Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida

A new insertion sequence (IS element), IS1411, was identified downstream of the phenol degradation genes pheBA that originated from plasmid DNA of Pseudomonas sp. strain EST1001. According to sequence analysis, IS1411 belongs to a new family of IS elements that has recently been named the ISL3 family (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725-774, 1998). IS1411 generates 8-bp duplication of the target DNA and carries 24-bp inverted repeats (IRs), highly homologous to the IRs of other IS elements belonging to this family. IS1411 was discovered as a result of insertional activation of promoterless pheBA genes in Pseudomonas putida due to the presence of outward-directed promoters at the left end of IS1411. Both promoters located on the IS element have sequences that are similar to the consensus sequence of Escherichia coli sigma70. IS1411 can produce IS circles, and the circle formation is enhanced when two copies of the element are present in the same plasmid.     

IS1634, a novel insertion element creating long, variable-length direct repeats which is specific for Mycoplasma mycoides subsp. mycoides small-colony type

A new insertion sequence, IS1634, has been identified in Mycoplasma mycoides subsp. mycoides small-colony type (SC). IS1634 shows structural and functional similarities to IS1549 of Mycobacterium smegmatis and with it seems to form a new class or family of insertion sequences. IS1634 has a size of 1,872 bp, including two 13-bp terminal inverted repeats. It contains an open reading frame (ORF) encoding a product of 533 amino acids which shows similarity to the transposase of IS1549 and to a lesser extent to the transposases of IS elements of the IS4 family. IS1634 is present at about 30 copies in the genome of all 22 different field strains of M. mycoides subsp. mycoides SC tested. Characteristic of IS1634 are the long and variable-length direct repeats at the sites of insertion which were found to reach up to about 500 bp. IS1634 is specific to M. mycoides subsp. mycoides SC and is not present in any of the other members of the M. mycoides cluster. Neither was it found in other closely related Mycoplasma species of ruminants.     

Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria

A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.     

Introduction of a distance cut-off into structural alignment by the double dynamic programming algorithm

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer.     

A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences

Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related Lactococcus lactis subsp. cremoris strains MG1363 and NCDO763 revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, the two inversion junctions of MG1363 and NCDO763 chromosomes were cloned and characterized. Nucleotide sequence analysis showed the presence of one copy of the lactococcal IS905 element in each junction. Each copy of this element contained the same nucleotide mutation that inactivates the putative transposase. Comparison of the sequences surrounding the insertion sequence demonstrated that the large inversion arose from a single-step homologous recombination event between the two defective copies of the IS905 element. The large inversion presumably conferred no selective disadvantage on strain NCDO763 because this rearrangement did not alter the oriC-terC symmetry of the chromosome and the local genetic environment.     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.     

Comparison of the leakage of carboxyfluorescein from symmetric- and asymmetric-acyl chain phosphatidylcholine vesicles

The Saccharopolyspora erythraea eryAI and eryAII genes, which, together with eryAIII, are responsible for the formation of the macrolactone portion of the antibiotic erythromycin, are separated by a 1.46-kb segment, designated IS1136, with the characteristics of an insertion sequence. It contains an open reading frame of 425 codons similar to that of the Anabaena IS891 and is present in four nonidentical copies in the Sac. erythraea genome. Inverted repeats were found near the ends of IS1136, and in the copy in eryA, one of the ends was found to overlap the 5' end of eryAII. Hybridization analysis suggests that IS1136 is confined to Saccharopolyspora species containing eryA-homologous DNA.     

Characterization of IS1547, a new member of the IS900 family in the Mycobacterium tuberculosis complex, and its association with IS6110

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS116, IS900, IS901, and IS1110 have been proposed as members of the IS900 family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS1547, which was first identified in a clinical isolate of Mycobacterium tuberculosis. Its structure, insertion site, and putative transposase all conform with the conventions of the IS900 family, suggesting that it is a new member of this family. IS1547 was detected only in isolates of the M. tuberculosis complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the M. tuberculosis complex and for distinguishing different strains of M. tuberculosis. ipl is a preferential locus for IS6110 insertion where there are eight known different insertion sites for IS6110. Surprisingly, the DNA sequence of ipl is now known to be a part of IS1547, meaning that IS1547 is a preferential site for IS6110 insertion.     

IS481 and IS1002 of Bordetella pertussis create a 6-base-pair duplication upon insertion at a consensus target site

The insertion sequence IS481 and its isoform IS1002 have been observed to transpose into the bvgAS locus of Bordetella pertussis, for which the DNA sequence has previously been determined. Upon insertion of IS481 at three different sites and IS1002 at one site, a 6-bp sequence originally present was found at the junction of bvg and insertion sequence DNA. This indicates that, contrary to prior reports, IS481 and IS1002 do create a duplication upon insertion. In this light, examination of these and other examples of IS481 and IS1002 reported in the literature leads to the observation that the 6-bp recognition sequence usually fits the consensus NCTAGN. The near-palindromic nature of this sequence, when directly repeated at the ends of IS481 or IS1002, apparently led to the interpretation that 5 of these base pairs were part of the terminal inverted repeats flanking these elements.     

Human cortical-evoked fields during detection, localisation, and identification of ''pop-out'' targets

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons.     

Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis

Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21. Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5' end of integrons was found. Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R. erythropolis SQ1.     

Identification of two specific prolactin binding sites on Nb2 rat lymphoma cells

IS1201, a 1387-bp insertion sequence isolated from Lactobacillus helveticus, was identified by its nucleotide (nt) sequence. It carries a single open reading frame encoding a 369-amino-acid protein, which shares homology with transposases found in a class of related IS, including ISRm3 from Rhizobium meliloti, IS256 from Staphylococcus aureus, IS6120 from Mycobacterium smegmatis, IS1081 from M. bovis, IST2 from Thiobacillus ferroxidans and IS406 from Pseudomonas cepacia. IS1201 has terminal inverted repeats of 24 bp in length and a target site duplication of 8 bp. Its copy number ranges from 3 to about 16 per L. helveticus genome. No homology was found between the nt sequence of IS1201 and those of the other bacterial IS from the same class. These results, together with previous observations [de los Reyes-Gavilán et al., Appl. Environ. Microbiol., 58 (1992) 3429-3432], confirm that IS1201 can be used as a specific DNA probe for the identification of L. helveticus strains.     

Characterization of IS1245 for strain typing of Mycobacterium avium

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159 M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had > or = 10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245 typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.     

A selective medium for the rapid detection by an impedance technique of Pseudomonas spp. associated with poultry meat

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobacter crescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISR1, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5' end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family.     

An active Ac-like transposable element in teleost fish

The i4/i4 genotype of the medakafish, Oryzias latipes, exhibits a quasi albino phenotype due to insertion of a novel transposable element, Tol2, into the tyrosinase gene. Tol2 is 4681 bp in length, has short inverted terminal repeats, and contains four ORFs with the potential to encode a transposase protein. Excision activity of the element has been detected by PCR analysis. Genomic Southern of the Tol2 element revealed that about 20 copies are present in the diploid genome. Dot-matrix comparisons of amino acid sequences of ORFs show relatively high similarity with transposases from Ac of maize, hobo of Drosophila, and Tam3 of snapdragon, which are all active transposable elements. Tol2 is thus concluded to be an active Ac-like transposable element probably encoding a transposase protein. It should therefore find application as a unique material for establishing a gene tagging system in fish.