair with respect to the other along the flat hydrophobic tetramer interaction surface. Rejoining occurs when the free 3’OH attack the 5’ phospho-serine bond.

This model is supported by the structure of a TnpR tetramer bound to two site I DNA molecules in a synaptic complex <ref><pubmed>15994378</pubmed></ref><ref name=":114"><pubmed>PMC1483221</pubmed></ref> which shows that each TnpR dimer bound to its DNA presents an unusual flat, hydrophobic surface to the other member of the pair (Fig. Tn3.17 G) with the suggestion that strand exchange indeed occurs by rotation around this interface.

Fig. Tn3.17G. Structure of the cleaved Site I synaptic complex: The figure shows the crystallographic structure of the Tn1000-resolvase tetramer covalently linked to cleaved DNA. This corresponds to the left panel in Fig. Tn3.17F. The two site I DNAs are shown in magenta and gold and blue and green. Each is bent at the point of cleavage. The resolvase tetramer is shown in grey (top) and brown (bottom) intersected by the dimer interface. The DNA binding and catalytic domains are indicated. The resolvase DNA binding domains are shown as small helix-turn-helix structures tucked into the major groove. 5’ phospho-serine bonds are shown at serine 10 – top green, bottom red. The flat hydrophobic interface at while strand exchange will occur by rotation is also indicated. This figure was taken from the PDB file: 1ZR2.
The Tn3 synaptosome

An understanding of the structure of the Tn1000/Tn3 synaptosome is important to validate the proposed resolution reaction pathway (Fig. Tn3.17 E). Visualization of the entire synaptosome has proved challenging due to its complexity: with two res sites each composed of three TnpR binding sites (the catalytic site I and the “architectural” sites II and III) and a total of six TnpR dimers. The first synaptosome structure obtained was that of Sin, which has simpler organization with only two TnpR binding sites <ref name=":91" /><nowiki><ref name=":95"><pubmed>PMC2428073</pubmed></ref><ref><pubmed>33405333</pubmed></ref>. A long awaited Tn3 synaptosome complex has now been described at the structural level <ref name=":107">Montaño SP, Rowland S-J, Fuller JR, Burke ME, MacDonald AI, Boocock MR, Stark WM, Rice PA. Structural basis for topological regulation of Tn3 resolvase. BioRxiv. 2021 Dec 8.</ref> (Fig. Tn3.17 H) and provides convincing evidence for the previous models (Fig. Tn3.17 Eiii).[[File:Fig.Tn3.17H.png|thumb|640x640px|Fig. Tn3.17H. The Tn3 Synaptosome. i) The Tn3 res site (see Fig. Tn3.17 Ciii). Arrangement of TnpR dimers at sites II and III and the dimer of dimers between the two res sites at site I. Double headed arrows indicate monomer/monomer interactions in the TnpR dimers and dimer/dimer interactions between the two res sites at site I <ref name=":107" />. '''iii)''' The synaptosome structure showing both DNA and TnpR molecules, from which the cartoon was drawn. Red bonds indicate hypersensitivity to Dnase in footprinting.|alt=|center]] =====The Tn''1721'', Tn''21'' and Tn''501'' ''res''.===== In contrast to those of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''], the ''tnpA'' and ''tnpR'' genes of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] are transcribed in the same orientation, with ''tnpR'' upstream of ''tnpA'' and their ''res'' sites located upstream of ''tnpR'' ([[:File:Fig.Tn3.17A.png|Fig. Tn3.17 '''Aii''']] and [[:File:Fig.Tn3.17C.png|Fig. Tn3.17 '''Cii''']]). They are relatively well conserved within the Tn''21'' clade ([[:File:Fig.Tn3.7F.PNG|Fig. Tn3.7 '''F''']]). Early experiments with [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] showed that it too underwent transposition using a [[wikipedia:Cointegrate|cointegrate]] intermediate <nowiki><ref name=":74"><pubmed>PMC345658</pubmed></ref>.

Like resTn3 and resTn1000, resTn1721 and resTn21 are composed of three TnpR binding sites (I, II and III) as determined by footprinting <ref name=":70" /> <nowiki><ref name=":97" /> with site III proximal to ''tnpR'' ([[:File:Fig.Tn3.17A.png|Fig. Tn3.17 '''Aii''']] and [[:File:Fig.Tn3.17C.png|Fig. Tn3.17 '''Cii''']]) and each site exhibits some degree of dyad symmetry. Moreover, there is considerable identity observed among the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''] ''tnpR'' genes and also the ''res''<sub>Tn''21''</sub> '', res''<sub>Tn''501''</sub> and ''res''<sub>Tn''1721''</sub> sites <nowiki><ref name=":62" />. All three elements complement a ''tnpR'' mutant of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] whereas [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] does not <nowiki><ref name=":62" />. This is perhaps not surprising since the ''res''<sub>Tn''3''</sub> sequence appeared to be quite different from those of this Tn group ([[:File:Fig.Tn3.17C.png|Fig. Tn3.17 '''C''']]) and the authors were unable to identify a ''res'' site homologous to that of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']. In addition, the TnpR amino acid sequence of Tn''3'' is somewhat distant from those of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721'']. These observations were reinforced by additional studies demonstrating that purified [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1721-X61367.1 Tn''1721''] TnpR can resolve [[wikipedia:Cointegrate|cointegrate]] substrates containing repeat copies of ''res''<sub>Tn''1721''</sub>, of ''res''<sub>Tn21</sub>, and of a substrate carrying both ''res''<sub>Tn21</sub> and ''res''<sub>Tn''1721''</sub> copies, but not of ''res''<sub>Tn''3''</sub> <nowiki><ref><pubmed>6318048</pubmed></ref> while Tn21 TnpR catalyzed site-specific recombination between directly repeated resTn21 and resTn1721 but not resTn3 <ref><pubmed>2993810</pubmed></ref>. The reaction required a supercoiled substrate with two directly oriented res sites.

Several studies explored the DNA sequence binding and recombination specificities between Tn3 and Tn21 using hybrid TnpR containing the DNA binding domain of one and the catalytic domain of the other <ref><pubmed>PMC310539</pubmed></ref><ref name=":75"><pubmed>2175363</pubmed></ref><ref><pubmed>2175362</pubmed></ref>. These studies showed that, while a Tn21 TnpR catalytic DNA domain spliced to the Tn3 DNA binding domain has a somewhat lower affinity for resTn21, it retained some ability to mediate recombination between resTn21 but was unable to recombine resTn3 sites in spite of the fact that the hybrid protein was able to bind resTn3. This led to the conclusion that although

“alterations in amino acid sequence of resolvase within the helix-turn-helix DNA binding domain modulate the affinity of the protein for its DNA target sequence, the specificity of resolvase for recombination at its cognate res sites is determined by the resultant organization of the DNA-protein complex” <ref name=":75" />.</blockquote> =====Tn ''res'' activity ''tnpR'' and ''tnpA'' gene expression.===== It was proposed <nowiki><ref name=":62" /> that in all three elements, ''tnpA'' may be transcribed independently of ''tnpR'' and that its promoter is located within ''tnpR''. Moreover, no [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] ''tnpR'' promoter could be found ''in vitro''. This is consistent with the observation that in interreplicon [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''] transposition into plasmid R388, resolution could be induced in the recipient by mercury selection <nowiki><ref name=":74" /> suggesting that ''tnpR'' may be expressed at least partially as part of the [http://parts.igem.org/Part:BBa_K1420000 mercury resistance operon] located upstream of ''tnpR''. Interestingly, a study using [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] revealed a gene, ''[https://www.uniprot.org/uniprot/P04162 tnpM]'' (for modulator), whose expression appeared to enhance transposition and suppress resolution <nowiki><ref name=":63" />. [https://www.uniprot.org/uniprot/P04162 TnpM] results from the insertion of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn402-U67194.4 Tn''402''] derivative, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5060-AJ551280.1 Tn''5060''] which led to the formation of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] ([[:File:Fig.Tn3.7G.PNG|Fig. Tn3.7 '''G''']]). This event interrupted the ''urfM'' gene, of unknown function but possibly part of the [http://parts.igem.org/Part:BBa_K1420000 mercury operon], generating the [[wikipedia:C-terminus|C-terminal]] fragment with a fortuitous translation initiation codon. Removal of the region in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''] resulted in a reduced transposition frequency and increased resolution activity and these activities were restored when the ''[https://www.uniprot.org/uniprot/P04162 tnpM]'' “gene” cloned into another compatible plasmid was provided ''in trans''. Moreover, transposition of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn501-Z00027 Tn''501''], which like [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn21-AF071413 Tn''21''], also includes a complete ''ufrM'' gene, was also affected. The mechanism by which the UfrM fragment, [https://www.uniprot.org/uniprot/P04162 TnpM], functions is unclear and has not been addressed since its initial description <nowiki><ref name=":63" />. =====The long serine recombinases===== TnpR proteins carrying an extended C-terminus (TnpR<sub>L</sub>) ([[:File:Fig.Tn3.17B.png|Fig. Tn3.17 '''B''']]) have been studied in only a single case, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISXca5 Tn''Xca5''] ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISXca5 IS''Xca5'']) from [[wikipedia:Xanthomonas|''Xanthomonas campestris pv. citri'' XAS450]] <nowiki><ref><pubmed>1327971</pubmed></ref>. Establishment of an in vitro system <ref name=":82"><pubmed>9663657</pubmed></ref> has shown that, as for the short forms of TnpR, recombination requires two directly repeated resTnXc5 copies in a supercoiled plasmid substrate and Mg2+ as a divalent cation (although the divalent cation presumably serves the same purpose as it does in the Tn3/Tn1000 and Tn21 systems viz to alter DNA conformation rather than actively participate as a co-factor <ref name=":61" /><nowiki><ref name=":108" />). Footprinting revealed three TnpR-binding subsites <nowiki><ref name=":82" />. The centres of subsite I and II are separated by seven helical turns (74 bp), similar to the ''res'' sites of Tn''917'' and Tn''522''. This is longer than those of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']/[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] (53 bp) although all share the same overall site configuration ([[:File:Fig.Tn3.17I.png|Fig. Tn3.17 '''I''']]'''i'''). It was also determined that at least six TnpR<sub>Tn''Xc5''</sub> monomers are required for recombination and are presumably composed of three dimers each binding to a ''res'' subsite. Moreover, the synaptosome formed between directly repeated Tn''Xc5'' ''res'' sites must be similar in overall architecture to the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] synaptosome since both trap three supercoils. There are significant amino acid sequence similarities between the serine resolvases and another type of serine recombinase, the invertases, such as [https://www.uniprot.org/uniprot/P03015 Gin] (involved in an inversion switch determining [[wikipedia:Bacteriophage_Mu|phage Mu tail fibers]] e.g. <nowiki><ref><pubmed>6232613</pubmed></ref> (Fig. Tn3.17 III) (see Serine-recombinases which use IHF/Hu: the Sin Synaptosome). Among these similarities are: conserved residues in two regions important in TnpRTn1000 function (A and B in Fig. Tn3.17 Iii) and the relative position of residues in the DNA recognition helix at the very C-terminal end of TnpRTn1000, Gin and TnpRTnXc5 (residues STLY; Fig. Tn3.17 Iii; <ref name=":82" /><nowiki><ref name=":109" />). However, although closely related, serine resolvases and invertases catalyze recombination using quite different nucleoprotein structures: called synaptosomes and invertasomes respectively. Instead of the three protein binding sites identified in ''res'', the invertion site is composed of two inverted sequences at which recombination occurs and an external site, the enhancer, which binds a small DNA bending protein, Fis (factor for inversion stimulation), to ensure the correct invertasome architecture. The functional similarities between serine resolvases and invertases have been probed using the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''] resolvase which, among the serine resolvases, is the most closely related to the [https://www.uniprot.org/uniprot/P03015 Gin] resolvase <nowiki><ref name=":82" />) ([[:File:Fig.Tn3.17I.png|Fig. Tn3.17 '''I''']]'''ii''') and [https://www.uniprot.org/uniprot/P03015 Gin]. Liu, et al <nowiki><ref name=":82" /> suggest that the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''] ''res'' site I at which recombination occurs is similar to the ''gix'' site recognized and recombined by [https://www.uniprot.org/uniprot/P03015 Gin]. Moreover it was argued from amino acid sequence considerations that the TnpR<sub>Tn''Xc5''</sub> dimer interface is more similar to that of the invertases than to that of TnpR<sub>Tn''1000 .''</sub> However, despite this apparent similarity, no TnpR<sub>Tn''Xc5''</sub> recombination on standard plasmid DNA “inversion substrates” containing two inverted intact [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''] ''res'' sites, was detected even in the presence of an appropriately positioned Fis site or inverted isolated copies of site I <nowiki><ref name=":82" />. However, strikingly, [https://www.uniprot.org/uniprot/P03015 Gin] was found capable of Fis-dependent inversion/deletion using a substrate composed of two isolated inverted or direct [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''] ''res'' site I copies ([[:File:Fig.Tn3.17I.png|Fig. Tn3.17 '''I''']]'''iiia, iiib''') both ''in vitro'' and ''in vivo'' <nowiki><ref name=":110"><pubmed>10656789</pubmed></ref>. On the other hand, it was not capable of using substrates with two complete res sites either in inverted or direct orientation (Fig. Tn3.17 Iiiic, iiid).The authors attributed this recombination inhibition to Gin binding to sites III (both site I and III exhibit about 53% identity with the gix site shown as circles in Fig. Tn3.17 Ii) since gel shift assays and footprinting using different substrates indicated that Gin recognizes res sitesI and III, but not site II. Furthermore, a chimeric recombinase, composed of the catalytic domain of Gin and the DNA-binding domain of TnpRTnXc5 was observed to efficiently recombine two res but unable to assemble a productive invertasome, suggesting that the C-terminal domain of Gin is instrumental in its formation.

These lines of analysis contribute to our understanding of the evolutionary relationship between these classes of recombinase.[[File:Fig.Tn3.17I.png|thumb|640x640px|Fig. Tn3.17I. The Sequence and organization of res sites used by long serine resolvases. i) the regions of resolvase binding defined by footprinting are shown as horizontal green lines. Sites I (core site) and sites II and III (accessory sites) are indicated and the generally accepted functional limits are boxed in blue were determined <ref name=":82" />. The probable crossover dinucleotide in site I is shown in red. The nucleotides in common with the ''gix'' site are indicated by circles. '''ii)''' Alignment of TnpRTn''Xc5'' with various invertases and TnpRTn''1000''. TnpRTn''Xc5'' and TnpRTn''1000'' are from TnCentral, Hinsty (''[[wikipedia:Salmonella|Salmonella typhimurium]]''; uniprot: [https://www.uniprot.org/uniprot/P03013 P03013]), Hinsae (''[[wikipedia:Salmonella|Salmonella abortus equi]]''; uniprot: [https://www.uniprot.org/uniprot/Q02869 Q02869]), Cin P1 (invertase from [[wikipedia:P1_phage|phage P1]]; uniprot: [https://www.uniprot.org/uniprot/P10311 P10311]), Cin P7 (invertase from phage P7; uniprot: [https://www.uniprot.org/uniprot/P21703 P21703]), Pin (invertase from ''[[wikipedia:Escherichia_coli|Escherichia coli]]''; uniprot: [https://www.uniprot.org/uniprot/P03014 P03014] ), [https://www.uniprot.org/uniprot/P03015 Gin] (from [[wikipedia:Bacteriophage_Mu|phage Mu]]; uniprot: [https://www.uniprot.org/uniprot/P03015 P03015]). Fully conserved residues are boxed in red, that commom between TnpRTn''Xc5'' and the invertases are boxed in green and those commons between TnpRTn''1000'' and the invertases are boxed in blue <nowiki><ref name=":82" />. '''iii)''' Inversion substrates with different configurations of Tn''Xc5'' ''res'' sites were used to test the activity of [https://www.uniprot.org/uniprot/P03015 Gin]. Sites are blue boxes labeled I, II, III with the orientation indicated by the arrowhead of site I. The green boxes represent a Fis binding site <nowiki><ref name=":110" />.|alt=|center]]Topological analysis of the recombination products suggests that the ''res''<sub>Tn''Xc5''</sub> synaptic complex must be very similar to those of ''res''<sub>Tn''3''</sub> and ''res''<sub>Tn''1000''</sub> since 4 supercoils are lost on recombination and the directionality of strand exchange is the same <nowiki><ref name=":82" />. No structural studies are at present available. =====Serine-recombinases which use IHF/Hu: the Sin Synaptosome.===== It is worth noting that certain [[wikipedia:Site-specific_recombination|serine recombinases]], such as '''[https://www.uniprot.org/uniprot/P03015 Gin]''' and '''[https://www.uniprot.org/uniprot/P03013 Hin]''' , involved in inversion switches <nowiki><ref name=":92" /><nowiki><ref><pubmed>3524854</pubmed><br /></ref><ref><pubmed>PMC413488</pubmed></ref><ref name=":109"><pubmed>11972771</pubmed></ref> or Sin which is involved in plasmid recombination <ref name=":91" /><nowiki><ref name=":96"><pubmed>15813731</pubmed></ref><ref><pubmed>16553879</pubmed></ref> use “simpler” recombination sites but depend on DNA bending proteins such as IHF, Fis, HU and HUB to achieve the correct architecture. These are not known to act in the resolution process of Tn3 family transposons.

The Staphylococcal Sin resolvase was identified as part of a multi-resistance staphylococcal plasmid, pI9789 <ref><pubmed>PMC401285</pubmed></ref>. While Sin is not part of a transposon or an invertible DNA segment, DNA sequences immediately upstream of the Sin binding site, sin, are favored integration sites for Tn554-family transposons. The sin site is composed of only two Sin binding sites I and II and an intervening region bound by an HU-like DNA bending protein such as the Bacillus subtilis Hbsu (Fig. Tn3.17 Ii). The functional site includes four imperfect: two inverted copies of a 12-bp motif repeat (sites I-L and I-R) and two in direct repeat (sites IIa and IIb) <ref name=":91" />. While '''[https://www.uniprot.org/uniprot/Q53757 Sin]''' on its own binds to sites I and II to generate DNase-protected regions, Hsbu on its own showed no protection. However, together, the two proteins result in protection of the entire site (note that protection is not even and additional sites of hypersensitivity are generated <nowiki><ref name=":91" />. Similar biochemical and topological and genetic approaches to those used in the analysis of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']/[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn1000-X60200.1 Tn''1000''] TnpR/''res'' function were used to investigate the '''[https://www.uniprot.org/uniprot/Q53757 Sin]'''/'''[https://www.uniprot.org/uniprot/Q53757 ''sin'']''' system <nowiki><ref name=":95" /><nowiki><ref name=":96" />. This showed that the synaptosome traps three supercoils between the points of strand exchange and requires both site I, where strand exchange occurs, and site II together with [https://www.uniprot.org/uniprot/A3F3E2 Hbsu]. A synaptosome structure, the first determined for a resolution system, has been solved <nowiki><ref name=":114" /><nowiki><ref name=":95" /> and a cartoon showing the arrangement of '''[https://www.uniprot.org/uniprot/Q53757 Sin]''' dimers and a dimer of dimers bridging the two '''[https://www.uniprot.org/uniprot/Q53757 s''in'']''' sites is shown in [[:File:Fig.Tn3.17J.png|Fig. Tn3.17 '''J''']]. This illustrates how bound non sequence-specific DNA bending proteins assist in synaptosome formation. [[File:Fig.Tn3.17J.png|center|thumb|640x640px|'''Fig. Tn3.17J.''' Serine-recombinases which use [[General Information/Transposase expression and activity#IHF.2C HU.2C HNS.2C and FIS|IHF/Hu]]: the Sin synaptosome '''i)''' the sin site showing the sequence repeats horizontal arrows indicate repeat orientation, blue Sin binding sites <nowiki><ref name=":91" />. General region of protection against Dnase digestion are shown as horizontal green lines. '''ii)''' The Sin synaptosome. The blue elipses represent the location of the DNA bending protein. The orange and pink elipses are Sin monomers. Double-headed arrows indicate monomer/monomer interactions in the Sin dimers and dimer/dimer interactions which occur between the two ''res'' sites at site I. '''iii)''' The sin synaptosome structure. Note that the DNA bending proteins are dimeric (pink and pale pink)<nowiki><ref name=":95" />. ]] =====The ''irs''/TnpI system===== A small group of known Tn''3'' family members which include the ''[[wikipedia:Bacillus_thuringiensis|Bacillus thuringiensis]]'' transposons [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] <nowiki><ref><pubmed>3018445</pubmed></ref> and Tn5401 <ref name=":83"><pubmed>PMC205437</pubmed></ref> encode a resolvase, TnpI, carrying a tyrosine residue at the active-site nucleophile <ref name=":43" /><nowiki><ref name=":83" /><nowiki><ref name=":84"><pubmed>7608077</pubmed></ref> (Fig. Tn3.17 Ki). The tnpI gene lies upstream of tnpA and both genes are transcribed in the same direction (Fig. Tn3.17 Aiii). Insertion mutagenesis showed that interruption of tnpI resulted in an increased level of cointegrate intermediates in Escherichia coli <ref name=":43" />. The [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4430-X07651.1 Tn''4430''] sequence <nowiki><ref name=":43" /> revealed a series of small sequence repeats directly upstream of ''tnpI'' as well as two smaller repeats abutting the inside border of IRL. The ''tnpI'' proximal repeat sequences include two 14bp inverted repeats, IR1 and IR2, together with two longer direct repeats, DR1 and DR2, related in sequence to IR1 and IR2 ([[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''Kii''']]). [[File:Fig.Tn3.17Ka.png|center|thumb|640x640px|'''Fig. Tn3.17Ka.''' Y recombinases: The First Strand exchange. The model of ordered strand exchange is based on that proposed for the Cre recombinase (Van Duyne, 2001). The two core ''irs'' sites at which strand crossover occurs (IR1/IR2), are shown as filled arrows interrupted at the point of recombination by the six base pair core site at which recombination occurs (red and blue). The repeated sequences to which resolvase binds are shown as blue arrows within the ''irs'' sites. The recombination sites are aligned in parallel ('''i''') and their strand polarities are shown. Only the C-terminal catalytic domain with its active site tyrosine (purple arrow) is shown for simplicity. In this system, each monomer in the tetramer undergoes activation by allosteric connections to its neighbour. ('''ii''') The C-terminal tyrosine nucleophile (dark green monomers) attacks the appropriate strand in each IR1 site to generate a 3’ phosphotyrosine bond (small yellow circle) generating a 5’ OH (small red and blue circles). ('''iii''') The 5’OH subsequently attacks the phospho-tyrosine bond (small yellow circle) from the opposite (dark green) monomer to complete strand transfer ('''iv''') and generate a [[wikipedia:Holliday_junction|Holliday junction]] ('''v''' next figure). ]]DNase footprinting revealed that TnpI bound to all four sites together called the internal resolution site (''irs'') <nowiki><ref name=":80" /> but not to the (unrelated) IRL proximal repeats ([[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''Kii''']]). Using a suicide substrate which contains a nick close to the point of recombination and which traps intermediates in the cleavage reaction, in an ''in vitro'' reaction TnpI was found to be able to bind to a linear DNA fragment containing IR1-IR2 and did not require assistance from the two DR repeats. DNA cleavage is staggered occurring six base pairs apart <nowiki><ref name=":80" /> ([[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''Kii''']]) forming a transient 3′-phosphotyrosyl bond leading to 3’OH in an identical way to other [[wikipedia:Site-specific_recombination|tyrosine recombinases]] (e.g. <nowiki><ref name=":85"><pubmed>26104563</pubmed></ref><ref><pubmed>PMC1170750</pubmed></ref><ref><pubmed>11340053</pubmed></ref><ref><pubmed>9288963</pubmed></ref><ref><pubmed>PMC450016</pubmed></ref><ref><pubmed>PMC2859247</pubmed></ref>. A complete in vitro resolution reaction requires supercoiled DNA substrate <ref name=":80" />. A similar overall sequence architecture was observed upstream of ''tnpI'' in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''] <nowiki><ref name=":84" /> ([[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''Kii''']]). Here, the repeated sequences are 12bp long with identical repeats abutting the inside border of IRL and of IRR. Footprinting also identified the TnpI ''irs'' binding sites but, in addition showed TnpI binding to the IR proximal site <nowiki><ref name=":84" />. =====The Mechanics of Resolution.===== In contrast to the requirements for the accessory sites I and II in [[wikipedia:Site-specific_recombination|serine recombinase]]-catalyzed resolution <nowiki><ref name=":81" />, there is no absolute requirement for the DR1 and DR2 accessory sites for activity in TnpI-catalyzed recombination. Instead, in their absence IR1-IR2 core site recombination can give rise to different recombination products such as deletions, inversions and intermolecular recombination in vivo and topologically complex products ''in vitro'' instead of the simple catenanes <nowiki><ref name=":80" />. In other words, the accessory sites channel recombination to generate resolutive recombination between two directly repeated ''irs'' sites on the same DNA molecule. This gave rise to the model shown in [[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''K''']]. [[File:Fig.Tn3.17K.png|center|thumb|640x640px|'''Fig. Tn3.17Kb.''' Y recombinases: The Second Strand exchange. The first strand transfer product ('''iii''', previous figure) in the form of a [[wikipedia:Holliday_junction|Holliday junction]] then isomerises ('''v''') and the second pair of recombinases (light green) are activated, the tyrosine (purple arrows) nucleophile (purple arrows) attacks the opposite strand ('''vi''') at IR2, creating a phospho-tyrosine bond (small yellow circle) and a 3’OH (small red and blue circles). The 3’OH then attacks the opposing 5’ phosphotyrosine bond ('''vii''') to complete strand transfer.]] More specifically, formation of synapses including DR1 and DR2 was found to stabilize recombination intermediates favoring the forward recombination reaction and to impose an order of cleavage at the IR1-IR2 core sites: activation of the IR1-bound TnpI subunits (those furthest from the accessory sites) occurs resulting in IR1 cleavage ([[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''Lii''']]) and first strand exchange [[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''Liii''']] to form a [[wikipedia:Holliday_junction|Holliday junction]] ([[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''Liv''']]) while the second pair, the IR2-bound subunits, are then activated to resolve the [[wikipedia:Holliday_junction|holliday junction]] [[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''Lv''']]) by cleavage and exchange of the second pair of strands ([[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''Lvi''']]) to resolve the cointegrate [[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''Lvii''']]) <nowiki><ref name=":80" /><nowiki><ref><pubmed>PMC2847244</pubmed></ref>.

Although the exact topology of the synaptic complex is unknown, two alternative models <ref name=":8" /> lead to the conclusion formation of the synaptic complex induces the same net change in substrate topology, trapping two negative supercoils between the crossover sites and converting them into catenation nodes in the product (see [[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''L''']]). [[File:Fig.Tn3.17L.png|center|thumb|640x640px|'''Fig. Tn3.17L.''' The S/T System '''i)''' Map of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''] ('''top''') and proposed ''res'' site ('''bottom'''); '''ii)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXo19-KR071788 Tn''XO19''] ([http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXo19-KR071788 Tn''7217'']) showing an insertion of two genes with potential ATPase activity between TnpT and TnpS.]] =====Irs, ''tnpR'' and ''tnpA'' and gene expression.===== Transcriptional start sites within [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''] were mapped by primer extension analysis and the -35 and -10 promoter elements were identified ([[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''Kiii''']]) <nowiki><ref name=":83" />. Two overlapping and divergent promoters were identified: one which would drive expression of tnpI and tnpA and the other which could drive the upstream but divergent [[wikipedia:Toxin-antitoxin_system|toxin antitoxin genes]] (see: [[Transposons families/Tn3 family#Tn3 family-associated TA passenger gene are located in a unique position.|Tn3 family-associated TA passenger genes are located in a unique position]]). =====The rst/TnpS/T system.===== The third major type of resolution system encoded by Tn''3'' family members is the TnpT-TnpS system which uses a resolution site, ''rst'' ('''r'''es site for Tnp'''S''' and Tnp'''T''') encoded by the catabolic transposon Tn''4651'' <nowiki><ref name=":76" />. Tn''4651'', isolated from a ''[[wikipedia:Pseudomonas|Pseudomonas]]'' plasmid carries a set of toluene degrading (''xyl'') passenger genes ([[:File:Fig.Tn3.3.png|Fig. Tn3.3]]) and is similar to the [http://parts.igem.org/Part:BBa_K1420000 mercury resistance] transposon [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''] ([[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''Ki''']]) <nowiki><ref name=":28" />. The ''tnpS'' and T genes are expressed divergently with the res site between the two. In some cases, tnpT and tnpS are separated by insertion of passenger genes ([[:File:Fig.Tn3.17L.png|Fig. Tn3.17 '''Lii''']]). Resolution of [[wikipedia:Cointegrate|cointegrates]] generated by Tn''4651'' was shown to require three Tn''4651''-encoded factors: the ''res'' site (now called ''rst'') and the ''tnpS'' and ''tnpT'' gene products which are located at a significant distance (48kb) away from the ''tnpA'' transposase gene. A similar long distance between transposase and resolvase is found in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''] <nowiki><ref name=":77" /> [[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''K''']]). Here, ''tnpS'' and ''tnpT'' are referred to as ''orfQ'' and ''orfI'' respectively and ''rst'' as ''att''<sub>Tn''5041''</sub>. [[File:Fig.Tn3.17M.png|thumb|640x640px|'''Fig. Tn3.17M.''' The TnpS Y Recombinase Domains I and II. The conserved catalytic motif of typical tyrosine recombinases is shown by vertical blue arrow heads. Data initially from <nowiki><ref name=":76" />. |alt=|center]] The 323 aa TnpS protein is a [[wikipedia:Site-specific_recombination|tyrosine recombinase]] ([[:File:Fig.Tn3.17M.png|Fig. Tn3.17 '''M''']]) with similarity to the [[wikipedia:Site-specific_recombination|Cre resolvase]] <nowiki><ref name=":8" /> while the 332 aa TnpT appears to enhance TnpS-mediated recombination <nowiki><ref name=":76" />. The sequence of the ''tnpS''/''T'' intergenic region is very similar in Tn''4651<nowiki><ref name=":80" />'' , [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4652-AF151431.1 Tn''4652''] (a Tn''4651'' deletion derivative lacking the [[wikipedia:Toluene|toluene]]-catabolic genes) <nowiki><ref><pubmed>2851712</pubmed></ref>, Tn4661 <ref name=":9" /> and Tn''4676'' <nowiki><ref name=":78" />. It is composed of a 203 bp sequence which includes two pairs of inverted repeats, IRL and IRR and IR1 and IR2 ([[:File:Fig.Tn3.17N.png|Fig. Tn3.17 '''Ni''']]) with overlapping promoters which drive TnpS and TnpT expression. The mRNA start point was identified by primer extension <nowiki><ref name=":76" />. The length of the functional ''rst'' site, 136 bp, was defined by the recombination activities of sequential deletion derivatives in an ''in vivo'' resolution system <nowiki><ref name=":8" />. This involved the construction of an artificial [[wikipedia:Cointegrate|cointegrate]] containing one complete ''rst'' copy and a second copy which carries the deletions. IR1 and IR2 are indispensable for the full resolution activity and [[wikipedia:Cointegrate|cointegrate]] resolution was shown to require both TnpS and TnpT. Moreover, the resolution reaction could be reversed to obtain [[wikipedia:Site-specific_recombination|site-specific integration]] (recombination between ''rst'' sites on different DNA molecules) in a reaction which requires TnpS but not TnpT. Suggesting that TnpT is a factor which determines the direction of recombination <nowiki><ref name=":8" /><nowiki><ref name=":76" />. Although the TnpS/T proteins of Tn''4651'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4661-AB375440.1 Tn''4661''] are highly similar and the Tn share highly similar sequences in the inverted repeat motif, IRL and IRR, of the ''rst'' core site, the 7bp spacer separating the repeats are somewhat different ([[:File:Fig.Tn3.17N.png|Fig. Tn3.17 '''Ni''']]). An artificial [[wikipedia:Cointegrate|cointegrate]] composed of an ''rst''<sub>Tn''4651''</sub> and an ''rst''<sub>Tn''4661''</sub> site could not be resolved using TnpS<sub>Tn''4651''</sub> and TnpT<sub>Tn''4651''</sub> <nowiki><ref name=":9" />. The mismatches in the IRL-IRR region concern principally the spacer region between IRL and IRR. ''rst''<sub>Tn''4651''</sub> and ''rst''<sub>Tn''4661''</sub> have six mismatches, with five located in the spacer ([[:File:Fig.Tn3.17N.png|Fig. Tn3.17 '''Nii''']]). In other [[wikipedia:Site-specific_recombination|tyrosine recombinase systems]] such as xerC/D or phage P1 Cre protein (see <nowiki><ref name=":8" /><nowiki><ref name=":85" /><nowiki><ref><pubmed>26104463</pubmed></ref>) this is the region where strand cleavages occur and sequence differences have a strong influence on the ability for two sites to recombine.

The effect of these sequence differences was investigated using a cointegrate, in which the spacer sequence of rstTn4661 was replaced with that from rstTn4651, (rstTn4661v1) (Fig. Tn3.17 Nii). In contrast to the cointegrate carrying both rstTn4651 and rstTn4661, that carrying rstTn4651 and rstTn4661v1 underwent TnpSTn4651 and TnpTTn4651-mediated resolution, demonstrating that it is the differences spacer sequence which prevents recombination <ref name=":9" />. Moreover, the sequence of this region in the resolved products confirmed that recombination occurred within the IRL-IRR region. It should be noted that neither the tnpS/T intergenic sequence nor the proposed core recombination site of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''] <nowiki><ref name=":28" /><nowiki><ref name=":77" /> ([[:File:Fig.Tn3.17K.png|Fig. Tn3.17 '''K''']]) shows significant similarity to those of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4661-AB375440.1 Tn''4661''] <nowiki><ref name=":9" />. Moreover, in depth analysis of the [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5041-X98999.3 Tn''5041''] resolution reaction has not been reported. No footprinting, binding stoichiometry or topology studies are yet available for the TnpS/T system and the exact role of TnpT in the resolution reaction is not known although it has been demonstrated to bind the DNA region containing IR1 and IR2 <nowiki><ref name=":9" />. [[File:Fig.Tn3.17N.png|center|thumb|640x640px|'''Fig.Tn3.17N.''' S/T Recombinase ''res'' Sites. '''i)''' Tn''4651''/[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4652-AF151431.1 Tn''4652''] ''res'' site showing the expression signals for the ''tnpS'' and ''tnpT'' genes with convergent (overlapping) promoter elements. mRNA initiation and the IRL-IRR (blue horizontal arrows) and core recombination sequence. The equivalent sequences of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4661-AB375440.1 Tn''4661''] and Tn''4676'' are shown below, with the variant bases marked in red. '''ii)''' ''res'' site sequences used in inter ''res'' recombination analyses. Data from <nowiki><ref name=":9" /><nowiki><ref name=":76" />.]] ====Toxin-Antitoxin genes: Special Passengers linked to the transposition process?==== Several studies had identified individual Tn''3'' family members with type II [[wikipedia:Toxin-antitoxin_system|toxin-antitoxin]] ('''TA''') passenger genes <nowiki><ref name=":30" /><nowiki><ref name=":79" /><nowiki><ref><pubmed>16122561</pubmed></ref><ref name=":86"><pubmed>PMC4840908</pubmed></ref> (see reference <ref name=":8" />). Unusually for passenger genes of this Tn family, the [[wikipedia:Toxin-antitoxin_system|T/A genes]] are consistently found adjacent to a '''resolvase gene'''. Some [[wikipedia:Toxin-antitoxin_system|type II TA systems]] are involved in plasmid maintenance in growing bacterial populations by a mechanism known as [[wikipedia:Toxin-antitoxin_system|post segregational killing]]. Upon plasmid loss, degradation of the labile antitoxin liberates the toxin from the inactive complex, which in turn is free to interact with its target and cause cell death. They were first identified in the mid-1980s in plasmids F <nowiki><ref><pubmed>PMC219208</pubmed></ref> and R1 <ref><pubmed>PMC323463</pubmed></ref> and it was recently shown that acquisition of a Tn3 family transposon Tn6231 carrying a type II TA gene pair was indeed able to “stabilize” an unstable target plasmid <ref name=":86" />. Many different [[wikipedia:Toxin-antitoxin_system|type II TA gene pairs]] have now been identified in bacterial chromosomes as well as plasmids <nowiki><ref name=":87"><pubmed>PMC3141249</pubmed></ref><ref name=":88"><pubmed>21819231</pubmed></ref><ref name=":89"><pubmed>34975154</pubmed></ref>. They are generally composed of 2 relatively short proteins: a stable toxin and a labile antitoxin that binds the toxin and inhibits its lethal activity (see reference <ref name=":88" />). The antitoxin includes a [[wikipedia:DNA-binding_domain|DNA binding domain]] involved in promoter binding and negative regulation of TA expression. =====Identification of TA gene pairs in Tn3 family members.===== Among nearly 200 Tn''3'' family members, 39 were observed to carry [[wikipedia:Toxin-antitoxin_system|type II T/A genes]] (colored squares; [[:File:Fig.Tn3.4.png|Fig. Tn3.4A]], [[:File:Fig.Tn3.18B.png|Fig. Tn3.18 '''B''']]) <nowiki><ref name=":30" />. The host transposons included examples from all known combinations and orientations of transposase and resolvase genes ([[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''A''']]) and were almost all located adjacent to the resolvase genes in family members with TnpR, with long serine TnpR, with TnpI (e.g. [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnBth4-CP010092.1 Tn''Bth4'']) and with TnpS/T (e.g. Tn''HdN1.1'') ([[:File:Fig.Tn3.4.png|Fig. Tn3.4A]], [[:File:Fig.Tn3.18B.png|Fig. Tn3.18 '''B''']]). Illustrative examples are shown in ([[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''A''']]). [[File:Fig.Tn3.18A.png|center|thumb|640x640px|'''Fig.Tn3.18A.''' Examples of [[wikipedia:Toxin-antitoxin_system|TA]] gene pair location in a variety of Tn''3'' family transposons. [[wikipedia:Toxin-antitoxin_system|Toxin-antitoxin gene pairs]] shown in bright orange, genes of unknown function in magenta, transposition-related genes in purple, heavy metal resistance genes in chrome, and antibiotic resistance genes in red. '''i)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5046-Y18360.1 Tn''5046''], accession [https://www.ncbi.nlm.nih.gov/nuccore/Y18360.1 Y18360.1], has an unusual structure with the ''mer'' passenger genes located downstream from the transposase gene. It carries a typical ''tnpR'' cognate res site. '''ii)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.6-MF487840.1 Tn''5501.6''], accession [https://www.ncbi.nlm.nih.gov/nuccore/MF487840.1 MF487840.1], carries a ''bla''NPS-1 passenger gene. It carries a typical ''tnpR'' cognate ''res'' site. '''iii)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''], accession [https://www.ncbi.nlm.nih.gov/nuccore/U03554.1 U03554.1], there are no known passenger genes apart from the [[wikipedia:Toxin-antitoxin_system|TA]] gene pair. It carries a typical ''tnpI'' cognate ''irs'' site with, in addition, a copy of the DR2 TnpI binding site close to each end. '''iv)''' Tn''HdN1.1'', accession [https://www.ncbi.nlm.nih.gov/nuccore/FP929140.1 FP929140.1], is treated as a partial copy since the ends of the transposon have not yet been identified. Consequently, no passenger genes except the [[wikipedia:Toxin-antitoxin_system|TA]] gene pair have been identified. However, Tn''HdN1.1'' carries a typical ''tnpT''/''tpnS'' resolvase pair and the [[wikipedia:Toxin-antitoxin_system|toxin/antitoxin genes]] are located between the resolvase and the ''tnpA'' gene. The ''rst'' site has not yet been defined, but for other transposons with a TnpS/T/''rst'' resolution system, it is located between the divergent ''tnpS'' and ''tnpT'' genes. '''v)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4662a-NC_014124.1 Tn''4662a'']: accession [https://www.ncbi.nlm.nih.gov/nuccore/NC_014124.1 NC_014124.1]. This transposon carries a potential metal-dependent phosphohydrolase passenger gene and a ''tnpR'' cognate ''res'' site. In this case, in contrast to the vast majority of cases, the toxin gene is located upstream of the antitoxin gene. ]] [[File:Fig.Tn3.18B.png|center|thumb|640x640px|'''Fig.Tn3.18B.''' Tn''3'' family [[wikipedia:Toxin-antitoxin_system|TA]] systems and configuration of the [[wikipedia:Toxin-antitoxin_system|TA]] operon with respect to the resolvase and TnpA genes (extracted from <nowiki><ref name=":30" />).]] =====TA diversity in Tn''3'' family members.===== The Tn''3''-associated [[wikipedia:Toxin-antitoxin_system|TA modules]] include a number of different types of [[wikipedia:Toxin-antitoxin_system|TA module]]: 5 toxin ([https://www.uniprot.org/uniprot/P0C077 RelE]/[https://www.uniprot.org/uniprot/P0C077 ParE], [http://pfam.xfam.org/family/PF13876 Gp49], [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR002850/ PIN_3], [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR002850/ PIN],and [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR008201/ HEPN]) and 6 antitoxin families ([https://www.uniprot.org/uniprot/P22995 ParD], [https://pfam.xfam.org/family/PF13744 HTH_37], [https://www.ebi.ac.uk/interpro/entry/pfam/PF09957/ RHH_6], [https://www.uniprot.org/uniprot/Q06253 Phd]/[https://www.uniprot.org/uniprot/P69346 YefM], AbrB/[https://www.uniprot.org/uniprot/P0AE72 MazE], and [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR008201/ MNT]) ([[:File:Fig.Tn3.4.png|Fig. Tn3.4A]]; [[:File:Fig.Tn3.18B.png|Fig. Tn3.18 '''B''']]). All, except [https://www.uniprot.org/uniprot/P0C077 ParE], are associated with [[wikipedia:Ribonuclease|RNase activity]] <nowiki><ref name=":87" /><nowiki><ref name=":88" /><nowiki><ref><pubmed>PMC3710099</pubmed></ref>, while ParE inhibits DNA gyrase activity by an unknown molecular mechanism <ref><pubmed>12010492</pubmed></ref>.

TA distribution and organization within the Tn3 family

The majority of examples occurred in two Tn3 subgroups: Tn3 (2 toxin families) and Tn3000 (3 toxin families), but other subgroups also included members with T/A modules (6 members of 5 different toxin families (ParE, Gp49, PIN_3, PIN, and HEPN). The majority of T/A-containing members of the Tn3 subgroup also encode a long serine recombinase, TnpRL as their resolvase and two (Tn5401 and TnBth4) encode the tyrosine TnpI resolvase, while those in the Tn3000 subgroup all encode a short serine resolvase, TnpRS (Fig. Tn3.4A; Fig. Tn3.18 B). It is also noteworthy that, a given toxin gene can be paired with different antitoxins forming 7 different toxin-antitoxin pairs: ParE-ParD, ParE-PhD, PIN_3-RHH_6 (??), Gp49-HTH_37, PIN-Phd, PIN-AbrB, and HEPN-MNT (Fig. Tn3.18 B).

Although TA genes are generally arranged with the antitoxin upstream of the toxin gene, TA systems of reverse order have been identified <ref name=":88" />. Among the Tn''3'' family-associated [[wikipedia:Toxin-antitoxin_system|TA systems]], in five [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3000-AF174129 Tn''3000''] subgroup members ([[:File:Fig.Tn3.18B.png|Fig. Tn3.18 '''B''']] and [[:File:Fig.Tn3.4.png|Fig. Tn3.4A]]) the [https://www.uniprot.org/uniprot/P0C077 RelE]/[https://www.uniprot.org/uniprot/P0C077 ParE] superfamily toxin [http://pfam.xfam.org/family/PF13876 Gp49] ([https://pfam.xfam.org/family/PF05973 PF05973]) toxin gene precedes that of a [https://www.uniprot.org/uniprot/P67701 HigA superfamily antitoxin], [https://pfam.xfam.org/family/PF13744 HTH_37] ([https://pfam.xfam.org/family/PF13744 PF13744]) <nowiki><ref name=":87" /><nowiki><ref name=":88" /><nowiki><ref name=":89" />. A similar situation is found in the unrelated Tn''4651'' subgroup member Tn''Posp1_p''. In addition to encoding a TnpI resolvase, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnBth4-CP010092.1 Tn''Bth4''], both encode a [https://www.uniprot.org/uniprot/P22995 ParD] antitoxin, which appears to lack the [[wikipedia:DNA-binding_domain|DNA-binding domain]]. =====Acquisition and exchange of TA modules.===== An important question is whether these systems have been repeatedly recruited or have evolved from a common ancestor. Putting aside the fact that several groups of [[wikipedia:Toxin-antitoxin_system|T/A encoding]] Tn (e.g. Tn''5051'' and its derivatives which differ essentially by their other passenger genes), clearly the fact the Tn collection also includes examples of different combinations of [[wikipedia:Toxin-antitoxin_system|T/A genes]] and examples in which the gene order has been inverted argue for a certain level of repeated acquisition. In cases where the [[wikipedia:Toxin-antitoxin_system|TA module]] is found in related transposons (with similar ''tnpA'' and/or resolvase genes), it is likely that it was first acquired by a transposon that subsequently diverged. Alternatively, for transposons which are generally not related (different ''tnpA'' family group, different resolvase) but which harbor [[wikipedia:Toxin-antitoxin_system|TA modules]] that are similar at the DNA level, it is likely that the [[wikipedia:Toxin-antitoxin_system|TA module]] was acquired by recombination with another transposon. Phylogenetic analysis suggested that [https://www.uniprot.org/uniprot/P0C077 ParE] had been acquired three times, [http://pfam.xfam.org/family/PF13876 Gp49] together with an [[wikipedia:Helix-turn-helix|HTH]] antitoxin on three occasions and [https://www.ebi.ac.uk/interpro/entry/InterPro/IPR002850/ PIN] on two occasions <nowiki><ref name=":30" />. Although it is unclear how most of the [[wikipedia:Toxin-antitoxin_system|T/A modules]] were initially acquired, it is important to underline that the ''res''/''rst''/''irs'' sites are highly recombinogenic in the presence of their cognitive resolvases producing transitory single (Y-recombinases) or double (s-recombinases) breaks. It seems possible that the modules were recruited via non-productive resolution events. Moreover, this recombination activity has clearly led to spread of [[wikipedia:Toxin-antitoxin_system|T/A modules]] to different Tn''3'' family members by inter-''res'' recombination ([[:File:Fig.Tn3.18B.png|Fig. Tn3.18 '''B''']]). There are two cases in the Tn''3'' library, both involving Tn''5051'' and its derivatives, which demonstrate this capacity ([[:File:Fig.Tn3.18C.png|Fig. Tn3.18 '''C''']]). In the first case ([[:File:Fig.Tn3.18C.png|Fig. Tn3.18 '''Ci''']]), comparison between Tn''5051'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnTsp1-NC_014154 Tn''Tsp1''] shows a clear break in the homology between the two Tn which occurs at the ''res'' site ([[:File:Fig.Tn3.18C.png|Fig. Tn3.18 '''Ci''']]). The identities towards the right of the ''res'' site III decrease rapidly within a short distance. In the second case ([[:File:Fig.Tn3.18C.png|Fig. Tn3.18 '''Cii''']]), comparison of Tn''5051'' and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4662a-NC_014124.1 Tn''4662a''] shows a clear break in identity at ''res'' site I suggesting that they have previously exchanged left and right ends ''via'' recombination at ''res'' site I. An additional transposon, Tn''5051.12'' is clearly a hybrid of these two since it carries the left end of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4662a-NC_014124.1 Tn''4662a''] and the right end of Tn''5051''. [[File:Fig.Tn3.18C.png|thumb|640x640px|'''Fig.Tn3.18C.''' Inter-transposon recombination at the res site exchanges [[wikipedia:Toxin-antitoxin_system|TA]] modules. '''i)''' Comparison of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501''] accession [https://www.ncbi.nlm.nih.gov/nuccore/JN648090.1 JN648090.1] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnTsp1-NC_014154 Tn''Tsp1''] accession [https://www.ncbi.nlm.nih.gov/nuccore/NC_014154 NC_014154] showing a possible recombination point between the two Tn where exchange at the [[wikipedia:Toxin-antitoxin_system|TA]] gene pair may have occurred. The bottom section shows the region of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnTsp1-NC_014154 Tn''Tsp1''] including the [[wikipedia:Toxin-antitoxin_system|TA]] gene module (orange), the ''res'' site (green), ''tnpR'' and part of ''tnpA'' (purple). The top segment shows the equivalent map of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501'']. Below is shown a DNA sequence alignment (magenta) with the equivalent region of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501'']. Both transposons have similar DNA sequences to the left of ''res'' site I. The level of sequence identity is reduced in ''tnpR'' and is insignificant in ''tnpA''. The res site I sequences (green) are shown between the two panels and the AT dinucleotide at which recombination probably occurs is indicated in red. Sequence non-identities are underlined. The two sequences are identical up to the probable recombination site and show some diversity to its right. '''ii)''' The region of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.12-CP017294.1 Tn''5501.12''] accession [https://www.ncbi.nlm.nih.gov/nuccore/CP017294.1 CP017294.1] showing the 5’ end of the ''tnpA'' gene, the ''tnpR'' gene, a ''res'' site typical of the ''tnpR'' ''res'' sites, and toxin/antitoxin gene pair (note that the toxin gene is upstream of the antitoxin gene. The horizontal magenta lines at the bottom show the alignment of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.12-CP017294.1 Tn''5501.12''] with [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4662a-NC_014124.1 Tn''4662a''] ([https://www.ncbi.nlm.nih.gov/nuccore/NC_014124.1 NC_014124.1]). The right half of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501''] is clearly highly homologous to the right side of Tn5501.12 whereas the left side of Tn4662a is homologous to the left side of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.12-CP017294.1 Tn''5501.12'']. The DNA sequences at the top show the res subsite I (green) with the dinucleotide at which recombination should occur in red together with flanking sequences. Underlined bases indicate regions of nucleotide identity. This suggests a scenario in which [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.12-CP017294.1 Tn''5501.12''] was generated by recombination at ''res'' I between transposons similar to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4662a-NC_014124.1 Tn''4662a'']. |alt=|center]] =====Tn3 family-associated TA passenger gene are located in a unique position.===== In most of the cases identified, the [[wikipedia:Toxin-antitoxin_system|T/A modules]] are embedded within the transposition module comprising transposase and resolvase genes and the ''res'' site at a position very close to the ''res'' sites ([[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''A''']]). This is in sharp contrast to all other [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] family passenger genes, which are generally located away from the resolution and transposon genes and, where known, have often been acquired as [[wikipedia:Integron|integron]] cassettes or by insertion of other transposons. Indeed, several TA-carrying transposons represent closely related derivatives with identical transposase, resolvase, and TA modules but contain different sets of passenger genes (e.g., [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.1-CP016447.1 Tn''5501.1''] and derivatives ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.3-GQ983559.1 5501.2]'', ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.3-GQ983559.1 5501.3]'', ''[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501.4-KY206932.1 5501.4]'', etc.). Most [[wikipedia:Toxin-antitoxin_system|T/A modules]] <nowiki><ref name=":30" /> are located directly upstream of the resolvase genes (''tnpR'', ''tnpR''<sub>L</sub> or ''tnpI'') ([[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''A''']]) with only three exceptions: a single example of a derivative with the TnpS/TnpT resolvase, TnHdN1.1 ([[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''Aiv''']]), where they are located between the resolvase ''tnpS'' and transposase genes; [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnSku1-CP002358.1 Tn''Sku1''] [[http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnSku1-CP002358.1 Tn''7197'']], where they are located downstream of and transcribed towards tnpR; and a partial transposon copy, Tn''Amu2_p'' with a short open reading frame (ORF) of unknown function between the divergently transcribed antitoxin and ''tnpR'' genes. =====Regulation of Tn''3'' family TA gene expression.===== An as yet unanswered question is how expression of the identified [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']-associated [[wikipedia:Toxin-antitoxin_system|T/A genes]] is regulated. It is possible that it occurs from their own promoters although it has not yet been demonstrated any of the [[wikipedia:Toxin-antitoxin_system|T/A modules]] carry their own promoters. Alternatively, the fact that the genes are embedded in the transposition modules, it is tempting to speculate that they may be regulated in a similar way to ''tnpR'' and ''tnpA'' expression. =====Tn''3'' family with TnpR and TnpR<sub>L</sub>===== In [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] itself, which has been examined in detail, transposase and resolvase gene expression is controlled by promoters found within the ''res'' site located between the two divergent genes ([[:File:Fig.Tn3.17C.png|Fig. Tn3.17C]]) which are regulated by resolvase binding. The location of the [[wikipedia:Toxin-antitoxin_system|TA genes]] in proximity to the ''res'' sites raises the possibility that their expression is also controlled by these promoters ([[:File:Fig.Tn3.18D.png|Fig. Tn3.18 '''Di''']]). Although few of the ''res'' sites in the collection of [[wikipedia:Toxin-antitoxin_system|TA-associated]] Tn''3'' family members have been defined either experimentally or by sequence comparison, 27 potential sites were identified <nowiki><ref name=":30" /> using the canonical ''tnpR''-associated ''res''-site organization schematized in <nowiki><ref name=":8" /> as a guide, a ''res'' site library (kindly provided by [https://www.gla.ac.uk/researchinstitutes/biology/staff/martinboocock/ Martin Boocock]), and [http://rsat.sb-roscoff.fr/ RSAT tools] ([http://rsat.sb-roscoff.fr/ Regulatory Sequence Analysis Tools]) <nowiki><ref name=":30" />. For transposons with a TnpR or TnpR<sub>L</sub> resolvase, the [[wikipedia:Toxin-antitoxin_system|TA genes]] are always located just downstream from ''res'' site I, whereas ''tnpR'' is located next to ''res'' site III ([[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''A''']] '''i, ii''' and '''v'''; [[:File:Fig.Tn3.18D.png|Fig. Tn3.18 '''Di''']]). In transposons with divergent ''tnpA'' and ''tnpR'' such as [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5563a-KJ920395.1 Tn''5563a''] ([[:File:Fig.Tn3.18D.png|Fig. Tn3.18 '''Di''']]), ''tnpA'', ''tnpR'' and ''res'' are organized similarly to those of [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''], which itself does not carry the [[wikipedia:Toxin-antitoxin_system|TA module]], except that ''tnpA'' is separated from ''res'' by the intervening [[wikipedia:Toxin-antitoxin_system|TA genes]]. This organization is also similar in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] members in which ''tnpA'' is downstream of ''tnpR'' and in the same orientation (e.g., [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5501-JN648090.1 Tn''5501''] and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4662a-NC_014124.1 Tn''4662a'']'';'' [[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''A''']] '''i''' and '''v'''). =====Tn''3'' family with TnpI===== Promoters have also been defined in the ''res'' (''irs'') site of the ''tnpI''-carrying [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''] <nowiki><ref name=":83" /><nowiki><ref name=":84" /><nowiki><ref name=":90"><pubmed>PMC103759</pubmed></ref>, and tnpI and tnpA expression is modulated by TnpI binding to the irs site <ref name=":90" /> (Fig. Tn3.17I). The other tnpI-carrying transposon with [[wikipedia:Toxin-antitoxin_system|TA genes]], [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnBth4-CP010092.1 Tn''Bth4''], has an identical ''irs'' site, and therefore expression is probably regulated in the same way. Again, the potential promoters are pertinently located for driving expression of the TA module ([[:File:Fig.Tn3.18D.png|Fig. Tn3.18 '''Dii''']]). [[File:Fig.Tn3.18D.png|thumb|640x640px|'''Fig.Tn3.18D.''' Relationship between the res site, known promoter elements, and [[wikipedia:Toxin-antitoxin_system|TA]] gene pairs. In addition, potential or proven minus-10 and minus-35 promoter elements are shown as red arrows. '''i)''' Res sites (green) with a structure related to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3'']. 300 bp including flanking DNA is shown. TnpR (purple) is expressed to the left, and TnpA (purple, Tn3) and [[wikipedia:Toxin-antitoxin_system|the toxin/antitoxin genes]] (orange, [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''], and [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5044-Y17691.1 Tn''5044'']) to the right. In this type of organization, the ''res'' III subsite is proximal to t''npR.'' Recombination leading to [[wikipedia:Cointegrate|cointegrate]] resolution occurs at a [[wikipedia:Toxin-antitoxin_system|TA]] dinucleotide within ''res'' site I. '''a)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn3-V00613 Tn''3''] ''res'' site. The ''res'' site was defined by footprinting using TnpR and by functional deletion analysis. The promoter elements are predicted. '''b)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''] ''res'' site, also called [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 IS''Xc5'']. The ''res'' site was defined by footprinting with TnpR. '''c)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5044-Y17691.1 Tn''5044''] . The ''res'' site was defined by comparison with [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnXc5-Z73593 Tn''Xc5''] and as described here. '''ii)''' Res site organization for transposons carrying ''res'' sites for the TnpI resolvase. '''a)''' [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''] ''res'' site. This was identified by footprinting with TnpI and by deletion analysis . '''b)''' Tn''Bth1'' ''res'' site ([https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP010092.1 NZ_CP010092.1]). Tn''Bth1'' is similar but not identical to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401''] over the ''res'' site but varies considerably in the ''tnpI'' and ''tnpA'' genes. It maintains the promoter elements (red arrows) identified in [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn5401-U03554.1 Tn''5401'']. ''tnpI'' and ''tnpA'' are expressed to the right. The [[wikipedia:Toxin-antitoxin_system|toxin-antitoxin pair]] is expressed to the left. '''iii)''' Gene organization for transposon Tn''HdN1.1'' carrying the TnpS/T resolvase.|alt=|center]] =====Tn''3'' family with TnpS/T===== Finally, transposon TnHdN1.1 ([[:File:Fig.Tn3.18A.png|Fig. Tn3.18 '''Aiv''']]) is the only example in our collection of a ''tnpS''/''tnpT'' transposon carrying a [[wikipedia:Toxin-antitoxin_system|TA module]]. The ''res'' (''rst'') site and relevant promoter elements for the divergently expressed ''tnpS'' and ''tnpT'' have been identified between the two genes in transposon Tn''4651'' ([[:File:Fig.Tn3.18D.png|Fig. Tn3.18 '''Diii''']]). In TnHdN1.1, the [[wikipedia:Toxin-antitoxin_system|TA gene pair]] is to the right of ''tnpS'', between ''tnpS'' and ''tnpA'', and all three genes are oriented in the same direction. Although the exact regulatory arrangement remains to be determined, it seems possible that the promoters in the ''rst'' site regulate expression of the TA gene pair. Thus, for all three types of resolvase-carrying Tn''3'' family members, the [[wikipedia:Toxin-antitoxin_system|T/A gene module]] is strategically placed so that it could be place under control of the resolvase/transposase transcriptional expression signals except for the two exceptions [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=TnSku1-CP002358.1 Tn''Sku1''] and Tn''Amu2''_p. [[wikipedia:Toxin-antitoxin_system|T/A activity]] could therefore be intimately linked to the transposition process itself rather than, or in addition to, simply providing a general addiction system that stabilizes the host replicon, generally a plasmid, carrying the transposon. =====A model for T/A activity in transposon transposition.===== [[wikipedia:Toxin-antitoxin_system|Type II TA expression]], like that of tnpA and tnpR, is tightly regulated at the transcriptional level (see <nowiki><ref name=":88" />). Where analyzed, the [[wikipedia:Toxin-antitoxin_system|toxin-antitoxin complex]] binds via the antitoxin [[wikipedia:DNA-binding_domain|DNA-binding domain]] to palindromic sequences located in the operon promoter and acts as a negative transcriptional regulator. This regulation depends critically on the relative levels of toxin and antitoxin in a process known as conditional cooperativity, a common mechanism of transcriptional regulation of prokaryotic [[wikipedia:Toxin-antitoxin_system|type II toxin-antitoxin]] operons in which, at low toxin/antitoxin ratios, the toxin acts as a corepressor together with the antitoxin. At higher ratios, the toxin behaves as a derepressor. It will be important to determine whether the Tn-associated [[wikipedia:Toxin-antitoxin_system|TA genes]] include their indigenous promoters <nowiki><ref name=":88" /><nowiki><ref><pubmed>27159580</pubmed></ref><ref><pubmed>20603017</pubmed></ref>.

Transposon Tn6231 <ref name=":86" /> (99% identical to [http://tncentral.ncc.unesp.br/cgi-bin/tn_report.pl?id=Tn4662a-NC_014124.1 Tn''4662'']) clearly provides a level of stabilization of its host plasmid implying that TA expression occurs in the absence of transposition. There are a number of ways in which this could take place ([[:File:Fig.Tn3.18E.png|Fig. Tn3.18 '''E''']]). Expression could occur from a resident TA promoter ([[:File:Fig.Tn3.18E.png|Fig. Tn3.18 '''Ei''']]) if present. However, this might lead to expression of the downstream tnpA gene by readthrough transcription. Alternatively, in the absence of a TA promoter, TA expression could occur stochastically from the ''res'' promoter ([[:File:Fig.Tn3.18E.png|Fig. Tn3.18 '''Eii''']]). However, this does not rule out the possibility that TA expression is regulated at two levels with a low-level “maintenance” expression, resulting in the plasmid stabilization properties described by Loftie-Eaton et al. <nowiki><ref name=":86" /> together with additional expression linked to derepression of the ''tnpA'' (and ''tnpR'') promoters that must occur during transposition ([[:File:Fig.Tn3.18E.png|Fig. Tn3.18 '''Eiii''']]). Regulation of ''tnpR'' and ''tnpA'' by TnpR is a mechanism allowing a burst of TnpA (and TnpR) synthesis, transitorily promoting transposition as the transposon invades a new host. Subsequent repression by newly synthesized TnpR would reduce transposition activity, reinstalling homeostasis once the transposon has been established, a process similar to [[wikipedia:Zygotic_induction|zygotic induction]] <nowiki><ref><pubmed>13373067</pubmed></ref> or plasmid transfer derepression as originally observed for the plasmid ColI <ref><pubmed>14482966</pubmed></ref> and subsequently for plasmids R100 <ref><pubmed>4598795</pubmed></ref> and R1 <ref><pubmed>PMC1202851</pubmed></ref>. An alternative but nonexclusive explanation stems from the observed enhanced plasmid stability afforded by Tn6231 TnpR, in addition to that afforded by the neighboring TA system <ref name=":86" />. Resolvase systems are known to promote resolution of plasmid [[wikipedia:Protein_dimer|dimers]] (see reference <nowiki><ref name=":14" />), and it was suggested that integration of the [[wikipedia:Toxin-antitoxin_system|TA system]] into Tn''6231'' “such that all the transposon genes shared a single promoter region” permits coordinated TA and TnpR expression and may facilitate temporary inhibition of cell division while resolving the multimers, promoting plasmid persistence. In this light, it is interesting that the ''ccd'' [[wikipedia:Toxin-antitoxin_system|TA system]] of [[wikipedia:Escherichia_coli|''Escherichia coli'' plasmid F]] is in an operon with a resolvase-encoding gene <nowiki><ref><pubmed>PMC341330</pubmed></ref><ref><pubmed>2511422</pubmed></ref>.

Expression of the TA module from the tnpA/tnpR promoter at the time of the transposition burst could transiently increase invasion efficiency (“addiction”) over and above that provided by the endogenous TA regulation system. If the transposon is on a molecule (e.g. a conjugative plasmid) that is unable to replicate vegetatively in the new host, expression of the TA module without transposition to a stable replicon would lead to loss of the transposon and consequent cell death, whereas cells in which transposition had occurred would survive and give rise to a new population in which all cells would contain the Tn. This might be seen as a “take me or die” mechanism <nowiki>[1], a notion which could be explored experimentally.

Fig. Tn3.18E. Working Model for the Integration of TA Activity into the Transposition Process. A hypothetical Tn3 family transposon carrying a TA gene pair is shown. i) Homeostasis on a plasmid stably established in the cell. Transcription (orange and blue dotted wavy line) occurs from a putative endogenous TA promoter (P, proximal to TA) and maintains low toxin (T) and antitoxin (A) levels to maintain the vector plasmid in the host cell population. Expression of tnpA and tnpR from the res site promoters is largely repressed by TnpR binding. However, readthrough transcription from the TA gene pair into tnpA would be expected to result in a level of background TnpA expression. ii) Stochastic expression If the TA genes do not have an endogenous promoter, stochastic expression (blue and orange dotted wavy lines) from the divergent res promoters (P, within the res site) would result in low TnpA and TnpR levels as well as low level TA expression. iii) Plasmid conjugation into a recipient cell resulting in derepression of the res promoters results in higher levels of tnpA, tnpR, and TA transcription (blue and orange wavy lines) and expression of TA proteins resulting in an increased level of “addiction”.

Conclusion and Future.

The Tn3 family is widely spread and diverse as we have underlined and illustrated here. There is some understanding of the different evolutionary pathways and mechanisms which have permitted family members to sequester a large set of passenger genes widely variable functions and to shuffle them between and within both plasmids and chromosomes. Although much is known about a number of model Tn3 family members, there remain a number of open questions. In particular, historically this has proved recalcitrant to analysis in spite of much effort from their discovery in the 1970s to the present day.

Recent studies with Tn4330 however may have unlocked a door to understanding Tn3 family transposition in molecular detail. The structural studies using cryo-em have provided precious information as to the location and function of a large number of domains in the exceptionally long transposases. The studies point to the way in which the transpososome may be assembled although additional analyses are essential to a full understanding of docking of target DNA and its place in the assembly pathway. In addition, it is at present unclear how duplication of this family occurs during transposition: how it may recruit replication enzymes, whether replication initiates from one particular end, or, indeed whether it involves parasitizing existing replication forks in the target. The phenomenon of immunity is also not understood although it is clear that, mutationally, it is linked to transposition activity. In the absence of an ATPase activity, it seems unlikely that it occurs with the same mechanism as does that of bacteriophage Mu or transposon Tn7.

Acknowledgments

We would like to thank Marshall Stark (University of Glasgow), Martin Boocock (University of Glasgow), Dave Sherratt (University of Oxford) and Phoebe Rice (University of Chigago) for critical comments.

Bibliography

  1. ↑ Cite error: Invalid <ref> tag; no text was provided for refs named :30


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