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.