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.     

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.     

Chromate resistance plasmid in Pseudomonas fluorescens.

Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified. Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance. Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re-acquisition of the chromate resistance phenotype and the plasmid. When pLHB1 was transferred by conjugation to Escherichia coli, the plasmid still conferred chromate resistance.     

Genetic and molecular characterization of the Pseudomonas plasmid pVS1

A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed. Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P. fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1. The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21. A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P. aeruginosa PAO but selective pressure was needed for plasmid maintenance. The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent. Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P. aeruginosa, P. putida, P. fluorescens, P. acidovorans, P. cepacia, P. mendocina, P. stutzeri, P. syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum.     

A natural mutant of plasmid RP4 that confers phage resistance and reduced conjugative transfer

A natural isolate of RP4 (PRC#116) acquired from the Stanford University Plasmid Reference Center differed from the wild-type Incompatibility Group P plasmid in several respects. Cells of Escherichia coli harboring PRC#116 were resistant to the IncP pili-specific bacteriophage PRD1 and GU5, and transferred this plasmid at a lower efficiency than the wild-type RP4. Phage sensitivity was restored, and transfer considerably improved in PRC#116+ bacteria transformed with plasmid constructs containing the origin of transfer (oriT region) of RP4. Mutant RP4 plasmids equivalent to PRC#116 were selected at a high frequency from an RP4+ E. coli population infected with PRD1 indicating that this RP4 variant may be the product of a very common mutation of the wild-type plasmid.     

Isolation of a nontransmissible antibiotic resistance plasmid by transductional shortening of R factor RP1.

A plasmid segregant carrying tetracycline and carbenicillin resistance markers has been isolated from R factor RP1 by transductional shortening with phage P22. The new plasmid RP1-S2, which has a molecular weight of 23 times 10-6, has lost the transfer, phage sensitivity, and neomycin resistance functions of RP1. It combines readily with a W group plasmid, R388, to form a transmissible carbenicillin and trimethoprim resistance plasmid, RWP1.     

Transfer of plasmid RP1 into chemolithotrophic Thiobacillus neapolitanus.

RP1, a broad-host-range incompatibility group P1 plasmid specifying multiple drug resistances, has been transferred into the chemolithotrophic bacterium Thiobacillus neapolitanus. The ability of T. neapolitanus to receive, express, and transmit RP1-encoded antibiotic resistances was examined. The data show that this obligate chemolithotroph can accept, replicate, and express heterologous plasmid DNA from a heterotrophic bacterium.     

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.     

Properties of 1-phosphofructokinase from Pseudomonas putida.

The 1-phosphofructokinase (1-PFK, EC 2.7.1.56) from Pseudomonas putida was partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. In its kinetic properties, this enzyme resembled the 1-PFK's from other bacteria. With the substrates fructose-1-phosphate (F-1-P) and adenosine triphosphate (ATP) Michaelis-Menten kinetics were observed, the Km for one substrate being unaffected by a variation in the concentration of the other substrate. At pH 8.0, the Km values for F-1-P and ATP were 1.64 X 10(-4) M and 4.08 X 10(-4) M, respectively. At fixed concentrations of F-1-P and ATP, an increase in the Mg2+ resulted in sigmoidal kinetics. Activity was inhibited by ATP when the ratio of ATP:Mg2+ was greater than 0.5 suggesting that ATP:2 Mg2+ was the substrate and free ATP was inhibitory. Activity of 1-PFK was stimulated by K+ and to a lesser extent by NH4+ and Na+. The reaction rate was unaffected by 2 mM K2HPO4, pyruvate, phosphoenolpyruvate, adenosine monophosphate, adenosine 3',5'-cyclic monophosphate, fructose-6-phosphate, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, or citrate. The results indicated that the 1-PFK from P. putida was not allosterically regulated by a number of metabolites which may play an important role in the catabolism of D-fructose.     

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.     

Transfer and expression of pseudomonas plasmid RP1 in Caulobacter.

This study demonstrates that the host range of Pseudomonas plasmid RP1 includes the genus Caulobacter. Caulobacter was shown to acquire three antibiotic resistance markers located in RP1. A fourth plasmid marker, susceptibility to an RNA bacteriophage, was not expressed, but could be transferred from Caulobacter to Escherichia coli. The lack of phenotypic expression of the phage marker was manifested by the inability of the phage to adsorb or to produce plaques on Caulobacter transcipients. Matings of Pseudomonas aeruginosa and Caulobacter vibrioides CV6 were carried out in the presence of bacteriophage phi6, a DNA phage that infects and kills only swarmer cells of Caulobacter. No decrease in plasmid transfer in the presence of phage phi6 was detected, suggesting that stalked cells, and not swarmer cells, serve as recipients. Our evidence suggests that transfer of chromosomal segments from Caulobacter may be mediated by plasmid RP1; such segments are not stably maintained.     

Fertility inhibition of RP1 by IncN plasmid pKM101.

IncN plasmids, including pKM101, strongly inhibit the conjugal transfer of cohabiting IncP plasmids. We localized the pKM101 DNA sufficient for this phenomenon to a 1.1-kilobase region (denoted fip). Two fip-deficient Tn5 insertion derivatives of pKM101 were isolated; neither affected other pKM101-mediated functions. fip did not inhibit either the synthesis of the IncP plasmid's sex pilus or its ability to mediate entry exclusion against other IncP plasmids.     

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.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa

Abstract A new integron, located on the R plasmid of Pseudomonas aeruginosa , was isolated in Japan. This integron was made up of two conserved segments (5'- and 3'-conserved segments) and a single streptomycin resistance gene as a gene cassette. The structure of this integron resembles that of integron InC, the existence of which was postulated by Bissonnette and Roy (J. Bacteriol. 174, 1248–1257, 1992).