Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of the insertion sequences IS1 and IS2 in F and R plasmids.

Heteroduplex experiments between the plasmid R6 and one strand of the deoxyribonucleic acid (DNA) of a lambda phage carrying the insertion sequence IS1 show that IS1 occurs on R6 at the two previously mapped junctions of resistance transfer factor (RTF) DNA with R-determinant DNA. From previous heteroduplex experiments, it then follows that IS1 occurs at the same junctions in R6-5, R100-1, and R1 plasmids. Heteroduplex experiments with the DNA from a lambda phage carrying the insertion sequence IS2 show that one copy of IS2 occurs in R6, R6-5, and R100-1 (but not R1) at a point within the RTF with coordinates 67.5 TO 68.9 kilobase units (kb). In an accompanying paper, Ptashne and Cohen (1975) show that the insertion sequence IS3 occurs on R6 and R6-5. R100-25, a traC mutant, differs from its parent R100-1 only in that it contains an additional copy of IS1 inserted within the tra gene region of 82.1 kb. R100-31, atraX, TC-s mutant of R100-1, is deleted in R100-1 sequences starting at one of the IS3 termini (46.9 kb) and extending with RTF to 61.0 kb. Heteroduplex studies of F plasmids with the DNA of a lambda phage bearing insertion sequence IS2 show that the sequence of F with coordinates 16.3-17.6F is IS2. The occurrence of IS1 at the two junctions of R-determinant DNA and RTF DNA in R plasmids provides a structural basis to explain the mechanism of the previously observed formation of molecules containing one RTF unit and several tandem copies of the R-determinant unit, when R plasmids in Proteus mirabilis are grown in the presence of antibiotics, and the segregation of an R plasmid into an RTF unit and an R-determinant unit. In general, correlation of our results with previous studies shows that insertion sequences play a role in a variety of F- and R-related intra- and intermolecular recombination phenomena.     

Transposition of IS91 does not generate a target duplication.

We determined the DNA sequences surrounding the junctions of IS91 in two insertion derivatives: pSU234 (pACYC184::IS91) and pSU240 (pBR322::IS91). The termini of IS91 consist of two imperfect inverted repeats eight base pairs long. Their sequence is 5'-TCGAGTAGG. . . CCTATCGA-3'. Insertion of IS91 did not generate direct repetitions in the target DNAs.     

Occurrence of insertion sequence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications.

Insertion sequence (IS) regions have been identified previously as a cause of strongly polar mutations in Escherichia coli and several bacteriophages. The present experiments indicate that genetically characterized IS regions occur on bacterial plasmid deoxyribonucleic acid (DNA) as both direct and inverted DNA sequence duplications. The DNA insertion which has been shown previously (Sharp et al., 1973) to control expression of tetracycline resistance in the R6-5 plasmid, and which occurs as directly and inversely repeated DNA sequences adjacent to the region believed to contain the tetracycline resistance gene, has been identified as IS3. A second genetically characterized insertion sequence (IS1) has been identified as a direct DNA duplication occurring at both junctions of the resistance transfer factor and R-determinant components of R6-5 and related plasmids. A model is presented for the reversible dissociation of resistance transfer factor and R-determinant components of co-integrate R plasmids at the sites of DNA sequence homology provided by the repeated IS regions.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Functional analysis of unique class II insertion sequence IS1071

Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS1071. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS1071 has remained unclear. Our study revealed that IS1071 was only able to transpose at high frequencies in two environmental beta-proteobacterial strains, Comamonas testosteroni and Delftia acidovorans, and not in any of the bacteria examined which belong to the alpha- and gamma-proteobacteria. IS1071 was found to have the functional features of the class II transposons in that (i) the final product of the IS1071 transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS1071, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a tnpA mutation of IS1071 was efficiently complemented by supplying the wild-type tnpA gene in trans. Deletion analysis of the IS1071 IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS1071 might be different from that of the other well-characterized class II transposons.     

Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis

Insertion sequence IS6120 from Mycobacterium smegmatis was identified by its ability to transpose into different sites in the lambda repressor gene, cl857, carried on an Escherichia coli/mycobacteria shuttle plasmid. IS6120 is a novel 1.5 kb insertion sequence, which has 24-bp imperfect terminal inverted repeats and generates 9-bp duplications of the target DNA following insertion. IS6120 is present in at least three copies in M. smegmatis but was not found in other species, including Mycobacterium tuberculosis. Nucleotide sequence analysis revealed that IS6120 contains two open reading frames, one of which encodes a putative transposase with similarities to those found in IS256 from Staphylococcus aureus, IST2 from Thiobacillus ferrooxidans, and ISRm3 from Rhizobium meliloti. The fact that IS6120 does not recognize a consensus target sequence for insertion and has no homologous sequences in the other strains studied makes IS6120 useful for transposon mutagenesis in mycobacteria.     

DNA sequence at the integration sites of the insertion element IS1.

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.     

An IS15 insertion generates an eight-base-pair duplication of the target DNA

In plasmid pIP1088 the transposable module IS15 is inserted at nucleotide position 1,430 of the vector plasmid pBR322. We have sequenced the termini of the IS15 element, which consists of two perfect inverted repeat sequences, 14 bp long. The sequence is 5′-GGCACTGTTGCAAA… TTTGCAACAGTGCC-3′. The integration event results in the duplication of 8 bp of target DNA.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.     

Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.     

Nucleotide sequence of IS492, a novel insertion sequence causing variation in extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica.

The complete nucleotide sequence of insertion element IS492, which causes reversible inactivation of extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica, is presented. Insertion of IS492 results in the EPS- phenotype, and excision results in restoration of EPS+. DNA sequencing of the site of insertion in the eps locus showed that insertion of IS492 generates a 5-base-pair repeat and that its excision is precise. IS492 is 1,202 nucleotides in length and contains one large open reading frame encoding a protein of 318 amino acids, a candidate for transposition function. No similarity between IS492 and other transposable elements has been found. Unlike the situation with other insertion sequences, no direct or inverted repeats exist at the termini of IS492.     

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.     

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.     

Anatomy of a preferred target site for the bacterial insertion sequence IS903

Like many transposons the bacterial insertion sequence IS903 was thought to insert randomly. However, using both genetic and statistical approaches, we have derived a target site for IS903 that is used 84% of the time. Computational and genetic analyses of multiple IS903 insertion sites predicted a preferred target consisting of a 21 bp palindromic pattern centered on the 9 bp target duplication generated during transposition. Here we show that targeting can be dissected into four components: the 5 bp flanking sequences, the most important sequences required for site-specific insertion; the 7 bp palindromic core within the target duplication; the dinucleotide pair at the transposon-target junction; and the local DNA context. Finally, using a substrate with multiple target sites we show that a target site is more likely found by a local bind-and-slide model and not by extended DNA tracking.     

Linearization and transposition of circular molecules of insertion sequence IS3.

IS3 transposase has been shown to promote production of characteristic circular and linear IS3 molecules from the IS3-carrying plasmid; IS3 circles have the entire IS3 sequence with terminal inverted repeats, IRL and IRR, which are separated by a three base-pair sequence originally flanking either end in the parental plasmid, whereas linear IS3 molecules have three nucleotide overhangs at their 5' ends. Here, we showed that a plasmid carrying an IS3 derivative, which is flanked by different sequences at both ends, generated IS3 circles and linear IS3 molecules owing to the action of transposase. Cloning and sequencing analyses of the linear molecules showed that each had the same 5'-protruding three nucleotide overhanging sequences at both ends, suggesting that the linear molecules were not generated from the parental plasmid by the two double-strand breaks at both end regions of IS3. The plasmid carrying IS3 with a two base-pair mutation in the terminal dinucleotide, which would be required for transposase to cleave the 3' end of IS3, could still generate linear molecules as well as circles. Plasmids bearing an IS3 circle were cleaved by transposase and gave linear molecules with the same 5'-protruding three nucleotide overhanging sequences. These show that the linear molecules are generated from IS3 circles via a double-strand break at the three base-pair intervening sequence. Plasmids carrying an IS3 circle with the two base-pair end mutation still were cleaved by transposase, though with reduced efficiencies, suggesting that IS3 transposase has the ability to cleave not only the 3' end of IS3, but a site three nucleotides from the 5' end of IS3. IS3 circles also were shown to transpose to the target plasmids. The end mutation almost completely inhibited this transposition, showing that the terminal dinucleotides are important for the transfer of the 3' end of IS3 to the target as well as for the end cleavage.     

Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411.

The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined. The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence. IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase. Three polypeptides of 29,000, 27,000, and about 10,000 molecular weight, determined by IS3411, were identified in minicells. Since Tn3411 generates a 3-base-pair repeat upon integration, the nucleotide sequences of IS3411 were compared with those of IS3.