DNA sequence of the transposable element IS1

Summary The nucleotide sequence of an IS1 element recently transposed into the lacI gene is reported. This sequence is nearly identical to one previously reported for another IS1 element (Ohtsubo and Ohtsubo, 1978). The implications of this similarity are discussed. The sizes of potential polypeptides encoded in the IS1 DNA have been determined and possible roles for these peptides in the illegitimate recombination events mediated by the element are considered.     

Identification of IS elements in <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains grown in a medium with ferrous iron or adapted to elemental sulfur

IS elements were identified in the genomes of five Acidithiobacillus ferrooxidans strains isolated from various media. IST2 elements were revealed in all the strains grown in a medium with ferrous iron, ISAfe1 elements were detected in four strains (TFBk, TFL-2, TFV-1 and TFO). Three strains (TFV-1, TFN-d and TFO) were found to contain IS elements, ~600 bp long. These were named preliminary as ISAfe600. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of an ISAfe1 element in TFBk and TFL-2 strains and complete sequencing of the ISAfe1 element in the TFBk strain has revealed nucleotide substitutions as compared to the prototype, i.e., the ISAfe1 element of an ATCC 19859 strain. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of the IST2 elements in TFO, TFBk and TFL-2 strains has shown numerous nucleotide substitutions when compared to the IST2 element of an ATCC 19859 strain. Complete sequencing of the IST2 element in the TFBk strain has revealed: the divergence between the IST2 elements in the TFBk strain and the prototype was 21.2%. Southern hybridization of EcoRI fragments of the chromosomal DNA from five A. ferrooxidans strains grown in a medium with ferrous iron using an internal region of ISAfe1, a full-length ISAfe1 or a full-length IST2 as probes has shown them to differ in the number of copies of IS elements and their localization on the chromosomes. Adaptation to elemental sulfur in A. ferrooxidans strains caused changes in the number, intensity and localization of hybridization bands. The authors discuss the role of IS elements in the adaptation of A. ferrooxidans to the new energy substrate. The nucleotide sequence data reported in this paper were deposited in GenBank under accession numbers: AY823401, the ISAfe1 from A. ferrooxidans TFBk; AY825254, the IST2 from TFBk; DQ002894, the 5′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ002895, the 3′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ005952, the 5′-terminal nucleotide sequence of IST2 from TFV-1; DQ005953, the 3′-terminal nucleotide sequence of IST2 from TFV-1.     

A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution

An insertion sequence (here called IS 1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS 1031A as well as the sequences of two novel iso-IS 1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS 1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS 1031-like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031-like elements. Taken together, the results suggest that A. xylinum contains a family of IS 1031 elements with considerably diversified nucleotide sequences.     

IS4 is found between eleven or twelve base pair duplications

Summary Three mutations caused by the integration of IS4 in galT in both possible orientations were shown by DNA sequence analysis to be integrated between a duplication of eleven base pairs of gene galT.IS4 has been cloned from its single position on the E. coli K12 chromosome. Here, 12 base pairs are duplicated adjacent to IS4. This sequence is unrelated to the duplicated sequence in galT.     

Multiple copies of IS10 in the Enterobacter cloacae MD36 chromosome.

Repetitive sequences were isolated and characterized as double-stranded DNA fragments by treatment with S1 nuclease after denaturation and renaturation of the total DNA of Enterobacter cloacae MD36. One repetitive sequence was identical to the nucleotide sequence of IS10-right (IS10R), which is the active element in the plasmid-associated transposon Tn10. Unexpectedly, 15 copies of IS10R were found in the chromosomal DNA of E. cloacae MD36. One copy of the central region of Tn10 was found in the total DNA of E. cloacae MD36. IS10Rs in restriction fragments isolated from the E. cloacae MD36 total DNA showed 9-bp duplications adjacent to the terminal sequences that are characteristic of Tn10 transposition. This result suggests that many copies of IS10R in E. cloacae MD36 are due to transposition of IS10R alone, not due to transposition of Tn10 or to DNA rearrangement. I also found nine copies of IS10 in Shigella sonnei HH109, two and four copies in two different natural isolates of Escherichia coli, and two copies in E. coli K-12 strain JM109 from the 60 bacterial strains that were examined. All dam sites in the IS10s in E. cloacae MD36 and S. sonnei HH109 were methylated. Tn10 and IS10 transpose by a mechanism in which the element is excised from the donor site and inserted into the new target site without significant replication of the transposing segment; thus, the copy numbers of the elements in the cell are thought to be unchanged in most circumstances. Accumulation of IS10 copies in E. cloacae MD36 has interesting evolutionary implications.     

Repeated sequences including RS 1100 from Pseudomonas cepacia AC1100 function as IS elements

Summary Several lines of evidence were obtained that the previously identified, repeated sequence RS 1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences or a newly identified 3400 by repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.[/ab]Abbreviations aphc aminoglycoside phosphotransferase gene - BSM basal salts medium - chq chlorohydroxy hydroquinone degradative gene(s) - dCTP deoxycytidine triphosphate - IS insertion sequence - Tft 2,4,5-T degradative phenotype     

Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.     

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.     

The regulatory role of the IS 1-encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1-mediated cointegrate formation and transposition of a synthetic IS 1-based transposon (‘omegon’Ω-on). These results also indicate that the Ω-on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1-encoded genes both in cis or in trans. The experiments involving Ω-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.     

A new type of IS1-mediated deletion

Summary Genetical tests and DNA sequence analysis revealed that the mechanism of formation of IS1-induced type I and type II deletions differs.IS1-mediated type II deletions occur at the termini of the integrated element and do not remove the element. This process is independent of the cellular recA system and does not involve DNA sequence homology.Conversely, the formation of IS1-induced type I deletions differs substantially. They require recA gene product, small DNA sequence duplications and a topological arrangement of the DNA molecule to allow alignment of duplications.     

IS2-61 and IS2-611 arise by illegitimate recombination from IS2-6

Summary A more stable derivative of IS2-6 has been isolated, which had lost 54 bp of the 108 bp long insert characteristic of IS2-6. This new allele of IS2, IS2-61, segregates the remaining 54 bp to yield allele IS2-611. DNA sequence analysis shows that the segregation products of IS2-6 arise by recA-independent, illegitimate recombination at 9 bp long direct sequence repetitions.     

IS30, a new insertion sequence of Escherichia coli K12

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

The sequence of IS4

Summary IS-elements are devoid of easily recognizable transacting functions and exert their visible effects in the position cis only (recent reviews Calos and Miller 1980; Starlinger 1980). It has been a matter of debate, whether these elements encode functions for their own transposition. In the case of the E. coli IS-elements this could not easily be determined by genetic methods, because most of these elements are present in several copies (Saedler and Heiss 1973; Deonier et al. 1979). In the case of the IS-elements flanking transposons, evidence has recently been brought forward that these carry the transposition specificity (Rothstein et al. 1980; Kleckner 1980; Grindley 1981).IS4 is present in one copy only in several E. coli K12 strains and should, therefore, be suitable for genetic and physiological studies (Chadwell et al. 1979). It has been cloned from several sites on the E. coli chromosome in pBR322 (Klaer and Starlinger 1980). Here we report the DNA sequence of IS4 which contains an open reading frame for 442 amino acids, and of the junctions of this element with surrounding DNA at three different sites in E. coli chromosome.     

Artificial transposable elements in the study of the ends of IS1

We have constructed artificial IS1-based transposons by attaching synthetic oligodeoxynucleotides, corresponding to the sequence of the ends of IS1, to a selectable DNA segment ['omega' fragment; Prentki and Krisch, Gene 29 (1984) 303-313]. These transposons were used to examine the sequence requirements at the ends for IS1 transposition. We show here that a 24- to 28-bp sequence from the left or right ends of IS1 is capable of transposition when present at both ends of the omega fragment in the correct orientation. Transposition activity requires the presence of an intact IS1 in cis on the same plasmid molecule. In trans, however, neither resident genomic copies of IS1, nor copies carried by a compatible, high-copy-number plasmid present in the same cell, complement the artificial transposons efficiently. Transposition frequencies in the presence of a cis-complementing IS1 are, however, similar to those of the naturally occurring IS1-based transposon, Tn9. In addition, transposition results in a 9-bp duplication in the target DNA molecule as is usually the case for insertion of the intact IS1. Using this system, we have obtained evidence indicating that the activity of a synthetic IS1 end is not determined exclusively by its sequence, but can be strongly enhanced by a second, wild-type end used in the transposition event. The data also show that single base pair mutations can exhibit a cumulative effect in reducing transposition activity.     

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

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

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

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

Effect of IS900 Gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis

Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A 3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M. smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 μM ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33.5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF. There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis. Received: 12 May 1998 / Accepted: 6 July 1998     

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.     

Characterization of IS 1110, a highly mobiie genetic element from Mycobacterium avium

A highly mobile Insertion sequence designated IS 1110 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. IS 1110 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 IS 1110. 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.