Conjugation-mediated genetic exchange in Legionella pneumophila.

Genetic exchange mechanisms, to our knowledge, have not been reported for Legionella pneumophila, and consequently, studies on the genetic organization of L. pneumophila have not appeared in the literature. Here, we describe gene transfer mediated by broad host range conjugative plasmids in Legionella spp. Escherichia coli strains carrying plasmids RP1 and R68.45 (IncP1), S-a (IncW), and R40a (IncC), but not plasmids of incompatibility groups FI, FII, and FV, served as donors in matings with L. pneumophila Knoxville 1 (LPK-1). Transconjugants selected by resistance to kanamycin (RP1, R68.45, and S-a) and carbenicillin (R40a) were observed at frequencies of 6.6 X 10(-3), 4.7 X 10(-3), 2.2 X 10(-4), and 5.4 X 10(-5), respectively. Plasmid transfer was not affected by DNase added to the mating medium. After plasmid transfer, LPK-1 stably maintained RP1, R68.45, and S-a, but not R40a. Plasmid-containing LPK-1 isolates also served as donors in agar plate matings with E. coli W1485-1 and naladixic acid-resistant mutants of LPK-1, Legionella micdadei, and Legionella longbeachii. Recombinational exchange of a chromosomal trait was demonstrated when a thymidine auxotroph of L. pneumophila was repaired by R68.45-mediated chromosomal mobilization of a prototrophic donor strain.     

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.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

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.     

Direct DNA repeat in plasmid R68.45 is associated with deletion formation and concomitant loss of chromosome mobilization ability.

Plasmid R68.45 has been useful in promoting the transfer of chromosomal markers in bacteria of many genera. In donors harboring R68.45, chromosome-mobilizing ability (Cma) may be lost without the loss of other plasmid markers. However, Cma can be somewhat stabilized by maintenance of the donors in the presence of kanamycin (Km). We isolated variants of R68.45 from four bacterial species of three genera. Plasmid variants isolated included those without Cma, without transfer function (Tra) and Cma, or without Tra, Cma, and Km resistance. In Erwinia carotovora subsp. atroceptica EA153, the loss of plasmid markers is dependent on the culture medium on which the donors are maintained. Restriction endonuclease analyses of the variant plasmids revealed that most are deletion mutants of R68.45. In all cases when the uncertainty in the ends of the deletions was not too great, one end of the deletion was shown to originate within or near the direct DNA duplication in R68.45 which is required for Cma and which maps close to the Km resistance determinant. Furthermore, the types of deletions observed are consistent with what might be expected for deletions generated by tandemly repeated insertion sequences. Therefore, we suggest that the DNA duplication is the source of much of the instability observed in R68.45. However, data are presented for E. carotovora subsp. atroceptica EA153 which suggest that another region of R68.45 may also play a role in its stability in this species.     

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.     

Genetic transformation of Rhizobium leguminosarum by plasmid DNA.

We demonstrated the genetic transformation of Rhizobium leguminosarum by R68.45 plasmid DNA by freezing and thawing cell suspensions in the presence of R68.45 plasmid DNA and 20 mM MgCl2. Clones resistant to kanamycin and tetracycline were recovered at a frequency of 10(-8) per recipient cell. No colonies that were doubly drug resistant were recovered in parallel control experiments.     

Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli

Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10^?3–10^?5 per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS21 in direct orientation. After resolution of the cointegrate in recA⁺ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid. Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.     

Chromosomal mapping in Erwinia carotovora subsp. carotovora with the IncP plasmid R68::Mu.

Conjugational gene transfer was established in Erwinia carotovora subsp. carotovora SCRI193 by using plasmid R68::Mu c+ to mobilize the chromosome into multiply mutant recipients. It was observed that although the plasmid alone mobilized markers randomly at a frequency of ca. 10(-5) chromosomal recombinants per donor, the presence of a Mu prophage on the chromosome of the donor increased the frequency of mobilization of markers adjacent to the prophage by up to 10-fold. Using this system it was possible to order 17 chromosomal mutations. The behavior of Mu in E. carotovora subsp. carotovora was also studied.     

Heterotrophic bacteria of the freshwater neuston and their ability to act as plasmid recipients under nutrient deprived conditions

Significantly higher numbers of Gram-negative heterotrophic bacteria were present at the air-water interface (neston) of freshwater lakes than in the bulk water. Neuston bacteria were distinguished as a population distinct from bacteria in the bulk water by a higher incidence of pigmented colony types and significantly greater levels of multiple resistance to antibiotics and heavy metals. The incidence of plasmids in 236 neuston and 229 bulk water strains were similar (14 and 16.2%, respectively). Nine of 168 plasmid-free strains and 2 of 14 plasmid carrying strains, isolated from both bulk water and neuston, acted as recipients of plasmid R68.45 in plate matings with aPseudomonas aeruginosa donor strain PAO4032 at 21°C, but at frequencies below that of matings with a restriction-minus recipient strain ofP. aeruginosa, strain PAO1168. In a model system composed of nutrient-free synthetic lake water, plasmid R68.45 was shown to transfer betweenP. aeruginosa strains at frequencies between 10⁻³ and 10⁻⁵. Transconjugants were detected about 100 times more frequently at the interface than in the bulk water, which in part reflected a greater enrichment of the donor at this site. None of the aquatic isolates were able to act as recipients of plasmid R68.45 in this model system with strain PAO4032 as donor. The results suggest that under nutrient deprived conditions, the spread of plasmid R68.45 and similar plasmids by lateral transfer into this particular aquatic population would be a rare event.     

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.     

Transmission of F'lac plasmid from Escherichia coli K-12 to bacteria of the genus Erwinia]

The F'lac plasmid was transferred by conjugation from Escherichia coli K-12 W1655 to 21 lac- strains of Erwinia spp. (5.2 . 10(-6) to 6.8 . 10(-2) lac+ exconjugants per donor cell). Erw. herbicola and Erw. chrysanthemi were the better recipients than others. The degree of the stability of lac+ genes in Erwinia exconjugants depends on the strains. Stable exconjugants of Erwinia, which harbored F'lac plasmid, were able to utilize lactose, to transfer lac genes by conjugation to Erwinia spp. and E. coli, and were sensitive to the F-specific phages f1, f2, Qbeta. The F'lac plasmid was eliminated from the exconjugants by the treatment with acridine orange, which indicates that this genetic element is not integrated into the Erwinia chromosome.     

Spontaneous deletions of the chromosome-mobilizing plasmid R68.45 in Pseudomonas aeruginosa PAO

In Pseudomonas aeruginosa strain PAO the chromosome-mobilizing IncP-1 plasmid R68.45 was unstable whereas the parent plasmid R68 was stable. The instability of R68.45 was observed in rec+ and rec strains within about 100 generations after conjugal transfer of the plasmid and, to a lesser extent, in established R68.45 donor strains. Two phenotypically distinct classes of R68.45 derivatives were obtained: (i) CbR (carbenicillin-resistant), TcR (tetracycline-resistant), KmR (kanamycin-resistant), Tra+ (transfer proficient), Cma- (chromosome-mobilizing ability), lacking the duplicated IS21 copy typical of R68.45 and indistinguishable from R68 by restriction enzyme analysis; (ii) CbR TcR KmS Tra- Cma-, due to deletion of one IS21 copy, the adjacent KmR gene, and a variable part of the Tra-1 region including, in most cases, the origin of transfer (oriT). Both types of deletion derivatives were stable. R68.45 derivatives lacking the Tra-2 region were not recovered spontaneously, but could be constructed in vitro and were stable in strain PAO. Deletion formation of type ii as well as Cma did not depend on homologous recombination and can be ascribed to functions of the duplicated IS21. Chromosome mobilization does not appear to require obligatory transfer of the entire R68.45 plasmid. Four ClaI restriction sites were mapped on R68 extracted from P. aeruginosa. One of these sites was cryptic, presumably because of methylation, when the plasmid was prepared from Escherichia coli (dam+).     

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.     

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.     

Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics.

Summary The conjugative plasmid R68.45 mobilizes the chromosome of Pseudomonas aeruginosa strain PAO from multiple sites located in different chromosome regions. In interrupted matings on the plate, selection for any single marker tested resulted in entry times of 3–5 min. When selection was imposed for two markers linked in R68.45-mediated conjugation, double recombinants appeared after a delay which corresponded approximately to the map distance between the two markers as measured by the sex factor FP2. Thus, R68.45 and FP2 appear to promote chromosome transfer at similar rates, but R68.45, unlike FP2, seems to give non-polarized transfer. R68.45 may be used to estimate map distances between linked markers located in those chromosome regions where other sex factors do not produce enough recombinants to permit accurate measurement of entry times.In R68.45 matings on the plate, most recombinants inherited short donor chromosome fragments (usually less than 10 min long) and lost the R plasmid during purification. Used like a large generalized transducing phage, R68.45 has proved valuable in construction of PAO strains with desired genotypes.     

Erwinia carotovora as a recipient system for the natural hybrid plasmid RP4::Mu cts62

The plasmid RP4::Mu cts62 is transferred from Escherichia coli cells into a recipient strain Erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. The maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. Structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of RP4, thermosensitivity to growth at 42 degrees C as well as spontaneous and thermally-induced production and zygotic induction of bacteriophage determined by the genome of Mu cts62, total length of the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid RP4::Mu cts62 in Erwinia carotovora transconjugant cells has been determined. A single bacteriophage genome has been shown to transpose into the chromosome of the cell with the elimination of RP4 fragment under the conditions of thermal induction.     

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.     

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.     

Conjugative Transfer of Chromosomal Genes between Fluorescent Pseudomonads in the Rhizosphere of Wheat

Bacteria released in large numbers for biocontrol or bioremediation purposes might exchange genes with other microorganisms. Two model systems were designed to investigate the likelihood of such an exchange and some factors which govern the conjugative exchange of chromosomal genes between root-colonizing pseudomonads in the rhizosphere of wheat. The first model consisted of the biocontrol strain CHA0 of Pseudomonas fluorescens and transposon-facilitated recombination (Tfr). A conjugative IncP plasmid loaded with transposon Tn5, in a CHA0 derivative carrying a chromosomal Tn5 insertion, promoted chromosome transfer to auxotrophic CHA0 recipients in vitro. A chromosomal marker (pro) was transferred at a frequency of about 10(sup-6) per donor on wheat roots under gnotobiotic conditions, provided that the Tfr donor and recipient populations each contained 10(sup6) to 10(sup7) CFU per g of root. In contrast, no conjugative gene transfer was detected in soil, illustrating that the root surface stimulates conjugation. The second model system was based on the genetically well-characterized strain PAO of Pseudomonas aeruginosa and the chromosome mobilizing IncP plasmid R68.45. Although originally isolated from a human wound, strain PAO1 was found to be an excellent root colonizer, even under natural, nonsterile conditions. Matings between an auxotrophic R68.45 donor and auxotrophic recipients produced prototrophic chromosomal recombinants at 10(sup-4) to 10(sup-5) per donor on wheat roots in artificial soil under gnotobiotic conditions and at about 10(sup-6) per donor on wheat roots in natural, nonsterile soil microcosms after 2 weeks of incubation. The frequencies of chromosomal recombinants were as high as or higher than the frequencies of R68.45 transconjugants, reflecting mainly the selective growth advantage of the prototrophic recombinants over the auxotrophic parental strains in the rhizosphere. Although under field conditions the formation of chromosomal recombinants is expected to be reduced by several factors, we conclude that chromosomal genes, whether present naturally or introduced by genetic modification, may be transmissible between rhizosphere bacteria.