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| − | Tn3 family.
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| − | <span style="color:#ff0000;">REMEMBER TO GO THROUGH TO ADD REGISTER Tn NAMES AT THE END! </span>
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| − | <span style="color:#ff0000;">New to add to Excel list:</span><span style="color:#ff0000;">, Tn4662a.2-MF495478, ISAs20, TnDsu1-NC_016616 (to do), TnDra1-CP015084 (to do), Tn4676-AB088420 (to do)</span>
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| − | <span style="color:#44546a;">Historical</span>
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| − | Members of the Tn''3'' 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 Tn''1'', was isolated from the plasmid RP4 [1] while the closely related TnB and TnC (later called Tn''2'' and Tn''3'' respectively) were isolated from plasmids RSF1010 [2] and R1 [3,4]. Tn''3'' proved to be inserted into another, larger Tn''3'' family transposon, Tn''4'' [4]. A number of early studies using electron microscope DNA heteroduplex analysis (e.g. [5–7] <span style="color:#5b9bd5;">Fig. Tn3.1</span>) 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) Tn''3'' 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 (<u>I</u>nternal <u>R</u>esolution <u>S</u>ite) (<span style="color:#5b9bd5;">Fig. Tn3.2i</span>). In its absence, insertion of two complete, directly repeated, Tn''3'' copies occurred [8]. 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'' [10] according to the Shapiro cointegrate model of replicative transposition (<span style="color:#5b9bd5;">Fig. Tn3.2ii</span><nowiki>; </nowiki>[11] <span style="color:#5b9bd5;">Fig. 2 Early models</span>). Indeed, Tn''3 ''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].
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| − | A related TE, or Tn1''000'', 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 Tn''3'' and to IS''101'', a small 200bp sequence carried by the pSC101 plasmid [21,22].
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| − | Many other related transposons have since been identified with a highly diverse range of passenger genes (see [23] and <span style="color:#44546a;">Fig. Tn3.4B</span>). The tetracycline resistance transposon, Tn''1721'' from plasmid pRSD1 [24] and the multi-resistance transposons, Tn''4'' from R6-5 and Tn''21'', a component of the 25 kb resistance determinant (r-det) of the plasmid NR1 (R100) [6] are two of many early examples.
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| − | <span style="color:#44546a;">General Organization</span>'''.'''
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| − | 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 (<span style="color:#5b9bd5;">Fig. 3.2i</span>) [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.
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| − | A second feature of members of this transposon family is that they carry short (~100-150bp) DNA segments, ''res'' (for ''res''olution) or ''rst'' (for <u>''r''</u>esolution site tnp<u>''S''</u> tnp<u>''T''</u> – <span style="color:#44546a;">see below</span><nowiki>; </nowiki>[27])<span style="color:#ff0000;"> </span>at<span style="color:#ff0000;"> </span>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 (<span style="color:#5b9bd5;">Fig. Tn3.2ii</span>)(see [23]). This highly efficient recombination system is assured by a transposon-specified sequence-specific recombinase enzyme: the resolvase.
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| − | There are at present three known major resolvase types: TnpR (which includes two subgroups, '''long''' and '''short''' <span style="color:#5b9bd5;">with and without a C-terminal extension; Resolution</span>), 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 <span style="color:#5b9bd5;">Fig. Tn3.3</span>. 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.<span style="color:#ff0000;"> </span>But where analyzed, ''res'' sites also include promoters which drive both transposase and resolvase expression. Indeed, TnpR from Tn''3'' was originally named for its ability to repress transposase expression by binding to these sites [8][10]. (see later: <span style="color:#5b9bd5;">Tn3 family resolution systems</span>)
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| − | <span style="color:#44546a;">Diversity: TnpA Tree.</span>
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| − | 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 Tn''3'' 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 (<span style="color:#5b9bd5;">Fig. Tn3.3</span>)<span style="color:#5b9bd5;"> </span>also form part of the Tn''3'' family arsenal of passenger genes.
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| − | <span style="color:#000000;">The diversity of Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family members was investigated using a library of carefully annotated examples in the ISfinder database </span><span style="color:#000000;">[32]</span><span style="color:#000000;">, those listed in Nicolas et al. </span><span style="color:#000000;">[23]</span><span style="color:#000000;">, those resulting from a search of NCBI for previously annotated Tn</span><span style="color:#000000;">''3 ''</span><span style="color:#000000;">family members (March 2018) and those obtained using a script, Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;">_TA_finder, which can searched for </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;">, </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;">, genes located in proximity to each other (Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;">finder, </span>[https://tncentral.proteininformationresource.org/TnFinder.html https://tncentral.proteininformationresource.org/TnFinder.html]<span style="color:#000000;"><nowiki>; Tn</nowiki></span><span style="color:#000000;">''3''</span><span style="color:#000000;">_TA_finder, </span><span style="color:#0000ff;">https://github.com/danillo-alvarenga/tn3-ta_finder</span><span style="color:#000000;">) in complete bacterial genomes in the RefSeq database at NCBI. This yielded 190 Tn</span><span style="color:#000000;">''3 ''</span><span style="color:#000000;">family transposons for which relatively complete sequence data (transposase, resolvase, and generally both IRs) were available. Full annotations can be found at TnCentral (</span>[https://tncentral.proteininformationresource.org/index.html https://tncentral.proteininformationresource.org/index.html]<span style="color:#000000;">). A tree based on the transposases of these transposons is shown in </span><span style="color:#5b9bd5;">Fig. Tn3.4A</span><span style="color:#ff0000;"> </span><span style="color:#000000;">[33]</span><span style="color:#000000;">.</span>
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| − | 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: Tn''3''<nowiki>; Tn</nowiki>''4651''<nowiki>; Tn</nowiki>''3000''<nowiki>; Tn</nowiki>''1071''<nowiki>; Tn</nowiki>''21''<nowiki>; Tn</nowiki>''163''<nowiki>; and Tn</nowiki>''4330''. As can be seen from <span style="color:#5b9bd5;">Fig. Tn3.4A</span>, the vast majority of Tn''3'' 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 (<span style="color:#5b9bd5;">Fig. Tn3.4A</span>). 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.
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| − | 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 <span style="color:#5b9bd5;">Fig. Tn3.4B</span>.
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| − | <span style="color:#44546a;">Tn3 family complementation groups </span>
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| − | 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 [34,35]. Transposition-deficient ''tnpA'' mutants of Tn''1721'' (Tn''21'' clade; <span style="color:#5b9bd5;">Fig. Tn3.4A</span>) and the mercury resistance transposon Tn''501'' [36–39] (close to Tn''1721'' in the Tn''21'' clade;) could be complemented ''in trans'' by co-resident wild type copies of either Tn''21'', Tn''501'', or Tn''1721'', while transposition of a Tn''21 tnpA'' mutant could only be restored by Tn''21''. Moreover, Tn''3'' was unable to complement either Tn''21'', Tn''501'', or Tn''1721'', and ''vice versa'' [35]. Similarly, a Tn''21'' ''tnpR'' mutant could be complemented by Tn''21'', Tn''501'' or Tn''1721'', but not by Tn''3''. Moreover, mutations in the Tn''2603'' tnpA and tnpR genes could be complemented by mercury resistance transposons Tn''2613'' and Tn''501'' (although 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 <span style="color:#5b9bd5;"><u>Fig. Tn3.7A</u></span>) [40]. In this context, it is perhaps useful to note the Tn''501'' and Tn''1721'' are located at some distance from Tn''21'' in the ''tnpA'' phylogenetic tree<span style="color:#44546a;">. </span>This reinforced the idea, based principally on the direction of transcription of their ''tnpA'' and ''tnpR ''genes, that the Tn''3'' family could be divided into 2 major groups: Tn''3'' and Tn''21 ''[41].
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| − | <span style="color:#44546a;">Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> and Tn</span><span style="color:#44546a;">''21''</span><span style="color:#44546a;"> groups </span>
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| − | Grinsted et al. [42] identified at least five Tn''3'' family subgroups which correspond to those shown in <span style="color:#5b9bd5;">Fig. Tn3.4A</span>. In addition to the Tn''3'' and Tn''21'' subgroups, the others included Tn''2501'' (Tn''163'' subgroup), Tn''917''/Tn''551'' (Tn''4430'' subgroup) and Tn''4556'' (Tn''3000'' subgroup). Tn''917'' and Tn''551'' are quasi-identical and Tn''4430'' 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 Tn''3'' family transposition modules (<span style="color:#5b9bd5;">Fig. Tn3.5</span>) 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 Tn''3'' subgroups by ''tnpR''/''res'' inversion and sequence divergence. For Tn such as Tn''4430'', 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.
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| − | The diversification of different Tn''21'' clade members was also examined [42] (<span style="color:#5b9bd5;">Fig. Tn3.6</span>) and forms two subclades. One includes Tn''21'', Tn''2613'' (whose sequence is not available but which may be identical to Tn''5060''-AJ551280.1) and Tn''3926'' (with only a partial sequence available but which complements a ''tnpA''-defective Tn''21'' but not Tn''1721'' or Tn''501'' mutants [43]). The other includes Tn''501'', Tn''1722'', Tn''1721'' and <span style="color:#000000;">Tn</span><span style="color:#000000;">''4653''</span>. Tn''501 ''and Tn''1721'' are located in a sub-clade distinct from Tn''21'' and Tn''5060'' (<span style="color:#5b9bd5;">Fig. Tn3.4A</span>). In this scheme, mercury resistance was proposed to have been acquired twice independently in each subclade, early in the Tn''21'' subclade lineage and later in the line leading to Tn''501''. The ancestor of Tn''21'' had acquired an integron platform transported by a <span style="color:#5b9bd5;">Tn</span><span style="color:#5b9bd5;">''402''</span><span style="color:#5b9bd5;"> family</span> transposon and Tn''1721'' was derived from Tn''1722'' by acquisition of a ''tet'' resistance gene.
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''21''</span><span style="color:#44546a;"> Clade </span>
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| − | The Tn''21'' is a large group with 49 members at present in TnCentral (most of these are shown in <span style="color:#5b9bd5;">Fig. Tn3.7A</span>). Like the entire Tn''3'' family, Tn''21'' clade members possess highly conserved IRL and IRR (<span style="color:#5b9bd5;">Fig. Tn3.7B, C and D</span>).
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| − | 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 (<span style="color:#5b9bd5;">Fig. Tn3.7E</span><span style="color:#44546a;">) </span>show a high degree of identity (<span style="color:#5b9bd5;">Fig. Tn3.7F</span>). However other ''tnpR''/''tnpA'' configurations also occur (<span style="color:#44546a;">Fig. Tn3.3; Fig. Tn3.7E</span>) and their ''res'' sites (see below: <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''1721''</span><span style="color:#44546a;">, Tn</span><span style="color:#44546a;">''21''</span><span style="color:#44546a;"> and Tn</span><span style="color:#44546a;">''501''</span><span style="color:#44546a;"> </span><span style="color:#44546a;">''res''</span>) show relatively good conservation (<span style="color:#44546a;">Fig. Tn3.7F</span>)
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| − | <span style="color:#44546a;">Derivatives with a simple mercury operon.</span>
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| − | In general, passenger genes in this clade are located upstream of ''tnpR'' and the ''res'' site (<span style="color:#44546a;">Figs. Tn3.7G-N</span>).<span style="color:#c00000;"> </span>Ten<span style="color:#c00000;"> </span>carry only genes for resistance to mercury salts. Two of these, Tn''5060'' (AJ551280.1) (<span style="color:#44546a;">Tn3.7G</span>), the proposed ancestor of the Tn''21'' integron group (<span style="color:#44546a;">Tn3.7I</span>) [44], and Tn''20'' (AF457211.1) are nearly identical except for a few SNP and a deletion of a few base pairs in ''ufrM'' (Tn''20'')''. ''These are quite different in sequence both in the ''mer'' operon and in ''tnpR''/''tnpA'' segments from the other transposons of similar organization. [https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn1696.1-CP047309 Tn][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn1696.1-CP047309 1696.1][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn1696.1-CP047309 (CP047309]) and [https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn5036-Y09025 Tn][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn5036-Y09025 5036][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn5036-Y09025 (Y09025]) differ by only a few SNPs while [https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn4378-CP000355 Tn][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn4378-CP000355 4378 ][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn4378-CP000355 (CP000355]), [https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6203-CP065412 Tn][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6203-CP065412 6203][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6203-CP065412 (CP065412]) and [https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6346-KM659090 Tn][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6346-KM659090 6346][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6346-KM659090 (KM659090]) are also quite different from the these. [https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn4378-CP000355 Tn][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn4378-CP000355 4378] and [https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6203-CP065412 Tn][https://tncentral.proteininformationresource.org/cgi-bin/tn_report.pl?id=Tn6203-CP065412 6203] show many sequence differences along their entire length as does Tn''As2'' (JN106175.1) while clearly, Tn''6346'' shares identity with Tn''4378 ''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 (<span style="color:#44546a;">Tn3.7H</span>). A similar recombination has occurred with Tn''501''. In addition, Tn''4380'' appears to have been derived from Tn''6346'' by deletion of the entire ''res'' site. Thus Tn''4378'', Tn''6436'' (Tn''4380'') and Tn''501'' share highly related ''mer'' operons but vary in the sequences of ''tnpR'' and ''tnpA''.
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| − | <span style="color:#44546a;">Derivatives with class I integrons: 2 events leading to multiple antibiotic resistance</span>
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| − | At least 22 Tn''21'' clade members carry class I integrons (<span style="color:#44546a;">Fig. Tn3.7A; </span><span style="color:#44546a;">Tn3.7I</span>) although the DNA sequence of some of these is not available. These are transmitted by Tn''402'' derivative transposons which exhibit pronounced target specificity (<span style="color:#44546a;">Tn</span><span style="color:#44546a;">''402''</span><span style="color:#44546a;"> family</span>) and show a preference for insertion into or close to Tn''3'' family ''res'' sites or into plasmid ''res'' sites. A major pathway for the acquisition of passenger genes was the initial integration of a Tn''402''-like transposon which carried a class I integron platform. The integron insertions have occurred at one of two positions in the Tn''5060'' /Tn''20'' related examples (<span style="color:#44546a;">Fig. Tn3.7G</span>). In one group, which all encode an identical ''mer'' operon, insertion occurred in a precise position in a gene of unknown function, ''ufrM'' (<span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''21 ''</span><span style="color:#44546a;">Lineage</span>) (<span style="color:#44546a;">Fig. Tn3.7I</span>). Since these occur at the same nucleotide, it seems possible that all diverged from a single insertion event.
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| − | In the others, the ''res'' site itself has been targeted: at two slightly different positions both in the Tn''1696'' (<span style="color:#44546a;">Fig. Tn3.7J</span>) (also carrying a ''mer'' operon) and Tn''1721'' (with an ''mcp'' gene) groups (<span style="color:#44546a;">Fig. Tn3.7K</span>) while a third example can be observed in Tn''5045''.1 carrying the ''tao'' gene cluster (<span style="color:#44546a;">Fig. Tn3.7L</span>). The fact that integrons In2 and In4 are located in different sequence environments in two distinct mercury resistance transposons, Tn''21'' and Tn1''696 ''has previously been noted [45].
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| − | Thus, although widespread in nature, class 1 integrons appear to have inserted in only six target sequences in the entire Tn''21'' 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 (<span style="color:#44546a;">Tn</span><span style="color:#44546a;">''21''</span><span style="color:#44546a;"> lineage</span>) of the Tn''401'' transposition genes ''tniA,B,Q'' and its resolvase ''tniR'' (<span style="color:#44546a;">Tn</span><span style="color:#44546a;">''402''</span><span style="color:#44546a;"> family</span>).
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| − | <span style="color:#44546a;">Derivatives with upstream passenger genes: colistin resistance.</span>
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| − | Of the four colistin resistant examples (<span style="color:#44546a;">Fig. Tn3.7M</span>): Tn''Sen1.1'' [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7191''</span>] and Tn''Sen1.2'' [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7192''</span>] are nearly identical except that Tn''Sen1.2'' carries an IS''Pa96'' insertion; both Tn''Ec026'' [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7159''</span>] and Tn''MCR5ECO26H11'' [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7163''</span>] 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 Tn''Sen11.2''/Tn''Sen1.2'' and Tn''EcO26''/ Tn''MCR5ECO26H11'' carry divergent ''tnpR'' and ''tnpA''.
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| − | <span style="color:#44546a;">Derivatives with upstream passenger genes: other passengers.</span>
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| − | There are a number of other Tn''21'' clade members with different upstream passenger genes. Analysis of these reveals that, although there has been some diversification of the ''tnpR'' and ''tnpA'' genes (<span style="color:#44546a;">Fig. Tn3.7N</span>), there is a clear breakpoint in identity which occurs at the ''res'' site. Sequence analysis (<span style="color:#44546a;">Fig. Tn3.7N</span>) indicates that the break in identity occurs at the potential AT recombination dinucleotide (<span style="color:#44546a;">Resolution</span> below) strongly suggesting that acquisition of various passenger genes frequently occurs by modular exchange via inter-''res'' recombination.
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| − | <span style="color:#44546a;">Derivatives with divergent tnpR and tnpA.</span>
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| − | There are a number of Tn''21'' clade members in which the ''tnpR'' and ''tnpA'' genes are expressed divergently. Several of these (e.g. Tn''4659'', Tn''Acsp1'' [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7133''</span><span style="color:#000000;">]</span>, Tn''Ec1'' [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7158''</span>] and Tn''Sba14 ''[<span style="color:#000000;">Tn</span><span style="color:#000000;">''7190''</span><span style="color:#000000;">]</span>) (<span style="color:#44546a;">Fig. Tn3.7O</span>) do not encode passenger genes and are not closely related, while others encode heavy metal resistance operons located between ''tnpR'' and ''tnpA'' (e.g. Tn''LfArs ''[<span style="color:#000000;">Tn</span><span style="color:#000000;">''7162''</span><span style="color:#000000;">]</span>, Tn''OtChr ''[<span style="color:#000000;">Tn</span><span style="color:#000000;">''7169''</span><span style="color:#000000;">]</span>) while Tn''Pa38'' [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7172''</span>] encodes genes of unknown function and Tn''Sod9'' [<span style="color:#000000;">Tn7199</span>] is the only example in the Tn''21'' clade to encode a <span style="color:#44546a;">Toxin/Antitoxin</span> gene pair. These are not closely related.
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''21 ''</span><span style="color:#44546a;">Lineage.</span>
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| − | The Tn''21'' lineage is an example of the plasticity of Tn''3 ''family transposons. Tn''21'' was originally identified in the multiple antibiotic resistance plasmid NR1/R100 [46], as part of the IS''1''-flanked r-determinant [5] and its component antibiotic resistance genes were first mapped by restriction enzyme digestion and cloning [47]. The Tn''21'' 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] (<span style="color:#44546a;">Fig. Tn3.7A; </span><span style="color:#44546a;">Fig. Tn3.7G</span>). Tanaka and collaborators proposed in the early 1980s that Tn''21''-like transposons which carry a variety of antibiotic resistance genes are related and evolved from an ancestor carrying a mercury resistance operon [49] (<span style="color:#44546a;">Fig. Tn3.5; Fig. Tn3.7P</span>).
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| − | Tn''21'' itself is a complex collection of intercalated TE and a comprehensive and detailed schemes for its formation has been proposed [42,48,49] (see <span style="color:#44546a;">Fig. Tn3.6.</span><nowiki>;</nowiki><span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.7P</span>). Unfortunately, although the DNA sequences of some of the component transposons are now available (e.g. Tn''4, ''Tn''21'', Tn''2411''), many are not and comparison was based on physical and functional maps (restriction, genetic features) [40,49–51].
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| − | This scheme was later expanded with the addition of more up-to-date information to include a number of potential Tn''21'' descendants (see [48]) (<span style="color:#44546a;">Fig. Tn3.7Q</span>). It was proposed that a Tn''21'' precursor (Tn''21'') acquired an integron platform such as is found in Tn''4'' (for convenience, called In_Tn''4'' here) which then received an insertion of IS''1353'' into a resident IS''1326'' copy to generate In2 found in Tn''21'' [49].
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| − | Although the Tn''21'' group ancestor prior to acquisition of the mercury resistance genes is at present unknown, the later identification of a mercury resistance transposon, Tn''5060 ''(AJ551280.1), isolated from the Siberian permafrost [44] (<span style="color:#44546a;">Fig. Tn3.7R</span>) provided a possible candidate for the hypothetical Tn21 precursor, Tn''21''. Other examples of this Tn such as Tn''20 ''(AF457211) (<span style="color:#44546a;">Fig. Tn3.7I</span>)<span style="color:#ff0000;"> </span>can be identified which share a number snips with other members of the group compared to Tn''5060'' [52] and therefore is perhaps a better candidate as an ancestor.
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| − |
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| − | An alternate view of the path from Tn''5060'' to Tn''21'' is that evolution of the integron platform occurred “''in situ''” by the gradual loss/accumulation of component TE. In this scheme (<span style="color:#44546a;">Fig. Tn3.7S</span><span style="color:#44546a;"> </span>and<span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.7P</span><span style="color:#44546a;">)</span>, a first step would be insertion into the ''ufrM'' (unknown function) gene of a <span style="color:#44546a;">Tn</span><span style="color:#44546a;">''402''</span><span style="color:#44546a;"> family </span>transposon to provide the integron platform (<span style="color:#44546a;">Fig. Tn3.7S</span>). Although it has been shown that transposition of defective Tn''402'' 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 Tn''402'' derivative with a complete functional set of Tn''402'' transposition genes. We have chosen a simple integron platform, In_Tn''1721.1'' from Tn''1721.1'' (HQ730118.1), for convenience. This carries ''tniA,B,Q,'' the resolvase ''tniR'' together with the Tn''402'' ''res'' site, both ends (IRt and IRi), the integron integrase ''int'' and a common ''qac'' gene cassette. Insertion into the Tn''5060'' ''urfM'' gene generates a 5 bp DR (<span style="color:#44546a;">Fig. Tn3.7S</span>) 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 <span style="color:#44546a;">Resolution </span><span style="color:#000000;">below</span>). Subsequent steps in the Tn''21'' lineage (<span style="color:#44546a;">Fig. Tn3.7T</span>) would then involve modification of the integron platform by acquisition of the typical GNAT (previously known as ''orf5'') and ''sul'' genes, decay of the Tn''402'' transposition genes and insertion, first of IS''1326'' (resulting in In0) followed by acquisition of the ''aadA'' integron cassette (generating In_Tn''4'') and, finally, insertion of IS''1353'' into IS''1326'' (IS''1326''::IS''1353'') between IRL and the start of the ''istA'' gene presumably not affecting IS''1326'' transposition functions (generating In2).
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| − | 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]).
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| − | Tn''2411'' is not only the precursor of Tn''21''. It was proposed to give rise to additional transposons (<span style="color:#44546a;">Fig. Tn3.7Q</span>)[48]: to Tn''4'' by insertion of a Tn''3'' transposon copy into the ''merP'' gene (<span style="color:#44546a;">Fig. Tn3.7U</span>)<nowiki>; to Tn</nowiki>''5086'' [57] by deletion of the In_Tn''4'' IS''1326'' copy to generate Tn''2608'' [49] and replacement of the ''aadA'' cassette and acquisition of ''dfrA7'' (<span style="color:#44546a;">Fig. Tn3.7V</span>); and to Tn''2410'' by replacement of the ''aadA'' cassette by an ''oxa'' cassette [51].
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| − | The complete DNA sequences of many of these Tn are not available but Tn''5086'' or Tn''2608 ''could be reconstructed from Tn''21'' using the limited sequence data in ref [57]. Moreover, using the reconstructed Tn''5086'' 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 IS''4321'', in ''E. coli'' plasmid pSCU-397-2 (CP054830) in addition to many closely related copies. This analysis suggests that deletion of IS''1326'' had occurred by nearly-precise excision [58] since the deletion junction observed in Tn''5086'' [57] is not the original sequence identified in Tn''2411''. Indeed, the DNA sequences of Tn''2411,'' Tn''2608 ''and'' ''Tn''5086'', (<span style="color:#44546a;">Fig. Tn3.7V</span>) suggest that In_Tn2608 and In22 were derived by deletion from a structure similar to In_Tn4 because neither carry an IS''1326'' 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 IS''1326'' end.
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| − | Tn''21'' was also proposed to give rise to a number of different transposons [48,51]: to Tn''1831'' by IS''1326''-mediated deletion (IS''1326'' in IS''1326''::IS''1353'' is almost certainly functional) rightwards towards or past the IRt end of the integron while retaining the IS (<span style="color:#44546a;">Fig. Tn3.7Q</span><span style="color:#44546a;"><nowiki>; </nowiki></span><span style="color:#44546a;">Fig. Tn3.7W</span>)<nowiki>; to Tn</nowiki>''2607'' by insertion of Tn''2601'' (probably similar to Tn''3'') into the ''mer'' genes; to Tn''2424'' by insertion of IS''161'' to first generate Tn''2425'' and subsequent acquisition of two integron cassettes ''aacA1 ''and'' catB2'' (<span style="color:#44546a;">Fig. Tn3.7Q</span><nowiki>;</nowiki><span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.7X</span>); and to Tn''2603'' by insertion of an ''oxaA1'' cassette.
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| − |
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| − | <span style="color:#44546a;">Tn</span><span style="color:#44546a;">''1721''</span><span style="color:#44546a;"> and (tandem) amplification </span><span style="color:#44546a;">of the </span><span style="color:#44546a;">''tet''</span><span style="color:#44546a;"> genes</span><span style="color:#44546a;"> </span>
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| − |
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| − | Tn''1721'' (<span style="color:#44546a;">Fig. Tn3.7K</span>) carries resistance to tetracycline (''tet''), is present on <span style="background-color:#ffffff;">plasmid pRSD1 and is capable of undergoing amplification to generate tandem repeats </span><span style="background-color:#ffffff;">[59]</span><span style="background-color:#ffffff;">. It was isolated by transposition to a lambda phage followed by a further transposition event onto plasmid R388 </span><span style="background-color:#ffffff;">[24]</span><span style="background-color:#ffffff;"> where it retained the ability to amplify </span><span style="background-color:#ffffff;">[24]</span><span style="background-color:#ffffff;">. Amplification was identified using restriction enzyme mapping </span>(<span style="color:#44546a;">Fig. Tn3.7Y</span>) which showed a duplication of an Eco''R''I fragment and <span style="background-color:#ffffff;">presumably occurs via replication slippage or unequal crossing over during replication between the full </span><span style="background-color:#ffffff;">''tnpA''</span><span style="background-color:#ffffff;"> gene and the 5’-end </span><span style="background-color:#ffffff;">''tnpA''</span><span style="background-color:#ffffff;"> segment at the right end of Tn</span><span style="background-color:#ffffff;">''1721''</span><span style="background-color:#ffffff;">. Indeed, amplification was shown to depend on the host </span><span style="background-color:#ffffff;">''recA''</span><span style="background-color:#ffffff;"> gene </span><span style="background-color:#ffffff;">[60]</span><span style="background-color:#ffffff;">. </span>
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| − |
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''163''</span><span style="color:#44546a;"> Clade</span>
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| − | There are 39 members of this clade (May 2021). Two (TnSku1 [<span style="color:#000000;">Tn</span><span style="color:#000000;">''7197''</span><span style="color:#000000;">]</span> CP 002358.1 and TnAmu_p NC_015188.1) have acquired <span style="color:#44546a;">toxin/antitoxin gene pairs </span>and most members (<span style="color:#44546a;">Fig. Tn3.8A</span><span style="color:#44546a;"><nowiki>; </nowiki></span><span style="color:#44546a;">Fig. Tn3.8B</span>) encode divergent ''tnpR'' and ''tnpA'' genes. There are a number of members without passenger genes as in the Tn''21'' clade (e.g. Tn''6137'', Tn''Mex22''[ <span style="color:#000000;">Tn7165]</span>, Tn''Mex38'' [<span style="color:#000000;">Tn7166],</span> Tn''Che1'', [<span style="color:#000000;">Tn7155],</span> Tn''Amu1'' [<span style="color:#000000;">Tn7138</span> ], Tn''Ali20 ''[<span style="color:#000000;">Tn7136],</span>'' ''Tn''6122, ''Tn''3434'').
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| − | One small related group (Tn''6137'', Tn''6136'', Tn''6134'', Tn''6138'') 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 (<span style="color:#44546a;">Fig. Tn3.8C</span>). Alignment against Tn''6136'' (<span style="color:#44546a;">Fig. Tn3.8Ci</span>) shows that Tn''6137'' carries the left half while Tn''6134'' carries the right section while Tn''6137'' carries the right while Tn''6134'' carries the left segments of Tn''6138'' (excluding the passenger gene insertion). Although the res sites have yet to be defined in detail, comparisons clearly show sequence divergence in this region (<span style="color:#44546a;">Fig. Tn3.8ii</span>). Both Tn''6134'' and Tn''6138'' carry the same passenger gene (<span style="color:#44546a;">Fig. Tn3.8Ciii</span>) whose insertion has occurred proximal to IRL (<span style="color:#44546a;">Fig. Tn3.8Civ</span>).
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| − | The ancestor of another group of related transposons, the Tn''5393'' group (<span style="color:#44546a;">Fig. Tn3.8D</span>), appears to be Tn''5393''c (AY342395.1; ''Pseudomonas syringae'' pv. syringae plasmid pPSR1) which underwent an insertion of Tn''5501.6'' to generate ''Tn5393.1'' (MF487840.1; ''Pseudomonas aeruginosa'' PA34), of IS''1133'' to generate Tn''5393'' (M95402; ''Erwinia amylovora'' plasmid pEa34) (<span style="color:#44546a;">Fig. Tn3.8E</span>) and of a complex set of mobile elements to generate Tn''5393.4'' (AJ627643; Alcaligenes faecalis). Tn''5393'' also gave rise to a number of other derivatives: Insertion of Tn''3'' into its transposase gene generated Tn''5393.7'' (LT827129; Escherichia coli strain K12 J53); insertion of Tn''10'' into IS''1133'' to generate Tn''5393.2'' (CP030921; ''Escherichia coli ''KL53 plasmid pKL53-M) (<span style="color:#44546a;">Fig. Tn3.8F</span>) followed by insertion of IS''903'' to generate Tn''5393.11 ''(CP000602; ''Yersinia ruckeri'' YR71 plasmid pYR1); insertion of Tn''10'' in ''res'' to generate Tn''5393.8'' (CP002090; ''Salmonella enterica'' subsp. enterica plasmid pCS0010A). There are also 4 examples carrying derivatives of Tn''5'' inserted into ''tnpA''. They have an identical 3’ junction. In Tn''5393.12'' (KM409652; ''Escherichia coli'' REL5382 plasmid pB15), carries a complete Tn''5''. A second, Tn''5393.13'' (AB366441; ''Salmonella enterica subsp. enterica'' serovar Dublin plasmid pMAK2) is derived from Tn''5393.12'' by insertion of Tn''2'' into the IS''1133'' copy. In Tn''5393.3'' (LT985287; ''Escherichia coli strain'' RPC3 plasmid: RCS69_pI) the Tn''5'' insertion is a partial head-to-head Tn''5 ''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 Tn''5'' sequence. Finally, Tn''5393'' also gave rise to Tn''5393.9'' (KU987453; ''Klebsiella pneumoniae'' 05K0261 plasmid F5111) by multiple insertion including a type II intron, IS''5708'', IS''CR1'', IS''Ec28'', IS''Ec29'' and Tn''2.'' A number of intermediate structures have yet to be identified but can probably be found in the large number of Tn''5393'' derivatives in the public databases. This group of Tn''163'' clade members have undergone a large number of modifications and constitute a broad network of related elements.
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''4430''</span><span style="color:#44546a;"> Clade</span>
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| − | At present (May 2021) this clade is composed of only 11 examples (<span style="color:#44546a;">Fig. Tn3.9A</span>). One example, ''Tn4430'' (X07651.1), encodes a <span style="color:#44546a;">''tnpI''</span><span style="color:#44546a;"> </span>gene and a ''res'' site with <span style="color:#44546a;">its associated organization</span> but no passenger genes. The others encode a ''tnpR'' gene (<span style="color:#44546a;">Fig. Tn3.9B</span>). There are two small groups: Tn''1546'' which carry vancomycin resistance genes, and Tn''6332'' which carry mercury resistance genes.
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''1564''</span><span style="color:#44546a;"> Vancomycin Resistance Group</span>
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| − | Resistance to Vancomycin in Enterococci appeared in 1988 [62], was shown to be transmissible [63,64] and carried by a transposon, Tn''1546'' (M97297.1) [65]. The relationship within the Tn''1546'' vancomycin resistant transposons is relatively simple and the result of insertions/deletions mediated by several different insertion sequences: Tn''1546.2'' (AB247327) is derived from Tn''1546'' [65,66] by insertion of IS''1216E'' between ''vanYA'' and ''vanXA'' and ''Tn1546.1_p'' (KR349520.1) appears to be derived from Tn''1546.2'' by insertion of IS''1251'' between ''vanHA'' and ''vanSA'' and a neighboring deletion to the right of IS''1216E'' 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 IS''Ef1'', IS''1542'' and IS''19'' [67], in addition to a number of other IS''1216'' 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] (<span style="color:#44546a;">Fig. Tn3.9C</span>)
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| − | <span style="color:#44546a;">The Mercury Resistance Group</span>
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| − | 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<nowiki>; </nowiki><span style="color:#44546a;">Fig. Tn3.9D</span>), 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 (<span style="color:#44546a;">Fig. Tn3.9E</span>). Comparison with Tn''6294'' suggests that the mercury gene set has been exchanged by recombination at the level of the ''res'' site (<span style="color:#44546a;">Fig. Tn3.9D</span>).
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| − | The sequences of two closely related members of the same group, Tn''5083'' and Tn''5085'', are incomplete [71].
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> Clade</span>
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| − | <span style="color:#000000;">This clade includes the classical Tn</span><span style="color:#000000;">''1''</span><span style="color:#000000;">, </span><span style="color:#000000;">''2''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''3''</span><span style="color:#000000;"> (see </span><span style="color:#44546a;">Historical</span><span style="color:#000000;">) as well as Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;">. There are </span>29 e<span style="color:#000000;">xamples of the Tn</span><span style="color:#000000;">''3 ''</span><span style="color:#000000;">clade (of </span>which 26 can<span style="color:#000000;"> be found in TnCentral) (</span><span style="color:#44546a;">Fig. Tn3.10A</span><span style="color:#000000;">) which fall into two subgroups. The majority have divergently expressed </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and most carry passenger genes (</span><span style="color:#44546a;">Fig. Tn3.10B</span><span style="color:#000000;">). The </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites of each sub-group show significant similarity (</span><span style="color:#44546a;">Fig. Tn3.10C</span><span style="color:#000000;">). A number carry </span><span style="color:#44546a;">toxin-antitoxin genes</span><span style="color:#000000;"> generally located between the divergent </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;">. These are of two types (</span><span style="color:#44546a;">Fig. Tn3.10A</span><span style="color:#000000;">) and appear to be specific for each subgroup. Passenger genes can be located upstream of downstream of the </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;">/</span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> transposition module </span><span style="color:#44546a;">(</span><span style="color:#44546a;">Fig. Tn3.10B</span><span style="color:#000000;">). All except two encode </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> type resolvases. The two which do not, Tn</span><span style="color:#000000;">''Bth4''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''5401''</span><span style="color:#000000;">, also encode a TA module.</span>
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| − | <span style="color:#44546a;">Importance of IS</span><span style="color:#44546a;">''Ecp1''</span><span style="color:#44546a;"> in </span><span style="color:#44546a;">''bla''</span><span style="color:#44546a;"> CTX-M-expression</span>
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| − | There are examples of members of the Tn3 clade which carry insertions of IS''Ecp1''-like sequences (<span style="color:#44546a;">IS</span><span style="color:#44546a;">''1380''</span><span style="color:#44546a;"> family</span>) 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 Tn''3'' 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].
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| − | <span style="color:#44546a;">The Tn3 group</span>
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| − | <span style="color:#000000;">Tn</span><span style="color:#000000;">''3,''</span><span style="color:#000000;"> Tn</span><span style="color:#000000;">''1''</span><span style="color:#000000;">, Tn</span><span style="color:#000000;">''1MER''</span><span style="color:#000000;">, Tn</span><span style="color:#000000;">''2''</span><span style="color:#000000;">, Tn</span><span style="color:#000000;">''2.1''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''3.1''</span><span style="color:#000000;">. all carry a probable internal IR upstream of the </span><span style="color:#000000;">''bla''</span><span style="color:#000000;"> gene (</span><span style="color:#44546a;">Fig. Tn3.10D</span><span style="color:#000000;">) which acts as a hotspot for IS</span><span style="color:#000000;">''231A''</span><span style="color:#000000;"> insertion and was initially observed in the </span><span style="color:#000000;">''bla''</span><span style="color:#000000;"> gene of plasmid pBR322 </span><span style="color:#000000;">[79]</span><span style="color:#000000;">. Tn</span><span style="color:#000000;">''2''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''2.1''</span><span style="color:#000000;"> are identical except for the </span><span style="color:#44546a;">IS</span><span style="color:#44546a;">''Ecp1''</span><span style="color:#000000;"> insertion which also carries an internal IS</span><span style="color:#000000;">''1''</span><span style="color:#000000;"> insertion (</span><span style="color:#44546a;">Fig. Tn3.10E</span><span style="color:#000000;">). Note that an IS</span><span style="color:#000000;">''Ecp1''</span><span style="color:#000000;"> promoter drives </span><span style="color:#000000;">''bla''</span><span style="color:#000000;"> CTX-M-expression. There are a number of closely related derivatives (e.g. Tn</span><span style="color:#000000;">''6339''</span><span style="color:#000000;">-MF344565) in which the IS</span><span style="color:#000000;">''1''</span><span style="color:#000000;"> copy appears to have been involved in small rearrangements of the IS</span><span style="color:#000000;">''Ecp1''</span><span style="color:#000000;"> copy while maintaining the IS</span><span style="color:#000000;">''Ecp1''</span><span style="color:#000000;"> promoter. Three examples carry a number of integron cassettes without </span><span style="color:#000000;">either the integrase gene, the Tn</span><span style="color:#000000;">''402''</span><span style="color:#000000;"> ends or the Tn</span><span style="color:#000000;">''402''</span><span style="color:#000000;"> transposition genes that are often associated with integrons in the Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> clade.</span>
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| − | <span style="color:#000000;">Inspection of the alignment (</span><span style="color:#44546a;">Fig. Tn3.10E</span><span style="color:#000000;">) 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 </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> as had been previously noted for Tn</span><span style="color:#000000;">''1''</span><span style="color:#000000;">, </span><span style="color:#000000;">''2''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''3''</span><span style="color:#000000;"> </span><span style="color:#000000;">[80]</span><span style="color:#000000;"> (for </span><span style="color:#000000;">''res''</span><span style="color:#000000;">, see </span><span style="color:#44546a;">Fig. Tn3.10C</span><span style="color:#000000;">) and an evolutionary pathway </span>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. Tn''1331'' and Tn''1332'')<span style="color:#000000;">. In this respect, the integron carrying</span><span style="color:#000000;"> </span><span style="color:#000000;">Tn</span><span style="color:#000000;">''6238''</span><span style="color:#000000;"> is more similar to Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> while Tn</span><span style="color:#000000;">''1''</span><span style="color:#000000;">MER, Tn</span><span style="color:#000000;">''1331, ''</span><span style="color:#000000;">and Tn</span><span style="color:#000000;">''1332 ''</span><span style="color:#000000;">are</span><span style="color:#000000;">'' ''</span><span style="color:#000000;">more similar to Tn</span><span style="color:#000000;">''1''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''2.1''</span><span style="color:#000000;"> resembles Tn</span><span style="color:#000000;">''2''</span><span style="color:#000000;">.</span>
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| − | <span style="color:#44546a;">The Xanthomonas group</span>
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| − | This group except for Tn''Psy39 ''(Tn''7187''), all members of this group in the tree<span style="color:#000000;"> carry the same TA pair and the passenger genes are located to the right of the transposition module. The Xanthomonas transposon cluster (</span><span style="color:#44546a;">Fig. Tn3.10F</span><span style="color:#000000;">) are closely related and differ essentially by insertion of IS</span><span style="color:#000000;">''Xac1''</span><span style="color:#000000;"> and IS</span><span style="color:#000000;">''Xac5''</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.10G</span><span style="color:#000000;">) as well as deletions (in particular of the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site in </span><span style="color:#000000;">Tn</span><span style="color:#000000;">''Xc4.2 ''</span><span style="color:#000000;">[Tn</span><span style="color:#000000;">''7212''</span><span style="color:#000000;">]). Tn</span><span style="color:#000000;">''Xc4.1 ''</span><span style="color:#000000;">[Tn</span><span style="color:#000000;">''7211''</span><span style="color:#000000;">], although having an organisation identical to that of Tn</span><span style="color:#000000;">''Xc4''</span><span style="color:#000000;"> [Tn</span><span style="color:#000000;">''7210''</span><span style="color:#000000;">] has undergone significant sequence divergence along its entire length. Tn</span><span style="color:#000000;">''Thsp9 ''</span><span style="color:#000000;">[Tn</span><span style="color:#000000;">''7202''</span><span style="color:#000000;">] also shows sequence variation within the region carrying transposition and TA functions (but includes mercury genes instead of plant pathogenicity functions while Tn</span><span style="color:#000000;">''Psy39 ''</span><span style="color:#000000;">[Tn</span><span style="color:#000000;">''7187''</span><span style="color:#000000;">] only exhibits similarity in the TnpA gene.</span>
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| − | <span style="color:#000000;">All members of the second cluster, which encode for the same TA gene pair as the Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> group </span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.33A</span><span style="color:#000000;">), also carry mercury resistance genes although these have undergone some rearrangements and sequence divergence (</span><span style="color:#44546a;">Fig. Tn3.10H</span><span style="color:#000000;">) and are also divergent from those present in </span><span style="color:#000000;">Tn</span><span style="color:#000000;">''Thsp9 ''</span><span style="color:#000000;">(Tn</span><span style="color:#000000;">''7202''</span><span style="color:#000000;">).</span><span style="color:#000000;"> </span>
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''3000''</span><span style="color:#44546a;"> Clade</span>
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| − | <span style="color:#000000;">This clade is composed of nearly 30 members (25 in TnCentral) all of which encode TnpR resolvases and carry </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;">-related </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites. Most also encode TA gene pairs and these are of three types (</span><span style="color:#44546a;">Fig. Tn3.11A</span><span style="color:#000000;">).</span>
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''5501''</span><span style="color:#44546a;"> cluster.</span>
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| − | There are a number of Tn''5501'' examples <span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.11B</span><span style="color:#000000;">). All have their passenger genes located upstream of the transposition module and all except Tn</span><span style="color:#000000;">''Pysy42''</span><span style="color:#000000;"> [</span><span style="color:#000000;">Tn</span><span style="color:#000000;">''7188''</span><span style="color:#000000;">] and Tn</span><span style="color:#000000;">''5501.12 ''</span><span style="color:#000000;">encode the same parE/parD TA genes </span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.11A</span><span style="color:#000000;">).</span><span style="color:#000000;"> Tn</span><span style="color:#000000;">''5501.12''</span><span style="color:#000000;"> appears to have acquired different TA genes (HTH_37, GP49) by recombination at the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site </span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.11C</span><span style="color:#000000;">)</span><span style="color:#000000;">. </span>
| |
| − |
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| − | <span style="color:#000000;">The relationship between members of the cluster is shown in </span><span style="color:#44546a;">Fig. Tn3.11C</span><span style="color:#000000;">. 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 Tn</span><span style="color:#000000;">''5051.3''</span><span style="color:#000000;">. For 8 of these, the right junctions of the deletions are close but not identical (</span><span style="color:#44546a;">Fig. Tn3.11Di and Dii)</span><span style="color:#000000;">. All leave the TA module intact. In only one example, the toxin gene has undergone deletion leaving the antitoxin intact (</span><span style="color:#44546a;">Fig. Tn3.11Diii). </span><span style="color:#000000;">The left junction is less clear and difficult to interpret.</span>
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| − | A number of Tn''5501'' derivatives are related by IS insertions and deletion (<span style="color:#44546a;">Fig. Tn3.</span>)
| |
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| − | <span style="color:#000000;">Finally, a small group of Tns which, like Tn</span><span style="color:#000000;">''5501.12, ''</span><span style="color:#000000;">all carry the HTH_37/GP49 TA pair is shown in </span><span style="color:#44546a;">Fig. Tn3.11F</span><span style="color:#000000;">. It appears that there has been an exchange between a Tn</span><span style="color:#000000;">''5501.5''</span><span style="color:#000000;">-like transposon and a derivative of Tn4</span><span style="color:#000000;">''662a''</span><span style="color:#000000;"> (lacking the IS</span><span style="color:#000000;">''As20''</span><span style="color:#000000;"> insertion) by recombination at the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site to generate Tn</span><span style="color:#000000;">''5501.12.''</span>
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| − | <span style="color:#44546a;">Clinical Importance of Tn</span><span style="color:#44546a;">''4401''</span>
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| − | <span style="background-color:#ffffff;">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 </span><span style="background-color:#ffffff;">[81]</span><span style="background-color:#ffffff;">.</span> The 10kb transposon, Tn''4401'' has been instrumental in the spread of the carbapenem resistance gene ''bla''<sub>KPC</sub>. It was described in 2008 in a number of clinical isolates of ''Klebsiella pneumoniae'' and ''Pseudomonas aeruginosa'' from the <span style="background-color:#ffffff;">United States, Colombia and Greece </span>[82,83]. Members of this small group have divergently expressed ''tnpR'' and ''tnpA'' genes located towards the left end and ''bla''<sub>KPC</sub> towards the right end downstream from ''tnpA'' (<span style="color:#44546a;">Fig. Tn3.11B</span>) flanked by two different insertion sequences, <span style="background-color:#ffffff;">IS</span><span style="background-color:#ffffff;">''Kpn6''</span><span style="background-color:#ffffff;"> and IS</span><span style="background-color:#ffffff;">''Kpn7 ''</span>(<span style="color:#44546a;">Fig. Tn3.11G)</span><span style="background-color:#ffffff;">. The IS</span><span style="background-color:#ffffff;">''Kpn7''</span><span style="background-color:#ffffff;"> insertion had occurred within an additional Tn</span><span style="background-color:#ffffff;">''4401''</span><span style="background-color:#ffffff;"> IR. It was further observed that there were two “isoforms” of Tn</span><span style="background-color:#ffffff;">''4401''</span><span style="background-color:#ffffff;">: Tn</span><span style="background-color:#ffffff;">''4401a''</span><span style="background-color:#ffffff;"> and Tn</span><span style="background-color:#ffffff;">''4401b''</span><span style="background-color:#ffffff;">. Tn</span><span style="background-color:#ffffff;">''4401a''</span><span style="background-color:#ffffff;">, isolated in the United States and Greece carried a 100bp deletion upstream of the </span><span style="background-color:#ffffff;">''bla''</span><span style="background-color:#ffffff;"> gene compared to Tn</span><span style="background-color:#ffffff;">''4401b''</span><span style="background-color:#ffffff;"> from Colombia. The Tn</span><span style="background-color:#ffffff;">''4401''</span><span style="background-color:#ffffff;"> backbone appears to have undergone a number of recombination events. A third derivative, Tn</span><span style="background-color:#ffffff;">''4401''</span><span style="background-color:#ffffff;">c </span><span style="background-color:#ffffff;">[84]</span><span style="background-color:#ffffff;">, was found to carry a deletion of about 200 bp upstream of </span><span style="background-color:#ffffff;">''bla''</span><span style="background-color:#ffffff;"> while in a fourth, Tn</span><span style="background-color:#ffffff;">''4401d''</span><span style="background-color:#ffffff;"> </span><span style="background-color:#ffffff;">[85]</span><span style="background-color:#ffffff;">, the IS</span><span style="background-color:#ffffff;">''Kpn7''</span><span style="background-color:#ffffff;"> copy along with flanking DNA has undergone deletion to leave a 3’ segment of </span>''bla''<sub>KPC</sub> <span style="background-color:#ffffff;">and a 5’ segment of </span><span style="background-color:#ffffff;">''tnpA''</span><span style="background-color:#ffffff;"> 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 </span>''bla''<sub>KPC</sub> [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 IS''Kpn7'' (as is characteristic for a member of the IS''21'' family; <span style="color:#44546a;">IS</span><span style="color:#44546a;">''21''</span><span style="color:#44546a;"> chapter</span><nowiki>; </nowiki><span style="color:#44546a;">formation of hybrid promoters</span> sections).
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''4651''</span><span style="color:#44546a;"> Clade</span>
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''4651 ''</span><span style="color:#44546a;">mix of radically different structures</span>
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| − |
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| − | This Tn''3'' family clade (<span style="color:#44546a;">Fig. Tn3.12A</span>) contains members with very diverse structures (<span style="color:#44546a;">Fig. Tn3.12B</span>). They fall into three major clusters. Two encode the ''tnpT''/''S''/''rst'' while the third encodes the ''tnpR''/''res'' system.
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| − | <span style="color:#44546a;">The </span><span style="color:#44546a;">''tnpT''</span><span style="color:#44546a;">/</span><span style="color:#44546a;">''S''</span><span style="color:#44546a;">/</span><span style="color:#44546a;">''rst''</span><span style="color:#44546a;"> clusters</span>
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| − | In the first ''tnpT''/''S''/''rst'' cluster, mostly from the plant pathogen ''Xanthomonas'' (<span style="color:#44546a;">Fig. Tn3.12C</span>), Tn''Xax1.1'' [Tn''7207''] 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. Tn''Xax1.3'' [Tn''7209''] differs from the others (Tn''Xax1'' [Tn''7206'']; Tn''Xax1.2 ''[Tn''7208''];'' ''Tn''Xax1.3 ''[Tn''7209''] in the 3’ region of ''tnpA'' and there is some variation in ''tnpS'' and ''tnpT''.
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| − | Tn''Xax1'' derivatives [25] are generally vehicles for pathogenicity genes such as Transcriptional Activator Like Effectors (<span style="color:#44546a;">TALE genes</span>), cell wall degrading enzymes (''mtl'') and genes (''xop'') involved in type III secretion system (TTSS) translocation of effector proteins into host plant cells [87]<span style="color:#44546a;"> (</span><span style="color:#1f3864;">Fig. Tn3.12C</span>). Tn''Xax1'' derivatives can include IR which are significantly longer (72/92 bp) than the 38-40bp characteristic of the Tn''3 ''family (<span style="color:#1f3864;">Fig. Tn3.12D</span>) 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 (<span style="color:#1f3864;">Fig. Tn3.12E</span>) while their right ends appear more homogeneous (<span style="color:#1f3864;">Fig. Tn3.12F</span>).
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| − | The second ''tnpT''/''S''/''rst'' cluster is characterized by Tn''4651'', 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, Tn''4652'', a Tn''4651'' 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 Tn''4652'' ends (<span style="color:#44546a;">Fig. Tn3.12G</span>) [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 (<span style="color:#44546a;">Fig. Tn3.12G</span>). Tn''4652'' 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 <span style="color:#44546a;">IS</span><span style="color:#44546a;">''10 ''</span>(<span style="color:#44546a;">IS</span><span style="color:#44546a;">''4''</span><span style="color:#44546a;"> family</span>). Moreover, Tn''1000'' (Tn''3'' clade) carries an IHF binding site proximal to each IR which acts copoperatively to increase TnpA binding and <span style="color:#44546a;">immunity</span> [94,95]. One additional interface with host physiology is the observation that the CorR/CorS two component system regulates transposition positively [96].
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| − | Other members of the cluster include: ''Pseudomonas sp''. mercury resistance transposon Tn''5041'' [97,98] <nowiki>; Tn</nowiki>''4676'', a long (72,752bp) and complex ''Pseudomonas resinovorans ''carbazole-catabolic transposon from plasmid pCAR1 [99,100]<nowiki>; and Tn</nowiki>''4661'', a ''Pseudomonas'' ''aeruginosa'' cryptic transposon [27]. All include ''tnpA'', ''tnpC'' and the<span style="color:#ff0000;"> </span><span style="color:#44546a;">''tnpS''</span><span style="color:#44546a;">/</span><span style="color:#44546a;">''T''</span><span style="color:#44546a;"> </span>resolution system.<span style="color:#ff0000;"> </span>
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| − | Tn''5041'' 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 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 Tn''5041''-Tn''4651'' ''tnpA'' genes. Tn''5041'' apparently acquired its ''mer'' operon from a derivative of Tn''21'' or Tn''501'' [101]. It is reported to be preceded by a 24 bp element with 75% sequence similarity to the outermost part of IRs typical for Tn''21''-like transposons.<span style="color:#ff0000;"> </span>
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| − | <div style="color:#ff0000;"></div>
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| − | <div style="color:#44546a;"></div>
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''1071''</span><span style="color:#44546a;"> Clade</span>
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''1071''</span><span style="color:#44546a;"> group. </span>
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| − | Members of this small group are often associated with xenobiotic catabolism and other “exotic” functions (<span style="color:#44546a;">Fig. Tn3.13A</span>).
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| − | Tn''1071'' itself <span style="background-color:#ffffff;">(</span><span style="background-color:#ffffff;color:#44546a;">Fig. Tn3.13Bi</span><span style="background-color:#ffffff;">)</span>, the founding member, was identified as part of a compound transposon, Tn''5271'', 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 <span style="background-color:#ffffff;">(</span><span style="background-color:#ffffff;color:#44546a;">Fig. Tn3.13Bii</span><span style="background-color:#ffffff;">) </span>and was first described as <u>IS</u>''1071. ''Two other members of this small group, IS''882 ''from <span style="background-color:#ffffff;">''Ralstonia eutropha''</span><span style="background-color:#ffffff;"> H16 megaplasmid pHG1 encoding key enzymes for H2-based lithoautotrophy and anaerobiosis</span> [103] and IS''Busp1'' (aka IS''Bmu13''<nowiki>; NC_007509.1) from the</nowiki>'' 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''.''
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| − | A limited functional analysis of Tn''1071'' transposition is available [105]. It <span style="background-color:#ffffff;">was only able to transpose at high frequencies in two environmental </span><span style="background-color:#ffffff;"></span><span style="background-color:#ffffff;">-proteobacteria </span><span style="background-color:#ffffff;">''Comamonas''</span><span style="background-color:#ffffff;"> </span><span style="background-color:#ffffff;">''testosteroni''</span><span style="background-color:#ffffff;"> and </span><span style="background-color:#ffffff;">''Delftia''</span><span style="background-color:#ffffff;"> </span><span style="background-color:#ffffff;">''acidovorans''</span><span style="background-color:#ffffff;"> but not in </span><span style="background-color:#ffffff;">''Agrobacterium tumefaciens''</span><span style="background-color:#ffffff;"> (</span><span style="background-color:#ffffff;"></span><span style="background-color:#ffffff;">-proteobacteria) or </span><span style="background-color:#ffffff;">''Escherichia coli''</span><span style="background-color:#ffffff;">,</span><span style="background-color:#ffffff;"> </span><span style="background-color:#ffffff;">''Pseudomonas alcaligenes''</span><span style="background-color:#ffffff;"> and </span><span style="background-color:#ffffff;">''Pseudomonas putida''</span><span style="background-color:#ffffff;"> (all </span><span style="background-color:#ffffff;"></span><span style="background-color:#ffffff;">-proteobacteria). These studies showed that Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> 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 Tn</span><span style="background-color:#ffffff;">''3''</span><span style="background-color:#ffffff;"> family members which only require the 38 bp IR. </span>
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| − | <span style="background-color:#ffffff;">The absence of a resolution system implies that, like </span><span style="background-color:#ffffff;color:#44546a;">IS</span><span style="background-color:#ffffff;color:#44546a;">''26''</span><span style="background-color:#ffffff;">, Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> probably forms </span><span style="background-color:#ffffff;color:#44546a;">“pseudo-compound transposons” </span><span style="background-color:#ffffff;color:#44546a;">[106–108]</span><span style="background-color:#ffffff;">. In these structures the flanking Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> copies must be in direct orientation as </span><span style="color:#131413;">a consequence of the homologous recombination event required to resolve the cointegrate structure. </span>T<span style="color:#131413;">ransposition is initiated by one of the flanking IS to generate a </span><span style="color:#131413;">cointegrate structure with three Tn</span><span style="color:#131413;">''1071''</span><span style="color:#131413;"> copies (similar to those generated by the IS6 family of insertion sequences; </span><span style="color:#44546a;">Fig. IS6.8B</span><span style="color:#131413;">). </span><span style="color:#131413;">“</span><span style="color:#131413;">Resolution</span><span style="color:#131413;">” </span><span style="color:#131413;">resulting in transfer of the transposon passenger gene requires recombination between the </span><span style="color:#131413;">“</span><span style="color:#131413;">new</span><span style="color:#131413;">” </span><span style="color:#131413;">IS copy and the copy which was not involved in generating the cointegrate. The implications of this model as for IS</span><span style="color:#131413;">''6''</span><span style="color:#131413;"> family members are that the transposon passenger gene(s) are simply transferred from donor to target molecules in the </span><span style="color:#131413;">“</span><span style="color:#131413;">resolution</span><span style="color:#131413;">” </span><span style="color:#131413;">event and are therefore lost from the donor </span><span style="color:#131413;">“</span><span style="color:#131413;">transposon</span><span style="color:#131413;">” leaving a single Tn</span><span style="color:#131413;">''1071''</span><span style="color:#131413;"> copy in the donor plasmid</span><span style="color:#131413;">. 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.</span>
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| − | <span style="background-color:#ffffff;">A significant number of Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;">-associated xenobiotic-degrading genes on many catabolic plasmids have been documented by population-based PCR </span><span style="background-color:#ffffff;">[109–111]</span><span style="background-color:#ffffff;"> and genetic studies </span><span style="background-color:#ffffff;">[112,113]</span><span style="background-color:#ffffff;">. Tn</span><span style="background-color:#ffffff;">''5271''</span><span style="background-color:#ffffff;"> itself is widely distributed in bacteria isolated from a large ground water bioremediation site </span><span style="background-color:#ffffff;">[111]</span><span style="background-color:#ffffff;"> and plasmid derivatives carrying the transposon together with a third Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> 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 </span><span style="background-color:#ffffff;">[111]</span><span style="background-color:#ffffff;"> (</span><span style="background-color:#ffffff;color:#ff0000;">Fig. for intramol transposition</span><span style="background-color:#ffffff;">).</span>
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| − | = <span style="background-color:#ffffff;">A number of additional potential compound transposons have been identified although these may be inactive: a >28kb transposon, Tn</span><span style="background-color:#ffffff;">5330</span><span style="background-color:#ffffff;"> (AF029344), from </span><span style="background-color:#ffffff;">Delftia acidicorans</span><span style="background-color:#ffffff;"> </span>[114] <span style="background-color:#ffffff;">carries the entire 2,4-dichlorophenoxyacetic acid degradation pathway and, although </span>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 <span style="background-color:#ffffff;">DR from </span><span style="background-color:#ffffff;">Achromobacter xylosoxidans</span><span style="background-color:#ffffff;"> plasmid, pEST4011, </span>also carries identical IS1471 inactivating insertions in each flanking Tn1071 copy [115] and a 7kb internal tandem duplication compared to the Delftia acidovorans transposon. =
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| − |
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| − | <span style="background-color:#ffffff;">When analyzed in more detail, these genes are sometimes flanked by Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> copies in direct repeat as in the original Tn</span><span style="background-color:#ffffff;">''5271''</span><span style="background-color:#ffffff;"> but are found in more complex Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;">-based structures. </span>
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| − |
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| − | <span style="background-color:#ffffff;">Tn</span><span style="background-color:#ffffff;">''Had2''</span><span style="background-color:#ffffff;"> </span><span style="background-color:#ffffff;">[116]</span><span style="background-color:#ffffff;"> (</span><span style="background-color:#ffffff;color:#44546a;">Fig. Tn3.13Ci</span><span style="background-color:#ffffff;">), for example, from a </span><span style="background-color:#ffffff;">''Delftia acidovorans''</span><span style="background-color:#ffffff;"> haloacetate-catabolic plasmid, pUO1, carries a nested copy of a potential Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;">-based compound transposon, Tn</span><span style="background-color:#ffffff;">''Had1''</span><span style="background-color:#ffffff;"> which does not carry flanking DR. Tn</span><span style="background-color:#ffffff;">''Had1''</span><span style="background-color:#ffffff;"> is inserted into a larger structure, Tn</span><span style="background-color:#ffffff;">''Had2''</span><span style="background-color:#ffffff;"> with flanking 5bp DR, typical Tn</span><span style="background-color:#ffffff;">''3''</span><span style="background-color:#ffffff;"> family ends related to those of Tn</span><span style="background-color:#ffffff;">''21''</span><span style="background-color:#ffffff;"> but no apparent dedicated transposase except that of the Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> copies. The authors state that Tn</span><span style="background-color:#ffffff;">''Had2''</span><span style="background-color:#ffffff;"> was unable to transpose as judged by a </span><span style="background-color:#ffffff;color:#44546a;">“mating out”</span><span style="background-color:#ffffff;"> assay using the plasmid R388 as a target. However, The TnHAD2 Tn</span><span style="background-color:#ffffff;">''21''</span><span style="background-color:#ffffff;">-like IRs were found to be active in transposition if supplied with Tn</span><span style="background-color:#ffffff;">''21''</span><span style="background-color:#ffffff;"> but not with Tn</span><span style="background-color:#ffffff;">''1722''</span><span style="background-color:#ffffff;"> transposition functions </span><span style="background-color:#ffffff;">[116]</span><span style="background-color:#ffffff;">. TnHad2 also appeared to carry a functional </span><span style="background-color:#ffffff;">''res''</span><span style="background-color:#ffffff;"> site. </span>
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| − |
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| − | <span style="background-color:#ffffff;">Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> also flanks atrazine degrading genes in plasmid </span><span style="background-color:#ffffff;">''Pseudomonas''</span><span style="background-color:#ffffff;"> pADP-1 (U66917) </span><span style="background-color:#ffffff;">[117]</span><span style="background-color:#ffffff;"> in a structure with three directly repeated Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> copies intercalated with three copies of an </span><span style="background-color:#ffffff;color:#44546a;">IS</span><span style="background-color:#ffffff;color:#44546a;">''91''</span><span style="background-color:#ffffff;color:#44546a;"> family</span><span style="background-color:#ffffff;"> member, IS</span><span style="background-color:#ffffff;">''Pps1.''</span><span style="background-color:#ffffff;"> These are apparently generated by duplication events since regions with identical sequence stretch from the </span><span style="background-color:#ffffff;">''oriIS''</span><span style="background-color:#ffffff;"> end of IS</span><span style="background-color:#ffffff;">''Pps1''</span><span style="background-color:#ffffff;"> through Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> and terminate just before the </span><span style="background-color:#ffffff;">''atz''</span><span style="background-color:#ffffff;"> genes (</span><span style="background-color:#ffffff;color:#44546a;">Fig. Tn3.13Cii</span><span style="background-color:#ffffff;">). The repeated regions also includes the DR sequences at each Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> except for that at the far right. </span>
| |
| − |
| |
| − | <span style="background-color:#ffffff;">There are a number examples of other structures with multiple Tn</span><span style="background-color:#ffffff;">''1071''</span><span style="background-color:#ffffff;"> copies and in a large proportion of these cases, the multiple copies occur in direct repeat. They are associated with plasmids which degrade the </span><span style="background-color:#ffffff;">phenylurea herbicide linuron e.g. pBPS33-2 (CP044551) </span><span style="background-color:#ffffff;">[118]</span><span style="background-color:#ffffff;"> and have been isolated from a variety of bacteria with </span><span style="background-color:#ffffff;">the capacity to degrade a wide range of chlorinated aromatics and pesticides </span><span style="background-color:#ffffff;">[109]</span><span style="background-color:#ffffff;"> or p-toluene sulfonate (TSA) where they flank the TSA genes in plasmid pTSA (AH010657) </span><span style="background-color:#ffffff;">[119]</span><span style="background-color:#ffffff;">. </span>
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| − | <span style="color:#44546a;">MITES, MICs and TALES</span>
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| − |
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| − | 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 <span style="color:#323e4f;">MICs (Minimal Insertion Cassette) </span>and <span style="color:#323e4f;">MITEs (</span><span style="color:#323e4f;">Miniature inverted-repeat </span><span style="color:#323e4f;">transposable</span><span style="color:#323e4f;">'' ''</span><span style="color:#323e4f;">elements)</span> respectively. For Tn''3'', related MITEs are known as TIMEs (Tn''3''-Derived Inverted-Repeat Miniature Elements) [120,121].
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| − |
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| − | Studies have shown that ''Xanthomonas'' genomes are often havens for MICs carrying genes involved in pathogenicity towards their host plants [25]. A number of Tn''3'' family structures were identified in a conjugative plasmid, pXac64 (CP024030), of the principal pathogen of citrus trees, ''Xanthomonas citri'', an important economic problem (e.g., reference [122]) (<span style="color:#44546a;">Fig. Tn3.14A</span>). The plasmid includes two Tn''3'' family transposons, Tn''Xc4.4'' (Tn''7210'') and Tn''Xac1.4'' (Tn''7206'') and a MIC (MIC XAC64.T1; 3948bp) which carries a TAL effector gene. Other TAL effector-carrying MICs can be identified in other ''Xanthomonas ''plasmids such as pXac33 (CP008996) [123] (two TAL-carrying MIC: MIC XAC33.T1, 3739bp, and MIC XAC33.T2, 3538bp; and the Tn''3'' family transposon Tn''Xc5'') and from the ''Xanthomonas fuscans'' plasmid pplc XAF (FO681497) [124] (a single MIC, MIC XAF.T1 ,3768bp, and a 10kb MIC with a number of virulence genes. Some MICs, e.g. MIC XAC33.T1 (<span style="color:#44546a;">Fig. Tn3.14B right</span>), are flanked by 5bp DR, a hallmark of Tn''3'' family transposition.
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| − |
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| − | A global analysis of TAL effector genes in (<span style="color:#44546a;">Fig. Tn3.14C</span>) within the ''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 [25].
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| − |
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| − | Inspection showed that the chromosome of ''X. citri'' strains do not carry identifiable TAL-carrying MICs but those of ''X. oryzae'' carry relatively high numbers [25]. A smaller number of MICs carrying other pathogenicity-related genes are also observed (e.g. Type III ''Xop'' genes) It is notable that the majority of the TAL-associated MICs occur as two or more tandem copies. These are listed for three example genomes, ''X. oryzae'' PXO99A, MAFF and KACC in <span style="color:#44546a;">Fig. Tn3.14D.1-3</span>. 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 (<span style="color:#44546a;">Fig. Tn3.14E</span>), suggesting that the tandem dimers and multimers arose by amplification possibly via replication slippage and unequal crossing over [25] (<span style="color:#44546a;">Fig. Tn3.14F</span>). 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.
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| − |
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| − | It is clear that this “radiation” of TAL-associated MICs does not only occur by transposition. In one case (<span style="color:#44546a;">Fig. Tn3.14Ei, Eii and Fig. Tn3.14G</span>) an entire DNA segment containing a tandem MIC dimer (MIC P.T11-MIC 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.
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| − |
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| − | This variability in MIC sequence can be observed within the longer arrays (e.g. <span style="color:#44546a;">Fig. Tn3.14E viii</span>) suggesting that diversification follows amplification. This is due to changes in the TAL genes. TAL proteins are composed of conserved N-terminal and C-terminal regions separated by a variable number of 34 amino acid repeats (<span style="color:#44546a;">Fig. Tn3.14H</span>) 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 (<span style="color:#44546a;">Fig. Tn3.14H; Fig. Tn3.14I</span>) e.g. [125–127]. 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 (<span style="color:#44546a;">Fig. Tn3.14J</span>) which includes 19.5 such repeats. The TAL effectors encoded by other members of this cluster (<span style="color:#44546a;">Fig. Tn3.14Eviii</span>), 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. 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.
| |
| − |
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| − | Diversification can also be observed between clusters in related ''X. oryzae'' strains such as MAFF and KACC.
| |
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| − | Strain PXO99A and MAFF share the cluster MIC P.T15, MIC P.T16, MIC P.T17 (<span style="color:#44546a;">Fig. Tn3.14Ki; Fig. Tn3.14L</span>). 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).
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| − |
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| − | 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.
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| − | <div style="color:#44546a;"></div>
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| − | <span style="color:#44546a;">Acquisition of Passenger Genes.</span>
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| − | <span style="color:#000000;">Tn3-family transposons </span><span style="color:#000000;">carry large and diverse and diverse sets of passenger genes (e.g. </span><span style="color:#000000;">Fig. Tn3.3). These have been acquired by a number of different processes. </span>
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| − |
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| − | <span style="color:#44546a;">Tn</span><span style="color:#44546a;">''402''</span><span style="color:#44546a;"> and integron platforms.</span>
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| − |
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| − | <span style="color:#000000;">One major source of antibiotic passenger genes has been by ancestral insertions of </span><span style="color:#44546a;">Tn</span><span style="color:#44546a;">''402''</span><span style="color:#000000;"> derivatives which have often “decayed” to lose their transposition properties but have retained their abilities to acquire (and lose) integron gene cassettes (</span><span style="color:#44546a;">Fig. Tn3.7G; Fig. Tn3.7I; Fig. Tn3.7J; Fig. Tn3.7R; Fig. Tn3.7S; Fig. Tn3.7U; Fig. Tn3.7V; Fig. Tn3.18C</span><span style="color:#000000;">). </span>
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| − |
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| − | <span style="color:#44546a;">Additional TE</span>
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| − |
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| − | <span style="color:#000000;">A second pathway to acquisition is by insertion of additional transposable elements with, or without rearrangement (</span><span style="color:#44546a;">Fig. Tn3.7U; Fig. Tn3.8E; Fig. Tn3.8F</span><span style="color:#000000;">). It is also interesting to note that there are a number of cases in which additional IR appear within certain structures (e.g. </span><span style="color:#44546a;">Fig. Tn3.10D; </span><span style="color:#44546a;">Fig. Tn3.11G</span>) such as Tn''3'' [79] and Tn''501'' [128] raising the possibility that these have been involved in generating the host transposon.
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| − | <span style="color:#44546a;">Recombination at </span><span style="color:#44546a;">''res''</span><span style="color:#44546a;">. </span>
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| − | A third major pathway to passenger gene acquisition is by inter-transposon exchange via ''res'' sites (<span style="color:#44546a;">Resolution</span>). This was first suggested to explain the formation of Tn''501'', by exchange of a transposition module with a Tn1721-related transposon [128]. It was later observed by Kholodii and coworkers [129,130] and called “shuffling”, by Yano et al., [131] and by others [33]. 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 (Tn''21'': <span style="color:#44546a;">Fig. Tn3.7H; Fig. Tn3.7M; Fig. Tn3.7N</span><nowiki>; Tn</nowiki>''163'': <span style="color:#44546a;">Fig. Tn3.8Ci and iii</span><nowiki>; Tn</nowiki>''4330'': <span style="color:#44546a;">Fig. Tn3.9D</span><nowiki>; Tn</nowiki>''3000'': <span style="color:#44546a;">Fig. Tn3.11C; Fig. Tn3.11F</span><nowiki>; and Tn</nowiki>''4561'': <span style="color:#44546a;">Fig. Tn3.12C</span>). 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 [132].
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| − | <span style="color:#44546a;">Mercury Resistance: a </span><span style="color:#44546a;">Major Passenger Gene Group </span>
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| − | <span style="color:#44546a;">The Mercury Operon and the Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> family </span>
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| − | Not surprisingly, bacteria carrying ''mer'' operons are particularly abundant in areas with increased mercury concentrations such as mercury mines and contaminated soil or water [133–135] and it was suggested that mercury resistance is an ancient system as reflected by a wide geographical, environment and species range and that it evolved as a response to increased levels of mercury in natural environments resulting, for example, from volcanic activity [136]. It is certainly present in the Murray collection [137], a collection of Enterobacteriaceae isolated in the pre-antibiotic era, as part of transposons Tn''5073'' and Tn''5074'' which show high homology to present day examples such as Tn''5036'' and Tn''1696'' (Tn''3'' family members of the Tn''21'' clade) and Tn''5053'' (a Tn''402'' family member of the Tn''5053'' clade) and Tn''5075'' respectively [52].
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| − | Although Tn''3'' family members carry a large variety of passenger genes, mercury resistance is found repeatedly within the family and is thought to be one of the first sets of passenger genes to be acquired (<span style="color:#44546a;">Fig. Tn3.6</span>) and appears in precursors of the major groups of antibiotic resistance carrying Tn''3'' family members (<span style="color:#44546a;">Fig. Tn3.7G</span>). Mercury resistance operons were proposed to have been acquired at least twice [42](<span style="color:#44546a;">Fig. Tn3.6</span>): once by an ancestor of Tn''21'' and once by an ancestor of Tn''501''. Their acquisition presumably predates the acquisition of antibiotic resistance integron platforms since a number of mercury resistance Tn''3'' family transposons have been identified and, in at least two cases, Tn''21'' and Tn''1696'' (whose ''mer'' genes appear to fall largely into different groups;<span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3Bi-vii</span>), clear precursors devoid of integrons (Tn''5060'' [44] and Tn''20'' and Tn''1696.1 ''respectively) have been identified. Mercury resistance genes are found in a number of Tn''3'' family clades (<span style="color:#44546a;">Fig. Tn3.15B and 15Bi-vii)</span>. These include Tn''3'', Tn''21'', Tn1''63'', Tn''4430'' and Tn''4651''. Those associated with the Tn''21'' clade occur upstream of, and are generally expressed towards, ''tnpR'' (<span style="color:#44546a;">Fig. Tn3.7G</span>); those of the Tn''3'' clade are located downstream of ''tnpA'' (<span style="color:#44546a;">Fig. Tn3.15C</span><span style="color:#000000;">) and in </span><span style="color:#000000;">those carrying the tnpS/T genes, they are between the transposase module and the tnpS/T module (</span><span style="color:#44546a;">Fig. Tn3.15D</span><span style="color:#000000;">).</span><span style="color:#ff0000;"> </span>
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| − | A survey of 29 functional 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 Tn''5053'', a member of the Tn''402'' family [130,138]. In addition, Yurieva et al [130]<span style="color:#ff0000;"> </span>identified a third group, related to Tn''5041'', a member of the Tn''4651'' clade They also identify “mosaic” ''mer'' operons which, they suggest, are generated by homologous recombination between short DNA sequences. While MerR appears to be very similar between different ''mer'' operons, while MerA showed a higher degree of mosaicism as did MerT and MerP to some extent [130].
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| − | <span style="color:#44546a;">The Mercury Operon: Organization, Regulation, and Resistance Mechanism </span>
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| − | The mechanism underlying mercury resistance has been extensively reviewed a number of times [48]. Briefly, 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 MerP, transferred across the periplasm to the inner membrane proteins MerC or MerT and then across the cytoplasmic membrane to the mercuric reductase, MerA which converts it to the volatile Hg<sub>0</sub>. The operon is regulated by two genes, ''merR'' and ''merD'' (<span style="color:#44546a;">Fig. Tn3.15A</span>). The order of these genes is generally ''merT'', ''merP'', ''merC'', merA, merD and merE. merR is located upstream and is transcribed in the opposite direction with overlapping promoters. Binding of MerR represses expression of the operon and of itself. Interaction with Hg(II) releases MerR repression of the ''mer'' structural genes permitting their expression without significantly impacting on its autorepression [139] and its interaction with RNA polymerase creates a pre-transcription initiation complex [140].
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| − | The product of the secondary regulator gene, ''merD'' [37], appears to play a role in down-regulating the ''mer'' operon [141]. It binds weakly but specifically to the ''merOP'' region and DNase I footprinting identified a common operator binding sequence for both MerR and MerD [141].
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| − |
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| − | The genes essential for mercury resistance were identified as ''merR'', ''merT'', ''merP'' and ''merA'' [142]. An additional mercury ion transmembrane transporter gene, ''merE'' (UniProtKB - D4N5J4) involved in the accumulation of methyl-mercury [48,143] is often present. Not all mercury operons include ''merC'' and some have a gene, ''merF'' [144], an alternative mercury ion transmembrane transporter (UniProtKB - Q1H9Y3). Some also include a mercury lyase gene, ''merB'', involved in resistance to organo-mercury [145,146].
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| − | <span style="color:#44546a;">The Mercury Operon: Diversity in</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">various Tn3 family clades.</span>
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| − |
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| − | The ''mer'' carrying Tn''3'' family members (<span style="color:#44546a;">Fig. Tn3.15B</span>) all lack ''merF''. Most examples carry a full ''mer'' gene complement although a small group (Tn''501'', Tn''511'', Tn''1412'', Tn''4378'' and Tn''4380'') lack the ''merC'' gene and only 3 (Tn''5084'', Tn''6294'', Tn''6332''), all members of the Tn''4430'' clade) carry a ''merB'' gene and have a duplicated or partially duplicated ''mer'' operon.
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| − |
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| − | Phylogenetic trees generated for MerR (<span style="color:#44546a;">Fig. Tn3.15Ci and Cii</span>), MerT (<span style="color:#44546a;">Fig. Tn3.15Ciii</span>), MerP (<span style="color:#44546a;">Fig. Tn3.15Civ</span>), MerA (<span style="color:#44546a;">Fig. Tn3.15Cvi</span>), MerD (<span style="color:#44546a;">Fig. Tn3.15Cvii</span>) and MerE (<span style="color:#44546a;">Fig. Tn3.15Cviii</span>) reveal that, in general, Tn''501''-related ''mer'' genes group separately from those of Tn''21'' 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 Tn''402'' family relatives.
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| − |
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| − | Within the Tn''21'' clade, all members carry the ''mer'' operon upstream of ''tnpR'' with the direction of transcription to the right (<span style="color:#44546a;">Fig. Tn3.15D top</span>). ''merR'', on the other hand, is transcribed in the opposite direction and terminates with a TAG codon within the IRL sequence (<span style="color:#44546a;">Fig. Tn3.15D bottom</span>) with one exception, Tn''6023''. On the other hand, for the few members of the Tn''3'' clade, the ''mer'' genes are located downstream of ''tnpA'' and are transcribed to the left (<span style="color:#44546a;">Fig. Tn3.15E top</span>) except for ''merR'' which is transcribed towards and terminates some distance from IRR (<span style="color:#44546a;">Fig. Tn3.15E bottom</span>), while for the unique Tn''4651'' member, the ''mer'' operon is located between ''tnpA'' and ''tnpS/T'' (<span style="color:#44546a;">Fig. Tn3.15F</span>).
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| − |
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| − | <span style="color:#44546a;">The Mercury Operon: Tn</span><span style="color:#44546a;">''21''</span><span style="color:#44546a;"> in </span><span style="color:#44546a;">''mer''</span><span style="color:#44546a;"> acquisition by Tn</span><span style="color:#44546a;">''402''</span><span style="color:#44546a;">?</span>
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| − |
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| − | It is worth noting that members of the Tn''402'' family Tn''5053'' mercury resistance subgroup carry a single copy of a sequence closely related to Tn''21'' IRL (<span style="color:#44546a;">Fig. Tn3.15G top</span>) located in a similar position with respect to the mercury operon as the resident IRL in the Tn''21'' group <span style="color:#44546a;">(Fig. Tn3.15D</span> <span style="color:#44546a;">Fig. Tn3.15G top and middle</span>) (see [144]). There is some variability in the 10 C-terminal amino acid tail of the neighboring MerR protein (<span style="color:#44546a;">Fig. Tn3.15G bottom</span>) although the major part of MerR amino acid sequence is highly conserved. This raises the possibility that the mercury resistance genes carried by the Tn''402'' family elements was derived from an ancestral Tn''21'' group transposon.
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| − | <span style="color:#44546a;">Transposition Mechanism Overview</span>
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| − | <span style="color:#44546a;">Early Studies</span>
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| − | In early studies of Tn''3'' (Tn''1'' and ''2'') [12–15], Tn''4651'' [88] and Tn''4430 ''[30,147] it was clearly demonstrated that Tn''3'' 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 cointegrate structure followed by a site-specific recombination step, resolution, catalyzed by a dedicated enzyme, the resolvase (<span style="color:#44546a;">Fig. Tn3.2</span>). While the resolution step for a number of Tn''3'' family members has been studied in exquisite detail (see <span style="color:#44546a;">Resolution </span>below), study of the initial strand transfer and replication steps have proved problematic.
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| − |
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| − | The consequences of these pathways are shown in greater detail in <span style="color:#44546a;">Fig. Tn3.16A</span>. This underlines why not all Tn3 family transposition events yield transposons flanked by 5bp direct repeats. Figure Tn3.16Ai shows intermolecular transposition generating a cointegrate 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. Tn''3''-mediated inversions and deletions of this type have been described a number of times with Tn''3'', Tn''1'', Tn''2660'' and Tn''1721'' [6,148–153].
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| − | Early studies also demonstrated that, like a number of transposons, the transposition frequency of Tn''3'' family transposons appears to decrease exponentially with increasing length [40] (<span style="color:#44546a;">Fig. Tn3.16B</span>). 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.
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| − | <span style="color:#44546a;">Replicative transposition</span>
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| − | 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.
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| − | <span style="color:#44546a;">Interaction of transposase and transposon ends</span>
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| − | The Tn''3'' transposase, TnpA<sub>Tn''3''</sub>, was first purified in 1981 and shown to bind DNA in a salt resistant way [154] and one of the first attempts to investigate TnpA<sub>Tn''3 ''</sub>activity ''in vitro'' [155] concluded that addition of ATP was necessary to obtain TnpA<sub>Tn''3 ''</sub>binding to the Tn''3'' ends. However, in a subsequent article this was shown to be erroneous and probably due to a pH effect of the added ATP solution [156]. Purified TnpA<sub>Tn''3 ''</sub>was observed to bind specifically to both IR<sub>Tn''3''</sub> and protect a sub-terminal DNA region within the IR (<span style="color:#44546a;">Fig. Tn3.16Ci</span>) in a heparin resistant manner a measure of its strong and highly sequence-specific DNA binding activity while another study using a different TnpA<sub>Tn''3 ''</sub>purification scheme and DNA binding conditions [157] showed a much less sequence-specific protection which included the entire IR<sub>Tn''3 ''</sub>and a significant region of flanking DNA. Further functional analysis of the Tn''3'' ends [158] demonstrated that mutations in the first 10 IR<sub>Tn''3 ''</sub>base pairs (domain A) did not influence TnpA<sub>Tn''3 ''</sub>binding while mutations in the 13-38 base pair region (domain B) inhibited binding (<span style="color:#44546a;">Fig. Tn3.16Ci</span>), behavior confirmed in a second study [159]. This is a similar functional architecture to the ends of other transposable elements (see <span style="color:#44546a;">General Information/IS Organization/Terminal Inverted Repeats</span>). In addition, the effects of mutations in the Tn''3'' ends on transposition ''in vivo'' [160] indicated that mutations in the TnpA<sub>Tn''3 ''</sub>binding site have a stronger effect when present at both transposon ends than when located at only one end.
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| − | Similar binding studies have been undertaken for Tn''1000'' () (<span style="color:#44546a;">Fig. Tn3.16Cii</span>). Protection against DNAse is more extensive than for Tn''3'' although this depends critically on the binding and digestion conditions [95]. 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 Tn''1000'' ends include a binding site for the host DNA architectural protein, <span style="color:#44546a;">IHF, </span>and both proteins were found to bind cooperatively<span style="color:#44546a;"> </span>[95]. However, IHF appeared to downregulate Tn''1000'' transposition [94]. The juxtaposition of IHF sites and transposon ends has been observed in several other TE (see [161–164]).
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| − | Binding studies have also been carried out with the transposase of Tn''4430'', TnpA<sub>Tn''4430''</sub>, a Tn''3'' derivative which encodes a TnpI resolvase [165]. Here, it was necessary to use a mutant transposase (<span style="color:#44546a;">Fig. Tn3.16Ei</span>) which had been selected for a reduction in its transposition immunity (see <span style="color:#44546a;">Transposition immunity </span>below) and which concomitantly showed an increase in transposition activity. Similar protection patterns (<span style="color:#44546a;">Fig. Tn3.16Eii</span>) were observed as with TnpA<sub>Tn''3''</sub> and TnpA<sub>Tn''1000''</sub>: transposase binding protects the distal IR<sub>Tn''44300''</sub> 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 IR<sub>Tn''3''</sub> (<span style="color:#44546a;">Fig. Tn3.16Ci</span>).
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| − | <span style="color:#44546a;">TnpA functional domains</span>
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| − | The TnpA<sub>Tn''3''</sub> is 1004 amino acid residues long. Like many other transposases, it carries a DDE catalytic motif (<span style="color:#44546a;">General Information/Reaction mechanisms/The main groups</span>). Characterization of a series of fusions of TnpA<sub>Tn''3''</sub> segments to -galactosidase [166,167] (<span style="color:#44546a;">Fig. Tn3.16Di</span>) 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.
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| − | 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 TnpA<sub>Tn''3''</sub> and TnpA<sub>Tn''1000''</sub> [167]. TnpA<sub>Tn''3''</sub> and TnpA<sub>Tn''1000''</sub> were found to share over 64% identity (<span style="color:#44546a;">Fig. Tn3.16Dii</span>). 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> [167] (<span style="color:#44546a;">Fig. Tn3.16Dii</span>). 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 (<span style="color:#44546a;">Fig. Tn3.16Diii</span>) [167].
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| − | A functional map of the Tn4330 transposase, TnpA<sub>Tn4430</sub>, was obtained by partial proteolysis with trypsin and chymotrypsin (<span style="color:#44546a;">Fig. Tn3.16Di</span>)[168]. This treatment indicated that, like TnpA<sub>Tn3</sub>, TnpA<sub>Tn4430</sub> has three major domains: an N-terminal domain (amino acids 1-152) similar to a CENP-B DNA binding domain [169]<nowiki>;</nowiki> 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 [170]. The presence of insertion domains between the D and E residues observed in other transposases does not disturb the catalytic RNAse fold [170] and, in both cases studied in detail [171,172], performs crucial functions in the transposition chemistry specific for each element.
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| − | <span style="color:#44546a;">Cleavage and Strand transfer.</span>
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| − | In spite of the extensive DNA binding studies, the biochemistry of Tn''3'' family transposition has proved refractory<span style="color:#ff0000;"> </span>to detailed analysis. A single study with Tn''3'' [173] ''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 Tn''3'' derivative was observed with a cell-free extract containing TnpA<sub>Tn3 </sub>in a reaction which required Mg2+ and was stimulated by a host factor determined to be acyl carrier protein (ACP) [174]. A similar observation had been made for the Tn7 transposition reaction [175]
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| − | In a more recent a study using the mutant TnpA<sub>Tn''4430</sub> ''[165] an ''in vitro'' system including both strand cleavage and strand transfer was developed. The mutant TnpA<sub>Tn''4430''</sub> carried 3 mutations (<span style="color:#44546a;">Fig. Tn3.16Ei</span>) selected for a reduced level of <span style="color:#44546a;">transposition immunity </span>[168] but exhibiting a hyper transposition efficiency [165]. 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 single end and paired end (SEC and PEC) species containing one or two IR<sub>Tn''4430''</sub> molecules bridged by the transposase. Footprinting both types of complex revealed an identical pattern of DNase protection (<span style="color:#44546a;">Fig. Tn3.16Eii</span>) except for some additional weak protection of flanking DNA in the PEC. When probed with the 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 (<span style="color:#44546a;">Fig. Tn3.16Eii</span>). 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 <span style="color:#44546a;">Tn10 </span>and 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 (<span style="color:#44546a;">Fig. Tn3.16Eiii</span>). Initial data have also suggested that TnpA<sub>Tn''4430''</sub> binds preferential to DNA structures which resemble replication forks ''in vitro ''(cited in [23])'' ''[176] and insertion appears to be influenced by replication of the target molecule in vivo (cited in [23]).
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| − | 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. [171,177,178]). Recent data both from Atomic Force Microscopy (AFM) and Cryoelectron microscopy demonstrates that the TnpA<sub>Tn''4430''</sub> is indeed a dimer (B. Hallet personal communication; [179,180]).
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| − | <span style="color:#44546a;">Mechanism in the Light of Structure</span>
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| − | A 3.6 Å average resolution cryoelectron microscopy structure has demonstrated that TnpA<sub>Tn''4430''</sub> is indeed dimeric and has provided some insight into how it might function in transposition [179]. Moreover, using the hyperactive immunity deficient TnpA mutant it was possible to resolve a structure for the PEC which was composed of the transposase dimer and two double strand Tn''4330'' ends.
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| − | The structural model permitted a refinement of the TnpA<sub>Tn''4430''</sub> functional modules obtained from partial proteolysis and footprinting (<span style="color:#44546a;">Fig. Tn3.16Ei and Eii</span>)<span style="color:#44546a;">. </span>Four DNA binding domains were identified<span style="color:#44546a;"> </span>(DBD1-4;<span style="color:#44546a;"> Fig. Tn3.16F top</span>). DBD1,2 and 4 bind the IR in a sequence-specific manner. The first (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 (<span style="color:#44546a;">Fig. Tn3.16F bottom</span>). There are also phosphate contacts across the IR/flank junction by residues in the catalytic RNH domain. When bound, there 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 Tn''3''/Tn''1000'' transposase organization (<span style="color:#44546a;">Fig. Tn3.16Di</span>).
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| − |
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| − | The apo-protein appears relatively compact (<span style="color:#44546a;">Fig. Tn3.16Gi</span>). The dimer is held together at the bottom by the DD domains and at the top by the C-terminal domain which docks onto the surface of the adjacent monomer. The CT interaction appears to be further stabilized by DNA binding (<span style="color:#44546a;">Fig. Tn3.16Gi</span>). The authors point out that this is an unusual dimer interface. IR binding is accompanied by large conformational change (<span style="color:#44546a;">Fig. Tn3.16Gi</span>). In this pre-cleavage complex, the protein “arms” align the 4 DBD along IR, bend the DNA at Site A (<span style="color:#44546a;">Fig. Tn3.16Fiii</span>) which moves the flank with respect to the IR tip and places the scissile phosphate bond at the catalytic site of the opposite monomer both LN and RNH residues are involved. Like other transposition systems cleavage appears to be “in trans” ([https://tnpedia.fcav.unesp.br/index.php/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 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 RNaseH fold is poorly defined in the apo-protein, it becomes more easily recognizable in the rearranged PEC. However, in this conformation only E881 (<span style="color:#44546a;">Fig. Tn3.16Ei</span>) 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 (<span style="color:#44546a;">Fig. Tn3.16Hi and iii</span>) representing a strand transfer product. The low-resolution structure suggests that the target segment of the branched molecule is located at the base (<span style="color:#44546a;">Fig. Tn3.16Hii</span>). These are proposed to be the position at which the target (<span style="color:#44546a;">Fig. Tn3.16Hiv</span>) 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.
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| − | <span style="color:#44546a;">Tn3 Transposition immunity, a poorly understood phenomenon.</span>
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| − | In some of the earliest studies on TnA (Tn''1'') [181] 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 10<sup>5</sup> fold reduction in the immune target). Two other Tn''3'' family transposons, Tn''501'' and Tn''1721'', also exhibited this inhibition phenomenon (cited as personal communication in [182]). However, other studies have identified plasmids having received two copies of TnA but these probably occurred at the same time rather than consecutively [183,184].
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| − | 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 Tn''7'' (e.g. [185–187]) where it involves proteins with ATPase activity, MuB [188,189] and TnsC [190,191] respectively. However, Tn''3'' 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 Tn''7''.
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| − | <div style="margin-left:0cm;margin-right:0cm;"><span style="color:#44546a;">Immunity Requires a Transposon End</span></div>
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| − | Further analyses of TnA [182] demonstrated that between 290 bp and 470 base pairs at the right end (<span style="color:#44546a;">Fig. Tn3.16Ii</span>) were sufficient to confer immunity [182]. 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 [182]. While plasmids carrying the right end showed immunity, those carrying the left end showed no immunity or only “partial-immunity”. 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 Tn''3'' derivative in which ''tnpR'' was inactivated by linker insertion. In this system [148,192], 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 [193]. Here, plasmids carrying restriction fragments containing one or other Tn''3'' 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 an immune and non-immune targets appeared to be different [193]. However, the study also indicated in some cases that the orientation of the Tn3 DNA fragment in the target affected the immunity level. Furthermore, it was observed that deletions within the IRs which eliminated transposition, also eliminated immunity (<span style="color:#44546a;">Fig. Tn3.16Iii</span>) [194]. However, studies comparing TnpA<sub>Tn''3''</sub> binding and immunity [159] 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 [195] was not supported by subsequent studies [194].
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| − | A finer scale analysis of the extent of the Tn''3'' IR sequence required for immunity was obtained by sequential deletion analysis of one IR [196] (<span style="color:#44546a;">Fig. Tn3.16Iiii</span>). 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; <span style="color:#44546a;">Fig. Tn3.16Ci</span>). This is also largely in agreement with the results from Huang et al.[194].
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| − | Interestingly, Bishop and Sherratt [153], using a plasmid system which allows identification of both inter- and intra-molecular 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 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.
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| − | For 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 Tn''1000'' insertion [197] while no other segment of Tn''1000'' 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 Tn''1000''). 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 [197].
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| − | Like Tn''4652'' (<span style="color:#44546a;">Fig. Tn3.12G</span>) [90,91] in which IHF binding to sites located close to the ends positively regulates TnpA binding [91] to the terminal IRs, Tn''1000'' also carries IHF sites proximal to the IRs. A more detailed analysis of the related Tn''1000'' IRR [94,198] using a mating-out assay [199] 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/Tn''1'',Tn''2'',Tn''3'') conferred a significantly higher level of immunity in the presence of IHF (<span style="color:#44546a;">Fig. Tn3.16Iiv</span>) while removal of the terminal 2 GC base pairs at the tip had no real effect. IHF has been shown to bind cooperatively with TnpA<sub>Tn''1000''</sub> [95]. This result strongly suggested that it is the IHF-enhanced binding strength TnpA<sub>Tn''1000'' </sub>which determines the level of immunity [94].
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| − | 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.
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| − | <span style="color:#44546a;">Immunity in Tn</span><span style="color:#44546a;">''4430''</span>
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| − | More recent studies on immunity of Tn''4430'' [200] have involved isolation of TnpA<sub>Tn''4430''</sub> mutants which escape immunity [168]. 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 (<span style="color:#44546a;">Fig. Tn3.16Ei</span>). 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 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 [179]
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| − | Although some data suggested that immunity could be observed in a relatively crude cell-free system [201], the establishment of a more defined and robust ''in vitro'' transposition system [165] might permit further experimental investigation into the molecular basis of Tn''3'' family transposition immunity.
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| − | <span style="color:#44546a;">On Ended Transposition.</span>
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| − | Early in the study of Tn''21'' and 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 [202,203] 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 <span style="background-color:#ffffff;"> 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 </span><span style="background-color:#ffffff;">[204]</span><span style="background-color:#ffffff;">. Some recombinants were observed to contain only short segments of the donor plasmid </span><span style="background-color:#ffffff;">[205]</span><span style="background-color:#ffffff;">. 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. </span>
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| − | <span style="color:#44546a;">Resolution</span>
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| − | <span style="color:#44546a;">The serine recombinases.</span>
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| − | <span style="color:#000000;">Efficient resolvase-catalyzed recombination between two directly repeated </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites is instrumental in completing transposition by physically separating donor and target molecules. This was first recognized in studies on complementation of transposition deficient Tn</span><span style="color:#000000;">''1''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> mutants where mutation of </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> resulted in accumulation of </span>cointegrates [8,10,192,206] (<span style="color:#44546a;">Fig. Tn3.2ii</span>). 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 [207]<span style="color:#000000;"> for both genes (see </span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">), but that it has an active function in the transposition process.</span>
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| − | <span style="color:#000000;">A number of resolvase enzymes have since been recognized (for a comprehensive review see </span>[23](<span style="color:#44546a;">Fig. Tn3.17Ai-iv</span><span style="color:#000000;">). </span>
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| − | <span style="color:#000000;">The majority so far identified appear to be recombinases which use a serine residue as the nucleophile during recombination (</span><span style="color:#44546a;">Fig. Tn3.17Ai and ii</span><span style="color:#000000;">). These serine recombinases can be divided into two major groups (</span><span style="color:#44546a;">Fig. Tn3. 17A</span><span style="color:#000000;">): the “classical” recombinases, TnpR encoded by Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;">, Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> and their relatives (~185 aa); and “long” serine recombinases </span>[23,208] <span style="color:#000000;">(~300aa) (</span><span style="color:#44546a;">Fig. Tn3.17B</span><span style="color:#000000;">) (see </span><span style="color:#000000;">[23,209,210]</span><span style="color:#000000;">. 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 Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> subgroup which includes Tn</span><span style="color:#000000;">''5044,''</span><span style="color:#000000;"> the Xanthomonas transposons Tn</span><span style="color:#000000;">''Xc4''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''Xc5''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''1412''</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.4A</span><span style="color:#000000;">). It is worth noting that all members of this Tn group also encode a </span><span style="color:#44546a;">toxin/antitoxin system </span><span style="color:#000000;">located between the divergent </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> genes (</span><span style="color:#44546a;">Fig. Tn3. 4</span><span style="color:#000000;">). </span>
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| − | <span style="color:#44546a;">Studies with Tn</span><span style="color:#44546a;">''1000 ''</span><span style="color:#44546a;">(</span><span style="color:#44546a;">γδ</span><span style="color:#44546a;">) and Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> </span><span style="color:#44546a;">''res''</span><span style="color:#44546a;">.</span>
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| − | <span style="color:#000000;">Early studies using the resolvase of Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;"> (aka </span><span style="color:#000000;">γδ</span><span style="color:#000000;">) </span><span style="color:#000000;">''in vitro''</span><span style="color:#000000;"> demonstrated that the enzyme could introduce double strand breaks in a </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site and, in the absence of the divalent cation Mg</span><span style="color:#000000;"><sup>2+</sup></span><span style="color:#000000;">, formed covalent TnpR-DNA intermediates </span>[211].<span style="color:#000000;"> Cleavage occurred at a </span><span style="color:#000000;">crossover point in a palindromic sequence to generate a </span><span style="color:#000000;">cleavage product with a free 3’OH group and a</span><span style="color:#000000;"> 2 base 3’ overhang </span><span style="color:#000000;">[211]</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">). </span><span style="color:#000000;">Furthermore, formation of a free 3’OH implied that the covalent protein-DNA linkage occurred at the 5’ end and was more efficient if the substrate carried 2 directly repeated </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> copies. This led to the hypothesis that although TnpR acts as a repressor at </span><span style="color:#000000;">''res''</span><span style="color:#000000;">, binding simultaneously to two </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> copies in some way changes the protein conformation allowing recombination to proceed </span><span style="color:#000000;">[211]</span><span style="color:#000000;">. It was further shown using DNase and footprinting that </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''3</sub></span><span style="color:#000000;"> ''</span><span style="color:#000000;">and </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1000''</sub></span><span style="color:#000000;"> carry three TnpR binding sites </span><span style="color:#000000;">[212]</span><span style="color:#000000;">, I, II and III (where sites II and III, known as accessory sites, are </span><span style="color:#000000;">closely spaced and site I known as the core site, is very slightly distanced) (</span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">) </span><span style="color:#000000;">[212]</span><span style="color:#000000;"> and that the recombination point (the dinucleotide TA) </span><span style="color:#000000;">[213]</span><span style="color:#000000;"> is included within site I. Each site shows some degree of two-fold symmetry </span>[212,214] <span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">). The </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn3</sub></span><span style="color:#000000;"> has an identical organization </span>[215] <span style="color:#000000;">and almost identical sequence and the Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;"> TnpR products are interchangeable </span><span style="color:#000000;">[215]</span><span style="color:#000000;">. These similarities were exploited to determine the crossover point using Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;"> TnpR-mediated resolution between </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''3</sub></span><span style="color:#000000;"> ''</span><span style="color:#000000;">and </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1000''</sub></span><span style="color:#000000;"> carried by a single plasmid </span>[213].<span style="color:#000000;"> </span>
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| − | <span style="color:#44546a;">Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> </span><span style="color:#44546a;">''res,''</span><span style="color:#44546a;"> </span><span style="color:#44546a;">''tnpR''</span><span style="color:#44546a;"> and </span><span style="color:#44546a;">''tnpA''</span><span style="color:#44546a;"> gene expression.</span>
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| − |
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| − | <span style="color:#000000;">In both Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;">, </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> are divergent and the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site is located in the intergenic space with subsite III proximal to tnpR (</span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">). Promoters for both </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpR,''</span><span style="color:#000000;"> were located by footprinting of RNA polymerase and lie within </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> </span>[207,216]<span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">). The -35 promoter elements of both gene are only 10 bp distant from each other and the -10 element of </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> is located within site I straddling the point of recombination crossover (</span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">). The transcription start point for both genes has been mapped. Clearly, tnpA and tnpR expression would be regulated by TnpR binding.</span>
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| − |
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| − | <span style="color:#000000;">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 </span><span style="color:#000000;">[23]</span><span style="color:#000000;">).</span>
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| − | <span style="color:#44546a;">The Mechanics of Resolution.</span>
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| − | <span style="color:#000000;">TnpR binding to </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> generates a highly compact protein-DNA complex as judged by electron microscopy </span>[217].<span style="color:#000000;"> This was explained by the observation that TnpR binding to </span><span style="color:#000000;">''res''</span><span style="color:#000000;">-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 </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites </span><span style="color:#000000;">[218]</span><span style="color:#000000;">.</span>
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| − |
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| − | <span style="color:#000000;">Gentle proteolysis of purified Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;"> 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 </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites </span><span style="color:#000000;">[219]</span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.17B</span><span style="color:#000000;">)</span><span style="color:#000000;">. Unlike full length TnpR which binds the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sub-sites with equal affinity, the C-terminal fragment binds to each of the half-sites but with different affinities suggesting that the N-terminal part of TnpR is involved in protein-protein interactions within the TnpR-</span><span style="color:#000000;">''res''</span><span style="color:#000000;"> complex </span><span style="color:#000000;">[219]</span><span style="color:#000000;">. Footprinting of small fragment binding indicated that the protection was centered on the 9bp half-sites (</span><span style="color:#44546a;">Fig. Tn3.17Ci</span><span style="color:#000000;">)</span><span style="color:#000000;">. 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 </span>[220]<span style="color:#000000;">.</span><span style="color:#000000;"> </span>
| |
| − |
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| − | <span style="color:#000000;">A model of the overall architecture of single TnpR-</span><span style="color:#000000;">''res''</span><span style="color:#000000;"> complexes was proposed </span><span style="color:#000000;">[218]</span><span style="color:#000000;"> based on results using a number of footprinting agents to reveal sensitive sites on the DNA and permutation experiments to identify DNA curvature </span>[221]<span style="color:#000000;"> in which each subsite binds a TnpR dimer (with one monomer recognizing each partial diad symmetry element called “half-sites”) </span>[212,214] <span style="color:#000000;">and introduces an “intra-site” bend in the DNA at each site while at another level, protein-protein interactions introduce inter-site bends (</span><span style="color:#44546a;">Fig. Tn3.17D</span><span style="color:#000000;">)</span><span style="color:#000000;">.</span><span style="color:#000000;"> Experimentally, this conformation requires all 3 sites and a correct spacing between sites I and II. </span>
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| − |
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| − | <span style="color:#000000;">''In vitro''</span><span style="color:#000000;"> resolution systems have been developed and require supercoiled DNA together with a divalent cation, Mg</span><span style="color:#000000;"><sup>2+</sup></span><span style="color:#000000;"> </span><span style="color:#000000;">[29,211,215,222]</span><span style="color:#000000;">. A number of laboratories have contributed to an understanding of how the complex 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. </span>
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| − |
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| − | <span style="color:#000000;">''In vitro''</span><span style="color:#000000;"> resolution requires a supercoiled DNA substrate carrying two directly repeated </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites and results in a simple concatenated recombination </span><span style="color:#000000;">product with a specific change in linking number (the number of time one DNA strand crosses another) (</span><span style="color:#44546a;">Fig. Tn3.17E</span><span style="color:#000000;">) </span><span style="color:#000000;">[29,211,215,222]</span><span style="color:#000000;"> indicating that the synaptic complex must have a very precise type of protein-DNA architecture. The </span><span style="color:#000000;">''in vitro''</span><span style="color:#000000;"> reaction is very inefficient when the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites are in an inverted orientation raising the question of how the two </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites are aligned for recombination (for review see </span><span style="color:#000000;">[28]</span><span style="color:#000000;">). Random collision between </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> 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 (</span><span style="color:#44546a;">Fig. Tn3.17Eii</span><span style="color:#000000;">). Alignment of the two </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> 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 suggests that this is not the case [123]. In particular the observation that </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> 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 (</span><span style="color:#44546a;">Fig. Tn3.17Eii</span><span style="color:#000000;">) wound DNA molecules</span><span style="color:#000000;"> </span><span style="color:#000000;">(for review see </span><span style="color:#000000;">[28]</span><span style="color:#000000;">)</span><span style="color:#000000;">.</span>
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| − |
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| − | <span style="color:#000000;">Intensive studies using both gel electrophoresis and electron microscopy to visualize TnpR recombination activities </span><span style="color:#000000;">[223,224]</span><span style="color:#000000;"> led to a model in which the two </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> </span><span style="color:#44546a;">Fig. Tn3.17Eiii</span><span style="color:#000000;">) sites are constrained in a configuration which entraps 3 supercoils (</span><span style="color:#44546a;">Fig. Tn3.17Eiiib</span><span style="color:#000000;">) and which takes into account the observation that Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> resolution (</span><span style="color:#44546a;">Fig. Tn3.17Eii</span><span style="color:#000000;">ic</span><span style="color:#000000;">)</span><span style="color:#000000;"> removes four negative supercoils on recombination (</span><span style="color:#44546a;">Fig. Tn3.17Eiiid</span><span style="color:#000000;">) </span><span style="color:#000000;">[29]</span><span style="color:#000000;">. The resulting energy change probably drives the reaction. In this model, it is TnpR interactions at the accessory sites II and III which are important for this allowing the recombining site I to finalize the recombination event (</span><span style="color:#44546a;">Fig. Tn3.17F</span><span style="color:#000000;">). This occurs by simple rotation at site I </span><span style="color:#000000;">[28]</span><span style="color:#000000;"> on the flat hydrophobic surface after simultaneous cleavage of all four strands in the synaptic complex. The TnpR monomers remain attached to the 5’ ends (</span><span style="color:#44546a;">Fig. Tn3.17F</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">left</span><span style="color:#000000;">) and the serine-DNA bond is then broken by attack by the 3’OH of the recombining site (</span><span style="color:#44546a;">Fig. Tn3.17F</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">right</span><span style="color:#000000;">) to complete recombination. More than a single round of recombination </span><span style="color:#000000;">can occur and this results in the generation of knots of increasing complexity with increasing numbers of recombination events (not shown).</span>
| |
| − |
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| − | <span style="color:#000000;">This model is supported by the structure of a TnpR tetramer bound to two site I DNA molecules in a synaptic complex </span><span style="color:#000000;">[225,226]</span><span style="color:#000000;"> which shows that each TnpR dimer bound to its DNA presents an unusual flat, hydrophobic surface to the other member of the pair (</span><span style="color:#44546a;">Fig. Tn3.17G</span><span style="color:#000000;">) with the suggestion that strand exchange indeed occurs by rotation around this interface. </span>
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| − | <span style="color:#44546a;">The Tn</span><span style="color:#44546a;">''1721''</span><span style="color:#44546a;">, Tn</span><span style="color:#44546a;">''21''</span><span style="color:#44546a;"> and Tn</span><span style="color:#44546a;">''501''</span><span style="color:#44546a;"> </span><span style="color:#44546a;">''res''</span><span style="color:#44546a;">.</span>
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| − |
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| − | <span style="color:#000000;">In contrast to those of Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;">, the </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> genes of Tn</span><span style="color:#000000;">''1721''</span><span style="color:#000000;">, Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''501''</span><span style="color:#000000;"> are transcribed in the same orientation, with </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> upstream of </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and their </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites located upstream of </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.17Aii and</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.17Cii</span><span style="color:#000000;">). They are relatively well conserved within the Tn21 clade</span><span style="color:#000000;">'' ''</span><span style="color:#000000;">(Fig. Tn3.7F). Early experiments with Tn</span><span style="color:#000000;">''501''</span><span style="color:#000000;"> showed that it too underwent transposition using a cointegrate intermediate </span><span style="color:#000000;">[227]</span><span style="color:#000000;">.</span>
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| − |
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| − | <span style="color:#000000;">Like </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''3</sub></span><span style="color:#000000;"> ''</span><span style="color:#000000;">and </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1000''</sub></span><span style="color:#000000;">, </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1721''</sub></span><span style="color:#000000;"> and </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''21''</sub></span><span style="color:#000000;"> are composed of three TnpR binding sites (I, II and III) as determined by footprinting </span><span style="color:#000000;">[228,229]</span><span style="color:#000000;"> with site III proximal to </span><span style="color:#000000;">''tnpR ''</span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.17Aii and</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.17Cii</span><span style="color:#000000;">) and each site exhibits some degree of dyad symmetry. Moreover, there is considerable identity observed the Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;">, Tn</span><span style="color:#000000;">''501''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''172''</span><span style="color:#000000;">1 </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> genes and also between the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''21</sub></span><span style="color:#000000;"> , res''</span><span style="color:#000000;"><sub>Tn50</span><span style="color:#000000;">''1''</sub></span><span style="color:#000000;"> and </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1721''</sub></span><span style="color:#000000;"> sites </span><span style="color:#000000;">[34]</span><span style="color:#000000;">. </span>
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| − |
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| − | <span style="color:#000000;">All three elements complement a </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> mutant of Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> whereas Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> does not </span><span style="color:#000000;">[34]</span><span style="color:#000000;">. This is perhaps not surprising since the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''3 ''</sub></span><span style="color:#000000;">sequence </span><span style="color:#000000;">appeared to be quite different from those of this Tn group (</span><span style="color:#44546a;">Fig. Tn3.17C</span><span style="color:#000000;">) and the authors were unable to identify a </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site homologous to that of Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;">. In addition, the TnpR amino acid sequence of Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> is somewhat distant from those of Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;">, Tn</span><span style="color:#000000;">''501''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''1721''</span><span style="color:#000000;">.</span>
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| − |
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| − | <span style="color:#000000;">These observations were reinforced by additional studies demonstrating that purified Tn</span><span style="color:#000000;">''1721''</span><span style="color:#000000;"> TnpR can resolve cointegrate substrates containing repeat </span><span style="color:#000000;">copies of </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1721''</sub></span><span style="color:#000000;">, of </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn21</sub></span><span style="color:#000000;">, and of a substrate carrying both </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn21</sub></span><span style="color:#000000;"> and</span><span style="color:#000000;">'' res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1721''</sub></span><span style="color:#000000;"> copies, but not of </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''3''</sub></span><span style="color:#000000;"> </span><span style="color:#000000;">[230]</span><span style="color:#000000;"> while </span><span style="color:#000000;">Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> TnpR catalyzed site-specific recombination between directly repeated </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn21</sub></span><span style="color:#000000;"> and</span><span style="color:#000000;">'' res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1721''</sub></span><span style="color:#000000;"> but not </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''3</span><span style="color:#000000;"> ''</sub></span><span style="color:#000000;">[231]</span><span style="color:#000000;">.</span><span style="color:#000000;"> </span><span style="color:#000000;">The reaction required a supercoiled substrate with two directly oriented </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites. </span>
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| − |
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| − | <span style="color:#000000;">Several studies explored the DNA sequence binding and recombination specificities between Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> using hybrid TnpR containing the DNA binding domain of one and the catalytic domain of the </span>other [232–234].<span style="color:#000000;"> These studies showed that, while a Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> TnpR catalytic DNA domain spliced to the Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> DNA binding domain has a somewhat lower affinity for </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn21</sub></span><span style="color:#000000;">, it retained some ability to mediate recombination between </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn21</sub></span><span style="color:#000000;"> but was unable to recombine </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn3</sub></span><span style="color:#000000;"> sites in spite of the fact that the hybrid protein was able to bind </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn3</sub></span><span style="color:#000000;">. 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 </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites is determined by the resultant organization of the DNA-protein complex</span>” [233]<span style="color:#000000;">.</span>
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| − |
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| − | <span style="color:#44546a;">Tn </span><span style="color:#44546a;">''res ''</span><span style="color:#44546a;">activity </span><span style="color:#44546a;">''tnpR''</span><span style="color:#44546a;"> and </span><span style="color:#44546a;">''tnpA''</span><span style="color:#44546a;"> gene expression.</span>
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| − |
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| − | <span style="color:#000000;">It was proposed </span>[34] <span style="color:#000000;">that in all three elements, </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> may be transcribed independently of </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> and that its promoter is located within </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;">. Moreover, no Tn</span><span style="color:#000000;">''501''</span><span style="color:#000000;"> </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> promoter could be found </span><span style="color:#000000;">''in vitro''</span><span style="color:#000000;">. This is consistent with the observation that in interreplicon Tn</span><span style="color:#000000;">''501''</span><span style="color:#000000;"> transposition into plasmid R388, resolution could be induced in the recipient by mercury selection </span>[227] <span style="color:#000000;">suggesting that </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> may be expressed at least partially as part of the mercury resistance operon located upstream of </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;">.</span>
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| − |
| |
| − | <span style="color:#000000;">Interestingly, a study using Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> revealed a gene, </span><span style="color:#000000;">''tnpM''</span><span style="color:#000000;"> (for modulator), whose expression appeared to enhance transposition and suppress resolution</span> [54]<span style="color:#000000;">. TnpM results from the insertion of the Tn</span><span style="color:#000000;">''402''</span><span style="color:#000000;"> derivative, Tn</span><span style="color:#000000;">''5060''</span><span style="color:#000000;"> which led to the formation of Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.7G</span><span style="color:#000000;">). This event </span><span style="color:#000000;">interrupted the </span><span style="color:#000000;">''urfM''</span><span style="color:#000000;"> gene, of unknown function but possibly part of the mercury operon, generating the C-terminal fragment with a fortuitous translation initiation codon. Removal of the region in Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;"> resulted in a reduced transposition frequency and increased resolution activity and these activities were restored when the </span><span style="color:#000000;">''tnpM''</span><span style="color:#000000;"> “gene” cloned into another compatible plasmid was provided </span><span style="color:#000000;">''in trans''</span><span style="color:#000000;">. Moreover, transposition of Tn</span><span style="color:#000000;">''501''</span><span style="color:#000000;">, which like Tn</span><span style="color:#000000;">''21''</span><span style="color:#000000;">, also includes a complete </span><span style="color:#000000;">''ufrM''</span><span style="color:#000000;"> gene, was also affected. The mechanism by which the UfrM fragment, TnpM, functions is unclear and has not been addressed since its initial description </span>[54]<span style="color:#000000;">.</span>
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| − |
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| − | <span style="color:#44546a;">The long serine recombinases</span>
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| − |
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| − | <span style="color:#000000;">TnpR proteins carrying an extended C-terminus (TnpR</span><span style="color:#000000;"><sub>L</sub></span><span style="color:#000000;">) (</span><span style="color:#44546a;">Fig. Tn3.17B</span><span style="color:#000000;">) have been studied in only a single case, Tn</span><span style="color:#000000;">''Xca5''</span><span style="color:#000000;"> (IS</span><span style="color:#000000;">''Xca5''</span><span style="color:#000000;">) from </span><span style="color:#000000;">''Xanthomonas campestris pv. citri''</span><span style="color:#000000;"> XAS450 1 </span>[235].<span style="color:#000000;"> Establishment of an </span><span style="color:#000000;">''in vitro''</span><span style="color:#000000;"> system </span>[236] <span style="color:#000000;">has shown that, as for the short forms of TnpR, recombination requires two directly repeated </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''Xc5''</sub></span><span style="color:#000000;"> copies in a supercoiled plasmid substrate and Mg</span><span style="color:#000000;"><sup>2+</sup></span><span style="color:#000000;"> as a divalent cation. Footprinting reveal three TnpR-binding subsites with a relative spacing and similar position with respect to </span><span style="color:#000000;">''tnpR</span><span style="color:#000000;"><sub>L''</sub></span><span style="color:#000000;"> as Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''1000''</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.17H</span><span style="color:#000000;">).</span>
| |
| − |
| |
| − | <span style="color:#000000;">Topological analysis of the recombination products suggests that the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''Xc5 ''</sub></span><span style="color:#000000;">synaptic complex must be very similar to those of </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''3''</sub></span><span style="color:#000000;"> and </span><span style="color:#000000;">''res''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''1000''</sub></span><span style="color:#000000;"> since 4 supercoils are lost on recombination and the directionality of strand exchange is the same </span>[236].<span style="color:#000000;"> No structural studies are at present available.</span>
| |
| − |
| |
| − | <span style="color:#44546a;">Serine-recombinases which use IHF/Hu. </span>
| |
| − |
| |
| − | <span style="color:#000000;">It is worth noting that certain serine recombinases, such as Gin and Hin , involved in inversion switches (refs) or Sin which is involved in plasmid recombination (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.</span>
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| − |
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| − |
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| − | <span style="color:#44546a;">The </span><span style="color:#44546a;">''irs''</span><span style="color:#44546a;">/TnpI system</span>
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| − |
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| − | <span style="color:#000000;">A small group of known Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family members which include the </span><span style="color:#000000;">''Bacillus thuriniensis''</span><span style="color:#000000;"> transposons Tn</span><span style="color:#000000;">''4430''</span><span style="color:#000000;"> </span>[237]<span style="color:#000000;"> and Tn</span><span style="color:#000000;">''5401''</span><span style="color:#000000;"> </span>[238] <span style="color:#000000;">encode a resolvase, TnpI, carrying a tyrosine residue at the active-site nucleophile </span>[30,238,239]<span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.17Ii</span><span style="color:#000000;">). The </span><span style="color:#000000;">''tnpI''</span><span style="color:#000000;"> gene lies upstream of </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and both genes are transcribed in the same direction (</span><span style="color:#44546a;">Fig. Tn3.17Aiii</span><span style="color:#000000;">). </span><span style="color:#000000;">Insertion mutagenesis showed that interruption of </span><span style="color:#000000;">''tnpI''</span><span style="color:#000000;"> resulted in an increased level of cointegrate intermediates in </span><span style="color:#000000;">''Escherichia coli''</span><span style="color:#000000;"> </span>[30].<span style="color:#000000;"> The Tn</span><span style="color:#000000;">''4330''</span><span style="color:#000000;"> sequence </span>[30] <span style="color:#000000;">revealed a series of small sequence repeats directly upstream of </span><span style="color:#000000;">''tnpI''</span><span style="color:#000000;"> as well as two smaller repeats abutting the inside border of IRL. The </span><span style="color:#000000;">''tnpI''</span><span style="color:#000000;"> 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 (</span><span style="color:#44546a;">Fig. Tn3.17Iii</span><span style="color:#000000;">). DNase footprinting revealed that TnpI bound to all four sites together called the internal resolution site (</span><span style="color:#000000;">''irs''</span><span style="color:#000000;">) </span>[147]<span style="color:#000000;"> but not to the (unrelated) IRL proximal repeats (</span><span style="color:#44546a;">Fig. Tn3.17Iii</span><span style="color:#000000;">). Using a suicide substrate which contains a nick close to the point of recombination and which traps intermediates in the cleavage reaction, in an </span><span style="color:#000000;">''in vitro''</span><span style="color:#000000;"> 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 </span>[147]<span style="color:#ff0000;"> </span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.17Iii</span><span style="color:#000000;">)) forming a transient 3′-phosphotyrosyl bond leading to 3’OH in an identical way to other tyrosine recombinases (e.g. </span>[240–245])<span style="color:#000000;">. A complete in vitro resolution reaction requires supercoiled DNA substrate </span>[147]<span style="color:#000000;">.</span>
| |
| − |
| |
| − | <span style="color:#000000;">A similar overall sequence architecture was observed upstream of </span><span style="color:#000000;">''tnpI''</span><span style="color:#000000;"> in Tn</span><span style="color:#000000;">''5401''</span><span style="color:#000000;"> </span>[239] <span style="color:#000000;">((</span><span style="color:#44546a;">Fig. Tn3.17Iii</span><span style="color:#000000;">i). Here, the repeated sequences are 12bp long with identical repeats abutting the inside border of IRL and of IRR. Footprinting also identified the TnpI </span><span style="color:#000000;">''irs''</span><span style="color:#000000;"> binding sites but, in addition showed TnpI binding to the IR proximal site </span>[239]<span style="color:#000000;">. </span>
| |
| − |
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| − | <span style="color:#44546a;">The Mechanics of Resolution.</span>
| |
| − |
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| − | <span style="color:#000000;">In contrast to the requirements for the accessory sites I and II in serine recombinase-catalyzed resolution </span>[221]<span style="color:#000000;">, 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 </span>[147].<span style="color:#000000;"> In other words, the accessory sites channel recombination to generate resolutive recombination between two directly repeated </span><span style="color:#000000;">''irs''</span><span style="color:#000000;"> sites on the same DNA molecule. This gave rise to the model shown in </span><span style="color:#44546a;">Fig. Tn3.17J</span><span style="color:#000000;">.</span>
| |
| − |
| |
| − | <span style="color:#000000;">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 (</span><span style="color:#44546a;">Fig. Tn3.17Kii</span>)<span style="color:#000000;"> and first strand exchange </span><span style="color:#44546a;">Fig. Tn3.17Kiii</span><span style="color:#000000;">''')'''</span><span style="color:#000000;"> to form a Holliday junction (</span><span style="color:#44546a;">Fig. Tn3.17Kiv</span><span style="color:#000000;">) while the second pair, the IR2-bound subunits, are then activated to resolve the holiday junction </span><span style="color:#44546a;">Fig. Tn3.17Kv</span><span style="color:#000000;">) by cleavage and exchange of the second strand (</span><span style="color:#44546a;">Fig. Tn3.17Kvi</span><span style="color:#000000;">) to resolve the cointegrate </span><span style="color:#44546a;">Fig. Tn3.17Kvii</span><span style="color:#000000;">)</span><span style="color:#000000;">''' '''</span>[147,246]<span style="color:#000000;">.</span>
| |
| − |
| |
| − | <span style="color:#000000;">Although the exact topology of the synaptic complex is unknown, two alternative </span>models [23]<span style="color:#ff0000;"> </span><span style="color:#000000;">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 </span><span style="color:#44546a;">Fig. Tn3.17J</span><span style="color:#000000;">). </span>
| |
| − |
| |
| − | <span style="color:#44546a;">Irs, </span><span style="color:#44546a;">''tnpR''</span><span style="color:#44546a;"> and </span><span style="color:#44546a;">''tnpA''</span><span style="color:#44546a;"> and gene expression.</span>
| |
| − |
| |
| − | <span style="color:#000000;">Transcriptional start sites within Tn</span><span style="color:#000000;">''5401''</span><span style="color:#000000;"> were mapped by primer extension analysis and the -35 and -10 promoter elements were identified (</span><span style="color:#44546a;">Fig. Tn3.17Iiii</span>) [238]. <span style="color:#000000;">Two overlapping and divergent promoters were identified: </span><span style="color:#000000;">one which would drive expression of tnpI and tnpA and the other which could drive the upstream but divergent toxin antitoxin genes (see </span><span style="color:#44546a;">Tn3 family-associated TA passenger genes are located in a unique position</span><span style="color:#000000;">).</span>
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| − |
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| − |
| |
| − | <span style="color:#44546a;">The rst/TnpS/T system.</span>
| |
| − |
| |
| − | <span style="color:#000000;">The third major type of resolution system encoded by Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family members is the TnpT-TnpS system which uses a resolution site, </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"> (</span><span style="color:#000000;">'''r'''</span><span style="color:#000000;">es site for Tnp</span><span style="color:#000000;">'''S'''</span><span style="color:#000000;"> and Tnp</span><span style="color:#000000;">'''T'''</span><span style="color:#000000;">) encoded by the catabolic transposons Tn</span><span style="color:#000000;">''4651 ''</span>[247]. Tn''4651''<span style="color:#000000;">, isolated from a Pseudomonas plasmid carries a set of toluene degrading (</span><span style="color:#000000;">''xyl''</span><span style="color:#000000;">) passenger genes (</span><span style="color:#44546a;">Fig. Tn3.3</span>)<span style="color:#000000;"> and is similar to the mercury resistance transposon Tn</span><span style="color:#000000;">''5041''</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.17Li</span>)<span style="color:#ff0000;"> </span>[97]<span style="color:#000000;">. 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 (</span><span style="color:#44546a;">Fig. Tn3.17Lii</span>).<span style="color:#ff0000;"> </span><span style="color:#000000;">Resolution of cointegrates generated by Tn</span><span style="color:#000000;">''4651''</span><span style="color:#000000;"> was shown to require three Tn</span><span style="color:#000000;">''4651''</span><span style="color:#000000;">-encoded factors: the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site (now called </span><span style="color:#000000;">''rst''</span><span style="color:#000000;">) and the </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpT''</span><span style="color:#000000;"> gene products which are located at a significant distance (48kb) away from the </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> transposase gene. A similar long distance between transposase and resolvase is found in Tn</span><span style="color:#000000;">''5041''</span><span style="color:#000000;"> </span>[98] <span style="color:#44546a;">Fig. Tn3.17L</span><span style="color:#000000;">). Here, </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpT ''</span><span style="color:#000000;">are referred to as </span><span style="color:#000000;">''orfQ''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''orfI''</span><span style="color:#000000;"> respectively and rst as </span><span style="color:#000000;">''att''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''5041''</sub></span><span style="color:#000000;">.</span>
| |
| − |
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| − | <span style="color:#000000;">The 323 aa TnpS protein is a tyrosine recombinase (</span><span style="color:#44546a;">Fig. Tn3.17M</span><span style="color:#000000;">) </span><span style="color:#000000;">with similarity to the Cre resolvase</span> [23]<span style="color:#000000;"> while the 332 aa TnpT appears to enhance TnpS-mediated recombination </span>[31].<span style="color:#000000;"> </span>
| |
| − |
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| − | <span style="color:#000000;">The sequence of the </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;">/</span><span style="color:#000000;">''T''</span><span style="color:#000000;"> intergenic region is very similar in Tn</span><span style="color:#000000;">''4651''</span><span style="color:#000000;"> [147], Tn</span><span style="color:#000000;">''4652''</span><span style="color:#000000;"> (a Tn</span><span style="color:#000000;">''4651''</span><span style="color:#000000;"> deletion derivative lacking the toluene-catabolic genes) </span>[248]<span style="color:#000000;">, </span>Tn''4661'' [27] <span style="color:#000000;">and Tn</span><span style="color:#000000;">''4676''</span><span style="color:#000000;"> </span><span style="color:#000000;">[99]</span><span style="color:#000000;">. It is composed of a 203 bp sequence which includes two pairs on inverted repeats, IRL and IRR and IR1 and IR2 (</span><span style="color:#44546a;">Fig. Tn3.17N</span><span style="color:#44546a;">i</span><span style="color:#000000;">) with overlapping promoters which drive TnpS and TnpT expression. The mRNA start point was identified by primer </span>extension [31].
| |
| − |
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| − | <span style="color:#000000;">The length of functional </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"> site, 136 bp, was defined by the recombination activities of sequential deletion derivatives in an </span><span style="color:#000000;">''in vivo''</span><span style="color:#000000;"> resolution system </span>[23].<span style="color:#000000;"> This involved the construction of an artificial cointegrate containing one complete </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"> copy and a second copy which carries the deletions. IR1 and IR2 are indispensable for the full resolution activity and cointegrate resolution was shown to require both TnpS and TnpT.</span><span style="color:#ff0000;"> </span><span style="color:#000000;">Moreover, the resolution reaction could be reversed to obtain site-specific integration (recombination between </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"> 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 </span>[23,31].
| |
| − |
| |
| − | <span style="color:#000000;">Although the TnpS/T proteins of Tn</span><span style="color:#000000;">''4651''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''4661''</span><span style="color:#000000;"> are highly similar and the Tn share highly similar sequences in the inverted repeat motif, IRL and IRR, of the </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"> core site, the 7bp spacer separating the repeats are somewhat different (</span><span style="color:#44546a;">Fig. Tn3.17N</span><span style="color:#44546a;">i</span><span style="color:#000000;">). An artificial cointegrate composed of an </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;"> and an </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4661''</sub></span><span style="color:#000000;"> site could not be resolved using TnpS</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;"> and TnpT</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;"> </span>[27].<span style="color:#000000;"> The mismatches in the IRL-IRL region concern principally the spacer region between IRL and IRR. </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;"> and </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4661''</sub></span><span style="color:#000000;"> have six mismatches, with five located in the spacer (</span><span style="color:#44546a;">Fig. Tn3.17N</span><span style="color:#44546a;">ii</span><span style="color:#000000;">). In other tyrosine recombinase systems such as xerC/D or phage </span>P1 Cre protein<span style="color:#000000;"> (see </span><span style="color:#000000;">[23,240,249]</span><span style="color:#000000;">) 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 </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4661''</sub></span><span style="color:#000000;"> was replaced with that from </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651,''</sub></span><span style="color:#000000;"> (</span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4661''</sub></span><span style="color:#000000;">v1) (</span><span style="color:#44546a;">Fig. Tn3.17N</span><span style="color:#44546a;">ii</span><span style="color:#000000;">). In contrast to the cointegrate carrying both </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;"> and </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4661''</sub></span><span style="color:#000000;">, that carrying </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;"> and </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4661''</sub></span><span style="color:#000000;">v1 underwent TnpS</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;"> and TnpT</span><span style="color:#000000;"><sub>Tn</span><span style="color:#000000;">''4651''</sub></span><span style="color:#000000;">-mediated resolution, demonstrating that it is the differences spacer sequence which prevents recombination </span>[27]. <span style="color:#000000;">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 </span><span style="color:#000000;">site of </span>Tn''5041'' [97,98]<span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.17L</span><span style="color:#000000;">) shows significant similarity to those of Tn</span><span style="color:#000000;">''4651 ''</span>[27]. <span style="color:#000000;">Moreover, in depth analysis of the Tn</span><span style="color:#000000;">''5041 ''</span><span style="color:#000000;">resolution reaction has not been reported.</span>
| |
| − |
| |
| − | <span style="color:#000000;">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 </span>[27].
| |
| − |
| |
| − | <span style="color:#44546a;">Toxin-Antitoxin genes: Special Passengers linked to the transposition process?</span>
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| − |
| |
| − | <span style="color:#000000;">Several studies had identified individual Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family members with type II toxin-antitoxin (TA) passenger </span>genes [33,121,250,251]<span style="color:#000000;"> (see reference </span>[23]<span style="color:#000000;">). Unusually for passenger genes of this Tn family, the T/A genes are consistently found adjacent to a resolvase gene.</span>
| |
| − |
| |
| − | <span style="color:#000000;">Some type II TA systems are involved in plasmid maintenance in growing bacterial populations by a mechanism known as 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 </span>[252] a<span style="color:#000000;">nd R1 </span>[253] <span style="color:#000000;">and it was recently shown that acquisition of a Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family transposon Tn</span><span style="color:#000000;">''6231 ''</span><span style="color:#000000;">carrying a type II TA gene pair was indeed able to “stabilize” an unstable target plasmid </span>[251].
| |
| − |
| |
| − | <span style="color:#000000;">Many different type II TA gene pairs have now been identified in bacterial chromosomes as well as plasmids </span><span style="color:#000000;">[254–256]</span><span style="color:#000000;">.</span><span style="color:#ff0000;"> </span><span style="color:#000000;">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 </span>[255]<span style="color:#000000;">). The antitoxin includes a DNA binding domain involved in promoter binding and negative regulation of TA expression.</span>
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| − |
| |
| − | <span style="color:#44546a;">Identification of TA gene pairs in Tn3 family members.</span>
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| − |
| |
| − | <span style="color:#000000;">Among nearly 200 Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family members, 39 were observed to carry type II T/A genes (colored squares; </span><span style="color:#44546a;">Fig. Tn3.4A, </span><span style="color:#44546a;">Fig. Tn3.18B</span><span style="color:#000000;">) </span>[33].<span style="color:#000000;"> The host transposons included examples from all known combinations and orientations of transposase and resolvase genes (</span><span style="color:#44546a;">Fig. Tn3.18A</span>)<span style="color:#ff0000;"> </span><span style="color:#000000;">and were almost all located adjacent to the resolvase genes in family members with TnpR, with long serine TnpR, with TnpI (e.g. Tn</span><span style="color:#000000;">''5401''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''Bth4''</span><span style="color:#000000;">) and with TnpS/T (e.g. Tn</span><span style="color:#000000;">''HdN1.1''</span><span style="color:#000000;">) (</span><span style="color:#44546a;">Fig. Tn3.4A</span><span style="color:#000000;">, </span><span style="color:#44546a;">Fig. Tn3.18B</span><span style="color:#000000;">). Illustrative examples are shown in (</span><span style="color:#44546a;">Fig. Tn3.18A</span><span style="color:#000000;">). </span>
| |
| − |
| |
| − | <span style="color:#44546a;">TA diversity in Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> family members.</span>
| |
| − |
| |
| − | <span style="color:#000000;">The Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;">-associated TA modules include a number of different types of TA module: 5 toxin (RelE/ParE, Gp49, PIN_3, PIN,and HEPN) and 6 antitoxin families (ParD, HTH_37, RHH_6, Phd/YefM, AbrB/MazE, and MNT) (</span><span style="color:#44546a;">Fig. Tn3.4A; </span><span style="color:#44546a;">Fig. Tn3.18B</span><span style="color:#000000;">). All, except ParE, are associated with RNase activit</span>y [254,255,257]<span style="color:#000000;">, while ParE inhibits gyrase activity by an unknown molecular mechanism </span>[258].
| |
| − |
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| − | <span style="color:#44546a;">TA distribution and organization within the Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> family</span><span style="color:#000000;"> </span>
| |
| − |
| |
| − | <span style="color:#000000;">The majority of examples occurred in two Tn</span><span style="color:#000000;">''3 ''</span><span style="color:#000000;">subgroups: Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> (2 toxin families) and Tn</span><span style="color:#000000;">''3000''</span><span style="color:#000000;"> (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, TnpR</span><span style="color:#000000;"><sub>L</sub></span><span style="color:#000000;"> as their resolvase and two (Tn</span><span style="color:#000000;">''5401''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''Bth4''</span><span style="color:#000000;">)</span><span style="color:#000000;"> </span><span style="color:#000000;">encode the tyrosine TnpI resolvase, while those in the Tn</span><span style="color:#000000;">''3000''</span><span style="color:#000000;"> subgroup all encode a short serine resolvase, TnpR</span><span style="color:#000000;"><sub>S</sub></span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.4A;</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.18B</span><span style="color:#000000;">). 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 (</span><span style="color:#44546a;">Fig. Tn3.18B</span><span style="color:#000000;">).</span>
| |
| − |
| |
| − | <span style="color:#000000;">Although TA genes are generally arranged with the antitoxin upstream of the toxin gene, TA systems of reverse order have been identified </span>[255].<span style="color:#000000;"> Among </span><span style="color:#000000;">the Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family-associated TA systems, in five Tn</span><span style="color:#000000;">''3000''</span><span style="color:#000000;"> subgroup members (</span><span style="color:#44546a;">Fig. Tn3.18B</span><span style="color:#000000;"> and </span><span style="color:#44546a;">Fig. Tn3.4A</span><span style="color:#000000;">) the RelE/ParE superfamily toxin Gp49 (PF05973) toxin gene</span><span style="color:#000000;">''' '''</span><span style="color:#000000;">precedes that of a HigA superfamily antitoxin, HTH_37 (PF13744) </span><span style="color:#000000;">[254–256]</span><span style="color:#000000;">. A similar situation is found in the unrelated Tn</span><span style="color:#000000;">''4651''</span><span style="color:#000000;"> subgroup member Tn</span><span style="color:#000000;">''Posp1_p''</span><span style="color:#000000;">.</span>
| |
| − |
| |
| − | <span style="color:#000000;">In addition to encoding a TnpI resolvase, Tn</span><span style="color:#000000;">''5401''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''Bth4''</span><span style="color:#000000;">, both encode a ParD antitoxin, which appears to lack the DNA-binding domain.</span><span style="color:#ff0000;"> </span>
| |
| − |
| |
| − | <span style="color:#44546a;">Acquisition and exchange of TA modules.</span>
| |
| − |
| |
| − | <span style="color:#000000;">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 T/A encoding Tn (e.g. Tn</span><span style="color:#000000;">''5051''</span><span style="color:#000000;"> and its derivatives which differ essentially by their other passenger genes), clearly the fact the Tn collection also includes examples of different combinations of T/A genes and examples in which the gene order has been inverted argue for a certain level of repeated acquisition. </span>
| |
| − |
| |
| − | <span style="color:#000000;">In cases where the TA module is found in related transposons (with similar </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> 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 </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> family group, different resolvase) but which harbor TA modules that are similar at the DNA level, it is likely that the TA module was acquired by recombination with another transposon.</span><span style="color:#000000;"> </span>
| |
| − |
| |
| − | <span style="color:#000000;">Phylogenetic analysis suggested that ParE had been acquired three times, Gp49 together with an HTH antitoxin</span><span style="color:#ff0000;"> </span><span style="color:#000000;">on three occasions and PIN on two occasions </span>[33].
| |
| − |
| |
| − | <span style="color:#000000;">Although it is unclear how most of the 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-</span><span style="color:#000000;">recombinases) or double (s-recombinases) breaks. It seems possible that the modules were recruited via non-productive resolution events. </span>
| |
| − |
| |
| − | <span style="color:#000000;">Moreover, this recombination activity has clearly led to spread of T/A modules to different Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family members by inter-</span><span style="color:#000000;">''res''</span><span style="color:#000000;"> recombination (</span><span style="color:#44546a;">Fig. Tn3.18B</span><span style="color:#000000;">). There are two cases in the Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> library, both involving Tn</span><span style="color:#000000;">''5051''</span><span style="color:#000000;"> and its derivatives, which demonstrate this capacity (</span><span style="color:#44546a;">Fig. Tn3.18C</span><span style="color:#000000;">). In the first case (</span><span style="color:#44546a;">Fig. Tn3.18Ci</span><span style="color:#000000;">), comparison between Tn</span><span style="color:#000000;">''5051''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''Tsp1''</span><span style="color:#000000;"> shows a clear break in the homology between the two Tn which occurs at the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site (</span><span style="color:#44546a;">Fig. Tn3.18Ci</span><span style="color:#000000;">). The identities towards the right of the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site III decrease rapidly within a short distance. In the second case (</span><span style="color:#44546a;">Fig. Tn3.18Cii</span><span style="color:#000000;">), comparison of Tn</span><span style="color:#000000;">''5051''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''4662a''</span><span style="color:#000000;"> shows a clear break in identity at </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site I suggesting that they have previously exchanged left and right ends </span><span style="color:#000000;">''via''</span><span style="color:#000000;"> recombination at </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site I. An additional transposon, Tn</span><span style="color:#000000;">''5051.12''</span><span style="color:#000000;"> is clearly a hybrid of these two since it carries the left end of Tn</span><span style="color:#000000;">''4662a''</span><span style="color:#000000;"> and the right end of Tn</span><span style="color:#000000;">''5051''</span><span style="color:#000000;">.</span>
| |
| − |
| |
| − | <span style="color:#44546a;">Tn3 family-associated TA passenger gene are located in a unique position.</span>
| |
| − |
| |
| − | <span style="color:#000000;">In most of the cases identified, the T/A modules are embedded within the transposition module comprising transposase and resolvase genes and the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site at a position very close to the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites</span><span style="color:#ff0000;"> </span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.18A</span><span style="color:#000000;">).</span><span style="color:#ff0000;"> </span><span style="color:#000000;">This is in sharp contrast to all other Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family passenger genes, which are generally located away from the resolution and transposon genes and, where known, have often been acquired as 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., Tn</span><span style="color:#000000;">''5501.1''</span><span style="color:#000000;">and derivatives </span><span style="color:#000000;">''5501.2''</span><span style="color:#000000;">, </span><span style="color:#000000;">''5501.3''</span><span style="color:#000000;">, </span><span style="color:#000000;">''5501.4''</span><span style="color:#000000;">, etc.). Most T/A modules </span><span style="color:#000000;">[33]</span><span style="color:#000000;"> are located directly upstream of the resolvase genes (tnpR, tnpR</span><span style="color:#000000;"><sub>L</sub></span><span style="color:#000000;"> or tnpI) (</span><span style="color:#44546a;">Fig. Tn3.18A</span><span style="color:#000000;">) with only three exceptions: a</span><span style="color:#ff0000;"> </span><span style="color:#000000;">single example of a derivative with the TnpS/TnpT resolvase, TnHdN1.1</span><span style="color:#ff0000;"> </span><span style="color:#000000;">(</span><span style="color:#44546a;">Fig. Tn3.18Aiv</span><span style="color:#000000;">), where they are located between the resolvase </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;"> and transposase genes; Tn</span><span style="color:#000000;">''Sku1''</span><span style="color:#000000;"> </span><span style="color:#000000;">[</span><span style="color:#000000;">Tn</span><span style="color:#000000;">''7197''</span><span style="color:#000000;">,</span><span style="color:#000000;"> where they are located downstream of and transcribed towards tnpR; and a partial </span><span style="color:#000000;">transposon copy, Tn</span><span style="color:#000000;">''Amu2_p''</span><span style="color:#000000;"> with a short open reading frame (ORF) of unknown function between the divergently transcribed antitoxin and </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> genes. </span>
| |
| − |
| |
| − | <span style="color:#44546a;">Regulation of Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> family TA gene expression</span><span style="color:#44546a;">. </span>
| |
| − |
| |
| − | <span style="color:#000000;">An as yet unanswered question is how expression of the identified Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;">-associated 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 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 </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> expression. </span>
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| − |
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| − | <div style="margin-left:0cm;margin-right:0cm;"><span style="color:#44546a;">Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> family with TnpR and TnpR</span><span style="color:#44546a;"><sub>L</sub></span></div>
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| − |
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| − | <span style="color:#000000;">In Tn</span><span style="color:#000000;">''3 ''</span><span style="color:#000000;">itself, which has been examined in detail, transposase and resolvase gene expression is controlled by promoters found within the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site located between the two divergent genes (</span><span style="color:#44546a;">Fig. Tn3.17C</span><span style="color:#000000;">) which are regulated by resolvase binding.</span><span style="color:#000000;"> The location of the TA </span><span style="color:#000000;">genes in proximity to the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites raises the possibility that their expression is also controlled by these promoters (</span><span style="color:#44546a;">Fig. Tn3.18Di</span><span style="color:#000000;">). Although few of the </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> sites in the collection of TA-associated Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;"> family members have been defined either experimentally or by sequence comparison, 27 potential sites were identified </span>[33] <span style="color:#000000;">using the canonical </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;">-associated </span><span style="color:#000000;">''res''</span><span style="color:#000000;">-site</span><span style="color:#000000;"> </span><span style="color:#000000;">organization schematized in </span>[23] <span style="color:#000000;">as a guide, a </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site library (kindly provided by Martin Boocock), and RSAT tools (Regulatory Sequence Analysis Tools; </span>[http://rsat.sb-roscoff.fr/ http://rsat.sb-roscoff.fr/]<span style="color:#000000;">) </span>[33]. <span style="color:#000000;">For transposons with a TnpR or TnpR</span><span style="color:#000000;"><sub>L</sub></span><span style="color:#000000;"> resolvase, the TA genes are always located just downstream from res site I, whereas </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> is located next to </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> site III (</span><span style="color:#44546a;">Fig. Tn3.18Ai</span><span style="color:#44546a;">, ii and v;</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.18Di</span><span style="color:#000000;">).</span><span style="color:#000000;"> </span><span style="color:#000000;">In transposons with divergent </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> such as Tn</span><span style="color:#000000;">''Xc5''</span><span style="color:#000000;"> and Tn</span><span style="color:#000000;">''5563a''</span><span style="color:#000000;"> (</span><span style="color:#44546a;">Fig. Tn3.18Di</span><span style="color:#000000;">), </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;">, </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> are organized similarly to those of Tn</span><span style="color:#000000;">''3''</span><span style="color:#000000;">, which itself does not carry the TA module, except that </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> is separated from </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> by the intervening TA genes.</span><span style="color:#000000;"> </span><span style="color:#000000;">This organization is also similar in Tn3 members in which </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> is </span><span style="color:#000000;">downstream of </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> and in the same orientation (e.g., Tn</span><span style="color:#000000;">''5501''</span><span style="color:#000000;">and Tn</span><span style="color:#000000;">''4662a;''</span><span style="color:#ff0000;"> </span><span style="color:#44546a;">Fig. Tn3.18Ai </span><span style="color:#44546a;">and v</span><span style="color:#000000;">)</span><span style="color:#000000;">. </span>
| |
| − |
| |
| − | <div style="margin-left:0cm;margin-right:0cm;"><span style="color:#44546a;">Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> family with TnpI </span></div>
| |
| − |
| |
| − | <span style="color:#000000;">Promoters have also been defined in the </span><span style="color:#000000;">''res ''</span><span style="color:#000000;">(</span><span style="color:#000000;">''irs''</span><span style="color:#000000;">) site of the </span><span style="color:#000000;">''tnpI''</span><span style="color:#000000;">-carrying Tn</span><span style="color:#000000;">''5401 ''</span><span style="color:#000000;">[238,239,259]</span><span style="color:#000000;">, and </span><span style="color:#000000;">''tnpI''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> expression is modulated by TnpI binding to the </span><span style="color:#000000;">''irs''</span><span style="color:#000000;"> site </span><span style="color:#000000;">[259]</span><span style="color:#000000;"> </span><span style="color:#44546a;">(</span><span style="color:#44546a;">Fig. Tn3.17I</span><span style="color:#000000;">). The other tnpI-carrying transposon with TA genes, Tn</span><span style="color:#000000;">''Bth4''</span><span style="color:#000000;">, has an identical </span><span style="color:#000000;">''irs''</span><span style="color:#000000;"> 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 (</span><span style="color:#44546a;">Fig. Tn3.18Dii</span><span style="color:#000000;">).</span>
| |
| − |
| |
| − | <div style="margin-left:0cm;margin-right:0cm;"><span style="color:#44546a;">Tn</span><span style="color:#44546a;">''3''</span><span style="color:#44546a;"> family with TnpS/T</span></div>
| |
| − |
| |
| − | <span style="color:#000000;">Finally, transposon TnHdN1.1 (</span><span style="color:#44546a;">Fig. Tn3.18Aiv</span><span style="color:#000000;">) is the only example in our collection of a </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;">/</span><span style="color:#000000;">''tnpT''</span><span style="color:#000000;"> transposon carrying a TA module. The </span><span style="color:#000000;">''res''</span><span style="color:#000000;"> (</span><span style="color:#000000;">''rst''</span><span style="color:#000000;">) site and relevant promoter elements for the divergently expressed </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpT''</span><span style="color:#000000;"> have been identified between the two genes in transposon Tn</span><span style="color:#000000;">''4651 ''</span><span style="color:#000000;">(</span><span style="color:#ff0000;">refs</span><span style="color:#000000;">) (</span><span style="color:#44546a;">Fig. Tn3.18Diii</span><span style="color:#000000;">). In TnHdN1.1, the TA gene pair is to the right of </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;">, between </span><span style="color:#000000;">''tnpS''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;">, 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 </span><span style="color:#000000;">''rst''</span><span style="color:#000000;"> site regulate expression of the TA gene pair.</span>
| |
| − |
| |
| − | <span style="color:#000000;">Thus, for all three types of resolvase-carrying Tn3 family members, the 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 Tn</span><span style="color:#000000;">''Sku1 ''</span><span style="color:#000000;">and Tn</span><span style="color:#000000;">''Amu2''</span><span style="color:#000000;">_p. 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. </span>
| |
| − |
| |
| − |
| |
| − | <span style="color:#44546a;">A model for T/A activity in transposon transposition</span><span style="color:#44546a;">. </span>
| |
| − |
| |
| − | <span style="color:#000000;">Type II TA expression, like that of tnpA and tnpR, is tightly regulated at the transcriptional level (</span>see [255]). <span style="color:#000000;">Where analyzed, the toxin-antitoxin complex binds via the antitoxin 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 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 TA genes include their indigenous promoters </span>[255,260,261].
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| − |
| |
| − | <span style="color:#000000;">Transposon Tn</span><span style="color:#000000;">''6231''</span><span style="color:#000000;"> </span>[251] (<span style="color:#000000;">99% identical to Tn</span><span style="color:#000000;">''4662''</span><span style="color:#000000;">) 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 (</span><span style="color:#44546a;">Fig. Tn3.18E</span><span style="color:#000000;">). Expression could occur from a resident TA promoter (</span><span style="color:#44546a;">Fig. Tn3.18Ei</span><span style="color:#000000;">) 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 </span><span style="color:#000000;">''res ''</span><span style="color:#000000;">promoter (</span><span style="color:#44546a;">Fig. Tn3.18Eii</span><span style="color:#000000;">). 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. </span>[251] <span style="color:#000000;">together with additional expression linked to derepression of the </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> (and </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;">) promoters that must occur during transposition (</span><span style="color:#44546a;">Fig. Tn3.18Eiii</span><span style="color:#000000;">). Regulation of </span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> and </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;"> 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 zygotic induction</span>[262] o<span style="color:#000000;">r plasmid transfer derepression as originally observed for the plasmid ColI </span>[263] <span style="color:#000000;">and subsequently for plasmids </span>R100 [264]<span style="color:#000000;"> and R1 </span>[265].<span style="color:#000000;"> An alternative but nonexclusive explanation stems from the observed enhanced plasmid stability afforded by Tn</span><span style="color:#000000;">''6231''</span><span style="color:#000000;"> TnpR, in addition to that afforded by the neighboring TA system </span>[251]. <span style="color:#000000;">Resolvase systems are known to promote resolution of plasmid dimers (see reference </span>[42])<span style="color:#000000;">, and it was suggested that integration of the TA system into Tn</span><span style="color:#000000;">''6231''</span><span style="color:#000000;"> “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 </span><span style="color:#000000;">''ccd''</span><span style="color:#000000;"> TA system of </span><span style="color:#000000;">''Escherichia coli''</span><span style="color:#000000;"> plasmid F is in an operon with a resolvase-encoding </span>gene [266,267]. <span style="color:#000000;">Expression of the TA module from the </span><span style="color:#000000;">''tnpA''</span><span style="color:#000000;">/</span><span style="color:#000000;">''tnpR''</span><span style="color:#000000;"> 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</span><span style="color:#000000;"> </span><span style="color:#000000;">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 </span>[33],<span style="color:#000000;"> a notion which could be explored experimentally. </span>
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| − |
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| − | <span style="background-color:#ffffff;color:#44546a;">Conclusion and Future.</span>
| |
| − |
| |
| − | <span style="background-color:#ffffff;">The Tn</span><span style="background-color:#ffffff;">''3''</span><span style="background-color:#ffffff;"> 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 Tn</span><span style="background-color:#ffffff;">''3''</span><span style="background-color:#ffffff;"> 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. </span>
| |
| − |
| |
| − | <span style="background-color:#ffffff;">Recent studies with Tn</span><span style="background-color:#ffffff;">''4330''</span><span style="background-color:#ffffff;"> however may have unlocked a door to understanding Tn</span><span style="background-color:#ffffff;">''3''</span><span style="background-color:#ffffff;"> 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 Tn</span><span style="background-color:#ffffff;">''7''</span><span style="background-color:#ffffff;">. </span>
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| − |
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| − | <div style="margin-left:1.199cm;margin-right:0cm;"><span style="background-color:#ffffff;">Bibliography</span></div>
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