Microbial degradation of hydrocarbons. Catabolism of 1-phenylalkanes by Nocardia salmonicolor

1. Nocardia salmonicolor grew on a variety of alkanes, 1-phenylalkanes and 1-cyclo-hexylalkanes as sole carbon and energy sources. 2. Growth on 1-phenyldodecane in batch culture was diauxic. Isocitrate lyase activity was induced during lag phase, reaching a maximum activity in the first growth phase, during which both the aromatic ring and the side chain were degraded. However, 4-phenylbutyrate, 4-phenylbut-3-enoate, 4-phenylbut-2-enoate, 3-phenylpropionate, cinnamate and phenylacetate accumulated in the growth medium. These compounds disappeared at the onset of diauxic lag and four hydroxylated compounds accumulated; one was 4-(o-hydroxyphenyl)but-3-enoate and another was identified as 4-(o-hydroxyphenyl)butyrate. These compounds were utilized during the second growth phase. 3. Washed 1-phenyldodecane-grown cells oxidized acetate, cinnamate, 3,4-dihydroxyphenylacetate, homogentisate, o-, m- and p-hydroxyphenylacetate, phenylacetate, and 4-phenylbutyrate rapidly without lag. 4. Extracts of such cells rapidly oxidized homogentisate,3,4-dihydroxyphenylacetate, catechol and protocatechuate. 5. The organism grew readily on 4-phenylbutyrate, phenylacetate, o-hydroxyphenylacetate, homogentisate and 3,4-dihydroxyphenylacetate as sole carbon energy sources, but growth was slow on cinnamate and 4-phenylbut-3-enoate. 6. When cinnamate and phenylacetate were sole carbon sources for growth, phenylacetate and o-hydroxyphenylacetate respectively were detected in culture supernatants. 4-Phenylbut-3-enoate and 4-phenylbutyrate both yielded a mixture of cinnamate and phenylacetate. 7. It is proposed that 1-phenyldodecane is catabolized by ω-oxidation of the terminal methyl group, side-chain β-oxidation to 4-phenylbutyrate, both β- and α-oxidation to phenylacetic acid, hydroxylation to homogentisate via o-hydroxyphenylacetate and ring cleavage to maleylacetoacetate. Catabolism via 3,4-dihydroxyphenylacetate may also occur. 8. Growth on 1-phenylnonane was also diauxic and cinnamic acid, phenylpropionic acid, benzoic acid and hydroxyphenylpentanoic acid accumulated in the medium. Respirometric data and ring-cleavage enzyme activities showed similar patterns to those obtained after growth on 1-phenyldodecane. The results suggest that the main catabolic routes for 1-phenyldodecane and 1-phenylnonane may converge at cinnamate. 9. Possible reasons for diauxie are discussed.     

Metabolism of arylsulphonates by micro-organisms

1. Species of Pseudomonas capable of degrading arylsulphonates and detergents of the alkylbenzenesulphonate type were isolated from sewage and river water. 2. Benzenesulphinate, benzenesulphonate and toluene-p-sulphonate were rapidly degraded by these organisms with the release of the sulphonate group as sulphite; detergent homologues with a chain length up to 16 carbon atoms (4-n-hexadecyl-benzenesulphonate) also released sulphite. Sulphite oxidation to sulphate in the medium can occur non-enzymically. 3. Growth on benzenesulphonate and toluene-p-sulphonate elicited a catechol 2,3-oxygenase, which effected a ;meta' cleavage of the ring. The metabolic route for benzenesulphonate was determined as: benzenesulphonate-->catechol-->2-hydroxymuconic semialdehyde-->formate and 4-hydroxy-2-oxovalerate-->acetaldehyde and pyruvate; the enzymes catalysing these steps were all inducible. 4. Toluene-p-sulphonate was degraded via 2-hydroxy-5-methylmuconic semialdehyde to formate and 4-hydroxy-2-oxohexanoate and the latter was cleaved to propionaldehyde and pyruvate. Propionaldehyde and propionate were oxidized rapidly by toluene-p-sulphonate-grown cells but slowly by fumarate-grown organisms. 5. The specificity of the catechol 2,3-oxygenase induced by the arylsulphonates, towards catechol and the methylcatechols, varied during the purification and suggested that 3-methylcatechol was probably oxidized by a separate enzyme. Detergents of the alkylbenzenesulphonate type also induced a catechol 2,3-oxygenase in these bacteria. 6. A few isolates, after growth on benzenesulphonate, opened the ring of catechol by an ;ortho' route to form cis-cis-muconate. The enzymes to degrade this intermediate to beta-oxoadipate were also present in induced cells.     

Biodegradation of phenanthrene by<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. strain PP2: novel metabolic pathway,role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation

Pseudomonas sp. strain PP2 isolated in our laboratory efficiently metabolizes phenanthrene at 0.3% concentration as the sole source of carbon and energy. The metabolic pathways for the degradation of phenanthrene, benzoate and p-hydroxybenzoate were elucidated by identifying metabolites, biotransformation studies, oxygen uptake by whole cells on probable metabolic intermediates, and monitoring enzyme activities in cell-free extracts. The results obtained suggest that phenanthrene degradation is initiated by double hydroxylation resulting in the formation of 3,4-dihydroxyphenanthrene. The diol was finally oxidized to 2-hydroxymuconic semialdehyde. Detection of 1-hydroxy-2-naphthoic acid, alpha-naphthol, 1,2-dihydroxy naphthalene, and salicylate in the spent medium by thin layer chromatography; the presence of 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-2,3-dioxygenase activity in the extract; O(2) uptake by cells on alpha-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate and catechol; and no O(2) uptake on o-phthalate and 3,4-dihydroxybenzoate supports the novel route of metabolism of phenanthrene via 1-hydroxy-2-naphthoic acid --> [alpha-naphthol] --> 1,2-dihydroxy naphthalene --> salicylate --> catechol. The strain degrades benzoate via catechol and cis,cis-muconic acid, and p-hydroxybenzoate via 3,4-dihydroxybenzoate and 3-carboxy- cis,cis-muconic acid. Interestingly, the culture failed to grow on naphthalene. When grown on either hydrocarbon or dextrose, the culture showed good extracellular biosurfactant production. Growth-dependent changes in the cell surface hydrophobicity, and emulsification activity experiments suggest that: (1) production of biosurfactant was constitutive and growth-associated, (2) production was higher when cells were grown on phenanthrene as compared to dextrose and benzoate, (3) hydrocarbon-grown cells were more hydrophobic and showed higher affinity towards both aromatic and aliphatic hydrocarbons compared to dextrose-grown cells, and (4) mid-log-phase cells were significantly (2-fold) more hydrophobic than stationary phase cells. Based on these results, we hypothesize that growth-associated extracellular biosurfactant production and modulation of cell surface hydrophobicity plays an important role in hydrocarbon assimilation/uptake in Pseudomonas sp. strain PP2.     

Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation

Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways.     

The microbial degradation of phenylalkanes. 2-Phenylbutane, 3-phenylpentane, 3-phenyldodecane and 4-phenylheptane

1. Two Pseudomonas strains capable of utilizing 2-phenylbutane, 3-phenylpentane and 4-phenylheptane as the sole carbon and energy source were isolated. 2. Two Nocardia strains capable of utilizing only 3-phenyldodecane as the sole carbon and energy source were isolated. 3. All the isolated strains were unable to grow on the corresponding phenylalkane-p-sulphonates. 4. From liquid cultures of Pseudomonas strains utilizing 2-phenylbutane, 2-(2,3-dihydro-2,3-dihydroxyphenyl)butane was isolated and identified. Evidence for a meta cleavage of the benzene ring was also obtained. 5. From liquid cultures of Pseudomonas strains utilizing 3-phenylpentane, 3-(2,3-dihydro-2,3-dihydroxyphenyl)pentane and 2-hydroxy-7-ethyl-6-oxonona-2,4-dienoic acid were isolated and identified. 6. Evidence for the formation of both a diol and a meta-cleavage compound was obtained from liquid cultures of both Pseudomonas strains utilizing 4-phenylheptane. 7. Liquid cultures of both Nocardia strains utilizing 3-phenyldodecane never formed a diol or a semialdehyde-related compound. 2-Phenylbutyric acid, 3-phenylvaleric acid and 4-phenylhexanoic acid were shown to be present in these cultures.     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Methanogenic Decomposition of Ferulic Acid, a Model Lignin Derivative

Ferulic acid, a model lignin derivative, was observed to be biodegradable to methane and carbon dioxide under strict anaerobic conditions. This conversion appears to be carried out by a consortium of bacteria similar to that previously described for the methanogenic degradation of benzoic acid. A temporary buildup of acetate in these cultures indicates that it is a likely intermediate and precursor for methane formation. An analog of coenzyme M, 2-bromoethanesulfonic acid (BESA), inhibited gas production and enhanced the buildup of propionate, butyrate, isobutyrate, and isovalerate. Phenylacetate, cinnamate, 3-phenylpropionate, benzoate, cyclohexane carboxylate, adipate, and pimelate were also detected in BESA-inhibited cultures. A pathway is proposed which includes these various acids as possible intermediates in the methanogenic degradation of ferulic acid. This model overlaps previously described benzoic acid degradation pathways, suggesting that this type of anaerobic degradation may be common for aromatic compounds.     

Metabolism of bismuth subsalicylate and intracellular accumulation of bismuth by Fusarium sp. strain BI

Enrichment cultures were conducted using bismuth subsalicylate as the sole source of carbon and activated sludge as the inoculum. A pure culture was obtained and identified as a Fusarium sp. based on spore morphology and partial sequences of 18S rRNA, translation elongation factor 1-alpha, and beta-tubulin genes. The isolate, named Fusarium sp. strain BI, grew to equivalent densities when using salicylate or bismuth subsalicylate as carbon sources. Bismuth nitrate at concentrations of up to 200 muM did not limit growth of this organism on glucose. The concentration of soluble bismuth in suspensions of bismuth subsalicylate decreased during growth of Fusarium sp. strain BI. Transmission electron microscopy and energy-dispersive spectroscopy revealed that the accumulated bismuth was localized in phosphorus-rich granules distributed in the cytoplasm and vacuoles. Long-chain polyphosphates were extracted from fresh biomass grown on bismuth subsalicylate, and inductively coupled plasma optical emission spectrometry showed that these fractions also contained high concentrations of bismuth. Enzyme activity assays of crude extracts of Fusarium sp. strain BI showed that salicylate hydroxylase and catechol 1,2-dioxygenase were induced during growth on salicylate, indicating that this organism degrades salicylate by conversion of salicylate to catechol, followed by ortho cleavage of the aromatic ring. Catechol 2,3-dioxygenase activity was not detected. Fusarium sp. strain BI grew with several other aromatic acids as carbon sources: benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, gentisate, d-mandelate, l-phenylalanine, l-tyrosine, phenylacetate, 3-hydroxyphenylacetate, 4-hydroxyphenylacetate, and phenylpropionate.     

A comparison of biodegradation of phenol and homologous compounds by Pseudomonas vesicularis and Staphylococcus sciuri strains

Pseudomonas vesicularis and Staphylococcus sciuri were isolated as dominant strains from phenol-acclimated activated sludge. P. vesicularis was an efficient degrader of phenol, catechol, p-cresol, sodium benzoate and sodium salicylate in a single substrate system. Under similar conditions S. sciuri degraded only phenol and catechol from among aromatic compounds that were tested. Cell-free extracts of P. vesicularis grown on phenol (376 mg l(-1)), sodium benzoate (576 mg l(-1)) and sodium salicylate (640 mg l(-1)) showed catechol 2,3-dioxygenase activity initiating an extradiol (meta) splitting pathway. The degradative intradiol (ortho) pathway as a result of catechol 1,2-dioxygenase synthesis was induced in P. vesicularis cells grown on catechol (440 mg l(-1)) orp-cresol (432 mg l(-1)). Catechol 1,2-dioxygenase and the ortho-cleavage has been also reported in S. sciuri cells capable of degrading phenol (376 mg l(-1)) or catechol (440 mg l(-1)). In cell-free extracts of S. sciuri no meta-cleavage enzyme activity was detected. These results demonstrated that gram-positive S. sciuri strain was able to effectively metabolize some phenols as do many bacteria of the genus Pseudomonas but have a different capacity for degrading of these compounds.     

Characterization of Pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of Pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal DNA fragment able to substitute for xylS in activation of the TOL lower-pathway promoter.

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represents by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression from OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.     

Nitrobenzoates and aminobenzoates are chemoattractants for Pseudomonas strains

Three Pseudomonas strains were tested for the ability to sense and respond to nitrobenzoate and aminobenzoate isomers in chemotaxis assays. Pseudomonas putida PRS2000, a strain that grows on benzoate and 4-hydroxybenzoate by using the beta-ketoadipate pathway, has a well-characterized beta-ketoadipate-inducible chemotactic response to aromatic acids. PRS2000 was chemotactic to 3- and 4-nitrobenzoate and all three isomers of aminobenzoate when grown under conditions that induce the benzoate chemotactic response. P. putida TW3 and Pseudomonas sp. strain 4NT grow on 4-nitrotoluene and 4-nitrobenzoate by using the ortho (beta-ketoadipate) and meta pathways, respectively, to complete the degradation of protocatechuate derived from 4-nitrotoluene and 4-nitrobenzoate. However, based on results of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase assays, both strains were found to use the beta-ketoadipate pathway for the degradation of benzoate. Both strains were chemotactic to benzoate, 3- and 4-nitrobenzoate, and all three aminobenzoate isomers after growth with benzoate but not succinate. Strain TW3 was chemotactic to the same set of aromatic compounds after growth with 4-nitrotoluene or 4-nitrobenzoate. In contrast, strain 4NT did not respond to any aromatic acids when grown with 4-nitrotoluene or 4-nitrobenzoate, apparently because these substrates are not metabolized to the inducer (beta-ketoadipate) of the chemotaxis system. The results suggest that strains TW3 and 4NT have a beta-ketoadipate-inducible chemotaxis system that responds to a wide range of aromatic acids and is quite similar to that present in PRS2000. The broad specificity of this chemotaxis system works as an advantage in strains TW3 and 4NT because it functions to detect diverse carbon sources, including 4-nitrobenzoate.     

Catechol ring-cleavage in Pseudomonas cepacia: the simultaneous induction of ortho and meta pathways

This work demonstrates the ring-cleavage pathways of catechol on Pseudomonas cepacia ATCC 29351, formed upon its growth on salicylate and benzoate, each as a sole carbon source. When grown on salicylate, P. cepacia induces only the catechol ortho pathway by its induction of catechol 1,2-dioxygenase. However, interestingly, benzoate-grown cells induce the ortho and meta pathways for the biodegradation of catechol, by inducing simultaneously catechol 1,2-dioxygenase and 2,3-dioxygenase, respectively, in the ratio of 7:1. The results indicate that P. cepacia ATCC 29351 possesses the genetic capacity for enzymes of both the ortho- and meta-cleavage pathways of benzoate degradation, although the phenotypic expression for the ortho pathway is higher. The simultaneous induction of catechol 1,2- and 2,3-dioxygenase is not detected in salicylate degradation. Although catechol is the metabolic intermediate for both salicylate and benzoate, catechol did not induce either pathway when used as a sole carbon source.     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.     

Anaerobic degradation of trans-cinnamate and ω-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a β-oxidation mechanism

The mechanism responsible for the initial steps in the anaerobic degradation of trans-cinnamate and -phenylalkane carboxylates by the purple non-sulphur photosynthetic bacterium Rhodopseudomonas palustris was investigated. Phenylacetate did not support growth and there was a marked CO₂ dependence for growth on acids with greater side-chain lengths. Here, CO₂ was presumably acting as a redox sink for the disposal of excess reducing equivalents. Growth on benzoate did not require the addition of exogenous CO₂. Aromatic acids with an odd number of side-chain carbon atoms (3-phenylpropionate, 5-phenylvalerate, 7-phenylheptanoate) gave greater apparent molar growth yields than those with an even number of side-chain carbon atoms (4-phenylbutyrate, 6-phenylhexanoate, 8-phenyloctanoate). HPLC analysis revealed that phenylacetate accumulated and persisted in the culture medium during growth on these latter compounds. Cinnamate and benzoate transiently accumulated in the culture medium during growth on 3-phenylpropionate, and benzoate alone accumulated transiently during the course of trans-cinnamate degradation. The transient accumulation of 4-phenyl-2-butenoic acid occurred during growth on 4-phenylbutyrate, and phenylacetate accumulated to a 1:1 molar stoichiometry with the initial 4-phenylbutyrate concentration. It is proposed that the initial steps in the anaerobic degradation of trans-cinnamate and the group of acids from 3-phenylpropionate to 8-phenyloctanoate involves -oxidation of the side-chain.Abbreviation 3-PP 3-phenylpropionic acid - 4-PB 4-phenylbutyric acid - 5-PV 5-phenylvaleric acid - 6-PH 6-phenylhexanoic acid - 7-PH 7-phenylheptanoic acid - 8-PO 8-phenyloctanoic acid - 4-P2B 4-phenyl-2-butenoic acid - GC/MS Gas chromatography/Mass spectrometry - HPLC High-pressure liquid chromatography     

Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol.

Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of Pseudomonas putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol, and 4-fluorocatechol. The apparent Michaelis constants for 3-methylcatechol and catechol were 10.6 and 22.0 muM, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. In addition, incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki = 0.14 muM) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 muM). The effect of the ion-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove iron from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.     

New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp.

A denitrifying Pseudomonas sp. is able to oxidize aromatic compounds compounds completely to CO2, both aerobically and anaerobically. It is shown that benzoate is aerobically oxidized by a new degradation pathway via benzoyl-coenzyme A (CoA) and 3-hydroxybenzoyl-CoA. The organism grew aerobically with benzoate, 3-hydroxybenzoate, and gentisate; catechol, 2-hydroxybenzoate, and protocatechuate were not used, and 4-hydroxybenzoate was a poor substrate. Mutants were obtained which were not able to utilize benzoate as the sole carbon source aerobically but still used 3-hydroxybenzoate or gentisate. Simultaneous adaptation experiments with whole cells seemingly suggested a sequential induction of enzymes of a benzoate oxidation pathway via 3-hydroxybenzoate and gentisate. Cells grown aerobically with benzoate contained a benzoate-CoA ligase (AMP forming) (0.1 mumol min-1 mg-1) which converted benzoate but not 3-hydroxybenzoate into its CoA thioester. The enzyme of 130 kDa composed of two identical subunits of 56 kDa was purified and characterized. Cells grown aerobically with 3-hydroxybenzoate contained a similarly active CoA ligase for 3-hydroxybenzoate, 3-hydroxybenzoate-CoA ligase (AMP forming). Extracts from cells grown aerobically with benzoate catalyzed a benzoyl-CoA- and flavin adenine dinucleotide-dependent oxidation of NADPH with a specific activity of at least 25 nmol NADPH oxidized min-1 mg of protein-1; NADH and benzoate were not used. This new enzyme, benzoyl-CoA 3-monooxygenase, was specifically induced during aerobic growth with benzoate and converted [U-14C]benzoyl-CoA stoichiometrically to [14C]3-hydroxybenzoyl-CoA.     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

Role of Catechol and the Methylcatechols as Inducers of Aromatic Metabolism in Pseudomonas putida

Pseudomonas putida NCIB 10015 metabolizes phenol and the cresols (methylphenols) by the meta pathway and metabolizes benzoate by the ortho pathway. Growth on catechol, an intermediate in the metabolism of both phenol and benzoate, induces both ortho and meta pathways; growth on 3- or 4-methylcatechols, intermediates in the metabolism of the cresols, induces only the meta pathway to a very limited degree. Addition of catechol at a growth-limiting rate induces virtually no meta pathway enzymes, but high levels of ortho pathway enzymes. The role of catechol and the methylcatechols as inducers is discussed. A method is described for assaying low levels of catechol 1,2-oxygenase in the presence of high levels of catechol 2,3-oxygenase and vice versa.     

Reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol dioxygenases

The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined. Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme. Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates. 3-Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl-cis,cis-muconic acid. However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM). The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported.