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This chapter has appeared in a modified form as: '''The IS6 family, a clinically important group of insertion sequences including IS26.''' Alessandro Varani, Susu He, Patricia Siguier, Karen Ross and Michael Chandler.Mobile DNA (2021) 12:11 '''[https://doi.org/10.1186/s13100-021-00239-x doi:10.1186/s13100-021-00239-x] <ref>{{#pmid:33757578}}</ref>'''


== General ==
There are at present nearly 160 family members in [https://isfinder.biotoul.fr/ ISfinder] from nearly 80 bacterial and archaeal species but this represents only a fraction of those present in the public databases. The family was named<ref name=":0">Galas DJ, Chandler M. Bacterial Insertion Sequences. In: Berg DE, Howe MM, editors. Mob DNA. Washington, D.C.: American Society for Microbiology; 1989. p. 109–162. </ref> after the directly repeated insertion sequences in transposon Tn''6'' <ref>Berg DE, Davies J, Allet B, Rochaix JD. Transposition of R factor genes to bacteriophage lambda. ProcNatlAcadSciUSA. 1975;72:3628–3632. </ref> to standardize the various names that had been attributed to identical elements (e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15DI IS''15Δ'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15D''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS46 IS''46''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS140 IS''140'']'','' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS160 IS''160''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS176 IS''176'']) <ref name=":16">Labigne-Roussel A, Courvalin P. IS15, a new insertion sequence widely spread in R plasmids of gram-negative bacteria. MolGenGenet. 1983;189:102–112.
<br /></ref><ref name=":17">Trieu-Cuot P, Courvalin P. Nucleotide sequence of the transposable element IS15. Gene. 1984;30:113–120. </ref><ref name=":18">{{#pmid:2994132
</ref><ref name=":6">{{#pmid:PMC326375
</ref><ref name=":7">{{#pmid:3003524
</ref><ref name=":2">{{#pmid:PMC215669
</ref><ref>Nucken EJ, Henschke RB, Schmidt FR. Nucleotide-sequence of insertion element IS15 delta IV from plasmid pBP11. DNA Seq. 1990;1:85–88.
<br /></ref><ref name=":32">{{#pmid:6304469
</ref><ref name=":28">{{#pmid:2999303
</ref><ref name=":3">Colonna B, Bernardini M, Micheli G, Maimone F, Nicoletti M, Casalino M. The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936- kanamycin resistance element. Plasmid. 1988;20:221–231.</ref><ref name=":4">{{#pmid:PMC162495
</ref><ref>{{#pmid:PMC305975}}</ref>, including one isolate, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15''], corresponding to an insertion of one iso-IS''6'' ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15DI IS''15Δ'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15D''], note that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15DI IS''15Δ''] is sometimes referred to as IS''15D'', IS''15-Δ'' or IS''15-delta'') into another <ref name=":17" /><ref name=":18" /> (Note that compared to [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']'','' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15''D] derivatives, have one or two base pair changes). More recently there has been some attempt to rename the family as the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] family (see <ref name=":22">{{#pmid:32871211}}</ref>), presumably because of accumulating experimental data from [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] itself and the importance of this IS in accumulation and transmission of multiple anti biotic resistance, although this might potentially introduce confusion in the literature. IS''6'' family members have a simple organization ([[:File:IS6.1.png|Fig. IS6.1]]) and generate 8bp direct target repeats on insertion.   
This family is very homogenous with an average length of about 800 bp and highly conserved short, generally perfect, '''IRs''' ([[:File:IS6.1.png|Fig. IS6.1]] and [[:File:Fig. IS6.2.png|Fig. IS6.2]]). There are two examples of MITES ('''M'''iniature '''I'''nverted repeat '''T'''ransposable '''E'''lements composed of both IS ends and no intervening orfs; <ref>{{#pmid:2842323}}</ref>of 227 and 336 bp), 7 members between 1230 and 1460 bp and three members between 1710 and 1760 bp. One member, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15''], of 1648 bp represents and insertion of one IS into another <ref name=":16" /><ref name=":18" />. Many are found as part of [[Transposons families/compound transposons|compound transposons]] (called [[Transposons families/compound transposons|pseudo-compound transposons]] <ref name=":0" /> described below) invariably as flanking ''direct'' ''repeats'' ([[:File:IS6.1.png|Fig. IS6.1]]) a consequence of their transposition mechanism <ref name=":6" /><ref name=":2" /><ref name=":3" /><ref name=":4" /><ref name=":48">{{#pmid:PMC1196216}}</ref><ref>{{#pmid:PMC3195058}}</ref><ref>{{#pmid:PMC3587239}}</ref><ref>{{#pmid:PMC219079}}</ref><ref name=":11">{{#pmid:PMC209129}}</ref><ref>Barberis-Maino L, Berger-Bachi B, Weber H, Beck WD, Kayser FH. IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris. Gene. 1987;59:107–113. </ref><ref>{{#pmid:PMC174916}}</ref><ref name=":33">{{#pmid:3033719
</ref><ref name=":34">{{#pmid:2543009
</ref><ref>Sundstrom L, Jansson C, Bremer K, Heikkila E, Olsson-Liljequist B, Skold O. A new dhfrVIII trimethoprim-resistance gene, flanked by IS26, whose product is remote from other dihydrofolate reductases in parsimony analysis. Gene. 1995;154:7–14. </ref><ref name=":8">{{#pmid:19074421
</ref><ref>{{#pmid:21393132}}</ref><ref name=":29">{{#pmid:PMC284528
</ref>. 
[[File:IS6.1.png|border|center|thumb|600x600px|'''Fig. IS6.1.''' IS''6'' family organization. '''A.''' Structure of IS''6'' family. The IS is represented by a yellow bar. Left ('''IRL''') and right ('''IRR''') terminal 14 bp IRs are shown as grey-filled arrows with the DNA sequence below. The 8 bp direct target repeats are shown as black-filled arrows. The transposase open reading frame is shown in purple and its orientation is indicated by the arrowhead. '''B.''' A Pseudo-compound transposon (see text for explanation). IS''6'' family characteristics are as above. A generic antibiotic resistance gene '''ABr''' is shown in red.  ]]
[[File:Fig. IS6.2.png|alt=|border|center|thumb|720x720px|'''Fig. IS6.2.''' The general characteristics of the IS''6'' family. '''A.''' Distribution of IS length (base pairs). The number of examples used in the sample is shown above each column. '''B.''' shows the domain structure of IS''6'' family transposases with a [[wikipedia:Helix-turn-helix|helix-turn-helix domain]] (HTH) and a catalytic domain with the Characteristic DDE triad followed by a K/R residue, and, in the case of the middle section, an additional zinc finger motif present in the longer members of the family ('''clade h''') while in the right-hand section an additional N-terminal domain is present ('''clade i'''). '''C.''' Secondary structure prediction of TnpA [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] by Jpred '''D.''' Left ('''IRL''') and right '''IRR''' inverted terminal repeats are shown in [http://weblogo.threeplusone.com/ WebLogo] format.]]
== Distribution and Phylogenetic Transposase Tree ==
A phylogenetic tree based on the transposase amino acid sequence of the [https://isfinder.biotoul.fr/ ISfinder] collection ([[:File:IS6.3.png|Fig. IS6.3]]) shows that the IS''6'' family members fall into a number of well-defined clades. This slightly more extensive set of IS corresponds well to the results of another wide-ranging phylogenetic analysis <ref name=":5">{{#pmid:PMC6807381
</ref>. These clades include one which groups all archaeal IS''6'' family members ([[:File:IS6.3.png|Fig. IS6.3]] '''a''') composed mainly of ''[[wikipedia:Euryarchaeota|Euryarchaeota]]'' (''[[wikipedia:Haloarchaea|Halobacteria]]'' ; [[:File:IS6.3.png|Fig. IS6.3]] '''ai-iii'''). '''Group''' '''aiv''' includes both ''[[wikipedia:Euryarchaeota|Euryarchaeota]]'' (''[[wikipedia:Thermococcales|Thermococcales]]'' and ''[[wikipedia:Methanococcales|Methanococcales]]'') and ''[[wikipedia:Crenarchaeota|Crenarchaeota]]'' (''[[wikipedia:Sulfolobales|Sulfolobales]]''). Of the 10 clades containing bacterial IS: clade b includes examples from the [[wikipedia:Alphaproteobacteria|Alpha]]-, [[wikipedia:Betaproteobacteria|Beta]]-, and [[wikipedia:Gammaproteobacteria|Gamma-''proteobacteria'']], ''[[wikipedia:Firmicutes|Firmicutes]]'', ''[[wikipedia:Cyanobacteria|Cyanobacteria]]'', ''[[wikipedia:Acidobacteria|Acidobacteria]]'' and [[wikipedia:Bacteroidetes|Bacteroidetes]] ; '''clade''' '''c''' is more homogenous and is composed of ''[[wikipedia:Alphaproteobacteria|Alphaproteobacteria]]'' (''[[wikipedia:Rhizobiaceae|Rhizobiaceae]]'' and ''[[wikipedia:Methylobacteriaceae|Methylobacteriaceae]]''); '''clade d''' includes some [[wikipedia:Actinobacteria|Actinobacteria]], [[wikipedia:Alphaproteobacteria|Alpha]]-, [[wikipedia:Betaproteobacteria|Beta]]-, and ''[[wikipedia:Gammaproteobacteria|Gamma-proteobacteria]]'' ; while '''clades e, f, g''' and '''h''' are composed exclusively of [[wikipedia:Firmicutes|Firmicutes]] (almost exclusively ''[[wikipedia:Lactococcus|Lactococci]]'' in the case of '''clades e and f'''). '''Clades I''' and '''j''' are more mixed.                               
Clearly, the  [https://isfinder.biotoul.fr/ ISfinder] collection does not necessarily reflect the true IS''6'' family distribution and these grouping should be interpreted with care. For example, although many do not form part of the [https://isfinder.biotoul.fr/ ISfinder]database, IS''6'' family elements are abundant in archaea and cover almost all of the traditionally recognized archaeal lineages (methanogens, halophiles, thermoacidophiles, and hyperthermophiles <ref>{{#pmid:PMC1847376}}</ref> ([[:File:IS6.3.png|Fig. IS6.3]]) .                               
[[File:IS6.3.png|border|center|thumb|920x920px|'''Fig. IS6.3.''' A dendrogram of IS''6'' family members. The figure shows '''11''' major clades. The surrounding colored circles and the insert indicate the clades identified by [38]. The insert shows the correspondence between the clades from Harmer and Hall and those defined here. Clades: '''A :''' composed almost entirely of '''archea''' ; '''Ai:''' (n=12) is composed of diverse Halobacterial species (''Halohasta'', ''Haloferax'', ''Natrinema'', ''Natrialba'', ''Halogeometricum'', ''Natronomonas'', ''Natronococcus'', and ''Haloarcula''); '''Aii:''' (n = 12) is composed uniquely of Halobacterial Euryarchaeota; '''Aiii:''' (n = 5) is composed entirely of Halobacterial Euryarchaeota (Haloarcula, Halomicrobium, Natronomonas, Natronobacterium, Natrinema); Aiv: (n = 9) which includes both Euryarchaeota and Crenarchaeota; '''b:''' (n=16) Actinobacteria, Alpha-, Beta-, and Gamma-proteobacteria; '''c:''' (n= 14) Alphaproteobacteria: Rhizobiaceae and Methylobacteriaceae); '''d:''' (n=24) (Alpha-, Beta-, and Gamma-proteobacteria, Firmicutes, Cyanobacteria, Acidobacteria and Bacteroidetes); '''e:''' (n=23) is composed mainly of IS from ''Lactococcus'', a single ''Leuconostoc'' and other bacilli (''Lysteria'', ''Enterococcus''); '''f:''' (n = 11) largely Staphylococci with 2 ''B. thuringiensis''; '''g:''' (n = 10) is heterogenous (Alpha proteobacteria: ''Methylobacterium'', ''Paracoccus'', ''Roseovarius'', ''Rhizobium'', ''Bradyrhizobium''; Deinococci and ''Halobacteria''); '''h:''' (n= 5) composed entirely of Firmicutes (''Natranaerobius'', ''Clostridium'' and ''Thermoanaerobacter'') ;  '''i:''' (n=3) is composed of ''Halanaerobia'' and ''Thermoanaerobacter''. TnpA protein sequences retrieved from ISfinder curated data set were aligned with MAFFT 7.309, and their best-fit evolutionary models were predicted with ProTest 3.2.4. A maximum-likelihood tree was reconstructed with RaxML 8.2.9 using a bootstrap value of 1,000. The final tree was visualized in FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Inkscape 0.92.4 (http://www.inkscape.org).|alt=]]       
====Terminal Inverted Repeats.====
The division into clades is also underlined to some extent by the '''IR''' sequences. As shown in [[:File:Fig. IS6.2.png|Fig. IS6.2]] ('''bottom'''), in spite of the wide range of bacterial and archaeal species in which family members are found, there is a surprising sequence conservation. In particular, the presence of a G dinucleotide at the IS tips and '''cTGTt''' and '''caaa''' internal motifs. Sequence motifs are more pronounced when each clade is considered separately ([[:File:FigIS6.4d.png|Fig. IS6.4]]). 
*'''Clade b'''
(n=16; ''[[wikipedia:Actinobacteria|Actinobacteria]]'', ''[[wikipedia:Alphaproteobacteria|Alpha]]''-, ''[[wikipedia:Betaproteobacteria|Beta]]''-, and ''[[wikipedia:Gammaproteobacteria|Gamma-proteobacteria]]'') includes a well conserved GG..cTGTTGCAAA signature with little conservation further into each end.
*'''Clade c'''
(n= 14; ''[[wikipedia:Alphaproteobacteria|Alphaproteobacteria]]'': ''[[wikipedia:Rhizobiaceae|Rhizobiaceae]]'' and ''[[wikipedia:Methylobacteriaceae|Methylobacteriaceae]]'') shows considerable conservation of an extended motif (GGG... TGTCGCAAA) and some conservation further into both '''IRL''' and '''IRR''', although these are different for each end.
*'''Clade d'''
(n=24; with [[wikipedia:Alphaproteobacteria|Alpha]]-, [[wikipedia:Betaproteobacteria|Beta]]-, and [[wikipedia:Gammaproteobacteria|Gamma-''proteobacteria'']], ''[[wikipedia:Firmicutes|Firmicutes]]'', ''[[wikipedia:Cyanobacteria|Cyanobacteria]]'', ''[[wikipedia:Acidobacteria|Acidobacteria]]'' and ''[[wikipedia:Bacteroidetes|Bacteroidetes]]'') maintains stronger traces of parts of these motifs (GG.. tcTGtt and CAaa).
*'''Clade e'''
(n=23; s composed mainly of IS from ''[[wikipedia:Lactococcus|Lactococcus]]'', a single ''[[wikipedia:Leuconostoc|Leuconostoc]]'' and other bacilli (''[[wikipedia:Listeria|Lysteria]]'', ''[[wikipedia:Enterococcus|Enterococcus]]'');
*'''Clade f'''
(n = 11; largely ''[[wikipedia:Staphylococcus|Staphylococci]]'' with 2 ''[[wikipedia:Bacillus_thuringiensis|B. thuringiensis]]'') also exhibit the typical GGTTCTGTTGCAAAGTTt signature and some internal conservation in IRL.
*'''Clade g'''
(n = 10; is more heterogenous ([[wikipedia:Alphaproteobacteria|Alpha proteobacteria]]'': [[wikipedia:Methylobacterium|Methylobacterium]], [[wikipedia:Paracoccus|Paracoccus]], [[wikipedia:Roseovarius|Roseovarius]], [[wikipedia:Rhizobium|Rhizobium]], [[wikipedia:Bradyrhizobium|Bradyrhizobium]] ; [[wikipedia:Deinococcus–Thermus|Deinococci]]'' and ''[[wikipedia:Haloarchaea|Halobacteria]]''). It contains a poorly conserved IR sequence but does include a prominent gG dinucleotide tip and a poorly pronounced tgtcaagtt signature).
*'''Clade h'''
(n= 5; composed entirely of ''[[wikipedia:Firmicutes|Firmicutes]]'' (''[[wikipedia:Natranaerobius|Natranaerobius]]'', ''[[wikipedia:Clostridium|Clostridium]]'' and ''[[wikipedia:Thermoanaerobacter|Thermoanaerobacter]]'') exhibits a moderately well-defined internal signature TcTgTtAAgTt).
*'''Clade i'''
Finally, clade I (n=3) is composed of Halanaerobia and [[wikipedia:Thermoanaerobacter|Thermoanaerobacter]].
'''The archaeal-specific clades also generally exhibit well-defined consensus sequences.'''
*'''Clade Ai'''
Is composed of diverse [[wikipedia:Haloarchaea|''Halobacterial'']] ''species'' (''Halohasta, [[wikipedia:Haloferax|Haloferax]], [[wikipedia:Natrinema|Natrinema]], [[wikipedia:Natrialba|Natrialba]], [[wikipedia:Halogeometricum|Halogeometricum]], [[wikipedia:Natronomonas|Natronomonas]], [[wikipedia:Natronococcus|Natronococcus]],'' and ''[[wikipedia:Haloarcula|Haloarcula]]''): GgcACtGTCTAGtT.
*'''Clade Aii'''
(n = 12) is composed uniquely of ''[[wikipedia:Haloarchaea|Halobacterial]]'' ''[[wikipedia:Euryarchaeota|Euryarchaeota]]'' with a ggtaGTGTTcagatAaG signature and significant internal conservation which is different for each end.
*'''Clade Aiii'''
(n = 5), is composed entirely of ''[[wikipedia:Haloarchaea|Halobacterial]]'' ''[[wikipedia:Euryarchaeota|Euryarchaeota]]'' (''[[wikipedia:Haloarcula|Haloarcula]], [[wikipedia:Halomicrobium|Halomicrobium]], [[wikipedia:Natronomonas|Natronomonas]], [[wikipedia:Natronobacterium|Natronobacterium]], [[wikipedia:Natrinema|Natrinema]]'') also has well conserved ends, ggtcgTGTTTaGTT, and significant internal conservation which is different for each end.
*'''Clade Aiv'''
(n = 9) which includes both [[wikipedia:Euryarchaeota|''Euryarchaeota'']] and [[wikipedia:Crenarchaeota|''Crenarchaeota'']], has poor conservation although on further analysis, an alignment shows significant conservation in the [[wikipedia:Sulfolobus|''Sulfolobus'']] and in the [[wikipedia:Pyrococcus|''Pyrococcus'']] groups with good interior conservation also in the 3 [[wikipedia:Pyrococcus|''Pyrococcal'']] members. It is possible that the IS ends in the [[wikipedia:Sulfolobus|''Sulfolobus'']] members have not been accurately identified.
[[General Information/IS Identification#IS identification|MCL analysis]] <ref>{{#pmid:PMC101833}}</ref> for the entire group of transposases using the criteria of ISfinder for classification  (IS identification) <ref>{{#pmid:19286454}}</ref> showed that all members fell within the definition of a single family (Inflation factor 1.2, score >30) and fell into 3 groups: clades b-I; clades Ai-Aiii; and Aiv using the appropriate filter (Inflation factor 2, score >140). The answer to the recent question “An analysis of the IS6/[http://weblogo.threeplusone.com/IS26 IS26] family of insertion sequences: is it a single family?”<ref name=":5" /> is therefore “Probably, '''yes'''” according to the ISfinder definition.
A recent study <ref name=":19">{{#pmid:30753435
</ref> identified a number of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] variants with specific mutations in their Tpases. In particular one variant, originally called [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15DI IS''15Δ'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15D''] <ref name=":17" /><ref>Trieu-Cuot P, Courvalin P. Transposition behavior of IS15 and its progenitor IS15-Δ: Are cointegrates exclusive end products? Plasmid. 1985 Jul;14(1):80–89. </ref> was observed to exhibit enhanced activity and it was suggested that such mutants, even though they satisfy ISfinder criteria attributing a new name for an IS (< 95% nucleotide identity and/or < 98% amino acid identity). It has been suggested that such variant should be suffixed as [http://weblogo.threeplusone.com/IS26 IS''26''].v1, .v2 etc. <ref name=":19" />. This makes sense if the mutation is not functionally neutral results in a change IS properties or behavior.
<gallery mode="slideshow" caption="'''Fig. IS6.4.'''">
File:FigIS6.4a.png|'''Fig. IS6.4.''' Clades '''b''' and '''c''' left (IRL) and right IRR and a combined IRL+IRR inverted terminal repeats for each clade are shown in WebLogo format (Crooks et al., 2004). The last slide shows an alignment of the ends of clade Aiv adjusted by hand
File:FigIS6.4b.png|'''Fig. IS6.4.''' Clades '''d''' and '''e''' left (IRL) and right IRR and a combined IRL+IRR inverted terminal repeats for each clade are shown in WebLogo format (Crooks et al., 2004). The last slide shows an alignment of the ends of clade Aiv adjusted by hand
File:FigIS6.4c.png|'''Fig. IS6.4.''' Clade '''h''' left (IRL) and right IRR and a combined IRL+IRR inverted terminal repeats for each clade are shown in WebLogo format (Crooks et al., 2004). The last slide shows an alignment of the ends of clade Aiv adjusted by hand
File:FigIS6.4d.png|'''Fig. IS6.4.''' Clade '''Ai''' and '''Aii''' left (IRL) and right IRR and a combined IRL+IRR inverted terminal repeats for each clade are shown in WebLogo format (Crooks et al., 2004). The last slide shows an alignment of the ends of clade Aiv adjusted by hand
File:FigIS6.4e.png|'''Fig. IS6.4.''' Clades '''Aiii''' and '''Aiv''' left (IRL) and right IRR and a combined IRL+IRR inverted terminal repeats for each clade are shown in WebLogo format (Crooks et al., 2004). The last slide shows an alignment of the ends of clade Aiv adjusted by hand
File:FigIS6.4f.png|'''Fig. IS6.4.''' Alignment of Clades Aiv.
</gallery>
== Genomic Impact and Clinical Importance ==
Activity resulting in horizontal dissemination is suggested, for example, by the observation that copies identical to ''[[wikipedia:Mycobacterium_fortuitum|Mycobacterium fortuitum]]'' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS6100 IS''6100''] <ref name=":12">{{#pmid:2163027}}</ref> ('''Clade b''') occur in other bacteria: as part of a plasmid-associated catabolic transposon carrying genes for nylon degradation in ''[[wikipedia:Arthrobacter|Arthrobacter sp.]]'' <ref>{{#pmid:PMC205175}}</ref>, from the ''[[wikipedia:Pseudomonas_aeruginosa|Pseudomonas aeruginosa]]'' plasmid R1003 <ref>{{#pmid:PMC196970}}</ref>, in integrons of the In4-type integrons from transposons such as [https://tncentral.ncc.unesp.br/report/te/Tn1696-U12338.3 Tn''1696''] <ref>{{#pmid:1648560}}</ref><ref>{{#pmid:PMC90453}}</ref> and within the ''[[wikipedia:Xanthomonas_campestris|Xanthomonas campestris]]'' transposon [https://tncentral.ncc.unesp.br/report/te/Tn5393-M95402.1 Tn''5393b''] <ref name=":13">{{#pmid:PMC167566}}</ref>. Similar copies have also been reported in ''[[wikipedia:Salmonella_enterica|Salmonella enterica]]'' (typhimurium) <ref>{{#pmid:10930753}}</ref>, and on plasmid pACM1 from ''[[wikipedia:Klebsiella_oxytoca|Klebsiella oxytoca]]'' ([https://www.ncbi.nlm.nih.gov/nuccore/AF107205.1/ AF107205]) <ref>{{#pmid:25291385}}</ref>.
==== Passenger Genes ====
A number of IS families contain members, called tIS which carry passenger genes. A single member of the family, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDsp3 IS''Dsp3''], present in a single copy in [[wikipedia:Dehalococcoides|''Dehalococcoides sp.'' BAV1]] carries a passenger gene annotated as a [[wikipedia:Hypothetical_protein|hypothetical protein]]..
=====Expression of neighboring genes=====
The formation of hybrid promoters on insertion, where the inserted element provides a '''-35 promoter''' component and the flanking sequence carries a '''-10 promoter''' component, is clearly a general property of members of the IS''6'' family <ref name=":11" /><ref name=":9">{{#pmid:PMC101884}}</ref><ref name=":10">{{#pmid:PMC284724}}</ref><ref name=":15">{{#pmid:PMC2443897}}</ref><ref name=":20">Allmansberger R, Brau B, Piepersberg W. Genes for gentamicin-(3)-N-acetyl-transferases III and IV. II. Nucleotide sequences of three AAC(3)-III genes and evolutionary aspects. MolGenGenet. 1985;198:514–520. </ref><ref name=":21">{{#pmid:3892230}}</ref>.
For example, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS257 IS''257''] <ref>Rouch D, Skurray R. IS257 from Staphylococcus aureus member of an insertion sequence superfamily Gram-positive and Gram-negative bacteria. Gene. 1989;76:195–205. </ref>('''Clade f''') (also known as [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS431 IS''431'']) has played an important role in sequestering a variety of antibiotic resistance genes in clinical isolates of [[wikipedia:Methicillin|methicillin]] resistant [[wikipedia:Staphylococcus_aureus|''Staphylococcus aureus'']] (MRSA) (e.g. <ref name=":9" /><ref name=":10" /><ref name=":14">Rouch DA, Messerotti LJ, Loo SL, Jackson CA, Skurray RA. Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257. Mol Microbiol. 1989;3:161–175. </ref><ref>Stewart PR, Dubin DT, Chikramane SG, Inglis B, Matthews PR, Poston SM. IS257 and small plasmid insertions in the mec region of the chromosome of Staphylococcus aureus. Plasmid. 1994;31:12–20. </ref>. It provides an outward oriented promoter which drives expression of genes located proximal to the left end. Moreover, both left and right ends appear to carry a –35 promoter component which would permit formation of hybrid promoters on insertion next to a resident –10 element <ref name=":10" /><ref name=":49">{{#pmid:PMC107441}}</ref>. Insertion of can result in activation of a neighboring gene using both a hybrid promoter and an indigenous promoter <ref name=":10" />. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS257 IS''257''] is also involved in expression of [https://www.wikigenes.org/e/gene/e/2716475.html ''tetA''] <ref name=":21" /> and [https://www.wikigenes.org/e/gene/e/2598376.html ''dfrA''] <ref name=":9" /> in  [[wikipedia:Staphylococcus_aureus|''S. aureus'']]''..'' This is also true of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] which forms hybrid promoters shown to drive antibiotic resistance genes such as [https://www.uniprot.org/uniprot/Q7WUG8 ''aphA7''] ''( [[wikipedia:Pasteurella|Pasteurella piscicida]]'' <ref>{{#pmid:8052160}}</ref> ''[[wikipedia:Klebsiella_pneumoniae|Klebsiella pneumoniae]] <ref name=":11" />'')'', bla<sub>SHV-2a</sub>'' ([[wikipedia:Pseudomonas_aeruginosa|''Pseudomonas aeruginosa'']] <ref>{{#pmid:PMC89260}}</ref>) and wide spectrum [[wikipedia:Beta-lactam|beta-lactam]] resistance gene ''bla''<sub>KPC</sub> <ref>{{#pmid:PMC7190074}}</ref><ref>{{#pmid:26104715}}</ref>. While [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS6100 IS''6100''] <ref name=":12" /> ('''Clade b''') drives [https://www.wikigenes.org/e/gene/e/2716516.html ''strA''] ''[https://www.wikigenes.org/e/gene/e/2716517.html strB]'' expression in [[wikipedia:Xanthomonas_campestris_pv._vesicatoria|''X. campestris'' pv. vesicatoria]] <ref name=":13" />
The formation of hybrid promoters on insertion ('''Table''' [[General Information/IS and Gene Expression|IS and Gene Expression]]) is clearly a general property of members of the IS''6'' family <ref name=":11" /><ref name=":9" /><ref name=":10" /><ref name=":15" /><ref name=":20" /><ref name=":21" />.
=====Pseudo-compound transposons=====
This IS family is able to form transposons which resemble compound transposons with the flanking IS in direct repeat but, because of the particular transposition mechanism of IS''6'' family members which involves the formation of cointegrates (see below), were called pseudo-compound transposons <ref name=":0" /><ref name=":22" />. These include Tn''610'' (flanked by [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS6100 IS''6100''] <ref name=":12" />), Tn''4003'' and others (flanked by [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS257 IS''257''] <ref name=":14" /><ref>{{#pmid:PMC172007}}</ref><ref name=":23">{{#pmid:PMC6148190}}</ref>), Tn''2680''<ref name=":6" /> (flanked by [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] <ref name=":24">{{#pmid:21702681}}</ref>).
=====IS''26'' and the Clinical Landscape=====
In view of the particular importance of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] in clinical settings it is worthwhile devoting a separate section to the contribution of this IS to the clinical landscape. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] <ref name=":6" /><ref name=":7" /> ('''clade b''') is encountered with increasing frequency in plasmids of clinical importance where it is involved in: sequestering antibiotic resistance genes and generating arrays of these genes in clinically important conjugative plasmids and in the host chromosome; expression of antibiotic resistance genes; and other plasmid rearrangements (see <ref name=":8" /><ref name=":23" /><ref name=":50">{{#pmid:PMC1913244}}</ref><ref name=":25">{{#pmid:23330672
</ref><ref>{{#pmid:16870645}}</ref><ref name=":26">{{#pmid:23169892
</ref><ref>{{#pmid:20093380}}</ref><ref name=":30">{{#pmid:PMC4471558
</ref>).
Recognition of its place as an important player has derived from the large number of sequences now available of multiple antibiotic resistance plasmids and chromosomal segments such as '''G'''enomic '''R'''esistance '''I'''slands (GRI). It is now no longer practical to provide a complete analysis of the literature (A PubMed search (19<sup>th</sup> November 2020) using [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] as the search term yielded nearly 450 citations). The references in the following are not exhaustive but simply provide examples.
=====IS Arrays=====
IS''6'' family members are often found in arrays ([[:File:FigIS6.5.png|Fig. IS6.5]] and [[:File:FigIS6.6.png|Fig. IS6.6]]) in direct and inverted repeat in multiple drug resistant plasmids (e.g. ''[[wikipedia:Salmonella|Salmonella. typhimurium]]'' <ref name=":8" /><ref name=":24" /><ref>{{#pmid:30336162}}</ref>, ''[[wikipedia:Klebsiella|Klebsiella quasipneumoniae]]'' <ref>{{#pmid:32145334}}</ref>, ''[[wikipedia:Acinetobacter_baumannii|Acinetobacter baumannii]]'' <ref name=":26" /><ref>{{#pmid:PMC2687260}}</ref>, ''[[wikipedia:Proteus_mirabilis|Proteus mirabilis]]'' <ref name=":31">{{#pmid:PMC7448864
</ref> and uncultured sewage bacteria <ref>{{#pmid:15817778}}</ref> (among many others). These are often intercalated in or next to other transposable elements rather than neatly flanking Antibiotic Resistance genes and can form units able to undergo tandem amplification.
<br />
[[File:FigIS6.5.png|center|thumb|680x680px|'''Fig. IS6.5.''' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] arrays. Genbank accession numbers for the DNA segments are shown in parentheses. Images were initially created using SnapGene. Open reading frames are shown as horizontal boxes, where the arrowheads indicate the direction of translation. Red, antibiotic resistance genes; lavender, transposase related genes; purple, other; yellow boxes, IS copies; green boxes, integron cassette recombination sites; the terminal IRsare also shown. Grey boxes show the overlap between potential transposons. The figure shows '''A)''' overlapping potential transposons from plasmid pRCS59. '''B)''' plasmid pO26-CRL-125 containing the well known [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352''] and a longer, overlapping, element called [https://tncentral.ncc.unesp.br/report/te/Tn6023-GU562437.2 Tn''6023''] <ref name=":53">{{#pmid:20701539}}</ref>.  '''C)''' The TnMB1860 DNA segment <ref name=":51">{{#pmid:33164081}}</ref>. The major amplified segment is indicated by a horizontal bracket below. The horizontal brackets in '''A)''' and '''B)''' and the bracket in '''C)''' indicate overlapping potential transposons. The horizontal bracket represents the amplified segment. It should be noted that defining IS''26''-based transposons is difficult, since it must be shown that each “Tn” can be found in other genetic contexts.  ]]
<br />
[[File:FigIS6.6.png|center|thumb|680x680px|'''Fig. IS6.6.''' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] arrays (continuation). Genbank accession numbers for the DNA segments are shown in parentheses. Images were initially created using SnapGene. Open reading frames are shown as horizontal boxes, where the arrowheads indicate the direction of translation. Red, antibiotic resistance genes; lavender, transposase related genes; purple, other; yellow boxes, IS copies; green boxes, integron cassette recombination sites; the terminal IRsare also shown. Grey boxes show the overlap between potential transposons. The figure shows '''A)''' overlapping potential transposons from plasmid pRCS59. '''B)''' plasmid pO26-CRL-125 containing the well known [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352''] and a longer, overlapping, element called [https://tncentral.ncc.unesp.br/report/te/Tn6023-GU562437.2 Tn''6023''] <ref name=":53" />.  '''C)''' The TnMB1860 DNA segment <ref name=":51" />. The major amplified segment is indicated by a horizontal bracket below. The horizontal brackets in '''A)''' and '''B)''' and the bracket in '''C)''' indicate overlapping potential transposons. The horizontal bracket represents the amplified segment. It should be noted that defining IS''26''-based transposons is difficult, since it must be shown that each “Tn” can be found in other genetic contexts.]]
<br />
=====IS26-mediated Gene Amplification=====
Early studies with Tn''1525'' (from ''[[wikipedia:Salmonella_enterica|Salmonella enterica]]'' serovar Panama), in which an [https://www.uniprot.org/uniprot/P00551 ''aphA1'' (''aph'' (3') (5")-I) gene] is flanked by two directly repeated copies of a the IS''6'' family member, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15''], reported tandem amplification of ''[https://www.uniprot.org/uniprot/P00551 aphA1]'' when the host was challenged by [[wikipedia:Kanamycin_A|kanamycin]] <ref>{{#pmid:6304464}}</ref>. Restriction enzyme mapping was used to demonstrate that the amplified segments were of the type IS-aph-IS-aph-IS-aph-IS but no direct sequence data is available. Amplification was thought to occur by homologous recombination between two flanking [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15''] copies since it occurred in a wildtype host but the transposon was stable in a ''recA'' genetic background. Another example was observed following treatment of a patient with [[wikipedia:Tobramycin|Tobramycin]] in clinical isolates of ''Acinetobacter baumannii'' from a single patient over a period of days with continued antibiotic treatment <ref name=":27" />''.'' Amplification occurred with Tn6''020'', an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-based transposon in which the flanking IS bracket a similar ''[https://www.uniprot.org/uniprot/P00551 aphA1]'' gene and could also be reproduced in bacterial culture <ref name=":27">{{#pmid:PMC3994513}}</ref>. In this case, the amplified unit was proposed to be '''IS-aph-IS-IS-aph-IS-IS-aph-IS'''. This structure would clearly be unusual but may be due to a misinterpretation of the depth of coverage of the region. In addition, the amplified transposon had inserted into a known target prior to amplification generating the expected eight base pair target repeat but an 8bp segment between the first DR and the first IS end ('''DR-8 bp-ISaph-IS-IS-aph-IS-ISaph-IS…DR'''). A third example <ref name=":51" /> was identified during a study of clinical isolates of non-carbapenemase-producing Carbapenem-Resistant Enterobacteria, non-CP-CRE, isolated from several patients with recurrent bacteraemia. An increase in carbapenem resistance occurred partially due to IS''26''-mediated amplification up to 10 fold of a DNA segment carrying ''blaOXA''-''1'' and ''blaCTX-M-1'' genes These form part of a larger chromosomal structure of [http://weblogo.threeplusone.com/IS26 IS''26''] arrays which they call Tn''MB1860'' ([[:File:FigIS6.6.png|Fig. IS6.6]]). It was unclear whether this cassette amplification was due to transposition activity or, as had been observed in similar, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1R IS''1'']-mediated, gene amplifications <ref name=":35">{{#pmid:385225}}</ref><ref>{{#pmid:PMC219339}}</ref><ref>{{#pmid:PMC215067}}</ref><ref name=":36">{{#pmid:4942895}}</ref><ref>{{#pmid:PMC247559}}</ref><ref>{{#pmid:7003302}}</ref> which may occur by replication slippage between direct repeats or by unequal crossing-over <ref>{{#pmid:231182}}</ref><ref>{{#pmid:393954}}</ref>. 
A more dramatic amplicon, was observed in the case of the IS''6'' family member [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISAba125 IS''Aba125''] in the transposon Tn''6924'' ([[:File:Fig.IS6.6A.png|Fig.IS6.6A]]) in which two blocks of 7 and 8 IS arrays are present flanking 14 copies of [[wikipedia:Aminoglycoside|aminoglycoside]] resistance APH(3')-VI genes (aka ''aphA6'', [[wikipedia:Amikacin|amikacin resistance]]) which gives rise to high levels of resistance, presumably facilitated by high [[wikipedia:Amikacin|amikacin]] selective pressure <ref>{{#pmid:35019774}}</ref>.
[[File:Fig.IS6.6A.png|center|thumb|680x680px|'''Fig.IS6.6A.''' Example of IS arrays in Tn''6924''. ]]
Another example has been revealed by Hubbard et al <ref name=":37">{{#pmid:PMC7530762}}</ref> who analysed a multi resistant derivative of the clinically important, globally dispersed pathogenic, ''[[wikipedia:Escherichia_coli|Escherichia coli]]'' ST131 subclade H30Rx, isolated from a number of bacteraemic patients and revealed that increased [[wikipedia:Piperacillin|piperacillin]]/[[wikipedia:Tazobactam|tazobactam]] resistance was due to [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-mediated amplification of ''blaTEM-1B''. A similar type of limited (tandem dimer) amplification of an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-flanked ''blaSHV''-5-carrying DNA segment found in plasmids from a number of geographically diverse enteric species was identified in a nosocomial ''[[wikipedia:Enterobacter_cloacae|Enterobacter cloacae]]'' strain <ref>{{#pmid:19519856}}</ref>. More extensive amplification (>10 fold) was observed with the same DNA segment located in a different plasmid in a well-characterised laboratory strain of ''[[wikipedia:Escherichia_coli|Escherichia coli]]'' and occurred in a ''recA''-independent manner <ref name=":25" /> while even higher levels of tandem amplification (~65 fold) of the ''[https://www.uniprot.org/uniprot/P00551 aphA1]'' gene in the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-based Tn''6020'' were identified in ''[[wikipedia:Acinetobacter_baumannii|Acinetobacter baumannii]]'' <ref name=":27" />. 
Extensive IS''26''-mediated drug resistance gene amplification has also recently been observed experimentally under controlled laboratory conditions during growth of the host strain in sub-lethal concentrations of [[wikipedia:Meropenem|meropenem]] or [[wikipedia:Tobramycin|tobramycin]] and gave rise to high [[wikipedia:Carbapenem|carbapenem]] resistance <ref>{{#pmid:35130728}}</ref>.<br />
=====IS''26''-mediated Plasmid Cointegration=====
The earliest studies on this family of IS demonstrated that they could generate cointegrates as part of the transposition mechanism (see [[IS Families/IS6 family#Cointegrate formation|Cointegrate formation below]]) <ref name=":18" /><ref name=":6" /><ref name=":2" /><ref name=":28" /><ref name=":3" /><ref name=":29" />.
Several studies have now demonstrated that this can occur in a clinical setting. For example, plasmid pBK32533 ([https://www.ncbi.nlm.nih.gov/nuccore/KP345882.1/ KP345882]) <ref>{{#pmid:PMC4394832}}</ref>, carried by ''[[wikipedia:Escherichia_coli|E. coli]]'' BK32533 isolated from a patient with a urinary tract infection is an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-mediated cointegrate between [[wikipedia:Klebsiella_pneumoniae|''Klebsiella pneumonia''e]] BK30661 plasmid pBK30661 ([https://www.ncbi.nlm.nih.gov/nuccore/KF954759 KF954759]) <ref>{{#pmid:PMC3294926}}</ref> and a relative of ''[[wikipedia:Salmonella_enterica|Salmonella enterica]]'' p1643_10 ([https://www.ncbi.nlm.nih.gov/nuccore/KF056330 KF056330]) <ref>{{#pmid:25465657}}</ref>. Interestingly, the flanks of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies at the junction of the two plasmids are TGTTTTTT-IS-TTATTAAT and TTATTAAT-IS-TGTTTTTT. The most parsimonious explanation would be that pBK32533 was generated in a multi-step inter-molecular transposition event: in one step, an IS''26'' copy from an unknown source used a TTATTAAT target sequence in pBK30661 and this cointegrate was then resolved resulting in pBK30661 containing an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy flanked by the target repeat (TTATTAAT-IS''26''-TTATTAAT) and, in a second step, a TGTTTTTT sequence in p1643_10  was targeted by the pBK30661  [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] to generate the final cointegrate in which the two [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies are flanked by the observed target sequences. Additional examples have been identified in KPC-producing ''[[wikipedia:Proteus_mirabilis|Proteus mirabilis]]'' <ref name=":31" /> and in ''[[wikipedia:Klebsiella_pneumoniae|Klebsiella pneumoniae]]'' also involving inversions <ref name=":30" /><ref name=":38">{{#pmid:PMC5142620}}</ref>.<br />
== Organization ==
IS''6'' family members range in length from 696 to 1761 bp ([https://tncentral.ncc.unesp.br/ISfinder/index.php ISFinder]) with the majority between 789 bp ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS257 IS''257'']) to 880 bp ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS6100 IS''6100'']) ([[:File:Fig. IS6.2.png|Fig. IS6.2]] '''A''') and generally create 8 bp direct flanking target repeats ('''DR''') on insertion <ref name=":6" />.<br />
==== The transposase ====
A single transposase ''orf'' is transcribed from a promoter at the left end and stretches across almost the entire IS. The putative transposases (Tpases) are between 234 ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15DI IS''15Δ'']) and 254 (e.g., [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS6100 IS''6100'']) amino acids long with a majority in the 220-250 amino acid range. They are very closely related and show identity levels ranging from 40 to 94% with a [[wikipedia:Helix-turn-helix|helix-turn-helix]] (HTH) and a typical catalytic motif (DDE) ([[:File:Fig. IS6.2.png|Fig. IS6.2]] '''C''' and [[:File:FigIS6.7.png|Fig. IS6.7]]). However, the 7 members of '''clade h''', all from Clostridia, are somewhat larger than other IS''6'' family members (approximately 1200bp, [[:File:Fig. IS6.2.png|Fig. IS6.2]] '''A''') with longer transposases (340-350 amino acids) as a consequence of an N-terminal extension with a predicted [[wikipedia:Zinc_finger|'''Z'''inc '''F'''inger]] (ZF) composed of several CxxC motifs ([[:File:Fig. IS6.2.png|Fig. IS6.2]] '''B'''; [[:File:FigIS6.7.png|Fig. IS6.7]]). A Blast analysis of the non-redundant protein database at NCBI revealed a large number of IS''6'' family transposases of this type (data not shown). The vast majority of these were from Clostridial species. In addition, the transposases of members of '''clade i''' (450 amino acids) have both the [[wikipedia:Zinc_finger|ZF]] domain and a supplementary N-terminal extension. 
[[File:FigIS6.7.png|center|thumb|720x720px|'''Fig. IS6.7.''' Alignment of IS''6'' family transposases illustrating the presence of a [[wikipedia:Zinc_finger|zinc finger]] domain in members of '''clade i'''. Alignment of a representative sample of the transposases of IS''6'' family members including members of each major clade. The alignment was by Clustal (Sievers F, Higgins DG. 2014) and the graphic output from SnapGene. The figure shows the probable [[wikipedia:Zinc_finger|zinc finger]] N-terminal extension (consecutive CxxC motifs), the [[wikipedia:Helix-turn-helix|helix-turn-helix domain]] (HTH), and the catalytic domain (DDE  K/R).|alt=]]
Several members (e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISRle39a IS''Rle39a''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISRle39b IS''Rle39b''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISEnfa1 IS''Enfa1'']) apparently require a frameshift for Tpase expression. It is at present unclear whether this is biologically relevant. However, alignment with similar sequences in the public databases suggests that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISEnfa1 IS''Enfa1''] itself has an insertion of 10 nucleotides and is therefore unlikely to be active.
<br />
==== Transposase expression ====
In the case of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''], the promoter is located within the first 82 bp of the left end and the intact ''orf'' is required for transposition activity <ref name=":7" />, and the predicted amino acid sequence of the corresponding protein exhibits a strong DDE motif ([[:File:Fig. IS6.2.png|Fig. IS6.2]] '''C'''; [[:File:FigIS6.7.png|Fig. IS6.7]]) Translation products of this frame have been demonstrated for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS240 IS''240''] <ref name=":34" />. Little is known concerning the control of transposase expression although transposition activity of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS6100 IS''6100''] in ''Streptomyces lividans'' <ref>{{#pmid:8825097}}</ref> is significantly increased when the element is placed downstream from a strong promoter. This is surprising since IS generally incorporate mechanisms to restrict transposition induced by insertion into highly transcribed genes (see [[:File:1.32.1.png|Fig 1.32.1]]).
<br />
==== Terminal Inverted Repeats ====
All carry short, related terminal '''IR''' of 13- 25bp. (depending on how mismatches are scored ).  As shown in  [[:File:Fig. IS6.2.png|Fig. IS6.2]] '''D''', in spite of the wide range of bacterial and archaeal species in which family members are found, there is a surprising sequence conservation. In particular, the presence of a G dinucleotide at the IS tips and cTGTt and caaa internal motifs (where uppercase letters are fully conserved and lowercase letters are strongly conserved nucleotides). Sequence motifs are more pronounced when each clade is considered separately ([[:File:FigIS6.4d.png|Fig. IS6.4]]).
== Mechanism: the state of play ==
Early studies suggested that IS''6'' family members give rise exclusively to replicon fusions (cointegrates) in which the donor and target replicons are separated by two directly repeated IS copies 
(e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15DI IS''15Δ'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS15 IS''15D''] <ref name=":18" />, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''46'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''140''] <ref name=":6" /><ref name=":2" /><ref name=":32" />, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS257 IS''257'']''<ref name=":49" />'', [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1936 IS''1936''] <ref name=":3" />, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISSd1 IS''S1''] <ref>{{#pmid:PMC213975}}</ref>). More recent results principally with [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] have suggested that this IS perhaps like [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1R IS''1''] ([[IS Families/IS1 family|IS''1'' family]]) <ref>{{#pmid:PMC394650}}</ref> and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS903 IS''903''] ([[IS Families/IS5 and related IS1182 families|IS''5'' family]]) <ref>{{#pmid:6099240}}</ref><ref name=":47">{{#pmid:PMC125483}}</ref>, members of this family may be able to transpose using alternative pathways <ref name=":22" /><ref name=":39">{{#pmid:PMC4894685}}</ref><ref name=":40">{{#pmid:PMC4676283}}</ref><ref name=":41">{{#pmid:PMC4196232}}</ref>.
=== Cointegrate formation ===
Transposition of IS''6'' family elements to generate cointegrates <ref name=":18" /><ref name=":2" /><ref name=":32" /><ref name=":28" /> presumably occurs in a replicative manner by '''Target Primed Transposon Replication''' (For a discussion see “[[General Information/Influence of transposition mechanisms on genome impact|Influence of transposition mechanisms on genome impact]]”; [[:File:1.23.1.png|Fig 17.1]] and [[:File:1.23.2.png|Fig. 17.2]]), and as expected, requires two functional IS''26'' ends <ref name=":52">{{#pmid:33504667}}</ref>. As shown in [[:File:FigIS6.8.png|Fig. IS6.8]] ('''top'''), intermolecular replicative transposition of this type generates fused donor and target replicons which are separated by two copies of the IS in direct repeat at the replicon boundaries. The initial '''D'''irect '''R'''epeats (DR) flanking the donor IS are distributed between each daughter IS in the cointegrate as is the DR generated in the target site. Recombination between the two IS then regenerates the donor molecule with the original DRs and a target molecule in which the IS is flanked by new DR. No known specific resolvase system such as that found in Tn''3''-related elements (see I[[General Information/ IS derivatives of Tn3 family transposons|S Derivatives of Tn''3'' family transposons]]) has been identified in this family but “Resolution” of IS''6''-mediated cointegrates was observed to depend on a functional ''recA'' gene in several cases and therefore occurs using the host homologous recombination pathway <ref name=":18" /><ref name=":2" />. 
[[File:FigIS6.8.png|center|thumb|820x820px|'''Fig. IS6.8.''' '''Intermolecular transposition models.''' '''A:''' classical replicative cointegration (Shapiro 1979)<ref>{{#pmid:287033}}</ref><ref name=":38" /> .Donor DNA is shown in black, target DNA is a red dotted line. Replication origins on each molecule are represented by a small oval.  The IS is shown as a blue box with a white arrow indicating the direction of expression of the transposase. The small directly repeated flanking sequences generated by insertion are shown as red arrows. The target sequence destined to become the new flanking repeat is indicated by white arrows. Transposition is initiated by cleavage at both terminal inverted repeats (marked '''1''' and '''2''') of the IS to generate 3’OH ends (small green circles) that attack the target site (red arrows) in what is called a strand transfer reaction. DNA replication generates a cointegrate containing two IS copies in direct repeat together with a new target site duplication (white arrows). This structure can be subsequently resolved into a plasmid identical to the original donor plasmid and a modified target plasmid carrying an IS copy flanked by target site duplications arranged as direct repeats. '''B:''' replicative cointegration by an IS6-family pseudo-transposon <ref name=":7" /> (modified from <ref name=":0" />). The symbols are identical to those above. The transposon is composed of two directly repeated copies of the IS flanking a DNA segment carrying passenger gene (green) with the internal flanks represented by yellow arrows. A target plasmid is distinguished by an open oval representing the origin of replication. Transposase-mediated replicon fusion of the two molecules using one of the two flanking IS copies generates a third copy of the IS in the same orientation as the original pair. Homologous recombination, using the ''recA'' system, between any two copies can in principle occur. This will either regenerate the donor plasmid, leaving a single IS copy in the target, delete the transposon, or transfer the transposon to the target (as shown), leaving a single copy of the IS in the donor molecule.]]
A systematic analysis of the cointegrate forming properties of an artificial [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-based pseudo-compound transposon with a [[wikipedia:Chloramphenicol|chloramphenicol]] [[wikipedia:Acetyltransferase|transacetylase]] passenger gene has demonstrated that if the inside ends of the two flanking IS are ablated, the full-length transposon can promote cointegrate formation at a low frequency. The sequence of the resulting cointegrates confirmed that the donor and target replicons were separated by a copy of the entire transposon at each junction with the appropriate 8 base pair target duplication ([https://scholar.google.com/citations?user=0igAN8sAAAAJ&hl=en He et al.,] pers. comm).
While the intermolecular cointegrate pathway leads to replicon fusion, transposition can also occur within the same replicon. Intramolecular transposition using the replicative mechanism gives rise to deletion or inversion of DNA located between the IS and its target site. The outcome depends on the orientation of the two attacking IS ends ([[:File:FigIS6.9.png|Fig. IS6.9]]). Intramolecular transposition of this type can explain the assembly of antibiotic resistance gene clusters (e.g. <ref name=":30" />).
[[File:FigIS6.9.png|center|thumb|820x820px|'''Fig. IS6.9.''' Intramolecular transposition. Symbols are identical to those in [[:File:FigIS6.5.png|Fig. IS6.5]].  The red dotted lines represent the DNA segment between the resident IS and its intramolecular target shown as a white arrow and marked “0”.  In addition, '''a''' and '''b''' represent two markers on this DNA segment. The 3’-OH groups generated by cleavage at both IS ends can either attack the target site on the same strand (cis) (top pathway) or the opposite strand (trans) (bottom pathway). When in '''cis''', DNA between the IS and the target site is deleted as a circle containing the markers “'''a'''” and “'''b'''”, one IS copy flanked by one copy of the original flank, 2, and one copy of the target flank, 0. The other partner also contains a single IS a copy with one copy of the original flank, 1, and one copy of the target flank, 0. When the reaction occurs in trans, DNA between IS and the target site is instead inverted (“'''a b'''” becomes “'''b''' '''a'''”), bracketed by the original IS and a new copy in an inverted orientation. The target site is also duplicated but in an inverted orientation, and one copy of the original flank and one copy of the target flank is associated with each IS copy. ]]
[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name= IS''6''] family members are known to generate structures that resemble composite transposons in which a passenger gene (such as a gene specifying antibiotic resistance) is flanked by two IS copies. Generally, other flanking IS in compound structures can occur as direct or inverted repeat copies ([[General Information/IS History|IS history]]; [[:File:1.2.3.png|Fig 2.3]], [[:File:1.2.4.png|Fig. 2.4]]). However, in the case of IS''6'' functional “compound transposons”, the flanking IS are always found as direct repeats. This is a direct consequence of the (homologous) recombination event required to resolve the cointegrate structure <ref name=":18" /><ref name=":2" />. As shown in [[:File:FigIS6.8.png|Fig. IS6.8]] ('''bottom''') <ref name=":42">{{#pmid:PMC98933}}</ref>, transposition is initiated by one of the flanking IS to generate a cointegrate structure with three IS copies. “Resolution” resulting in transfer of the transposon passenger gene requires recombination between the “new” IS copy and the copy which was not involved in generating the cointegrate. The implications of this model <ref name=":0" /><ref name=":42" /> are that the transposon passenger gene(s) are simply transferred from donor to target molecules in the “resolution” event and are therefore lost from the donor “transposon”. Clearly this pathway could initiate from a donor in which the flanking IS''6'' family members were inverted with respect to each other. However, transposition would be arrested at the cointegrate stage because there is no suitable second IS to participate in recombination. It is for this reason that compound IS''6''-based transposons carry directly repeated flanking IS copies. These previously published models (e.g.<ref name=":0" /><ref name=":30" /><ref name=":38" /><ref name=":42" /> have been revisited and it has been recently proposed <ref name=":22" /> that the term pseudo-compound transposons first used over 30 years ago <ref name=":0" /> should be resurrected to describe these [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS6 IS''6''] family structures.
== Circular transposon molecules: translocatable units (TU) ==
Although [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] transposition appears to be replicative with formation of cointegrate molecules, results from ''in vivo'' experiments suggest that its transposition may be more complex <ref name=":41" />. The idea that IS''26'' might mobilize DNA in an unusual way arose from the observation that IS''6'' family members can often be found in the form of arrays <ref name=":40" /><ref name=":41" /> which could be interpreted as overlapping pseudo-compound transposons <ref name=":22" /> ([[:File:FigIS6.5.png|Fig. IS6.5]]). Note that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] and potential [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-based transposons do not necessarily carry flanking direct target repeats but, as is the case for other TE, which transpose by replicative transposition such as members of the Tn''3'' family, intramolecular transposition would lead to loss of the flanking repeats ([[:File:FigIS6.9.png|Fig. IS6.9]]). This led to the suggestion that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] might be able to transpose via a novel circular form called '''T'''ranslocatable '''U'''nits (TU) <ref name=":40" /><ref name=":41" /> (not to be confused with those originally described in the sea urchin and other eukaryotes <ref>Liebermann D, Hoffman-Liebermann B, Troutt AB, Kedes L, Cohen SN. Sequences from Sea Urchin TU Transposons Are Conserved among Multiple Eucaryotic Species, Including Humans. Mol Cell Biol. 1986;6:218–226. </ref>) such as those shown in [[:File:FigIS6.10.png|Fig. IS6.10]]. These potential circular transposition intermediates which were proposed to include a single [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy along with neighboring DNA are structurally similar to IS''1''-based circles observed in the 1970s (e.g. <ref name=":35" /><ref name=":36" />). Translocatable units differ from the transposon circles identified during copy-out-paste-in transposition by IS of the [[IS Families/IS3 family|IS''3'']] ([[:File:Fig. IS3.9A.png|Fig. IS3. 9A]]; [[IS Families/IS3 family#The Transposition Pathway|IS3 family transposition pathway]]), [[IS Families/IS21 family|IS''21'']] ([[:File:Fig. IS21.7.png|Fig. IS21.7]]), [[IS Families/IS30 family|IS''30'']], [[IS Families/IS256 family|IS''256'']] and [[IS Families/ISL3 family|IS''L3'']] families where the circular IS transposition intermediate has abutted left and right ends separated by a few base pairs and is extremely reactive to the cognate transposase. In stark contrast, for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''], the IS ends would be separated by the neighboring DNA sequence rather than by a few base pairs ([[:File:FigIS6.11.png|Fig. IS6.11]]).   
[[File:FigIS6.10.png|center|thumb|650x650px|'''Fig. IS6.10.''' Summary of analysis of TU formation from the mutant transposon Tn''4352B''. The authors <ref name=":39" /> used an IS''26''-based transposon, Tn''4352B'', carrying the ''[https://www.uniprot.org/uniprot/P00551 aphA1]'' gene in which the right hand IS fortuitously carried an additional '''GG''' dinucleotide at the left internal end of one of the components [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies to generate a '''GGGGG''' pentanucleotide at the IS tip. This appears to render the transposon unstable resulting in the excision of a non-replicative circle, called a translocatable element (TU), carrying a single IS a copy and the ''[https://www.uniprot.org/uniprot/P00551 aphA1]'' gene. The other partner, the parental plasmid from which the TU had been excised, retained one IS copy and the original 8 base pair direct target repeat (framed in red). The sequence of the IS flanks in the TU was not reported. Symbols are the same as those used in [[:File:FigIS6.6.png|Fig. IS6.6]].]] 
Evidence for the excision step of translocatable units was obtained <ref name=":40" /> from the study of the stability of two [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-based pseudo-compound transposons, “wildtype” [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352''] <ref name=":33" /> and “mutant” [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352B''] <ref name=":43">{{#pmid:PMC149023}}</ref> which carry the ''[https://www.uniprot.org/uniprot/P00551 aphA1]'' gene specifying resistance to [[wikipedia:Kanamycin_A|kanamycin]]. [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352B''] is a special mutant derivative of [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352''] including an additional GG dinucleotide at the left internal end of one of the component [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies to generate a string of 5 G nucleotides at the IS tip which appears to render the transposon unstable. Cells carrying the plasmid lose the resistance gene from the mutant [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352B''] at an appreciable rate in the absence of selection. This generates a “donor” plasmid with one copy of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] flanked by the original [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352B'']-associated 8bp direct repeats and an excision product with the size expected for a TU containing the second IS flanked by the sequences of the original central segment presumably including the additional GG dinucleotide together with the ''[https://www.uniprot.org/uniprot/P00551 aphA1]'' gene. TU formation, as judged by a PCR reaction, appeared to be dependent on the GG insertion (since, surprisingly, no TU could be detected from the wildtype [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352'']) but not on the surrounding sequence environment. Excision required an active transposase. In a test in which the target plasmid also carried an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy (a [[IS Families/IS6 family#Targeted transposition.|targeted integration reaction]] – see below), there appeared to be no difference in cointegrate formation frequencies between single [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies with or without the additional GG dinucleotide. However, results from a standard integration test into a plasmid without a resident [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy were not reported. The excision process occurs in a ''recA'' background and therefore does not require the host homologous recombination system. Moreover, frameshift mutations in both IS, which should produce severely truncated transposase, eliminated activity. This implies that the process is dependent on transposition. However, excision continued to occur if the transposase of the GG-IS copy was inactivated but was eliminated when the same transposase mutation was introduced into the ”wildtype” IS copy. This is curious since it implies that the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] transposase must act exclusively ''in cis'' on the IS from which it is expressed (see [[General Information/Transposase expression and activity#Co-translational%20binding%20and%20multimerization|Co-translational binding and multimerization]]).   
[[File:FigIS6.11.png|center|thumb|820x820px|'''Fig. IS6.11.''' '''Two models for TU formation.''' Formally, both models would result in the formation of a TU. '''Top:''' ''[[wikipedia:RecA|recA]]''-dependent simple homologous recombination from an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-based pseudo-compound transposon leading to excision and transposase-dependent replicative transposition leads to a cointegrate. '''Bottom:''' Intramolecular transposition in cis from a donor with a single IS''26'' leads to excision and transposase-dependent replicative transposition leads to a cointegrate. ]]A summary of these results is shown in [[:File:FigIS6.10.png|Fig. IS6.10]]. These data suggest that excision is driven by the wildtype [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] (L), leaving the right hand IS in the excisant. At present, there is no obvious mechanistic explanation for this phenomenon. It should be noted that recombination between directly repeated copies of IS''1'' which flank the majority of Antibiotic Resistance genes in the plasmid R100.1 (NR1) generates a non-replicative circular molecule, the r-determinant (r-det), with a single [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1R IS''1''] copy. In this case too, this “constitutive” circle production is due to a (uncharacterized) mutation in the plasmid, although in this case, circle production requires ''[[wikipedia:RecA|recA]]'' <ref>{{#pmid:6285398}}</ref>.
However, “Classical” recombination and transposition models do not fit the data The results appear to rule out two obvious models ([[:File:FigIS6.11.png|Fig. IS6.11]]): since, although both would generate the correct TU and “excisant”, the first (top panel) requires homologous recombination between two directly repeated [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies (mechanistically equivalent to the “resolution” step in intermolecular IS''6'' transposition) and the second (bottom panel), which requires a functional transposase as observed <ref name=":40" /><ref name=":41" />, would not generate the correct flanking sequences. Modification of the transposition model to take into account the entire transposon ([[:File:FigIS6.12.png|Fig. IS6.12]]) in which the active [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''L] uses either of flanking sequences of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''R] does not generate the correct structures. Thus the observed structures must be generated by another, and at present unknown, pathway. One possibility is that TU are generated by reversing a non-replicative targeted insertion mechanism presented below ([[:File:FigIS6.14.png|Fig. IS6.14]]; see [[IS Families/IS6 family#Targeted transposition.|Targeted Transposition]]).<br />[[File:FigIS6.12.png|center|thumb|820x820px|'''Fig. IS6.12.''' '''Two Models for TU formation from the Pseudo-compound Transposon [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4325B''].''' Symbols are as in the previous figures. The small filled circle within one of the internal IS flanks (white arrow) indicates the additional GG dinucleotide carried by [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4325B'']. Both use an intramolecular replicative transposition pathway in a cis configuration. In the top panel, the wildtype IS uses the flank of the mutated IS as a target. This would generate a TU with a single IS and both internal flanking sequences and an excisant with two tandem IS copies separated by a mutant flank. In the lower panel, the TU carries two tandem IS copies and the excisant.]]
=== '''Tn''4352''B Instability is Dependent on Specific-''recA'' Alleles.''' ===
Curiously, although a [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352''B] copy carried by a low copy number ''IncC'' plasmid was stable in two different ''[[wikipedia:Klebsiella_pneumoniae|K. pneumoniae]]'' hosts <ref>{{#pmid:28833671}}</ref> and in two recombination proficient ''[[wikipedia:Escherichia_coli|E. coli]]'' hosts <ref name=":1">{{#pmid:36481310}}</ref>, it became unstable in certain ''[[wikipedia:RecA|recA]]''-deficient ''[[wikipedia:Escherichia_coli|E. coli]]'' hosts. The instability appeared to depend on the particular ''[[wikipedia:RecA|recA]]'' allele involved: it occurred in the presence of the ''[[wikipedia:RecA|recA1]]'' allele <ref>{{#pmid:14294081}}</ref> but not in the presence of ''[[wikipedia:RecA|recA13]]'' <ref>{{#pmid:4898990}}</ref> or of a novel ''[[wikipedia:RecA|recA]]'' mutant fortuitously identified during the analysis <ref name=":1" />.
The necessity of a specific defective [[wikipedia:RecA|RecA]] protein derivative to accomplish the formation of a proposed TU transposition intermediate is intriguing and certainly merits further investigation.<blockquote>'''To summarize:''' It has been clearly demonstrated that circular DNA species carrying a single [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy together with flanking “passenger” DNA can be generated efficiently ''in vivo'' from a variant plasmid replicon <ref name=":43" /> and also that replicons carrying a single [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy are capable of integrating into a second replicon to form a cointegrate. This occurs at a frequency 10<sup>2</sup>-fold higher if the target plasmid contains a single IS copy and in a targeted manner not involving IS duplication.</blockquote>The TU insertion pathway was addressed by transforming TU, constructed ''in vitro'' taking advantage of a unique [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] restriction site, into recombination deficient cells carrying an appropriate target plasmid <ref name=":39" />. Establishment of the ''[https://www.uniprot.org/uniprot/P00551 aphA1]''-carrying TU was dependent on the presence of a resident plasmid carrying an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy and occurred next to the resident [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy. The DNA of two TU each with a different antibiotic resistance gene was shown to undergo this type of targeted integration and, moreover, were able to consecutively insert to generate a typical [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] array.  It was also shown that the TU circles produced from the mutant [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352''B] were able to subsequently undergo insertion <ref name=":47" />. Therefore, artificially produced TU and those produced from [https://tncentral.ncc.unesp.br/report/te/Tn4352-M20306 Tn''4352''B] are capable of subsequent insertion. In addition, Zienkiewicz et <ref name=":30" /> have presented evidence that “an IS''26'' -''bla'' <sub>SHV-5</sub> module that was capable of amplification ''in cis'' could also move in trans and concluded that: “consistent with earlier observations <ref name=":48" /><ref name=":50" /><ref>{{#pmid:19188377}}</ref>, it did not copy by transposition but via IS''26''-mediated recombination.
== Targeted transposition ==
Targeted [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] transposition, was also observed in intermolecular cointegrate formation where cointegrate formation frequency was significantly increased about 100 fold if the target replicon also contained an IS''26'' copy <ref name=":41" />. A similar result was obtained in ''[[wikipedia:Escherichia_coli|Escherichia coli]]'' with a related IS, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1216 IS''1216''] <ref>{{#pmid:PMC6952201}}</ref> whereas a third member of the family, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS257 IS''257''] (e.g., [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS431 IS''431'']) showed a much lower level of activity using the same assay. As for TU integration, this phenomenon does not appear to be the result of homologous recombination between the IS copies carried by donor and target molecules since the reaction was independent of [[wikipedia:RecA|RecA]]. Using a PCR-based assay to identify the replicon fusions between [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-containing donor and target plasmids, it was observed that the resulting cointegrate ([[:File:FigIS6.13.png|Fig. IS6.13]]) did not contain an additional copy of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] which would be expected if replicative transposition were involved ([[:File:FigIS6.12.png|Fig. IS6.12]]). This suggests that the phenomenon results from a conservative recombination mechanism. Despite the absence of [[wikipedia:RecA|RecA]], the observed cointegrate is structurally equivalent to the recombination product between the two [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies in the donor and target plasmids. However, it indeed appears to be transposition related since the phenomenon requires an active transposase in both donor and target replicons <ref name=":41" />. When each of the triad of conserved DDE residues were mutated individually in the donor plasmid, the targeted insertion frequency decreased significantly.
Another characteristic of the products was that the flanking 8 bp repeats carried by the donor and recipient [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copies are in some way exchanged <ref name=":41" /> ([[:File:FigIS6.13.png|Fig. IS6.13]]). This suggests a model in which transposase might catalyze an exchange of flanking DNA during the fusion process. [[File:FigIS6.13.png|center|thumb|600x600px|'''Fig. IS6.13.''' '''IS''26'' Non-replicative Targeted Transposition.''' Symbols are identical to those in previous figures. The diagram shows the fate of flanking sequences following a targeted integration event resulting in the formation of a cointegrate. ]]
====A Model for Targeted Integration====
One possibility ([[:File:FigIS6.13.png|Fig. IS6.13]]) is that two IS ends from different IS copies in separate replicons are synapsed intermolecularly in the same transpososome ([[:File:FigIS6.14.png|Fig. IS6.14]] '''i'''). Strand exchange would then couple the donor and target replicons ([[:File:FigIS6.14.png|Fig. IS6.14]] '''ii'''). A similar mechanism has been invoked to explain “targeted” insertion of [[IS Families/IS3 family|IS''3'']] and [[IS Families/IS30 family|''IS30'' family]] members into related IRs ([[:File:Fig. IS3.14.png|Fig. IS3.14]]) <ref name=":44">{{#pmid:14756780}}</ref><ref>{{#pmid:8389976}}</ref>. Branch migration ([[:File:FigIS6.14.png|Fig. IS6.14]] '''iii''') would lead to exchange of an entire IS strand ([[:File:FigIS6.14.png|Fig. IS6.14]] '''iv''') and cleavage at the distal IS end and strand transfer ([[:File:FigIS6.14.png|Fig. IS6.14]] '''v''') would result in the observed cointegrate ([[:File:FigIS6.14.png|Fig. IS6.14]] '''vi''') containing a single strand nick on opposite strands at each end of the donor DNA molecule. These could simply be repaired or eliminated by plasmid replication. Each IS would be composed of complementary DNA strands from each of the original donor and target IS copies. This proposed mechanism would retain the DNA flanks of the IS in the original target replicon, be dependent on an active transposase and independent of the host ''[[wikipedia:RecA|recA]]'' system. It seems probable that mismatches between the two participant IS would inhibit the strand migration reaction. This may be the reason for the observation that introducing a frameshift mutation by insertion of additional bases into the transposase gene of either participating [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] copy reduces the frequency of targeted cointegration <ref name=":41" /> since, not only does this produce a truncated transposase but also introduces a mismatch. As in the case of intermolecular targeting of the [[IS Families/IS3 family|IS''3'' family member]], [[IS Families/IS3 family#The IS911 transpososome|IS''911'']] <ref name=":45">{{#pmid:15306008}}</ref>, might require the RegG [[wikipedia:Helicase|helicase]] to promote strand migration.
The model shown in [[:File:FigIS6.14.png|Fig. IS6.14]] presents the transposition process as a progression involving two consecutive, temporally separated, strand cleavages interrupted by a strand migration. However, it seems equally probable that both cleavage reactions are coordinated within a single transpososome ([[General Information/Reaction mechanisms#The transpososome|The Transpososome]]) including both donor IS ends and the target IS ends. This would be compatible with the known properties of ''trans'' cleavage of several transposases in which a transposase molecule bound to one transposon end catalyses cleavage of the opposite end ([[General Information/Transposase expression and activity#Cleavage in Trans: A Committed Complex|Cleavage in Trans: A Committed Complex]]). Recently, evidence have been presented supporting this type of “rolling in” model <ref name=":52" />.  In addition, it has been observed that targeted integration does not necessarily require two functional ends of the donor IS <ref>{{#pmid:28833671}}</ref>. This indicates the strand migration process could be terminated (probably via HJ resolution) prior to encountering the second '''IR'''. Moreover, using two IS, IS''1006'' and IS''1008'' <ref>{{#pmid:15073307}}</ref> which have significant identity to [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''], at their ends together with a hybrid molecule IS''1006''/''1008'' constructed ''in vitro'', it was shown that targeted integration required both identical transposases and identical DNA sequences at the reacting ends. The authors propose a model in which a single IS end is cleaved and transferred to the flank of the target IS end, an event which creates a [[wikipedia:Holliday_junction|Holliday junction]] ('''HJ''') which, on branch migration, is resolved. This differs from the model shown here ([[:File:FigIS6.14.png|Fig. IS6.14]]) since it does not involve transposase-mediated cleavage at the second IS end. It is similar to that proposed for targeted insertion of [[IS Families/IS3 family#The IS911 transpososome|IS''911'']] <ref name=":44" /><ref name=":45" /><ref>{{#pmid:PMC126149}}</ref><ref>{{#pmid:PMC2546779}}</ref> which requires the [[wikipedia:Helicase|RecG helicase]] and, presumably [[wikipedia:RuvABC|RuvC]].  However, it has now been demonstrated that neither ''recG'', ''ruvA'' or ''ruvC'' genes influence this type of IS''26'' activity. This therefore implies that '''[[wikipedia:Holliday_junction|HJ]]''' structures, if they are indeed formed, must be processed by a different pathway, as yet to be deciphered <ref>{{#pmid:37358447}}</ref>.
<br />
[[File:FigIS6.14.png|center|thumb|850x850px|'''Fig. IS6.14. A model for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-mediated conservative targeted integration.''' '''i)''' Two IS ends from different IS copies in separate replicons are synapsed intermolecularly in the same transpososome, one end is cleaved to generate a 3’OH (shown as a dark blue circle) leaving a 5’ and on the flank (3 white boxes). This attacks the end of the second IS in the transpososome (shown as two dotted circles joined by a dotted line). '''ii)''' strand transfer would then couple the donor and target replicons via the target IS flank (3 bright red squares) leaving a 3’OH on the target IS (light blue circle). '''iii)''' strand migration can then occur in which one strand of the door IS and one strand of the target IS invade their partners. '''iv)''' following exchange of the entire partner strands, only a single physical strand cleavage would have occurred leaving a single single-strand break (three white squares). '''v)''' a second strand cleavage at the distal end of the donor IS occurs (dark blue circle) leaving its free 5’ flank (three orange squares). The 3’OH then attacks the distal target IS end (shown as two dotted circles joined by a dotted line). '''vi)''' strand transfer then generates a cointegrate with single-strand nicks at each end on opposite strands (white and orange squares) which could then be repaired. Note that the cointegrate retains the original flanking repeats of the target IS (three bright red and three dark red squares). ]]
====Emerging Biochemistry: Transposase binding.====
Some initial studies have begun to address binding of the IS''26'' transposase, Tnp26, to its cognate IS <ref name=":46">{{#pmid:PMC8477213}}</ref>. Tnp26 carries a typical DDE motif with the conserved triad located at codons 82, 142 and 177 ([[:File:FigIS6.7.png|Fig. IS6.7]]) and, in addition, includes the often conserved K/R residue 7 amino acids downstream from the final E(177) ([[General Information/Major Groups are Defined by the Type of Transposase They Use|Major Groups are Defined by the Type of Transposase They Use]]). As expected, the DDE triad is required for transposition activity <ref name=":41" />.
Moreover, the DNA binding domain of a number of transposases of different IS families is located in the N-terminal region which often contains multiple [[wikipedia:Alpha_helix|alpha-helices]], in particular, [[wikipedia:Helix-turn-helix|helix-turn-helix]] motifs (e.g.  [[IS Families/IS1 family|IS''1'' family]], [[IS Families/IS3 family|IS''3'' family]], [[IS Families/IS30 family|IS''30'' family]], [[IS Families/IS1595 family|IS''1595'' family]], [[IS Families/IS256 family|IS''256'' family]]). This analysis <ref name=":46" /> identified a potential N-terminal triple helix ([[:File:FigIS6.15.png|Fig. IS6.15 '''A''']]).
The binding studies used the full length 234 amino acid (27.9kD) Tnp26 or various fragments cloned as a fusion with '''M'''altose '''b'''inding '''p'''rotein (MBP) and were used with the MBP tag since its removal resulted in precipitation. The Tnp fragments composed of amino acids 1-56 (Tnp1-56, 6.95kD) and 1-72 (Tnp1-72, 9.11kD) were chosen following a secondary structure prediction of the transposase. They contain the potential triple helix. This results in fusion proteins of 72.6kd, 51.6 kD and 53.7 kD respectively. 
TheMBP-tagged proteins were used in EMSA assays with 50bp Cy5-labeled double strand DNA fragments containing the 14 bp IRs ([[:File:FigIS6.15.png|Fig. IS6.15 '''B''']]). The wildtype tnp-MPB and ''tnp''1-56 fusions generated a relatively weak retarded single band with both '''IRL''' and '''IRR''', consistent with binding to the '''IR'''. That obtained with tnp1-56 was significantly weaker than the wildtype while Tnp1-72 failed to exhibit the complex. At higher protein concentrations, DNA was retained in the well a phenomenon which did not occur with “random” DNA and could represent paired end complexes or simply sequence-specific aggregation. A number of mutations in Tnp26 including two within helix 3 and two within a few residues of the N-terminal end ([[:File:FigIS6.15.png|Fig. IS6.15 '''A''']]) eliminated the retarded complex as well as reducing the material in the well. <br />
[[File:FigIS6.15.png|center|thumb|820x820px|'''Fig. IS6.15.''' Transposase binding to IS26 ends. '''A)''' an alignment of the N-terminal end of transposases of the IS''6'' family. The IS names are shown to the left. They are those chosen by Pong et al. 2021 <ref name=":46" />  representing a single member from each major clade. A Weblogo indicating conservation is shown below. The figure was generated using Jalview (https://www.jalview.org/). The Tnp fragments used are indicated by horizontal blue lines above and the three potential helices (1, 2, 3) by horizontal blue double-headed arrows. The first '''D''' of the '''DDE''' triad is also indicated. The mutations used are shown below by arrows, with the mutant amino acids indicated within circles. '''B)''' DNA oligonucleotide sequences used. Only the top strand is shown for the left '''IRL''' and right ('''IRR''') ends. The '''IRs''' are enclosed in a blue box, with the mutations indicated above by “.” and shown below by arrows.]]
Each of the Tnp mutations were observed to reduced cointegrate formation ''in vivo'' by over 2 orders of magnitude ''in vivo''.
To determine the '''IR''' nucleotides important for Tnp recognition, a number of mutations were introduced in the IRs ([[:File:FigIS6.15.png|Fig. IS6.15 '''B''']]). A double mutant (GG>TT) at the '''IR''' tip only slightly affected complex formation as might be expected since in general, the ends of several IS have been found to be divided into two functional domains: the tip at which transposition chemistry occurs and a region inside the IR to which transposase binds in a sequence-specific way ([[General Information/IS Organization|IS Organization]]). A G>T change at position 7 appeared to abolish complex formation whereas a G>T change at position 10 did not.
Clearly, additional and more systematic mutagenesis experiments would be able to define the transposase binding site in more detail and determine whether external DNA might be required to assist in binding in a non-sequence-specific manner. Moreover, it cannot at present be ruled out that the relatively large MBP addition affects overall behavior of transposase and its derivatives. It will be of interest to determine whether Tnp26 can form dimeric or multimeric species and can form one or a number of different paired end complexes ([[General Information/Transposase expression and activity|PEC; Transposase expression and activity]]) with IS''26'' '''IR''' in which two ends are bridged by transposase, whether the wildtype transposase acts as a dimer and how end cleavages occur.
<br />
== Conclusions and Future Directions ==
We have presented a survey of our present knowledge concerning the properties, distribution and activities of IS''6'' family members and their importance, in particular that of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''], in gene acquisition and gene flow of antibacterial resistance in enterobacteria. There are many questions which remain to be answered and we feel that some care should be exercised in interpreting some of the very interesting results in the absence of formal proof. For example, the notion that the basic IS''6'' family transposition unit is a non-replicative circular DNA molecule carrying a single IS copy is attractive and would provide a nice parallel to the integron antibiotic resistance gene cassette intermediates <ref>{{#pmid:PMC1356339}}</ref><ref>{{#pmid:20707672}}</ref><ref>{{#pmid:26104695}}</ref> but such a molecule, a TU, has thus far been formally observed in only a single case. It was generated ''in vivo'' from an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-flanked peudo-transposon in which one of the two flanking IS involved included a mutation and rendered the transposon unstable. The “wildtype” transposon was stable <ref name=":40" />. Since “TU” is now being used in the literature to describe [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26'']-flanked DNA segments in multimeric arrays (e.g. <ref name=":37" />, it is essential to provide more formal evidence that these non-replicative DNA circles are indeed general intermediates in the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] transposition pathway and are not simply amplified units (AU). The fact that a replicating plasmid containing a single IS copy is able to form cointegrates does not à priori support a model for TU transposition and is not necessarily simply a TU that has the capacity to replicate <ref name=":41" /> although the observation that artificially constructed TU can undergo targeted insertion when introduced into a suitable cell by transformation <ref name=":39" /> supports the TU hypothesis. A second important question to be answered is how targeted integration occurs. We have suggested one model and suggested ways it might be tested ([[:File:FigIS6.14.png|Fig. IS6.14]]). The answers to many of these fascinating outstanding questions will be provided when the biochemistry of the reactions is known.
<br />
== Acknowledgements ==
We would like to thank [https://scholar.google.com/citations?user=0igAN8sAAAAJ&hl=en Susu He] ([https://www.nju.edu.cn/EN/ Nanjing University]) for stimulating discussions concerning the transposition models and for supplying the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS26 IS''26''] transposase secondary structure predictions in [[:File:Fig. IS6.2.png|Fig. IS6.2]] '''C'''.
[https://www.niddk.nih.gov/about-niddk/staff-directory/biography/dyda-frederick Fred Dyda] and [https://www.niddk.nih.gov/about-niddk/staff-directory/biography/hickman-alison Alison Hickman] ([https://www.niddk.nih.gov/about-niddk NIDDK], [https://www.nih.gov/ NIH], Bethesda, Maryland, USA) for reading this chapter and for suggestions and [https://www.sydney.edu.au/medicine-health/about/our-people/academic-staff/sally-partridge.html Sally Partridge] ([https://www.sydney.edu.au/ The University of Sydney] and [https://www.wslhd.health.nsw.gov.au/Westmead-Hospital Westmead Hospital], Australia) for critical comments.
==Bibliography==
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