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

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.     

New transposable elements identified as insertions in rice transposon Tnr1

Tnr1 (235 bp long) is a transposable element in rice. Polymerase chain reactions (PCRs) done with a primer(s) that hybridizes to terminal inverted repeat sequences (TIRs) of Tnr1 detected new Tnr1 members with one or two insertions in rice genomes. Six identified insertion sequences (Tnr4, Tnr5, Tnr11, Tnr12, Tnr13 and RIRE9) did not have extensive homology to known transposable elements, rather they had structural features characteristic of transposable elements. Tnr4 (1767 bp long) had imperfect 64-bp TIRs and appeared to generate duplication of a 9-bp sequence at the target site. However, the TIR sequences were not homologous to those of known transposable elements, indicative that Tnr4 is a new transposable element. Tnr5 (209 bp long) had imperfect 46-bp TIRs and appeared to generate duplication of sequence TTA like that of some elements of the Tourist family. Tnr11 (811 bp long) had 73-bp TIRs with significant homology to those of Tnr1 and Stowaway and appeared to generate duplication of sequence TA, indicative that Tnr11 is a transposable element of the Tnr1/Stowaway family. Tnr12 (2426 bp long) carried perfect 9-bp TIRs, which began with 5'-CACTA- -3' from both ends and appeared to generate duplication of a 3-bp target sequence, indicative that Tnr12 is a transposable element of the En/Spm family. Tnr13 (347 bp long) had 31-bp TIRs and appeared to generate duplication of an 8-bp target sequence. Two sequences, one the transposon-like element Crackle, had partial homology in the Tnr13 ends. All five insertions appear to be defective elements derived from autonomous ones encoding the transposase gene. All had characteristic tandem repeat sequences which may be recognized by transposase. The sixth insertion sequence, named RIRE9 (3852 bp long), which begins with 5'-TG- -3' and ends with 5'- -CA-3', appeared to generate duplication of a 5-bp target sequence. These and other structural features indicate that this insertion is a solo LTR (long terminal repeat) of a retrotransposon. The transposable elements described above could be identified as insertions into Tnr1, which do not deleteriously affect the growth of rice cells.     

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.     

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.     

IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum.

An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide sequence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of Mr 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.     

A new insertion sequence, IS231M, in an autoagglutinable isolate of Bacillus thuringiensis

An insertion sequence was isolated from an autoagglutinable strain of Bacillus thuringiensis. Analysis of its DNA sequence revealed high homology to the IS231 family. The name IS231M is proposed for this new insertion sequence. IS231M is 1652 bp long and is delimited by two imperfect 20-bp inverted repeat sequences with two mismatches, which are flanked by two perfect 11-bp direct repeats (DRs). The region upstream of the open reading frame, presumed to be able to form a stable hairpin structure, is particularly well conserved in IS231M. Based on primary nucleotide sequences, IS231M is most homologous to IS231F and IS231G and most distant from IS231V and IS231W. However, as opposed to the single transposase A ORF found in IS231A, -B, -C, -D, -F, and -G, IS231M has two overlapping open reading frames, ORF1 and ORF2, that could code for polypeptides of 334 and 143 amino acids, respectively. Whether IS231M is a functional transposable element remains to be determined.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Genetic organization of insertion element IS2 based on a revised nucleotide sequence

We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.     

IS861, a group B streptococcal insertion sequence related to IS150 and IS3 of Escherichia coli.

A 1,442-base-pair (bp) insertion sequence (IS861) was identified in the type III group B streptococcal (GBS) strain COH-1. It is flanked by 26-bp imperfect inverted repeats and contains two open reading frames, 1 and 2, encoding 141- and 277-amino-acid proteins, respectively. A 3-bp target sequence, ACA, is duplicated and flanks each inverted repeat. IS861 shares greater than 30% homology with IS3 and IS150 of Escherichia coli, primarily in the region of their putative transposases. Northern (RNA) analysis revealed that RNA is actively transcribed in vivo by IS861 and 17- and 36-kilodalton proteins were synthesized in E. coli maxicell assays. Multiple copies of IS861 were observed throughout the chromosome of COH-1, and one of the copies is located near genes involved in GBS capsule synthesis. IS861 is the first insertion sequence identified in GBS. Its role in GBS and the significance of its relationship to the phylogenetically similar insertion sequences typified by IS150 and IS3 of E. coli are unknown.     

Transposition of IS10 from the host Escherichia coli genome to a plasmid may lead to cloning artefacts

During recloning of Nicotiana tabacum L. repetitive sequence R8.3 in Escherichia coli, a modified clone that differed from the original by the insertion of an IS10 sequence was unintentionally produced. The insert was flanked by a 9-bp direct repeat derived from the R8.3 sequence, the 9-bp duplication of acceptor DNA in the site of insertion being a characteristic of IS10 transposition events. A database search using the FASTA program showed IS10 and other prokaryotic IS elements inserted into numerous eukaryotic clones. Unexpectedly, the IS10, which is not a natural component of the E. coli genome, appeared to be by far the most frequent contaminant of DNA databases among several IS sequences tested. In the GenEMBL database, the IS10 query sequence yielded positive scores with more than 500 eukaryotic clones. Insertions of shortened IS10 sequences having only one intact terminal inverted repeat were commonly found. Most full-length IS10 insertions (32 out of 40 analyzed) were flanked by 9-bp direct repeats having the consensus 5'-NPuCNN-NGPyN-3' with a strong preference for 5'-TGCTNA-GNN-3'. One insertion was flanked by an inverted repeat of more than 400 bp in length. PCR amplification and Southern analysis revealed the presence of IS10 sequences in E. coli strains commonly used for DNA cloning, including some reported to be Tn10-free. No IS10-specific PCR product was obtained with N. tabacum or human DNA. Our data suggest that transposition of IS10 elements may accompany cloning steps, particularly into large BAC vectors. This might lead to the relatively frequent contamination of DNA databases by this bacterial sequence. It is estimated that one in approximately every thousand eukaryotic clone in the databases is contaminated by IS-derived sequences. We recommend checking submitted sequences for the presence of IS10 and other IS elements. In addition, DNA databases should be corrected by removing contaminating IS sequences.     

Structural and genetic organization of IS232, a new insertion sequence of Bacillus thuringiensis.

In the Bacillus thuringiensis strains toxic for the lepidopteran larvae, the delta-endotoxin genes cryIA are frequently found within a composite transposonlike structure flanked by two inverted repeat sequences. We report that these elements are true insertion sequences and designate them IS232. IS232 is a 2,184-bp element and is delimited by two imperfect inverted repeats (28 of 37 bp are identical). Two adjacent open reading frames, overlapping for three codons, span almost the entire sequence of IS232. The potential encoded polypeptides of 50 and 30-kDa are homologous to the IstA and IstB proteins of the gram-negative insertion sequence IS21. The N-terminal part of the 50-kDa polypeptide contains a helix-turn-helix DNA-binding motif. The junctions at the insertion sites of three IS232 elements were analyzed. Each case was different, with 0, 4, or 6 bp of the target DNA being duplicated. Transposition of IS232 in Escherichia coli was demonstrated by using a genetic marker inserted upstream of the two open reading frames.     

The minimum internal and external sequence requirements for transposition of the eukaryotic transformation vector piggyBac

The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.     

Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences.

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of elements.     

Identification and characterization of IS1112 and IS1113 insertion element sequences in Xanthomonas oryzae pv. oryzae.

Two new insertion sequences (IS1112 and IS1113) were identified in the genome of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Three copies of IS1112 were trapped, one containing 1052-bp and the other two with 1055-bp. They all have 25-bp imperfect inverted repeats with a 3-bp duplication at the site of insertion. They contain an open reading frame (ORF) of 317 and 318 amino acid residues, respectively. IS1113 is 1306-bp, contains 25-bp imperfect terminal inverted repeats, and is flanked by a 9-bp direct repeat at the site of insertion. It contains an ORF of 395 amino acid residues.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.     

The novel insertion sequences IS1417, IS1418, and IS1419 from Burkholderia glumae and their strain distribution

Three insertion sequences, IS1417, IS1418, and IS1419, were isolated from Burkholderia glumae (formerly Pseudomonas glumae), a gram-negative rice pathogenic bacterium, on the basis of their abilities to activate the expression of the neo gene of the entrap vector pSHI1063. The 1335-bp IS1417 element with 17-bp imperfect terminal inverted repeats was found to be flanked by 5-bp direct repeats of the vector sequence. IS1418 is 865 bp in length and carries 15-bp inverted repeats with a target duplication of 3 bp. The 1215-bp IS1419 sequence is bounded by the 36-bp terminal inverted repeats of the element and 7-bp direct repeats of the vector sequence. IS1417 and IS1418 belong to the IS2 subgroup of the IS3 family and the IS427 subgroup of the IS5 family, respectively, whereas IS1419 does not appear to be a member of any known IS family. Southern blot analysis of DNAs from B. glumae field isolates indicated that those IS elements are widely distributed, but the host range of the three IS elements appears to be limited to B. glumae and some other related species such as B. plantarii. The polymorphisms exhibited in B. glumae isolates suggest that those elements are useful for molecular epidemiological studies of B. glumae infections.     

IS231 D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family

IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231F). Like the previously described IS231A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478-amino-acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso-IS231 terminal inverted repeats are strongly conserved 20 bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231F is bordered by a 12 bp direct repeat. No direct repeats were found flanking IS231D or IS231E.