A cleavage map of the TOL plasmid of Pseudomonas putida mt-2.

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.     

Toluene Elicits a Carbon Starvation Response in Pseudomonas putida mt-2 Containing the TOL Plasmid pWW0

Pseudomonas putida mt-2(pWWO) exhibited a carbon starvation response in the presence of toluene, a utilizable carbon source. When growth-supporting (4-mg/liter), inhibitory (130-mg/liter), and lethal (267-mg/ liter) levels of toluene were provided as the sole carbon source, P. putida responded by rapidly inhibiting protein synthesis and by producing 26 new proteins, 22 of which overlapped with those induced by carbon starvation. P. putida produced the same proteins when cultures were starved by depleting their carbon source or were downshifted into a carbon-free medium. Carbon supplementation of toluene-exposed cells suppressed the production of the toluene-induced proteins. The level of toluene provided as the sole carbon source influenced the length of time that this response was observed. Following 1.5 to 3 h in a basal salts medium with 4 mg of toluene per liter, protein synthesis increased, the production of the majority of the toluene-induced proteins ceased, and the cells began to grow. In cells provided with 130 mg of toluene per liter, protein synthesis remained inhibited over a 6.5-h experimental period. At this concentration, the production of 15 toluene-induced proteins was prolonged, with nine still detectable in the profiles at 6.5 h. In cells provided with 267 mg of toluene per liter, there was a rapid loss of viability and the toluene-induced proteins were detected prior to death. In cells provided with 4 mg of toluene per liter, the carbon starvation response is transient and likely reflects a period of induction and/or adaptation prior to growth on toluene. At the toluene concentrations which inhibit growth, P. putida exhibits a prolonged starvation response despite the presence of an excess of a utilizable carbon source.     

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.     

Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2

Pseudomonas putida mt-2 carries a plasmid (TOL, pWWO) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. Characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m-toluate, whereas the inducers of the enzymes responsible for oxidation of m-xylene to m-toluate include m-xylene and m-methylbenzyl alcohol but not m-toluate. A regulatory mutant is described in which m-xylene and m-methylbenzyl alcohol no longer induce any of the pathway enzymes, but m-toluate is still able to induce the enzymes responsible for its own degradation. Among revertants of this mutant are some strains in which all the enzymes are expressed constitutively and are not further induced by m-xylene. A model is proposed for the regulation of the pathway in which the enzymes are in two regulatory blocks, which are under the control of two regulator gene products. The model is essentially the same as proposed earlier for the regulation of the isofunctional pathway on the TOL20 plasmid from P. putida MT20.     

Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2.

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS.     

Metabolism of Benzoate and the Methylbenzoates by Pseudomonas putida (arvilla) mt-2: Evidence for the Existence of a TOL Plasmid

Mutant strains of Pseudomonas putida (arvilla) mt-2 which have lost the ability to grow at the expense of m- or p-toluate (methylbenzoate) but retain the ability to grow with benzoate arise spontaneously during growth on benzoate; this genetic loss occurs to a lesser extent during growth on nonaromatic carbon sources in the presence of mitomycin C. The mutants have totally lost the activity of the enzymes of the divergent meta pathway with the possible exception of 2-oxopent-4-enoate hydratase and 4-hydroxy-2-oxovalerate aldolase; unlike the wild type they utilize benzoate by the ortho pathway. Evidence is presented that these mutants have lost a plasmid coding for the enzymes of the meta pathway, which may be transmitted back to them or into other P. putida strains. Preliminary results from these mutants and from a mutant defective in the regulation of the plasmid-carried pathway suggest that the wild type contains two benzoate oxidase systems, one on the plasmid which is nonspecific in both its catalysis and its induction and one on the chromosome which is more specific to benzoate as substrate and is specifically induced by benzoate.     

An endonuclease cleavage map of the plasmid pWWO-8, a derivative of the TOL plasmid of Pseudomonas putida mt-2.

Summary Cleavage sites on the pWWO-8 plasmid were determined for the restriction endonucleases HindIII and XhoI. Terminal labelling using DNA polymerase I was particularly useful both for the characterisation of the smaller cleavage products and for confirmation of the order of fragments in the intact plasmid.     

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.     

Adaptation of Pseudomonas putida mt-2 to growth on aromatic amines

Pseudomonas putida mt-2 (ATCC 33015) carrying the TOL plasmid pWW0 could adapt to growth on the aromatic amines aniline and m- and p-toluidine. In strain UCC2, a derivative adapted to rapid growth on these compounds, they were oxidatively deaminated to catechol or 4-methylcatechol, which in turn were dissimilated by a meta-cleavage pathway. The aniline/toluidine oxygenase and the meta-cleavage pathway enzymes were inducible by aromatic amines. Evidence is presented that in strain UCC2, plasmid pWW0 has undergone deletion of its catabolic genes, and that it is a novel plasmid, pTDN1, which is involved in the catabolism of aniline and m- and p-toluidine. The meta-cleavage pathway genes which are carried by pTDN1 were shown not to have originated in pWW0.     

Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.     

Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.

Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene.     

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K₂) were 1.62 × 10⁻³ sec⁻¹ for 3-chlorocatechol and 2.38 × 10⁻³ sec⁻¹ for 3-fluorocatechol. The inhibitor constants (K_i) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.     

Catabolism of pseudocumene and 3-ethyltoluene by Pseudomonas putida (arvilla) mt-2: evidence for new functions of the TOL (pWWO) plasmid.

Pseudocumene (1,2,4-trimethylbenzene) and 3-ethyltoluene were found to serve as growth substrates for Pseudomonas putida (arvilla) mt-2, in addition to toluene, m-xylene, and p-xylene as previously described. Similar observations were made with several additional P. putida strains also capable of growth with toluene and the xylenes. Additional substrates which supported the growth of these organisms included 3,4-dimethylbenzyl alcohol, 3,4-dimethylbenzoate, and 3-ethylbenzoate. P. putida mt-2 cells grown either with toluene or pseudocumene rapidly oxidized toluene, pseudocumene, and 3-ethyltoluene as well as 3,4-dimethylbenzoate, 3-ethylbenzoate, 3,4-dimethylcatechol, and 3-ethylcatechol. Cell extracts from similarly grown P. putida mt-2 cells catalyzed a meta fission of 3,4-dimethylcatechol and 3-ethylcatechol to compounds having the spectral properties of 2-hydroxy-5-methyl-6-oxo-2,4-heptadienoate and 2-hydroxy-6-ox-2,4-octadienoate, respectively. The further metabolism of these intermediates was shown to be independent of oxidized nicotinamide adenine dinucleotide (NAD+) and resulted in the formation of essentially equimolar amounts of pyruvate, indicating that each ring fission product was degraded via the hydrolytic branch of the meta fission pathway. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine led to the isolation of a mutant, which when grown with succinate in the presence of pseudocumene or 3-ethyltoluene accumulated 3,4-dimethylcatechol or 3-ethylcatechol. Cells unable to utilize toluene, m-xylene, and p-xylene, obtained by growth in benzoate, also lost the ability to utilize pseudocumene and 3-ethyltoluene. The ability to utilize these substrates could be reacquired by incubation with a leucine auxotroph otherwise able to grow on all of the aromatic substrates.