IS870 requires a 5'-CTAG-3' target sequence to generate the stop codon for its large ORF1.

The TB regions of the Agrobacterium vitis octopine/cucumopine Ti plasmids constitute a family of related structures. All contain a bacterial insertion element downstream of the TB-iaaM gene, IS870.1. Whereas 43 isolates with octopine/cucumopine Ti plasmids carry only one IS870 copy, strain Ag57 carries a second copy (IS870.2) 3.9 kb to the right of IS870.1 and part of the same TB region. Two other octopine/cucumopine strains carry an IS870 copy on their chromosome (IS870.3). A study of the unmodified insertion sites of IS870.2 and IS870.3, cloned from closely related strains, enabled us to delimit the IS870 elements. IS870 has a size of 1,152 bp and is terminated by inverted repeats. It contains a large open reading frame without a stop codon. However, a stop codon is generated by insertion into the target sequence 5'-CTAG-3'. IS870 is related to five other insertion sequence elements. For two of these, the stop codon of the largest open reading frame is also created by insertion into a CTAG target site.     

Isolation and characterization of IS 31831, a transposable element from Corynebacterium glutamicum

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS 31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS 1096 from Mycobacterium smeg-matis. Both IS 31831 and IS 1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS 31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/ Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.     

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.     

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.     

Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120.

A family of repetitive elements from the cyanobacterium Anabaena sp. strain PCC 7120 was identified through the proximity of one element to the psbAI gene. Four members of this seven-member family were isolated and shown to have structures characteristic of bacterial insertion sequences. Each element is approximately 1,200 bp in length, is delimited by a 30-bp inverted repeat, and contains two open reading frames in tandem on the same DNA strand. The four copies differ from each other by small insertions or deletions, some of which alter the open reading frames. By using a system designed to trap insertion elements, one of the elements, denoted IS895, was shown to be mobile. The target site was not duplicated upon insertion of the element. Two other filamentous cyanobacterial strains were also found to contain sequences homologous to IS895.     

Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.     

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.     

Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.     

ISWpi1 from Wolbachia pipientis defines a novel group of insertion sequences within the IS5 family

Insertion sequences are transposable elements that can represent substantial proportions of prokaryotic genomes and play a substantial role in shaping host genome evolution. As such, evaluating and understanding insertion sequence diversity is an important task to fulfill, because it is expected to yield new insight into the evolution of bacterial transposable elements and contribute to improve genome annotations. Here, I characterized an insertion sequence, termed ISWpi1, for which the taxonomic distribution appears to be restricted to the obligate intracellular alpha-Proteobacterium Wolbachia pipientis. ISWpi1 exhibits approximately 46% identity at the amino acid level with members of the IS1031 group of insertion sequences from the IS5 family. However, the IS1031 group is characterized by a transposase gene encoded by a single open reading frame, whereas the ISWpi1 transposase gene consists of two overlapping open reading frames presumably translated as a single protein via programmed translational frameshifting. Such structure suggests that ISWpi1 may instead be related to the IS427 group of insertion sequences from the IS5 family. Altogether, these data indicate that ISWpi1 extends the known spectrum of diversity of the IS5 family, and I propose to define a novel group of insertion sequences within the IS5 family typified by ISWpi1. Probable transpositional activity, relevant insertion site preferences and taxonomic specificity make ISWpi1 a promising tool for experimentally manipulating W. pipientis bacteria, especially in light of the increasing interest in developing these bacteria as tools for controlling insect disease vectors and agricultural pests.     

Sequence and characteristics of IS900, an insertion element identified in a human Crohn''s disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.     

IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.     

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.     

Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria.

We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy number of IS1165 varies from 4 to 13 in L. mesenteroides subsp. cremoris strains allowing genetic fingerprinting of strains based on location and number of bands on hybridization. IS1165 or closely related elements have been detected by hybridization in L. lactis, L. oenos, Pediococcus sp., Lactobacillus helveticus, and Lb. casei but not in Lactococcus.     

Isolation of a new insertion element of Yersinia intermedia closely related to remnants of mobile genetic elements present on Yersinia plasmids harboring the Yop virulon

A new insertion element present in two alleles, designated IS1635.1 and IS1635.2, was identified on a plasmid of a Yersinia intermedia strain by hybridization with the Yersinia enterocolitica pYV virulence plasmid. IS1635.1 and IS1635.2 are 861 bp long, carry imperfect inverted terminal repeats and possess a single open reading frame encoding a putative transposase of the IS6 family. A truncated IS1635 element is present immediately downstream of element IS1635.2. The capacity of the IS1635 elements to mediate transposition in Yersinia was demonstrated with a R6K-derived suicide vector, where a kanamycin resistance gene had been inserted between IS1635.1 and IS1635.2. Hybridization and sequence alignments showed that remnants of IS1635-like insertion elements harboring large deletions and point mutations are present on the Yop virulon harboring plasmids of pathogenic Yersinia strains. In a few cases, the IS1635 element has also been found on plasmids of apathogenic Yersinia strains.     

Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences?

Members of the IS3 family of insertion sequences are found in a wide range of bacteria. At least 10 members of this family carry two major open reading frames: a small upstream frame (0 phase), and a longer downstream frame in the -1 phase. The downstream frame shows significant similarity at the amino acid level. A highly conserved region of this frame also exhibits notable similarity with a region of the integrase (endonuclease) domain of retroviruses. Although the overall transposition mechanism of the insertion sequence and retroviral elements is certainly different, the two groups may share additional common features, including a -1 frameshift resulting in the production of a fusion protein.     

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.     

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.