Detailed comparison between the substrate specificities of two angular dioxygenases, dibenzofuran 4,4a-dioxygenase from Terrabacter sp. and carbazole 1,9a-dioxygenase from Pseudomonas resinovorans

The preferred substrates in angular dioxygenation, monooxygenation, and lateral dioxygenation by dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 and carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas resinovorans strain CA10 are shown to be distinctly different. The preferred oxygenation reactions suggest that DFDO evolved from a polycyclic aromatic hydrocarbon dioxygenase and that its most preferred substrates were fluorene and 9-fluorenone. The angular dioxygenases involved in the degradation pathway of dibenzofuran (dioxin) and fluorene are closely related in function, while CARDO is a novel enzyme not only phylogenetically but also functionally.     

Degradation of fluorene by Brevibacterium sp. strain DPO 1361: a novel C-C bond cleavage mechanism via 1,10-dihydro-1,10-dihydroxyfluoren-9-one.

Angular dioxygenation has been established as the crucial step in dibenzofuran degradation by Brevibacterium sp. strain DPO 1361 (V. Strubel, K. H. Engesser, P. Fischer, and H.-J. Knackmuss, J. Bacteriol. 173:1932-1937, 1991). The same strain utilizes biphenyl and fluorene as sole sources of carbon and energy. The fluorene degradation sequence is proposed to be initiated by oxidation of the fluorene methylene group to 9-fluorenol. Cells grown on fluorene exhibit pronounced 9-fluorenol dehydrogenase activity. Angular dioxygenation of the 9-fluorenone thus formed yields 1,10-dihydro-1,10-dihydroxyfluoren-9-one (DDF). A mechanistic model is presented for the subsequent C-C bond cleavage by an NAD(+)-dependent DDF dehydrogenase, acting on the angular dihydrodiol. This enzyme was purified and characterized as a tetramer of four identical 40-kDa subunits. The following Km values were determined: 13 microM for DDF and 65 microM for 2,3-dihydro-2,3-dihydroxybiphenyl. The enzyme also catalyzes the production of 3-(2'-carboxyphenyl)catechol, which was isolated, and structurally characterized, in the form of the corresponding lactone, 4-hydroxydibenzo-(b,d)-pyran-6-one. Stoichiometry analysis unequivocally demonstrates that angular dioxygenation constitutes the principal pathway in Brevibacterium sp. strain DPO 1361.     

Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63

A gram-positive bacterium Terrabacter sp. strain DBF63 is able to degrade dibenzofuran (DF) via initial dioxygenation by a novel angular dioxygenase. The dbfA1 and dbfA2 genes, which encode the large and small subunits of the dibenzofuran 4,4a-dioxygenase (DFDO), respectively, were isolated by a polymerase chain reaction-based method. DbfA1 and DbfA2 showed moderate homology to the large and small subunits of other ring-hydroxylating dioxygenases (less than 40%), respectively, and some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands were conserved in DbfA1. DFDO activity was confirmed in Escherichia coli cells containing the cloned dbfA1 and dbfA2 genes with the complementation of nonspecific ferredoxin and ferredoxin reductase component of E. coli. Under this condition, these cells exhibited angular dioxygenation of DF and dibenzo-p-dioxin, and monooxygenation of fluorene, but not angular dioxygenation of carbazole, xanthene, and phenoxathiin. Phylogenetic analysis revealed that DbfA1 formed a branch with recently reported large subunits of polycyclic aromatic hydrocarbon (PAH) dioxygenase from gram-positive bacteria but did not cluster with that of other angular dioxygenases, i.e., DxnA1 from Sphingomonas sp. strain RW1 [Armengaud, J., Happe, B., and Timmis, K. N. J. Bacteriol. 180, 3954-3966, 1998] and CarAa from Pseudomonas sp. strain CA10 [Sato, S., Nam, J.-W., Kasuga, K., Nojiri, H., Yamane, H., and Omori, T. J. Bacteriol. 179, 4850-4858, 1997].     

Dioxygenolytic cleavage of aryl ether bonds: 1, 10-dihydro-1, 10-dihydroxyfluoren-9-one, a novel arene dihydrodiol as evidence for angular dioxygenation of dibenzofuran

Two dibenzofuran degrading bacteria, Brevibacterium strain DPO 1361 and strain DPO 220, were found to utilize fluorene as sole source of carbon and energy. Cells which were grown on dibenzofuran, transformed fluorene into a number of products. For five of the seven metabolites isolated, the structure could be established unequivocally. Accumulation of one metabolite, 1,10-dihydroxy-1,10-dihydrofluoren-9-one, indicated the presence of a novel type of dioxygenase, attacking polynuclear aromatic systems in the unusual angular position. Debenzofuran degradation is proposed to likewise proceed via initial angular dioxygenation. One aryl oxygen ether bond, which normally is extremely stable, is thus transformed to a hemiacetal. After spontaneous cleavage and subsequent rearomatization by dehydration, 2,2',3-trihydroxybiphenyl [3-(2-hydroxyphenyl)-catechol] thus results as the immediate product of the first enzymatic reaction in the degradation sequence.     

Bacterial degradation of aromatic compounds via angular dioxygenation

Dioxygenation is one of the important initial reactions of the bacterial degradation of various aromatic compounds. Aromatic compounds, such as biphenyl, toluene, and naphthalene, are dioxygenated at lateral positions of the aromatic ring resulting in the formation of cis-dihydrodiol. This "normal" type of dioxygenation is termed lateral dioxygenation. On the other hand, the analysis of the bacterial degradation of fluorene (FN) analogues, such as 9-fluorenone, dibenzofuran (DF), carbazole (CAR), and dibenzothiophene (DBT)-sulfone, and DF-related diaryl ether compounds, dibenzo-p-dioxin (DD) and diphenyl ether (DE), revealed the presence of the novel mode of dioxygenation reaction for aromatic nucleus, generally termed angular dioxygenation. In this atypical dioxygenation, the carbon bonded to the carbonyl group in 9-fluorenone or to heteroatoms in the other compounds, and the adjacent carbon in the aromatic ring are both oxidized. Angular dioxygenation of DF, CAR, DBT-sulfone, DD, and DE produces the chemically unstable hemiacetal-like intermediates, which are spontaneously converted to 2,2',3-trihydroxybiphenyl, 2'-aminobiphenyl-2,3-diol, 2',3'-dihydroxybiphenyl-2-sulfinate, 2,2',3-trihydroxydiphenyl ether, and phenol and catechol, respectively. Thus, angular dioxygenation for these compounds results in the cleavage of the three-ring structure or DE structure. The angular dioxygenation product of 9-fluorenone, 1-hydro-1,1a-dihydroxy-9-fluorenone is a chemically stable cis-diol, and is enzymatically transformed to 2'-carboxy-2,3-dihydroxybiphenyl. 2'-Substituted 2,3-dihydroxybiphenyls formed by angular dioxygenation of FN analogues are degraded to monocyclic aromatic compounds by meta cleavage and hydrolysis. Thus, after the novel angular dioxygenation, subsequent degradation pathways are homologous to the corresponding part of that of biphenyl. Compared to the bacterial strains capable of catalyzing lateral dioxygenation, few bacteria having angular dioxygenase have been reported. Only a few degradation pathways, CAR-degradation pathway of Pseudomonas resinovorans strain CA10, DF/DD-degradation pathway of Sphingomonas wittichii strain RW1, DF/DD/FN-degradation pathway of Terrabacter sp. strain DBF63, and carboxylated DE-degradation pathway of P. pseudoalcaligenes strain POB310, have been investigated at the gene level. As a result of the phylogenetic analysis and the comparison of substrate specificity of angular dioxygenase, it is suggested that this atypical mode of dioxygenation is one of the oxygenation reactions originating from the relaxed substrate specificity of the Rieske nonheme iron oxygenase superfamily. Genetic characterization of the degradation pathways of these compounds suggests the possibility that the respective genetic elements constituting the entire catabolic pathway have been recruited from various other bacteria and/or other genetic loci, and that these pathways have not evolutionary matured.     

Mineralization of 4-Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and Burkholderia sp. Strain JWS

The dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) attacks 4-chlorodibenzofuran on the unsubstituted aromatic ring via distal dioxygenation adjacent to the ether bridge to produce 3(prm1)-chloro-2,2(prm1),3-trihydroxybiphenyl, which was identified by nuclear magnetic resonance spectroscopy and mass spectrometry. The compound is subsequently meta cleaved, and the respective intermediate is hydrolyzed to form a C-5 moiety, which is further degraded to Krebs cycle intermediates and to 3-chlorosalicylate. This dead-end product is released into the culture medium. A coculture of strain RW1 and the 3,5-dichlorosalicylate-degrading strain Burkholderia sp. strain JWS (A. Schindowski, R.-M. Wittich, and P. Fortnagel, FEMS Microbiol. Lett. 84:63-70, 1991) is able to completely degrade 4-chlorodibenzofuran with concomitant release of Cl(sup-) and formation of biomass.     

Degradation of polycyclic aromatic hydrocarbons by a newly isolated dibenzofuran-utilizing<Emphasis Type="Italic"> Janibacter</Emphasis> sp. strain YY-1

The dibenzofuran (DF)-utilizing bacterium strain YY-1 was newly isolated from soil. The isolate was identified as Janibacter sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various physiological characteristics. In addition to DF, strain YY-1 could grow on fluorene and dibenzothiophene as sole sources of carbon and energy. It was also able to cometabolize a variety of polycyclic aromatic hydrocarbons including dibenzo-p-dioxin, phenanthrene, and anthracene. The major metabolites formed from DF, biphenyl, dibenzothiophene, and naphthalene were identified by using gas chromatography-mass spectrometry as 2,3,2-trihydroxybiphenyl, biphenyl-dihydrodiol, dibenzothiophene 5-oxide, and coumarin, respectively. These results indicate that strain YY-1 can catalyze angular dioxygenation, lateral dioxygenation, and sulfoxidation.     

New metabolites in the degradation of fluorene by Arthrobacter sp. strain F101.

Identification of new metabolites and demonstration of key enzyme activities support and extend the pathways previously reported for fluorene metabolism by Arthrobacter sp. strain F101. Washed-cell suspensions of strain F101 with fluorene accumulated 9-fluorenone, 4-hydroxy-9-fluorenone, 3-hydroxy-1-indanone, 1-indanone, 2-indanone, 3-(2-hydroxyphenyl) propionate, and a compound tentatively identified as a formyl indanone. Incubations with 2-indanone produced 3-isochromanone. The growth yield with fluorene as a sole source of carbon and energy corresponded to an assimilation of about 34% of fluorene carbon. About 7.4% was transformed into 9-fluorenol, 9-fluorenone, and 4-hydroxy-9-fluorenone. Crude extracts from fluorene-induced cells showed 3,4-dihydrocoumarin hydrolase and catechol 2,3-dioxygenase activities. These results and biodegradation experiments with the identified metabolites indicate that metabolism of fluorene by Arthrobacter sp. strain F101 proceeds through three independent pathways. Two productive routes are initiated by dioxygenation at positions 1,2 and 3,4, respectively. meta cleavage followed by an aldolase reaction and loss of C-1 yield the detected indanones. Subsequent biological Baeyer-Villiger reactions produce the aromatic lactones 3,4-dihydrocoumarin and 3-isochromanone. Enzymatic hydrolysis of the former gives 3-(2-hydroxyphenyl) propionate, which could be a substrate for a beta oxidation cycle, to give salicylate. Further oxidation of the latter via catechol and 2-hydroxymuconic semialdehyde connects with the central metabolism, allowing the utilization of all fluorene carbons. Identification of 4-hydroxy-9-fluorenone is consistent with an alternative pathway initiated by monooxygenation at C-9 to give 9-fluorenol and then 9-fluorenone. Although dioxygenation at 3,4 positions of the ketone apparently occurs, this reaction fails to furnish a subsequent productive oxidation of this compound.     

Biotransformation of fluorene, diphenyl ether, dibenzo-p-dioxin and carbazole by Janibacter sp.

Fluorene, diphenyl ether, dibenzo-p-dioxin, and carbazole were used by a dibenzofuran-utilizing Janibacter sp. strain YY-1. Metabolites were identified by GC-MS. Angular dioxygenation was the major pathway for degradation of fluorene, diphenyl ether, and dibenzo-p-dioxin but not for carbazole. Lateral dioxygenation of all tested compounds was indicated by the detection of mono- or di-hydroxylated compounds. The bacterium also catalyzed the monooxygenation of fluorene at the C9 position.     

Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in <Emphasis Type="Italic">Terrabacter</Emphasis> sp. strain DBF63

Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.     

Metabolism of Hydroxydibenzofurans, Methoxydibenzofurans, Acetoxydibenzofurans, and Nitrodibenzofurans by Sphingomonas sp. Strain HH69

The metabolism of 11 substituted dibenzofurans by the dibenzofuran-degrading Sphingomonas sp. strain HH69 was investigated. Strain HH69 utilizes 2-, 3-, and 4-acetoxydibenzofuran as well as 2-, 3-, and 4-hydroxydibenzofuran as sole sources of carbon and energy. The degradation of acetoxydibenzofurans is initiated by hydrolysis of the ester bonds, yielding the corresponding hydroxydibenzofurans and acetate. Strain HH69 grew on 2-methoxydibenzofuran only after it was adapted to the utilization of 5-methoxysalicylic acid, whereas 3- and 4-methoxydibenzofuran as well as 2- and 3-nitrodibenzofuran were only cooxidized. During the breakdown of all eight hydroxy-, methoxy-, and nitrodibenzofurans studied here, the corresponding substituted salicylic acids accumulated in the culture broth. In the cases of 2- and 3-hydroxydibenzofuran as well as 2- and 3-nitrodibenzofuran, salicylic acid was also formed. Those four dibenzofurans which did not serve as carbon sources for strain HH69 were converted to a nonutilizable salicylic acid derivative. From turnover experiments with the mutant HH69/II, which is deficient in meta-cleavage, 2,2(prm1),3,4(prm1)-tetrahydroxybiphenyl, 2,2(prm1),3-trihydroxy-5(prm1)-methoxybiphenyl, 2,2(prm1),3-trihydroxy-5(prm1)-nitrobiphenyl, and 2,2(prm1),3-trihydroxy-4(prm1)-nitrobiphenyl were isolated as the main products formed from 3-hydroxydibenzofuran, 2-methoxydibenzofuran, and 2- and 3-nitrodibenzofuran, respectively. These results indicate significant regioselectivity for the dioxygenolytic cleavage of the ether bond of these monosubstituted dibenzofurans, with a preference for the nonsubstituted aromatic nucleus. Substituted trihydroxybiphenyls are converted further by meta-cleavage followed by the removal of the side chain of the resulting product. A stepwise degradation of this side chain was found to be involved in the metabolism of 2-hydroxydibenzofuran.     

Degradation of chlorinated biphenyl,dibenzofuran, and dibenzo-p-dioxin by marine bacteria that degrade biphenyl,carbazole, or dibenzofuran

Marine bacterial strains (BP-PH, CAR-SF, and DBF-MAK) were isolated using biphenyl, carbazole (CAR), or dibenzofuran (DF) respectively as substrates for growth. Their 16S ribosomal DNA sequences showed that the species closest to strain BP-PH, strain CAR-SF, and strain DBF-MAK are Alteromonas macleodii (96.3% identity), Neptunomonas naphthovorans (93.1% identity), and Cycloclasticus pugetii (97.3% identity), respectively. The metabolites produced suggested that strain CAR-SF degrades CAR via dioxygenation in the angular position and by the meta-cleavage pathway, and that strain DBF-MAK degrades DF via both lateral and angular dioxygenation. Polychlorinated biphenyl (KC-300) and 2,3-dichlorodibenzo-p-dioxin were partially degraded by strain BP-PH and strain DBF-MAK, while 2,7-dichlorodibenzo-p-dioxin and 2,4,8-trichlorodibenzofuran remained virtually unchanged.     

Oxidation of Naphthenoaromatic and Methyl-Substituted Aromatic Compounds by Naphthalene 1,2-Dioxygenase

Oxidation of acenaphthene, acenaphthylene, and fluorene was examined with recombinant strain Pseudomonas aeruginosa PAO1(pRE695) expressing naphthalene dioxygenase genes cloned from plasmid NAH7. Acenaphthene underwent monooxygenation to 1-acenaphthenol with subsequent conversion to 1-acenaphthenone and cis- and trans-acenaphthene-1,2-diols, while acenaphthylene was dioxygenated to give cis-acenaphthene-1,2-diol. Nonspecific dehydrogenase activities present in the host strain led to the conversion of both of the acenaphthene-1,2-diols to 1,2-acenaphthoquinone. The latter was oxidized spontaneously to naphthalene-1,8-dicarboxylic acid. No aromatic ring dioxygenation products were detected from acenaphthene and acenaphthylene. Mixed monooxygenase and dioxygenase actions of naphthalene dioxygenase on fluorene yielded products of benzylic 9-monooxygenation, aromatic ring dioxygenation, or both. The action of naphthalene dioxygenase on a variety of methyl-substituted aromatic compounds, including 1,2,4-trimethylbenzene and isomers of dimethylnaphthalene, resulted in the formation of benzylic alcohols, i.e., methyl group monooxygenation products, which were subsequently converted to the corresponding carboxylic acids by dehydrogenase(s) in the host strain. Benzylic monooxygenation of methyl groups was strongly predominant over aromatic ring dioxygenation and essentially nonspecific with respect to the substitution pattern of the aromatic substrates. In addition to monooxygenating benzylic methyl and methylene groups, naphthalene dioxygenase behaved as a sulfoxygenase, catalyzing monooxygenation of the sulfur heteroatom of 3-methylbenzothiophene.     

Diverse Oxygenations Catalyzed by Carbazole 1,9a-Dioxygenase from Pseudomonas sp. Strain CA10

Carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp. strain CA10 is a multicomponent enzyme that catalyzes the angular dioxygenation of carbazole, dibenzofuran, and dibenzo-p-dioxin. It was revealed by gas chromatography-mass spectrometry and 1H and 13C nuclear magnetic resonance analyses that xanthene and phenoxathiin were converted to 2,2',3-trihydroxydiphenylmethane and 2,2',3-trihydroxydiphenyl sulfide, respectively. Thus, for xanthene and phenoxathiin, angular dioxygenation by CARDO occurred at the angular position adjacent to the oxygen atom to yield hetero ring-cleaved compounds. In addition to the angular dioxygenation, CARDO catalyzed the cis dihydroxylation of polycyclic aromatic hydrocarbons and biphenyl. Naphthalene and biphenyl were converted by CARDO to cis-1, 2-dihydroxy-1,2-dihydronaphthalene and cis-2,3-dihydroxy-2, 3-dihydrobiphenyl, respectively. On the other hand, CARDO also catalyzed the monooxygenation of sulfur heteroatoms in dibenzothiophene and of the benzylic methylenic group in fluorene to yield dibenzothiophene-5-oxide and 9-hydroxyfluorene, respectively. These results indicate that CARDO has a broad substrate range and can catalyze diverse oxygenation: angular dioxygenation, cis dihydroxylation, and monooxygenation. The diverse oxygenation catalyzed by CARDO for several aromatic compounds might reflect the differences in the binding of the substrates to the reaction center of CARDO.     

Emergence of multifunctional oxygenase activities by random priming recombination

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.     

Identification of a novel metabolite in the degradation of pyrene by Mycobacterium sp. strain AP1: actions of the isolate on two- and three-ring polycyclic aromatic hydrocarbons.

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6'-dihydroxy-2,2'-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.     

Metabolism of fluoranthene by Mycobacterium sp. strain AP1

The pyrene-degrading Mycobacterium strain AP1 was found to utilize fluoranthene as a sole source of carbon and energy. Identification of metabolites formed from fluoranthene (by growing cells and washed-cell suspensions), the kinetics of metabolite accumulation, and metabolite-feeding studies all indicated that strain AP1 oxidizes fluoranthene using three alternative routes. The first route is initiated by dioxygenation at C-7 and C-8 and, following meta cleavage and pyruvate release, produces a hydroxyacenaphthoic acid that is decarboxylated to acenaphthenone (V). Monooxygenation of this ketone to the quinone and subsequent hydrolysis generates naphthalene-1,8-dicarboxylic acid (IV), which is further degraded via benzene-1,2,3-tricarboxylic acid (III). A second route involves dioxygenation at C-1 and C-2, followed by dehydrogenation and meta cleavage of the resulting diol. A two-carbon fragment excision of the meta cleavage product yields 9-fluorenone-1-carboxylic acid (II), which appears to undergo angular dioxygenation and further degradation to produce benzene-1,2,3-tricarboxylic acid (III), merging this route with the 7,8-dioxygenation route. Decarboxylation of benzene-1,2,3-tricarboxylic acid to phthalate (VIII), as well as further oxidation of the latter, would connect both routes with the central metabolism. The identification of Z-9-carboxymethylenefluorene-1-carboxylic acid (I) suggests a third route for fluoranthene degradation involving dioxygenation at C-2, C-3, and ortho cleavage. There is no evidence of any further degradation of this compound.     

Two angular dioxygenases contribute to the metabolic versatility of dibenzofuran-degrading Rhodococcus sp. strain HA01

Rhodococcus sp. strain HA01, isolated through its ability to utilize dibenzofuran (DBF) as the sole carbon and energy source, was also capable, albeit with low activity, of transforming dibenzo-p-dioxin (DD). This strain could also transform 3-chlorodibenzofuran (3CDBF), mainly by angular oxygenation at the ether bond-carrying carbon (the angular position) and an adjacent carbon atom, to 4-chlorosalicylate as the end product. Similarly, 2-chlorodibenzofuran (2CDBF) was transformed to 5-chlorosalicylate. However, lateral oxygenation at the 3,4-positions was also observed and yielded the novel product 2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran. Two gene clusters encoding enzymes for angular oxygenation (dfdA1A2A3A4 and dbfA1A2) were isolated, and expression of both was observed during growth on DBF. Heterologous expression revealed that both oxygenase systems catalyze angular oxygenation of DBF and DD but exhibited complementary substrate specificity with respect to CDBF transformation. While DfdA1A2A3A4 oxygenase, with high similarity to DfdA1A2A3A4 oxygenase from Terrabacter sp. strain YK3, transforms 3CDBF by angular dioxygenation at a rate of 29% +/- 4% that of DBF, 2CDBF was not transformed. In contrast, DbfA1A2 oxygenase, with high similarity to the DbfA1A2 oxygenase from Terrabacter sp. strain DBF63, exhibited complementary activity with angular oxygenase activity against 2CDBF but negligible activity against 3CDBF. Thus, Rhodococcus sp. strain HA01 constitutes the first described example of a bacterial strain where coexpression of two angular dioxygenases was observed. Such complementary activity allows for the efficient transformation of chlorinated DBFs.     

Biodegradation of dioxin by a newly isolated Rhodococcus sp. with the involvement of self-transmissible plasmids

A newly isolated Rhodococcus sp. strain p52 could aerobically utilize dibenzofuran as the sole source of carbon and energy, and completely remove dibenzofuran at 500 mg?l^?1 within 48 h. The strain metabolizes dibenzofuran by initial angular dioxygenation to yield 2,2′,3-trihydroxybiphenyl. Strain p52 could also remove 70 % of 100 mg?l^?1 2-chlorodibenzofuran within 96 h and could metabolize a variety of aromatic compounds, namely dibenzo-p-dioxin, 2,8-dichlorodibenzofuran, dibenzothiophene, biphenyl, naphthalene, fluorene, phenanthrene, anthracene, carbazole, indole, xanthene, phenoxathiin, xanthone, and 9-fluorenone. Two distinct gene clusters encoding angular dioxygenases (DbfA and DfdA) were amplified and sequenced. The dbfA and dfdA gene clusters are located on two circular plasmids, pDF01 and pDF02, respectively. Both plasmids are self-transmissible; that is, they can transfer to the Gram-positive bacterium Bacillus cereus by conjugation.     

Characterization of a Novel Angular Dioxygenase from Fluorene-Degrading Sphingomonas sp. Strain LB126

In this study, the genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The α and β subunits of a dioxygenase complex (FlnA1-FlnA2), showing 63 and 51% sequence identity, respectively, to the subunits of an angular dioxygenase from the gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in Escherichia coli, FlnA1-FlnA2 was responsible for the angular oxidation of fluorene, 9-hydroxyfluorene, 9-fluorenone, dibenzofuran, and dibenzo-p-dioxin. Moreover, FlnA1-FlnA2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. The quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1-FlnA2.