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.     

Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium, Paenibacillus sp. strain YK5

Spore-forming bacterial strains capable of utilizing dibenzofuran (DF) as a sole source of carbon and energy were isolated. Characteristics of the isolates justified their classification into the genus Paenibacillus, and their closest relative was P. naphthalenovorans. Degenerate primers for aromatic hydrocarbon dioxygenase alpha subunit (AhDOa) genes and genomic DNA of the strain YK5 were used for gene isolation. The nucleotide sequences of clones of the PCR products revealed that the strain YK5 carries at least five different AhDOa genes. Northern hybridization analysis showed that one of the AhDOa genes was transcribed under DF-containing culture conditions. A gene cluster encoding the AhDOa was isolated. The genes predicted to encode extradiol dioxygenase (dbfB) and hydrolase (dbfC) were found to be an upstream of genes encoding the alpha and beta subunit of the AhDO (dbfA1 and dbfA2, respectively); the latter two gene products showed 60 and 53% identity to the amino acid sequences of DbfA1 and DbfA2 of Terrabacter sp. DBF63, respectively. Two Paenibacillus validus JCM 9077 strains transformed with the dbf gene clusters acquired the ability to convert DF to 2,2′,3-trihydroxybiphenyl (THBP) and salicylic acid (SAL). These results suggest that the enzymes encoded by the gene cluster isolated in this study are involved in DF metabolism in YK5.     

Biodegradation of Dibenzofuran by Janibacter terrae Strain XJ-1

The dibenzofuran (DF)-degrading bacterium, Janibacter terrae strain XJ-1, was isolated from sediment from East Lake in Wuhan, China. This strain grows aerobically on DF as the sole source of carbon and energy; it has a doubling time of 12 hours at 30°C; and it almost completely degraded 100 mg/L⁻¹ DF in 5 days, producing 2,2′,3-trihydroxybiphenyl, salicylic acid, gentisic acid, and other metabolites. The dbdA (DF dioxygenase) gene cluster in the strain is almost identical to that on a large plasmid in Terrabacter sp. YK3. Unlike Janibacter sp. strain YY-1, XJ-1 accumulates gentisic acid rather than catechol as a final product of DF degradation.     

Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Two Types of Bacteria Having Angular Dioxygenases with Different Features

Two kinds of bacteria having different-structured angular dioxygenases—a dibenzofuran (DF)-utilizing bacterium, Terrabacter sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp. strain CA10—were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.     

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.     

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.     

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.     

Genetic characterization of the dibenzofuran-degrading Actinobacteria carrying the dbfA1A2 gene homologues isolated from activated sludge

Thirteen dibenzofuran (DF)-utilizing bacteria carrying the DF terminal dioxygenase genes homologous to those of Terrabacter sp. strain DBF63 (dbfA1A2) were newly isolated from activated sludge samples. The amplified ribosomal DNA restriction analysis and the hybridization analyses showed that these strains were grouped into five genetically different types of bacteria. The sequence analyses of the 16S rRNA genes and the dbfA1A2 homologues from these five selected isolates revealed that the isolates belonged to the genus Rhodococcus, Terrabacter or Janibacter and that they shared 99-100% conserved dbfA1A2 homologues. We investigated the genetic organizations flanking the dbfA1A2 homologues and showed that the minimal conserved DNA region present in all five selected isolates consisted of an approximately 9.0-kb region and that their outer regions became abruptly non-homologous. Among them, Rhodococcus sp. strain DFA3 possessed not only the 9.0-kb region but also the 6.2-kb region containing dbfA1A2 homologues. Sequencing of their border regions suggested that some genetic rearrangement might have occurred with insertion sequence-like elements. Also, within their conserved regions, some insertions or deletions were observed.     

Isolation and characterization of dibenzofuran-degrading actinomycetes: analysis of multiple extradiol dioxygenase genes in dibenzofuran-degrading Rhodococcus species

Sixteen actinomycetes capable of utilizing dibenzofuran as a sole source of carbon and energy were isolated, including Rhodococcus, Microbacterium, and Terrabacter genera. Heretofore, no dibenzofuran-utilizing strain belonging to the genus Microbacterium has been reported. Five extradiol dioxygenase genes (dfdB, and edil to 4) of the strain Rhodococcus sp. YK2 were cloned and analyzed. The nucleotide sequence of dfdB gene was almost identical to the bphC1 gene of Terrabacter sp. DPO360, which was involved in dibenzofuran metabolism in this strain. Southern and Northern hybridization analyses using these extradiol dioxygenase genes as probes suggest that the dfdB gene in YK2 was conserved in diverse dibenzofuran-utilizing actinomycetes; also, the dfdB gene was the only expressed gene among five extradiol dioxygenase genes in the medium containing DF as a sole carbon source. These results suggest that the dfdB gene is important for dibenzofuran metabolism not only in the strain YK2, but also in diverse dibenzofuran-degrading actinomycetes.     

Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63

Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min^–1 mg^–1) and convert dibenzofuran to 2,2,3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.     

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.     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.     

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 (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α 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 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].     

Molecular Characterization and Substrate Specificity of Nitrobenzene Dioxygenase from Comamonas sp. Strain JS765

Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase_NBZ, Ferredoxin_NBZ, Oxygenase_NBZα, and Oxygenase_NBZβ, with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase_NBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.     

3-Nitrotoluene dioxygenase from Diaphorobacter sp. strains: cloning,sequencing and evolutionary studies

The first step in the degradation of 3-nitrotoluene by Diaphorobacter sp. strain DS2 is the dihydroxylation of the benzene ring with the concomitant removal of nitro group. This is catalyzed by a dioxygenase enzyme system. We report here the cloning and sequencing of the complete dioxygenase gene with its putative regulatory sequence from the genomic DNA of Diaphorobacter sp. strains DS1, DS2 and DS3. Analysis of the 5 kb DNA stretch that was cloned, revealed five complete open reading frames (ORFs) encoding for a reductase, a ferredoxin and two dioxygenase subunits with predicted molecular weights (MW) of 35, 12, 50 and 23 kDa respectively. A regulatory protein was also divergently transcribed from the reductase subunit and has a predicated MW of 34 kDa. Presence of parts of two functional ORFs in between the reductase and the ferredoxin subunits reveals an evolutionary route from a naphthalene dioxygenase like system of Ralstonia sp. strain U2. Further a 100 % identity of its ferredoxin subunit reveals its evolution via dinitrotoluene dioxygenase like system present in Burkholderia cepacia strain R34. A modeled structure of oxygenase_3NT from strain DS2 was generated using nitrobenzene dioxygenase as a template. The modeled structure only showed minor changes at its active site. Comparison of growth patterns of strains DS1, DS2 and DS3 revealed that Diaphorobacter sp. strain DS1 has been evolved to degrade 4-nitrotoluene better by an oxidative route amongst all three strains.