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.     

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.     

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.     

Identification of insertion sequence element IS427 in pTiT37 plasmid DNA of an Agrobacterium tumefaciens T37 isolate

The isolation and characterization of an insertion sequence (IS) element, IS427, from Agrobacterium tumefaciens T37 is described. IS427 is present in three nonidentical copies on the pTiT37 plasmid. The copy that was isolated through transposition on the entrapment vector pUCD800 contains at its ends a 16-bp imperfect inverted repeat and generates a 2-bp duplication of the target DNA. IS427 does not show homology with previously characterized IS elements of A. tumefaciens, based on hybridization experiments and/or sequence comparison.     

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.     

Is103, a New Insertion Element in Escherichia Coli: Characterization and Distribution in Natural Populations

IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.     

ISPst9, an ISL3-like insertion sequence from Pseudomonas stutzeri AN10 involved in catabolic gene inactivation

A novel insertion sequence (IS), ISPst9, from Pseudomonas stutzeri AN10 was cloned and characterized. ISPst9 is a typical bacterial IS, consisting of a 2472-bp element flanked by 24-bp perfect inverted repeats that generates 8-bp AT-rich target duplications upon insertion. The sequence also contains a gene that encodes an active transposase (TnpA) with significant amino acid identity to members of the ISL3 family. Southern blot analysis of digested genomic DNA of strain AN10 and its 4-chlorosalicylate-degrading derivative strain AN142 demonstrated that native ISPst9 transposes in multiple copies, with one of them responsible for the nahH insertional inactivation observed in strain AN142. Precise excision of ISPst9 yielded NahH+ revertants of AN142 at high frequencies (up to 10-6). In vivo transposition, mainly in multiple copies, of an ISPst9 derivative containing a KmR cassette cloned into a suicide vector was also demonstrated. Hybridization experiments carried out with different strains of P. stutzeri and with 292 phylogenetically distinct environmental isolates suggested that the presence of an ISPst9-like IS occurs in diverse bacteria together with the presence of aromatic hydrocarbon-degrading determinants.     

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.     

Structural and Functional Characterization of IS679 and IS66-Family Elements

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB, and tnpC). A plasmid carrying a composite transposon (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679. A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn679 duplicated an 8-bp sequence at the target site. Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS66 family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS679, but many have a mutation(s) in an ORF(s). In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tnpA. In contrast, tnpC, separated from tnpB by a space of ca. 20 bp, is located in any one of three frames relative to tnpB. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.     

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.     

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.     

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.     

Functional analysis of unique class II insertion sequence IS1071

Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS1071. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS1071 has remained unclear. Our study revealed that IS1071 was only able to transpose at high frequencies in two environmental beta-proteobacterial strains, Comamonas testosteroni and Delftia acidovorans, and not in any of the bacteria examined which belong to the alpha- and gamma-proteobacteria. IS1071 was found to have the functional features of the class II transposons in that (i) the final product of the IS1071 transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS1071, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a tnpA mutation of IS1071 was efficiently complemented by supplying the wild-type tnpA gene in trans. Deletion analysis of the IS1071 IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS1071 might be different from that of the other well-characterized class II transposons.     

Tourist: a large family of small inverted repeat elements frequently associated with maize genes.

The wx-B2 mutation results from a 128-bp transposable element-like insertion in exon 11 of the maize Waxy gene. Surprisingly, 11 maize genes and one barley gene in the GenBank and EMBL data bases were found to contain similar elements in flanking or intron sequences. Members of this previously undescribed family of elements, designated Tourist, are short (133 bp on average), have conserved terminal inverted repeats, are flanked by a 3-bp direct repeat, and display target site specificity. Based on estimates of repetitiveness of three Tourist elements in maize genomic DNA, the copy number of the Tourist element family may exceed that of all previously reported eukaryotic inverted repeat elements. Taken together, our data suggest that Tourist may be the maize equivalent of the human Alu family of elements with respect to copy number, genomic dispersion, and the high frequency of association with genes.     

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.     

A 10- rather than 9-bp duplication associated with insertion of Tn5 in Escherichia coli K-12

The composite transposable element Tn5, which is made up of two inverted IS50 elements surrounding genes encoding drug resistance, generally generates 9-bp duplications at the site of insertion. In our studies of three Tn5 insertion mutants at one location in the Escherichia coli chromosome, we have observed that one contains a duplication of 10 bp, while the other two have the usual 9-bp duplication. Three other insertion elements, IS1, IS4, and IS186, give variable-sized target site sequence duplications. We observed a similarity of amino acid sequence in a small region of the putative transposases among IS4, IS186, and Tn5 suggesting a conservation of function in this group of transposases.     

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.     

Isolation and characterization of IS elements repeated in the bacterial chromosome

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.     

Structural and functional analyses of the fosfomycin resistance transposon Tn2921.

The fosfomycin resistance transposon Tn2921 is flanked by directly repeated sequences homologous to the Tn10-related insertion sequence IS10. The nonrepeated DNA sequences of Tn2921 can be deleted without affecting the transposition ability of the element, showing that at least one of the direct repeats is an active insertion sequence. Transposition of Tn2921 seems to occur through direct transposition, since cointegrates have not been observed. The evolutionary relatedness of Tn2921 and IS10 is discussed.