A novel IS element, IS621, of the IS110/IS492 family transposes to a specific site in repetitive extragenic palindromic sequences in Escherichia coli

Abstract

An Escherichia coli strain, ECOR28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome. This insertion sequence (named IS621) has one large open reading frame encoding a putative protein that is 326 amino acids in length. A computer-aided homology search using the DNA sequence as the query revealed that IS621 was homologous to the piv genes, encoding pilin gene invertase (PIV). A homology search using the amino acid sequence of the putative protein encoded by IS621 as the query revealed that the protein also has partial homology to transposases encoded by the IS110/IS492 family elements, which were known to have partial homology to PIV. This indicates that IS621 belongs to the IS110/IS492 family but is most closely related to the piv genes. In fact, a phylogenetic tree constructed on the basis of amino acid sequences of PIV proteins and transposases revealed that IS621 belongs to the piv gene group, which is distinct from the IS110/IS492 family elements, which form several groups. PIV proteins and transposases encoded by the IS110/IS492 family elements, including IS621, have four acidic amino acid residues, which are conserved at positions in their N-terminal regions. These residues may constitute a tetrad D-E(or D)-D-D motif as the catalytic center. Interestingly, IS621 was inserted at specific sites within repetitive extragenic palindromic (REP) sequences at 10 loci in the ECOR28 genome. IS621 may not recognize the entire REP sequence in transposition, but it recognizes a 15-bp sequence conserved in the REP sequences around the target site. There are several elements belonging to the IS110/IS492 family that also transpose to specific sites in the repeated sequences, as does IS621. IS621 does not have terminal inverted repeats like most of the IS110/IS492 family elements. The terminal sequences of IS621 have homology with the 26-bp inverted repeat sequences of pilin gene inversion sites that are recognized and used for inversion of pilin genes by PIV. This suggests that IS621 initiates transposition through recognition of their terminal regions and cleavage at the ends by a mechanism similar to that used for PIV to promote inversion at the pilin gene inversion sites.

FIG. 1.

Physical maps of the DNA segments around the insertion sites of IS621 in the genomes of nine E. coli strains. The solid lines show the sequences of E. coli K-12 MG1655 and EHEC O157:H7. For the other E. coli strains, sequences identified by gel electrophoresis of PCR-amplified fragments and by sequencing are shown. Positions of IS element insertions (IS621, IS4, and IS200) and REP sequences (small rectangles) are shown. IS4 and IS200 have been identified in the regions shown. Numbers with or without K are coordinates given to the E. coli K-12 MG1655 sequence in kilobases or base pairs, respectively. The positions of the primers used for PCR are shown by small arrowheads; the solid arrowheads represent primers that were used to analyze polymorphism of the fragments with or without IS621 or REP. Note that IS621 is present in REP sequences in ECOR28 and that ECOR36 and ECOR46 have no REP sequences.

FIG. 2.

Nucleotide sequences with IS621 at 10 loci in the ECOR28 chromosome. (A) Nucleotide sequences with IS621 at three loci in the region corresponding to the 0- to 10-min region of the E. coli K-12 map. The nucleotide sequence without IS621 at each position in the E. coli K-12 (MG1655) chromosome is shown for comparison. Two types of REP sequences that have the highest homology to the sequences at the 10 loci are shown in bold. Palindromic sequences in REP are indicated by short horizontal arrows. Note that each element is flanked by 2-bp sequences (in boxes). (B) Nucleotide sequences with IS621 at seven loci located outside of the 0- to 10-min region. IS621 is inserted into the 2-bp sequences shown in boxes. The nucleotide sequence without IS621 at each position in the E. coli K-12 (MG1655) chromosome is shown for comparison. Note that REP sequences at five positions (kb 1814.3, 1952.5, 2840.5, 4372.1, and 4468.0) are in a reverse orientation.

FIG. 3.

(A) The nucleotide sequence of IS621. The amino acid sequence of transposase encoded by one large open reading frame (orf) in IS621 is shown below the nucleotide sequence. A possible Shine-Dalgarno sequence preceding the initiation codon ATG is underlined. Palindromic sequences found in the region downstream of the coding region are shown by a pair of arrows. The CT sequences that appear at the junction regions of IS621 with the target sequence (Fig. 2) are underlined. Note that IS621 has no terminal inverted repeats. Four acidic residues constituting a catalytic motif are shown in circles. (B) Comparison of the pilin gene inversion site sequences and terminal sequences of IS621 and three other IS110/IS492 family elements that are closely related to IS621. Pilin gene inversion site sequences (horizontal arrows) and flanking sequences are indicated by uppercase and lowercase letters, respectively. Terminal sequences of IS621 at kb 5.6 (Fig. 1), IS492 (7), IS110 (9, 26), and IS1000 (3) and their flanking regions are shown by uppercase and lowercase letters, respectively. The CT sequences duplicated at the junction regions with IS621 are underlined. Homologous sequences between pilin gene inversion site sequences and terminal sequences of IS621 are shown in boxes.

FIG. 4.

An alignment of PIV proteins and transposases. Only three regions that are well conserved are shown. A RuvC Holliday junction resolvase (accession no. P24239) is aligned to show that four acidic amino acid residues [D, E (or D), D, and D, shown in boxes] in the PIV and transposase proteins are present in positions corresponding to those constituting the catalytic center in the RuvC protein. Amino acid residues present in all the proteins are indicated by asterisks. Other conserved amino acid residues are indicated by dots.

FIG. 5.

A phylogenetic tree of piv genes and IS110/IS492 family elements. The tree was constructed by the neighbor-joining method based on amino acid sequences of PIV proteins and transposases (Fig. 4). The scale bar equals a distance of 0.1.

FIG. 6.

Comparison of secondary structures of RuvC, IS621 transposase, and PIV proteins. The secondary structure of RuvC, based on the tertiary structure (PDB code 1HJR), is shown above the polypeptide sequence. α helices are indicated by ribbons, and β sheets are indicated by arrows. Other features are indicated by straight solid lines. Positions of acidic amino acids constituting the D-E-D-D motif are indicated by thin vertical lines. The secondary structures of RuvC, IS621 transposase, and PIV proteins were predicted by PSIPRED and are shown under the polypeptide sequence of each protein.

FIG. 7.

Nucleotide sequences with or without another IS110/IS492 family element, ISSt1232 or IS1594. (A) Nucleotide sequences of ISSt1281 with or without ISSt1232 at two loci. ISSt1281 sequences are shown in bold. (B) Nucleotide sequences with or without IS1594 at three loci. REP-like sequences are shown in bold for comparison. The two types of REP-like sequences that have the highest homology to the sequences at the three loci are shown. Palindromic sequences in the REP-like sequences are indicated by horizontal arrows. Note that REP-like sequences at kb 266.6 and 4388.6 are identical but are in a reverse orientation. Note also that each element is flanked by 2-bp sequences shown in boxes.