R-plasmid-mediated chromosome mobilization in Bordetella pertussis

Antibiotic-resistant and auxotrophic mutants of Bordetella pertussis were isolated. These were used as recipients for the uptake from Escherichia coli of broad-host-range R plasmids R68.45, RP1, and RP1 and RP4 carrying transposons Tn501 and Tn7 respectively. B. pertussis transconjugants from these crosses were used as donors to mobilize StrR, NalR, thr+ and gly+ chromosomal markers to B. pertussis or to B. parapertussis recipient strains. The frequency of plasmid transfer varied and depended on the donor and recipient strains used. Differences in chromosome mobilization frequencies of individual markers were observed and appeared to depend on the presence or absence of transposons Tn501 and Tn7 on the plasmid. Linkage was detected between the gly+ and NalR markers.     

Transfer and properties of some natural and suicide replicons in Pasteurella multocida

We tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013--a narrow-host-range plasmid with the broad-host-range IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1011, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.     

Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria.

A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.     

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.     

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

Use of transposons for studying the genetic organization of Vibrio cholerae non 01]

The nonagglutinating vibrions having Tn-elements inserted into the chromosome were obtained as a result of conjugal transfer of vector plasmids carrying the different transposons (Tn9, Tn10, Tn601, Tn5-Mob) and of the consequent isolation of plasmid-free clones of Vibrio cholerae non OI. Identification of auxotrophic mutations induced by the transposons inserted into the bacterial genome made possible the construction of the primary chromosomal map of Vibrio cholerae non OI. The efficient donor strains of Vibrio cholerae non OI were constructed by introducing the transposon Tn5-Mob and the helper plasmid RP-4. The donors are capable of oriented conjugal transfer of chromosome.     

Specificity of transposition of Tn7 in Vibrio parahaemolyticus

Tn7 was found to transpose at a high frequency from the plasmid, RP4::Tn7, to the chromosome of Vibrio parahaemolyticus. Seven isolates carrying Tn7 insertions were derived from three wild-type strains isolated from geographically distinct areas, and HindIII and BstEII DNA digests of these strains were probed with a ColE1::Tn7 biotinylated probe. The results indicated that V. parahaemolyticus is similar to several other species which have been studied in having a highly preferred site of insertion of Tn7 in the chromosome.     

Transposon mutagenesis of the Pseudomonas aeruginosa PAO chromosome and the isolation of high frequency of recombination donors

Abstract The transposons Tn 5 and Tn 2521 have been loaded on to a temperature-sensitive, replication-defective mutant of the promiscuous IncP-1 plasmid R68 and used for transposon mutagenesis of the Pseudomonas aeruginosa PAO chromosome. Tn 5 was found to be particularly useful for the generation of new auxotrophic mutations and Tn 2521 for the generation of a range of high frequency of recombination donors by plasmid integration into the chromosome. The new origins and directions of chromosome transfer mediated by this conditional plasmid replication system will greatly facilitate improved genetic mapping of PAO.     

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.     

High-frequency chromosome transfer in Rhodopseudomonas sphaeroides promoted by broad-host-range plasmid RP1 carrying mercury transposon Tn501.

Insertion of the mercury resistance transposon Tn501 into broad-host-range plasmid RP1 greatly enhanced the ability of this plasmid to promote chromosome transfer in the photosynthetic bacterium Rhodopseudomonas sphaeroides. Compared with the wild-type RP1, which produced less than 10(-8) recombinants per donor cell, RP1::Tn501 produced between 10(-3) and 10(-7) recombinants per donor cell depending upon the marker selected. Plasmid RP1::Tn501 promoted polarized transfer of the chromosome from one or perhaps two origins on the chromosome, giving rise to two linkage groups. All of the biosynthetic and antibiotic resistance genes that have been mapped, including those involved in photosynthesis, occur on one or another of these linkage groups.     

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.     

A directional, high-frequency chromosomal mobilization system for genetic mapping of Rhizobium meliloti.

A system for mapping of the Rhizobium meliloti chromosome that utilizes transposon Tn5-Mob, which carries the mobilization site of IncP plasmid RP4 (R. Simon, Mol. Gen. Genet. 196:413-420, 1984), was developed. Insertions of Tn5-Mob that were located at particular sites on the R. meliloti chromosome were isolated and served as origins of high-frequency chromosomal transfer when IncP tra functions were provided in trans. This approach is, in principle, applicable to any gram-negative bacterium in which Tn5 can transpose and into which IncP plasmids can conjugate.     

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.     

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.     

Transposition of Tn7 in Pseudomonas aeruginosa and Isolation of alk::Tn7 Mutations

Conjugal crosses with Pseudomonas aeruginosa donors carrying the CAM-OCT and RP4::Tn7 plasmids result in transfer of the Tn7 trimethoprim resistance (Tp(r)) determinant independently of RP4 markers. All Tp(r) exconjugants which lack RP4 markers have CAM-OCT genes and therefore must have received CAM-OCT::Tn7 plasmids formed by transposition of Tn7 from RP4::Tn7 to CAM-OCT. Most crosses yield exconjugants carrying mutant CAM-OCT plasmids which no longer determine either camphor or alkane utilization and thus appear to carry Tn7 inserts in the cam or alk loci, respectively. Transduction and reversion experiments indicated that at least 13 alkane-negative, camphor-positive, Tp(r) CAM-OCT::Tn7 plasmids carry an alk::Tn7 mutation. Determination of linkage between the alk mutation and the Tp(r) determinant of Tn7 on these plasmids is complicated by the presence of multiple copies of the Tn7 element in the genome. Generalized transduction will remove Tn7 from a CAM-OCT alk::Tn7 plasmid to yield alk(+) cells which carry no Tp(r) determinant on the CAM-OCT plasmid (as shown by transfer of the plasmid to a second strain). But the transduction to alk(+) does not remove all Tp(r) determinants from the genome of the recipient cell because the alkane-positive transductants remain trimethoprim resistant. Thus, it appears that copies of Tn7 can accumulate in the genome of P. aeruginosa (CAM-OCT alk::Tn7) strains without leaving their original site. This result is consistent with transposition models that involve replication of the transposable element without excision from the original site.     

Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens.

We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome. First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45. We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5. By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations. We also constructed isolates of A. tumefaciens containing an insertion of Tn5 in the chromosome. We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients. Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested. Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies. Donors were shown to transfer chromosomal markers in a polarized fashion. Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent. The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A. tumefaciens chromosomal functions.     

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.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

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.     

Transposition of Tn904 encoding streptomycin resistance into the octopine Ti plasmid of Agrobacterium tumefaciens.

A transfer-deficient derivative of plasmid RP1-pMG1 was isolated after insertion of Mu cts62. The Tra- R plasmid was used to donate Tn904, encoding streptomycin resistance, to Ti plasmid pAL102 harbored by Agrobacterium tumefaciens Ach5. Under conditions promoting high Ti transfer frequencies, 155 strains were isolated in which the streptomycin marker coupled with Ti plasmid in further transfer experiments. These isolates represent stable insertions of Tn904 into the Ti plasmid. In addition, 19 strains were isolated in which the insertion of Tn904 was apparently unstable. The frequency of stable Tn904 transpositions was estimated to be 3 x 10(4-) per transferred Ti plasmid. Evidence was obtained that Tn904 readily may transpose from the Ti plasmid into the bacterial chromosome. The strains carrying Ti plasmids with stable insertions were characterized with respect to virulence, octopine degradation, octopine synthesis in induced tumors, and Ti plasmid transfer. Thirteen of the strains were found to be affected in tumor-inducing ability.