Fertility factors in Pseudomonas putida: selection and properties of high-frequency transfer and chromosome donors.

The octane plasmid (OCT) in Pseudomonas putida strains has been shown to be transferred at low frequency. However, bacteria which had newly received this plasmid showed a transient increase in donor ability. Using Octane+ P. putida as the donor, the transfer of most chromosomal markers was shown to be independent of OCT transfer, whereas the mobilization of the octanoate catabolism genes (octanoic and acetate) was dependent on OCT plasmid transfer. The presence of a fertility factor termed FPo has been postulated to explain these results. Strains carrying only this fertility factor have been obtained from strains carrying both OCT and FPo plasmids. Strains in which the OCT plasmid was transferred at high frequencies have also been isolated, and chromosome mobilization by OCT and FPo has been compared. A different gradient of transmission by OCT and FPo has been observed. It has also been shown that chromosome transfer by OCT was dependent on the bacterial recombination system, whereas the chromosome transfer by FPo was unaffected by the presence of a rec mutation in the donor strain.     

Integration of R91-5::Tn501 into the Pseudomonas putida PPN chromosome and genetic circularity of the chromosomal map

Derivatives of the Pseudomonas aeruginosa plasmid R91-5, loaded with the transposon Tn501, were transferred to P. putida PPN. Over 90% of exconjugants, which arose at a frequency of ca. 10(-6) per donor cell, exhibited high-frequency (greater than 10(-2) per donor cell) polarized transfer of chromosomal markers. In one instance it was demonstrated by transduction that the plasmid had been inserted into a gene required for serine biosynthesis. The integrated nature of the plasmid in this and other P. putida (R91-5::Tn501) derivatives was supported by the failure to detect covalently closed circular DNA in these strains. The transfer origins of six different Hfr donors have been characterized genetically, and time-of-entry kinetics obtained from interrupted matings have enabled the construction of a circular genetic map 103 min in length and containing 35 markers. The genetic map of P. putida PPN shows significant differences in marker order to that of P. aeruginosa PAO.     

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.     

Isolation and characterization of Pseudomonas putida R-prime plasmids

A number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient. For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved. Physical analysis of one R-prime showed that 3.5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.     

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.     

Transfer of chromosomal genes and formation of R'-derivatives using PR4::D312cts15 plasmids in Pseudomonas aeruginosa cells

Several hybrid RP4 plasmids containing the genome of heat-inducible D3112cts15 phage integrated into 2 different sites of RP4 were selected. It was shown that the plasmids RP4::D3112cts15 mobilized the chromosome of Pseudomonas aeruginosa from many sites located in different chromosome regions. Chromosomal recombinants are, formed at frequencies of about 10(-4) per recipient cell. Analysis of coinheritance of unselected markers showed that the majority of recombinants inherited short donor chromosome fragments (about 5 min). R' plasmids can be easily selected by mating with a rec- recipient. For instance, the frequency of selection of R' plasmids containing argH+ locus was about 10(-5) per donor cell. Conjugative transfer of RP4::D3112cts15 into nonlysogenic strains PAO P. aeruginosa results in partial or complete loss of prophage from a hybrid plasmid. The RP4::D3112cts15 plasmids appear to have retained the broad host range of the original RP4 (they are maintained in P. putida and Escherichia coli).     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

Genetic mapping of the obligate methylotroph Methylobacillus flagellatum: characteristics of prime plasmids and mapping of the chromosome in time-of-entry units.

A pULB113 (RP4::mini-Mu cts) plasmid was used to generate a library of prime plasmids carrying fragments of the Methylobacillus flagellatum genome. The genes carried by these prime plasmids were identified by complementation after transfer to suitably marked Escherichia coli and Pseudomonas aeruginosa strains. The hybrid plasmids were used for complementation mapping with a range of E. coli, M. flagellatum, and P. aeruginosa mutants. A preliminary map of the M. flagellatum genome section with seven groups of linked markers was obtained. Three of seven groups contain an overlapping sequence of cloned genes and can be considered as one large group of linked genes. A high-frequency-of-recombination donor of M. flagellatum (strain MFK64) mobilized the chromosome in a polarized manner from a single transfer origin. The donor was used to construct a time-of-entry map of the M. flagellatum chromosome. This was achieved by determining the time of entry of six randomly dispersed markers, four of which are included in known groups of linked markers. The linear map of M. flagellatum reported here consists of 44 markers.     

Chromosomal map of Pseudomonas putida PPN, and a comparison of gene order with the Pseudomonas aeruginosa PAO chromosomal map

The generalized transducing phage Pf16h2 has been used to confirm linkage relationships of chromosomal markers of Pseudomonas putida previously determined from their time-of-entry in Hfr crosses, and to map new auxotrophic mutations. By means of spot matings using Hfr donors of known origin of transfer, catabolic markers forming part of a closely linked group of operons referred to as a superoperonic cluster have been shown to be chromosomally located and their map positions determined. R-prime-mediated interspecific complementation has been used to equate functionally 21 auxotrophic loci in P. putida and P. aeruginosa, and the distribution of these loci on the two genetic maps has been compared. While both maps reveal that auxotrophic markers are largely restricted to about 40% of the chromosome and that auxotrophic markers of similar phenotype are not clustered, there is evidence of at least seven chromosomal rearrangements since divergence from a presumed common ancestor.     

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.     

Physical and genetic mapping of the catA region of Pseudomonas aeruginosa.

A prime plasmid has been used as the basis for the construction of a physical and genetic map of a 125 kb segment of the Pseudomonas aeruginosa PAO chromosome. Using pMO1811, a prime plasmid selected for the catA region, a series of Tn5 insertions were obtained which identified two new markers gcu (glycine utilization) and oap (organic acids and alcohols permeability) in the 125 kb region and located them in relation to other known markers of this region. A cosmid bank was constructed from the prime plasmid and an ordered array of cosmid clones for this region identified by restriction endonuclease mapping with EcoRI, HindIII and KpnI, as well as complementation mapping and chromosome walking. By Southern hybridization analyses, it was confirmed that the chromosomal insert carried by pMO1811 was flanked by single, tandemly arranged copies of IS21 and the orientation of the insert on this prime was determined. This cosmid bank provides a resource for the further analysis of this region of the P. aeruginosa genome.     

Incompatibility groups of epsilon-caprolactam biodegradation plasmids from bacteria of Pseudomonas genus

Incompatibility of epsilon-caprolactam biodegradation plasmids pBS262, pBS263, pBS264, pBS265, pBS266, pBS267, pBS268, pBS270, pBS276, pBS269 with the tester plasmids of P-1, P-2, P-7, P-9 incompatibility groups in the system of strains of P. putida line BSA, as well as the character of plasmid interaction with the number of P. aeruginosa and P. putida bacteriophages have been studied. The majority of the studied plasmids belongs to IncP-7, IncP-9 or simultaneously to IncP-7 and IncP-9 incompatibility groups. The ability to restrict the growth of some bacteriophages of P. aeruginosa and P. putida has been demonstrated for some plasmids.     

Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light.

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded.     

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.     

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.     

Transduction of Low-Copy Number Plasmids by Bacteriophage P22

The generalized transducing bacteriophage of Salmonella typhimurium, P22, can transduce plasmids in addition to chromosomal markers. Previous studies have concentrated on transduction of pBR322 by P22 and P22HT, the high transducing mutant of P22. This study investigates the mechanism of P22HT transduction of low-copy number plasmids, namely pSC101 derivatives. We show that P22HT transduces pSC101 derivatives that share homology with the chromosome by two distinct mechanisms. In the first mechanism, the plasmid integrates into the chromosome of the donor by homologous recombination. This chromosomal fragment is then packaged in the transducing particle. The second mechanism is a size-dependent mechanism involving a putative plasmid multimer. We propose that this multimer is formed by interplasmidic recombination. In contrast, P22HT can efficiently transduce pBR322 by a third mechanism, which is independent of plasmid homology with the chromosome. It has been proposed that the phage packages a linear concatemer created during rolling circle replication of pBR322, similar in fashion to phage genome packaging. This study investigates the role of RecA, RecD, and RecF recombination proteins in plasmid/plasmid and plasmid/chromosome interactions that form packageable substrates in the donor. We also examine the resolution of various transduced plasmid species in the recipient and the roles of RecA and RecD in these processes.     

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.     

Recruitment of a chromosomally encoded maleylacetate reductase for degradation of 2,4-dichlorophenoxyacetic acid by plasmid pJP4.

When Pseudomonas aeruginosa PAO1c or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes enzymes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not grow on TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6- and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1.     

Transposon-mediated integration of the RP1 plasmid into chromosomes of methylotrophic bacteria

The homology region between the DNA of plasmid RP1ts::Tn601 and chromosome of the thermotolerant methylotrophic bacterium Methylobacterium sp. SKF240 has been constructed by transposon Tn601 translocation into the chromosome. The clones of Methylobacterium sp. SKF240 having integrated the plasmid RP1 into the chromosome have been obtained by conjugation on the basis of above mentioned genetic technique. The integration of plasmid RP1 into the chromosomal DNA of the methylotroph has been confirmed by the genetic and electrophoretic methods. Clones harbouring the integrated plasmid are able to transfer the chromosomal genes for methionine and isoleucine-valine synthesis to the recipient cells of P. aeruginosa PAO ML4262 by conjugation.     

Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains.