Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Transfer and integration of chromosomal genes from Pseudomonas glycinea into Pseudomonas aeruginosa.

The plasmids FP2 and R68.45 were shown to function as chromosome-mobilizing plasmids in a series of interspecific crosses between the phytopathogen Pseudomonas glycinea and the human pathogen Pseudomonas aeruginosa. At least four of seven loci tested were transferred from P. glycinea donors to P. aeruginosa auxotrophic recipients. Transductional analysis indicates that a leu+ locus of the P. glycinea chromosome transferred is stably integrated into the P. aeruginosa chromosome.     

Interactions between the transposable element IS21 on R68.45 and TN7 in Pseudomonas aeruginosa PAO

Tn7 transposes from the chromosome of Pseudomonas aeruginosa into the plasmid R68.45 with tandem IS21, at up to 400 times the frequency that it transposes into R68, which has only one copy of IS21. While R68::TN7 derivatives are stable, R68.45::Tn7 isolates undergo frequent deletions. Instability of R68.45 occurs whether Tn7 is inserted into the plasmid (cis configuration) or into the bacterial chromosome (trans configuration). The deletions of R68.45 start at the junction between the tandem IS21 copies and proceed clockwise, ending in the region of oriT. It appears that Tn7 and IS21 can mutually stimulate transposition of each other.     

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.     

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.     

Isolation and characteristics of a temperature-sensitive plasmid pRP19.6--an RP1 derivative containing the duplicated IS21 sequence

When analysing the antibiotic resistant, temperature-independent derivatives of Proteus mirabilis cells, carrying the plasmid RP1ts12, a derivative of the latter (pRP19.6) with an elevated frequency of integration into E. coli K12 chromosome, has been isolated. The structure and properties of pRP19.6 was studied. As revealed from the data of structural and genetic analyses pRP19.6 is identical to the factor R68.45 described earlier by Haas and Holloway. Similarly to R68.45, the plasmid under study contains two copies of IS21 sequence and mobilises nonconjugative plasmid pBR325 with high efficiency. Using the temperature sensitive replication of pRP19.6, frequency of it's integration into the chromosomes of E. coli rec+ and recA- stains is determined. It is demonstrated that the clones carrying the plasmid in integrated state are Hfr-strains. The possibilities to use the temperature sensitive R68.45 like plasmid for isolation of Hfr-strains in the broad range of gram-negative bacteria and for insertional inactivation of chromosomal genes are discussed.     

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.     

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+).     

Chromosome mapping in Pseudomonas aeruginosa PAT.

A linkage map of Pseudomonas aeruginosa PAT has been derived from the results of conjugation experiments using the plasmids FP2-2, R68, R91-5, and R68.45. FP2-2 and R68 each mobilize the chromosome from single, distinct transfer origins. R91-5 appears to mobilize the chromosome from two such origins, and R68.45 utilizes a number of transfer origins. R68 and R91-5 have both been shown to mobilize the chromosome with a polarity opposite to that by FP2-2. The locations of the transfer origins of these plasmids are such that it has not been possible to demonstrate chromosomal circularity by means of interrupted mating experiments. However, the available time-of-entry data combined with linkage data from plate mating experiments support the conclusion that the chromosome of P. aeruginosa is circular.     

Analysis of flagellar genes in Pseudomonas aeruginosa by use of Rfla plasmids and conjugations.

Over 300 flagellar mutants were isolated in Pseudomonas aeruginosa PAO. R-prime plasmids carrying segments of bacterial chromosome which can complement the mutant phenotypes were isolated by means of plasmid R68.45. Among the R-prime plasmids, pMT6 complemented 167 out of 307 mutants examined, and pMT19 complemented the remaining 140 mutants. We found no mutant which was complemented by both of these plasmids. Hence, the flagellar genes were divided into two clusters by these two plasmids, namely, region I on pMT19 and region II on pMT6. By FP5- and R68.45-mediated conjugation, these two regions were located on the P. aeruginosa PAO chromosome with an order of puuF--region I--region II--oru-325.     

Isolation of High Frequency of Recombination Donors from Tn5 Chromosomal Mutants of Pseudomonas Putida Ppn and Recalibration of the Genetic Map

A Tn5 loaded derivative of the IncP-10 plasmid R91-5 (pMO75) was used as a suicide vector to generate random chromosomal insertion mutations in Pseudomonas putida PPN. Reintroduction of pMO75 into such mutants resulted in integration of the plasmid at the site of Tn5 insertion, giving rise to two classes of high frequency of donors recombination (Hfr) donors, transferring chromosome at high frequency (greater than 10(-1) per donor cell) in opposite directions. Consequently, Tn5 induced auxotrophic mutations could be equated with or distinguished from previously mapped mutations, and closely linked markers ordered, on the basis of marker recovery using the two classes of Hfr donor. The isolation of many new transfer origins allowed more accurate time-of-entry analysis than previously possible and resulted in the reduction of the genetic map from 103 min to 88 min.     

Isolation and characterization of Pseudomonas aeruginosa R'' plasmids constructed by interspecific mating

Plasmid R68.45 was used to construct R' plasmids carrying a maximum of 4 to 5 map minutes of the Pseudomonas aeruginosa PAO chromosome by interspecific mating, using P. putida PPN as the recipient. These R' plasmids were used to determine the map location of the amiE locus and to identify tentatively a number of P. putida auxotrophic mutations. Some of these R' plasmids could not be maintained in recombination-deficient P. aeruginosa strains.     

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.     

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.     

Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO. II. Physical characterization of pyocin R2 genes using R-prime plasmids constructed from R68.45

The chromosome segment which contains the genes responsible for production of pyocin R2 in P. aeruginosa PAO was defined physically using R-prime plasmids constructed in vivo from R68.45. The previous conclusion from genetic mapping that the cluster of pyocin R2 genes is located in between trpC and trpE genes was confirmed by deletion mapping of various R prime plasmids bearing the trpC gene. The pyocin R2 gene cluster was further localized on two contiguous HindIII fragments of 16 kb and 8.0 kb. PML14 strain, in which R-type pyocin genes were completely deleted, had only one 11 kb HindIII fragment instead. Heteroduplexes between this 11 kb fragment with the two HindIII fragments of PAO revealed that the cluster of pyocin R2 genes was an insertion 13 kb long.     

Integration of plasmid RP1 with the chromosome of Escherichia coli K-12 recA. 2 classes of Hfr strains

Integration of broad host range RP1 plasmid into the chromosome of Escherichia coli K-12 recA- cells has been studied. Using temperature-sensitive for replication plasmids pVD1 and pVD3, the derivatives of RP1, it has been shown that integration of RP1 into the bacterial chromosome results in formation of two classes of Hfr strains. Properties of these Hfrs have been examined. From the data obtained, it has been concluded that the plasmid integration and formation of one of the Hfrs classes appear to be mediated by transposon Tn1 residing on RP1. The other class of Hfr strains is formed due to a stable integration of RP1. In the course of analysis of R+ transconjugants arising at low frequency in crosses between stable Hfrs and E. coli rec+ recipients, it has been found that the significant part of them contain plasmid-chromosome hybrids (R-prim plasmids). On the basis of the latter results, a new simple method for R' plasmids selection has been proposed. Using restriction endonuclease analysis, the structure of plasmids that were excised from chromosomes of the stable Hfr strains and were comparable in their size to RP1, has been investigated. Probable mechanisms of the stable Hfr strains formation are discussed.