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PubMed1994

Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium.

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Amino Acid Sequence↗
PubMed1994

IS231A insertion specificity: consensus sequence and DNA bending at the target site.

In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430. Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli. Sequence analysis reveals that IS231A insertion sites match the 5'-GGG(N)5CCC-3' consensus. However, this consensus is not the only determinant of IS231A insertion specificity. Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random. We demonstrate that this preference is determined by the flanking regions of the site. These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly 'S'-shaped structure centered on the target consensus. DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions. We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA.

Bacillus thuringiensis↗
PubMed1995

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron.

The complete nucleotide sequence of an 8447 bp-long mercury-resistance transposon (Tn5053) has been determined. Tn5053 is composed of two modules: (i) the mercury-resistance module and (ii) the transposition module. The mercury-resistance module carries a mer operon, merRTPFAD, and appears to be a single-ended relic of a transposon closely related to the classical mercury-resistance transposons Tn21 and Tn501. The transposition module of Tn5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ, and tniR. Transposition of Tn5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site-specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR. The same pathway of transposition is used by Tn402 (Tn5090) which carries the integron of R751. Transposition genes of Tn5053 and Tn402 are interchangeable. Sequence analysis suggests that Tn5053 and Tn402 are representatives of a new family of transposable elements, which fall into a recently recognized super-family of transposons including retroviruses, insertion sequences of the IS3 family, and transposons Tn552 and Tn7. We suggest that the tni genes were involved in the dissemination of integrons.

Amino Acid Sequence↗
PubMed1995

Characterization of IS1221 from Mycoplasma hyorhinis: expression of its putative transposase in Escherichia coli incorporates a ribosomal frameshift mechanism.

Seven complete and two partial copies of IS1221 variants from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae characterized to date have established a consensus IS1221 as a 1513 bp element with unique structural characteristics resembling the IS3 family of bacterial insertion sequences. Each IS1221 copy contains highly conserved 28 bp imperfect terminal inverted repeats and three distinctive internal inverted repeats (LIR, RIR and IIR). IIR is located within the coding region of the element and it is proposed that it plays a critical role in the regulation of putative transposase expression. Consensus IS1221 and one particular copy, G1135.2, contain a single long open reading frame (ORF). Two potential initiation codons are present at nucleotide 46 (AUG46) and nucleotide 397 (AUG397) and both are preceded by strong ribosome-binding sites. Both initiation codons can be used efficiently in an Escherichia coli T7 expression system. The LIR has a negative regulatory effect on translation initiation from AUG46. A-1 translational frameshift event is shown to be involved in expression of the IS1221 ORF and results in the production of 20 kDa and 6 kDa truncated proteins from the respective upstream initiation codons of the IS1221 ORF. Base substitution and deletion mutations in sequences resembling characterized motifs in documented examples of translational frameshifting resulted in a significant increase in the full-length products and a corresponding decrease in the truncated products from the IS1221 ORF. In contrast to the usual -1 frameshift regulatory event in the IS3 family, which produces a transframe fusion product as the active transposase, IS1221 may have evolved a high-frequency -1 frameshift mechanism that produces a truncated product from the upstream coding domain and thereby results in the regulated low-level production of the full-length presumptive transposase.

Amino Acid Sequence↗
PubMedDate not supplied

Identification of single nucleotide mutations that prevent developmentally programmed DNA elimination in Paramecium tetraurelia.

The excision of internal eliminated sequences (IESs) occurs during the differentiation of a new somatic macronuclear genome in ciliated protozoa. In Paramecium tetraurelia, IESs show few conserved features with the exception of an invariant 5'-TA-3' dinucleotide that is part of an 8-bp inverted terminal repeat consensus sequence with similarity to the ends of mariner/Tc1 transposons. We have isolated and analyzed two mutant cell lines that are defective in excision of individual IESs in the A-51 surface antigen gene. Each cell line contains a mutation in the flanking 5'-TA-3' dinucleotide of IES6435 and IES1835 creating a 5'-CA-3' flanking sequence that prevents excision. The results demonstrate that the first position of the 5'-TA-3' is required IES excision just as previous mutants have shown that the second position (the A residue) is required. Combining these results with other Paramecium IES mutants suggests that there are few positions essential for IES excision in Paramecium. Analysis of many IESs reveals that there is a strong bias against particular nucleotides at some positions near the IES termini. Some of these strongly biased positions correspond to known IES mutations, others correlate with unusual features of excision.

Animals↗
PubMed1995

Virulence-associated fyuA/irp2 gene cluster of Yersinia enterocolitica biotype 1B carries a novel insertion sequence IS1328.

The fyuA/irp2 gene cluster, which is part of the Yersinia pestis pigmentation (pgm) locus encoding genes involved in iron uptake and virulence, is present in all pesticin-sensitive bacteria. In Y. enterocolitica biotype 1B strains (serotypes O8, O20, O21), the fyuA/irp2 gene cluster carries an insertion of a novel repetitive sequence, IS1328. It was also found in the genome of Y. enterocolitica O5 (biotype 1A) and O13 (biotype 1B), but not in pesticin-sensitive Y. pseudotuberculosis O1 and Escherichia coli Phi. The 1353-bp repetitive element has 12-bp perfect inverted terminal repeats. A single open reading frame is capable of encoding a 334-amino acid polypeptide. IS1328 DNA has high homology with the DNA sequences located downstream of the aggR gene from the enteroaggregative E. coli (EAggEC), to the region of the R751 plasmid flanking Tn501, to the sequence following the merR gene of S. marcescens pDU1358 plasmid, and to the sequences of K. pneumoniae plasmid pCFF04. The putative polypeptide has 36.4% identity with the transposase encoded by the Coxiella burnetii IS1111a insertion sequence. The IS1328 insertion sequence could be responsible for the deletions of the fyuA/irp2 gene cluster observed in Y. enterocolitica O8 and could represent a member of a new group of widely distributed repetitive elements.

Amino Acid Sequence↗
PubMed1997

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis.

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (> 75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2000

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.

Bacterial Outer Membrane Proteins↗
PubMed2001

IS1550 from Mycoplasma fermentans is transposable in Escherichia coli.

An insertion sequence (IS)-like element (ISMil) was previously isolated from the incognitus strain of Mycoplasma fermentans. With polymerase chain reaction primers corresponding to the left and right terminal inverted repeats of ISMil, a 1.4-kb DNA fragment was amplified from the genome of the M64 strain of M. fermentans. This DNA fragment has structural characteristics similar to those of ISMil and is designated IS1550. One copy of IS1550 encoded two considerable overlapping open reading frames (ORFs), ORF1 and ORF2. A putative translation frame-shift signal AAAAAAG (A6G) was located near the 3'-end of ORF1. This signal might cause a -1 frame-shift to form a fused product of ORF1 and ORF2 with 444 amino acids, which has a significant similarity to the putative transposase of the IS3 family. This copy of IS1550 was shown to be transposable in Escherichia coli ISM612. Its transposition caused a 1465-bp deletion immediately adjacent to the 3'-end of the element and the creation of a pair of 3-bp direct repeats flanking the element at the new insertion site. On the basis of these results, IS1550 was considered a typical transposable element.

Base Sequence↗
PubMed2002

Representational difference analysis uncovers a novel IS10-like insertion element unique to pathogenic strains of Yersinia enterocolitica.

The method of suppressive subtractive hybridization was employed to map out genomic differences between the highly pathogenic Yersinia enterocolitica (Ye) biogroup 1B, serotype O:8 strain (WA-314) and the closely related apathogenic Y. enterocolitica biogroup 1A, serotype O:5 strain (NF-O). A novel IS10-like element, IS1330, uncovered by this technique was found to be uniquely present in high copy numbers among the highly pathogenic Y. enterocolitica 1B strains, while a single copy of the element was found in the low pathogenic Ye biogroup 4 serotype O:3 strain. The 1321-bp repetitive element has 19-bp imperfect inverted terminal repeats and is bracketed by a 10-bp duplication of the target sequence. The predicted transposase shares high homology with the IS10 open reading frame of the large virulence plasmid pWR501, of Shigella flexneri, with IS10 transposase of Salmonella typhi, and with IS1999 (tnpA) of Pseudomonas aeruginosa. The IS1330 tnp gene is transcribed in vitro and in vivo in HeLa cells. At least one copy of IS1330 flanks the recently described chromosomal type III secretion cluster in Y. enterocolitica WA-314, O:8, and future studies should shed light on whether this novel transposase mediates transposition events in highly pathogenic Y. enterocolitica strains, thus enhancing the genetic plasticity of this species.

Base Sequence↗
PubMed2002

Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2.

We have analyzed the structure of two extrachromosomal elements of the methylenomycin producing actinomycete Streptomyces violaceoruber SANK95570. The presence of the circular plasmid pSV1 which was supposed to contain the genes for methylenomycin biosynthesis could be verified. Physical mapping of pSV1 revealed a size of 175.35 kb for this plasmid. In addition we generated a restriction map for the 100-kb linear plasmid pSV2. Cloning and sequencing of the terminal ends of pSV2 indicated the presence of 426-bp terminal inverted repeats. Both pSV2 termini show significant homology to the chromosome ends of Streptomyces coelicolor A3(2) which is a closely related strain to S. violaceoruber SANK95570.

Amino Acid Sequence↗
PubMed2006

Novel macrolide resistance module carried by the IncP-1beta resistance plasmid pRSB111, isolated from a wastewater treatment plant.

The macrolide resistance plasmid pRSB111 was isolated from bacteria residing in the final effluents of a wastewater treatment plant. The 47-kb plasmid confers resistance to azithromycin, clarithromycin, erythromycin, roxithromycin, and tylosin when it is carried by Pseudomonas sp. strain B13 and is very similar to prototype IncP-1beta plasmid pB3, which was previously isolated from an activated-sludge bacterial community of a wastewater treatment plant. The two plasmids differ in their accessory regions, located downstream of the conjugative transfer module gene traC. Nucleotide sequence analysis of the pRSB111 accessory region revealed that it contains a new macrolide resistance module composed of the genes mphR(E), mph(E), and mrx(E), which putatively encode a transcriptional regulator, a macrolide phosphotransferase, and a transmembrane transport protein, respectively. Analysis of the contributions of the individual genes of the macrolide resistance module revealed that mph(E) and mrx(E) are required for high-level macrolide resistance. The resistance genes are flanked by two insertion sequences, namely, ISPa15 and ISRSB111. Two truncated transposable elements, IS6100 and remnants of a Tn3-like transposon, were identified in the vicinity of ISRSB111. The accessory element of pRSB111 apparently replaced the Tn402-like element present on the sister plasmid, pB3, as suggested by the conservation of Tn402-specific terminal inverted repeats on pRSB111.

Bacterial Proteins↗
PubMed1991

Characterization of the gentamicin resistance transposon Tn5281 from Enterococcus faecalis and comparison to staphylococcal transposons Tn4001 and Tn4031.

In Enterococcus faecalis, the genetic determinant encoding gentamicin resistance (Gmr) on the conjugative plasmid pBEM10 previously has been shown to be on a mobile element. In the current study, this element, termed Tn5281, was shown to relocate in the absence of homologous recombination in E. faecalis UV202. On the basis of restriction endonuclease analysis and DNA-DNA hybridization studies, Tn5281 was shown to be similar, if not identical, to the Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in U.S. isolates of Staphylococcus epidermidis, since all three of these transposons have symmetrically located HindIII (2.5 kb apart), ClaI (slightly more than 2.5 kb apart), and HaeIII (3.9 kb apart) sites. Restriction endonuclease digestion patterns of Tn5281 generated with HincII, ScaI, and AluI were also consistent with Tn4001 and Tn4031. By using a probe specific for the external portion of the terminal inverted repeat of Tn4031, it was determined that each terminus of Tn5281 contained a 0.35-kb HaeIII fragment and a 0.7-kb HindIII-HaeIII fragment. The sizes of these fragments are identical to those found in the staphylococcal transposons, which is a further indication that inverted repeats like IS256 are present in Tn5281. A 1-kb HaeIII fragment in pBEM10 also hybridized with this probe, which indicates that Tn5281 in pBEM10 contains a double copy of the inverted repeat at one end.

Conjugation, Genetic↗
PubMed1996

Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus.

We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.

Amino Acid Sequence↗
PubMed1998

Characterization of IS18, an element capable of activating the silent aac(6')-Ij gene of Acinetobacter sp. 13 strain BM2716 by transposition.

Insertion sequence IS18 was detected by analysis of the spontaneous aminoglycoside resistant mutant Acinetobacter sp. 13 strain BM2716-1. Insertion of the element upstream from the silent acetyltransferase gene aac(6')-Ij created a hybrid promoter that putatively accounts for the expression of the aminoglycoside resistance gene. The 1, 074-bp IS18 element contained partially matched (20 out of 26 bases) terminal inverted repeats, one of which overlapped the 3' end of a 935-bp open reading frame potentially encoding a protein related to the transposases of the IS30 family. IS18 was found in 6 out of 29 strains of Acinetobacter sp. 13 but not in 10 strains each of A. baumannii and A. haemolyticus.

Acinetobacter↗
PubMed1999

Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315.

In methicillin-resistant Staphylococcus aureus, the methicillin resistance gene mecA is localized within a large chromosomal region which is absent in the methicillin-susceptible S. aureus chromosome. The region, designated mec DNA, is speculated to have originated from the genome of another bacterial species and become integrated into the chromosome of the S. aureus cell in the past. We report here cloning and determination of the structure of the entire mec DNA sequence from a Japanese S. aureus strain, N315. The mec DNA was found to be 51,669 bp long, including terminal inverted repeats of 27 bp and a characteristic pair of direct repeat sequences of 15 bp each: one is situated in the right extremity of mec DNA, and the other is situated outside the mec DNA and abuts the left boundary of mec DNA. The integration site of mec DNA was found to be located in an open reading frame (ORF) of unknown function, designated orfX. Clusters of antibiotic resistance genes were noted in mec DNA carried by transposon Tn554 and an integrated copy of plasmid pUB110. Both the transposon and plasmid were integrated in the proximity of the mecA gene, the latter being flanked by a pair of insertion sequence IS431 elements. Many ORFs other than those encoding antibiotic resistance were considered nonfunctional because of the acquired mutations or partial deletions found in the ORFs. Two ORFs potentially encoding novel site-specific recombinases were found in mec DNA. However, there was no ORF that might encode mec DNA-specific transposase or integrase proteins, indicating that the mec DNA is not a transposon or a bacteriophage in nature.

Amino Acid Sequence↗
PubMed2001

Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus.

The beta-lactam resistance gene mecA of Staphylococcus aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec), identified in the chromosome of a Japanese methicillin-resistant S. aureus (MRSA) strain. We now report identification of two additional types of mecA-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the elements and the SCCmec. However, new elements shared the chromosomal integration site with the SCCmec. Structural analysis of the new elements revealed that they possessed all of the salient features of the SCCmec: conserved terminal inverted repeats and direct repeats at the integration junction points, conserved genetic organization around the mecA gene, and the presence of cassette chromosome recombinase (ccr) genes responsible for the movements of SCCmec. The elements, therefore, were considered to comprise the SCCmec family of staphylococcal mobile genetic elements together with the previously identified SCCmec. Among 38 epidemic MRSA strains isolated in 20 countries, 34 were shown to possess one of the three typical SCCmec elements on the chromosome. Our findings indicated that there are at least three distinct MRSA clones in the world with different types of SCCmec in their chromosome.

Amino Acid Sequence↗