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PubMed2000

Effects of the movement of insertion sequences on the structure of VanA glycopeptide resistance elements in Enterococcus faecium.

A Tn1546-related element with IS1216V at position 8839 underwent a structural change after storage of the host strain of Enterococcus faecium at 4 degrees C. The element acquired IS1542 at position 3932, nucleotides 8732 to 8831 were deleted, and the first 3417 nucleotides were lost and replaced by an inverted copy of the IS1216V-vanY-vanZ-inverted-repeat block from the 3' end. Insertion sequence movement is likely to play a key role in the evolution of VanA resistance elements.

Anti-Bacterial Agents↗

PubMed1996

Identification of IS1356, a new insertion sequence, and its association with IS402 in epidemic strains of Burkholderia cepacia infecting cystic fibrosis patients.

Burkholderia cepacia is now recognized as an important opportunistic pathogen in cystic fibrosis (CF) and other compromised patients. Epidemicity among CF patients has been attributed to at least one particularly infectious strain (strain ET12), and both genetic evidence and anecdotal evidence suggest that this strain, currently endemic in Ontario, and those causing an epidemic in the United Kingdom, are indeed the same. Our study was conducted to determine whether there was any association between the presence of various insertion sequence (IS) elements, the cable pilin subunit gene (cblA), electrophoretic type (ET), and ribotype (RT) in a collection of 97 clinical and 2 environmental isolates of B. cepacia. No apparent linkage was found for IS elements IS401, IS402, IS406, IS407, and IS408 with ET or RT. The cblA target, said to be a marker for high infectivity, was detected in 100% (38 of 38) of strains of B. cepacia ET12 and in a single strain of ET13 that differed in a single enzyme allele. A new IS, IS1356, identified during the investigation, was present in 71.7% of all isolates, and 50.7% of these isolates harbored IS1356 as a hybrid IS element inserted into IS402. IS1356 is 1,353 bp in length, and when it is inserted into IS402 it results in a 10-bp duplication at the site of insertion. IS1356 contains one major open reading frame of 1,260 bp coding for a putative transposase which has significant homology to ISRm3 in Rhizobium meliloti (59%) and to an undesignated IS element in Corynebacterium diphtheriae (49%). The IS402-IS1356 element was found exclusively in the epidemic strains from Ontario and the United Kingdom, being detected in 94.7% (36 of 38 isolates) of B. cepacia ET12 isolates. Of the two ET12 isolates found to be devoid of the IS402-IS1356 element, both contained IS1356 unassociated with IS402, one was temporally unrelated to the epidemic, and the other was from a CF patient in a geographic area remote from Ontario and the United Kingdom. It is evident that the IS402-IS1356 hybrid element, the cblA pilin subunit gene, and the allelic suite represented by multilocus enzyme electrophoretic type ET12 may provide useful markers for the epidemic, highly transmissible transatlantic strain isolated in Ontario and the United Kingdom.

Amino Acid Sequence↗

PubMed1992

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Insertion sequence IS200 can differentiate drug-resistant and drug-sensitive Salmonella typhi of Vi-phage types E1 and M1.

The type strains of Vi-phage types E1, M1 and A of Salmonella typhi, together with drug-resistant and drug-sensitive strains of phage types E1 and M1 isolated in 1992 from patients associated with India or Pakistan, and a drug-resistant strain of phage type A isolated in South Africa in 1991, were characterised with respect to the presence of plasmids conferring resistance to antimicrobial drugs and their chromosomal insertion sequence IS200 profiles. The three type strains, the drug-sensitive strains of Vi-phage types E1 and M1, and a strain of phage type M1 resistant to ampicillin and trimethoprim but not to chloramphenicol, did not contain plasmids. In contrast, for strains of phage types E1 and M1 resistant to chloramphenicol, ampicillin and trimethoprim, and for the drug-resistant strain of phage type A, the complete spectrum of resistance was encoded by high molecular mass plasmids belonging to the H1 incompatibility group. Characterisation of IS200 profiles demonstrated that at least 13 IS200 copies were distributed on the chromosome of all strains tested. Although the IS200 profiles of the type strains of Vi-phage types A, E1 and M1 were identical, it was possible to distinguish between drug-sensitive and drug-resistant strains of Vi-phage types E1 and M1 isolated from patients infected in India and Pakistan by this method. It was concluded that although IS200 typing is not as discriminatory as phage typing for the primary subdivision of S. typhi, it may be useful for certain epidemiological investigations and, in particular, for investigating the origins of strains with multiple drug resistance.

Bacteriophage Typing↗

PubMed1984

Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion sequences flank operon in plasmid-mediated systems.

Large plasmids were detected in two aerobactin-producing enteric bacterial species (Aerobacter aerogenes 62-I, Salmonella arizona SA1, and S. arizona SL5301) and designated pSMN1, pSMN2, and pSMN3, respectively. Other Salmonella spp., namely, S. arizona SL5302, S. arizona SLS, Salmonella austin, and Salmonella memphis, formed aerobactin but contained no detectable large plasmids. S. arizona SL5283 made no aerobactin. A probe consisting of the aerobactin biosynthetic genes cloned on plasmid pABN5 hybridized to a HindIII digest of pSMN1 but not to digests of pSMN2 or pSMN3. A larger probe, the insert of pABN1 containing the complete aerobactin operon, hybridized to four fragments in HindIII digests of the parent plasmid, pColV-K30. A 2.0-kilobase PvuII fragment responsible for this multiple-hybridization pattern was cloned into vector pUC9 to form pSMN30. The latter was mapped and shown to correspond to either IS1 or to a closely related insertion sequence.

Chromosomes, Bacterial↗

PubMed2002

Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland.

BACKGROUND: Since Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from intestinal tissue of a human patient suffering Crohn's disease, a controversial discussion exists whether MAP have a role in the etiology of Crohn's disease or not. Raw milk may be a potential vehicle for the transmission of MAP to human population. In a previous paper, we have demonstrated that MAP are found in raw milk samples obtained from a defined region in Switzerland. The aim of this work is to collect data about the prevalence of MAP specific IS900 insertion sequence in bulk-tank milk samples in different regions of Switzerland. Furthermore, we examined eventual correlation between the presence of MAP and the somatic cell counts, the total colony counts and the presence of Enterobacteriaceae. RESULTS: 273 (19.7%) of the 1384 examined bulk-tank milk samples tested IS900 PCR-positive. The prevalence, however, in the different regions of Switzerland shows significant differences and ranged from 1.7% to 49.2%. Furthermore, there were no statistically significant (p >> 0.05) differences between the somatic cell counts and the total colony counts of PCR-positive and PCR-negative milk samples. Enterobacteriaceae occur as often in IS900 PCR-positive as in PCR-negative milk samples. CONCLUSION: This is the first study, which investigates the prevalence of MAP in bulk-tank milk samples all over Switzerland and infers the herd-level prevalence of MAP infection in dairy herds. The prevalence of 19.7% IS900 PCR-positive bulk-milk samples shows a wide distribution of subclinical MAP-infections in dairy stock in Switzerland. MAP can therefore often be transmitted to humans by raw milk consumption.

Animals↗

PubMed1990

Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia.

We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRP1::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-beta-D-thiogalactopyranoside. Lac+ variants of the pGC91.14-containing strains which formed beta-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.

Blotting, Southern↗

PubMed1994

Use of an insertion sequence for laboratory diagnosis and epidemiologic studies of tuberculosis.

A dramatic improvement in the rapidity and accuracy of laboratory testing for tuberculosis is anticipated in the near future from the application of molecular biology techniques. The polymerase chain reaction and other nucleic acid amplification methodologies have the potential to detect, amplify, and identify very small quantities of Mycobacterium tuberculosis DNA directly in a clinical specimen, even on the same day it is collected. Within the past 3 years, a number of polymerase chain reaction-based assays for tuberculosis have emerged. The development and evaluations of the polymerase chain reaction assay based on the insertion sequence IS6110 are described. For practical application in the clinical setting, amplification assays require a simple, reliable sample preparation method; an internal positive control to monitor for inhibitors; a method for eliminating contamination with amplicon (a polymerase chain reaction product) to prevent false-positive results; and a simple, sensitive detection method. The DNA fingerprinting method that uses IS6110 probes provides a means of differentiating individual strains of M tuberculosis and is a powerful tool for epidemiologic studies. The method and its applications are described.

DNA Fingerprinting↗

PubMed1991

Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences.

Halobacterium-halobium NRC-1 harbors a 200-kb plasmid, pNRC100, which contains a cluster of genes for synthesis of buoyant gas-filled vesicles. Physical mapping of pNRC100 by using pulsed-field gel electrophoresis showed the presence of a large (35 to 38-kb) inverted repeat (IR) sequence. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single-copy region and the large single-copy region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. Additionally, the identities and approximate positions of 17 insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. The large IRs terminated at an ISH2 element at one end and an ISH3 element at the other end. pNRC100 is similar in structure to chloroplast and mitochondrial genomes, which contain large IRs and other large halobacterial and prokaryotic plasmids that are reservoirs of IS elements but lack the large IRs.

Base Sequence↗

PubMed2001

Optimal estimation of transposition rates of insertion sequences for molecular epidemiology.

Outbreaks of infectious disease can be confirmed by identifying clusters of DNA fingerprints among bacterial isolates from infected individuals. This procedure makes assumptions about the underlying properties of the genetic marker used for fingerprinting. In particular, it requires that each fingerprint changes sufficiently slowly within an individual that isolates from separate individuals infected by the same strain will exhibit similar or identical fingerprints. We propose a model for the probability that an individual's fingerprint will change over a given period of time. We use this model together with published data in order to estimate the fingerprint change rate for IS6110 in human tuberculosis, obtaining a value of 0.0139 changes per copy per year. Although we focus on insertion sequences (IS), our method applies to other fingerprinting techniques such as pulsed-field gel electrophoresis (PFGE). We suggest sampling intervals that produce the least error in estimates of the fingerprint change rate, as well as sample sizes that achieve specified levels of error in the estimate.

Bias↗

PubMed1995

Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content.

Molecular typing allowed the separation of the species Bacteroides fragilis into two genotypically distinct groups. A unique set of 50 strains of B. fragilis carrying the chromosomal metallo-beta-lactamase gene cfiA was subjected to a comparative analysis with respect to sets of up to 250 randomly collected strains devoid of this gene. The two groups were found to be distinct on the basis of the following results: (i) ribotyping, after DNA digestion with AvaI, revealed a practically homogeneous DNA fragment pattern for the cfiA-positive strains and distinct multiple patterns for the cfiA-negative strains; (ii) PCR, arbitrarily primed with an experimentally selected decamer, generated fragment patterns typical for the strains of each group; (iii) the three insertion sequences described to date in the species B. fragilis, i.e., IS4351, IS942, and IS1186, were all but confined to the cfiA-positive group, in which they were capable of providing promoter sequences for the transcription of cfiA; and (iv) the cepA gene, encoding the so-called endogenous cephalosporinase of B. fragilis, was found exclusively in the cfiA-negative group, in which it was present in ca. 70% of the strains. The cfiA-, cepA-negative fraction was not characterized further. In a natural population of 500 randomly selected strains of B. fragilis, the cfiA-positive and cfiA-negative groups represented ca. 3 and 97% of the strains, respectively. Analysis of 82 metabolic traits revealed no difference between the two groups.

Bacterial Proteins↗

PubMed1991

Chlorobenzoate catabolic transposon Tn5271 is a composite class I element with flanking class II insertion sequences.

The structure of a transposon specifying the biodegradation of chlorobenzoate contaminants is described. Tn5271 is a 17-kilobase (kb) transposon that resides in the plasmid or chromosome of Alcaligenes sp. strain BR60 and allows this organism to grow on 3- and 4-chlorobenzoate. The transposon is flanked by a directly repeated sequence of 3201 base pairs (bp), which in turn is flanked by 110-bp inverted repeats. The 3.2-kb repeated sequence, designated IS1071, exists in multiple copies in the genome of Alcaligenes sp. strain BR60 and is involved in recombination of the catabolic genes into the chromosome of this strain. Sequence analysis revealed that the inverted repeat of IS1071 and the derived amino acid sequence of the single open reading frame within IS1071 are related to the inverted repeats and transposase (TnpA) proteins of the class II (Tn3 family) transposable elements. The absence of a resolvase gene within IS1071 suggests that this element is capable of determining the first step in class II transposition only. This was confirmed by observations on the IS1071-dependent formation of stable cointegrates in a recombination-deficient Escherichia coli. These results support an evolutionary scheme in which the class II transposable elements descended from simple insertion sequences.

Alcaligenes↗

PubMed1999

mRNA stability and selenocysteine insertion sequence efficiency rank gastrointestinal glutathione peroxidase high in the hierarchy of selenoproteins.

The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.

Animals↗

PubMed1997

Bacterial interspersed mosaic elements (BIMEs) are a major source of sequence polymorphism in Escherichia coli intergenic regions including specific associations with a new insertion sequence.

A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS 1397. Remarkably, 14 out of IS 1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS 1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed.

Amino Acid Sequence↗

PubMed2001

Transposition of IS1397 in the family Enterobacteriaceae and first characterization of ISKpn1, a new insertion sequence associated with Klebsiella pneumoniae palindromic units.

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.

Base Sequence↗

PubMed1999

The Caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence (SECIS) element that differs from mammalian SECIS elements but directs selenocysteine incorporation.

Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS elements from worm to man.

Amino Acid Sequence↗

PubMed1999

Functional expression of rat thioredoxin reductase: selenocysteine insertion sequence element is essential for the active enzyme.

Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3'-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [75Se]selenite. 75Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.

3' Untranslated Regions↗

PubMed2000

FimE-catalyzed off-to-on inversion of the type 1 fimbrial phase switch and insertion sequence recruitment in an Escherichia coli K-12 fimB strain.

We have investigated the capacity of a well-defined Escherichia coli fimB strain, AAEC350 (a derivative of MG1655), to express type 1 fimbriae under various growth conditions. The expression of type 1 fimbriae is phase-variable due to the inversion of a 314-bp DNA segment. Two tyrosine recombinases, FimB and FimE, mediate the inversion of the phase switch. FimB can carry out recombination in both directions, whereas the current evidence suggests that FimE-catalyzed switching is on-to-off only. We show here that AAEC350 is in fact capable of off-to-on phase switching and type 1 fimbrial expression under aerobic static growth conditions. The phase switching is mediated by FimE, and allows emerging fimbriate AAEC350 to outgrow their non-fimbriate counterparts by pellicle formation. Following inversion of the phase switch, this element can remain phase-locked in the on orientation due to integration of insertion sequence elements, viz. IS1 or IS5, at various positions in either the fimE gene or the phase switch.

Agglutination↗