IS Families/IS66 family

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IS66 was first identified in the Ti plasmid pTi66 of Agrobacterium tumefaciens Sciaky[1][2] and, soon after in the symbiotic plasmid, pSym, of Rhizobium fredii[3]. The vast majority of IS66 members originate from the Proteobacteria with several from the Bacteroidetes/Chlorobi and the Firmicutes. A second group of closely related ISs, widely spread among both bacteria and archaea are thought to represent a sub-group within the IS66 family[4]. These are relatively well distributed (Fig. IS66.1). The founder member, ISBst12, originally isolated from Bacillus stearothermophilus, was described as a novel family[5], but identification of many additional examples suggests that the ISBst12 and IS66 groups should be considered a single family (Fig. IS66.2). Several examples of IS derivatives with passenger genes have been identified (Fig. IS66.3; Table IS66.1). One important example is a potential tIS in which IRL is located downstream from an MCR-3 (colistin resistance gene) [6]. Members of the ISBst12 group are found in Actinobacteria, Cyanobacteria, Deinococcus/Thermus, Firmicutes, and Planctomycetes as well as in Proteobacteria. They are also found in the phylum of archaea (but have not yet been identified in the Crenarchaeota).

Fig IS66.1. Distance tree computed by neighbor-joining, using the JTT matrix-based[7] with gamma-distribution and bootstrap of 500 replicates. The results are visualized with TreeView[8]. Pink boxes indicate Firmicutes, blue boxes indicate Bacteroidetes/Chlorobi, and green boxes with white characters indicate Archaeal hosts.

Fig IS66.2. Each circle represents an individual IS transposase (TnpC) amino acid sequence. Left) Inflation factor of 1.2, score >60 (links with scores of less than 60 were removed). These values should indicate the division between families but show that the IS66 and ISBst12 groups belong to the same family. Right) Inflation factor of 2, score >60. These settings define IS groups (Siguier et al., 2009) which are automatically assigned different colors.

Fig IS66.3. Organization of different classical IS66 family members and of the ISBst12 group. The IS is represented as a rectangle with flanking Direct Repeats (DR) in red and terminal inverted repeats in blue (triangles). The orfs are shown in red (tnpA), orange (tnpB), dark blue (the transposase, tnpC), and green (passenger genes, P). The length of each orf is indicated in base pairs together with the total length of the example IS. From top to bottom: IS679) The "classic" IS66 organization. tISBf10) IS66 with associated passenger genes. To denote these special cases and underline their close relationship to classic ISs, we refer to them as ‘‘tIS’’ signifying the presence of transposable passenger genes. ISPpu15) IS66 variants lacking tnpA. ISBst12) carrying the single orf equivalent to tnpC of IS66. tISMasp4) ISBst12 with passenger genes (tIS).

Genome Impact

Members of this family are involved in insertional gene inactivation (e.g. inactivation of the liaFSR operon leading to hypersensitivity to daptomycin[9]; loss of loss of S-layer-gene expression in Bacillus stearothermophilus[10]; gene disruption in the cyanobacterium Fremyella duplosiphon involved in regulation of phycoerythrin synthesis[11] and in the gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase) of Acinetobacter baumannii leading to changes in the antibacterial resistance profile[12]).

It has also been shown (using RACE) that IS66 insertion can create hybrid promoters[13].


Fig IS66.4. The left (IRL) and right (IRR) inverted terminal repeats are shown in the WebLogo format. They are defined by the direction of transcription of the transposase gene. IRL, by definition, is located on the 5’ side of the transposase orf.

The IS66 reference copy from a plasmid of the enteropathogenic Escherichia coli B171, IS679[14] is defined by three orfs (Fig. IS66.3): tnpA, tnpB and tnpC and relatively well conserved terminal IR of about 20-30 bp flanked by an 8 bp DR at their insertion sites (Fig. IS66.4). Orf tnpC (Fig. IS66.5) is 1572 bp and its predicted product includes an N-terminal region with potential leucine zipper and zinc finger motifs (Fig. IS66.5; Fig. IS66.6a; Fig. IS66.7a) and a typical DDE motif (Fig. IS66.5; Fig. IS66.6b; Fig. IS66.7b; outlined in Fig. IS66.8). It also carries an insertion domain between the second D and the E of the DDE motif (e.g.IS679, ISPsy5 and ISMac8)[15] (see Fig. 1.8.3) (Table Transposases examined by secondary structure prediction programs).

The role of the products of tnpA (651 bp) and tnpB (345 bp) is less clear. TnpA carries a potential HTH motif ((Fig. IS66.9) while TnpB shows no marked potential secondary structure motifs. Mutation of each orf separately (by introduction of an in-frame deletion) reduced transposition by at least two orders of magnitude [16]. The three frames are disposed in a pattern suggesting translational coupling: tnpB is in general in translational reading frame -1 compared to tnpA and in most cases the termination codon of tnpA and the initiation codon of tnpB overlap (ATGA). An initiation codon for tnpC occurs slightly downstream separated from tnpB by about 20 bp.

However, rather surprisingly, in the light of a requirement for all three orfs for transposition of the canonical IS66 family member IS679, members of the ISBst12 group are devoid of tnpA and tnpB and carry only the tnpC reading frame. Although both ISBst12 and IS66 members contain IRs which start with 5’GTAA3’, they are clearly distinguishable due to a single conserved A at bp 11 In ISBst12 which is not conserved in IS66 (Fig. IS66.1; Fig. IS66.4).

IS66 members can be grouped into three classes based on their organization: those including all three orfs, A, B and C transcribed in the same direction; those with additional passenger genes invariably present downstream of orfC and transcribed in the same direction; and those which lack orfA but retain both orfs B and C (Fig. IS66.1; Fig. IS66.3). Each of these organizations includes members with multiple copies, implying that they are active in transposition. In addition to the DDE catalytic domain (Fig. IS66.5; Fig. IS66.6b; Fig. IS66.7b)[17], TnpC also exhibits a highly conserved CwAH-rR motif downstream of the second D residue, a relatively conserved CX2(C)X33CX2C motif characteristic of a zinc finger (ZF) further upstream and a leucine-rich region which might form a leucine zipper (LZ) necessary in multimerisation of other Tpases [18], at the N-terminus (Fig. IS66.5; Fig. IS66.6a; Fig. IS66.7a).

Fig IS66.5. IS66 family tranposase TnpC domain schema.
Fig IS66.6A. TnpC N-terminal region alignment and motifs.
Fig IS66.6B. TnpC DDE motif.
Fig IS66.7A. TnpC N-terminal region alignment and motifs
Fig IS66.7B. TnpC DDE motif from ISBst12 group.

Fig IS66.8. Conserved active site residues are shown in red. The positions of the IS679 residues (top line) are shown between brackets. The numbers between square brackets indicate the distance in amino acids between each sequence block. The upper case indicates fully conserved residues. The Lowercase indicates partial conservation.
Fig IS66.9. Alignment of TnpA showing the HTH domain.
A list of representative IS66 family members and the ISBst12 group
Table IS66.1. A list of representative IS66 family members and the ISBst12 group. The table summarises from left to right: the IS name; group defined in the MCL analysis; accession number; host from which it was identified; kingdom (archaea, A, or bacteria, B); phylum; organisation (org) A,B,C,P show the presence of TnpA, TnpB, TnpC and Passenger genes respectively; length of the IS in base pairs (bp); length of the terminal Inverted Repeats (IR); length of flanking Direct Repeats (DR); number of examples identified in the host genome.
IS Name Group Accession number Host A/B G+/G- Org. L (bp) IR (bp) DR (bp)
IS66-1 AF242881 Agrobacterium tumefaciens B G- ABC 2556 18/20 8 2
IS679 NC_002142 Escherichia coli B G- ABC 2704 17/25 8 6
ISAde1 NC_011891 Anaeromyxobacter dehalogenans 2CP-1 B G- ABC 2957 20/27 8 2
ISAtu6 NC_010929 Agrobacterium tumefaciens B G- ABCP 2798 23/24 8 3
ISAzo15 NC_006513 Azoarcus sp. EbN1 or Aromatoleum aromaticum EbN1 B G- ABC 2441 20/21 8 4
ISAzo19 NC_006513 Azoarcus sp. EbN1 or Aromatoleum aromaticum EbN1 B G- ABC 2423 23/26 8 3
ISAzo21 NC_006513 Azoarcus sp. EbN1 or Aromatoleum aromaticum EbN1 B G- ABC 2423 23/26 8 2
ISBcen14 NC_011001 Burkholderia cenocepacia J2315 B G- ABC 2516 18/22 8 4
ISBf10 NC_006347 Bacteroides fragilis YCH46 B G- ABCP 2939 20/21 8 4
ISBj7 NC_004463 Bradyrhizobium japonicum USDA 110 B G- ABC 2865 40/50 8 2
ISBthe6 NC_004663 Bacteroides thetaiotaomicron B G- ABC 2544 24 8 2
ISBvu4 NC_009614 Bacteroides vulgatus ATCC 8482 B G- BC 2371 30/32 8 5
ISEc8 NC_004431 Escherichia coli CFT073 B G- ABC 2442 18/22 8 7
ISPpu15 NC_002947 Pseudomonas putida KT2440 B G- BC 2041 22/28 8 4
ISPre3 NC_004444 Pseudomonas resinovorans B G- ABCP 2957 17/24 8 2
ISPsy5 AE016853 Pseudomonas syringae pv. tomato str. DC3000 B G- BC 2059 21/28 8 33
ISRle3 NC_008382 Rhizobium leguminosarum bv. viciae 3841 B G- ABC 2500 14/15 8 2
ISRsp1 U00090 Rhizobium sp. NGR234 B G- ABCP 3481 17/22 8 2
ISSfl3 AL391753 Shigella flexneri B G- ABC 2729 11 0 2
ISShes9 NC_008750 Shewanella sp. W3-18-1 B G- ABC 2370 18/24 8 3
ISAma4 ISBst12 NC_011138 Alteromonas macleodii 'Deep ecotype' B G- C 1529 28/29 9 3
ISAva2 ISBst12 NC_007410 Anabaena variabilis ATCC 29413 B G- C 1547 14 8 9
ISBrsp5 ISBst12 NC_009445 Bradyrhizobium sp. ORS278 B G- C 1541 18/19 8 2
ISBst12 ISBst12 AF162268 Bacillus stearothermophilus B G+ C 1612 15/16 8 15
ISCysp3 ISBst12 NZ_AAXW00000000 Cyanothece sp. CCY 0110 B G- C 1522 14 8 18
ISCysp4 ISBst12 NC_011884 Cyanothece sp. PCC 7425 B G- C 1585 14/17 8 12
ISDge4 ISBst12 NC_008025 Deinococcus geothermalis DSM 11300 B G+ C 1453 18/25 8 6
ISGka4 ISBst12 NC_006509 Geobacillus kaustophilus HTA426 B G+ C 1635 21/24 8 3
ISGob3 ISBst12 NZ_ABGO00000000 Gemmata obscuriglobus UQM 2246 B G- C 1597 11/12 8 8
ISGob4 ISBst12 NZ_ABGO00000000 Gemmata obscuriglobus UQM 2246 B G- C 1576 14/15 8 6
ISGst1 ISBst12 NC_010420 Geobacillus stearothermophilus B G+ C 1613 18/26 8 3
ISH10 ISBst12 NC_002607 Halobacterium sp. NRC-1 A C 1584 16/18 8 5
ISMac8 ISBst12 NC_003552 Methanosarcina acetivorans C2A A C 1603 13/15 8 3
ISMasp4 ISBst12 NC_008576 Magnetococcus sp. MC-1 B G- PC 1969 19 8 7
ISMbu5 ISBst12 NC_007955 Methanococcoides burtonii DSM 6242 A C 1696 18/19 8/0 9
ISMhu3 ISBst12 NC_007796 Methanospirillum hungatei JF-1 A PC 1727 9/12 0 2
ISMma14 ISBst12 NC_003901 Methanosarcina mazei Go1 A C 1529 22/30 8 9
ISMno2 ISBst12 NC_011894 Methylobacterium nodulans ORS 2060 B G- C 1419 21/27 8 8
ISPpr14 ISBst12 NC_006371 Photobacterium profundum SS9 B G- C 1568 19/27 8/9 9
ISWen3 ISBst12 NZ_AAGB00000000 Wolbachia endosymbiont of Drosophila ananassae B G- C 1482 18/20 8-0 30

Mechanism and Insertion Specificity

Nothing is known about the transposition mechanism of this group of IS and they exhibit no substantial target sequence specificity.


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