The IS1111 family members IS4321 and IS5075 have subterminal inverted repeats and target the terminal inverted repeats of Tn21 family transposons

Abstract

IS5075 and IS4321 are closely related (93.1% identical) members of the IS1111 family that target a specific position in the 38-bp terminal inverted repeats of Tn21 family transposons and that are inserted in only one orientation. They are 1,327 bp long and have identical ends consisting of short inverted repeats of 12 bp with an additional 7 bp (TAATGAG) or 6 bp (AATGAG) to the left of the left inverted repeats and 3 bp (AGA) or 4 bp (AGAT) to the right of the right inverted repeat. Circular forms of IS5075 and IS4321 in which the inverted repeats are separated by abutting terminal sequences (AGATAATGAG) were detected. A similar circular product was found for the related ISPa11. Transposition of IS4321 into the 38-bp target site was detected, but a flanking duplication was not generated. The precisely reconstituted target site was also identified. Over 50 members of the IS1111 family were identified. They encode related transposases, have related inverted repeats, and include related bases that lie outside these inverted repeats. In some, the flanking bases number 5 or 6 on the left and 4 or 3 on the right. Specific target sites were found for several of these insertion sequence (IS) elements. IS1111 family members therefore differ from the majority of IS elements, which are characterized by terminal inverted repeats and a target site duplication, and from members of the related IS110 family, which do not have obvious inverted repeats near their termini.

FIG. 1.

Locations of known copies of IS4321 and IS5075. The 38-bp TIR of Tn21 family transposons are represented by black bars; arrowheads below the bars indicate their orientations. The identities of the adjacent sequences are indicated; e.g., tnp₂₁ indicates the tnp end of Tn21, mer_pDU indicates one of the mer regions in plasmid pDU1358, and so forth. The IS4321 and IS5075 elements are represented by open boxes; arrows in the boxes indicate the positions and directions of the transposase genes. The plasmid, transposon, or organism where the IS were found are indicated on the left. Sequences were as follows (GenBank accession numbers): R751 from Enterobacter aerogenes (U67194), pRMH760 from K. pneumoniae (AY123253, AY242531, AY242532, and AY242533), pHCM1 from S. enterica serovar Typhi (AL513383), Tn5075 from E. coli (AF457211), A. baumannii (AY196695), and K. pneumoniae (NC_002941).

FIG. 2.

Boundaries of IS4321, IS5075, and ISPa11. (A and C) Extents of IS indicated by bars. The IR are boxed (thin lines); arrows indicate the directions of the transposase genes. The base that may originate from either the left or the right end of the IS and the base in the target adjacent to which the IS is inserted are shown in bold type. The 38-bp transposon TIR is boxed (thick lines), and residues that differ in the two common alternate types are shown. (B and D) Sequences of circular intermediates. IRr and IRl are boxed, and potential −35 and −10 regions are underlined. The sequence of the IS1383 circular intermediate (22) is shown for comparison.

FIG. 3.

Movement of IS4321. (A) Substrates used to detect the movement of IS4321. Features of the IS, TIR, and adjacent regions are as described in the legend to Fig. 1. pACYC184 and pRMH777 backbone sequences are indicated by broken and thin lines, respectively. The positions and orientations of primers used are indicated by arrowheads with numbers to identify the RH primer (Table 1). The extents of PCR products are indicated by bars; their lengths are shown below the bars. (B) Sequences of PCR products. PCR products are named by the primer pairs giving rise to them. TIR, IR, and ambiguous bases are indicated as described in the legend to Fig. 2; the name of each region is shown above the sequence. The plasmid backbone sequence is shown in lowercase type.

FIG. 4.

Alignment of IS1111 family transposases. Twenty transposases from the IS shown in Table 2 are aligned. Only one example was included for pairs of proteins that were >90% identical (IS4321L and IS5075, IS1618 and ISSm1, and IS1492 and ISPsy16). An alignment of all 46 transposases without frameshifts from Tables 2 and 3 was used to define conserved amino acids. Residues conserved in all or all but 1 of the 46 sequences are indicated by asterisks above the sequence. Residues differing in 6 or fewer of the 46 sequences are shown as white on black and are indicated by uppercase letters below the sequence. Where only two alternative amino acids in 45 or 46 sequences are found, the two alternatives are indicated by lowercase letters below the sequence. The sequences used are from translations of nucleotide sequences identified by the GenBank accession numbers listed in Table 2.

FIG. 5.

Sequences of predicted circular forms of IS1111 family elements. The sequences shown are deduced from comparisons of sequences flanking the IS and uninterrupted target sequences. Features are indicated as described in the legend to Fig. 2B. The distances to the initiation codons of the transposase genes are also shown. Where the comparison predicts the origins of all central bases, a vertical arrow indicates the junction of the left and right ends.

FIG. 6.

Targets for additional IS. The IS is inserted to the right or left of the base in bold type. (A) Stem-loop structures targeted by IS1111. (B) The 38-bp TIR and related sequences recognized by members of the IS1618 group are compared to the 38-bp TIR recognized by Tn4321 and Tn5075. Dots indicate unknown sequences. (C) Sequences targeted by additional family members. Bases in lowercase letters are not completely conserved.