The conjugational transfer of plasmids to Legionella strains

In experiments on conjugation the transfer of a number of R-plasmids having a wide range of hosts, such as plasmids RP1, R68.45, RP4, N3, RK2, S-a, those having a narrow range of hosts, such as plasmid R64, to strains of different Legionella species was shown. The frequency of transfer varied from 3.1 X 10(-3) to 9.4 X 10(-7). The fact that the conjugation transfer was confirmed by the reverse transfer of plasmids from Legionella transconjugates to Escherichia coli strain K12, as well as by the detection of the DNA of the transferred plasmid by means of electrophoresis in agar gel. Plasmid RP1 showed different behavior in transconjugates of various Legionella species after several passages in a medium free of antibiotics. In the Legionella strain under study the unstable preservation of plasmid R64 was observed.     

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.     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

Plasmid R68.45 and S-a transduction by the temperate bacteriophage 59 of Erwinia carotovora 268

Temperature bacteriophage 59 of Erwinia carotovera 268 had transduced extrachromosomal DNA: plasmids of R68.45 and S-a. Before plasmid transduction experiments the suitable donor strains of indicator culture Erwinia horticola 450 harbouring R68.45 and S-a were created. The frequency of plasmid R68.45 transfer from Pseudomonas putida to E. horticola 450-8 by conjugation was equal to 5 x 10(-8) per a donor cell and in the case of S-a--from E. coli C600 for the same recipient cells--was 2 x 10(-6). Bacteriophage 59 has transduced only separate markers of plasmid R68.45, since plasmid S-a is probably transduced by the phage as an intact unit.     

R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa

Summary The R factor R68 readily promotes chromosome transfer in Pseudomonas aeruginosa strain PAT, but shows little such sex factor activity in strain PAO. A variant of this plasmid, R68.45, has been isolated which produces recombinants in PAO plate matings at frequencies of 10^-3–10^-5 per donor cell for markers in the 0–60 min region of the chromosome. Little or no chromosome transfer was shown in liquid media. The kinetics of chromosome transfer were studied by interrupting matings on solid media with nalidixic acid. Five chromosomal markers, mapping in widely spaced regions of the chromosome all entered 3–5 min after initiation of mating. These results, combined with linkage studies, indicate that R68.45, unlike the Pseudomonas sex factors FP2 and FP39, promotes chromosome transfer from a range of origin sites and can thus be used for mapping the region of the P. aeruginosa chromosome later than 40 min.R68.45 and other similar variants were isolated from rare chromosomal recombinants appearing in crosses between PAO(R68) donors and PAO recipients in which selection for argB ⁺ was made. Selection for other chromosomal markers did not result in such variants suggesting that plasmids of the R68.45 type arise by recombination of genetic material between the R68 plasmid and certain regions of the bacterial chromosome.     

Transfer and maintenance of small, mobilizable plasmids with ColE1 replication origins in Legionella pneumophila

With the mutagenesis of specific, virulence-associated genes of Legionella pneumophila as the eventual goal, methods for gene transfer to these bacteria were developed. Following the observations of others that conjugative, broad-host-range plasmids could be transferred from Escherichia coli to L. pneumophila at low frequency, we constructed a small mobilizable vector, pTLP1, which carries oriV from pBR322, oriT from pRK2, Kmr from Tn5, and an L. pneumophila-derived fragment to permit chromosomal integration. In triparental matings including an E. coli with a conjugative (Tra+) helper plasmid, kanamycin-resistance was transferred from E. coli to L. pneumophila. Southern hybridization of L. pneumophila transconjugants showed that pTLP1 was replicated autonomously. Additional matings of plasmids having deletions or substitutions of pTLP1 sequences confirmed that replication in L. pneumophila requires oriV only. pTLP1 was maintained in L. pneumophila with passage on medium containing kanamycin but was rapidly lost after passage on nonselective medium. This plasmid instability in L. pneumophila is most likely due to rapid generation of plasmid-free segregants because of plasmid multimerization and low plasmid copy number. We conclude that mobilizable pBR322-derived plasmids can be used as shuttle vectors to transfer cloned genes to L. pneumophila, a feature that can be exploited for the purposes of mutagenesis or genetic complementation.     

R-plasmid-mediated chromosomal gene transfer in Agrobacterium tumefaciens.

Although several techniques are available for transferring the Ti plasmids from one strain of agrobacterium tumefaciens to another, there are no reproducible methods for analysis of chromosomal markers in this phytopathogen. The R plasmid, R68.45, is known to show chromosomal mobilizing ability in several bacterial genera including the closely related Rhizobia. R68.45 was transferred into the prototrophic A. tumefaciens strain 15955. Ten kanamycin-resistant transconjugant clones were tested for chromosomal mobilizing ability by mating with strain SA10, rifampin- and streptomycin-resistant histidine auxotroph of strain 15955. Of the 10 donor clones, 2 showed high chromosomal mobilizing ability. Between 1,000 and 2,000 His+ colony-forming units per ml were obtained, a value 10 to 20 times greater than can be accounted for by spontaneous reversion. Sequential recloning and matings resulted in the isolation of relatively stable donor cultures. Chromosome gene transfer is dependent upon the presence in the donor of R68.45. Donors lacking an R plasmid or harboring the closely related plasmid RP4 failed to yield His+ transconjugants. With strain SA11, a methionine auxotroph of strain SA10, coinheritance of histidine and methionine independence could be demonstrated. Approximately half of the transconjugants also inherited R68.45. These results indicate that A. tumefaciens 15955 is capable of undergoing host chromosomal genetic exchange.     

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.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.     

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.     

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.     

Transfer of Drug Resistance Factors to the Dimorphic Bacterium CAULOBACTER CRESCENTUS

The P-type drug resistance factors RP4, RK2, R702, R68.45, and the N-type drug resistance factor R46 are transferred to Caulobacter crescentus at high frequencies. They are stably maintained and their antibiotic resistances are expressed. Experiments with RP4 have shown that intergeneric transfer of RP4 occur at a frequency of 10(-1). C. crescentus strains maintain RP4 as a plasmid, are sensitive to RP4-specific phage, and segregate phage-resistant cells at a frequency of 10(-4) to 10(-5). The RP4 plasmid can be used in several ways: (1) the RP4 plasmid will promote chromosomal exchange between C. crescentus strains at frequencies ranging from 10(-6) to 10(-8); (2) RP4 will promote the transfer of nonconjugative colE1 plasmids from E. coli to C. crescentus; once transferred, the colE1 plasmid is stably maintained under nonselective conditions, can be transferred serially, and segregates independently from RP4; and (3) RP4 can be used to introduce transposons into the C. crescentus chromosome, providing the basis for additional genetic techniques.     

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 of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

Transfer of RP4 and R68.45 factors to Rhizobium.

Two R factor were introduced by conjugation into Rhizobium trifolii and Rhizobium meliloti strains at a frequency of 10(-5) to 10(-6). Plasmids RP4 from Escherichia coli J53 and R68.45 from Pseudomonas aeruginosa PAO.25 were maintained stably in Rhizobium hosts and could be retransferred to other Rhizobium recipients. Some of the transconjugants were able to mobilize chromosome and transfer his or met genes in intra-, and interspecies matings.     

Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.     

Identification and mobilization by cointegrate formation of a nodulation plasmid in Rhizobium trifolii.

A nodulation plasmid, pRtr-514a, of molecular size 180 megadaltons (Mdal) was identified in Rhizobium trifolii strain NZP514. This plasmid was absent in both spontaneous and heat-cured Nod- derivatives of NZP514, and these strains were unable to induce root hair curling. The ability to nodulate clover was transferred from the wild-type strain to a Nod- derivatives, PN104, with the broad-host-range plasmid R68.45 (39 megadaltons) at a cotransfer frequency of about 4 X 10(-3). Most of the Nod+ transconjugants were resistant to kanamycin, tetracycline, and carbenicillin and had received a plasmid approximately 36 or 70 Mdal larger than pRtr514a but did not contain a plasmid of the size of R68.45, indicating that pRtr-514a was mobilized as a cointegrate plasmid containing either one or possibly two copies of R68.45. Use of these cointegrate-containing strains as donors in further crosses with the Nod- derivative strain PN118 resulted in high-frequency transfer of Nod+ (10(-3) to 10(-4), with cotransfer frequencies with kanamycin of up to 100%. Introduction of R68.45 into a derivative of NZP514 containing the broad-host-range plasmid pJP4 (52 Mdal) resulted in a high frequency of transconjugants carrying a cointegrate plasmid composed of pRtr-514a and pJP4. When used as donors to Nod- derivatives, such strains cotransferred Nod+ with kanamycin plus mercury at a frequency of 67%. The identification of stable cointegrates between pRtr-514a and the broad-host-range plasmids R68.45 and pJP4 should enable several genetic manipulations to be carried out with this nodulation plasmid, including the transfer of the plasmid to most gram-negative bacterial genera.     

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.     

Transfer of R factors to and between genetically marked sublines of Rhizobium japonicum.

Plasmids R1822 and pRD1 of the P-1 incompatibility group, for which Rhizobium japonicum had not previously been shown to serve as host, were introduced into a strain of R. japonicum. Acquisition of R68 and R68.45 plasmids by this Rhizobium was equivocal. Transfer of R1822 from Pseudomonas aeruginosa and of pRD1 from Escherichia coli to R. japonicum was unambiguous, because the exconjugants subsequently cotransferred the three R-factor resistance determinants (kanamycin, tetracycline, and penicillin) between genetically marked sublines of strain I-110. Under optimal conditions the transfer of R1822 and pRD1 occurred at frequencies of approximately 10(-3) in plate matings of strains bearing as many as five dissimilar genetic markers. In matings with R1822 on membrane filters, recombinants were formed at incidences as high as 4%.     

Mapping of pgi and gpd genes involved in C-1 assimilation in the obligate methylotroph Methylobacillus flagellatum

Homologous matings with plasmids R68.45 and pULB113, and also with Hfr type donor were employed for mapping pgi and gpd genes involved in C-1 metabolism in the obligate methylotroph Methylobacillus flagellatum. A preliminary map of the late chromosomal region was constructed on the basis of these experimental results. The C-1 markers were linked to methionine and leucine auxotrophy and nalidixic acid resistance markers. The phenomenon of retrotransfer, or shuttle transfer of chromosomal markers by Inc P1 plasmids, revealed earlier, was demonstrated for M. flagellatum.