Benzoate-dependent induction from the OP2 operator-promoter region of the TOL plasmid pWWO in the absence of known plasmid regulatory genes.

Expression of the lower catabolic pathway of the TOL plasmid pWWO requires an aromatic acid inducer and the product of the xylS regulatory gene. Pseudomonas putida cells transformed with a plasmid containing the operator-promoter region of the lower pathway (OP2 [or Pm]), upstream from the catechol 2,3-dioxygenase structural gene, showed enzyme induction in the absence of known TOL plasmid regulatory genes. Induction was not seen in transformed Escherichia coli cells or in a P. putida mutant lacking chromosomally encoded benzoate catabolic functions.     

Regulatory mutants affecting the use of the TOL catabolic plasmid in strain construction

Summary The pathway encoded by the TOL catabolic pathway has been reported to be in two regulons. Attempts to isolate fully constitutive mutants of the plasmid encoded catechol meta cleavage pathway (the lower regulon) were unsuccessful. However mutants were obtained with altered inducer specificity of this regulon. This was accompanied by loss or alteration of inducer specificity with both regulons and could cause problems with the use of the TOL plasmid in specific strain construction work.     

A comparative study of the NAH and TOL catabolic plasmids in Pseudomonas putida

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives. The NAH and TOL plasmids were found to be incompatible. Under the conditions used in these experiments the TOL plasmid transferred into some strains into which NAH was unable to transfer. The use of mutants to remove certain catabolic activities encoded by the bacterial host cell facilitated the allocation of growth genotypes to the NAH and TOL plasmids. TOL encoded the degradation of benzoate, m-toluate and p-toluate, whereas NAH encoded the degradation of naphthalene and salicylate. The other plasmid-associated growth phenotypes were partly plasmid-specified and partly specified by the host cell. The pH optimum of the catechol 2,3-dioxygenase specified by the TOL plasmid was approximately 6.7, whereas that of the NAH-encoded enzyme was approximately 8.3.     

Stability and conjugal transfer kinetics of a TOL plasmid in Pseudomonas aeruginosa PAO 1162

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.     

Excision and integration of degradative pathway genes from TOL plasmid pWW0.

WR211 is a transconjugant resulting from transfer of the 117-kilobase (kb) TOL degradative plasmid pWW0 into Pseudomonas sp. strain B13. The plasmid of this strain, pWW01211, is 78 kb long, having suffered a deletion of 39 kb. We show that WR211 contains the 39 kb that is missing from its plasmid, together with at least an additional 17 kb of pWW0 DNA integrated in another part of the genome, probably the chromosome. The ability of WR211 to grow on the TOL-specific substrate m-toluate is the result of expression of the TOL genes in this alternative location, whereas its inability to grow on m-xylene is caused by insertional mutagenesis by 3 kb of DNA of unknown origin in the xylR gene of this DNA. The resident plasmid pWW01211 plays no part in the degradative phenotype of WR211 since it can be expelled by mating in incompatible IncP9 resistance plasmid R2 or pMG18 without loss of the phenotype. This alternatively located DNA can be rescued back into the R2 and pMG18 plasmids as R2::TOL and pMG18::TOL recombinants by mating out into plasmid-free recipients and selecting for Mtol+ transconjugants. In all cases examined, these plasmids contained the entire R plasmid into which is inserted 59 kb of DNA, made up of 56 kb of pWW0 DNA and the 3-kb xylR insertion. Selection for faster growth on benzoate can lead to precise excision of the 39 kb from the TOL region of an R2::TOL recombinant, leaving a residual and apparently cryptic 17-kb segment of pWW0 DNA in the R plasmid.     

TOL plasmid in Pseudomonas aeruginosa PAO: thermosensitivity of self-maintenance and inhibition of host cell growth.

The TOL plasmid originally isolated in Pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of Pseudomonas, i.e., P. putida, P. fluorescens, and P. aeruginosa, except for a strain of P. aeruginosa, strain PAO. The same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. In addition, the transmissibility of the TOL plasmid from strain PAO to P. putida was low when the plasmid was modified by the donor. By using P. aeruginosa PAO carrying the TOL plasmid, the stability and genetic expression of the plasmid as well as its effect on the host cell growth were examined. Thus the self-maintenance of the plasmid was found to be thermosensitive. Furthermore, the TOL plasmid inhibited the growth of strain PAO at high temperature, accompanied by the formation of some filamentous cells. These thermosensitive properties of the TOL plasmid were host dependent and not exhibited in another strain of P. aeruginosa.     

The specific growth rate of Pseudomonas putida PAW1 influences the conjugal transfer rate of the TOL plasmid.

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.     

Isolation of a mutant TOL plasmid with increased activity and transmissibility from Pseudomonas putida (arvilla) mt-2.

Strains with greater ability to dissimilate m-toluate were obtained from the wild-type Pseudomonas putida (arvilla) mt-2 that harbors the TOL plasmid. Increased growth of a mutant strain on aromatic substrates was coupled with simultaneous increase in the activity of metapyrocatechase, an enzyme coded by the TOL plasmid, without changing its catalytic properties. In the mutant and the wild-type strains, the inducer specificity and the induction kinetics of metapyrocatechase synthesis were the same, and a half-maximal effect of m-toluate on the enzyme synthesis was observed at 0.25 mM. Thus, the increased utilizability seen in a mutant strain appeared to be due to an increased quantity of the enzymes coded by the TOL plasmid. The properties of the mutant strain were dependent upon the mutation on the TOL plasmid but not on the chromosome mutation. Transfer experiments with a strain carrying the mutant TOL (TOL-H) or the wild-type TOL plasmid revealed that the TOL-H transfer was 1,000 times greater than that of the wild type.     

Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene

In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates. A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P. putida mt-2. The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate. These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator. Such gene cassettes (mini-Tn5 [UPP2] and mini-Tn5 [TOD2]) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P. aeruginosa JB2. GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene. However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source. These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process. Choices to address this general problem are discussed.     

The TOL plasmid is naturally derepressed for transfer

Pseudomonas putida mt-2, formerly known as Pseudomonas arvilla mt-2, which carries the wild-type TOL plasmid, and P. putida strain AC37 carrying TOL, were completely lysed by the pilus-adsorbing plasmid-specific bacteriophages PR4 and PRD1. Pseudomonas putida strain PpS388, also harbouring the plasmid, was not lysed. In a P. putida mt-2 host, TOL transferred 18-fold better on a surface (2.5 X 10(-1) transconjugants per donor h-1) than in liquid; when P. putida PpS388 was the host, however, a frequency of only 2.3 X 10(-4) transconjugants per donor h-1 was obtained. Thus, TOL was derepressed for transfer in P. putida mt-2 and P. putida AC37, but not in P. putida PpS388. Electron microscopy revealed that TOL determined thick (8.5-10 nm diameter) flexible pili in large numbers, suggesting constitutive expression in its derepressed state.     

An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression.

Pseudomonas aeruginosa strain 9169 has been reported to contain a plasmid that expresses resistance to carbenicillin (Cb), kanamycin (Km), and tetracycline (Tc) in Escherichia coli but resistance only to Cb in certain Pseudomonas recipients. The triply resistant plasmid in E. coli belonged to incompatibility (Inc) group P or P-1, whereas the singly resistant plasmid in P. aeruginosa was compatible with IncP-1 plasmids and other plasmids of established Inc specificity but incompatible with plasmid pSR1 that is here used to define a new Pseudomonas Inc group P-10. Additional physical and genetic studies showed that strain 9169 contained not one but two plasmids: IncP-1 plasmid R91a, determining the Cb Km Tc phenotype, and IncP-10 plasmid R91, determining Cb that differed in molecular weight and in EcoRI and BamHI restriction endonuclease recognition sites. Plasmid multiplicity rather than host effects on plasmid gene expression can account for differences in the phenotype of strain 9169 transconjugants to E. coli and P. aeruginosa.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

Functionality of the TOL plasmid under varying environmental conditions following conjugal transfer

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.     

Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture

Pseudomonas putida mt-2, harbouring the TOL plasmid pWW0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates. The fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and xylene (TOL- cells), was determined. Genetic analysis revealed that all TOL- cells isolated harboured partially deleted plasmids, lacking the TOL catabolic genes. The growth-rate advantage of the TOL- cells was quantified from the kinetics of their increase as a fraction of the total population. At a dilution rate of 0.1 h-1 no growth-rate advantage of TOL- cells was found when phosphate or ammonium were limiting. Under sulphate-limitation, ingrowth of TOL- cells was evident but did not follow a straightforward pattern. Under succinate-limitation the growth-rate advantage was the highest, particularly at low dilution rates (about 50% at D = 0.05 h-1). In phauxostat culture, at the maximal growth rate, the growth-rate advantage of TOL- cells was less than 1%. The specific activity in TOL+ cells of the plasmid-encoded enzyme catechol 2,3-dioxygenase was relatively high at a low growth rate.     

Transfer-deficient mutants of the narrow-host-range plasmid R91-5 of Pseudomonas aeruginosa.

Three methods have been successful in the isolation of transfer-deficient mutants of the narrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa: (i) selection for donor-specific phage resistance; (ii) direct screening after mutagenic treatment with either ethyl methane sulfonate or N-methyl-N'-nitro-N-nitrosoguanidine; (iii) in vitro mutagenesis of plasmid DNA by hydroxylamine followed by transformation and direct screening. The majority of transfer-deficient mutants were donor-specific phage resistant, supporting the view that sex pili and other surface components are essential for conjugal transfer (since the phages PRD1 and PR4 adsorb to these sites). Some of the transfer-deficient mutants were also unable to inhibit the replication of phage G101 or lost entry exclusion or both phenotypes. The ability to revert these pleiotropic mutants to wild type implicates the latter two functions in R91-5 transfer. Suppressor mutations in P. aeruginosa enabled the detection of suppressor-sensitive, transfer-deficient mutants. Such mutants should prove useful in conjugational complementation tests for the identification of the transfer cistrons of R91-5.     

Quantification of the effect of substrate concentration on the conjugal transfer rate of the TOL plasmid in short-term batch mating experiments

Batch mating experiments with Pseudomonas putida PAW 1 (TOL) as a donor and Pseudomonas aeruginosa PAO 1162 as a recipient strain were performed to quantify the effect of the substrate concentration in the mating medium on the observed plasmid transfer rate coefficient. The impact of the substrate concentration in the mating medium was highly correlated with the growth history of the donor strain. When the donor strain was harvested in exponential growth phase, no impact was observed; when the donor strain was taken from the stationary phase, however, a strong impact of the substrate concentration was measured: a 10-fold reduction in the substrate concentration decreased the observed plasmid transfer rate by 55%.     

The TOL Plasmid pWW0 xylN Gene Product from Pseudomonas putida Is Involved in m-Xylene Uptake

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K(s) value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.     

Physical and genetic analysis of deletion mutants of plasmid R91-5 and the cloning of transfer genes in Pseudomonas aeruginosa.

We isolated deletion mutants of Pseudomonas aeruginosa plasmid R91-5 by both in vitro and in vivo means. Many of the deletion mutants selected on the basis of resistance to donor-specific phages fell into a few groups of apparently identical mutants, although the mutants were nonsibs. By analyzing plasmids with large deletions, we found that the essential replication genes of R91-5 were within a 3.85-kilobase region between coordinates 45.5 and 48.9. The origin of plasmid transfer (oriT) was mapped to a 4.5-kilobase region between coordinates 1.7 and 6.2. We indirectly determined the direction of plasmid transfer from oriT. By combining the data from our analysis of the deletions with data from complementation tests between cloned R91-5 fragments and known reference mutants, we ordered and mapped the 10 known transfer (tra) cistrons of R91-5. All of the tra cistrons mapped within the Tra2 region, and their order was as follows: traX, -Y, -T, -Q, -(V, R), -U, -(S, Z), -W (the cistrons in parentheses could not be ordered with respect to each other).     

Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid

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.