Genetic transfer of Pseudomonas aeruginosa R factors to plant pathogenic Erwinia species.

The R factors RP1, R68 and R91 were freely transmissible to and from Pseudomonas aeruginosa, Salmonella typhimurium, and various plant pathogenic Erwinia spp. The antibiotic resistance spectrum of R+ Erwinia recipients was similar to those of other bacteria harboring these R factors, but maximum resistance levels differed with each recipient. The sponstaneous elimination of these factors from the Erwinia strains and the ability to transfer multiple antibiotic resistance suggest that these exist as plasmids in these hosts. Several, but not all, RP1-carrying Erwinia strains were sensitive to the RP1 specific phage PRR1. The R factor R18-1 was also transferred from P. aeruginosa to Erwinia spp. R18-1 was unstable in all Erwinia strains. Stable strains were isolated in which R18-1 could not be eliminated by sodium dodecyl sulfate and could not be transferred to other strains.     

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.     

Plasmid transduction using the D3112DeltaH miniphage in Pseudomonas aeruginosa cells]

Plasmid DNA transduction with mini-D3112 delta H, deletion derivative of phage D3112, which lost the genes essential for phage growth but retained the sites required for transposition and packaging was studied. Unlike D3112, mini-D3112 delta H element can transduce plasmids and plasmid markers at frequencies of 10(-5)-10(-8) in rec+ cells of Pseudomonas aeruginosa. Plasmids R1162 and R388 of the size smaller than phage genome were transduced intact. Large plasmids, like RP4 and R151, were deleted under transduction. By this way, we isolated deletion derivatives of RP4. The smallest derivative pN2 contained a 4.5 kb fragment of RP4. Unlike the latter, pN2 plasmid had narrow host range and did not maintain in Escherichia coli cells.     

Phenotypic expression of mutations in a wide-host-range R plasmid in Escherichia coli and Rhizobium meliloti.

Eight different derivatives of R plasmid RP1 with thermosensitive mutations affecting maintenance in Escherichia coli and Pseudomonas aeruginosa were introduce into Rhizobium meliloti. None of the plasmids showed a thermosensitive character in R. meliloti. On the other hand, a certain deletion mutation in RP1 was found to cause plasmid instability in rhizobia and agrobacteria, but not in E. coli.     

Transformation by extracellular DNA produced by Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains are capable of producing extracellular DNA. Very closely linked chromosomal markers (leu+ and trp+) were co-transferred to P. aeruginosa PAO1819 (leu9001, trp9008) by the extracellular DNA produced by P. aeruginosa strains IFO3445 and PAO1 at a frequency of 10(-7) to 10(-8). Treatment of the extracellular DNA with DNase, heating at 95 C or sonication completely destroyed its transforming ability. The R plasmid in the extracellular DNA produced by P. aeruginosa IFO3445 (RP4) or PAO2142 (RLb679) could be transferred to Escherichia coli ML4901 or P. aeruginosa PAO1819. The resultant transformants showed identical resistance patterns in the respective donors, and the sizes of the DNAs of RLb679 and RP4 isolated from the transformants were the same as those in the respective donors. These results demonstrate that the extracellular DNA contains both chromosomal DNA and plasmid DNA, and that it exhibits transforming ability. This implies that transformation by the extracellular DNA produced by P. aeruginosa may occur in nature and this seems to be of clinical importance in view of the spread of R plasmids among pathogens.     

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.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

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.     

Reversion of an ilvB mutation in Pseudomonas aeruginosa in the presence of a derivative of RP1 plasmid

We have isolated a derivative of RP1, a broad-host-range plasmid, in whose presence the ilvB112 mutation of Pseudomonas aeruginosa strain PU21 reverts at a high frequency. This derivative of RP1 (RP1-ilvB+ complex) may have arisen by a fusion of the P. aeruginosa ilvB gene with RP1 during their co-transfer into strain PU21. The RP1 derivative is not very stable in the PU21 background but it can apparently be stabilized by its integration into the host chromosome, resulting in an Hfr-type donor strain, SP500.     

Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. The establishment of a lysogenic state and the effectiveness of lytic development

Expression of transposable phages (TP) of Pseudomonas aeruginosa in the cells of P. putida was studied. The high efficiency of phage lytic development was shown both as a consequence of zygotic induction after transfer of the RP4::TPc+ plasmid into nonlysogenic recipients, and as a result of heat induction of lysogens PpG1 (D3112cts15). The high phage yield (20-25 particles of D3112cts phage per one cell of P. putida) is an evidence for a high level of transposition in the cells of this bacterial species. Plasmids RP4::TP are transferred into cells of PpG1 and PAO1 with similar frequency. However, the efficiency of establishment of the lysogenic state is lower in PpG1. Transposable phages of P. aeruginosa can integrate into the chromosome of PpG1 producing stable inducible lysogens. The presence of RP4 in the P. putida cells is not necessary for expression of transposable phages. The transposable phage D3112cts15 can be used in experiments of interspecies transduction of plasmids and chromosomal genes.     

Structural instability of IncP-1 plasmids in Pseudomonas aeruginosa PAT involves interaction with plasmid pVS1

The structural instability exhibited by IncP-1 plasmids in Pseudomonas aeruginosa strain PAT was shown to be Rec+ dependent and involved interaction with the resident plasmid pVS1. Structural instability resulted from deletion of plasmid deoxyribonucleic acid at a frequency of ca. 10(-2)/cell per generation. Deletants could be stabilized by transduction into P. aeruginosa strain PAO, but in strain PAT deletants had only a transient existence, as continued deletion led eventually to the loss of the entire plasmid. The patterns of markers lost in PAT were used to demonstrate a marker order for R68 similar to that published elsewhere for RP4 (Barth and Grinter, J. Mol. Biol. 113:455-474, 1977), except that only one Tra region was found. R68 also exhibited Rec+-dependent structural instability in PAO(pVS1) derivatives but, unlike the case in PAT, instability was not accompanied by chromosome mobilization. We isolated deletants of pVS1 which were unable to promote structural instability.     

Genetic basis of non-transposition of Tn5 in Pseudomonas aeruginosa following mobilisation of RP4 Mob::Tn5 from Escherichia coli

Abstract A Tn 5 transposon mutagenesis system based on mobilization of the narrow-host-range plasmid pACYC184 from Escherichia coli by a chromosomally integrated promiscuous plasmid RP4 was found to be non-applicable to Pseudomonas aeruginosa recipients. Transposition following mobilization was based on cloning an RP4 DNA fragment (/ RP4 Mob) into pACYC184 and Tn 5 transposition into the fragment (/ RP4 Mob::Tn5). It was shown by DNA sub-cloning of RP4 Mob::Tn 5 on to a wide-host-range plasmid vector that mobilization was unaffected but that reduced survival of the vector or host following mobilization was responsible. However, mutagenesis was achieved by the provision of cloned RP4 Mob DNA in the P. aeruginosa recipients.     

Expression of Pseudomonas aeruginosa phage transposons in Pseudomonas putida PpG1 cells. II. Zygotic induction--a necessary condition for the formation of defective lysogens

The transfer of hybrid plasmid RP4::PT (where PT is the genome of a transposable phage specific for Pseudomonas aeruginosa) into recipient cells of P. putida strain PpG1 occurs with the same frequency as into P. aeruginosa, the homologous host for PT. Approximately 1/3 of all PpG1 exconjugants carrying RP4 markers lost the capability to produce viable PT phage. In contrast, in a cross with homologous recipient P. aeruginosa all exconjugant clones contained nondefective prophages in the hybrid plasmids. Zygotic induction is an obligatory condition for detection of PpG1 exconjugants with defective phages. The defective prophages in RP4::PT hybrid plasmids have deletions of different size; the other carry mutations indistinguishable from point mutations in an essential phage gene. Some of deletions also cover plasmid genes. At least some of the defective prophages, including deleted ones, have arisen in the recipient cells of P. putida after transfer of the hybrid plasmid.     

Compatibility of transposable phages of Escherichia coli and Pseudomonas aeruginosa. I. Co-development of phages Mu and D3112 and integration of phage D3112 into RP4::Mu plasmid in Pseudomonas aeruginosa cells

The possibility of using a model system (which included RP4::Mu plasmid and D3112 phage in Pseudomonas aeruginosa cells) for analysis of compatibility of transposable Escherichia coli phage Mu and P. aeruginosa phage D3112, as phages and transposons, was studied. No interaction was observed during the vegetative growth of phages. The majority of the hybrid RP4::Mu plasmids lost the Mu DNA after insertion of D3112 into RP4::Mu. The phenomenon was not a result of transposition immunity. We consider the loss of the Mu DNA as a consequence either of plasmid RP4::Mu instability in P. aeruginosa cells, because of the lack of functional Mu repressor, or of some D3112-encoded activity involved in its transposition. For the inambiguous conclusion on compatibility of two phages as transposons, it is necessary to modify the model system, eliminating the possibility of Mu phage replication--transposition.     

R Plasmids R91 and R91a from Pseudomonas aeruginosa Share Only the Gene for Carbenicillin Resistance

Plasmid R91a of Pseudomonas aeruginosa strain 9169 is homologous with RP1. Plasmid R91 carried by the same strain is related in the Tn1 region but is otherwise unrelated to R91a.     

Recombination between plasmids of incompatibility groups P-1 and P-2.

R plasmids of incompatibility group P-2 are readily transmissible between Pseudomonas strains, but not to Escherichia coli or other enterobacteria, whereas those of group P-1 have a broad host range. Pseudomonas aeruginosa donor strains carrying both a P-1 plasmid (RP1, RP4, or R751) and a P-2 plasmid (pMG1, pMG2, pMG5, or RPL11) were mated with E. coli K-12, and selection was imposed for resistance markers on the P-2 plasmids. Transconjugants were obtained at a low frequency, in which P-2 markers were expressed and were serially transmissible in E. coli together with P-1 markers. These plasmids had P-1 incompatibility properties, conferred susceptibility to phages active on P-1 carrying strains, and behaved on sucrose gradient centrifugation as unimolecular species of higher molecular weights than the P-1 parent. Recombinant plasmid formation was independent of a functional Rec gene in both donor and recipient and, with R751, had a preferred site leading to loss of trimethoprim resistance. Interaction between insertion sequences may be involved. Thus, plasmids of group P-2 can recombine with R factors of another group quite separate in compatibility properties, host range, and pilus type. Formation of such recombinants provides one pathway by which the genetic diversity of plasmids may have evolved.     

Hybrid plasmid pBS251 containing genes for n-alkane degradation

The strain of Pseudomonas aeruginosa BS316 utilizing H-alkanes of the C6-C12 series (Alk+) harbours the 96 Md plasmid pBS250. The use of plasmid RP4 to mobilize Alk+ markers in conjugal transfer to Pseudomonas aeruginosa and Pseudomonas putida has resulted in isolation of transconjugants resistant to antibiotics (due to genes coded by plasmid RP4) and capable of growth on H-alkanes. A transconjugant from this series harbours plasmid pBS251, a derivative of plasmid RP4 containing the genes for octane and octanol catabolism. A fragment of DNA inserted into RP4 has a mol mass 3.8 Md, possesses two restriction sites for EcoRI, one site for PstI, is not restricted by SmaI and BamHI restriction endonucleases, and is localized in the region 4.5-5.7 Md on the physical map of plasmid RP4.     

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.