Naphtho[1,2-b]thiophene Degradation by Pseudomonas sp. HKT554: Involvement of Naphthalene Dioxygenase

Pseudomonas sp. strain HKT554 degrades naphtho[1,2-b]thiophene and two other isomers, naphtho[2,1-b]thiophene and naphtho[2,3-b]thiophene, by cometabolism, in the absence of any specific inducer, at similar degradation rates. A mutant of strain HKT554, deficient in dibenzothiophene degradation, was generated by using a recently developed transposition system. Sequence analysis of the mutant revealed that the knocked out gene was almost identical to naphthalene dioxygenase (EC 1.14.12.12). The mutant, HKT554M, degraded neither the naphthothiophene isomers nor dibenzothiophene, suggesting that the naphthalene dioxygenase is responsible for the initial catabolic reactions onto naphthothiophenes and dibenzothiophene. Received: 28 January 2002 / Accepted: 28 March 2002     

Mutagenic activity of methyl-substituted tri- and tetracyclic aromatic sulfur heterocycles

A number of polycyclic aromatic sulfur heterocycles have been identified in coal-derived products and in shale oils. The mutagenic activity of some of these compounds, including dibenzothiophene, benzo[b]naphtho[1,2-d]thiophene, benzo[b]naphtho[2,1-d]thiophene and benzo[b]naphtho[2,3-d]thiophene have been determined using the Salmonella/microsome mutagenicity test. These compounds demonstrated either very weak or no mutagenic activity. The methyl derivatives of each of these four compounds were assayed for mutagenic activity. Salmonella typhimurium TA98 was used as the tester strain. All assays required a rat-liver homogenate metabolic activator. Five of the methylated derivatives, 1-methylbenzo[b]naphtho[1,2-d]thiophene, 3-methylbenzo[b]naphtho[1,2-d]thiophene, 1-methylbenzo[b]-naphtho[2,1-d]thiophene, 6-methylbenzo[b]naphtho[2,1-d]thiophene and 4-methylbenzo[b]naphtho[2,3-d]thiophene demonstrated mutagenic activity. However, activity was observed only at high concentrations of the metabolic activator.     

Selective cleavage of 10-methyl benzo[b]naphtho[2,1-d]thiophene by recombinant Mycobacterium sp. strain

Recombinant Mycobacterium sp. strain MR65 carrying dszABCD genes was used for desulfurization of 10-methylbenzo[b]naphtho[2,1-d]thiophene (10-methyl BNT) in the hexadecane phase. The specific activity was 25% of that of dibenzothiophene (DBT). One of two major metabolites of 10-methyl BNT produced by strain MR65 was identified as 1-methoxy-2-(3-methylphenyl)naphthalene by ¹H and ¹³C NMR. The other major metabolite and two minor metabolites were determined as 1-hydroxy-2-(3-methylphenyl)naphthalene, 2-(2-methoxy-3-methylphenyl)naphthalene and 2-(2-hydroxy-3-methylphenyl)naphthalene, respectively, by HPLC and GC-MS. The production ratio of the two desulfurization metabolite isomers was 0.99:0.01, calculated on the basis of peak GC areas. These results indicated that the C-S bond adjacent to the naphthalene skeleton was selectively cleaved to form the two major compounds.     

Bacterial Transformations of Naphthothiophenes

Naphthothiophenes are minor components of fossil fuels, and they can enter the environment from oil spills. Naphtho[2,1-b]thiophene, naphtho[2,3-b]thiophene, and 1-methylnaphtho[2,1-b]thiophene were synthesized and used in biodegradation studies with 1-methylnaphthalene (1-MN)-degrading Pseudomonas strains W1, F, and BT1. Cultures were incubated with one of the naphthothiophenes with or without 1-MN, acidified, and extracted with CH(inf2)Cl(inf2). The extracts were analyzed by gas chromatography with flame photometric and mass detectors to characterize sulfur-containing metabolites and with an atomic emission detector for quantification. Only strain W1 was able to grow on naphtho[2,1-b]thiophene, but strains F and BT1 cometabolized this compound if 1-MN was present. 1-MN was required by all three strains to metabolize naphtho[2,3-b]thiophene, which was more resistant to biodegradation than the [2,1-b] isomer. Two metabolites of naphtho [2,1-b]thiophene were purified, analyzed by (sup1)H nuclear magnetic resonance spectroscopy, and found to be 4-hydroxybenzothiophene-5-carboxylic acid (metabolite I) and 5-hydroxybenzothiophene-4-carboxylic acid (metabolite II). In cultures of strain W1 grown for 7 days on 52 (mu)mol of naphtho[2,1-b]thiophene, >84% of the substrate was degraded and metabolites I and II accounted for 19 and 9%, respectively, of the original amount of naphtho[2,1-b]thiophene. When 1-MN was present, strain W1 degraded >97% of the naphtho[2,1-b]thiophene and similar amounts of metabolite II were produced, but metabolite I did not accumulate. 1-MN was shown to promote the further degradation of metabolite I, but not of metabolite II, by strain W1. Thus, 1-MN enhanced the biodegradation of naphtho[2,1-b]thiophene. Approximately 70% of the 1-methylnaphtho [2,1-b]thiophene added to cultures of strain W1 with 1-MN was recovered as 4-hydroxy-3-methylbenzothiophene-5-carboxylic acid, the 3-methyl analog of metabolite I. The methyl substitution hindered further metabolism of 3-methyl-metabolite I even in the presence of 1-MN. Cometabolism of naphtho[2,3-b]thiophene yielded two products that were tentatively identified as 5-hydroxybenzothiophene-6-carboxylic and 6-hydroxybenzothiophene-5-carboxylic acids.     

Aspartate 205 in the catalytic domain of naphthalene dioxygenase is essential for activity

The naphthalene dioxygenase enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The crystal structure of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571-586, 1998) indicates that aspartate 205 may provide the most direct route of electron transfer between the Rieske [2Fe-2S] center of one alpha subunit and mononuclear iron in the adjacent alpha subunit. In this study, we constructed four site-directed mutations that changed aspartate 205 to alanine, glutamate, asparagine, or glutamine to test whether this residue is essential for naphthalene dioxygenase activity. The mutant proteins were very inefficient in oxidizing naphthalene to cis-naphthalene dihydrodiol, and oxygen uptake in the presence of naphthalene was below detectable levels. The purified mutant protein with glutamine in place of aspartate 205 had identical spectral properties to wild-type naphthalene dioxygenase and was reduced by NADH in the presence of catalytic amounts of ferredoxinNAP and reductaseNAP. Benzene, an effective uncoupler of oxygen consumption in purified naphthalene dioxygenase, did not elicit oxygen uptake by the mutant protein. These results indicate that electron transfer from NADH to the Rieske center in the mutant oxygenase is intact, a finding consistent with the proposal that aspartate 205 is a necessary residue in the major pathway of electron transfer to mononuclear iron at the active site.     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.     

Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene. Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.     

Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans

Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene, biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)≤0.2 ppm] conditions (Hirano et al., Biosci Biotechnol Biochem 68:557–564, 2004). A major protein fraction carrying dibenzothiophene degradation activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also impaired degradation activity of the strain under extremely low oxygen conditions (DO≤0.2 ppm). These results indicate that Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions.     

Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the alpha subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (-)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (-)-biphenyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.     

Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Biotransformation of benzothiophene by isopropylbenzene-degrading bacteria.

Isopropylbenzene-degrading bacteria, including Pseudomonas putida RE204, transform benzothiophene to a mixture of compounds. Induced strain RE204 and a number of its Tn5 mutant derivatives were used to accumulate these compounds and their precursors from benzothiophene. These metabolites were subsequently identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. When strain RE204 was incubated with benzothiophene, it produced a bright yellow compound, identified as trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate, formed by the rearrangement of cis-4-(3-keto-2,3-dihydrothienyl)-2-hydroxybuta-2,4-dieno ate, the product of 3-isopropylcatechol-2,3-dioxygenase-catalyzed ring cleavage of 4,5-dihydroxybenzothiophene, as well as 2-mercaptophenylglyoxalate and 2'-mercaptomandelaldehyde. A dihydrodiol dehydrogenase-deficient mutant, strain RE213, converted benzothiophene to cis-4,5-dihydroxy-4,5-dihydrobenzothiophene and 2'-mercaptomandelaldehyde; neither trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate nor 2-mercaptophenylglyoxalate was detected. Cell extracts of strain RE204 catalyzed the conversion of cis-4,5-dihydroxy-4,5-dihydrobenzothiophene to trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate in the presence of NAD+. Under the same conditions, extracts of the 3-isopropylcatechol-2,3-dioxygenase-deficient mutant RE215 acted on cis-4,5-dihydroxy-4,5-dihydrobenzothiophene, forming 4,5-dihydroxybenzothiophene. These data indicate that oxidation of benzothiophene by strain RE204 is initiated at either ring. Transformation initiated at the 4,5 position on the benzene ring proceeds by three enzyme-catalyzed reactions through ring cleavage. The sequence of events that occurs following attack at the 2,3 position of the thiophene ring is less clear, but it is proposed that 2,3 dioxygenation yields a product that is both a cis-dihydrodiol and a thiohemiacetal, which as a result of this structure undergoes two competing reactions: either spontaneous opening of the ring, yielding 2'-mercaptomandelaldehyde, or oxidation by the dihydrodiol dehydrogenase to another thiohemiacetal, 2-hydroxy-3-oxo-2,3-dihydrobenzothiophene, which is not a substrate for the ring cleavage dioxygenase but which spontaneously opens to form 2-mercaptophenylglyoxaldehyde and subsequently 2-mercaptophenylglyoxalate. The yellow product, trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate, is a structural analog of trans-o-hydroxybenzylidenepyruvate, an intermediate of the naphthalene catabolic pathway; extracts of recombinant bacteria containing trans-o-hydroxybenzylidenepyruvate hydratase-aldolase catalyzed the conversion of trans-4-[3-hydroxy-2-thienyl]-2-oxobut-3-enoate to 3-hydroxythiophene-2-carboxaldehyde, which could then be further acted on, in the presence of NAD+, by extracts of recombinant bacteria containing the subsequent enzyme of the naphthalene pathway, salicylaldehyde dehydrogenase.     

Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp. strain WU-K2R

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.     

Desaturation, dioxygenation, and monooxygenation reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp. strain 9816-4.

The stereospecific oxidation of indan and indene was examined with mutant and recombinant strains expressing naphthalene dioxygenase of Pseudomonas sp. strain 9816-4. Pseudomonas sp. strain 9816/11 and Escherichia coli JM109(DE3)[pDTG141] oxidized indan to (+)-(1S)-indanol, (+)-cis-(1R,2S)-indandiol, (+)-(1S)-indenol, and 1-indanone. The same strains oxidized indene to (+)-cis-(1R,2S)-indandiol and (+)-(1S)-indenol. Purified naphthalene dioxygenase oxidized indan to the same four products formed by strains 9816/11 and JM109(DE3)[pDTG141]. In addition, indene was identified as an intermediate in indan oxidation. The major products formed from indene by purified naphthalene dioxygenase were (+)-(1S)-indenol and (+)-(1R,2S)-indandiol. The results show that naphthalene dioxygenase catalyzes the enantiospecific monooxygenation of indan to (+)-(1S)-indanol and the desaturation of indan to indene, which then serves as a substrate for the formation of (+)-(1R,2S)-indandiol and (+)-(1S)-indenol. The relationship of the desaturase, monooxygenase, and dioxygenase activities of naphthalene dioxygenase is discussed with reference to reactions catalyzed by toluene dioxygenase, plant desaturases, cytochrome P-450, methane monooxygenase, and other bacterial monooxygenases.     

Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α₃β₃ hexamer. The apparent K_m of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM⁻¹ min⁻¹ for 2NT and 1.2 μM⁻¹ min⁻¹ for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Isolation and Characterization of a Subsurface Bacterium Capable of Growth on Toluene, Naphthalene, and Other Aromatic Compounds

A bacterium, designated F199, utilized toluene, naphthalene, dibenzothiophene, salicylate, benzoate, p-cresol, and all isomers of xylene as a sole carbon and energy source. This bacterium was isolated from Middendorf sediments, a Cretaceous age formation that underlies the Southeast Coastal Plain in South Carolina, at a depth of approximately 410 m. F199 is a gram-positive, irregular-shaped bacterium that has a varied cell morphology that is dependent on culture medium type and growth stage. F199 required microaerobic conditions (40 to 80 μM O₂) for growth on hydrocarbons, glucose, acetate, and lactate in mineral salts medium but not for growth on rich media. [¹⁴C]naphthalene mineralization by F199 was induced by either naphthalene or toulene; however, [¹⁴C]toluene mineralization by this strain was induced by toluene but not naphthalene. F199 was also found to harbor two plasmids larger than 100 kb. Restricted F199 plasmid and genomic DNA did not hybridize with toluene (pWW0) or naphthalene (NAH7) catabolic plasmid DNA probes. The presence in the Middendorf formation of bacteria with the capacity for degrading a variety of aromatic compounds suggests that indigenous microorganisms may have potential for in situ degradation of organic contaminants.     

High cell density cultivation of Pseudomonas putida strain HKT554 and its application for optically active sulfoxide production

Culture conditions with Pseudomonas putida strain HKT554, expressing naphthalene dioxygenase, known as the biocatalyst showing wide substrate specificity, were optimized for high cell density cultivation (HCDC). Culture in a medium TK-B modified from that for HCDC of Escherichia coli with glucose fed-batch and dissolved oxygen stat resulted in a high cell density growth of 114 g dry cell/l at 40 h of cultivation. This system was further applied for S-(+)-methyl phenyl sulfoxide (MPSO) production from methyl phenyl sulfide. Addition of nonpolar organic solvent, such as n-hexadecane, greatly enhanced the MPSO production due to the prevention of substrate evaporation, resulting in a MPSO production up to 39 mM in 30 h with a conversion rate of 95.7 mol%.     

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.     

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(NBZalpha), and Oxygenase(NBZbeta), 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.     

Mutagenicity of C24H14 PAH in human cells expressing CYP1A1

Relatively little is known about the mutagenicity of C24H14 PAH, a diverse group of five- and six-ring PAH, some of which are present at trace levels in the environment. To better understand the mutagenicity of this class of compounds, 11 C24H14 PAH, including benzo[a]perylene, benzo[b]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,f]fluoranthene, dibenzo[j,l]fluoranthene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, dibenzo[e,l]pyrene, naphtho[1,2-b]fluoranthene, naphtho[2,3-a]pyrene, and naphtho[2,3-e]pyrene, were tested in a mutagenicity assay based on human h1A1v2 cells. h1A1v2 cells are a line of human B-lymphoblastoid cells that have been engineered to express cytochrome P4501A1 (CYP1A1), an enzyme capable of metabolizing promutagenic PAH. Mutagenicity was measured at the thymidine kinase (tk) locus following a 72-h exposure period. Our results show that nine of the compounds were mutagenic. Benzo[a]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,i]pyrene, and naphtho[2,3-a]pyrene were the most potent mutagens, having minimum mutagenic concentrations (MMC) (i.e., the dose at which the induced response was twice that of the negative controls) in the 1-5 ng/ml range. Benzo[b]perylene, dibenzo[a,h]pyrene, dibenzo[a,f]fluoranthene, and naphtho[2,3-e]pyrene were somewhat less potent mutagens, having MMC in the 10-30 ng/ml range. Dibenzo[e,l]pyrene, which had an MMC of 280 ng/ml, was the least potent mutagen. Dibenzo[j,l]fluoranthene and naphtho[1,2-b]fluoranthene were not mutagenic at the doses tested (1-3000 ng/ml). The most mutagenic compounds were also quite toxic. At the highest doses tested, benzo[a]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,i]pyrene, dibenzo[a,h]pyrene, and dibenzo[a,f]fluoranthene induced > 60% killing, and naphtho[2,3-a]pyrene and naphtho[2,3-e]pyrene induced > 50% killing. Benzo[b]perylene, dibenzo[e,l]pyrene, dibenzo[j,l]fluoranthene, and naphtho[1,2-b]fluoranthene induced < 50% killing at the highest doses tested. Comparing these results to a previous study in which nine other C24H14 PAH were tested for mutagenicity in this same assay, it was found that dibenzo[a]pyrene isomers were generally more mutagenic than the other groups of C24H14 PAH tested. These observations are discussed with emphasis given to identifying C24H14 PAH that may be important environmental mutagens.