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).