Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.     

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.     

Plasmid and transposon transfer to Thiobacillus ferrooxidans.

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.     

Localization of camphor degradative plasmids on the chromosome of Pseudomonas putida strains PaW]

Camphor degradative plasmids (CAM, pRK1) are preferentially situated on chromosomes of Pseudomonas putida strains PaW. After having been transferred into Cam+ strains, the TOL plasmid pWWO dissociates into the cryptic plasmid pWWO-8 and chromosome-borne transposon Tn4651. The opposite situation, i.e. reconstruction of the TOL plasmid pWWO from the cryptic plasmid pWWO-8 and chromosome-borne catabolic operons of the pWWO plasmid has been described. Cam- derivatives of the CAM plasmid were obtained in vivo which contain the TOL plasmid transposons Tn4651 or Tn4652 as obligatory structural elements. These plasmids as well as pWWO-8 determine conjugational mobilization of chromosome-located cam operons followed by their integration into the chromosome of recipient.     

Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

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.     

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.     

Transposition of a beta-lactamase locus from RP1 into Pseudomonas putida degradative plasmids.

The beta-lactamase gene from the RP1 plasmid transposes into at least two Pseudomonas putida degradative plasmids. Donor strains that carry RP1 (bla+ tet+ aphA+) and a degradative plasmid yield transconjugants that have only the bla+ marker of RP1. This occurs in up to 80% of all bla+ transconjugants. Segregation of the bla+ marker requires the presence of a degradative plasmid in the donor and is only observed in transconjugants that have received degradative markers. The bla+ tet aphA transconjugants show 100% linkage of bla+ to degradative markers in conjugation,transduction, and transformation crosses. A transduction cross of an (RP1), (SAL) donor shows that 8% of all SAL plasmids also carry the transposed bla+ marker. Tn401 is the name we assign to the bla+ transposon from RP1 observed in Pseudomonas. Its identity with the RP1 bla+ transposon observed in Escherichia coli is not known. In four cases, Tn401 has inserted into the camphor genes of the CAM-OCT plasmid.     

Characterization of a translocation unit encoding resistance to mercuric ions that occurs on a nonconjugative plasmid in Pseudomonas aeruginosa.

The nonconjugative plasmid, pVS1, has a molecular weight of 18.5 X 10(6) and confers resistance to sulfonamides and to mercuric ions. In Pseudomonas aeruginosa PAO, the transfer can be mobilized by a variety of conjugative plasmids, and the process does not require a functional recombination system in the donor. Hybrid plasmids that arise by the relocation of the mer gene onto the mobilizing plasmid can be isolated readily, and, as far as can be determined, these hybrids retain the genome of the conjugative plasmid in toto. The relocation of mer occurs by a Rec-independent process and leads to a constant increase (about 6 X 10(6) daltons) in the size of the recipient plasmid. This suggests that the mer gene in pVS1 is located on a translocation unit, designated Tn501, of a molecular weight of about 6 X 10(6). The translocation of Tn501 into RP1 is not usually associated with the loss of any known plasmid-mediated function, but transfer-defective or tetracycline-sensitive derivatives do occur at frequencies of about 4%, whereas carbenicillin-sensitive or kanamycin-sensitive variants arise with a frequency of about 0.2% each. It seems therefore that the integration of Tn501 can occur at any one of a minimum of five sites in RP1.     

Transposon mutagenesis in Caulobacter crescentus

Transposons Tn5 (Km) and Tn7 (Tp and Sm) were transferred to Caulobacter crescentus via P-type antibiotic resistance factors. Transposition was demonstrated by the isolation of chromosomal insertions of each transposon. With C. crescentus strains harboring RP4 aphA::Tn7, the introduction of a wild-type RP4 resulted in the loss of the resident plasmid. Simultaneous selection for Kmr and Smr yielded colonies with chromosomal insertions of Tn7. Examination of over 10,000 chromosomal insertions of Tn7 indicated no auxotrophic or motility mutants. Thus, Tn7 appears to have a high specificity of insertion in C. crescentus. The Mu-containing plasmid pJB4JI transferred Tn5 to C. crescentus, but the plasmid was not maintained. Control experiments showed that recovery of Mu-containing plasmids occurred at very low frequencies in C. crescentus and that the plasmids which were recovered had undergone extensive deletion of plasmid DNA. Presumably, some part of the Mu genome was not tolerated by C. crescentus. The instability of the Mu-containing plasmids makes them excellent vectors for the introduction of transposons, and we have used pJB4JI to isolated chromosomal insertions of Tn5. When several thousand of these insertion mutants were examined, we found auxotrophic and motility mutants at frequencies of 1 and 2%, respectively. These results indicate that Tn5 had a low specificity of insertion in C. crescentus and therefore would be a useful mutagen for obtaining a variety of mutant phenotypes.     

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.     

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.     

Construction of transposons carrying the transfer functions of RP4

The transfer genes and origin of transfer of the wide host range plasmid RP4 have been cloned into the transposons Tn1 and Tn5. The newly constructed transposons can be used to mutagenize bacterial plasmids or the chromosome in species such as Escherichia coli or Rhizobium. It is then possible to mobilize the plasmid or chromosome using the transfer functions provided in cis by the transposon. These constructs may aid chromosome mapping in many gram-negative species by allowing the wider use of the RP4 conjugal transfer system combined with the potential ability to select the site of insertion and thus the site of the origin of transfer.     

Regional preference of insertion of Tn501 and Tn802 into RP1 and its derivatives

Summary The sites of insertion of Tn501 into RP1 and into derivatives of this plasmid that either lack the Tn801 (TnA) element or contain it in a different location have been determined. Similarly, the sites of insertion of Tn802 into a derivative of RP1 that lacks the Tn801 element and into recombinants of this plasmid with Tn501 were determined. Hot spots for insertion were observed with both transposons; but it is clear that a particular DNA sequence is not sufficient to define a hot spot, since a particular region does contain many insertions when present in one plasmid but does not do so when part of another.     

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.     

Transfer of IncP Plasmids to Extremely Acidophilic Thiobacillus thiooxidans

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10^-5 to 10^-7 per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 × 10^-3 per recipient.     

Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7

Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27. The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc-. Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C. These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible. Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible. Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour). Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids. The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa). A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site. Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites. Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27. Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27.