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     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

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.     

RecE-independent recombination of plasmids in Bacillus subtilis cells

The pUB110 and pE194 plasmid cointegrates have been isolated and examined in rec+ and recE4 strains of Bacillus subtilis. Cointegrates were shown to be formed by recombination at the specific site present on both parental plasmids as a short region of homology designated RSA. The RSA consists of 63 nucleotides in pE194 and 49 in pUB110; the length of its fully conserved core segment is 10 nucleotides. All cointegrates examined were formed by single crossover event taking place within the core segment, and as a result they have identical nucleotide sequences of recombination junctions. No conversion of mismatched base pairs to nucleotide sequences originally belonging to one of the parental plasmids was found. Though the action of RecE gene did not affect the frequency of cointegrate formation, it was reduced in rec149 host by one order of magnitude. Cointegrates retained their stability during transformation.     

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.     

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.     

Gene organization and target specificity of the prokaryotic mobile genetic element IS26

Summary The 820-bp mobile genetic element IS26 loses its ability to promote transpositional cointegration (1) by short deletions near the middle of the element causing shifts in both reading frames ORFI (left to right) and ORFII (right to left) and (2) by deletions causing substitutions of the C-terminus of ORFI but not affecting ORFII. The 702-bp ORFI is thus likely to code for the IS26 transposase. An 82-bp long sequence from the left end of IS26 contains a promoter-like structure in front of the start of ORFI at coordinate 64. In appropriately constructed plasmids, this sequence promotes the expression of the galK structural gene. The observation provides additional evidence for the functional relevance of ORFI. Neither the presence nor the absence of an intact IS26 element on the same plasmid affects measurably the degree of the galK gene expression by the IS26 promoter. Sequence comparison of 14 independent integration sites of IS26 and its relatives reveals no striking rules for target selection by the element, and the distrubtion of integration sites of IS26 on small multicopy plasmids is nearly random and independent of the local AT-content.     

Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22

Summary Recombinant plasmids having PstI fragments of P22 DNA inserted in the vector pBR322 can be transduced efficiently by Salmonella phage P22, irrespective of the cloned phage sequences. When the rec function of the donor cells and the corresponding recombination system erf of the infecting phage are simultaneously inactivated, only plasmids containing the P22 pac site can be transduced. By this selective, generalized transduction an EcoRV DNA fragment of the P22 related phage L has been identified that carries a base sequence recognized by phage P22 as a packaging signal. Experiments in which only one of the two recombination systems was inactivated, showed that the bacterial rec system obviously promotes cointegrate formation between plasmid and phage DNA much more efficiently than the phage-coded erf system, allowing the specialized plasmid transduction observed by Orbach and Jackson (1982).     

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     

Characterization of P1argF derivatives from Escherichia coli K12 transduction. I. IS1 elements flank the argF gene segment

Summary Specialized transducing derivatives of the temperate bacteriophage P1 (P1std) are selected by transduction into recipients with deletions in the corresponding genes (Stodolsky 1973). When Escherichia coli K12 strains are used as donors in such transduction experiments, P1argF derivatives can be selected. The argF gene is unique to these strains (Glansdorff et al. 1967). Under these experimental conditions P1argF are formed with frequencies 10,000 times greater than other P1std. The majority of the P1argF derivatives that have been analyzed are indistinguishable by cleavage analyses. One such derivative, P1argF5 has been characterized in detail. Heteroduplex analysis against P1, P7, and P1CmO identified an 11 kb insertion of DNA precisely at the naturally occurring IS1 locus of P1. Cleavage analysis with EcoRI, BamHI and PstI confirmed this finding. To further define the argF insertion, a P1Cm13argF derivative was constructed having the IS1 sequences of Cm13 and argF in opposite orientation. Intrastrand annealing of P1Cm13argF5 DNA established that the argF segment is flanked by directly repeated IS1 sequences. The IS1-argF-IS1 segment is desigmated Tn2901. The assignment of the map position of the argF gene within the 11 kb insert of P1argF5 is discussed. The evolutionary significance of this finding and a model for P1argF formation is also presented.     

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)₂ structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA ^– bacteria, replicated copies of the introduced (IS30)₂ structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)₂ are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)₂, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)₂ intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)₂ may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)₂. Evolutionary implications of these findings are discussed.     

A cloned DNA fragment from bacteriophage P1 enhances IS2 insertion

Summary A 1.75 kb DNA segment of the bacteriophage P1 genome is known to serve as a preferred target for IS2 insertions. The presence of this fragment in a plasmid expressing the galK gene dramatically increases the proportion of IS2 insertions among spontaneous galK ^- mutants. Subfragments from two different parts of the 1.75 kb segment independently stimulate IS2 insertion, while another subfragment does not. In the plasmids studied IS2 elements not only insert into the cloned P1 fragment but also into parts of the galK gene with similar probability and mostly in one orientation. Many insertion sites are unique but several specific sites within the preferred target are repeatedly used for IS2 integration. The experimental data are compatible with a proposed cooperative mechanism, according to which more than one attracting sequence on the same plasmid might significantly enhance the probability of a particular target region to attract IS2.     

Targeted conservative formation of cointegrates between two DNA molecules containing IS26 occurs via strand exchange at either IS end

We recently proposed a model for targeted, conservative cointegrate formation between DNA molecules each containing a copy of IS26, that involves Tnp26‐catalyzed strand exchange occurring at either the two left ends or the two right ends of the IS. Here, this model was validated by altering the bases at the outer left terminus, right terminus or both termini of one IS26. The correct bases at both ends were required in the untargeted replicative mode. However, when only one end was altered in one participating IS the frequency of targeted, conservative cointegrate formation was not reduced. The distribution of the altered bases in the cointegrates confirmed that the reaction occurred at the end where the terminal bases of both IS were correct, and cointegrates were not formed when both ends of the same IS were altered. The terminal bases of the active IS26 were also required to support deletion of the aphA1a translocatable unit (TU) from Tn4352B. The choices made by an incoming TU with a wild‐type IS26 when the target plasmid included one wild‐type IS26 and one with a frameshift in tnp26 demonstrated that Tnp26 exhibits a strong preference for cis action.     

Characterization of the new insertion sequence IS91 from an alpha-hemolysin plasmid of Escherichia coli

Summary IS91 is a 1.85 kb insertion sequence originally resident in the -hemolytic plasmid pSU233. The element was transposed sequentially from this plasmid to pA-CYC184, to R388, and to pBR322. Both cointegrates and simple insertions of the element were obtained. A detailed restriction enzyme map of the element is presented. This does not bear any relationship to the maps of previously described insertion sequences. Furthermore, hybridization between these sequences and IS91 could not be demonstrated.Deletion derivatives of IS91 were constructed which are unable to transpose. However, their transposition can be complemented in Trans by wild-type elements. One of these deletion derivatives has been genetically labeled with a kanamycin resistance marker from Tn5. When this new element was complemented for transposition, only about 2% of the transposition products were cointegrates. Thus, the behavior of IS91 is better explained by transposition models that allow direct transposition.Part of this work was carried out by E. Diaz-Aroca as a requirement for her degree in Sciences. The work is published (Esmeralda Diaz-Aroca, Tesina de Licenciatura, Universidad Autónoma de Madrid, 1983) and it contains the complete details of procedures and results of the cloning experiments and the restriction maps of the plasmids shown in this work. It is available from the authors upon request     

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.     

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.     

Structure and stability of cointegrating plasmids mediated by the IS1 elements in transposon delta Tn9'

In order to elucidate the structural features of the transposon Tn9', representative of the Tn9 family, which define the ability of the transposon to produce unstable cointegrates, we have obtained a derivative of this transposon carrying a deletion in its central region. The deletion in the obtained transposon delta Tn9' covers a DNA segment of about 50 bp in length, occupying the most distal position in relation to the cat gene, at its junction with the right copy of the IS1. The structure and stability of the IS1/delta Tn9'-mediated cointegrates between the plasmids pDK57.1 (pBR322::delta Tn9') and pRP3.1, a deletion derivative of RP1, have been studied. The three types of cointegrates were found. Those of the type I are predominantly formed, due to the left copy of the IS1 which in delta Tn9' occupies proximal position to the promoter of the cat gene. These cointegrates contain three copies of IS1 and are of high stability. The cointegrates of the type II contain two entire copies of delta Tn9' (i.e. four copies of IS1) as well as the structures of the type II, representing the cointegrate equivalent of inverse transposition and also containing four copies of IS1. Cointegrates of the type II and III dissociate efficiently in the rec+ cells but, in contrast to the cointegrates mediated by the original transposon Tn9', are unable to dissociate efficiently in the recA- cells. It was concluded that a DNA segment in the central region of Tn9' may be essential for the expression of the IS1-specific resolvase encoded by the right copy of IS1.     

Staphylococcal plasmid cointegrates are formed by host- and phage-mediated general rec systems that act on short regions of homology

Summary Cointegrates involving pairs of compatible staphylococcal plasmids can be isolated either by co-selection during transduction (Novick et al. 1981) or by selection for survival at the restrictive temperature of a thermosensitive, replication defective plasmid in the presence of a stable one. Cointegrates are formed by recombination at two specific sites, RS_A and RS_B. RS_B is present on each of six plasmids analyzed, namely pT181, pE194, pC194, pS194, pUB110, and pSN2, and RS_A is present on two of these, pT181 and pE194. In this communication, it is shown that the RS represent short regions of homology (RS_A is some 70 bp in length and RS_B is about 30) embedded in largely non-homologous contexts and that the crossovers take place within these homologous regions. The pT181 and pE194 RS_A sequences contain several mismatches which permit the localization of the crossover events to several different sites within the overall RS segment. The recombination system involved is therefore general (homology-specific) rather than site-specific (sequence-specific). Mismatches included within the crossover region are always corrected to the pT181 configuration. The cointegrates are therefore formed by a relatively efficient general rec system that recognizes short regions of homology and gives rise to Holliday junctions that probably involve very short heteroduplex overlaps. The sequence results are consistent with asymmetric single-strand invasion of a contralateral gap with nucleotide conversion by copying. It is noted that RS_B has substantial homology with the par sequence of plasmid pSC101, suggesting that it may be involved in plasmid partitioning.     

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2)

Summary On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperature phage C31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.     

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.