Growth phase dependent substrate utilization by Pseudomonas strain PH1

Pseudomonas strain PH1 can utilize nitro-, chloro-, and aminophenols and has been used in this study. The enzymes of the two-pathway viz., phenol, and meta-aminophenol (MAP) were analyzed under different growth conditions. The enzymes responsible for phenol to catechol conversion followed by the ring cleavage enzyme for catechol; and also the enzymes responsible for MAP oxidation and hydroxylation of resorcinol, were studied. Enzyme and respirometric assays were carried out with cells harvested from log phase and stationary phase from medium with different carbon sources and nitrogen levels. It was observed that the first step for utilization of both the substrates requires the same physiological state of the cells; whereas, the subsequent step follows independent approach to intermediates, based on cellular physiology.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Hydroxylation of phenol to catechol by Candida tropicalis: involvement of cytochrome P450

Microsomal preparations isolated from yeast Candida tropicalis (C. tropicalis) grown on three different media with or without phenol were isolated and characterized for the content of cytochrome P450 (CYP) (EC 1.14.15.1). While no CYP was detected in microsomes of C. tropicalis grown on glucose as the carbon source, evidence was obtained for the presence of the enzyme in the microsomes of C. tropicalis grown on media containing phenol. Furthermore, the activity of NADPH: CYP reductase, another enzyme of the microsomal CYP-dependent system, was markedly higher in cells grown on phenol. Microsomes of these cells oxidized phenol. The major metabolite formed from phenol by microsomes of C. tropicalis was characterized by UV/vis absorbance and mass spectroscopy as well as by the chromatographic properties on HPLC. The characteristics are identical to those of catechol. The formation of catechol was inhibited by CO, the inhibitor of CYP, and correlated with the content of cytochrome P450 in microsomes. These results, the first report showing the ring hydroxylation of phenol to catechol with the microsomal enzyme system of C. tropicalis, strongly suggest that CYP-catalyzed reactions are responsible for this hydroxylation. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by the C. tropicalis strain.     

Isolation and Properties of Phenol Utilizing Microorganisms with Special Reference to Catechol 1,2-Oxygenase

Phenol utilizing yeasts were isolated from soil. The relationship were examined between distribution of phenol uptake rate using intact cells and distribution of the activities of catechol 1,2-oxygenase which is one of the key enzymes in phenol metabolism. Two of the isolates showed catechol 1,2-oxygenase activity even when grown in glucose medium, though the enzyme activity was about 1% of the full activity induced by phenol. Partially constitutive mutants for catechol 1,2-oxygenase were obtained by mutagenesis of an inducible strain. The level of mutant enzyme activity was close to that of the isolated constitutive strain. One isolate, Trichosporon cutaneum, preferentially utilized phenol to glucose in medium containing both phenol (200 ppm) and glucose (0.1%), until the concentration of phenol decreased to 10–20 ppm.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.     

Induction of phenol-metabolizing enzymes in Trichosporon cutaneum

Some aspects of the induction of enzymes participating in the metabolism of phenol and resorcinol in Trichosporon cutaneum were studied using intact cells and cell-free preparations.Activities of phenol hydroxylase (1.14.13.7), catechol 1,2-oxygenase (1.13.11.1), cis,cis-muconate cyclase (5.5.1.-), delactonizing enzyme(s) and maleolylacetate reductase were 50–400 times higher in fully induced cells than in noninduced cells.In addition to phenol and resorcinol, also catechol, cresols and fluorophenols could induce phenol hydroxylase.The induction was severely inhibited by phenol concentrations higher than 1 mM. Using optimum inducer concentrations (0.01–0.10 mM), it took more than 8 h to obtain full induction, whether in proliferating or in nonproliferating cells.Phenol hydroxylase, catechol 1,2-oxygenase and cis,cis-muconate cyclase were induced simultaneously. The synthesis of the de-lactonizing activity was delayed in relation to these three preceeding enzymes of the pathway.High glucose concentration (over 15 mM) inhibited completely the induction of phenol oxidation by nonproliferating cells. It also inhibited phenol oxidation by pre-induced cells.Among the NADPH-generating enzymes, the activity of iso-citrate dehydrogenase was elevated in cells grown on phenol and resorcinol instead of glucose.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.Non-standard abbreviations MCPA 4-chloro-2-methylphenoxyacetic acid - 2MPA 2-methylphenoxyacetic acid - PA phenoxyacetic acid     

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.     

Characteristics of a New Catechol 1,2-Oxygenase from Trichosporon cutaneum WY 2-2

Successive feeding of phenol at concentrations of less than 5.5 mM into a thick suspension of Trichosporon cutaneum WY 2-2 precultured in MPY-medium resulted in a high yield (approximately 28.7 g wet cells/liter) of intact cells capable of decomposing phenol actively (3.7 μmol/min/g of wet cells).

The effects of pH and additions of ethanol and 2-mercaptoethanol were tested on the stability of crude extracts from the strain grown on phenol. The crude extracts were stable at a pH range of 7.6 and 8.3, and were stable for 35 days when 10% ethanol and 5 mM 2-mercaptoethanol were added.

A highly purified preparation of catechol 1,2-oxygenase was obtained from strain WY 2-2 grown on phenol. The purified enzyme was homogeneous on polyacrylamide disc-gel electrophoresis. The enzyme had a molecular weight of about 105,000 and gave rise to subunits of molecular weight of 35,000 by SDS gel electrophoresis. Therefore, the enzyme appears to be a trimer of subunits with identical molecular weight. The Michaelis constants were 9.0 μM for catechol and 6.8 μM for 4-methylcatechol. The enzyme exhibited higher activities towards 4-methylcatechol and hydroxyquinol than towards catechol, and had essentially the same substrate specificity as the crude extracts. 4-Methylcatechol completely inhibited the enzyme activity towards catechol.     

Characterization of the phenol monooxygenase gene from Chromobacterium violaceum: Potential use for phenol biodegradation

In this work, the biodegradation mechanism of phenol and sub products (such as catechol and hydroquinone) in Chromobacterium violaceum was investigated by cloning and molecular characterization of a phenol monooxygenase gene in Escherichia coli. This gene (Cvmp) is very similar (74 and 59% of similarity and identity, respectively) to the ortholog from Ralstonia eutropha bacteria capable of utilizing phenol as the sole carbon source. The phenol biodegradation ability of E. coli recombinant strains was tested by cell-growth in a minimal medium containing phenol as the sole source of carbon and release of intermediary metabolites (catechol and hydroquinone). Interestingly, during the growth of these strains on phenol, catechol, and hydroquinone accumulated transiently in the medium. These metabolites were further analyzed by HPLC. These results indicated that phenol can be initially orto or para hydroxylated to produce cathecol or hydroquinone, respectively, followed by meta-cleavage of aromatic rings. To verify this information, the metabolites obtained from HPLC were submitted to LC/MS to confirm their chemical structure, thereby indicating that the recombinant strains utilize two different routes simultaneously, leading to different ring-fission substrates for the metabolism of phenol.     

Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus

Aspergillus fumigatus (ATCC 28282), a thermotolerant fungus, has been shown to be capable of growth on phenol as the sole carbon and energy source. During growth of the organism on phenol, catechol and hydroquinone accumulated transiently in the medium; cells grown on phenol oxidised these compounds without a lag period. Two different routes operating simultaneously, leading to different ring-fission substrates, are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol, which is then cleaved by an intradiol mechanism leading to 3-oxoadipate. In the other route, phenol is hydroxylated in the para-position to produce hydroquinone, which is then converted into 1,2,4-trihydroxybenzene for ring fission by ortho-cleavage to give maleylacetate. Cell-free extracts of phenol-grown mycelia were found to contain enzymic activities for the proposed steps. Two ring-fission dioxygenases, one active towards 1,2,4-trihydroxybenzene, but not catechol, and one active towards both ring-fission substrates, were separated by FPLC. Succinate-grown mycelia did not oxidise any of the intermediates until a clear lag period had elapsed and did not contain any of the enzymic activities for phenol metabolism.     

Mineralization of phenol and its derivatives by Pseudomonas sp. strain CP4

A Pseudomonas sp. strain, CP4, was isolated that used phenol up to 1.5 g/l as sole source of carbon and energy. Optimal growth on 1.5 g phenol/l was at pH 6.5 to 7.0 and 30°C. Unadapted cells needed 72 h to decrease the chemical oxygen demand (COD) of about 2000 mg/l (from 1 g phenol/l) to about 200 mg/l. Adapted cells, pregrown on phenol, required only 65 h to decrease the COD level to below 100 mg/l. Adaptation of cells to phenol also improved the degradation of cresols. Cell-free extracts of strain CP4 grown on phenol or o-, m- or p-cresol had sp. act. of 0.82, 0.35, 0.54 and 0.32 units of catechol 2,3-dioxygenase and 0.06, 0.05, 0.05 and 0.03 units of catechol 1,2-dioxygenase, respectively. Cells grown on glucose or succinate had neither activity. Benzoate and all isomers of cresol, creosote, hydroxybenzoates, catechol and methyl catechol were utilized by strain CP4. No chloroaromatic was degraded, either as sole substrate or as co-substrate.The authors are with the Department of Microbiology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India     

The catechol 2,3-dioxygenase gene and toluene monooxygenase genes from Burkholderia sp. AA1, an isolate capable of degrading aliphatic hydrocarbons and toluene

Burkholderia sp. AA1 isolated from a diesel fuel-contaminated site degraded toluene, as well as a wide range of alkanes from decane (C₈) to pentacosane (C₂₅) as sole carbon and energy sources. This strain also utilized m-toluate, p-toluate, o-toluate, and m-cresol as sole carbon and energy sources. Toluene- and toluate-grown cells showed catechol 2,3-dioxygenase activity and indole oxidation activity that is exhibited by some toluene oxygenation enzymes. The catechol 2,3-dioxygenase gene (catB) was cloned and sequenced. Its deduced amino acid sequence is analogous to the extradiol dioxygenases cloned from a variety of microorganisms. A DNA fragment containing the genes for the indole oxidation activity was cloned and sequenced. A seven-gene cluster designated as tbhABCDEFG was identified. Significant similarities were found with multicomponent monooxygenase systems for toluene, benzene and phenol from different bacterial strains. Journal of Industrial Microbiology & Biotechnology (2000) 25, 127–131. Received 28 July 1999/ Accepted in revised form 28 June 2000     

Soil and Plant Contamination with <Emphasis Type="Italic">Mycobacterium Avium</Emphasis> subsp. <Emphasis Type="Italic">Paratuberculosis</Emphasis> After Exposure to Naturally Contaminated Mouflon Feces

The aim of this study was to demonstrate the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in soil and colonization of different plant parts after deliberate exposure to mouflon feces naturally contaminated with different amounts of MAP. Samples of aerial parts of plants, their roots, and the soil below the roots were collected after 15 weeks and examined using IS900 real-time quantitative PCR (qPCR) and cultivation. Although the presence of viable MAP cells was not demonstrated, almost all samples were found to be positive using qPCR. MAP IS900 was not only found in the upper green parts, but also in the roots and soil samples (from 1.00 × 10⁰ to 6.43 × 10³). The level of soil and plant contamination was influenced mainly by moisture, clay content, and the depth from which the samples were collected, rather than by the initial concentration of MAP in the feces at the beginning of the experiment.     

Kinetic studies of phenol degradation by Rhodococcus sp. P1 II. Continuous cultivation

The degradation of phenol by Rhodococcus sp. P1 was studied in continuous culture systems. The organism could be adapted by slowly increasing concentration, step by step, up to 30.0 g · 1^-1 phenol in the influent. The degradation rate reached values of about 0.3 g · g dry mass^-1 ·h^-1. Large step increases in phenol concentration and addition of further substrates (e.g., catechol) were tolerated up to a certain concentration. With increasing dilution rate and increasing inlet phenol concentration the stability of the system decreased.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Phenol metabolism by two microorganisms isolated from Amazonian forest soil samples

Two microorganisms isolated from Amazonian forest soil samples and identified as Candida tropicalis and Alcaligenes faecalis were capable of degrading phenol (16 and 12 mM, respectively) at high salt concentrations (15% and 5.6%, respectively). Chromatographic and enzymatic studies revealed that each microorganism cleaved phenol at the ortho position with total phenol mineralization. ¹⁴C-phenol mineralization assays showed that both microorganisms assimilated about 30% of the total label. No phenol degradation metabolite (i.e., catechol, cis, cis-muconic acid) was detected in the intercellular medium. The presence of phenol hydroxylase (EC 1.14.13.7) and catechol 1,2-dioxygenase (EC 1.13.11.1) extracellular activity suggested that these microorganisms may secrete these enzymes into the extracellular medium. Journal of Industrial Microbiology & Biotechnology (2000) 24, 403–409. Received 02 November 1999/ Accepted in revised form 08 March 2000     

Oxidation of phenols by cells and cell-free enzymes from Candida tropicalis

A yeast strain isolated from soil by enrichment on phenol as major carbon source was identified as Candida tropicalis. Washed cell suspensions of this strain and cell-free preparations obtained from mechanically disrupted cells oxidized phenol via catechol and cis, cis-muconate. In addition to phenol and the three isomeric diphenols, a number of phenol derivatives, amongst them fluoro-, nitro- and short-chain alkyl-phenols, were oxidized by the organism. However, no significant oxygen uptake could be demonstrated in the presence of pyrogallol, phloroglucinol, the cresols, the m-and p-hydroxy-benzoates, methoxylated phenol derivatives, benzene or toluene. Cell-free preparations from the yeast strain exhibited activity of phenol hydroxylase and of catechol 1,2-oxygenase. Both enzymes appeared in the soluble cell fraction. Both exhibit broad substrate specificities. The relative specific activity of the ring-cleaving enzyme towards various substrates seems to be dependent on the phenolic inducer.