Physical Analysis of Tn10- and Is10-Promoted Transpositions and Rearrangements

We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10(-4) per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.     

IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.     

Transposition behavior of IS15 and its progenitor IS15-Δ: Are cointegrates exclusive end products?

We report that the major product of IS15-promoted transposition is a cointegrate. When present in the multicopy plasmid pBR322, IS15 and its progenitor IS15-delta mediate the formation of cointegrates at frequencies of 3.5 X 10(-4) and 2.9 X 10(-5), respectively. We have studied the stability of the cointegrates generated by IS15 and IS15-delta. While these structures are resolved in a rec+ host, they were stable in a rec- host. These observations suggest that neither IS15 nor IS15-delta encode a resolvase and that cointegration is an end product of their transposition process. These properties of IS15-delta and IS15 can explain the transitions from IS15-delta to IS15 and from IS15 to IS15-delta observed in vivo.     

Nucleotide sequence and characterization of a new insertion element, IS240, from Bacillus thuringiensis israelensis

The nucleotide sequence of two repeated sequences (RS) in opposite orientations flanking the 125-kDa toxin gene of Bacillus thuringiensis israelensis (C. Bourgouin et al., J. Bacteriol. 170, 3575-3583, 1988) is reported in this paper. The analysis of these sequences indicates that these two RS display characteristic features of bacterial insertion sequences (IS) and are therefore referred to as IS240. IS240 B is 865 bp long and has two perfect terminal-inverted repeats of 16 bp; IS240 A is 99% identical to IS240 B. A long open reading frame encoding a polypeptide of 235 amino acids spans almost the entire sequence of both IS240 elements. Both the sequence of the inverted repeats and the putative transposases are homologous to IS26 of Proteus vulgaris, IS15-delta of Salmonella panama, IS431 of Staphylococcus aureus, and ISS1 of Streptococcus lactis.     

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.     

Frequency of IS1-mediated molecular events in different members of the family Enterobacteriaceae.

The numbers of chromosomal copies of the insertion sequence IS1 in strains of Salmonella typhimurium (0 to 8 copies), Shigella sonnei (56 copies), and Shigella flexneri (41 copies) isolated in Mexico City, Mexico, were similar to those reported for these genera isolated in other countries. Of the 11 Shigella strains studied, all carried several small plasmids; however, in only one of these strains did a small plasmid contain IS1, IS1 recombination, cointegrate formation mediated by IS1 or by the IS1-flanked transposon Tn9, and transposition of Tn9 occurred at a higher frequency in S. typhimurium than in either Escherichia coli or S. sonnei strains. The frequencies of IS1 recombination in S. typhimurium strains containing either zero or eight copies of IS1 were similar.     

Structural and Functional Characterization of IS679 and IS66-Family Elements

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB, and tnpC). A plasmid carrying a composite transposon (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679. A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn679 duplicated an 8-bp sequence at the target site. Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS66 family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS679, but many have a mutation(s) in an ORF(s). In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tnpA. In contrast, tnpC, separated from tnpB by a space of ca. 20 bp, is located in any one of three frames relative to tnpB. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.     

Genetic organization of transposon Tn10

Transposon Tn10 is 9300 bp in length, with 1400 bp inverted repeats at its ends. The inverted repeats are structurally intact IS-like sequences (Ross et al., 1979). Analysis of deletion mutants and structural variants of Tn10, reported below, shows that the two IS10 segments contain all of the Tn10-encoded genetic determinants, both sites and functions, that are required for transposition. Furthermore, the two repeats (IS10-Right and IS10-Left) are not functionally equivalent: IS10-Right is fully functional and is capable by itself of promoting normal levels of Tn10 transposition; IS10-Left functions only poorly by itself, promoting transposition at a very low level when IS10-Right is inactivated. Complementation analysis shows that IS10-Right encodes at least one function, required for Tn10 transposition, which can act in trans and which works at the ends of the element. Also, all of the sites specifically required for normal Tn10 transposition have been localized to the outermost 70 bp at each end of the element; there is no evidence that specific sites internal to the element play an essential role. Finally, Tn10 modulates its own transposition in such a way that transposition-defective point mutants, unlike deletion mutants, are not complemented by functions provided in trans; and wild-type Tn10, unlike deletion mutants, is not affected by functions provided in trans from a "high hopper" Tn10 element.     

The IS1 elements in Shigella boydii: horizontal transfer,vertical inactivation and target duplication

IS1(SB) and its two variants were identified as the major and minor IS1 elements in Shigella boydii. The nucleotide sequences of IS1(SB), IS1(O157:H7) from Escherichia coli O157:H7 and IS1F from E. coli K12 suggest that these IS1 elements had been horizontally transferred among S. boydii and E. coli O157:H7 and K12. The two IS1(SB) variants and IS1(O157:H7) have transposition activities 7- to 86-fold less than that of IS1(SB), whereas IS1F has little transposition activity. Analysis of the flanking sequences of IS1(SB) and its two variants in S. boydii revealed the nature of regional specificity of the target sites and the sequence dependence of 8 and 9 bp target duplications, for which a model is presented.     

IS186 Insertion at a Hot Spot in the lon Promoter as a Basis for Lon Protease Deficiency of Escherichia coli B: Identification of a Consensus Target Sequence for IS186 Transposition

The radiation sensitivity of Escherichia coli B was first described more than 50 years ago, and the genetic locus responsible for the trait was subsequently identified as lon (encoding Lon protease). We now show that both E. coli B and the first reported E. coli K-12 lon mutant, AB1899, carry IS186 insertions in opposite orientations at a single site in the lon promoter region and that this site represents a natural hot spot for transposition of the insertion sequence (IS) element. Our analysis of deposited sequence data for a number of other IS186 insertion sites permitted the deductions that (i) the consensus target site sequence for IS186 transposition is 5'-(G)(> or =4)(N)(3-6)(C)(> or =4)-3', (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N(3-6) sequence, and (iii) in a majority of instances, at least one end of the duplication is at the G-N (or N-C) junction. IS186-related sequences were absent in closely related bacterium Salmonella enterica serovar Typhimurium, indicating that this IS element is a recent acquisition in the evolutionary history of E. coli.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences.

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.     

Four different integrative recombination events involved in the mobilization of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in Escherichia coli

We identified and characterized four different recombination mechanisms involved in the cointegrative transfer of the Neisseria gonorrhoeae beta-lactamase plasmid pSJ5.2 by the gonococcal 41 kb tet(M) and the Gram negative self-transmissible plasmids N3 and R64 drd-33 using an Escherichia colirecA-background. Mobilization of pSJ5.2 by the tet(M) plasmid occurred by cointegration through a replicative transposition of two IS1 elements inserted upstream from the beta-lactamase gene of pSJ5.2 and creating a IS1::beta-lactamase hybrid promoter. Two types of recombinational events occurred within the 1.8 kb BamH1-HindIII fragment of pSJ5.2 with the N3 and R64 plasmids. A non-homologous recombination was found at coordinates 1817 and 2849 of pSJ5.2 with sequences from R64. A non-homologous recombination combined with an IS26-mediated one-ended transposition was found at coordinates 1817 and 3010 of pSJ5.2 with N3. In both recombinational events, a deletion of over 1 kb of pSJ5.2 occurred. The fourth recombination event was detected in the 1.0 kb BamH1-HindIII fragment of pSJ5.2 by homologous recombination between DNA from the truncated Tn3 resolvase gene of pSJ5.2 and the resolvase sequences from R64 and N3.     

Determination of the minimal length DNA homologous region required for plasmid integration into the Bacillus subtilis chromosome via homologous recombination]

With a view to determine a minimal sequence length of homology necessary for RecE-dependent homologous recombination in Bacillus subtilis cells, we developed a system, based on interaction between plasmid replicon and bacterial chromosome. Recombination frequencies were measured between ts plasmid pE194 derivatives carrying chromosomal beta-glucuronidase gene (bglS) fragments of various length, and a bacterial chromosome. The homologous recombination events resulted in bglS gene disruption. Approx. 70 bp of homology were found to be necessary for detectable homologous recombination. Homologous recombination was not detected when homology was equal 25 bp. These data indicate that homology requirement for recombination in B. subtilis differs from that in Escherichia coli.     

IS-elements and their role in genetic recombination]

The data concerning the biological functions and properties of short specific polynucleotide sequences (so called insertion sequences--IS) are reviewed. IS elements integrated in a genome can lead to strongly polar mutations in Escherichia coli, its bacteriophages and plasmids, while some IS (IS2) being integrated in inverted orientation turn on the gene activity. Several copies of the IS elements are present in the E. coli chromosome. A characteristic feature of IS is their ability to recA-independent migration along the bacterial chromosome. Possible mechanisms of IS integration are discussed. IS elements play the key role in the majority of recA-independent recombinational events: F-prime and partially Hfr-formation, plasmid recombination and dissociation, some cases of deletion formation etc. IS elements participate in recombination in the form of direct or inverted repeats. Direct repeats probably determine the processes of dissociation of the complete multicomponent R-factors and other plasmids. Inverted repeats (some of them are palindromes) are responsible for the migration of several drug-resistance determinants called transposons. Possible mechanisms of IS-dependent and probably IS-controlled recombination are discussed.     

Insertion Sequence-Related Genetic Variation in Resting Escherichia Coli K-12

Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.     

Two frameshift products involved in the transposition of bacterial insertion sequence IS629

IS629 is 1,310 bp in length with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, TTTTG (T4G) and AAAAT (A4T), are located near the 3'-end of orfA. With the lacZ gene as the reporter, both T4G and A4T motifs are determined to be a -1 frameshift signal. Two peptides representing the two transframe products designated OrfAB' and OrfAB, are identified by a liquid chromatography-tandem mass spectrometric approach. Results of transposition assays show that OrfAB' is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and Escherichia coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB', thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition.     

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.