Novel system for recognizing and eliminating foreign DNA in Pseudomonas putida.

Derivatives of pQSR49 (R1162::Tn1) containing cloned fragments of Escherichia coli chromosomal DNA are stable in E. coli but unstable in Pseudomonas putida. Similar derivatives containing P. putida chromosomal DNA are stable in both species. Instability is a consequence of plasmid loss during growth and is not due to death or inhibition of growth of plasmid-containing cells. Average copy numbers per cell of the unstable hybrid plasmids are similar to that of pQSR49, indicating that instability is not the result of reduced replication of plasmid DNA. A plasmid unrelated to pQSR49, RK2, becomes unstable in the presence of the unstable hybrids but only when it also contains foreign DNA. The data suggest an inducible mechanism in P. putida active in the elimination of plasmid-borne foreign DNA from the cell.     

TOL is a broad-host-range plasmid.

We readily isolated insertions of the carbenicillin resistance element Tn401 into the TOL plasmid in Pseudomonas putida. Hybrid TOL::Tn401 plasmids stably express the Cbr phenotype in Pseudomonas aeruginosa and Escherichia coli. Whereas the replicative and conjugative functions are expressed in both hosts, the ability to grow on m-toluate is only expressed in the Pseudomonas species.     

Transposition of Tn904 encoding streptomycin resistance into the octopine Ti plasmid of Agrobacterium tumefaciens.

A transfer-deficient derivative of plasmid RP1-pMG1 was isolated after insertion of Mu cts62. The Tra- R plasmid was used to donate Tn904, encoding streptomycin resistance, to Ti plasmid pAL102 harbored by Agrobacterium tumefaciens Ach5. Under conditions promoting high Ti transfer frequencies, 155 strains were isolated in which the streptomycin marker coupled with Ti plasmid in further transfer experiments. These isolates represent stable insertions of Tn904 into the Ti plasmid. In addition, 19 strains were isolated in which the insertion of Tn904 was apparently unstable. The frequency of stable Tn904 transpositions was estimated to be 3 x 10(4-) per transferred Ti plasmid. Evidence was obtained that Tn904 readily may transpose from the Ti plasmid into the bacterial chromosome. The strains carrying Ti plasmids with stable insertions were characterized with respect to virulence, octopine degradation, octopine synthesis in induced tumors, and Ti plasmid transfer. Thirteen of the strains were found to be affected in tumor-inducing ability.     

Tn7 and Tn501 Insertions into Pseudomonas aeruginosa plasmid R91-5: mapping of two transfer regions.

We constructed a restriction endonuclease map of the Pseudomonas aeruginosa narrow-host-range plasmid R91-5. Insertions of transposons Tn7 and Tn501 into the plasmid DNA were characterized physically and genetically. The distribution of sites of insertion showed some regional specificity for the insertion of these transposons, especially TN501. The insertion of Tn7 was unusual in that all 42 of 43 insertions were in the same orientation. By relating phenotypic changes to the site of insertion, the Tn1 transposon that was already present on R91-5 and coded for carbenicillin resistance was mapped, and its orientation was determined. Two major transfer regions were identified. We believe that Tra1 is involved in conjugal DNA metabolism, whereas Tra2 is involved mainly in production of the sex pili.     

Analysis of mini-F plasmid replication by transposition mutagenesis

Derivatives of a mini-F plasmid in which Tn3 is inserted in F deoxyribonucleic acid were obtained, and the sites of insertion for 40 of the derivatives were mapped. Tn3 was found to insert at many sites within mini-F, but most insertions were within the 43.0- to 43.7-kilobase (kb), 44.2- to 44.7-kb, and 45.9- to 46.3-kb segments. Hence, these segments are unnecessary for mini-F replication. Most of the Tn3 derivatives were similar to their parent miniplasmid with respect to copy number, stability, and incompatibility. Insertions at 45.15 kb and near 46.0 kb caused a moderate disruption of copy number control, and insertion at 47.6 kb resulted in unstable maintenance. Deletion derivatives lacking deoxyribonucleic acid between 40.3 and 44.76 kb and between 45.92 and 49.4 kb were obtained. This observation suggests either that mini-F contains a third origin, in addition to those already reported to be at 42.6 and 44.4 kb, or that the reported position of the secondary origin, 44.4 kb, is incorrect and that this origin is between 44.76 and 45.92 kb.     

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.     

Genetic analysis of insertion mutations of the promiscuous IncP-1 plasmid R18 mapping near oriT which affect its host range

Transposon Tn7 insertion mutations of the promiscuous IncP-1 plasmid R18 which affect its conjugational transmissibility from Pseudomonas aeruginosa to Escherichia coli C, a strain of E. coli K12, Salmonella typhimurium and P. maltophilia have been mapped physically. They map to coordinate 53.5 kb in the Tral region of the plasmid. An 800-bp fragment mapping between R18 coordinates 52.85 and 53.65 kb, which complemented the host range defect of the mutants when tested with E. coli C as recipient, has been identified. However, complementation occurred only when the 800-bp cloned fragment was provided in the E. coli C recipient but not when situated in the P. aeruginosa donor. It is concluded that a trans-acting gene product of R18 is required, in the transcipient, for conjugative DNA metabolism during, or immediately following, the conjugational transfer of this plasmid between certain donor and recipient hosts.     

Cloning and transposon vectors derived from satellite bacteriophage P4 for genetic manipulation of Pseudomonas and other gram-negative bacteria.

We developed transposon and cloning shuttle vectors for genetic manipulation of Pseudomonas and other gram-negative bacteria, exploiting the unique properties and the broad host range of the satellite bacteriophage P4. P4::Tn5 AP-1 and P4::Tn5 AP-2 are suicide transposon vectors which have been used for efficient Tn5 mutagenesis in Pseudomonas putida. pKGB2 is a phasmid vector with a cloning capacity of about 7.5 kb; useful unique cloning sites are SacI and SacII in the streptomycin resistance determinant and PvuI and XhoI in the kanamycin resistance determinant. pKGB4 is a cosmid derived from pKGB2 and carries the additional cloning site SmaI in the kanamycin resistance determinant; its cloning capacity is about 18 kb. These vectors and their recombined derivatives were transferred from Escherichia coli to P. putida by transduction and may be used for other bacterial species susceptible to P4 infection.     

Physical and genetic structure of the IncN plasmid R15

Restriction sites for seven hexanucleotide-specific endonucleases were located on the map of the conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty-four sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI were located close to or within the sequences of an IS5-like element and the transposons Tn2353 and Tn2354. By analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring R15 fragments, the genetic determinants for the streptomycin, sulfonamide, and mercury resistances were mapped, as well as the regions necessary for EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed.     

Mapping of transfer regions within incompatibility group HI plasmid R27.

Plasmids of incompatibility group HI are large (greater than 150 kilobases [kb]) and possess an unusual thermosensitive mode of conjugative transfer. R27, the prototype IncHi1 plasmid, encodes resistance to tetracycline via a determinant which is related to transposon Tn10. A restriction endonuclease map of R27 (size, 182 kb) was recently constructed with ApaI, PstI, and XbaI. Transfer genes within R27 were mapped by insertion of Tn5 and Tn7. At least two different regions of the plasmid were concerned with transfer functions. Insertions into either region completely abolished transfer. None of the insertions had any effect on entry exclusion (Eex) of other IncH plasmids. However, a deletion mutant which lacked the Eex function was obtained, allowing us to map the probable site of the gene encoding Eex to one of the two transfer regions. The tetracycline resistance determinant in R27 was located within an 8-kb region between the two main transfer regions. The transfer genes, therefore, are not located together in R27 but are situated in at least two major widely separated transfer regions.     

Selected translocation of plasmid genes: frequency and regional specificity of translocation of the Tn3 element.

A procedure is described that selects for the insertion of transposable antibiotic resistance elements in a variety of recipient replicons. The selected translocation procedure, which employs a plasmid having a temperature-sensitive defect in replication as a donor of transposable genetic elements, was used to investigate certain characteristics of the translocation process. Our results indicate that translocation of the Tn3 element from plasmid to plasmid occurs at a 10(3)- to 10(4)-times-higher frequency than from plasmid to chromosome. In both cases, continued accumulation of Tn3 on recipient genomes is prevented by development of an apparent equilibrium when only a small fraction of molecules in the recipient population contain Tn3. An alternative method for estimation of translocation frequency has shown that the translocation process is temperature sensitive and that its frequency is unaffected by the presence of host recA mutation. Insertions of Tn3 onto the 65 X 10(6)-dalton R6-5 plasmid in Escherichia coli are clustered on EcoRI fragments 3 (8 of 23 insertions) and 9 (7 of 23 insertions), which contain 12 and 5%, respectively, of the R6-5 genome. The occurrence of multiple insertions of Tn3 within EcoRI fragment 9, which contains the IS1 element and a terminus of the Tn4 element, is consistent with earlier evidence indicating that terminal deoxyribonucleic acid sequences of already present transposable elements may provide recognition sequences for subsequent illegitimate recombinational events.     

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.     

质粒RSF1010寄主范围特性的遗传学分析

采用Tn5插入诱变、限制性核酸内切酶作图以及DNA转化等方法,对广泛寄主范围型质粒SF 1010的衍生体－pKT 2 40进行研究。证实质粒的寄主围决定于它在遗传背景不同的寄主中复制并保存自身的能力,而repA,rcpB和repC基因为该质拉复制所必需。     

Molecular genetic organization and origin of plasmid pBS52 with a broad range of bacterial hosts

The data are presented on the localization of genetic determinants of resistance to streptomycin, ampicillin and sulfanilamides on the physical map of conjugative R plasmid pBS52 of 38,000 bp which has a broad bacterial host range and belongs to a new incompatibility group. The plasmid has a natural "polylinker" site (less than 200 bp) containing (in the order of arrangement) the recognition sites for restriction enzymes: BamHI-EcoRI-PstI-EcoRV-BglII (PvuII). The comparative analysis shows that pBS52 contains a segment homologous to DNA of plasmid RSE1010 (IncP-4). The evolutionary origin of plasmid pBS52 is discussed. The recA-independent formation of the mini-derivatives of pBS373 and pBS374 types during the transformation of Escherichia coli with pBS52 plasmid DNA has been shown. Plasmids pBS373 and yBS374 are capable of autonomous replication in Pseudomonas putida and P. aeruginosa cells, which is provided by the rep system of IncP-4 replicon.     

Isolation of the replication region of an indigenous plasmid of Rhodobacter sphaeroides

Abstract The isolation of the replication region of an indigenous plasmid of 42 kb of the phototrophic bacterium Rhodobacter sphaeroides is described. This plasmid was digested with the Bgl II restriction enzyme, ligated to the 2.7 Bgl II fragment of transposon Tn 10 , which contains the tet genes conferring tetracycline resistance, and the mixture was transformed into the Escherichia coli MC1061 strain. One of several chimeric plasmids harboring the replication region of the 42-kb plasmid obtained by this process was named pUA33 and further characterized. Plasmid pUA33 is approx. 8.3 kb. A partial restriction map has been constructed. Plasmid pUA33 is stable in E. coli cells growing under non-selective conditions and is non-self-transmissible. All these data suggest that the pUA33 plasmid may be a very useful tool for gene cloning in R. spheroides .     

Broad-host-range plasmid pRK340 delivers Tn5 into the Legionella pneumophila chromosome.

Transposon Tn5 was introduced into Legionella pneumophila on plasmid pRK340, which is temperature sensitive for plasmid maintenance. The presence of plasmid DNA was confirmed by agarose gel electrophoresis and by conjugal transfer of the plasmid to Escherichia coli. Tn5 insertions were obtained by culturing L. pneumophila at the nonpermissive temperature (43 degrees C) on buffered charcoal-yeast extract agar containing kanamycin. Of the 260 kanamycin-resistant colonies picked, 220 failed to conjugate pRK340 to E. coli. Plasmid DNA was not visualized from eight randomly picked Tn5-containing strains, and Southern hybridizations indicated that Tn5, but not pRK340, inserted into multiple sites in the Legionella chromosome. In addition, the streptomycin resistance determinant on Tn5 was expressed in L. pneumophila.     

Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7

Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27. The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc-. Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C. These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible. Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible. Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour). Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids. The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa). A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site. Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites. Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27. Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27.     

The dehalogenase gene dehI from Pseudomonas putida PP3 is carried on an unusual mobile genetic element designated DEH.

As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy. The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain PP3. DEH recombined with target plasmid DNAs at high frequencies (e.g. 3.8 x 10(-4) per RP4.5 plasmid transferred). The regulated expression of dehI was detected in P. putida, Pseudomonas aeruginosa, and Escherichia coli strains containing derivative plasmids of RP4.5 and pWW0 recombined with DEH. Movement of DEH from the unstable RP4 derivatives pNJ5000 and pMR5 resulted in the insertion of DEH into the chromosome of RecA+ strains of P. putida but not in RecA+ nor RecA- strains of E. coli. Rescue of DEH from the chromosome of P. putida KT2441 onto plasmid RP4 involved recombination at a frequency (2.7 x 10(-4) per RP4 plasmid transferred) comparable to that observed in strain PP3. The DEH element was not classified as a conventional transposon because it did not move as a discrete DNA fragment: dehI-containing inserts in plasmid DNA targets varied in size between 6 and 13 kb. In addition, DEH exhibited a marked preference for insertion into a specific site on the plasmid pWW0, but its transposition, independent of host recombinational systems, remains to be demonstrated. However, the transposonlike characteristics of DEH included the conservation of restriction endonuclease sites, high-frequency recombination with different target replicons (plasmid and chromosomal DNA), and promiscuous insertion into plasmid RP4-based replicons. Therefore, it is proposed that DEH is an unusual mobile genetic element.