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 and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.     

IS421, a new insertion sequence in Escherichia coli

The nucleotide sequence of a new insertion sequence (IS) in Escherichia coli, IS421, was determined. It is 1340 bp long and contains inverted repeats of 22 bp at its termini. It is flanked by 13 bp direct repeats apparently generated upon insertion. There are two ORFs longer than 200 bp in IS421. One can encode a polypeptide of 371 amino acids (aa) and the other, which is on the other strand, can encode a polypeptide of 102 aa. The C-terminal part of the 371 aa polypeptide shows some homology to that of transposases encoded in some other known IS elements. The copy number of IS421 in chromosomal DNA was 4 for E. coli K-12 and B, and 5 for E. coli C, as determined by the Southern hybridization of restriction fragments.     

Identification, distribution, and sequence analysis of new insertion elements in Caulobacter crescentus.

We describe two insertion elements isolated from Caulobacter crescentus that are designated IS298 and IS511. These insertion elements were cloned from spontaneous flagellar (fla) gene mutants SC298 and SC511 derived from the wild-type strain CB15 (ATCC 19089), in which they were originally identified as insertions in the flbG operon of the hook gene cluster (N. Ohta, E. Swanson, B. Ely, and A. Newton, J. Bacteriol. 158:897-904, 1984). IS298 and IS511 were each present in C. crescentus CB2 and CB15 in at least four different positions, but neither was present in strain CB13 or in several Caulobacter species examined, including C. vibrioides, C. leidyia, and C. henricii. Nucleotide sequence analysis across the chromosome-insertion element junctions showed that IS298 is located 152 base pairs (bp) upstream from the ATG translation start of the hook protein gene flaK, where it is bounded by a 4-bp direct repeat derived from the site of insertion, and that IS511 is inserted at codon 186 of the flaK coding sequence, where it is also bounded by a 4-bp direct repeat duplicated from the site of insertion. The ilvB102 mutation in strain SC125 was also shown to result from insertion sequence IS511, but no duplication of the genomic sequence was present at the insertion element junctions. IS298 contains an imperfect terminal inverted repeat 16 bp long, and IS511 contains a 32-bp inverted repeat at the termini. IS298 and IS511 are the first insertion elements described in C. crescentus.     

IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element.

Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis.

An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis. IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus. IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.     

Identification of IS 1206, a Corynebacterium glutamicum IS3-related insertion sequence and phylogenetic analysis

Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3-related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.     

Identification and characterisation of IS1383, a new insertion sequence isolated from Pseudomonas putida strain H

A new insertion sequence (IS1383) was identified on plasmids from Pseudomonas putida strain H and its nucleotide sequence was determined. IS1383 contains perfect terminal inverted repeats of 13-bp flanking a 1.4-kb internal sequence. A single significant open reading frame was identified that can encode a 342-amino acid polypeptide which was predicted to be highly basic and to have homology to polypeptides known from several other bacterial insertion sequences. At least six copies of IS1383 are present on the plasmids pPGH1 and pPGH2, whereas no copy could be detected on the chromosome of P. putida strain H. Target duplications did not flank the inverted repeats of any of the six IS1383 copies examined. Analysis of the integration sites of IS1383 revealed hints for a target specificity. Multiple sequence alignments of the transposases, the inverted repeats and the integration sites pointed to the assignment of IS1383 into a putative new family of insertion sequences defined as the IS1111 family.     

Method for selection of transposable DNA and characterization of a new insertion sequence, IS493, from Streptomyces lividans.

A method to select for transposable elements from Streptomyces spp. by using insertional inactivation of a repressor gene that functions in Escherichia coli was developed. Plasmid pCZA126, which can replicate in Streptomyces spp. or E. coli, contains a gene coding for the lambda cI857 repressor and a gene, under repressor control, coding for apramycin resistance. E. coli cells containing the plasmid are apramycin sensitive but become apramycin resistant if the cI857 repressor gene is disrupted. Plasmids propagated in Streptomyces spp. can be screened for transposable elements that have disrupted the cI857 gene by transforming E. coli cells to apramycin resistance. This method was used to isolate a new 1.6-kilobase insertion sequence, IS493, from Streptomyces lividans CT2. IS493 duplicated host DNA at the target site, had inverted repeats at its ends, and contained two tandem open reading frames on each strand. IS493 was present in three copies in the same genomic locations in several S. lividans strains. Two of the copies appeared to be present in regions of similar DNA context that extended at least 11.5 kilobases. Several other Streptomyces spp. did not appear to contain copies of IS493.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis

Abstract: An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis . IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus . IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.     

IS30, a new insertion sequence of Escherichia coli K12

Summary Three independent spontaneous mutations of prophage P1 affecting the ability of the phage to reproduce vegetatively are due to the insertion of a mobile genetic element, called IS 30. The same sequence is also carried in the R plasmid NR 1-Basel, but not in the parental plasmid NR 1. Southern hybridisation study indicates that the Escherichia coli K 12 chromosome carries several copies of IS 30 as a normal resident. IS 30 is 1.2 kb long and contains unique restriction cleavage sites for Bg/II, ClaI, HindIII, NciI and HincII, and it is cleaved twice by the enzymes HpaII and TaqI. The ends of IS 30 are formed by 26 bp long inverted repeats with 3 bases mismatched. Upon transposition IS 30 generates a duplication of only 2 bp of the target. The following observations suggest a pronounced specificity in target selection by IS 30. In transposition to the phage P 1 genome a single integration site was used three times independently, and in both orientations. A short region of sequence homology has been identified between the P 1 and NR 1-Basel insertion sites. IS 30 has mediated cointegration as well as deletion. The entire IS 30 sequences were duplicated in the cointegrates between a pBR 322 derivative containing IS 30 and the genome of phage P 1–15, and several loci on the P1–15 genome served as fusion sites, some of which were used more than once.     

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.     

Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis

Insertion sequence IS6120 from Mycobacterium smegmatis was identified by its ability to transpose into different sites in the lambda repressor gene, cl857, carried on an Escherichia coli/mycobacteria shuttle plasmid. IS6120 is a novel 1.5 kb insertion sequence, which has 24-bp imperfect terminal inverted repeats and generates 9-bp duplications of the target DNA following insertion. IS6120 is present in at least three copies in M. smegmatis but was not found in other species, including Mycobacterium tuberculosis. Nucleotide sequence analysis revealed that IS6120 contains two open reading frames, one of which encodes a putative transposase with similarities to those found in IS256 from Staphylococcus aureus, IST2 from Thiobacillus ferrooxidans, and ISRm3 from Rhizobium meliloti. The fact that IS6120 does not recognize a consensus target sequence for insertion and has no homologous sequences in the other strains studied makes IS6120 useful for transposon mutagenesis in mycobacteria.     

Identification and sequencing of a novel insertion sequence, IS1471, in Burkholderia cepacia strain 2a

Abstract Eighty-one isolates of Rhizoctonia solani AG4 were obtained from soil samples with diverse geographic origins in Korea. Forty-five out of 81 isolates (56%) contained at least one dsRNA molecule with their sizes ranging from 2.3 to > 23.1 kb. Nine different sizes of dsRNA molecules were found and extensive variation in banding patterns was observed among isolates. By comparing the sizes and combinations of dsRNAs, 21 distinct banding patterns were observed. Cytoplasmic fractions from 3 isolates showed the same dsRNA band patterns as those from the total cell extracts. The dsRNAs were stable through 10 successive hyphal tip cultures and serial transfers onto the potato dextrose agar supplemented with cycloheximide or emetine. The presence or absence of dsRNAs was not apparently correlated with disease severity, phenol oxidase activity, and geographic origin.     

IS15, a new insertion sequence widely spread in R plasmids of gram-negative bacteria

We have shown that the IS15 element, first detected in Salmonella ordonez and previously designated IS1522 (Labigne-Roussel et al. 1981), could transpose, with an approximate frequency of 5 X 10(-5), to various sites of different replicons in an Escherichia coli host deficient for general homologous recombination. Physical mapping with restriction endonucleases of this 1,500 base pairs (bp) transposable module indicated the presence of two, possibly contiguous, directly repeated internal sequences, at least 480 bp in size. IS15 could generate in vivo, by intramolecular recombination between the two direct repeats, IS15-delta, which is 830 bp in size. The reverse transition, IS15-delta to IS15, was not observed. The two related structural forms of IS15 were detected, by Southern hybridization, on plasmids belonging to various incompatibility groups (Inc6-C, I1, 7-M, and Y) isolated from phylogenetically remote pathogenic bacterial genera (Escherichia coli, Salmonella panama, Enterobacter cloacae, and Acinetobacter calcoaceticus). Whereas IS15 could promote its own transposition and transposition of DNA fragments it flanked, IS15-delta resulting from the 670 bp 'clean' deletion and representing the most common natural deletion derivative could only induce replicon fusion. It appears, therefore, that the two structural configurations of IS15 have evolved to play, by transposition, distinct and complementary roles in bacterial evolution.     

Sequence and distribution of IS866, a novel T region-associated insertion sequence from Agrobacterium tumefaciens

We have identified a new insertion sequence, IS866, located in the auxin synthesis gene TA iaaH of Tm4, a wide host range biotype III octopine/cucumopine type Agrobacterium tumefaciens strain with two T regions on its tumor-inducing (Ti) plasmid, TA, and TB. IS866 is 2716 bp long, has inverted repeats of 27 bp with three mismatches, and generates 8-bp direct repeats upon integration. In addition to IS866, pTiTm4 carries two copies of a related element, IS867, associated with TA and TB, respectively. A systematic study of 92 virulent Agrobacterium strains has shown that among the three biotypes all octopine/cucumopine and vitopine biotype III isolates contain IS866-like elements. The various octopine/cucumopine Ti plasmids always carry IS866 and IS867 at the same position as in pTiTm4. The chromosomes of the bacteria which contain these Ti plasmids also carry IS866 and IS867 copies but in varying numbers and locations.     

IS200: a Salmonella-specific insertion sequence

A new IS element (IS200) has been identified in Salmonella. The sequence was identified as an IS element by the following criteria: its insertion caused the mutation hisD984; six copies of the sequence are present in strain LT2 of S. typhimurium; and transposition of the sequence has been observed on several occasions. IS200 is found in almost all Salmonella species examined but is absent from most other enteric bacteria. The specificity of this element for Salmonella (and the absence of IS1-IS4 from Salmonella) suggest that transfer of insertion sequences between bacterial groups may be less extensive than is commonly believed. Alternatively, the distribution may suggest that these elements play a selectively important role in bacteria.     

DNA sequence at the integration sites of the insertion element IS1.

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.     

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.