Identification of metabolites from the degradation of fluoranthene by Mycobacterium sp. strain PYR-1.

Mycobacterium sp. strain PYR-1, previously shown to extensively mineralize high-molecular-weight polycyclic aromatic hydrocarbons in pure culture and in sediments, degrades fluoranthene to 9-fluorenone-1-carboxylic acid. In this study, 10 other fluoranthene metabolites were isolated from ethyl acetate extracts of the culture medium by thin-layer and high-performance liquid chromatographic methods. On the basis of comparisons with authentic compounds by UV spectrophotometry and thin-layer chromatography as well as gas chromatography-mass spectral and proton nuclear magnetic resonance spectral analyses, the metabolites were identified as 8-hydroxy-7-methoxyfluoranthene, 9-hydroxyfluorene, 9-fluorenone, 1-acenaphthenone, 9-hydroxy-1-fluorenecarboxylic acid, phthalic acid, 2-carboxybenzaldehyde, benzoic acid, phenylacetic acid, and adipic acid. Authentic 9-hydroxyfluorene and 9-fluorenone were metabolized by Mycobacterium sp. strain PYR-1. A pathway for the catabolism of fluoranthene by Mycobacterium sp. strain PYR-1 is proposed.     

Metabolism of hydroxybiphenyl and chloro-hydroxybiphenyl by biphenyl/chlorobiphenyl degradingPseudomonas testosteroni,strain B-356

Summary A biphenyl (BP) and chlorobiphenyl (CBP) metabolizingPseudomonas testosteroni, strain B-356 was also capable of utilizing 2-, 3-, and 4-hydroxybiphenyl. Data presented here suggest that utilization of biphenyl and mono-subtituted biphenyls involves the enzymes of the same pathway. Chloro-hydroxybiphenyls were also metabolized by strain B-356. The unsubstituted ring is first hydroxylated in position 2 and 3 and then cleaved in ameta 1, and 2, position to ultimately generate the benzoic acid derivatives. Since strain B-356 was capable of utilizing benzoic acid and mono-hydroxybenzoic acids, the utilization of biphenyl, 2-, 3-, and 4-hydroxybiphenyl is complete at non-toxic concentrations of the substrates. Chlorobenzoic acids and chloro-hydroxybenzoic acids were not metabolized further by this strain. Studies usingPseudomonas putida, strain KT2440 carrying cloned BP/CBP genes from strain B-356 provided further evidence for the presence of a common pathway for the metabolism of the above compounds inP. testosteroni, strain B-356. Suggestions are made on significance of the broad substrate specificity of the enzymes of biphenyl/chlorobiphenyl pathway in regard to their possible origin and in relation to PCB mixture degradation.     

Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric,and ferulic acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60 degrees C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The beta-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-beta-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-beta-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.     

Isolation and Characterization of Thermophilic Bacilli Degrading Cinnamic, 4-Coumaric, and Ferulic Acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60°C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The β-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-β-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

Degradation of biphenyl by a Micrococcus species

Summary A bacterium capable of utilizing biphenyl as the sole source of carbon was isolated from soil and identified as a Micrococcus species. The organism also utilized 4-chlorobiphenyl and several other aromatic compounds as growth substrates. 2,3-Dihydroxybiphenyl and benzoic acid were identified as intermediates by physico-chemical methods. The bacterium degraded biphenyl to 2,3-dihydroxybiphenyl followed by its meta-ring cleavage to yield 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, which was then hydrolysed to give benzoic acid. Benzoate was further metabolised via a catechol meta-cleavage pathway by a Micrococcus sp.Correspondence to: H. Z. Ninnekar     

Oxidation of biphenyl by the Cyanobacterium,Oscillatoria sp., strain JCM

The oxidation of biphenyl by Cyanobacterium, Oscillatoria sp., strain JCM was studied. The organism grown photoautotrophically in the presence of biphenyl oxidized biphenyl to form 4-hydroxybiphenyl. The structure of the metabolite was elucidated by ultraviolet and mass spectra and shown to be identical to authentic 4-hydroxybiphenyl. In addition this metabolite had properties indentical to 4-hydroxybiphenyl when analyzed by thin-layer and high-pressure liquid chromatography. Experiments with [¹⁴C]-biphenyl showed that over a 24 h period the organism oxidized 2.9% of the added biphenyl to ethyl acetate-soluble products.Abbreviations tlc thin-layer chromatography - hplc high pressure liquid chromatography     

Degradation of 2-sec-butylphenol: 3-sec-butylcatechol,2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid,and 2-methylbutyric acid as intermediates

Pseudomonas sp. strain HBP1 Prp, a mutant of strain HBP1 that was originally isolated on 2-hydroxybiphenyl, was able to grow on 2-sec-butylphenol as the sole carbon and energy source. During growth on 2-sec-butylphenol, 2-methylbutyric acid transiently accumulated in the culture medium. Its concentration reached a maximum after 20 hours and was below detection limit at the end of the growth experiment. The first three enzymes of the degradation pathway — a NADH-dependent monooxygenase, a metapyrocatechase, and ameta-fission product hydrolase — were partially purified. The product of the the monooxygenase reaction was identified as 3-sec-butylcatechol by mass spectrometry. This compound was a substrate for the metapyrocatechase and was converted to 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid which was identified by gas chromatography-mass spectrometry of its trimethylsilyl-derivative. The cofactor independentmeta-cleavage product hydrolase used 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid as a substrate. All three enzymes showed highest activities for 2-hydroxybiphenyl and its metabolites, respectively, indicating that 2-sec-butylphenol is metabolized via the same pathway as 2-hydroxybiphenyl.     

Bacterial metabolism of hydroxylated biphenyls.

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

Bacterial metabolism of hydroxylated biphenyls

Isolates able to grow on 3- or 4-hydroxybiphenyl (HB) as the sole carbon source were obtained by enrichment culture. The 3-HB degrader Pseudomonas sp. strain FH12 used an NADPH-dependent monooxygenase restricted to 3- and 3,3'-HBs to introduce an ortho-hydroxyl. The 4-HB degrader Pseudomonas sp. strain FH23 used either a mono- or dioxygenase to generate a 2,3-diphenolic substitution pattern which allowed meta-fission of the aromatic ring. By using 3-chlorocatechol to inhibit catechol dioxygenase activity, it was found that 2- and 3-HBs were converted by FH23 to 2,3-HB, whereas biphenyl and 4-HB were attacked by dioxygenation. 4-HB was metabolized to 2,3,4'-trihydroxybiphenyl. Neither organism attacked chlorinated HBs. The degradation of 3- and 4-HBs by these strains is therefore analogous to the metabolism of biphenyl, 2-HB, and naphthalene in the requirement for 2,3-catechol formation.     

Degradation of fluoranthene by Pasteurella sp. IFA and Mycobacterium sp. PYR-1: isolation and identification of metabolites

The findings from a biodegradability study of fluoranthene using two pure bacterial strains, Pasteurella sp. IFA (B-2) and Mycobacterium sp. PYR-1 (AM) are reported. Of total fluoranthene, 24% (B-2) and 46% (AM) was biodegraded in an aqueous medium during 14 d of incubation at room temperature. During this period the bacteria were capable of mineralizing approximately two-thirds (B-2) and four-fifths (AM) of biodegraded fluoranthene to CO₂, while one-third (B-2) and one-fifth (AM) of the original fluoranthene remained as stable metabolic products. These metabolites were isolated using liquid–liquid extraction and identified using gas chromatography – mass spectrometry (GC–MS) and derivatization techniques. Two metabolites (9-fluorenone-1-carboxylic acid and 9-fluorenone) were identified by GC–MS directly, while the metabolites 9-fluorenone-1-carboxylic acid, 9-hydroxyfluorene, 9-hydroxy-1-fluorene-carboxylic acid, 2-carboxybenzaldehyde, benzoic acid and phenylacetic acid were determined in their derivatized forms. From the identified metabolites, a fluoranthene biodegradation pathway was proposed for Pasteurella sp. IFA.     

Cloning, expression, and characterization of the katG gene, encoding catalase-peroxidase, from the polycyclic aromatic hydrocarbon-degrading bacterium Mycobacterium sp. strain PYR-1

A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.     

Bacterial Decarboxylation of o-Phthalic Acids

The decarboxylation of phthalic acids was studied with Bacillus sp. strain FO, a marine mixed culture ON-7, and Pseudomonas testosteroni. The mixed culture ON-7, when grown anaerobically on phthalate but incubated aerobically with chloramphenicol, quantitatively converted phthalic acid to benzoic acid. Substituted phthalic acids were also decarboxylated: 4,5-dihydroxyphthalic acid to protocatechuic acid; 4-hydroxyphthalic and 4-chlorophthalic acids to 3-hydroxybenzoic and 3-chlorobenzoic acids, respectively; and 3-fluorophthalic acid to 2-and 3-fluorobenzoic acids. Bacillus sp. strain FO gave similar results except that 4,5-dihydroxyphthalic acid was not metabolized, and both 3- and 4-hydroxybenzoic acids were produced from 4-hydroxyphthalic acid. P. testosteroni decarboxylated 4-hydroxyphthalate (to 3-hydroxybenzoate) and 4,5-dihydroxyphthalate but not phthalic acid and halogenated phthalates. Thus, P. testosteroni and the mixed culture ON-7 possessed 4,5-dihydroxyphthalic acid decarboxylase, previously described in P. testosteroni, that metabolized 4,5-dihydroxyphthalic acid and specifically decarboxylated 4-hydroxyphthalic acid to 3-hydroxybenzoic acid. The mixed culture ON-7 and Bacillus sp. strain FO also possessed a novel decarboxylase that metabolized phthalic acid and halogenated phthalates, but not 4,5-dihydroxyphthalate, and randomly decarboxylated 4-hydroxyphthalic acid. The decarboxylation of phthalic acid is suggested to involve an initial reduction to 1,2-dihydrophthalic acid followed by oxidative decarboxylation to benzoic acid.     

Molecular characterization of a phenanthrene degradation pathway in Mycobacterium vanbaalenii PYR-1

Mycobacterium vanbaalenii PYR-1 is capable of degrading a number of polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites via multiple pathways. Genes for the large and small subunits of a pyrene dioxygenase, nidA and nidB, respectively, were previously identified in M. vanbaalenii PYR-1 [Appl. Environ. Microbiol. 67 (2001) 3577]. A library of the M. vanbaalenii PYR-1 genome was constructed in a fosmid vector to identify additional genes involved in PAH degradation. Twelve fosmid clones containing nidA were identified by Southern hybridization. Sequence analysis of one nidA-positive clone, pFOS608, revealed a number of additional genes involved in PAH degradation. At this locus, one putative operon contained genes involved in phthalate degradation, and another contained genes encoding a putative ABC transporter(s). A number of the genes found in this region are homologous to those involved in phenanthrene degradation via the phthalic acid pathway. The majority of phenanthrene degradation genes were located between putative transposase genes. In Escherichia coli, pFOS608 converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, and converted 1-hydroxy-2-naphthoic acid into 2'-carboxybenzalpyruvate, 2-carboxybenzaldehyde, and phthalic acid. A subclone containing nidA and nidB converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, suggesting that the NidAB dioxygenase is responsible for an initial attack on phenanthrene. This study is the first to identify genes responsible for the degradation of phenanthrene via the phthalic acid pathway in Mycobacterium species.     

Metabolism of Aromatic Amino Acids in Microorganisms

It was found that when Rhodotorula rubra IFO 0911 was grown in a phenylalanine medium, benzoic acid and p-hydroxybenzoic acid besides cinnamic acid were formed in the cultured both. The conversions of cinnamic acid into benzoic acid and of benzoic acid into p-hydroxybenzoic acid, and the degradation of p-hydroxybenzoic acid were demonstrated in intact cells of Rhodotorula rubra. These activities were observed in the cells grown on various media, including the medium containing no phenylalanine, and were found to be distributed widely in Rhodotorula. The cells of Rhodotorula rubra were also able to degrade p-coumaric acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), p-hydroxyphenyl-acetic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). From these results, the metabolic pathways for phenylalanine and tyrosine in Rhodotorula were discussed.     

A high-performance liquid chromatography assay for measuring integrated biphenyl metabolism by intact cells: its use with rat liver and human liver and kidney

A rapid, sensitive high-performance liquid chromatography assay with fluorescence detection for measuring biphenyl metabolism by intact cells has been developed. The assay does not require organic solvent extraction or enzymatic digestion for the measurement of hydroxybiphenyl conjugates. The lower limit of detectability for 4-hydroxybiphenyl is 5 pmol injected. Rat hepatocytes incubated with biphenyl form predominantly 4-hydroxybiphenyl sulfate with lesser amounts of 4-hydroxybiphenyl glucuronide and free hydroxybiphenyls, and small amounts of 3-hydroxybiphenyl sulfate and 3-hydroxybiphenyl glucuronide. Slices of fresh human liver incubated with biphenyl form predominantly 4-hydroxybiphenyl glucuronide with some free hydroxybiphenyl and small amounts of 4-hydroxybiphenyl sulfate. 4-Hydroxybiphenyl glucuronide formation by human liver shows a lag time that is not abolished by preincubating the liver without substrate. Human kidney slices incubated with biphenyl form 4-hydroxybiphenyl glucuronide and 4-hydroxybiphenyl sulfate at rates less than one-tenth those seen with human liver. Human kidney slices do not form detectable free hydroxybiphenyl. There is wide intersubject variability in the rates of hydroxybiphenyl metabolite formation by human liver and kidney.     

Use of Fluorinated Compounds To Detect Aromatic Metabolites from m-Cresol in a Methanogenic Consortium: Evidence for a Demethylation Reaction

Anaerobic sewage sludge was used to enrich a methanogenic m-cresol-degrading consortium. 6-Fluoro-3-methylphenol was synthesized and added to subcultures of the consortium with m-cresol. This caused the accumulation of 4-hydroxy-2-methylbenzoic acid. In a separate experiment, the addition of 3-fluorobenzoic acid caused the transient accumulation of 4-hydroxybenzoic acid. Inhibition with bromoethanesulfonic acid caused the accumulation of benzoic acid. Thus, the proposed degradation pathway was m-cresol → 4-hydroxy-2-methylbenzoic acid → 4-hydroxybenzoic acid → benzoic acid. The m-cresol-degrading consortium was able to convert exogenous 4-hydroxybenzoic acid and benzoic acid to methane. In addition, for each metabolite of m-cresol identified, the corresponding fluorinated metabolite was detected, giving the following sequence: 6-fluoro-3-methylphenol → 5-fluoro-4-hydroxy-2-methylbenzoic acid → 3-fluoro-4-hydroxybenzoic acid → 3-fluorobenzoic acid. The second step in each of these pathways is a novel demethylation which was rate limiting. This demethylation reaction would likely facilitate the transformation of the methyl group to methane, which is consistent with the results of a previous study that showed that the methyl carbon of m-[methyl-¹⁴C]cresol was recovered predominantly as [¹⁴C]methane (D. J. Roberts, P. M. Fedorak, and S. E. Hrudey, Can. J. Microbiol. 33:335-338, 1987). The final aromatic compound in the proposed route for m-cresol metabolism was benzoic acid, and its detection in these cultures merges the pathway for the methanogenic degradation of m-cresol with those for the anaerobic metabolism of many phenols.     

Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2'-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

Cometabolic Degradation of Dibenzofuran by Biphenyl-Cultivated Ralstonia sp. Strain SBUG 290

Cells of the gram-negative bacterium Ralstonia sp. strain SBUG 290 grown in the presence of biphenyl are able to cooxidize dibenzofuran which has been 1,2-hydroxylated. Meta cleavage of the 1,2-dihydroxydibenzofuran between carbon atoms 1 and 9b produced 2-hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid, which was degraded completely via salicylic acid. The presence of these intermediates indicates a degradation mechanism for dibenzofuran via lateral dioxygenation by Ralstonia sp. strain SBUG 290.