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.     

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.     

Mobilization and transfer of Azospirillum lipoferum plasmid by the Tn5-Mob transposon into a plasmid-free Agrobacterium tumefaciens strain

Azospirillum lipoferum 4B harbors five cryptic plasmids. Several suicide plasmids were used to transfer Tn5-Mob to A. lipoferum 4B. Tn5-Mob insertion mutations of this strain could be obtained at frequencies of 10(-8)-10(-7) per recipient cell. One hundred Tn5-Mob A. lipoferum 4B mutants were used in bacterial matings with a plasmid-free Agrobacterium tumefaciens recipient strain. This is the first report of mobilization, transfer, and replication of an Azospirillum plasmid in Agrobacterium tumefaciens. One transconjugant was found which had lost an indigenous plasmid.     

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.     

A chromosomal linkage map of Azotobacter vinelandii

Summary A chromosomal map of Azotobacter vinelandii strain UW was constructed. The map was based on measures of cotransfer of various markers mediated by plasmids R68.45 and pJB3JI, on results obtained from conjugal experiments with R-primes, and on recombinants obtained by chromosomal transfer mediated by RP4/Tn5-Mob.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

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.     

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.     

Chromosome mobilization of Legionella pneumophila with RK2::Mu and Tn5-Mob.

RK2::Mu plasmids and transposon Tn5-Mob were used to mobilize the Legionella pneumophila chromosome. Plate matings between L. pneumophila donors that contained RK2::Mu plasmids and auxotrophic recipients yielded recombinants at frequencies ranging from 10(-6) to 10(-7) per recipient for the markers tested. The presence of a Mu insertion in the chromosome of donors that harbored RK2::Mu plasmids increased the frequency of chromosome transfer of certain selected markers as compared with strains that contained RK2::Mu alone. Cotransfer experiments with Mu-containing donors and a thymidine and tryptophan auxotroph failed to reveal any linkage between the thy and trp loci in L. pneumophila. A strain that contained a chromosomal Tn5-Mob insertion and helper plasmid pRK24.4 transferred chromosomal markers at frequencies of 10(-7) per recipient. These findings suggest that RK2::Mu plasmids and Tn5-Mob may be useful for genetic mapping experiments with L. pneumophila.     

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.     

Mini-Mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mulac derivative carrying the site for conjugal transfer of plasmid RP4 (oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid (oriRiHRI) was constructed to enable one-step lac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mulac derivatives with oriT and oriRiHRI in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS571 and Agrobacterium tumefaciens C58 was examined.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

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.     

The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Ti-plasmids

The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.     

Conjugative transfer of plasmid RP1 to soil isolates of Pseudomonas fluorescens is facilitated by certain large RP1 deletions

Abstract The broad-host-range IncP plasmid RP1 could not be transferred by conjugation from Escherichia coli to Pseudomonas fluorescens strain CHA0. However, this conjugative transfer was possible with RP1 derivatives which had large deletions extending from the primase gene towards the Tra-2 region, thus lacking the kanamycin resistance gene and IS 21 . Such RP1 deletion derivatives permitted IncP cosmid mobilization to P. fluorescens CHA0 and could be used as vectors for transposon mutagenesis with a newly constructed Tn 5 derivative (carrying kanamycin and mercury resistance determinants) in strain CHA0 and another P. fluorescens soil isolate, strain S9.     

Conjugal retrotransfer of chromosomal markers inAzotobacter vinelandii

The IncP plasmids R68.45 and pJB3JI mediate retrotransfer (i.e., transfer of chromosomal markers from the recipient bacterium to the original donor) in homologous matings withAzotobacter vinelandii. Retrotransfer is not an early event inAzotobacter. On the contrary, it begins after genetic transfer in the usual direction, i.e., from the donor bacterium to the recipient, has been taking place for some time. Transfer of chromosomal markers mediated by the RP4/Tn5-Mob system does not undergo retrotransfer. The simplest hypothesis compatible with our results is that IncP plasmids that confer a high chromosome-mobilizing ability promote retrotransfer when transferred to the recipient bacteria.     

Involvement of Azopirillum brasilense plasmid DNA in the productio of indole acetic acid

Indole acetic acid (IAA) production in Azospirillum brasilense strain Sp245 is controlled by a 85 MDa plasmid naturally present in this bacterium. In the presence of L-tryptophan, anthranilic acid production and almost no IAA production occurs in a derivative strain harbouring a Tn5-Mob insertion in the 85 MDa plasmid. Agrobacterium tumefaciens strain GM19023, upon transfer of Tn5-Mob labelled 85 MDa plasmid of A. brasilense Sp245, gains the ability to produce anthranilic acid.     

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.     

Genetic mapping with Tn5-derived auxotrophs of Caulobacter crescentus.

Chromosomal insertions of Tn5 in Caulobacter crescentus displayed complete stability upon transduction and proved useful in strain building on complex media. RP4-primes constructed in vitro containing C. crescentus genomic sequences in the HindIII site of the kanamycin resistance gene failed to show enhanced or directed chromosome mobilization abilities. One of these kanamycin-sensitive RP4 derivatives, pVS1, was used as a mobilization vector in conjugation experiments on complex media where chromosomal Tn5 transfer to the recipient was selected. pVS1-mediated transfer of Tn5-induced auxotrophic mutations occurred at frequencies of 10(-6) to 10(-8) per donor cell. During conjugation with Tn5-encoded kanamycin resistance as the selected marker, Tn5 remained in its donor-associated locus in 85 to 100% of the transconjugants. A collection of eight temperature-sensitive donor strains bearing Tn5 insertion mutations from various regions of the C. crescentus genetic map were used to provide a rapid means for the determination of the map location of a new mutation. Use of the techniques described in this paper allowed an expansion of the C. crescentus genetic map to include the relative locations of 32 genes.     

Incompatibility between a Rhizobium Sym plasmid and a Ri plasmid of Agrobacterium

The symbiotic plasmid of Rhizobium trifolii G1008 was mobilized to other Rhizobium strains and to Agrobacterium using Tn5-Mob, a transposon that confers on a host replicon the ability to be mobilized in trans by RP4. Incompatibility was observed between pSymG1008 and the hairy-root-inducing plasmid pRi1855. Agarose gel electrophoresis revealed that pRi1855 was eliminated as an autonomous element in the presence of pSymG1008 and its absence was correlated with loss of the ability to induce hairy root disease. This indicates a close ancestral relationship between a Rhizobium symbiotic plasmid and a plant pathogenic plasmid of Agrobacterium. pSymG1008 and pRi1855 can be assigned to the IncRh-3 incompatibility group. Furthermore, pSymG1008 was mobilized at low frequency to R. phaseoli 51E and the transconjugants isolated had lost the indigenous Sym plasmid and the ability to nodulate beans.