IS1-mediated mobility of the aerobactin system of pColV-K30 in Escherichia coli

Summary Genes determining the high affinity iron transport system mediated by the siderophore aerobactin are flanked in the enterobacterial plasmid pColV-K30 by inverted repeats of IS1 sequences, suggesting that the aerobactin genes are part of a transposon. To study this possibility, the entire region between the two IS1 sequences was cloned as an 18 kb HindIII-BamHI restriction fragment in pUC8 giving plasmid pMO1. A number of derivatives of pMO1, in which aerobactin genes were tagged with a kanamycin resistance gene, were prepared in order to assess the ability of both IS1s to promote the formation of cointegrates with pCJ105, an F derivative devoid of insertion sequences. Mating-out assays indicated that both flanking IS1s were active in cointegrate formation at detectable frequencies. In some cases, the cointegrates could be resolved, the final result being a transposition-like event for the entire aerobactin system.     

Escherichia coli alpha-haemolysin synthesis and export genes are flanked by a direct repetition of IS91-like elements

Summary A revised physical map of the -haemolysin plasmid pHly152 has been constructed. The known position of the hly genes in the restriction map of pHly152 allowed us to locate in it a direct repeat of IS elements flanking the hly genes of pHly152. These elements are IS92L, which is a derivative of the previously characterised element IS91 (1.85 kb) by insertion of a sequence of 1.2 kb, and IS92R, an element related to IS91 by a deletion of 0.7 kb and substitution of a 0.2 kb sequence of IS91 by a 1.2 kb heterologous sequence. IS92L is, in turn, flanked by an inverted repetition of sequences of 1.4 kb. These and previously published data strongly suggest that the hly genes spread at some time in evolution by means of the recombinational activity of IS91-like elements.     

Inter- and intramolecular transposition of Tn903.

Summary We have performed a detailed analysis of intra-and intermolecular endproducts of transposition of the compound transposon Tn903 and we show that, in our system, the transposition activity is almost entirely driven by one of the flanking insertion sequences, IS903L. The relatively inactive state of IS903R can be conferred on IS903L by changing the orientation of the internal Tn region. IS903L mediates the formation of the majority of adjacent deletions, insertion/inversions nd cointegrates, all of which are representative of replicative transposition; only a very low level of conservative transposition can be observed. Our results are discussed in relation to those showing that Tn903 uses predominantly the conservative pathway.     

IS<Emphasis Type="Italic">Ppy1</Emphasis>, a novel mobile element of the ancient <Emphasis Type="Italic">Psychrobacter maritimus</Emphasis> permafrost strain: Translocations in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 cells and formation of composite transposons

It was shown that IS element ISPpy1 isolated earlier in the permafrost strain Psychrobacter maritimus MR29-12 has a high level of functional activity in cells of the heterologous host Escherichia coli K-12. ISPpy1 can be translocated in E. coli cells by itself and mobilize adjacent genes and can also form composite transposons flanked by two copies of this element. Apart from translocations between different plasmids, the composite ISPpy1-containing transposon Tn5080a is capable of translocation from the plasmid into the E. coli chromosome with high frequency and from the chromosome into the plasmid. Among products of Tn5080a transposition into plasmid R388, simple insertions were predominantly formed together with cointegrates. Upon mobilization of adjacent genes with the use of one ISPpy1 copy, only cointegrates arise.     

Cointegration and resolution mediated by IS101 present in plasmid pSC101

Summary A certain class of cointegrate plasmids was found to occur between a pSC101 derivative and a second plasmid pBV320 in E. coli F^- cells. Cleavage analysis and DNA sequencing showed that the cointegrate plasmid contained direct repeats of an insertion sequence IS101 at the recombination junctions, indicating that formation of cointegrates was mediated by IS101, which is a natural constitutent of pSC101. These cointegrates were formed only in cells which contained the transposon gamma-delta, suggesting that the gamma-delta sequence, which provides transposase, is responsible for cointegration. Whenever the cointegrate plasmids were present in cells containing gamma-delta or its related transposon Tn3, the cointegrates were dissolved to give pBV320::IS101 due to recombination at duplicated IS101 sequences in the cointegrates, suggesting that both gamma-delta and Tn3, which provide a resolvase, are responsible for the resolution of the cointegrates. Comparison between the nucleotide sequence of IS101 and those of gamma-delta and Tn3 shows a high degree of homology in the regions that have been shown to be the binding sites of resolvases, as well as in the terminal inverted repeats. However, there is no homology between IS101 and the other element, gamma-delta or Tn3, in the internal resolution site, at which the resolution event may occur.Abbreviations Tc tetracycline - Cm chloramphenicol - Ap ampicillin - bp base pairs - kb kilobase pairs     

Cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives suggest molecular mechanisms for P1-mediated transduction of small plasmids

Summary We characterized cointegrates formed in an Escherichia coli rec ^– strain between bacteriophage P1 genomes and small plasmids related to pBR322. The partners were, on the one hand, either phage P1 DNA, which carries one copy of IS1, or phage P1-15 DNA, a derivative which lacks the IS1, and, on the other hand, plasmids containing either a split IS1 or no IS1. In the presence of IS1 sequences on both partners, cointegrates were usually formed by reciprocal recombination between IS1 sequences. Cointegrates between P1 and a plasmid carrying no IS1 sequence were formed by transpositional cointegration mediated by IS1 of P1. Cointegrates between P1-15 and small plasmids containing a split IS1 were formed by one of three ways: (a) acquisition of an IS1 by P1-15 followed by reciprocal recombination between IS1 sequences, (b) transpositional cointegration mediated by the split IS1 element, Tn2657, or (c) involvement of the invertible segment carried on P1-15 DNA. Most cointegrates segregated into the small plasmids and phage P1 derivatives. A comparison of the phenomena studied and of their frequencies allowed us to conclude that cointegrate formation is a molecular mechanism involved in the transduction of plasmids smaller than those packageable into P1 virions, although it does not seem to be the only process used.     

Cointegrate formation by IS50 requires multiple donor molecules

Summary The insertion sequence, IS50R, promotes cointegrate formation between a lambda::IS50R phage and the chromosome of Escherichia coli strain C. We show that formation of cointegrates mediated by IS50R between the non-replicating phage genome and the bacterial chromosome requires multiple donor molecules and depends on homologous recombination functions. We conclude that the two copies of IS50 present in the cointegrate originate in two different molecules. Thus, the existence of the cointegrate structure cannot be used as evidence for replication of IS50 sequences during IS50 transposition.     

Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points

Summary A pBR322-derived plasmid pKEN221 carrying a Serratia marcescens lpp gene overproduces the outer membrane lipoprotein in an Escherichia coli lpp ^–cell. However, when this strain was continuously cultured in a rich medium for about thirty generations, many Lpp^– mutants were accumulated. Out of six mutants analyzed, three were found to carry insertion mutation in the lpp gene in pKEN221. From restriction enzyme mapping and hybridization analysis of the mutant plasmid DNA, it was found that two mutants were caused by insertion sequence IS1 and one by IS5. Nucleotide sequence analysis of these mutant DNAs revealed that both IS1 and IS5 insertions occured in the A-T rich 5 untranslated region of the lpp gene. While the IS1 insertion resulted in a direct duplication of a nine-base-pair sequence in the original pKEN221 DNA at the junction with IS1, the IS5 insertion resulted in a direct duplication of a four-base-pair sequence. IS5 was found to contain inverted-repeat sequences of twelve nucleotides at its exact ends. This is the first example of the nucleotide sequence analysis of an IS5 insertion mutation. By Southern blot hybridization, the E. coli chromosomal DNA was found to contain about ten copies of IS5.     

Comparative analysis of 18 sex pheromone plasmids fromEnterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3-terminus of the surface exclusion gene,sep1, of sex pheromone plasmid pPD1 inEnterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.     

IS8- and Tn2353-mediated cointegration of the plasmids R15 and RP4::Tn1

Summary The plasmids R15 and RP4:: Tn1 form fused structures (85 Md and 92 Md cointegrates). The cointegrates do not resolve practically in recA ^– Escherichia coli cells and have a mean life-time of more than 50 generations in a recA ⁺ background.The 85 Md cointegrates were generated at a frequency of 4×10^–4 per R15 transconjugant during a mating between E. coli [R15; RP4:: Tn1] and E. coli [FColVBtrp:: Tn1755]. These plasmids carry two directly repeated copies of the mobile element IS8 at the junctions between R15 and RP4:: Tn1. The transposition of IS8 from RP4:: Tn1 to the R15 plasmid and the formation of hybrid molecules promoted by this process appear to be induced by the IS8 element of the Tn1755 structure during or after conjugal transfer of FColVBtrp:: Tn1755 into E. coli [R15; RP4:: Tn1] cells.The formation of the 92 Md cointegrates occurs at a frequency of 2×10^–5. The fused molecules of R15 and RP4:: Tn1 carry two direct copies of an 8.65 Md R15 fragment at the junctions between these replicons. The fragment has specific features of a new transposon. This element designated Tn2353 determines resistance to Hg, Sm and Su and contains two sites for each BamHI, BglII and SalI and three sites for both EcoRI and PstI. The physical map and some other characteristics of Tn2353 are presented.Abbreviations Ap ampicillin - EtBr ethidium bromide - Km kanamycin - Md megadaltons - Sm streptomycin - Su sulfanilamide - Tc tetracycline - [] brackets indicate plasmid-carrier state     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1

Summary A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H⁺-ATPase) of Escherichia coli causes growth inhibition of wild-type cells. Insertion of a transposable element in this plasmid released this inhibitory effect. In analyzing this inhibitory effect, we determined the insertion points at the nucleotidesequence level of transposable elements on 30 independent derivatives of pKY159. Insertions of IS1, IS5, and were found between the promoter and the gene for a possible component of 14,000 daltons of the H⁺-ATPase. Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 of in this region. Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence (AAAAACGT and AAACGTTG) was generated, while in the other two a 9 bp duplicate was found. In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K. In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence (AAACGT) was found. These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al. (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp. A 6 bp sequence (GTGATG) homologous to the end portion of IS1 was found at the hotspot, but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion. The direction of IS1 insertion at the hotspot was the same in 25 of 26 instances, suggesting that the direction of IS1 insertion is determined by the structure of the target and/or its nearby sequence.Abbreviations bp base pairs - 14 K protein a possible component of the H⁺-ATPase with molecular weight of 14,000 (see Kanazawa and Futai 1982 for details) - EDTA ethylenediaminetetraacetate     

Isolation, characterization and transposition of an (IS2)2 intermediate

We have isolated and characterized a dimer derivative of the extensively studiedEscherichia coli insertion sequence IS2. The dimer structure — called (IS2)₂ — consists of two IS2 elements arranged as a direct repeat, separated by 1 bp. The junction between the (IS2)₂ dimer and target sequences is located at various positions in independent isolates; however, one position was preferred. The transposition of (IS2)₂ into a target plasmid resulted in cointegrate-type structures. The transposition frequency of the (IS2)₂ dimer itself was significantly higher than that of the isogenic monomer IS2 insertion. The poor stability and high activity of (IS2)₂ indicates that this is an active transposition intermediate. The mode of transposition of (IS2)₂ is analogous to the joined dimer model described in the case of (IS21)₂ and (IS30)₂.     

Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6

Summary Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47° C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome.Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401.The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements.     

Genetic and molecular characterization of Tn21, a multiple resistance transposon from R100.1.

Tge transposon Tn21 has been transposed from R100.1 to plasmid pACYC184 and, from the resulting recombinants, to plasmid R388. The sites of insertion and the orientation of the element in several pACYC184::Tn21 recombinants have been examined. Restriction enzyme analysis of these recombinants has resulted in a detailed map of Tn21; this is compared with the published maps of the relevant part of R100.1. Heteroduplex analysis has shown short inverted repeat sequences at the ends of the element. With various in vitro-generated deletion mutants of Tn21, the internal gene necessary for transposition (tnpA) was localized within the terminal 4.3 kilobases of the right-hand end of the element. Genetic analysis of transposition of Tn21 suggests that the process proceeds via cointegrates. Since the end products of transposition are simple recombinants of the element and the recipient replicon, Tn21 must contain a gene that codes for a resolvase type of activity (tnpR gene).     

Insertion specificity and trans -activation of IS801

The transposable element IS801, isolated from plasmid pMMC7105 of Pseudomonas syringae pv. phaseolicola, transposes in Escherichia coli to plasmid targets, expressing a relatively relaxed target specificity. The target sequences are tetramers with homology with the left terminus (GAAC) of the transposing unit, the alternative targets being GAAC, GGAC, CAAG, and CGAC. In the areas flanking IS801 in 13 different locations, no similarities other than the target tetramer were observed. The transposase is physically and functionally separable from the transposing unit since transposition of constructs carrying marker genes occurs with the transposase expressed in trans. The IS801 transposase shows amino acid sequence homology to the transposases of the E. coli elements IS91 and IS1294. These tranposases contain conserved amino acid motifs found in the replicases of certain plasmids that replicate as rolling circles. Received: 18 March 1998 / Accepted: 15 August 1998     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

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 insertion sequence IS21 of R68.45 and the molecular basis for mobilization of the bacterial chromosome

The IncP-1 plasmid mutant R68.45, which is able to mobilize the chromosomes of many Gram-negative bacteria, was shown to carry a 2.10-kb insertion sequence designated IS21. This sequence transposed to the small multicopy plasmid pED815 at a high frequency (2 × 10^?3) and in two pED815::IS21 derivatives inactivated the tetracycline-resistance and replication functions, respectively. We propose that the chromosome-mobilizing ability of R68.45 is due to the formation of an R68.45-chromosome cointegrate during transposition of IS21. This would account for its high efficiency and the absence of a fixed chromosomal origin of transfer in Pseudomonas aeruginosa PAO, and its ability to function in a variety of bacterial hosts. R68.45 is formed from R68 by duplication of a 2.1-kb DNA segment including a distinctive cluster of seven restriction endonuclease sites. The two copies of the duplicated segment are probably contiguous and so might have arisen by a transition type of mechanism. IS21 is similar in length to the duplicated segment and includes the same set of seven cleavage sites located at similar distances from the two termini. However, the single copy of the duplicated segment in R68 transposed at an undetectably low frequency (<6 × 10^?8); either the duplicated segment and IS21, although overlapping, are not identical, or they are identical but the transposition system is nonfunctional in R68. Our further investigations of R68.45 and of several independently isolated chromosome-mobilizing derivatives of R68 demonstrated that these were indistinguishable from each other and that they did not include any P. aeruginosa PAO DNA. Furthermore, we searched without success for sequences corresponding to IS21, and to the Escherichia coli K-12 insertion sequences IS1, IS2, and IS3, on the chromosomes of P. aeruginosa PAO and PAT and P. putida PPN, and on several Pseudomonas plasmids. The contribution of homology to low-frequency chromosomal mobilization by these plasmids is discussed.     

Integration specificity of an artificial kanamycin transposon constructed by the in vitro insertion of an internal Tn5 fragment into IS2

Summary IS2 has been marked genetically by the in vitro insertion into its HindIII site of a 3.3 Kb HindIII fragment of Tn5 conferring resistance to kanamycin. The transposition of the IS2::Km, thus obtained, to has been found and insertion sites were characterised. Each of ten independent IS2::Km insertions were found at the same site at 61.2% of the map, always in the same orientation (orientation II relative to the xis gene). The integration sites of IS2::Km in five of the kanamycin-transducing phages were determined by DNA sequence analysis, and were found to be identical at the nucleotide level. Further transposition of IS2::Km from to the bacterial chromosome was demonstrated.