Difference between revisions of "IS Families/IS6 family"

From TnPedia
Jump to navigation Jump to search
(Replaced content with "==General== There are at present nearly 160 family members in ISfinder (<nowiki>https://www-is.biotoul.fr/scripts/search-db.php</nowiki>) from nearly 80 bacterial and arch...")
Tags: Visual edit Replaced
Line 2: Line 2:
 
There are at present nearly 160 family members in ISfinder (<nowiki>https://www-is.biotoul.fr/scripts/search-db.php</nowiki>) from nearly 80 bacterial and archaeal species but this represents only a fraction of those present in the public databases. The family was named ��[1]� after the directly repeated insertion sequences in transposon Tn''6 ��''[2]''�'' to standardize the various names that had been attributed to identical elements (e.g. IS''15'', IS''26'', IS''46'', IS''140,'' IS''160'', IS''176'') ��[3–15]�, including one isolate, IS''15'', corresponding to an insertion of one iso-IS''6'' (IS''15''D) into another ��[4,5]�. More recently there has been some attempt to rename the family as the IS''26'' family (see ��[16]�), presumably because of accumulating experimental data from IS''26'' itself and the importance of this IS in accumulation and transmission of multiple antibiotic resistance, although this might potentially introduce confusion in the literature. IS''6'' family members have a simple organization (Fig. IS6.1) and generate 8bp direct target repeats on insertion. This family is very homogenous with an average length of about 800 bp for the majority (between 700 and 890 bp) and highly conserved short, generally perfect, IRs (Fig. IS6.1 and 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; ��[17]� of 227 and 336 bp), 7 members between 1230 and 1460 bp and three members between 1710 and 1760 bp. One member, IS''15'', of 1648 bp represents and insertion of one IS into another ��[3,5]�. Many are found as part of compound transposons (called pseudo-compound transposons ��[1]� described below ��[16]�) invariably as flanking ''direct'' ''repeats'' (Fig. IS6.1) a consequence of their transposition mechanism ��[7,9,13,14,18–30]�.
 
There are at present nearly 160 family members in ISfinder (<nowiki>https://www-is.biotoul.fr/scripts/search-db.php</nowiki>) from nearly 80 bacterial and archaeal species but this represents only a fraction of those present in the public databases. The family was named ��[1]� after the directly repeated insertion sequences in transposon Tn''6 ��''[2]''�'' to standardize the various names that had been attributed to identical elements (e.g. IS''15'', IS''26'', IS''46'', IS''140,'' IS''160'', IS''176'') ��[3–15]�, including one isolate, IS''15'', corresponding to an insertion of one iso-IS''6'' (IS''15''D) into another ��[4,5]�. More recently there has been some attempt to rename the family as the IS''26'' family (see ��[16]�), presumably because of accumulating experimental data from IS''26'' itself and the importance of this IS in accumulation and transmission of multiple antibiotic resistance, although this might potentially introduce confusion in the literature. IS''6'' family members have a simple organization (Fig. IS6.1) and generate 8bp direct target repeats on insertion. This family is very homogenous with an average length of about 800 bp for the majority (between 700 and 890 bp) and highly conserved short, generally perfect, IRs (Fig. IS6.1 and 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; ��[17]� of 227 and 336 bp), 7 members between 1230 and 1460 bp and three members between 1710 and 1760 bp. One member, IS''15'', of 1648 bp represents and insertion of one IS into another ��[3,5]�. Many are found as part of compound transposons (called pseudo-compound transposons ��[1]� described below ��[16]�) invariably as flanking ''direct'' ''repeats'' (Fig. IS6.1) a consequence of their transposition mechanism ��[7,9,13,14,18–30]�.
  
<br />
+
== Acknowledgements ==
 
+
We would like to thank Susu He (Nanjing University) for stimulating discussions concerning the transposition models and for supplying the IS''26'' transposase secondary structure predictions in Fig. IS6.2C).
== Distribution and Phylogenetic Transposase Tree ==
 
A phylogenetic tree based on the transposase amino acid sequence of the ISfinder collection (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 ��[31]�. These clades include one, A, which groups all archaeal IS''6'' family members (Fig. IS6.3a) composed mainly of ''Euryarchaeota'' (''Halobacteria'' ; Fig. IS6.3Ai-iii). Group Aiv includes both ''Euryarchaeota'' (''Thermococcales'' and ''Methanococcales'') and ''Crenarchaeota'' (''Sulfolobales''). Of the 8 clades containing bacterial IS: clade b includes some Actinobacteria, Alpha-, Beta-, and ''Gamma-proteobacteria''clade; clade c is more homogenous and is composed of ''Alphaproteobacteria'' (''Rhizobiaceae'' and ''Methylobacteriaceae''); clade d includes examples from the Alpha-, Beta-, and Gamma-''proteobacteria'', ''Firmicutes'', ''Cyanobacteria'', ''Acidobacteriia'' and Bacteroidetes ; clades e and f are composed exclusively of Firmicutes (almost exclusively ''Lactococci'' in the case of clade e); Clades g and h are more mixed and clade I contains only three examples. As might be expected, transposase length is approximately correlated with the clades. For example, family members from the archaea tend to be slightly smaller, in the range of 700-750 for clades Ai and Aii while members of clades h and i all carry the longest transposase genes (1230 to 1460 bp and 1710 to 1760 bp respectively).
 
 
 
Clearly, the ISfinder collection does not necessarily reflect the true IS''6'' family distribution and these grouping should be interpreted with care. For example, although many are not included in the 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 ��[32]�(Fig 1.6.3).
 
<br />
 
 
 
== Terminal Inverted Repeats. ==
 
The division into clades is also underlined to some extent by the IR sequences. As shown in 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 (where uppercase letters are fully conserved and lowercase letters are strongly conserved nucleotides). Sequence motifs are more pronounced when each clade is considered separately (Fig. IS6.4).
 
 
 
Clade b (n=16; ''Actinobacteria'', ''Alpha''-, ''Beta''-, and ''Gamma-proteobacteria'') includes a well conserved GG..cTGTTGCAAA signature with little conservation further into each end.
 
 
 
Clade c (n= 14; ''Alphaproteobacteria'': ''Rhizobiaceae'' and ''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 Alpha-, Beta-, and Gamma-''proteobacteria'', ''Firmicutes'', ''Cyanobacteria'', ''Acidobacteria'' and Bacteroidetes) maintains stronger traces of parts of these motifs (GG.. tcTGtt and CAaa).
 
 
 
Clade '''e:''' (n=23) is composed mainly of IS from Lactococcus, a single ''Leuconostoc'' and other bacilli (Lysteria, Enterococcus);
 
 
 
Clade f (n = 11; largely ''Staphylococci'' with 2 ''B. thuringiensis'') also exhibit the typical GGTTCTGTTGCAAAGTTt signature and some internal conservation in IRL.
 
 
 
Clade g (n = 10) is more heterogenous (''Alpha proteobacteria: Methylobacterium, Paracoccus, Roseovarius, Rhizobium, Bradyrhizobium ; Deinococci'' and ''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 ''Firmicutes'' (''Natranaerobius'', ''Clostridium'' and ''Thermoanaerobacter'' ) exhibits a moderately well-defined internal signature TcTgTtAAgTt.
 
 
 
Finally, clade I (n=3) is composed of Halanaerobia and Thermoanaerobacter.
 
 
 
The archaeal-specific clades also generally exhibit well-defined consensus sequences.
 
 
 
Clade Ai, is composed of diverse ''Halobacterial species'' (''Halohasta, Haloferax, Natrinema, Natrialba, Halogeometricum, Natronomonas, Natronococcus,'' and ''Haloarcula''): GgcACtGTCTAGtT.
 
 
 
Clade Aii (n = 12) is composed uniquely of ''Halobacterial'' ''Euryarchaeota'' with a ggtaGTGTTcagatAaG signature and significant internal conservation which is different for each end.
 
 
 
Clade Aiii (n = 5), is composed entirely of ''Halobacterial'' ''Euryarchaeota'' (''Haloarcula, Halomicrobium, Natronomonas, Natronobacterium, Natrinema'') also has well conserved ends, ggtcgTGTTTaGTT, and significant internal conservation which is different for each end.
 
 
 
Clade Aiv (n = 9) which includes both ''Euryarchaeota'' and ''Crenarchaeota'', has poor conservation although on further analysis, an alignment shows significant conservation in the ''Sulfolobus'' and in the ''Pyrococcus'' groups with good interior conservation also in the 3 ''Pyrococcal'' members. It is possible that the IS ends in the ''Sulfolobus'' members have not been accurately identified.
 
 
 
==== MCL analysis ��[33]� for the entire group of transposases using the criteria of ISfinder for classification  (IS identification)��[34]� 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/IS26 family of insertion sequences: is it a single family?”��[31]� is therefore “Probably, yes” according to the ISfinder definition. ====
 
A recent study ��[35]� identified a number of IS''26'' variants with specific mutations in their Tpases. In particular one variant, originally called IS''15D'' ��[4,36]� 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 IS''26''.v1, .v2 etc. ��[35]�. This makes sense if the mutation is not functionally neutral results in a change IS properties or behavior.
 
 
 
 
 
== '''Genomic Impact and Clinical Importance''' ==
 
Activity resulting in horizontal dissemination is suggested, for example, by the observation that copies identical to ''Mycobacterium fortuitum'' IS''6100'' ��[37]�(Clade b) occur in other bacteria: as part of a plasmid-associated catabolic transposon carrying genes for nylon degradation in ''Arthrobacter sp. ��''[38]''�'', in the ''Pseudomonas aeruginosa'' plasmid R1003 ��[39]�, in integrons of the In4-type integrons from transposons such as Tn''1696'' ��[40,41]� and within the ''Xanthomonas campestris'' transposon Tn''5393b ��''[42]''�''. Similar copies have also been reported in ''Salmonella enterica'' (typhimurium) ��[43]�, and on plasmid pACM1 from ''Klebsiella oxytoca'' (AF107205)��[44]�.
 
 
 
Passenger Genes
 
 
 
A number of IS families contain members, called tIS which carry passenger genes. A single member of the family, IS''Dsp3'', present in a single copy in ''Dehalococcoides sp.'' BAV1 carries a passenger gene annotated as a 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 ��[22,45–49]�.
 
 
 
For example, IS''257'' ��[50]�(Clade f) (also known as IS''431'') has played an important role in sequestering a variety of antibiotic resistance genes in clinical isolates of methicillin resistant ''Staphylococcus aureus'' (MRSA) (e.g. ��[45,46,51,52]�. 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 ��[46]�, ��[53]�. Insertion of can result in activation of a neighboring gene using both a hybrid promoter and an indigenous promoter ��[46]�. IS''257'' is also involved in expression of ''tetA ��''[54]''�'' and ''dfrA ��''[45]''�'' in  ''S. aureus.'' This is also true of IS''26'' which forms hybrid promoters shown to drive antibiotic resistance genes such as ''aphA7 ( Pasteurella piscicida ��''[55]''� Klebsiella pneumoniae ��''[22]''�'')'', bla<sub>SHV-2a</sub>'' (''Pseudomonas aeruginosa ��''[56]''�'') and wide spectrum beta-lactam resistance gene ''bla''<sub>KPC ��</sub>[57,58]<sub>�</sub>. While IS''6100'' ��[37]� (clade b)'','' often used as an aid in classifying mycobacterial isolates ��[59–61]� drives ''strA strB'' expression in ''X. campestris'' pv. vesicatoria ��[42]�
 
 
 
The formation of hybrid promoters on insertion (Table IS and gene expression) is clearly a general property of members of the IS''6'' family ��[22,45–49]�.
 
 
 
== '''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 ��[1,16]�. These include Tn''610'' (flanked by IS''6100'' ��[37]�), Tn''4003'' and others (flanked by IS''257'' ��[51,62,63]�), Tn''2680'' ��[6]� and Tn''6023'' (flanked by IS''26'' ��[64]�).
 
 
 
== '''IS''26'' and the Clinical Landscape''' ==
 
In view of the particular importance of IS''26'' in clinical settings it is worthwhile devoting a separate section to the contribution of this IS to the clinical landscape. IS''26'' ��[6–8]�(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 ��[28,63,65–70]�).
 
 
 
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 Genomic Resistance Islands (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 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 (Fig. IS6.5 and Fig. IS6.6) in direct and inverted repeat in multiple drug resistant plasmids (e.g. ''Salmonella. typhimurium'' ��[28,64,71]�, ''Klebsiella quasipneumoniae'' ��[72]�, ''Acinetobacter baumannii'' ��[68,73]�, ''Proteus mirabilis'' ��[74]� and uncultured sewage bacteria ��[75]� (among many others). These are often intercalated in or next to other transposable elements rather than neatly flanking ABR genes and can form units able to undergo tandem amplification.
 
 
 
'''IS26-mediated Gene Amplification:''' Early studies with Tn''1525'' (from ''Salmonella enterica'' serovar Panama), in which an ''aphA1'' (''aph'' (3') (5")-I) gene is flanked by two directly repeated copies of a the IS''6'' family member, IS''15'', reported tandem amplification of ''aphA1'' when the host was challenged by kanamycin ��[76]�. 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 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 Tobramycin in clinical isolates of ''Acinetobacter baumannii'' from a single patient over a period of days with continued antibiotic treatment''.'' Amplification occurred with Tn6''020'', an IS''26''-based transposon in which the flanking IS bracket a similar ''aphA1'' gene and could also be reproduced in bacterial culture ��[77]�. 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 ��[78]� 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 IS''26'' arrays which they call TnMB1860 (Fig. IS6.6). It was unclear whether this cassette amplification was due to transposition activity or, as had been observed in similar, IS''1''-mediated, gene amplifications ��[79–84]� which may occur by replication slippage between direct repeats or by unequal crossing-over ��[85,86]�.
 
 
 
Another example has been revealed by Hastak et al ��[87]� who analysed a multi resistant derivative of the clinically important, globally dispersed pathogenic, ''Escherichia coli'' ST131 subclade H30Rx, isolated from a number of bacteraemic patients and revealed that increased piperacillin/tazobactam resistance was due to IS''26''-mediated amplification of blaTEM-1B. A similar type of limited (tandem dimer) amplification of an IS''26''-flanked blaSHV-5-carrying DNA segment found in plasmids from a number of geographically diverse enteric species was identified in a nosocomial ''Enterobacter cloacae'' strain ��[88]�. More extensive amplification (>10 fold) was observed with the same DNA segment located in a different plasmid in a well-characterised laboratory strain of ''Escherichia coli'' and occurred in a ''recA''-independent manner ��[66]� while even higher levels of tandem amplification (~65 fold) of the ''aphA1'' gene in the IS''26''-based Tn''6020'' were identified in ''Acinetobacter baumannii''  ��[77]�.
 
 
 
= 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 Cointegrate formation below) ��[5,7,9,12,13,30]�. =
 
 
 
= Several studies have now demonstrated that this can occur in a clinical setting. For example, plasmid pBK32533 (KP345882)��[89]�, carried by ''E. coli'' BK32533 isolated from a patient with a urinary tract infection is an IS''26''-mediated cointegrate between ''Klebsiella pneumonia''e BK30661 plasmid pBK30661 (KF954759)��[90]� and a relative of ''Salmonella enterica'' p1643_10 (KF056330)��[91]�. Interestingly, the flanks of the 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 IS26 copy flanked by the target repeat (TTATTAAT-IS26-TTATTAAT) and, in a second step, a TGTTTTTT sequence in p1643_10  was targeted by the pBK30661  IS26 to generate the final cointegrate in which the two IS26 copies are flanked by the observed target sequences. Additional examples have been identified in KPC-producing ''Proteus mirabilis'' ��[74]� and in ''Klebsiella pneumoniae'' also involving inversions ��[70,92]� =
 
Organization
 
 
 
IS''6'' family members range in length from 789 bp (IS''257'') to 880 bp (IS''6100'') (Fig. IS6.2A) and generally create 8 bp direct flanking target repeats (DR) on insertion. ��[6]�.
 
 
 
'''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 213 (IS''15'') and 254 (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 helix-turn-helix (HTH) and a typical catalytic motif (DDE) (Fig. IS6.2C and IS6.7). However, the 7 members of clade h, all from Clostridia, are somewhat larger than other IS6 family members (approximately 1200bp, Fig. IS6.2A) with longer transposases (340-350 amino acids) as a consequence of an N-terminal extension with a predicted Zinc Finger (ZF) an N-terminal extension with a predicted Zinc Finger (ZF) composed of several CxxC motifs (Fig. IS6.2B; Fig. IS6.7). A Blast analysis of the non-redundant protein database at NCBI revealed a large number of IS6 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 ZF domain and a supplementary N-terminal extension.
 
 
 
Several members (e.g. IS''Rle39a'', IS''Rle39b'' and 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 IS''Enfa1'' itself has an insertion of 10 nucleotides and is therefore unlikely to be active.
 
 
 
'''Transposase expression'''
 
 
 
In the case of IS''26'', the promoter is located within the first 82 bp of the left end and the intact ''orf'' is required for transposition activity ��[8]�, and the predicted amino acid sequence of the corresponding protein exhibits a strong DDE motif (Fig. IS6.2C; Fig. IS6.7) Translation products of this frame have been demonstrated for IS''240'' ��[26]�. Little is known concerning the control of transposase expression although transposition activity of IS''6100'' in ''Streptomyces lividans'' ��[93]� 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 Fig 1.32.1).
 
 
 
'''Terminal Inverted Repeats'''
 
 
 
All carry short related (15-20 bp) terminal IR.  As shown in Fig. 2D, 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 (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. IS''15D'', IS''26'', IS''257'', IS''1936'') ��[5,7,9,13,94]�. More recent results principally with IS''26'' have suggested that this IS perhaps like IS''1'' (IS''1'' family) ��[95]� and IS''903'' (IS''5'' family) ��[96,97]�, members of this family may be able to transpose using alternative pathways ��[16,98–100]�.
 
 
 
= Cointegrate formation =
 
 
 
= Transposition of IS''6'' family elements to generate cointegrates ��[5,9,11,12]� presumably occurs in a replicative manner by Target Primed Transposon Replication (For a discussion see “Influence of transposition mechanisms on genome impact”; Fig 17.1 and Fig. 17.2). As shown in 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 direct repeats (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 IS 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 ��[5,9]�. =
 
 
 
= A systematic analysis of the cointegrate forming properties of an artificial IS''26''-based pseudo-compound transposon with a chloramphenicol 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 (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 (Fig. IS6.9). Intramolecular transposition of this type can explain the assembly of antibiotic resistance gene clusters (e.g. ��[70]�). =
 
 
 
= 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 (IS history; Fig 2.3, 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 ��[5,9]�. As shown in Fig. IS6.8 (bottom) ��[101]�, 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 ��[1]���[101]� 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. ��[1,70,92,101]� have been revisited and it has been recently proposed ��[16]� that the term pseudo-compound transposons first used over 30 years ago ��[1]� should be resurrected to describe these IS''6'' family structures. =
 
Circular transposon molecules: translocatable units (TU)
 
 
 
Although 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 ��[100]�. 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 ��[99,100]� which could be interpreted as overlapping pseudo-compound transposons ��[16]� (Fig. IS6.5). Note that IS''26'' and potential 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 (Fig. IS6.9). This led to the suggestion that IS''26'' might be able to transpose via a novel circular form called translocatable units (TU) ��[99,100]� (not to be confused with those originally described in the sea urchin and other eukaryotes ��[102]�) such as those shown in Fig. IS6.10. These potential circular transposition intermediates which were proposed to include a single IS''26'' copy along with neighboring DNA are structurally similar to IS''1''-based circles observed in the 1970s (e.g. ��[79,82]�). Translocatable units differ from the transposon circles identified during copy-out-paste-in transposition by IS of the IS''3'' (Fig. IS3. 9A; IS3 family transposition pathway), IS''21'' (Fig. IS21.7), IS''30'', IS''256'' and 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 IS''26'', the IS ends would be separated by the neighboring DNA sequence rather than by a few base pairs (Fig. IS6.11).
 
 
 
Evidence for the excision step of translocatable units was obtained ��[99]� from the study of the stability of two IS''26''-based pseudo-compound transposons, “wildtype” Tn''4352'' ��[25]� and “mutant” Tn''4352B'' ��[103]� which carry the ''aphA1'' gene specifying resistance to kanamycin. Tn''4352B'' is a special mutant derivative of Tn''4352'' including an additional GG dinucleotide at the left internal end of one of the component 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 Tn''4352B'' at an appreciable rate in the absence of selection. This generates a “donor” plasmid with one copy of IS''26'' flanked by the original 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 ''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 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 IS''26'' copy (a targeted integration reaction – see below), there appeared to be no difference in cointegrate formation frequencies between single IS''26'' copies with or without the additional GG dinucleotide. However, results from a standard integration test into a plasmid without a resident 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 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]]).
 
 
 
A summary of these results is shown in Fig. IS6.10. These data suggest that excision is driven by the wildtype 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 ABR genes in the plasmid R100.1 (NR1) generates a non-replicative circular molecule, the r-determinant (r-det), with a single 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 ''recA'' ��[104]�.
 
 
 
However, “Classical” recombination and transposition models do not fit the data The results appear to rule out two obvious models (Fig. IS6.11): since, although both would generate the correct TU and “excisant”, the first (top panel) requires homologous recombination between two directly repeated 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 ��[99,100]�, would not generate the correct flanking sequences. Modification of the transposition model to take into account the entire transposon (Fig. IS6.12) in which the active IS''26''L uses either of flanking sequences of 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 (Fig. IS6.14; see Targeted Transposition).
 
 
 
To summarize: it has been clearly demonstrated that circular DNA species carrying a single IS''26'' copy together with flanking “passenger” DNA can be generated efficiently ''in vivo'' from a variant plasmid replicon ��[103]� and also that replicons carrying a single 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.
 
 
 
The TU insertion pathway was addressed by transforming TU, constructed ''in vitro'' taking advantage of a unique IS''26'' restriction site, into recombination deficient cells carrying an appropriate target plasmid ��[98]�. Establishment of the ''aphA1''-carrying TU was dependent on the presence of a resident plasmid carrying an IS''26'' copy and occurred next to the resident 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 IS''26'' array. Therefore, artificially produced TU are capable of insertion.
 
 
 
Targeted transposition.
 
 
 
Targeted 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 ��[100]�. A similar result was obtained in ''Escherichia coli'' with a related IS, IS''1216'' ��[105]� whereas a third member of the family, IS''257'' (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 RecA. Using a PCR-based assay to identify the replicon fusions between IS''26''-containing donor and target plasmids, it was observed that the resulting cointegrate (Fig. IS6.13) did not contain an additional copy of IS''26'' which would be expected if replicative transposition were involved (Fig. IS6.12). This suggests that the phenomenon results from a conservative recombination mechanism. Despite the absence of RecA, the observed cointegrate is structurally equivalent to the recombination product between the two 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 ��[100]�. 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 IS''26'' copies are in some way exchanged ��[100]� (Fig. IS6.13). This suggests a model in which transposase might catalyze an exchange of flanking DNA during the fusion process.
 
 
 
A Model for Targeted Integration
 
 
 
One possibility (Fig. IS6.13) is that two IS ends from different IS copies in separate replicons are synapsed intermolecularly in the same transpososome (Fig. IS6.14i). Strand exchange would then couple the donor and target replicons (Fig. IS6.14ii). A similar mechanism has been invoked to explain “targeted” insertion of IS''3'' and ''IS30'' family members into related IRs (Fig. IS3.14) ��[106,107]�. Branch migration (Fig. IS6.14iii) would lead to exchange of an entire IS strand (Fig. IS4.13iv) and cleavage at the distal IS end and strand transfer (Fig. IS6.14v) would result in the observed cointegrate (Fig. IS6.14vi) 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 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 IS26 copy reduces the frequency of targeted cointegration ��[100]� since, not only does this produce a truncated transposase but also introduces a mismatch. As in the case of intermolecular targeting of the IS''3'' family member, IS''911 ��''[108]''�'', might require the RegG helicase to promote strand migration.
 
 
 
The model shown in 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 (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 (Cleavage in Trans: A Committed Complex). Recently, evidence have been presented supporting this type of model ��[109]�. Using two IS, IS''1006'' and IS''1008'' ��[110]� which have significant identity to 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 Holliday junction which, on branch migration, is resolved. This differs from the model shown here (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''911'' ��[106,108,111,112]� which requires the RecG helicase and, presumably RuvC.
 
 
 
'''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 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 ��[113–115]� but such a molecule, a TU, has thus far been formally observed in only a single case. It was generated ''in vivo'' from an 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 ��[99]�. Since “TU” is now being used in the literature to describe IS''26''-flanked DNA segments in multimeric arrays (e.g. ��[87]�, it is essential to provide more formal evidence that these non-replicative DNA circles are indeed general intermediates in the 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 ��[100]� although the observation that artificially constructed TU can undergo targeted insertion when introduced into a suitable cell by transformation ��[98]� 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 (Fig. IS6.14). The answers to many of these fascinating outstanding questions will be provided when the biochemistry of the reactions is known.
 
 
 
Acknowledgements: We would like to thank Susu He (Nanjing University) for stimulating discussions concerning the transposition models and for supplying the IS''26'' transposase secondary structure predictions in Fig. IS6.2C).
 

Revision as of 15:28, 1 March 2021

General

There are at present nearly 160 family members in ISfinder (https://www-is.biotoul.fr/scripts/search-db.php) from nearly 80 bacterial and archaeal species but this represents only a fraction of those present in the public databases. The family was named ��[1]� after the directly repeated insertion sequences in transposon Tn6 ��[2] to standardize the various names that had been attributed to identical elements (e.g. IS15, IS26, IS46, IS140, IS160, IS176) ��[3–15]�, including one isolate, IS15, corresponding to an insertion of one iso-IS6 (IS15D) into another ��[4,5]�. More recently there has been some attempt to rename the family as the IS26 family (see ��[16]�), presumably because of accumulating experimental data from IS26 itself and the importance of this IS in accumulation and transmission of multiple antibiotic resistance, although this might potentially introduce confusion in the literature. IS6 family members have a simple organization (Fig. IS6.1) and generate 8bp direct target repeats on insertion. This family is very homogenous with an average length of about 800 bp for the majority (between 700 and 890 bp) and highly conserved short, generally perfect, IRs (Fig. IS6.1 and IS6.2). There are two examples of MITES (Miniature Inverted repeat Transposable Elements composed of both IS ends and no intervening orfs; ��[17]� of 227 and 336 bp), 7 members between 1230 and 1460 bp and three members between 1710 and 1760 bp. One member, IS15, of 1648 bp represents and insertion of one IS into another ��[3,5]�. Many are found as part of compound transposons (called pseudo-compound transposons ��[1]� described below ��[16]�) invariably as flanking direct repeats (Fig. IS6.1) a consequence of their transposition mechanism ��[7,9,13,14,18–30]�.

Acknowledgements

We would like to thank Susu He (Nanjing University) for stimulating discussions concerning the transposition models and for supplying the IS26 transposase secondary structure predictions in Fig. IS6.2C).