Transposon-mediated integration of the RP1 plasmid into chromosomes of methylotrophic bacteria

The homology region between the DNA of plasmid RP1ts::Tn601 and chromosome of the thermotolerant methylotrophic bacterium Methylobacterium sp. SKF240 has been constructed by transposon Tn601 translocation into the chromosome. The clones of Methylobacterium sp. SKF240 having integrated the plasmid RP1 into the chromosome have been obtained by conjugation on the basis of above mentioned genetic technique. The integration of plasmid RP1 into the chromosomal DNA of the methylotroph has been confirmed by the genetic and electrophoretic methods. Clones harbouring the integrated plasmid are able to transfer the chromosomal genes for methionine and isoleucine-valine synthesis to the recipient cells of P. aeruginosa PAO ML4262 by conjugation.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.     

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.     

Relationships among some R plasmids found in Haemophilus influenzae.

Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.     

Controlled Integration into the Lactococcus Chromosome of the pCI829-Encoded Abortive Infection Gene from Lactococcus lactis subsp. lactis UC811

The phage insensitivity gene of lactococcal plasmid pCI829 which encodes an abortive infection defense mechanism (Abi) was inserted into the Lactococcus lactis subsp. lactis CH919 chromosome by utilizing the integration plasmid pCI194, which contains 4.2 kb of homology with the conjugative transposon Tn919. Chloramphenicol-resistant transformants expressed phage insensitivity to the prolate-headed phage c2 and the small isometric-headed phage 712, and hybridization analysis indicated that transformants contained pCI194 integrated in single copy. The level of phage insensitivity expressed by the transformants was reduced from that observed when the abi gene was located on a replicating plasmid, as determined by plaque assay and burst size analysis. Amplification of the integrated structure after growth in increased concentrations of chloramphenicol resulted in an increase in the expression of phage insensitivity. Hybridization analysis revealed that while pCI194 was stably maintained in an integrated state over 100 generations in the absence of selective pressure, the ability to express phage insensitivity was lost. Hybridization analysis also revealed that DNA flanking the abi gene contains homology to the CH919 chromosome.     

Transfer of IncP plasmids into Stigmatella aurantiaca leading to insertional mutants affected in spore development

Summary Derivatives of the broad-host-range plasmid RP4, containing the wild-type or modified transposon Tn5 were transferred by conjugation to various Stigmatella aurantiaca isolates. The transposons and in some cases fragments of the plasmid as well were integrated into the chromosome. Thus, insertional mutants have been obtained affected in spore formation in liquid culture.     

Translocation of ampicillin transposon Tn1 in Escherichia coli cells during sexual reproduction

The efficiency of Tn1 transposition has been shown to increase considerably in course of bacterial conjugation. Usually, the frequency of Tn1 transposition from plasmid pSA2001, a derivative of RP4, into the chromosome never exceeded 0.1% per cell. Percentage of His+ transconjugants, marked by transposon Tn1 during conjugation between Hfr donor, carrying plasmid pSA2001, and auxotrophic recipient, was about 30%. Transposon Tn1 transfer into the recipient cells does not depend on the recA+ gene function in donor cells or on conjugative transfer of plasmid pSA2001. The transfer requires the recA+ gene function in recipients as well as the Hfr function in donor cells. Southern's blot-hybridization revealed the insertion of transposon Tn1 into the different sites of the chromosome of His+ transconjugants. The transposon inserted during conjugation retains the ability to potential further translocation into new sites on the chromosomal DNA.     

A mini-Mu transposon-based method for multiple DNA fragment integration into bacterial genomes

A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette containing selection marker and conditional replicative origin (R6Kγori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol, tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which could be applicable to a variety of microorganisms.     

Transposition of Tn1545-delta 3 in the pathogenic Neisseriae: a genetic tool for mutagenesis.

The ability to study the virulence of pathogenic Neisseria spp. has been greatly limited by the absence of genetic tools which allow the construction of defined mutants. We have engineered a transposon system which allows random mutagenesis of the Neisseria genome at relatively high frequency. Tn1545-delta 3 is a 3.4-kb derivative of the gram-positive transposon Tn1545 encoding resistance to kanamycin. Tn1545-delta 3 was subcloned into an erythromycin-resistant derivative of the mobilizable shuttle vector pLES2 to yield the plasmid pMGC20. This latter plasmid was introduced by conjugation from Escherichia coli S17-1 into Neisseria meningitidis 8013N and Neisseria gonorrhoeae 15063G. Kanamycin-resistant 8013N and 15063G transconjugants appeared at frequencies of 10(-5) and 10(-6), respectively. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that, in Neisseria spp., the transposon excised spontaneously from pMGC20 and integrated into chromosomal DNA. Our studies revealed that (i) transposition of Tn1545-delta 3 was in numerous, apparently distinct sites, (ii) in most cases, for each transconjugant a single copy of Tn1545-delta 3 was integrated into the chromosome, and (iii) several passages on selective media did not induce secondary transposition. The kanamycin resistance marker expressed by the transconjugants was subsequently transformed into a parental background without change in the chromosomal location of the transposon. To assess the role of the general recombination system in the transposition of Tn1545-delta 3, the recA gene of N. meningitidis has been cloned and a rec derivative of 8013N has been engineered. Similar results were obtained when this latter strain was used as recipient, suggesting that recA function were not required for Tn1545-delta 3 transposition in N. meningitidis. Transposition with Tn1545-delta 3 may be an important technique for mutagenesis of the pathogenic neisseriae.     

Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.     

Transposon-mediated insertion of R factor into bacterial chromosome

Summary Insertion of transposon Tn1 into the E. coli JC411 chromosome results in a sharp increase of plasmid RP4 integration frequency. This effect is absent in JC1553 recA cells. The RP4 integration with the chromosome is probably accomplished via recA-dependent recombination between transposon Tn1 inserted into the chromosome and the same transposon in the RP4 plasmid.     

Integration of plasmid RP1 with the chromosome of Escherichia coli K-12 recA. 2 classes of Hfr strains

Integration of broad host range RP1 plasmid into the chromosome of Escherichia coli K-12 recA- cells has been studied. Using temperature-sensitive for replication plasmids pVD1 and pVD3, the derivatives of RP1, it has been shown that integration of RP1 into the bacterial chromosome results in formation of two classes of Hfr strains. Properties of these Hfrs have been examined. From the data obtained, it has been concluded that the plasmid integration and formation of one of the Hfrs classes appear to be mediated by transposon Tn1 residing on RP1. The other class of Hfr strains is formed due to a stable integration of RP1. In the course of analysis of R+ transconjugants arising at low frequency in crosses between stable Hfrs and E. coli rec+ recipients, it has been found that the significant part of them contain plasmid-chromosome hybrids (R-prim plasmids). On the basis of the latter results, a new simple method for R' plasmids selection has been proposed. Using restriction endonuclease analysis, the structure of plasmids that were excised from chromosomes of the stable Hfr strains and were comparable in their size to RP1, has been investigated. Probable mechanisms of the stable Hfr strains formation are discussed.     

Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells

The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

Integration and intramolecular transposition of the TnBP3 Bordetella pertussis transposon in the Escherichia coli K-12 cells -- mutant for the phosphoenolpyruvate-dependent phosphotransferase system Hpr protein

Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussis into the chromosome of Escherichia coli and transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsH devoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metY gene sequence, which resulted in restoration of the Met+ phenotype. The integration and transposition events were only observed in the E. coli cells carrying the ptsH+ allele. The ptsH mutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsH mutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coli K12. The results obtained indicate that the ptsH mutants of E. coli can serve as the optimal host for cloning of fragments carrying repeated sequences of B. pertussis, which may apply to the repeated sequences of other microorganisms.     

A chromosomally located transposon in Pseudomonas aeruginosa

A new transposon, Tn2521, coding for carbenicillin, streptomycin, spectinomycin, and sulfanilamide resistance, has been identified in Pseudomonas aeruginosa. The transposon occurs naturally in the chromosome of clinical strains of P. aeruginosa isolated in geographically separated hospitals. This has been demonstrated by its transductional linkage to the pur-136 marker and also by Southern hybridization. Tn2521 is 6.8 kilobases, can transpose from the chromosome to both IncP-1 and IncP-2 plasmid genomes, and has a pattern of restriction endonuclease sites unlike that of any previously described transposon. The carbenicillin resistance carried by Tn2521 is due to the PSE-4 type of beta-lactamase.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

Gene transfer in magnetic bacteria: transposon mutagenesis and cloning of genomic DNA fragments required for magnetosome synthesis.

Broad-host-range IncP and IncQ plasmids have been transferred to the aerobic magnetic bacterium Aquaspirillum sp. strain AMB-1. Conjugal matings with Escherichia coli S17-1 allowed high-frequency transfer of the RK2 derivative pRK415 (4.5 x 10(-3) transconjugant per recipient cell) and the RSF1010 derivative pKT230 (3.0 x 10(-3) transconjugant per recipient). These plasmids successfully formed autonomous replicons in transconjugants and could be isolated and transformed back into E. coli, illustrating their potential as shuttle vectors. A mobilizable plasmid containing transposon Tn5 was transferred to Aquaspirillum sp. strain AMB-1 and also to the obligately microaerophilic magnetic bacterium Aquaspirillum magnetotacticum MS-1. Five nonmagnetic kanamycin-resistant mutants of Aquaspirillum sp. strain AMB-1 in which Tn5 was shown to be integrated into the chromosome were obtained. Different genomic fragments containing the mutagenized regions were cloned into E. coli. Two genomic fragments were restriction mapped, and the site of Tn5 insertion was determined. They were shown to be identical, although derived from independent transposon insertions. One of these clones was found to hybridize strongly to regions of the A. magnetotacticum MS-1 chromosome. This is the first report of gene transfer in a magnetic bacterium.     

Use of the plasmid pRK 2013 as a vehicle for transposition inAzotobacter vinelandii

Plasmid stability inAzotobacter vinelandii has been determined and a way to introduce transposon into these cells using the plasmid pRK 2013 has been devised. Transposition of both Tn3 and Tn10 has been attained.     

Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene.

The Escherichia coli heat-stable enterotoxin STII gene in P307 is flanked by inverted repeat sequences, suggesting that the STII gene is part of a transposon. To study the transposability, a DNA fragment containing the putative STII transposon has been cloned. Results of transposition assays indicated that the STII gene can transpose from one plasmid to another. The size of the transposon has been determined to be approximately 9 kilobases. The structure and the location of the STII gene in clinical isolates of Escherichia coli have been investigated by restriction enzyme analyses. The structural genes of STII from different clinical isolates appear to be uniform in size, but the flanking sequences are heterogeneous. This result suggests that the STII genes in different isolates are not on the same transposon as observed in P307.