Transposons families/Tn3 family

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 ��[1]�: “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 ��[1]� while the closely related TnB and TnC (later called Tn2 and Tn3 respectively) were isolated from plasmids RSF1010 ��[2]� and R1 ��[3,4]�. Tn3 proved to be inserted into another, larger Tn3 family transposon, Tn4 ��[4]�. A number of early studies using electron microscope DNA heteroduplex analysis (e.g. ��[5–7]� Fig. Tn3.1) demonstrated that movement of ampicillin resistance was accompanied by insertion of a DNA segment of about 4-5 kilobases (kb). The DNA sequence of the 4957 base pair (bp) Tn3 was obtained in 1979 ��[8]� 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 ��[9]� which acts on a specific site, IRS (Internal Resolution Site) (Fig. Tn3.2i). In its absence, insertion of two complete, directly repeated, Tn3 copies occurred ��[8]�. It was suggested that this type of structure was an intermediate in Tn3 transposition and that the IRS site was required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 ��[10]� according to the Shapiro cointegrate model of replicative transposition (Fig. Tn3.2ii; ��[11]� Fig. 2 Early models). Indeed, Tn3 was shown to be instrumental in permitting transfer of a non-transmissible plasmid by a co-resident conjugative plasmid ��[12]� resulting in fusion of the two plasmids which were separated at their junctions by two directly repeated Tn copies ��[12–15]�.

A related TE, or Tn1000, was identified as part of the plasmid F and appeared as an insertion loop in heteroduplex analysis ��[15,16]�. It was also implicated in the integration of the F plasmid into the Escherichia coli host chromosome ��[16]� and deletion of chromosomal DNA in F’ plasmids ��[17,18]� derived from F-excision with flanking chromosomal DNA ��[19]�. It generates 5bp direct target repeat (DR) on insertion ��[20]� and carries similar ends to those of Tn3 and to IS101, a small 200bp sequence carried by the pSC101 plasmid ��[21,22]�.

Many other related transposons have since been identified with a highly diverse range of passenger genes (see ��[23]� and Fig. Tn3.4B). The tetracycline resistance transposon, Tn1721 from plasmid pRSD1 ��[24]� 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) ��[6]� are two of many early examples.

General Organization.

Members of the Tn3 transposon family form a tightly knit group with related transposases and DNA sequences at their ends. The basic Tn3 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 (Fig. 3.2i) ��[25]�. There is a large (~1000 aa) DDE transposase, TnpA, significantly longer than the DDE transposases normally associated with Insertion Sequences (IS) (see ��[26]�). 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; ��[27]�) 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. Tn3.2ii)(see ��[23]�). 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 C-terminal extension; 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 classic serine (S)-site-specific recombinase (e.g. ��[28,29]�); TnpI, a tyrosine (Y) recombinase similar to phage integrases ��[30]� (see ��[23]�); and a heteromeric resolvase combining a tyrosine recombinase, TnpS, and a divergently expressed helper protein, TnpT, with no apparent homology to other proteins ��[27,31]�. 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 ��[8]���[10]� (see later: Tn3 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 Insertion Sequences (IS) and integrons as well as other Tn3 family members – see ��[23]� - 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 (Fig. Tn3.3) also form part of the Tn3 family arsenal of passenger genes.

The diversity of Tn3 family members was investigated using a library of carefully annotated examples in the ISfinder database ��[32]�, those listed in Nicolas et al. ��[23]�, those resulting from a search of NCBI for previously annotated Tn3 family members (March 2018) and those obtained using a script, Tn3_TA_finder, which can searched for tnpA, tnpR, genes located in proximity to each other (Tn3finder, https://tncentral.proteininformationresource.org/TnFinder.html; Tn3_TA_finder, https://github.com/danillo-alvarenga/tn3-ta_finder) in complete bacterial genomes in the RefSeq database at NCBI. This yielded 190 Tn3 family transposons for which relatively complete sequence data (transposase, resolvase, and generally both IRs) were available. Full annotations can be found at TnCentral (https://tncentral.proteininformationresource.org/index.html). A tree based on the transposases of these transposons is shown in Fig. Tn3.4A ��[33]�.

The tree defines 7 deeply branching clades which supports the divisions proposed by Nicolas et al., ��[23]�. They were named after a representative Tn from each clade: Tn3; Tn4651; Tn3000; Tn1071; Tn21; Tn163; and Tn4330. As can be seen from Fig. Tn3.4A, the vast majority of Tn3 family members encode a tnpR/res resolution system and encode a TnpR without the C-terminal extension (shown by blue circles) and a small group which encodes a TnpR derivative with the C-terminal extension (Fig. Tn3.4A). However, a significant sub-group of the Tn4651 clade encodes the tnpS/tnpT/rst resolution system (pink circles) while the tnpI/irs is represented in only three cases.

An overview, extracted from TnCentral, of the diversity and distribution of different passenger genes within the Tn3 family and their presence in different bacterial hosts is shown in Fig. Tn3.4B.

Tn3 family complementation groups

Early studies on the relationship between different Tn3 family members revealed that they could be divided into different functional groups by genetic complementation of their tnpA and tnpR genes ��[34,35]�. Transposition-deficient tnpA mutants of Tn1721 (Tn21 clade; Fig. Tn3.4A) and the mercury resistance transposon Tn501 ��[36–39]� (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 ��[35]�. Similarly, a Tn21 tnpR mutant could be complemented by Tn21, Tn501 or Tn1721, but not by Tn3. Moreover, mutations in the Tn2603 tnpA and tnpR genes could be complemented by mercury resistance transposons Tn2613 and Tn501 (although Tn501 was much less efficient in complementation than Tn2613) but not by gamma delta, Tn2601 or Tn2602 (both of which resemble the Tn3 group – see Fig. Tn3.7A) ��[40]�. 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 ��[41]�.

Tn3 and Tn21 groups

Grinsted et al. ��[42]� 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.

The diversification of different Tn21 clade members was also examined ��[42]� (Fig. Tn3.6) and forms two subclades. One includes Tn21, Tn2613 (whose sequence is not available but which may be identical to Tn5060-AJ551280.1) and Tn3926 (with only a partial sequence available but which complements a tnpA-defective Tn21 but not Tn1721 or Tn501 mutants ��[43]�). The other includes Tn501, Tn1722, Tn1721 and Tn4653. Tn501 and Tn1721 are located in a sub-clade distinct from Tn21 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.

The Tn21 Clade

The Tn21 is a large group with 49 members at present in TnCentral (most of these are shown in Fig. Tn3.7A). Like the entire Tn3 family, Tn21 clade members possess highly conserved IRL and IRR (Fig. Tn3.7B, C and D).

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 (Fig. Tn3.7E) show a high degree of identity (Fig. Tn3.7F). However other tnpR/tnpA configurations also occur (Fig. Tn3.3; Fig. Tn3.7E) and their res sites (see below: The Tn1721, Tn21 and Tn501 res) show relatively good conservation (Fig. Tn3.7F)

Derivatives with a simple mercury operon.

In general, passenger genes in this clade are located upstream of tnpR and the res site (Figs. Tn3.7G-N). Ten carry only genes for resistance to mercury salts. Two of these, Tn5060 (AJ551280.1) (Tn3.7G), the proposed ancestor of the Tn21 integron group (Tn3.7I) ��[44]�, and Tn20 (AF457211.1) are nearly identical except for a few SNP and a deletion of a few base pairs in ufrM (Tn20). 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 (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.

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

At least 22 Tn21 clade members carry class I integrons (Fig. Tn3.7A; Tn3.7I) although the DNA sequence of some of these is not available. These are transmitted by Tn402 derivative transposons which exhibit pronounced target specificity (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. Tn3.7G). In one group, which all encode an identical mer operon, insertion occurred in a precise position in a gene of unknown function, ufrM (The Tn21 Lineage) (Fig. Tn3.7I). Since these occur at the same nucleotide, it seems possible that all diverged from a single insertion event.

In the others, the res site itself has been targeted: at two slightly different positions both in the Tn1696 (Fig. Tn3.7J) (also carrying a mer operon) and Tn1721 (with an mcp gene) groups (Fig. Tn3.7K) while a third example can be observed in Tn5045.1 carrying the tao gene cluster (Fig. Tn3.7L). 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 ��[45]�.

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 (Tn21 lineage) of the Tn401 transposition genes tniA,B,Q and its resolvase tniR (Tn402 family).

Derivatives with upstream passenger genes: colistin resistance.

Of the four colistin resistant examples (Fig. Tn3.7M): 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.

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. Tn3.7N), there is a clear breakpoint in identity which occurs at the res site. Sequence analysis (Fig. Tn3.7N) indicates that the break in identity occurs at the potential AT recombination dinucleotide (Resolution below) strongly suggesting that acquisition of various passenger genes frequently occurs by modular exchange via inter-res 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. Tn3.7O) 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.

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 ��[46]�, as part of the IS1-flanked r-determinant ��[5]� and its component antibiotic resistance genes were first mapped by restriction enzyme digestion and cloning ��[47]�. The Tn21 group of transposons appear to be very successful as judged by their distribution. This is arguably the result of acquisition of an integron platform permitting incorporation of various resistance genes as integron cassettes ��[42,48]� (Fig. Tn3.7A; Fig. Tn3.7G). 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 ��[49]� (Fig. Tn3.5; Fig. Tn3.7P).

Tn21 itself is a complex collection of intercalated TE and a comprehensive and detailed schemes for its formation has been proposed ��[42,48,49]� (see Fig. Tn3.6.; Fig. Tn3.7P). Unfortunately, although the DNA sequences of some of the component transposons are now available (e.g. Tn4, Tn21, Tn2411), many are not and comparison was based on physical and functional maps (restriction, genetic features) ��[40,49–51]�.

This scheme was later expanded with the addition of more up-to-date information to include a number of potential Tn21 descendants (see ��[48]�) (Fig. Tn3.7Q). It was proposed that a Tn21 precursor (Tn21) acquired an integron platform such as is found in Tn4 (for convenience, called In_Tn4 here) which then received an insertion of IS1353 into a resident IS1326 copy to generate In2 found in Tn21 ��[49]�.

Although the Tn21 group ancestor prior to acquisition of the mercury resistance genes is at present unknown, the later identification of a mercury resistance transposon, Tn5060 (AJ551280.1), isolated from the Siberian permafrost ��[44]� (Fig. Tn3.7R) provided a possible candidate for the hypothetical Tn21 precursor, Tn21. Other examples of this Tn such as Tn20 (AF457211) (Fig. Tn3.7I) can be identified which share a number snips with other members of the group compared to Tn5060 ��[52]� 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. Tn3.7S and Fig. Tn3.7P), a first step would be insertion into the ufrM (unknown function) gene of a Tn402 family transposon to provide the integron platform (Fig. Tn3.7S). Although it has been shown that transposition of defective Tn402 transposons (e.g. In0 and In2) can be complemented by a related, wildtype copy ��[53]�, 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. Tn3.7S) and leads to the formation of tnpM from the 3’ end of ufrM (serendipitously generating an ATG initiation codon) ��[48,54]�. TnpM has been suggested to be a transposition regulatory gene (but see Resolution below). Subsequent steps in the Tn21 lineage (Fig. Tn3.7T) 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).

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 ��[55]� (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 ��[56]�).

Tn2411 is not only the precursor of Tn21. It was proposed to give rise to additional transposons (Fig. Tn3.7Q)��[48]�: to Tn4 by insertion of a Tn3 transposon copy into the merP gene (Fig. Tn3.7U); to Tn5086 ��[57]� by deletion of the In_Tn4 IS1326 copy to generate Tn2608 ��[49]� and replacement of the aadA cassette and acquisition of dfrA7 (Fig. Tn3.7V); and to Tn2410 by replacement of the aadA cassette by an oxa cassette ��[51]�.

The complete DNA sequences of many of these Tn are not available but Tn5086 or Tn2608 could be reconstructed from Tn21 using the limited sequence data in ref ��[57]�. Moreover, using the reconstructed Tn5086 sequence in a BLAST search revealed an identical sequence in the E. coli SCU-164 chromosome (CP054343) and a nearly identical copy, in which the IRL had been interrupted by an insertion of IS4321, in E. coli plasmid pSCU-397-2 (CP054830) in addition to many closely related copies. This analysis suggests that deletion of IS1326 had occurred by nearly-precise excision ��[58]� since the deletion junction observed in Tn5086 ��[57]� is not the original sequence identified in Tn2411. Indeed, the DNA sequences of Tn2411, Tn2608 and Tn5086, (Fig. Tn3.7V) suggest that In_Tn2608 and In22 were derived by deletion from a structure similar to In_Tn4 because neither carry an IS1326 copy although they both retain the tip of the IRL (4 bp for In_Tn2608 and 3bp for In22) at one end and are missing 5bp of In_Tn4 DNA flanking the right IS1326 end.

Tn21 was also proposed to give rise to a number of different transposons ��[48,51]�: to Tn1831 by IS1326-mediated deletion (IS1326 in IS1326::IS1353 is almost certainly functional) rightwards towards or past the IRt end of the integron while retaining the IS (Fig. Tn3.7Q; Fig. Tn3.7W); to Tn2607 by insertion of Tn2601 (probably similar to Tn3) into the mer genes; to Tn2424 by insertion of IS161 to first generate Tn2425 and subsequent acquisition of two integron cassettes aacA1 and catB2 (Fig. Tn3.7Q; Fig. Tn3.7X); and to Tn2603 by insertion of an oxaA1 cassette.

Tn1721 and (tandem) amplification of the tet genes

Tn1721 (Fig. Tn3.7K) carries resistance to tetracycline (tet), is present on plasmid pRSD1 and is capable of undergoing amplification to generate tandem repeats ��[59]�. It was isolated by transposition to a lambda phage followed by a further transposition event onto plasmid R388 ��[24]� where it retained the ability to amplify ��[24]�. Amplification was identified using restriction enzyme mapping (Fig. Tn3.7Y) which showed a duplication of an EcoRI 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 Tn1721. Indeed, amplification was shown to depend on the host recA gene ��[60]�.

The Tn163 Clade

There are 39 members of this clade (May 2021). Two (TnSku1 [Tn7197] CP 002358.1 and TnAmu_p NC_015188.1) have acquired toxin/antitoxin gene pairs and most members (Fig. Tn3.8A; Fig. Tn3.8B) encode divergent tnpR and tnpA genes. There are a number of members without passenger genes as in the Tn21 clade (e.g. Tn6137, TnMex22[ Tn7165], TnMex38 [Tn7166], TnChe1, [Tn7155], TnAmu1 [Tn7138 ], TnAli20 [Tn7136], Tn6122, Tn3434).

One small related group (Tn6137, Tn6136, Tn6134, Tn6138) all identified within the hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26 genome ��[61]� 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. Tn3.8C). Alignment against Tn6136 (Fig. Tn3.8Ci) 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. Tn3.8ii). Both Tn6134 and Tn6138 carry the same passenger gene (Fig. Tn3.8Ciii) whose insertion has occurred proximal to IRL (Fig. Tn3.8Civ).

The ancestor of another group of related transposons, the Tn5393 group (Fig. Tn3.8D), 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. Tn3.8E) and of a complex set of mobile elements to generate Tn5393.4 (AJ627643; Alcaligenes faecalis). 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. Tn3.8F) 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). 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.

The Tn4430 Clade

At present (May 2021) this clade is composed of only 11 examples (Fig. Tn3.9A). 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. Tn3.9B). There are two small groups: Tn1546 which carry vancomycin resistance genes, and Tn6332 which carry mercury resistance genes.

The Tn1564 Vancomycin Resistance Group

Resistance to Vancomycin in Enterococci appeared in 1988 ��[62]�, was shown to be transmissible ��[63,64]� and carried by a transposon, Tn1546 (M97297.1) ��[65]�. 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 ��[65,66]� 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 ��[67]�, in addition to a number of other IS1216 insertions (often in multiple copies and accompanied by neighboring deletions) ��[66,68]�. A number of these insertion/deletion derivatives have been identified from several sources and different geographical locations ��[66–69]� (Fig. Tn3.9C)

The Mercury Resistance Group

Within the mercury resistance group (Tn6294-LC015492.1, Tn5084-AB066362.1, Tn6332-LC155216.1 and TnMERI1-LC152290 – note that we have reconstituted the left end by comparison with Y08064; Fig. Tn3.9D), the mercury resistance genes are expressed to the left while TnpR and TnpA are expressed to the right. All four carry additional copies of merB and merR. Huang et al ��[70]� have shown that expression of the mercury resistance genes of TnMERI1 is driven by three promoters (Fig. Tn3.9E). Comparison with Tn6294 suggests that the mercury gene set has been exchanged by recombination at the level of the res site (Fig. Tn3.9D).

The sequences of two closely related members of the same group, Tn5083 and Tn5085, are incomplete ��[71]�.

The Tn3 Clade

This clade includes the classical Tn1, 2 and 3 (see Historical) as well as Tn1000. There are 29 examples of the Tn3 clade (of which 26 can be found in TnCentral) (Fig. Tn3.10A) which fall into two subgroups. The majority have divergently expressed tnpR and tnpA and most carry passenger genes (Fig. Tn3.10B). The res sites of each sub-group show significant similarity (Fig. Tn3.10C). A number carry toxin-antitoxin genes generally located between the divergent tnpR and tnpA. These are of two types (Fig. Tn3.10A) and appear to be specific for each subgroup. Passenger genes can be located upstream of downstream of the tnpR/tnpA transposition module (Fig. Tn3.10B). All except two encode tnpR type resolvases. The two which do not, TnBth4 and Tn5401, also encode a TA module.

Importance of ISEcp1 in bla CTX-M-expression

There are examples of members of the Tn3 clade which carry insertions of ISEcp1-like sequences (IS1380 family) closely upstream of a bla-CTX-M gene. Indeed, upstream insertion of ISEcp1 derivatives have been identified associated with a number of different bla-CTX-M variants in both Tn3 and other groups ��[72–77]�. In some examples, this is limited to an isolated right end ��[77]� which is responsible for expression of the bla-CTX-M gene by providing a mobile promoter ��[78]�.

The Tn3 group

Tn3, Tn1, Tn1MER, Tn2, Tn2.1 and Tn3.1. all carry a probable internal IR upstream of the bla gene (Fig. Tn3.10D) which acts as a hotspot for IS231A insertion and was initially observed in the bla gene of plasmid pBR322 ��[79]�. Tn2 and Tn2.1 are identical except for the ISEcp1 insertion which also carries an internal IS1 insertion (Fig. Tn3.10E). 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.

Inspection of the alignment (Fig. Tn3.10E) 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 Tn1, 2 and 3 ��[80]� (for res, see Fig. Tn3.10C) and an evolutionary pathway involving a combination of homologous and resolvase-mediated recombination has been proposed. This can be detected by the distribution of SNIPs 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. Tn3.10F) are closely related and differ essentially by insertion of ISXac1 and ISXac5 (Fig. Tn3.10G) 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.

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. Tn3.10H) 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. Tn3.11A).

The Tn5501 cluster.

There are a number of Tn5501 examples (Fig. Tn3.11B). 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. Tn3.11A). Tn5501.12 appears to have acquired different TA genes (HTH_37, GP49) by recombination at the res site (Fig. Tn3.11C).

The relationship between members of the cluster is shown in Fig. Tn3.11C. 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. Tn3.11Di and Dii). All leave the TA module intact. In only one example, the toxin gene has undergone deletion leaving the antitoxin intact (Fig. Tn3.11Diii). The left junction is less clear and difficult to interpret.

A number of Tn5501 derivatives are related by IS insertions and deletion (Fig. Tn3.)

Finally, a small group of Tns which, like Tn5501.12, all carry the HTH_37/GP49 TA pair is shown in Fig. Tn3.11F. 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.

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 the antibiotics of last resort ��[81]�. The 10kb transposon, Tn4401 has been instrumental in the spread of the carbapenem resistance gene bla_KPC. It was described in 2008 in a number of clinical isolates of Klebsiella pneumoniae and Pseudomonas aeruginosa from the United States, Colombia and Greece ��[82,83]�. Members of this small group have divergently expressed tnpR and tnpA genes located towards the left end and bla_KPC towards the right end downstream from tnpA (Fig. Tn3.11B) flanked by two different insertion sequences, ISKpn6 and ISKpn7 (Fig. Tn3.11G). 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 ��[84]�, was found to carry a deletion of about 200 bp upstream of bla while in a fourth, Tn4401d ��[85]�, the ISKpn7 copy along with flanking DNA has undergone deletion to leave a 3’ segment of bla_KPC and a 5’ segment of tnpA and therefore would not be capable of autonomous transposition. 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 bla_KPC ��[86]�. Closer analysis using RACE 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; IS21 chapter; formation of hybrid promoters sections).

The Tn4651 Clade

The Tn4651 mix of radically different structures

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

The tnpT/S/rst clusters

In the first tnpT/S/rst cluster, mostly from the plant pathogen Xanthomonas (Fig. Tn3.12C), 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.

TnXax1 derivatives ��[25]� are generally vehicles for pathogenicity genes such as Transcriptional Activator Like Effectors (TALE genes), cell wall degrading enzymes (mtl) and genes (xop) involved in type III secretion system (TTSS) translocation of effector proteins into host plant cells ��[87]� (Fig. Tn3.12C). TnXax1 derivatives can include IR which are significantly longer (72/92 bp) than the 38-40bp characteristic of the Tn3 family (Fig. Tn3.12D) 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. Tn3.12E) while their right ends appear more homogeneous (Fig. Tn3.12F).

The second tnpT/S/rst cluster is characterized by Tn4651, a toluene-catabolic transposon identified in from Pseudomonas putida plasmid pWW0 ��[88]�. 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 ��[88]�, TnpC was shown to regulate TnpA expression post-transcriptionally ��[89]�. Moreover, the host protein IHF binds to sites in both Tn4652 ends (Fig. Tn3.12G) ��[90,91]�. These overlap the region protected by TnpA binding ��[91]� 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 ��[91]�. TnpA protects an extensive region encompassing the IRs and 8-9 bp of flanking DNA (Fig. Tn3.12G). Tn4652 transposition appears to be elevated in stationary phase, involves the stationary phase sigma factor, sigma S ��[92]�, and is limited by the levels of IHF ��[91]� whose level is increased in stationary phase. Another DNA chaperone host factor, FIS, has a negative effect on transposition, apparently by competing for IHF binding ��[91,93]�. IHF and FIS have been implicated in other transposition systems such as IS10 (IS4 family). Moreover, Tn1000 (Tn3 clade) carries an IHF binding site proximal to each IR which acts copoperatively to increase TnpA binding and immunity ��[94,95]�. One additional interface with host physiology is the observation that the CorR/CorS two component system regulates transposition positively ��[96]�.

Other members of the cluster include: Pseudomonas sp. mercury resistance transposon Tn5041 ��[97,98]� ; Tn4676, a long (72,752bp) and complex Pseudomonas resinovorans carbazole-catabolic transposon from plasmid pCAR1 ��[99,100]�; and Tn4661, a Pseudomonas aeruginosa cryptic transposon ��[27]�. All include tnpA, tnpC and the tnpS/T resolution system.

Tn5041 transposition has also been addressed experimentally ��[97,101]� and was observed to be host-dependent ��[101]�: it occurred in the original Pseudomonas sp. KHP41 host but not in P. aeruginosa PAO-R or in Escherichia coli K12. Interestingly, transposition in these strains was found to be complemented by the Tn4651 transposase gene (tnpA) and the region which determines this host dependence was mapped to a 5’ tnpA gene segment by construction of hybrid Tn5041-Tn4651 tnpA genes. Tn5041 apparently acquired its mer operon from a derivative of Tn21 or Tn501 ��[101]�. It is reported to be preceded by a 24 bp element with 75% sequence similarity to the outermost part of IRs typical for Tn21-like transposons.

The Tn1071 Clade

The Tn1071 group.

Members of this small group are often associated with xenobiotic catabolism and other “exotic” functions (Fig. Tn3.13A).

Tn1071 itself (Fig. Tn3.13Bi), the founding member, was identified as part of a compound transposon, Tn5271, in Comamonas testosteroni where it flanks a chlorobenzoate catabolic operon in ��[102]�. It is unusual since it carries only tnpA and not tnpR, has unusually long (110bp) IR (Fig. Tn3.13Bii) 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 ��[103]� and ISBusp1 (aka ISBmu13; NC_007509.1) from the Burkholderia multivorans ATCC 17616 genome ��[104]�, 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.

A limited functional analysis of Tn1071 transposition is available ��[105]�. It was only able to transpose at high frequencies in two environmental -proteobacteria Comamonas testosteroni and Delftia acidovorans but not in Agrobacterium tumefaciens (-proteobacteria) or Escherichia coli, Pseudomonas alcaligenes and Pseudomonas putida (all -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, Tn1071 probably forms “pseudo-compound transposons” ��[106–108]�. In these structures the flanking Tn1071 copies must be in direct orientation as a consequence of the homologous recombination event required to resolve the cointegrate 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. IS6.8B). “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 cointegrated would be expected to contain two directly repeated copies of the entire transposon sat the donor/target junctions.

A significant number of Tn1071-associated xenobiotic-degrading genes on many catabolic plasmids have been documented by population-based PCR ��[109–111]� and genetic studies ��[112,113]�. Tn5271 itself is widely distributed in bacteria isolated from a large ground water bioremediation site ��[111]� and plasmid derivatives carrying the transposon together with a third Tn1071 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 ��[111]� (Fig. for intramol transposition).

A number of additional potential compound transposons have been identified although these may be inactive: a >28kb transposon, Tn5330 (AF029344), from Delftia acidicorans ��[114]� carries the entire 2,4-dichlorophenoxyacetic acid degradation pathway and, although the sequence data for the flanking IS1071 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 ��[115]� 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 ��[116]� (Fig. Tn3.13Ci), 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 ��[116]�. TnHad2 also appeared to carry a functional res site.

Tn1071 also flanks atrazine degrading genes in plasmid Pseudomonas pADP-1 (U66917) ��[117]� in a structure with three directly repeated Tn1071 copies intercalated with three copies of an IS91 family member, ISPps1. These are apparently generated by duplication events since regions with identical sequence stretch from the oriIS end of ISPps1 through Tn1071 and terminate just before the atz genes (Fig. Tn3.13Cii). The repeated regions also includes the DR sequences at each Tn1071 except for that at the far right.

There are a number examples of other structures with multiple Tn1071 copies and in a large proportion of these cases, the multiple copies occur in direct repeat. They are associated with plasmids which degrade the phenylurea herbicide linuron e.g. pBPS33-2 (CP044551) ��[118]� and have been isolated from a variety of bacteria with the capacity to degrade a wide range of chlorinated aromatics and pesticides ��[109]� or p-toluene sulfonate (TSA) where they flank the TSA genes in plasmid pTSA (AH010657) ��[119]�.