IS1491fromPseudomonas alcaligenesNCIB 9867: Characterization and Distribution amongPseudomonasSpecies

A new insertion sequence, IS1491,has been cloned and sequenced. The 2489-bp IS1491was isolated from aPseudomonas alcaligenesNCIB 9867 (strain P25X) 4.8-kbPstI chromosomal fragment. IS1491is flanked by an imperfect inverted repeat of 23 bp and carries two overlapping open reading frames, ORF1 and ORF2. Both ORF1 and ORF2 displayed homology to the IstA-like and IstB-like transposases encoded by the IS21family of insertion sequences, which include two IS elements previously isolated fromP. alcaligenesP25X, IS1474,and IS1475(Yeo, C. C., and Poh, C. L. (1997).FEMS Microbiol. Lett.149,257–263). Transposition assays showed that IS1491transposed at a frequency of approximately 1.4 × 10⁻⁶. Transposition of IS1491into the target pRK415 replicon was observed but when ORF2 was disrupted, a fusion between the donor and target replicons was detected. IS1491-like sequences were detected in total DNA ofPseudomonas putidaNCIB 9869 (strain P35X),Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas mendocina, Comomonas acidovorans,andComomonas testosteroniby hybridization with IS1491DNA.     

A New Insertion Variant, IS231I, Isolated from a Mosquito-Specific Strain of Bacillus thuringiensis

A new insertion variant belonging to the family IS231, designated IS231I, was isolated from a mosquito larvicidal strain of the Bacillus thuringiensis serovar sotto (H4ab). IS231I was 1653 bp long and delimited by two 20 bp inverted repeats with one mismatch, flanked by two perfect 11 bp direct repeats. The element contained a single open reading frame (ORF) encoding 478 amino acids and five conserved domains: N1, N2, N3, C1, and C2. The 5′ noncoding region upstream of the ORF, presumed to form a stable stem-and-loop structure, was highly conserved in IS231I. The secondary structure conformation had a deduced free energy (ΔG = 25°C) of −17.2 kcal/mol. Comparison of the IS231I amino acid sequence with those of the 10 existing IS variants revealed that the new variant shares 89% identity with IS231A and IS231B, 65–66% with IS231M and IS231N, and 38% with IS231W.     

A new insertion sequence from Sinorhizobium meliloti with homology to IS1357 from Methylobacterium sp. and IS1452 from Acetobacter pasteurianus

The insertion sequence ISRm8 was identified by sequence analysis of the cryptic plasmid pRmeGR4b of Sinorhizobium meliloti GR4. ISRm8 is 1451 bp in length and carries 22/24-bp terminal imperfect inverted repeats with seven mismatches and a direct target site duplication of 3 bp. ISRm8 carries a unique open reading frame whose putative protein showed significant similarity to the insertion sequences IS1357 and IS1452, isolated from Methylobacterium sp. and Acetobacter pasteurianus, respectively. Two copies of this IS element were found in strain GR4; one of them is linked to plasmid pRmeGR4b, whereas the other is localized out of the non-pSym plasmids. In S. meliloti field populations ISRm8 shows a limited distribution (50% of the strains tested carry the IS element), with a copy number ranging from 1 to 6.     

Identification and characterization of IS 1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS 1138, and the target site into which it inserted were determined. IS1138 consists of 1288bp with 18bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS 1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS 1138 are present in most, if not all, strains of M. pulmonis, but Is1138–like sequences were not detected in other mycoplasmal species.     

Characterization of Insertions of IS476 and Two Newly Identified Insertion Sequences, IS1478 and IS1479, in Xanthomonas campestris pv. campestris

Thirty-two plasmid insertion mutants were independently isolated from two strains of Xanthomonas campestris pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS3 family member, IS476, and two new insertion sequences (IS), IS1478 and IS1479. While IS1478 does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS1479 demonstrated 73% sequence homology with IS1051 in X. campestris pv. dieffenbachiae, 62% homology with IS52 in Pseudomonas syringae pv. glycinea, and 60% homology with IS5 in Escherichia coli. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS1478 by itself constitutes a new subfamily of the widespread IS5 family, whereas IS1479, along with IS1051, IS52, and IS5, belongs to the IS5 subfamily of the IS5 family. All but one of the IS476 insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS476 insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS1478 and IS1479, on the other hand, were not random, and IS1478 and IS1479 each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS476, IS1478, and IS1479 are present in the genomes of all seven X. campestris pv. campestris strains tested and several X. campestris pathovars.     

Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli.

Summary Transposition events mediated by plasmid-borne copies of the insertion sequence IS3 of Escherichia coli are difficult to detect because of a low frequency of cointegrate formation. We found that cointegration activity could be strongly enhanced by using plasmid constructions in which a second IS3 element, disabled by a large deletion, was placed adjacent to an intact IS3 copy. Attempts to construct plasmids containing two adjacent intact IS3 copies were unsuccessful, probably because of instability. Transpositional hyperactivity of tandemly duplicated IS sequences was previously described for spontaneous duplications of IS21 and IS30 and may well be a more general phenomenon. The frequency of cointegration events was also strongly increased in an E. coli strain deficient in Dam methylation, suggesting that IS3, like some other Dam site-containing IS elements, is regulated by the Dam methylation system. Insertion sites were strongly clustered within the target lambda repressor gene; however no sequence specificity determinants could be identified. All insertions analyzed carried the IS element in the same orientation; target sequence duplications were mostly 3 bp, but in some cases 4 by long. To obtain information about the roles of the open reading frames (ORFs) in IS3, we constructed plasmid-borne mutant elements in which potentially functional reading frames were inactivated by site-directed mutations; the mutants were introduced into partial tandem constructions and tested in cointegration assays. Mutations inactivating the putative initiation codons of ORF I and 11 in the intact element reduced insertion activity to less than 4% of the wild type, whereas the introduction of a termination codon into ORF IV had no effect on cointegration frequency. We conclude that translation of ORFs I and II is essential for cointegration activity and that the mutagenized ATG codons most probably serve as the normal initiation codons in the wild-type element. In contrast, ORF IV could either be non-functional or its gene product could be supplied in trans from chromosomal elements.     

The Sinorhizobium meliloti Insertion Sequence (IS) Element ISRm14 Is Related to a Previously Unrecognized IS Element Located Adjacent to the Escherichia coli Locus of Enterocyte Effacement (LEE) Pathogenicity Island

ISRm14 is 2695 basepairs (bp) in size and bordered by 22 bp imperfect inverted repeats (IRs). A 9-bp target sequence is duplicated upon ISRm14 transposition. The DNA strand that putatively encodes the transposase enzyme carries three open reading frames (ORFs) designated ORFs1 to 3, which specify putative proteins of 15.9 kDa, 13.1 kDa, and 61.1 kDa, respectively. According to its structural characteristics, ISRm14 belongs to the recently proposed IS66 family of IS elements. The ORFs1 to 3 encoded putative proteins displayed significant similarities to ORFs of the previously unrecognized IS element ISEc8, which is inserted adjacent to the locus of enterocyte effacement (LEE) pathogenicity island of Escherichia coli EDL933. Analyses of the distribution of ISRm14 in a natural S. meliloti population showed its widespread occurrence in 66% of the strains tested with a copy number ranging from 1 to 6. Received: 13 May 1999 / Accepted: 14 June 1999     

IS1631 Occurrence in Bradyrhizobium japonicum Highly Reiterated Sequence-Possessing Strains with High Copy Numbers of Repeated Sequences RSα and RSβ

From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSα and RSβ (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845–1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSα or RSβ. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.     

IS1549 from Mycobacterium smegmatis Forms Long Direct Repeats upon Insertion

A new insertion element, IS1549, was identified serendipitously from Mycobacterium smegmatis LR222 during experiments using a vector designed to detect the excision of IS6110 from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the aph gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS231, which is a member of the IS4 family of insertion elements. IS1549 contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS1549 are present in LR222, but not all M. smegmatis strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of M. smegmatis 607 contain IS1549. None of 13 other species of Mycobacterium tested by PCR with two sets of primers specific for IS1549 were positive for the expected amplified product.     

Isolation and Characterization of IS1416fromPseudomonas glumae,a New Member of the IS3Family

Isolation and characterization of four different insertion sequence (IS) elements fromPseudomonas glumaeMAFF 302744 through transposition into the entrapment vector pSHI1063 are described. One of the elements, IS1416,was further characterized. IS1416is 1322 bp long and carries 29-bp terminal inverted repeats flanked by a 3-bp direct duplication. IS1416contains three open reading frames (ORFs), which are designated ORFA1, ORFA2, and ORFB, on one strand. Both DNA sequence of IS1416and the deduced amino acid sequences of its ORFs strongly suggest that IS1416is a member of the IS3family, and is closely related to IS401fromPseudomonas cepaciaand IS51fromPseudomonas syringae.To our knowledge, IS1416is the first IS element isolated fromP. glumae.The gene organization and possible regulation of transposition functions of IS1416are also discussed.     

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.     

Genetic organization and transposition properties of IS511

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobactercrescentus 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 ISRI, 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.     

Characterization and distribution of two insertion sequences,IS1191 and iso-IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co-cultures?

A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191. This is the first IS element characterized in this species. This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8bp. The single large open reading frame of IS1191 encodes a 391-amino-acid protein which displays homologies with transposases encoded by IS1201 from Lactobacillus helveticus (44.5% amino-acid sequence identity) and by the other ISs of the IS256 family. One of the copies of IS 1191 is inserted into a truncated iso-IS981 element. The nucleotide sequences of two truncated iso-IS981 s from S. thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity. The distribution of these insertion sequences in L. lactis and S. thermophilus strains suggests that intergeneric transfers occur during co-cultures used in the manufacture of cheese.     

ISLpl1 Is a Functional IS30-Related Insertion Element in Lactobacillus plantarum That Is Also Found in Other Lactic Acid Bacteria

We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.     

DNA sequence of IS91 and identification of the transposase gene.

IS91 is a 1,830-bp insertion sequence that inserts specifically at the sequence CAAG or GAAC of the target and does not duplicate any sequence upon insertion (23). By transposon mutagenesis, we have identified open reading frame 426 (ORF426; bp 454 to 1731) as the putative ORF for the transposase. It displays a cysteine-rich, potential metal-binding domain in its N-terminal region. Adjacent to ORF426, there is an ORF (ORF121) which precedes and terminally overlaps ORF426 by one amino acid. Tn1732 insertions in ORF121 do not affect the transposition frequency. IS91 has sequence similarities to IS801 from Pseudomonas syringae. Their putative transposases are 36% identical, including conservation of the cysteine-rich cluster. The information concerning IS801 insertion specificity and target duplication has been reevaluated in the light of our results.     

Sub-terminal sequences modulating IS30 transposition in vivo and in vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.     

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 20kDa and 6kDa 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.     

Cloning and Nucleotide Sequence of a New Insertion Sequence, IS231N, from a Non-Serotypable Strain of Bacillus thuringiensis

A new IS231 variant, IS231N, has been isolated from an autoagglutinable, non-serotypable strain of B. thuringiensis. IS231N is 1654 bp in length and is delimited by two incomplete 20-bp inverted repeats (IR_L and IR_R) with two mismatches. No direct repeats (DRs) were found at the right and left borders of IS231N. Surprisingly, IS231N contains three open reading frames (ORFs) that could code for polypeptides of 329 (ORF1), 118 (ORF2), and 17 (ORF3) amino acids, respectively. IS231N lacks the 5th conserved amino acid domain, called C2, owing to the addition of an adenine residue at nucleotide 1319. IS231N shows the highest nucleotide identity (99%) with IS231M, another insertion sequence previously isolated from the same bacterial strain. IS231N, however, shares only 83% amino acid identity with IS231M because of nucleotide substitutions and additions. The ORF1 of IS231N has five fewer amino acids than ORF1 of IS231M. Furthermore, the ORF2-3 putative fusion product in IS231N contains eight fewer amino acids than ORF2 in IS231M. The dendrogram showing the evolutionary relationship between members of the IS231 family and IS231N indicates that IS231N is phylogenetically more closely related to IS231M (83%), followed by IS231F(74%), and is more distant from IS231V and W(46%). Received: 4 January 2001/Accepted: 6 February 2001     

Characterization of IS1676 from Rhodococcus erythropolis SQ1

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5′-(C/T)TA(A/G)-3′ in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacteriumkansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available. Received: 1 April 1999 / Received revision: 6 July 1999 / Accepted: 1 August 1999