IS Families/IS66 family

General

IS66 was first identified in the Ti plasmid pTi66 of Agrobacterium tumefaciens Sciaky and, soon after in the symbiotic plasmid, pSym, of Rhizobium fredi. The vast majority of IS66 members originate from the Proteobacteria with several from the Bacteriodetes/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. These are relatively well distributed (Fig. IS66.1). The founder member, ISBst12, originally isolated from Bacillus stearothermophilus, was described as a novel family, 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). 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 Euryarchaeota phylum of archaea (but have not yet been identified in the Crenarchaeota).

Fig IS66.1. Distance tree computed by neighbour-joining, using the JTT matrix-based with gamma-distribution and bootstrap of 500 replicates. The results are visualzsed with TreeView. Pink boxes indicate Firmicutes, blue boxes indicate Bacteroidetes/Chlorob,i and green boxes with white characters indicate Archaeal hosts.

Fig IS66.2. Each circle represents an individual IS transposase (TnpC) amino acid sequence. a) 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. b) Inflation factor of 2, score >60. These settings define IS groups (Siguier et al., 2009) which are automatically assigned different colours.

Fig IS66.3. Organisation 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: a) The "classic" IS66 organisation. b) 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 (20). c) IS66 variants lacking tnpA. d) ISBst12 carrying the single orf equivalent to tnpC of IS66. e) 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; loss of loss of S-layer-gene expression in Bacillus stearothermophilus; gene disruption in the cyanobacterium Fremyella duplosiphon involved in regulation of phycoerythrin synthesis and in the gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase) of Acenitobacter baumannii leading to changes in the antibacterial resistance profile).

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

Organization

Fig IS66.4. The left (IRL) and right (IRR) inverted terminal repeats are shown in the WebLogo format (5). 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. For the number of sequences used in this analysis see Table S1.

The IS66 reference copy from a plasmid of the enteropathogenic Escherichia coli B171, IS679 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) (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. 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. IS66.4; Table IS66.1).

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. IS66.3; Table IS66.1). 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), 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 , at the N-terminus (Fig. IS66.5; Fig. IS66.6a; Fig. IS66.7a).

Fig IS66.5

Fig IS66.6A.

Fig IS66.6B.

Fig IS66.7A.

Fig IS66.7B.

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.

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)	N°
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

Mechanism and Insertion Specificity

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