TOL-like Plasmid Research Articles

Promotion mechanism of self-transmissible degradative plasmid transfer in maize rhizosphere and its application in naphthalene degradation in soil

Rhizospheres can promote self-transmissible plasmid transfer, however, the corresponding mechanism has not received much attention. Plant-microbe remediation is an effective way to promote pollutant biodegradation; however, some pollutants, such as naphthalene, are harmful to plants and result in inefficient plant-microbe remediation. In this study, transfer of a TOL-like plasmid, a self-transmissible plasmid loaded with genetic determinants for pollutant degradation, among different bacteria was examined in bulk and rhizosphere soils as well as addition of maize root exudate and its artificial root exudate (ARE). The results showed that the numbers of transconjugants and recipients as well as bacterial metabolic activities, such as xylE mRNA expression levels and catechol 2,3-dioxygenase (C23O) activities of bacteria, remained high in rhizosphere soils, when compared with bulk soils. The number of transconjugants and bacterial metabolic activities increased with the increasing exudate and ARE concentrations, whereas the populations of donor and recipient bacteria were substantially unaltered at all concentrations. All the experiments consistently showed that a certain number of bacteria is required for self-transmissible plasmid transfer, and that the increased plasmid transfer might predominantly be owing to bacterial metabolic activity stimulated by root exudates and ARE. Furthermore, ARE addition increased naphthalene degradation by transconjugants in both culture medium and soil. Thus, the combined action of a wide variety of components in ARE might contribute to the increased plasmid transfer and naphthalene degradation. These findings suggest that ARE could be an effectively alternative for plant-microbe remediation of pollutants in environments where plants cannot survive.

The rhizosphere and root exudates of maize seedlings drive plasmid mobilization in soil

Plasmid mobilization is important for environments. However, the effects of rhizospheres and root exudates on plasmid mobilization have received little attention. To address this problem, mobilization of the pSUP106 plasmid by a TOL-like plasmid from Pseudomonas fluorescens P13 into rifampicin-resistant P13 cells was investigated by biparental and triparental matings in the rhizosphere soils of maize seedlings and bulk soils. Mobilization frequencies were higher in the rhizosphere than in bulk soils. Plasmid mobilization frequencies decreased with vertical and horizontal distance away from plant roots. Furthermore, plasmid mobilization frequencies increased with growing contents of root exudates and artificial root exudate (ARE) of maize seedlings for 1 or 5d of incubation time; however, donor and recipient population remained basically stable for all contents. Individual components of the exudates failed to promote mobilization of pSUP106 but a combination of sugars and amino acids of the exudates did. Removal of individual components from the ARE reduced the plasmid mobilization-promoting activity but simultaneous removal of several components destroyed the plasmid mobilization. All the data suggest that the rhizosphere of maize seedlings promoted the mobilization of the pSUP106 plasmid by the TOL-like plasmid and that root exudate of maize seedlings was the important driver of plasmid mobilization. The individual components of root exudates were necessary to increase plasmid mobilization but their combination was important for plasmid mobilization.

Factors affecting transfer of degradative plasmids between bacteria in soils

Horizontal gene transfer is useful for enhancing bioremediation through gene bioaugmentation. However, factors affecting transfer of degradative plasmids have not been systematically addressed. To this end, plasmid transfer experiments were performed using a TOL-like plasmid carrying the gene encoding for catechol 2,3-dioxygenase (C23O) between two soil bacteria under different conditions. Transfer frequency increased with air temperature in the range of 10–35°C and reached 6×10−4 transconjugants per donor cell at 35°C. The transfer frequency detected at soil depth 5–10cm was significantly higher (p<0.05) compared with other depths. Addition of 5–75% LB in the microbial inoculum promoted plasmid transfer frequencies. Addition of phenol to the experimental system resulted in significantly higher transfer frequency (p<0.05) compared with no addition. Transfer frequency heat-moisture in loam was significantly higher (p<0.05) than in other soils. The highest transfer frequency was found in the experiment containing tomato seedlings, with up to about 1.3×10−3 transconjugants per donor cell. Corn and wheat seedlings also led to significantly higher transfer frequencies (p<0.05) compared with no plants. Furthermore, C23O activities of transconjugants formed under different conditions were measured, as a surrogate measure of the activity of transconjugant. Transfer temperature, soil and plant types had a minor influence on activities of transconjugants. Topsoil (0–5cm) transconjugants expressed C23O more efficiently under normal incubation condition, but less efficiently when soils incubated with excessive LB medium concentrations, and in the absence of phenol in soil. These results suggested that transfer temperature, soil depth, dilutions of LB broth, phenol content, and soil and plant types had important effects on transfer of the TOL-like plasmid in soil, and some factors also affected activities of transconjugants.

Pseudomonas stutzeri Strain Possessing a Self-Transmissible TOL-Like Plasmid Degrades Phenol and Promotes Maize Growth in Contaminated Environments

Phenol is volatile organic pollutant that plants can little degrade. For complete degradation of volatile pollutants, we introduced Pseudomonas stutzeri strain P7 to phenol-contaminated soils. The strain effectively degraded phenol and even promoted plant growth. A TOL-like plasmid was detected in the strain and found to be responsible for phenol degradation and self-transmissible. In addition, phenol degradation by strain P7 was more rapid in the contaminated soils with than without plants over the full course of the experiment; especially by 5days, the phenol concentration was reduced by about 30% in soil without plants and reduced by about 50-65% in soil with plants. This situation also occurred when inoculated with different transconjugants. Furthermore, transfer frequencies of TOL-like plasmid were significantly higher in soil with than without plants. Populations of rifampin-resistant P7 strain remained relatively constant for 20days, while the number of rhizosphere bacteria that contained the degradative plasmids gradually increased at the later stages, suggesting that plants might stimulate plasmid transfer from strain P7 to indigenous bacteria, one possible reason for plant enhancing microbial degradation. This is attractive for implementation of combinations of phytoremediation and bioaugmentation in degradation of volatile pollutants that plants can little degrade.

Continuous cultures of Pseudomonas putida mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h(-1) under toluene loading rates of 259 +/- 23 and 801 +/- 78 g m(-3) h(-1) (inlet toluene concentrations of 3.5 and 10.9 g m(-3), respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol(-) population at day 4 to approx. 100% Tol(+) cells from day 13 onward, sustaining elimination capacities of 232 +/- 10 g m(-3) h(-1) at 3.5 g Tol m(-3) and 377 +/- 13 g m(-3) h(-1) at 10.9 g Tol m(-3), which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol(-) variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.

Conjugal transfer of a TOL-like plasmid and extension of the catabolic potential ofPseudomonas putidaF1

Strain mX was isolated from a petrol-contaminated soil, after enrichment on minimal medium with 0.5% (v/v) meta-xylene as a sole carbon source. The strain was tentatively characterized as Pseudomonas putida and harboured a large plasmid (pMX) containing xyl genes involved in toluene or meta-xylene degradation pathways via an alkyl monooxygenase and a catechol 2,3-dioxygenase. This new TOL-like plasmid was stable over two hundred generations and was self-transferable. After conjugal transfer to P. putida F1, which possesses the Tod chromosomal toluene biodegradative pathway, the transconjugant P. putida F1(pMX) was able to grow on benzene, toluene, meta-xylene, para-xylene, and ethylbenzene compounds as the sole carbon sources. Catechol 2,3-dioxygenases of the transconjugant cells presented a more relaxed substrate specificity than those of parental cells (strain mX and P. putida F1).

Phenol degradation by an enterobacterium: aKlebsiellastrain carries a TOL-like plasmid and a gene encoding a novel phenol hydroxylase

Although phenol catabolism is described for many different microorganisms, there is no example for such a pathway in an enterobacterial strain. Here we characterize a Klebsiella oxytoca strain that grows on phenol as the only source of carbon and energy. As the key enzyme of phenol degradation, phenol hydroxylase was purified to apparent homogeneity. Compared with other phenol hydroxylases, the Klebsiella enzyme differs with respect to several properties: (i) SDS-PAGE and gel-filtration analysis of the purified protein revealed that the enzyme is a monomer with a molecular mass of 156 kDa; (ii) steady-state kinetic measurements resulted in a K(m) value of 0.22 mM for phenol; and (iii) the enzyme is both dependent on NADPH/FAD and sensitive to EDTA. Further degradation of catechol, the reaction product of phenol hydroxylase, may occur via the effective meta-fission pathway often located on TOL or TOL-like plasmids. Such a plasmid was prepared from the Klebsiella strain and further characterized. The given data demonstrate that the isolated strain exhibits all characteristics of an efficient phenol-degrading microorganism.

Phenol degradation by an enterobacterium: a &lt;i&gt;Klebsiella&lt;/i&gt; strain carries a TOL-like plasmid and a gene encoding a novel phenol hydroxylase

Phenol degradation by an enterobacterium: a &lt;i&gt;Klebsiella&lt;/i&gt; strain carries a TOL-like plasmid and a gene encoding a novel phenol hydroxylase

Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida

Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU. Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s). Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose. Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor. Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O. Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants. Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes. No variants formed in identical ethylbenzene-exposed controls. The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pathway.

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9.

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.

Characterization of a novel TOL-like plasmid from Pseudomonas putida involved in 1,2,4-trimethylbenzene degradation.

A strain of Pseudomonas putida (TMB) was found to resemble P. putida mt-2 (PaW1) in its ability to degrade 1,2,4-trimethylbenzene, toluene, m-xylene, and p-xylene via oxidation of a methyl substituent and reaction of the meta fission pathway, but a different regulatory model is suggested. The ability of P. putida TMB to degrade these substrates was encoded by plasmid pGB (85 kilobase pairs), which showed considerable differences in size, restriction patterns, and DNA sequence from those of plasmid pWWO of strain PaW1.

The Purification and Properties of Two Isofunctional 2-Hydroxy-6-oxohepta-2,4-dienoate Hydrolases from Alcaligenes eutrophus Strain 345

SUMMARY: Two 2-hydroxy-6-oxohepta-2,4-dienoate (HOD) hydrolases have been purified to homogeneity from Alcaligenes eutrophus ATCC 17707. One, HODHII, is encoded by the TOL-like plasmid, pRA1000, and the other, HODHI, is chromosomally-encoded. HODHI and HODHII have an M r of 104 × 103 and 116 × 103, respectively, and both are dissociated by SDS into three subunits of equal size which are not linked by disulphide bonds. Both hydrolases are active against a similar range of substrates formed by the action of catechol 2,3-oxygenases, generally being more active against substrates formed from 3-alkyl- rather than 4-alkyl-substituted catechols. HODHI and HODHII had similar K m values for HOD, but HODHI had a significantly lower K m value for, and a higher activity against, 2-hydoxymuconic semialdehyde than HODHII. It was shown by immunodiffusion studies that the two hydrolases were immunologically distinct and that HODHI had no common antigenic determinants with HOD hydrolases from the Pseudomonas putida strains NCIB 9865, NCIB 10015 and mt-2; HODHII gave a line of identity with antiserum to the hydrolase from P. putida NCIB 9865. A comparison of the physico-chemical properties of HODHI and HODHII with the same properties of the HOD hydrolases from the three P. putida strains demonstrated several similarities between the five HOD hydrolases which have been purified.

Characterization of a TOL-like plasmid from Alcaligenes eutrophus that controls expression of a chromosomally encoded p-cresol pathway

Alcaligenes eutrophus wild-type strain 345 metabolizes m- and p-toluate via a catechol meta-cleavage pathway. DNA analysis, curing studies, and transfer of this phenotype by conjugation and transformation showed that the degradative genes are encoded on a self-transmissible 85-kilobase plasmid, pRA1000. HindIII and XhoI restriction endonuclease analysis of pRA1000 showed it to be similar to the archetypal TOL plasmid, pWWO, differing in the case of HindIII only by the absence of fragments B and D present in pWWO. In strain 345, the presence of pRA1000 prevented the expression of chromosomally encoded enzymes required for the degradation of p-cresol, whereas these enzymes were expressed in strains cured of pRA1000. On the basis of studies with an R68.45-pRA1000 cointegrate plasmid, pRA1001, we conclude that the gene(s) responsible for the effect of p-cresol degradation resides within or near the m- and p-toluate degradative region on pRA1000.