IS Families/IS66 family

General

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 fredi �[3]�. 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 �[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 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 �(13)� with gamma-distribution and bootstrap of 500 replicates. The results are visualzsed with TreeView �(18)�. 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

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 fredi [3]. 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 [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 Euryarchaeota phylum of archaea (but have not yet been identified in the Crenarchaeota).

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, �(��[11]� 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) ���[12]� (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 ���[11]�. 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)���[4]�, 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 �(Haren, et al., 1998)�, at the N-terminus (Fig. IS66.5; Fig. IS66.6a; Fig. IS66.7a).

A list of representative IS66 family members and the ISBst12 group

Table IS66.1. 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.

A list of representative IS66 family members and the ISBst12 group

IS name	Group	Accession Number	Host	A/B	G+/G-	Org.	Length (bp)	IR (bp)	DR (bp)	N°
IS66-1	-	AF242881	Agrobacterium tumefaciens	B	G-	ABC	2556	18/20	8	2

Mechanism and Insertion Specificity

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