Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1.

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing 2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation, hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain RW1 when this organism was grown with dibenzo-p-dioxin.     

Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1.

A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and 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) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.     

Biodegradation of diphenyl ether and its monohalogenated derivatives by Sphingomonas sp. strain SS3.

The bacterium Sphingomonas sp. strain SS3, which utilizes diphenyl ether and its 4-fluoro, 4-chloro, and (to a considerably lesser extent) 4-bromo derivatives as sole sources of carbon and energy, was enriched from soil samples of an industrial waste deposit. The bacterium showed cometabolic activities toward all other isomeric monohalogenated diphenyl ethers. During diphenyl ether degradation in batch culture experiments, phenol and catechol were produced as intermediates which were then channeled into the 3-oxoadipate pathway. The initial step in the degradation follows the recently discovered mechanism of 1,2-dioxygenation, which yields unstable phenolic hemiacetals from diphenyl ether structures. Oxidation of the structure-related dibenzo-p-dioxin yielded 2-(2-hydroxyphenoxy)-muconate upon ortho cleavage of the intermediate 2,2',3-trihydroxydiphenyl ether. Formation of phenol, catechol, halophenol, and halocatechol from the conversion of monohalogenated diphenyl ethers gives evidence for a nonspecific attack of the dioxygenating enzyme system.     

Biodegradation of diphenyl ether and its monohalogenated derivatives by Sphingomonas sp. strain SS3.

The bacterium Sphingomonas sp. strain SS3, which utilizes diphenyl ether and its 4-fluoro, 4-chloro, and (to a considerably lesser extent) 4-bromo derivatives as sole sources of carbon and energy, was enriched from soil samples of an industrial waste deposit. The bacterium showed cometabolic activities toward all other isomeric monohalogenated diphenyl ethers. During diphenyl ether degradation in batch culture experiments, phenol and catechol were produced as intermediates which were then channeled into the 3-oxoadipate pathway. The initial step in the degradation follows the recently discovered mechanism of 1,2-dioxygenation, which yields unstable phenolic hemiacetals from diphenyl ether structures. Oxidation of the structure-related dibenzo-p-dioxin yielded 2-(2-hydroxyphenoxy)-muconate upon ortho cleavage of the intermediate 2,2',3-trihydroxydiphenyl ether. Formation of phenol, catechol, halophenol, and halocatechol from the conversion of monohalogenated diphenyl ethers gives evidence for a nonspecific attack of the dioxygenating enzyme system.     

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.     

Molecular bases of aerobic bacterial degradation of dioxins: involvement of angular dioxygenation

In the last decade, extensive investigation has been done on the bacterial degradation of dioxins and its related compounds, because this class of chemicals is highly toxic and has been widely distributed in the environment. These studies have revealed the primary importance of a novel dioxygenation reaction, called angular dioxygenation, in the aerobic bacterial degradation pathway of dioxin. Accompanied by the electron transport proteins, Rieske nonheme iron oxygenase catalyzes the incorporation of oxygen atoms to the ether bond-carrying carbon (the angular position) and an adjacent carbon, resulting in the irreversible cleavage of the recalcitrant aryl ether bond. The 2,2',3-trihydroxybiphenyl or 2,2',3-trihydroxydiphenyl ether derivatives formed are degraded through meta cleavage. In addition to the degradation system of dibenzofuran and dibenzo-p-dioxin (the nonchlorinated model compounds of dioxin), those of fluorene and carbazole were shown to function in dioxin degradation. Some dioxin degradation pathways have been studied biochemically and genetically. In addition, feasibility studies have shown that some dioxin-degrading strains can function in actual dioxin-contaminated soil. These studies provide useful information for the establishment of a bioremediation method for dioxin contamination. This review summarizes recent progress on molecular and biochemical bases of the bacterial aerobic degradation of dioxin and related compounds.     

Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10.

Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.     

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.     

Stability of mutations in a Sphingomonas strain.

Sphingomonas sp. strain RW1 is able to mineralise dibenzofuran and dibenzo-p-dioxin. Three mutants were constructed that could not use dibenzofuran or dibenzo-p-dioxin as a carbon source but were able to grow with the succeeding metabolites of the pathway. Two different mutagenic agents were applied, a chemical treatment with 1-methyl-3-nitro-1-nitrosoguanidine, resulting in mutants RW1-N6 and RW1-N7, and a biological insertion mutagenesis with the mini-Tn5 transposon pBSL118, resulting in mutant RW1-M3. Southern blot analysis and PCR experiments confirmed a single insertion of the mini-Tn5 into one of the genes coding for the oxygenase component of the dibenzofuran 4,4a-dioxygenase system. The genetic stability of these mutants was examined after growth with complex medium under nonselective conditions. All three mutants failed to revert to wild-type metabolic functions.     

Biodegradation of Dibenzo-p-dioxin, Dibenzofuran, and Chlorodibenzo-p-dioxins by Pseudomonas veronii PH-03

The dioxin-degrading strain Pseudomonas veronii PH-03 was isolated from contaminated soil by selective enrichment techniques. Strain PH-03 grew on dibenzo-p-dioxin and dibenzofuran as a sole carbon source. Further, 1-chlorodibenzo-p-dioxin, 2-chlorodibenzo-p-dioxin and other dioxin metabolites, salicylic acid, and catechol were also metabolized well. Resting cells of strain PH-03 transformed dibenzo-p-dioxin, dibenzofuran, 2,2',3-trihydroxybiphenyl, and some chlorodioxins to their corresponding metabolic intermediates such as catechol, salicylic acid, 2-hydroxy-(2-hydroxyphenoxy)-6-oxo-2,4-hexadienoic acid, and chlorocatechols. The formation of these metabolites was confirmed by comparison of gas chromatography-mass spectrometry (GC-MS) data with those of authentic compounds. Although we did observe the production of 3,4,5,6-tetrachlorocatechol (3,4,5,6-TECC) from 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) with resting cell suspensions of PH-03, growth of strain PH-03 in the presence of 1,2,3,4-TCDD was poor. This result suggests that strain PH-03 is unable to utilize 3,4,5,6-TECC, even at very low concentration (0.01 mM) due to its toxicity. In cell-free extracts of DF-grown cells, 2,2',3-trihydroxybiphenyl dioxygenase, 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase, and catechol-2,3-dioxygense activities were detected. Moreover, the activities of meta-pyrocatechase and 2,2',3-trihydroxybiphenyl dioxygenase from the crude cell-free extracts were inhibited by 3-chlorocatechol. However, no inhibition was observed in intact cells when 3-chlorocatechol was formed as intermediate.     

Novel organization of catechol <Emphasis Type="Italic">meta</Emphasis> pathway genes in the nitrobenzene degrader <Emphasis Type="Italic">Comamonas</Emphasis> sp. JS765 and its evolutionary implication

The catechol meta cleavage pathway is one of the central metabolic pathways for the degradation of aromatic compounds. A novel organization of the pathway genes, different from that of classical soil microorganisms, has been observed in Sphingomonas sp HV3 and Pseudomonas sp. DJ77. In a Comamonas sp. JS765, cdoE encoding catechol 2,3-dioxygenase shares a common ancestry only with tdnC of a Pseudomonas putida strain, while codG encoding 2-hydroxymuconic semialdehyde dehydrogenase shows a higher degree of similarity to those genes in classical bacteria. Located between cdoE and cdoG are several putative genes, whose functions are unknown. These genes are not found in meta pathway operons of other microorganisms with the exception of cdoX2, which is similar to cmpX in strain HV3. Therefore, the gene cluster in JS765 reveals a third type of gene organization of the meta pathway.     

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.     

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.     

Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin from soils inoculated with Sphingomonas sp. strain RW1

Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin (2-CDD) (10 ppm each) from soil microcosms to final concentrations in the parts-per-billion range was affected by the addition of Sphingomonas sp. strain RW1. Rates and extents of removal were influenced by the density of RW1 organisms. For 2-CDD, the rate of removal was dependent on the content of soil organic matter (SOM), with half-life values ranging from 5.8 h (0% SOM) to 26.3 h (5.5% SOM).     

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.     

Degradation of 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1.

Pseudomonas sp. strain HBP1 was found to grow on 2-hydroxy- and 2,2'-dihydroxy-biphenyl as the sole carbon and energy sources. The first step in the degradation of these compounds was catalyzed by an NADH-dependent monooxygenase. The enzyme inserted a hydroxyl group adjacent to the already existing hydroxyl group to form 2,3-dihydroxybiphenyl when acting on 2-hydroxybiphenyl and to form 2,2',3-trihydroxybiphenyl when acting on 2,2'-dihydroxybiphenyl. To be substrates of the monooxygenase, compounds required a 2-hydroxyphenyl-R structure, with R being a hydrophobic group (e.g., methyl, ethyl, propyl, sec-butyl, phenyl, or 2-hydroxyphenyl). Several chlorinated hydroxybiphenyls served as pseudosubstrates by effecting consumption of NADH and oxygen without being hydroxylated. Further degradation of 2,3-dihydroxy- and 2,2',3-trihydroxybiphenyl involved meta cleavage, with subsequent formation of benzoate and salicylate, respectively.     

Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360.

The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K.-H. Engesser, V. Strubel, K. Christoglou, P. Fischer, and H. G. Rast, FEMS Microbiol. Lett. 65:205-210, 1989; V. Strubel, Ph.D. thesis, University of Stuttgart, Stuttgart, Germany, 1991; V. Strubel, H. G. Rast, W. Fietz, H.-J. Knackmuss, and K.-H. Engesser, FEMS Microbiol. Lett. 58:233-238, 1989). Two 2,3-dihydroxybiphenyl-1,2-dioxygenases (BphC1 and BphC2) and one catechol-2,3-dioxygenase (C23O) were shown to be expressed in Terrabacter sp. strain DPO360 growing with dibenzofuran as a sole source of carbon and energy. These enzymes exhibited strong sensitivity to oxygen. They were purified to apparent homogeneity as homodimers (BphC and BphC2) and as a homotetrameric catechol-2,3-dioxygenase (C23O). According to their specificity constants kcat/Km, both BphC1 and BphC2 were shown to be responsible for the cleavage of 2,2',3-trihydroxybiphenyl, the first metabolite in dibenzofuran mineralization along the angular dioxygenation pathway. With this substrate, BphC2 exhibited a considerably higher kcat/Km, value (183 microM/min) than BphC1 (29 microM/min). Catechol-2,3-dioxygenase was recognized to be not involved in the ring cleavage of 2,2',3-trihydroxybiphenyl (kcat/Km, 1 microM/min). Analysis of deduced amino acid sequence data of bphC1 revealed 36% sequence identity to nahC from Pseudomonas putida PpG7 (S. Harayama and M. Rekik, J. Biol. Chem. 264:15328-15333, 1989) and about 40% sequence identity to various bphC genes from different Pseudomonas and Rhodococcus strains. In addition, another 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (bphC3) was cloned from the genome of Terrabacter sp. strain DPO360. Expression of this gene, however, could not be detected in Terrabacter sp. strain DPO360 after growth with dibenzofuran.     

Triclosan susceptibility and co-metabolism--a comparison for three aerobic pollutant-degrading bacteria

The antimicrobial agent triclosan is an emerging and persistent environmental pollutant. This study evaluated the susceptibility and biodegradation potential of triclosan by three bacterial strains (Sphingomonas wittichii RW1, Burkholderia xenovorans LB400 and Sphingomonas sp. PH-07) that are able to degrade aromatic pollutants (dibenzofuran, biphenyl and diphenyl ether, respectively) with structural similarities to triclosan. These strains showed less susceptibility to triclosan when grown in complex and mineral salts media. Biodegradation experiments revealed that only strain PH-07 was able to catabolize triclosan to intermediates that included hydroxylated compounds (monohydroxy-triclosan, and dihydroxy-triclosan) and the ether bond cleavage products (4-chlorophenol and 2,4-dichlorophenol), indicating that the initial dihydroxylation occurred on both aromatic rings of triclosan. Additional growth inhibition tests demonstrated that the main intermediate, 2,4-dichlorophenol, was less toxic to strain PH-07 than was triclosan. Our results indicate that ether bond cleavage might be the primary mechanism of avoiding triclosan toxicity by this strain.     

Degradation of 2-hydroxybiphenyl and 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1

Pseudomonas sp. strain HBP1 was found to grow on 2-hydroxy- and 2,2'-dihydroxy-biphenyl as the sole carbon and energy sources. The first step in the degradation of these compounds was catalyzed by an NADH-dependent monooxygenase. The enzyme inserted a hydroxyl group adjacent to the already existing hydroxyl group to form 2,3-dihydroxybiphenyl when acting on 2-hydroxybiphenyl and to form 2,2',3-trihydroxybiphenyl when acting on 2,2'-dihydroxybiphenyl. To be substrates of the monooxygenase, compounds required a 2-hydroxyphenyl-R structure, with R being a hydrophobic group (e.g., methyl, ethyl, propyl, sec-butyl, phenyl, or 2-hydroxyphenyl). Several chlorinated hydroxybiphenyls served as pseudosubstrates by effecting consumption of NADH and oxygen without being hydroxylated. Further degradation of 2,3-dihydroxy- and 2,2',3-trihydroxybiphenyl involved meta cleavage, with subsequent formation of benzoate and salicylate, respectively.     

A second [2Fe-2S] ferredoxin from Sphingomonas sp. Strain RW1 can function as an electron donor for the dioxin dioxygenase

The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.