Metabolism of resorcinylic compounds by bacteria: alternative pathways for resorcinol catabolism in Pseudomonas putida.

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol. One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway. After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase. Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate. Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction. P. putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates. The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described. Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P. putida ORC grown with resorcinol. Its further metabolism was reduced nicotinamide adenine dinucleotide dependent.     

Metabolism of resorcinylic compounds by bacteria. Purification and properties of acetylpyruvate hydrolase from Pseudomonas putida 01.

Acetylpyruvate hydrolase, the terminal inducible enzyme of the pathway of orcinol catabolism in Pseudomonas putida, catalyzes the quantitative conversion of acetylpyruvate into acetate and pyruvate. The enzyme has been purified approximately 40-fold from extracts of Ps. putida grown on orcinol. Disc gel electrophoresis of the preparations show one major and one minor band of protein. The molecular weight of the enzyme is approximately 38,000 by sodium dodecyl sulfate electrophoresis. Acetylpyruvate is the only known substrate for the enzyme; maleylpyruvate, fumarylpyruvate, acetoacetate, oxalacetate, and acetylacetone are not hydrolyzed by acetylpyruvate hydrolase. Several divalent cations, includ-Mg2+, Mn2+, Co2+, Ca2+, and Zn2+, enhanced hydrolytic activity, but Cu2+ was inhibitory. The enzyme shows a sharp pH optimum at 7.4. Acetylpyruvate hydrolase has an apparent K-m of 0.1 mM for acetylpyruvate with a molecular activity of 36 min minus 1 at 25 degrees. Pyruvate, oxalacetate, and oxalate are competitive inhibitors of acetylpyruvate hydrolysis by the enzyme with K-i values of 6.0, 4.5, and 0.45 mM, respectively.     

Bacterial metabolism of resorcinylic compounds: purification and properties of orcinol hydroxylase and resorcinol hydroxylase from Pseudomonas putida ORC.

The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.     

Metabolism of resorcinylic compounds by bacteria: new pathway for resorcinol catabolism in Azotobacter vinelandii.

We present evidence to document a third pathway for the microbial catabolism of resorcinol. Resorcinol is converted to pyrogallol by resorcinol-grown cells of Azotobacter vinelandii. Pyrogallol is the substrate for one of two ring cleavage enzymes induced by growth with resorcinol. Oxalocrotonate, CO2, pyruvate, and acetaldehyde have been identified as products of pyrogallol oxidation catalyzed by extracts of resorcinol-grown cells. The enzymes pyrogallol 1,2-dioxygenase, oxalocrotonate tautomerase (isomerase), oxalocrotonate decarboxylase, and vinylpyruvate hydratase are present in extracts from resorcinol-grown cells but not in succinate-grown cells.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.     

p-cymene pathway in Pseudomonas putida: initial reactions.

Initial reactions of the p-cymene pathway induced in Pseudomonas putida PL have been reinvestigated. Oxidation of the methyl group attached to the nucleus occurs in three steps to give p-cumic acid. The substrate for the ring cleavage of 2,3-dihydroxy-p-cumate is formed from p-cumate in two reactions via a dihydrodiol intermediate (2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate) and not as previously postulated via 3-hydroxy-p-cumate. There are three pieces of evidence for the physiological role of the dihydrodiol intermediate. (i) a mutant of P. putida PL-pT-11/43, which is unable to grow with p-cumate, accumulates a compound from p-cumate, which was identified as 2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate. (II) This metabolite is enzymically oxidized by a nicotinamide adenine dinucleotide-dependent dehydrogenase that is present in crude extracts of the wild type and a revertant strain (PL-pT-11/43-R1) but not in the mutant. (iii) 3-Hydroxy-p-cumate does not support growth of P . putida PL-W, and it is not oxidized by cells or extracts. 3-Hydroxy-p-cumate was readily isolated as before from culture supernatants, due to its ready formation from the dihydrodiol in acid solution. Mass spectral analysis of the dihydrodiol accumulated in 18O2-enriched atmospheres showed that both hydroxyl atoms are derived from the same molecule of O2. The formation and absorbance maxima of dihydrodiols that accumulated during the growth of the mutant PL-pT-11/43 in the presence of various benzoates (or toluenes) that have substituents at the carbon 4 atom also is reported.     

Degradation of aromatic compounds by Pseudomonas putida

The influence of different process kinetics on the course of phenol degradation has been studied as well as the influence of axial dispersion in the liquid phase on the reactor height with relatively large biofilm thickness in a conventional fluidized bed and air-lift bioreactor. The object of this was to achieve a high conversion of substrate in a device of real size in real process time. For calculating the mathematical model, the method of orthogonal collocation with the STIFF integration routine has been used.     

Transport of C5 dicarboxylate compounds by Pseudomonas putida.

Induced glutarate and 2-oxoglutarate uptake and transport by Pseudomonas putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,2-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent Km--1.22 mM, Vmax--400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent Km--131 microM, Vmax--255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent Km and Ki values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate greater than glutarate greater than D-2-hydroxyglutarate and L-2-hydroxyglutarate greater than glutaconate.     

Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111.

Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.     

Plasmid Involvement in Acyclic Isoprenoid Metabolism by Pseudomonas putida

An organism identified as Pseudomonas putida was found to utilize citronellol or geraniol as the sole carbon and energy source. The ability to degrade these acyclic isoprenols was associated with pSRQ50, a 50-megadalton transmissible plasmid.     

Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111.

Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.     

Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor.     

Pathways of Assimilative Sulfur Metabolism in Pseudomonas putida

Cysteine and methionine biosynthesis was studied in Pseudomonas putida S-313 and Pseudomonas aeruginosa PAO1. Both these organisms used direct sulfhydrylation of O-succinylhomoserine for the synthesis of methionine but also contained substantial levels of O-acetylserine sulfhydrylase (cysteine synthase) activity. The enzymes of the transsulfuration pathway (cystathionine gamma-synthase and cystathionine beta-lyase) were expressed at low levels in both pseudomonads but were strongly upregulated during growth with cysteine as the sole sulfur source. In P. aeruginosa, the reverse transsulfuration pathway between homocysteine and cysteine, with cystathionine as the intermediate, allows P. aeruginosa to grow rapidly with methionine as the sole sulfur source. P. putida S-313 also grew well with methionine as the sulfur source, but no cystathionine gamma-lyase, the key enzyme of the reverse transsulfuration pathway, was found in this species. In the absence of the reverse transsulfuration pathway, P. putida desulfurized methionine by the conversion of methionine to methanethiol, catalyzed by methionine gamma-lyase, which was upregulated under these conditions. A transposon mutant of P. putida that was defective in the alkanesulfonatase locus (ssuD) was unable to grow with either methanesulfonate or methionine as the sulfur source. We therefore propose that in P. putida methionine is converted to methanethiol and then oxidized to methanesulfonate. The sulfonate is then desulfonated by alkanesulfonatase to release sulfite for reassimilation into cysteine.     

Phenol and Benzoate Metabolism by Pseudomonas putida: Regulation of Tangential Pathways

Catechol occurs as an intermediate in the metabolism of both benzoate and phenol by strains of Pseudomonas putida. During growth at the expense of benzoate, catechol is cleaved ortho (1,2-oxygenase) and metabolized via the beta-ketoadipate pathway; during growth at the expense of phenol or cresols, the catechol or substituted catechols formed are metabolized by a separate pathway following meta (2,3-oxygenase) cleavage of the aromatic ring of catechol. It is possible to explain the mutually exclusive occurrence of the meta and ortho pathway enzymes in phenol- and benzoate-grown cells of P. putida on the basis of differences in the mode of regulation of these two pathways. By use of both nonmetabolizable inducers and blocked mutants, gratuitous synthesis of some of the meta pathway enzymes was obtained. All four enzymes of the meta pathway are induced by the primary substrate, cresol or phenol, or its analogue. Three enzymes of the ortho pathway that catalyze the conversion of catechol to beta-ketoadipate enol-lactone are induced by cis,cis-muconate, produced from catechol by 1,2-oxygenase-mediated cleavage. Observations on the differences in specificity of induction and function of the two pathways suggest that they are not really either tangential or redundant. The meta pathway serves as a general mechanism for catabolism of various alkyl derivatives of catechol derived from substituted phenolic compounds. The ortho pathway is more specific and serves primarily in the catabolism of precursors of catechol and catechol itself.     

Metabolism of Phenol and Cresols by Mutants of Pseudomonas putida

Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD(+))-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD(+)-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD(+)-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD(+)-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed.     

p-Hydroxyphenylacetic Acid Metabolism in Pseudomonas putida F6

Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage.     

Degradation of phenol and phenolic compounds by Pseudomonas putida EKII

Summary The phenol-degrading strain Pseudomonas putida EKII was isolated from a soil enrichment culture and utilized phenol up to 10.6 mM (1.0 g·1 ^-1) as the sole source of carbon and energy. Furthermore, cresols, chlorophenols, 3,4-dimethylphenol, and 4-chloro-m-cresol were metabolized as sole substrates by phenol-grown resting cells of strain EKII. Under conditions of cell growth, degradation of these xenobiotics was achieved only in co-metabolism with phenol. Phenol hydroxylase activity was detectable in whole cells but not in cell-free extracts. The specificity of the hydroxylating enzyme was found during transformation of cresols and chlorophenols: ortho- and meta-substituted phenols were degraded via 3-substituted catechols, while degradation of para-substituted phenols proceeded via 4-substituted catechols. In cell-free extracts of phenol-grown cells a high level of catechol 2,3-dioxygenase as well as smaller amounts of 2-hydroxymuconic semialdehyde hydrolyase and catechol 1,2-dioxygenase were detected. The ring-cleaving enzymes were characterized after partial purification by DEAE-cellulose chromatography.