Degradation of 3-O-methylgallate in Sphingomonas paucimobilis SYK-6 by pathways involving protocatechuate 4,5-dioxygenase

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate and 3-O-methylgallate (3MGA), respectively. 3MGA is metabolized via multiple pathways involving 3MGA 3,4-dioxygenase, protocatechuate 4,5-dioxygenase (LigAB), and gallate dioxygenase whereas protocatechuate is degraded via the protocatechuate 4,5-cleavage pathway. Here the secondary role of LigAB in syringate metabolism is investigated. The reaction product of 3MGA catalyzed by His-tagged LigAB was identified as 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD) and 2-pyrone-4,6-dicarboxylate (PDC), indicating that 3MGA is transformed to CHMOD and PDC by both reactions catalyzed by DesZ and LigAB. Mutant analysis revealed that the 3MGA catabolic pathways involving LigAB are functional in SYK-6.     

2-pyrone-4,6-dicarboxylic acid, a catabolite of gallic acids in Pseudomonas species.

2-Pyrone-4,6-dicarboxylate hydrolase was purified from 4-hydroxybenzoate-grown Pseudomonas testosteroni. Gel filtration and electrophoretic measurements indicated that the preparation was homogeneous and gave a molecular weight of 37,200 for the single subunit of the enzyme. Hydrolytic activity was dependent upon a functioning sulfhydryl group(s) and was freely reversible; the equilibrium position was dependent upon pH, with equimolar amounts of pyrone and open-chain form present at pH 7.9. Since the hydrolase was strongly induced when the nonfluorescent organisms P. testosteroni and P. acidovorans grew with 4-hydroxybenzoate, it is suggested that 2-pyrone-4,6-dicarboxylate is a normal intermediate in the meta fission degradative pathway of protocatechuate. Laboratory strains of fluorescent pseudomonads did not metabolize 2-pyrone-4,6-dicarboxylate, but a strain of P. putida was isolated from soil that utilized this compound for growth; the hydrolase was then induced, but it was absent from extracts of 4-hydroxybenzoate-grown cells that readily catabolized protocatechuate by ortho fission reactions. 2-Pyrone-4,6-dicarboxylic acid was the major product formed when gallic acid was oxidized by purified protocatechuate 3,4-dioxygenase. Protocatechuate 4,5-dioxygenase gave only the open-chain ring fission product when gallic acid was oxidized, but the enzyme attacked 3-O-methylgallic acid, giving 2-pyrone-4,6-dicarboxylic acid as the major product. Cell suspensions of 4-hydroxybenzoate-grown P. testosteroni readily oxidized 3-O-methylgallate with accumulation of methanol.     

The 4-oxalomesaconate hydratase gene, involved in the protocatechuate 4,5-cleavage pathway, is essential to vanillate and syringate degradation in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by the O-demethylase and converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligJ gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The ligJ gene is transcribed in the same direction as ligABC genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream from ligA. The ligJ gene product (LigJ), expressed in Escherichia coli, was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30 degrees C. The K(m) for OMA and the V(max) were determined to be 138 microM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. The ligJ gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the ligJ gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.     

4-sulfomuconolactone hydrolases from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

The 4-carboxymethylen-4-sulfo-but-2-en-olide (4-sulfomuconolactone) hydrolases from Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 are part of a modified protocatechuate pathway responsible for the degradation of 4-sulfocatechol. In both strains, the hydrolase-encoding genes occur downstream of those encoding the enzymes that catalyze the lactonization of 3-sulfomuconate. The deduced amino acid sequences of the 4-sulfomuconolactone hydrolases demonstrated the highest degree of sequence identity to 2-pyrone-4,6-dicarboxylate hydrolases, which take part in the meta cleavage pathway of protocatechuate. The 4-sulfomuconolactone hydrolases did not convert 2-pyrone-4,6-dicarboxylate, and the 2-pyrone-4,6-dicarboxylate hydrolase from Sphingomonas paucimobilis SYK-6 did not convert 4-sulfomuconolactone. Nevertheless, the presence of highly conserved histidine residues in the 4-sulfomuconolactone and the 2-pyrone-4,6-dicarboxylate hydrolases and some further sequence similarities suggested that both enzymes belong to the metallo-dependent hydrolases (the "amidohydrolase superfamily"). The 4-sulfomuconolactone hydrolases were heterologously expressed as His-tagged enzyme variants. Gel filtration experiments suggested that the enzymes are present as monomers in solution, with molecular weights of approximately 33,000 to 35,000. 4-Sulfomuconolactone was converted by sulfomuconolactone hydrolases to stoichiometric amounts of maleylacetate and sulfite. The 4-sulfomuconolactone hydrolases from both strains showed pH optima at pH 7 to 7.5 and rather similar catalytic constant (k(cat)/K(M))values. The suggested 4-sulfocatechol pathway from 4-sulfocatechol to maleylacetate was confirmed by in situ nuclear magnetic resonance analysis using the recombinantly expressed enzymes.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6

Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.     

Efficient production of 2-pyrone 4,6-dicarboxylic acid as a novel polymer-based material from protocatechuate by microbial function

Sphingomonas paucimobilis SYK-6, which can degrade various low molecular weight compounds derived from plant polyphenols such as lignin, lignan, and tannin, metabolizes these substances via 2-pyrone-4,6-dicarboxylic acid (PDC). We focused on this metabolic intermediate as a potential raw material for novel, bio-based polymers. We cloned the ligAB and ligC genes of SYK-6, which respectively encode protocatechuate 4,5-dioxygenase and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, into a broad host range plasmid vector, pKT230MC. The resulting plasmid, pDVABC, was introduced into the PpY1100 strain of Pseudomonas putida, and we found that PDC could be stably produced from protocatechuate and accumulated. In addition, we examined the efficiency of production of PDC from protocatechuate on a 5-L scale in a Luria–Bertani medium containing 100 mM glucose and determined that PDC was stably produced from protocatechuate to yield 10 g/L or more.     

A novel tetrahydrofolate-dependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate

Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H(4)folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H(4)folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H(4)folate, forming 5-methyl-H(4)folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H(4)folate. The possible role of ligH is discussed.     

Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (k_cat/K_M) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O₂ consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.     

Genetic and biochemical characterization of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6

Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that ligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase. The ligC gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da. The deduced amino acid sequence of ligC showed 19 to 20% identity with 3-chlorobenzoate cis-dihydrodiol dehydrogenase of Alcaligenes sp. strain BR60 and phthalate cis-dihydrodiol dehydrogenases of Pseudomonas putida NMH102-2 and Burkholderia cepacia DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13-56, 1994). The ligC gene was expressed in Escherichia coli and LigC was purified to near homogeneity. Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP(+) by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25 degrees C, respectively. The K(m) for NADP(+) was estimated to be 24.6 +/- 1.5 microM, which was approximately 10 times lower than that for NAD(+) (252 +/- 3.9 microM). The K(m)s for CHMS in the presence of NADP(+) and NAD(+) are 26.0 +/- 0.5 and 20.6 +/- 1.0 microM, respectively. Disruption of ligC in S. paucimobilis SYK-6 prevented growth with vanillate. Only PCA was accumulated during the incubation of vanillate with the whole cells of the ligC insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC. However, the introduction of ligC into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for ligAB gene expression.     

Characterization of the 3-O-methylgallate dioxygenase gene and evidence of multiple 3-O-methylgallate catabolic pathways in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by the tetrahydrofolate-dependent O-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the ligB insertion mutant, which encodes the beta subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the desZ gene, which confers 3MGA degradation activity on Escherichia coli. The deduced amino acid sequence of desZ showed ca. 20 to 43% identity with the type II extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both desZ and ligB in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the desZ ligB double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which ligAB, desZ, 3MGA O-demethylase, and gallate dioxygenase are participants.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study, Corynebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glutamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

Characterization of the gallate dioxygenase gene: three distinct ring cleavage dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase beta and alpha subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The K(m) for gallate and the V(max) were determined to be 66.9 +/- 9.3 microM and 42.7 +/- 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.     

Bacterial degradation of m-nitrobenzoic acid.

Pseudomonas sp. strain JS51 grows on m-nitrobenzoate (m-NBA) with stoichiometric release of nitrite. m-NBA-grown cells oxidized m-NBA and protocatechuate but not 3-hydroxybenzoate, 4-hydroxy-3-nitrobenzoate, 4-nitrocatechol, and 1,2,4-benzenetriol. Protocatechuate accumulated transiently when succinate-grown cells were transferred to media containing m-NBA. Respirometric experiments indicated that the conversion of m-NBA to protocatechuate required 1 mol of oxygen per mol of substrate. Conversions conducted in the presence of 18O2 showed the incorporation of both atoms of molecular oxygen into protocatechuate. Extracts of m-NBA-grown cells cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde. These results provide rigorous proof that m-NBA is initially oxidized by a dioxygenase to produce protocatechuate which is further degraded by a 4,5-dioxygenase.     

Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B.

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.     

Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT.

A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway.