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1.
The kinetics of the conjugal transfer of a TOL plasmid were investigated by using Pseudomonas putida PAW1 as the donor strain and P. aeruginosa PAO 1162 as the recipient strain. Short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. The experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. This method allowed analysis of the mating experiments by a single intrinsic kinetic parameter for conjugal plasmid transfer. Further results indicated that the specific growth rate of the donor strain antecedent to the mating experiment had a strong impact on the measured intrinsic plasmid transfer rate coefficient, which ranged from 1 x 10(-14) to 5 x 10(-13) ml per cell per min. Preliminary analysis suggested that the transfer rates of the TOL plasmid are large enough to maintain the TOL plasmid in a dense microbial community without selective pressures.  相似文献   

2.
Abstract Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO 1162 (TOL) as the plasmid donor and Pseudomonas putida PB 2442 and Pseudomonas aeruginosa PAO 1162N as the plasmid recipients. Transfer rates from PAO 1162 (TOL) to PAO 1162N and PB 2442 measured for exponentially grown PAO 1162 (TOL) were 1.81 × 10−14 (standard error (S.E.) 1.25 × 10−15) ml·cell−1min−1 and 3.32 × 10−13 (S.E. 4.42 × 10−14) ml·cell−1min−1, respectively. The instability of the TOL plasmid in PAO 1162 (TOL) was evaluated under conditions that were non-selective for maintenance of the TOL catabolic functions. The measured rates of instability were 6.7 10−6 to 8.3 10−6 min−1, and the loss of the catabolic functions was mainly caused by structural instability of the plasmid.  相似文献   

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Chromosomal location of TOL plasmid DNA in Pseudomonas putida.   总被引:5,自引:6,他引:5       下载免费PDF全文
The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid. By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB. A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18. Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.  相似文献   

6.
Conjugation of catabolic plasmids in contaminated environments is a naturally occurring horizontal gene transfer phenomenon, which could be utilized in genetic bioaugmentation. The potentially important parameters for genetic bioaugmentation include gene regulation of transferred catabolic plasmids that may be controlled by the genetic characteristics of transconjugants as well as environmental conditions that may alter the expression of the contaminant-degrading phenotype. This study showed that both genomic guanine–cytosine contents and phylogenetic characteristics of transconjugants were important in controlling the phenotype functionality of the TOL plasmid. These genetic characteristics had no apparent impact on the stability of the TOL plasmid, which was observed to be highly variable among strains. Within the environmental conditions tested, the addition of glucose resulted in the largest enhancement of the activities of enzymes encoded by the TOL plasmid in all transconjugant strains. Glucose (1 g/L) enhanced the phenotype functionality by up to 16.4 (±2.22), 30.8 (±7.03), and 90.8 (±4.56)-fold in toluene degradation rates, catechol 2,3-dioxygenase enzymatic activities, and xylE gene expression, respectively. These results suggest that genetic limitations of the expression of horizontally acquired genes may be overcome by the presence of alternate carbon substrates. Such observations may be utilized in improving the effectiveness of genetic bioaugmentation.  相似文献   

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The Pseudomonas putida TOL plasmid pWW0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). Transconjugants are recipient cells that have received DNA from donor cells, whereas retrotransconjugants are donor bacteria that have received DNA from a recipient. The TOL plasmid pWW0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrated into the chromosome of other P. putida strains, a process that appears to involve a single conjugational event. The rate of retrotransfer (as well as of direct transfer) of the chromosomal marker is influenced by the location of the kanamycin marker on the chromosome and ranges from 10(-3) to less than 10(-8) retrotransconjugants per donor (transconjugants per recipient). The mobilized DNA is incorporated into the chromosome of the retrotransconjugants (transconjugants) in a process that seems to occur through recombination of highly homologous flanking regions. No interspecific mobilization of the chromosomal marker in matings involving P. putida and the closely related Pseudomonas fluorescens, which belongs to rRNA group I, was observed.  相似文献   

9.
Summary A cleavage map of the TOL plasmid pWWO has been determined for the restriction endonucleases HindIII and XhoI. A number of techniques were employed including (i) digestion of purified cleavage products with a second enzyme; (ii) hybridisation of purified XhoI fragments to Southern blots of HindIII digest products and (iii) analysis of a number of deletion mutants.  相似文献   

10.
Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida   总被引:3,自引:0,他引:3  
The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas. It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653. Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames. Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition. All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions. The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins. pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids. In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events. Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified. Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds. The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.  相似文献   

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We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.  相似文献   

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Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.  相似文献   

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Regulation of the xyl gene operons of the Pseudomonas putida TOL plasmid is mediated by the products of the downstream clustered and divergently oriented xylR and xylS regulatory genes. The xylR-xylS intergenic region contains the xylR and xylS promoters Pr and Ps, respectively. A binding site for the XylR activator protein is located upstream of Ps and overlapping Pr. DNase I footprint experiments showed that one of these sites, which overlaps the recognition site for XylR activator, as well as an AT-rich region comprising the Ps promoter consensus were protected by integration host factor (IHF). IHF was found to act negatively in the in vivo activation of the Ps promoter, since the activity of a Ps promoter::lacZ fusion was elevated in an Escherichia coli mutant lacking IHF. In contrast, no alteration in the synthesis of XylR protein in the E. coli IHF-deficient mutant was detected.  相似文献   

18.
Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.  相似文献   

19.
We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.  相似文献   

20.
Continuous culture fermentation of Pseudomonas putida PPK1, containing the naturally occurring TOL plasmid pTK0, was carried out in either benzoate- or succinate-limited chemostats. The apparent stability of the plasmid decreased with increasing the dilution rate. An established model was used to predict the contribution of segregational instability and growth rate difference to the apparent plasmid stability.  相似文献   

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