An analysis of the IS 6/IS 26 family of insertion sequences: is it a single family?

Abstract

The relationships within a curated set of 112 insertion sequences (ISs) currently assigned to the IS6 family, here re-named the IS6/IS26 family, in the ISFinder database were examined. The encoded DDE transposases include a helix-helix-turn-helix (H-HTH) potential DNA binding domain N-terminal to the catalytic (DDE) domain, but 10 from Clostridia include one or two additional N-terminal domains. The transposase phylogeny clearly separated 75 derived from bacteria from 37 from archaea. The longer bacterial transposases also clustered separately. The 65 shorter bacterial transposases, including Tnp26 from IS26, formed six clades but share significant conservation in the H-HTH domain and in a short extension at the N-terminus, and several amino acids in the catalytic domain are completely or highly conserved. At the outer ends of these ISs, 14 bp were strongly conserved as terminal inverted repeats (TIRs) with the first two bases (GG) and the seventh base (G) present in all except one IS. The longer bacterial transposases are only distantly related to the short bacterial transposases, with only some amino acids conserved. The TIR consensus was longer and only one IS started with GG. The 37 archaeal transposases are only distantly related to either the short or the long bacterial transposases and different residues were conserved. Their TIRs are loosely related to the bacterial TIR consensus but are longer and many do not begin with GG. As they do not fit well with most bacterial ISs, the inclusion of the archaeal ISs and the longer bacterial ISs in the IS6/IS26 family is not appropriate.

Keywords: IS26; ISFinder; insertion sequence; phylogenetic analysis.

Fig. 1.

Un-rooted neighbour-joining phylogeny of 112 IS6/IS26 family transposases. The consensus tree was reconstructed by resampling the analysis 10 000 times. ISs derived from bacteria are coloured green (Gram-negative), red (Gram-positive) or purple (divergent longer bacterial ISs derived from Clostridium species), and ISs of archaeal origin are blue. Clades encompassing ISs of bacterial origin are labelled I to VIII, and clades encompassing ISs of archaeal origin are labelled a to d.

Fig. 2.

Structure of representative bacterial and archaeal IS6/IS26 family members. The extent and orientation of the transposases are shown with the N-terminal (N) and C-terminal (C) marked. Protein secondary structures predicted by JPred are marked, including predicted catalytic domains (blue ovals), H-HTH motifs (red boxes), zinc fingers (brown zig-zags) and an unknown domain (yellow triangle), and a signal peptide (S, green star). TIRs are shaded in grey. Drawn to scale in Gene Construction Kit (GCK, version 4.0) from the corresponding entries in the ISFinder database before transposing into Adobe Illustrator.

Fig. 3.

Alignment of the amino acid sequences of representative transposases of bacterial IS6/IS26 family members. The transposases of three representatives from clades I to VI, and ISFal2 are shown. The extents of the H-HTH putative DNA binding domain and the DDE catalytic domain are marked above. Residues in the catalytic domain that are conserved in at least 62 of the 65 transposases in the complete alignment (Fig. S1) are marked with a capital letter. Residues for which only two variations exist are marked with smaller letters. The completely conserved DDE residues are marked by red letters. Amino acids are shaded as follows: black, 100 % similarity; dark grey, 80–99 % similarity; light grey, 60–79 % similarity; unshaded, less than 59 % similarity.

Fig. 4.

Alignment of the TIRs of the short bacterial IS6/IS26 family members. The first 20 nucleotides and the complement of the last 20 nucleotides of each of the bacterial ISs from clades I to VI are aligned on the left and right, respectively, with each base coloured specifically. The consensus is shown above.

Fig. 5.

Alignment of the ten long ISs and ten representatives of the short bacterial ISs from clades I to VI. A ClustalW alignment (BLOSUM matrix, gap open cost 10, gap extend cost 0.1) was generated in Geneious (version 7.1.9) to align the transposases of the ten long bacterial ISs and ten representatives of the short bacterial ISs from clades I to VI. Amino acids are shaded as follows: black, 100 % similarity; dark grey, 80–99 % similarity; light grey, 60–79 % similarity; unshaded, less than 59 % similarity.

Fig. 6.

Alignment of the TIRs of the long bacterial IS6/IS26 family members. The first 20 nucleotides and the complement of the last 20 nucleotides of each of the bacterial ISs from clades VII and VIII are aligned on the left and right, respectively, with each base coloured specifically. The consensus is shown above.

Fig. 7.

Alignment of representative transposases from archaeal IS6/IS26 family members. The transposases of four representatives from archaeal clades a to d are shown. Amino acids are shaded as follows: black, 100 % similarity; dark grey, 80–99 % similarity; light grey, 60–79 % similarity; unshaded, less than 59 % similarity.

Fig. 8.

Alignment of the TIRs of the archaeal IS6/IS26 family members. The first 20 nucleotides and the complement of the last 20 nucleotides of each of the archaeal ISs from clades a to d are aligned on the left and right, respectively, with each base coloured specifically. The consensus is shown above.