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.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.     

Properties of transposon Tn2555 carrying the genes for sucrose utilization

Tn2555, a new transposon coding for genes of sucrose utilization was studied. Tn2555 was shown to integrate into the plasmids RP4 and R6K, phage P1CmClr100 and Escherichia coli K12 chromosome. Tn2555 frequency of transposition to RP4 and R6K DNA is (2-5) X 10(-7) in Rec+-strain, (3-6) X 10(-8) in Rec--strain. Tn2555 integration site in phage P1CmClr100 Sac+-derivative studied has been localised within the C-segment of P1 DNA. In three independent cases of Tn2555 transposition to the chromosome the transposon was found to be integrated in the region between 29 and 32 min of Escherichia coli K12 linkage map. The restriction endonuclease analysis of seven independent isolates of RP4::Tn2555 has shown the grouping of Tn2555 integration sites in the Tn1 region of RP4. Frequent rearrangements occurring within Tn2555 have been revealed by the analysis. However, an invertible DNA segment of about 6-7 kb was preserved in all transposon structures.     

Identification and characterization of Tn4656, a novel class II transposon carrying a set of toluene-degrading genes from TOL plasmid pWW53

It has been reported that the toluene-degrading (xyl) genes from Pseudomonas putida plasmid pWW53 are able to translocate to broad-host-range drug resistance plasmid RP4, and pWW53-4 is one of the smallest RP4 derivatives (H. Keil, S. Keil, R. W. Pickup, and P. A. Williams, J. Bacteriol. 164:887-895, 1985). Our investigation of pWW53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. This mobile element, Tn4656, was classified as a class II transposon since its transposition occurred by a two-step process: transposase (TnpA)-mediated formation of the cointegrate and resolvase (TnpR)-mediated site-specific resolution of the cointegrate at the two copies of the res site. The Tn4656 TnpA and TnpR functions encoded in the rightmost 4-kb region were found to be exchangeable with those specified by other Tn1721-related class II transposons, including another toluene transposon, Tn4653. Sequence analysis of the transposition-related genes and sites of Tn4656 also supported the hypothesis that this transposon is closely related to the Tn1721-related transposons. The lower transposition frequency of Tn4656 has been suggested to be due to the unique nucleotide sequence of one of the terminal 39-bp inverted repeats.     

Vibrio cholerae hybrid sex factor that contains ampicillin transposon Tn1.

The ampicillin resistance transposon Tn1 was translocated from the R plasmid RP4 to the Vibrio cholerae conjugative plasmid, P. The hybrid sex factor P::Tn1 was highly transmissible and expressed the biological activities of the P factor. In addition, P::Tn1 facilitated transfer of RP4 to V. cholerae recipients. Physical studies of P::Tn1 indicated that the Tn1 transposon was added to the otherwise unaltered P plasmid.     

Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another km transposon, Tn903

Summary A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.     

A directional, high-frequency chromosomal mobilization system for genetic mapping of Rhizobium meliloti.

A system for mapping of the Rhizobium meliloti chromosome that utilizes transposon Tn5-Mob, which carries the mobilization site of IncP plasmid RP4 (R. Simon, Mol. Gen. Genet. 196:413-420, 1984), was developed. Insertions of Tn5-Mob that were located at particular sites on the R. meliloti chromosome were isolated and served as origins of high-frequency chromosomal transfer when IncP tra functions were provided in trans. This approach is, in principle, applicable to any gram-negative bacterium in which Tn5 can transpose and into which IncP plasmids can conjugate.     

Transposable plasmid deoxyribonucleic acid sequence in Pseudomonas aeruginosa which mediates resistance to gentamicin and four other antimicrobial agents.

A 9.1 x 10(6)-dalton transposable deoxyribonucleic acid sequence resides within Pseudomonas aeruginosa plasmid R1033 and mediates resistance to gentamicin, streptomycin, sulfamethoxazole, chloramphenicol, and mercuric chloride. Transposability was demonstrated in Escherichia coli when this sequence, designated Tn1696, excised from R1033 and integrated into plasmid pMB8. Excision and insertion of Tn1696 occurred independently of the host Rec phenotype and may involve the 140-base pair, inverted deoxyribonucleic acid repeated region that flanks this sequence. Occurrence of a multiresistance transposon on a transferrable plasmid that has a broad host range may have serious epidemiological and therapeutic consequences.     

Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical.

The molecular relationships of the IncP1 plasmids RP1, RP4, R68 and RK2 were tested by electron microscopic examination of heteroduplexes. In several hybridization experiments molecules were detected which had a 7.8% portion of incomplete reannealing. This 'heterologous region' could be explained by the typical renaturation behaviour of the transposon Tn1. The identity of the Tn1 transposon present in RP1 and RP4 was proved by heteroduplex experiments with lambda phage DNA containing this transposon. These results indicated that the plasmids RP1 and RP4 are identical. Additional heteroduplex experiments between plasmids R68.45 and RP8 and between R68.45 and RK2 were performed. R68.45, a derivative of R68, has a small DNA insertion and RP8 can be regarded as a large insertion mutant of RP4; both insertions were used as single-stranded hybridization markers. From the hybrid molecules formed, it was deduced that R68 and RK2 are identical with RP1 and RP4 as far as molecular structure is revealed by the technique used.     

Adenine + thymine content of different genes located on the broad host range plasmid RP4

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.     

Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.     

Tn292l, a transposon encoding fosfomycin resistance.

The determinant of resistance to fosfomycin of the Serratia marcescens plasmid pOU900 was transposed into the plasmid ColE1 and into the plasmid RP4 in the absence of the RecA function of the host. In each case, the acquisition of fosfomycin resistance was correlated with the presence of a discrete piece of DNA, uniform in size and in restriction pattern, This new, 7.5-megadalton transposable element encoding resistance to fosfomycin has been called Tn2921. A preliminary map of the transposon is presented.     

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.     

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.     

Characteristics of RP4 tellurite-resistance transposon Tn521

A restriction map of the tellurite-resistance (Ter) transposon Tn521 (parent plasmid RP4Ter) was prepared. Five sites from RP4Ter, including the EcoRI origin, were found in pIN25::Tn521. Tn521 was inserted into a transferable 27.5 kb vector (pCU109) to make three different insertion mutants, in which the size of Tn521 was measured accurately at 4.5 kb. Unlike the Ter of IncHI2 plasmids, that of Tn521 in RP4Ter was non-inducible. Ter was expressed in five widely differing bacterial species to which RP4Ter was transferred from Escherichia coli. Electron micrographs of bacteria expressing the Ter of RP4Ter, H complex plasmids, and chromosomal mutants, all revealed similar tellurium metal crystallites when the bacteria were grown in potassium tellurite medium. No other Ter determinants were found amongst 54 plasmids representing most incompatibility groups (excluding the H complex).     

Transposition of DNA fragments flanked by two inverted Tn1 sequences: translocation of the plasmid RP4::Tn1 region harboring the Tcr marker

We have demonstrated the possibility of transposition of the plasmid RP4::Tn1 fragment (21.2 kb) carrying the tetracycline resistance (Tcr) gene and flanked by two Tn1 copies. The new transposon, designated Tn1756, bears lethal genes that kill host cells. Therefore, its transposition can only be revealed in the presence of lethality-compensating helper regions of the plasmid RP4. Thus, RP4::Tn1 consists of two transposons, Tn1755 (Tn1-Kmr-Tn1) and Tn1756 (Tn1-Tcr-Tn1), sharing the Tn1 sequences. Both of these transposons are capable of recA-independent translocation to other plasmids. Therefore, transposition of DNA fragments flanked by two inverted Tn1 sequences does not depend on Tn1 orientation.     

Tn2001, a transposon encoding chloramphenicol resistance in Pseudomonas aeruginosa.

We isolated a new transposon, Tn2001, from the group P-2 plasmid Rms159-1 in Pseudomonas aeruginosa. Tn2001-encoded chloramphenicol resistance did not result from the formation of chloramphenicol acetyltransferase. Tn2001 was transposable between temperate phages and conjugative and nonconjugative plasmids belonging to various incompatibility groups, including P-1, P-3, P-4, P-5, P-7, and P-8 in P. aeruginosa. Transposition occurred independently of the general recombination ability of the Pseudomonas host, and its frequency varied between 10(-1) and 10(-8), depending upon the donor and recipient replicons. Tn2001 transposition also occurred in a recombination-deficient strain of Escherichia coli. Agarose gel electrophoresis and electron microscopic observations revealed that Tn2001 could transpose to different sites in the RP4 replicon and that the transposed deoxyribonucleic acid fragment was 2.1 kilobases long.     

Transposition of Tn1831 to sym plasmids of Rhizobium leguminosarum and Rhizobium trifolii

Abstract All transposon-induced symbiotic mutants of Rhizobium described so far have been obtained using Tn 5 , which codes for kanamycin resistance (Km^R). To enable genetic complementation studies, we tried to find an effective transposon carrying another resistance marker. We report here a method for the apparent random transposition in Rhizobium of Tn 1831 , which codes for resistance against spectinomycin (Sp), streptomycin (Sm) and mercury chloride. When the suicide plasmid pMP12 (RP4::Tn 1831 , Km::Mu) was transferred to Rhizobium , in almost all cases the exconjugants harbour a deleted transfer-deficient R plasmid. From this deleted R plasmid transposition occurred to self-transmissible Sym-plasmids of R. leguminosarum and R. trifolii . Using this method a number of Tn 1831 -induced symbiotic mutants of pRL1JI were isolated.     

A molecular analysis of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans

Abstract: A 12.4-kb plasmid, pTF-FC2, that was isolated from Thiobacillus ferrooxidans and which is capable of replication in a wide range of Gram-negative bacteria, has been sequenced. The extent of the regions involved in both replication and mobilization have been delineated. The site of initiation of replication ( oriV ) has been localized on a 185-bp fragment and the origin of transfer ( oriT ) on a 138-bp fragment. Three proteins that were essential for replication and four that were essential for mobilization have been identified. The origin of replication was clearly similar to that of the IncQ plasmids although no complementation or incompatibility between pTF-FC2 and the IncQ plasmid, R300B, was detected. There was a clear similarity in the size,location and amino acid sequence of the proteins of the pTF-FC2 mobilization region with those of the TraI region of the IncP plasmids, RP4 and R751.Two inverted repeated sequences which had 37/38-bp and 38/38-bp sequence identity with the Tn 21 transposon were identified. The C-terminal part of a transposase and the N-terminal portion of a resolvase were located between the inverted repeats. These open reading frames are most likely the remnants of a defective transposon. A protein with homology to a mercury- resistance regulator was also present within the transposon-like element although no gene encoding for mercury reductase could be indentified.     

Restriction and electron microscopic analyses of deletion derivatives thermosensitive with respect to maintaining plasmid pEG1

Temperature-independent deletion mutants of temperature-sensitive in self-maintenance plasmid pEG-1, derived from R-factor RP4, are mapped using the restriction endonucleases PstI, SmaI, EcoRI, BamHI and the heteroduplex analysis. The pEG1 derivatives under study are found to have deletions in the area of ampicillin transposon Tn1 and nearby genes. The left and the right ends of these deletions boundaries are localized between 37.5 MD and 7.6 MD in the RP4 map. Thus far, the area of plasmid RP4 (37.5-7.6 MD) with Tn1 and, presumably, inc gene(s) in it does not have any genes needed for stable maintenance of R-factor. A conclusion is made that the gene RP4, which carries the mutation determining thermosensitive character of pEG1 maintenance and of inhibition of the cell growth, is localized between the EcoRI site and transposon Tn1 at a distance less than 1.0 MD from the latter.