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.     

p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon.

Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate.

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

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.     

Bacterial Degradation of 4-Hydroxyphenylacetic Acid and Homoprotocatechuic Acid

A species of Acinetobacter and two strains of Pseudomonas putida when grown with 4-hydroxyphenylacetic acid gave cell extracts that converted 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid) into carbon dioxide, pyruvate, and succinate. The sequence of enzyme-catalyzed steps was as follows: ring-fission by a 2,3-dioxygenase, nicotinamide adenine dinucleotide-dependent dehydrogenation, decarboxylation, hydration, aldol fission, and oxidation of succinic semialdehyde. Two new metabolites, 5-carboxymethyl-2-hydroxymuconic acid and 2-hydroxyhepta-2,4-diene-1,7-dioic acid, were isolated from reaction mixtures and a third, 4-hydroxy-2-ketopimelic acid, was shown to be cleaved by extracts to give pyruvate and succinic semialdehyde. Enzymes of this metabolic pathway were present in Acinetobacter grown with 4-hydroxyphenylacetic acid but were effectively absent when 3-hydroxyphenylacetic acid or phenylacetic acid served as sources of carbon.     

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.     

Identification and expression of the cym, cmt, and tod catabolic genes from Pseudomonas putida KL47: expression of the regulatory todST genes as a factor for catabolic adaptation

Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene (C(1)-C(4)), biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99%) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.     

Bacterial degradation of N,N-diethyl-m-toluamide (DEET): cloning and heterologous expression of DEET hydrolase

Pseudomonas putida DTB grew aerobically with N,N-diethyl-m-toluamide (DEET) as a sole carbon source, initially breaking it down into 3-methylbenzoate and diethylamine. The former was further metabolized via 3-methylcatechol and meta ring cleavage. A gene from DTB, dthA, was heterologously expressed and shown to encode the ability to hydrolyze DEET into 3-methylbenzoate and diethylamine.     

Incidence and Identification of Pseudomonas fluorescens and Pseudomonas putida in the Clinical Laboratory

Strains of Pseudomonas producing fluorescin but no pyocyanin or pyorubrin were studied by biochemical and antibiotic sensitivity testing. A rapid nitrate test was found to be useful in distinguishing P. aeruginosa (positive) from P. fluorescens and P. putida (both negative). A shortened gelatin test differentiated P. fluorescens (positive) from P. putida (negative). P. fluorescens and P. putida were very sensitive to low levels of kanamycin and resistant to carbenicillin, a pattern just the opposite of that obtained with P. aeruginosa.     

Bioconversion of limonene to increased concentrations of perillic acid by Pseudomonas putida GS1 in a fed-batch reactor

Pseudomonas putida GS1 is able to convert limonene to perillic acid (up to 64 mM,(11 g/l) when the bacteria is cultivated in fed-batch culture with non-limiting amounts of glycerol. ammonium, and limonene. P. putida GS1 can use p-cymene as a single source of carbon and energy, and the enzymes that are responsible for the conversion of limonene to perillic acid belong to the degradation pathway of p-cymene. The p-cymene pathway of P putida GS1 is very similar, if not identical, to the cym pathway of P. putida F1. The latter strain, and a recombinant Escherichia coli strain that carried the genes of the cym pathway of P. putida Fl, also converted limonene to perillic acid. However, the final concentrations that were obtained in batch cultures with these two strains were lower than those obtained with P. putida GS1.     

Peripheral metabolism of Pseudomonal putida transconjugants degrading chloro- and methylaromatic compounds

Peripheral metabolism was studied in the Pseudomonas putida 37cc transconjugant. In the strain grown on benzoate, pyrocatechase (PC) I with a low activity to chlorocatechols was induced, whereas PCII actively decomposing chlorocatechols was induced during its growth on 3-chlorobenzoic acid. The P. putida 37cc transconjugant grown on alpha-methylstyrene (MS) exerted the activity of both metapyrocatechase (MPC) and PC, whereas in the parent strain P. putida R-1 only MPC was involved in the degradation of alpha-MS. The substrate specificity of the enzymes involved in the ring cleavage by P. putida 37cc was compared to show that, apparently, MPC of the transconjugant was similar to this enzyme in the strain R-1 while PC decomposing chlorocatechols was similar to PC of the P. putida 87 donor. The regulation of the enzymes mediating the ring cleavage was studied in the parent strains and transconjugants.     

Identification of cis-diols as intermediates in the oxidation of aromatic acids by a strain of Pseudomonas putida that contains a TOL plasmid.

Pseudomonas putida BG1 was isolated from soil by enrichment with p-toluate and selection for growth with p-xylene. Other hydrocarbons that served as growth substrates were toluene, m-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene. The enzymes responsible for growth on these substrates are encoded by a large plasmid with properties similar to those of TOL plasmids isolated from other strains of Pseudomonas. Treatment of P. putida BG1 with nitrosoguanidine led to the isolation of a mutant strain which, when grown with fructose, oxidized both p-xylene and p-toluate to (-)-cis-1,2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylic acid (cis-p-toluate diol). The structure of the diol was determined by conventional chemical techniques including identification of the products formed by acid-catalyzed dehydration and characterization of a methyl ester derivative. The cis-relative stereochemistry of the hydroxyl groups was determined by the isolation and characterization of an isopropylidene derivative. p-Xylene-grown cells contained an inducible NAD+-dependent dehydrogenase which formed catechols from cis-p-toluate diol and the analogous acid diols formed from the other hydrocarbon substrates listed above. The catechols were converted to meta ring fission products by an inducible catechol-2,3-dioxygenase which was partially purified from p-xylene-grown cells of P. putida BG1.     

Influence of Side-Chain Substituents on the Position of Cleavage of the Benzene Ring by Pseudomonas fluorescens

Pseudomonas fluorescens was grown on mineral salts media with phenol, p-hydroxybenzoic acid, p-hydroxy-phenylacetic acid, or p-hydroxy-trans-cinnamic acid as sole carbon and energy source. Each compound was first hydroxylated, ortho to the hydroxyl group on the benzene ring, to give catechol, protocatechuic acid (3,4-dihydroxy-benzoic acid), homoprotocatechuic acid (3,4-dihydroxy-phenylacetic acid), and caffeic acid (3,4-dihydroxy-trans-cinnamic acid), respectively, as the ultimate aromatic products before cleavage of the benzene nucleus. Protocatechuic acid and caffeic acid were shown to be cleaved by ortho fission, via a 3,4-oxygenase mechanism, to give beta-substituted cis, cis-muconic acids as the initial aliphatic products. However, catechol and homoprotocatechuic acid were cleaved by meta fission, by 2,3-and 4,5-oxygenases, respectively, to give alpha-hydroxy-muconic semialdehyde and alpha-hydroxy-gamma-carboxymethyl muconic semialdehyde as initial aliphatic intermediates. Caffeic acid: 3,4-oxygenase, a new oxygenase, consumes 1 mole of O(2) per mole of substrate and has an optimal pH of 7.0. The mechanism of cleavage of enzymes derepressed for substituted catechols by P. fluorescens apparently changes from ortho to meta with the increasing nephelauxetic (electron donor) effect of the side-chain substituent.     

Isolation of a Pseudomonas stutzeri strain that degrades o-xylene

A Pseudomonas stutzeri strain capable of growing on o-xylene was isolated from enrichment cultures. The organism grew on 2,3- and 3,4-dimethylphenol but not on 2-methylbenzyl alcohol, o-tolualdehyde, or o-toluate. P. stutzeri was not able to utilize m-xylene, p-xylene, or 1,2,4-trimethylbenzene, but growth was observed in the presence of the corresponding alcohols and acids. From the Pseudomonas cultures supplied with o-xylene, 2,3-dimethylphenol was isolated and identified. When resting P. stutzeri cells were incubated with 2,3-dimethylphenol, the reaction mixture turned greenish yellow and showed spectral properties identical to those of the 3,4-dimethylcatechol meta ring fission product. Catechol 2,3-oxygenase was induced by growth on o-xylene or on 2,3- or 3,4-dimethylphenol. The suggested hypothesis is that the first metabolic steps of growth on o-xylene involve the direct oxygenation of the aromatic nucleus, followed by meta pathway reactions.     

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.     

Plasmid-assisted morpholine degradation by Pseudomonas fluorescens CAS 102

A fast-growing Pseudomonas fluorescens CAS102, isolated by enrichment technique from polluted soil, effectively utilized morpholine as the energy source. The strain was able to grow in high concentrations of morpholine but accumulation of ammonia inhibited its growth and complete mineralization. The molar conversion ratio of morpholine to ammonia was 1:0.82. The organism harboured a single, multiple antibiotic- and heavy metal-resistance 140kb plasmid which was resistant to curing. Transformation studies showed that the morpholine degradative phenotype was expressed only in Pseudomonas putida and not in Escherichia coli. Growth studies on different degradative intermediates of morpholine suggested that plasmid-encoded genes were involved in the heterocyclic ring cleavage and the remaining reactions were mediated by chromosomal genes.     

Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250

Abstract Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho -substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.