Assessment of Toluene/Biphenyl Dioxygenase Gene Diversity in Benzene-Polluted Soils: Links between Benzene Biodegradation and Genes Similar to Those Encoding Isopropylbenzene Dioxygenases

The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic α subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant α subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase α subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an α subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.     

Crystal structure of the terminal oxygenase component of cumene dioxygenase from Pseudomonas fluorescens IP01

The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 A by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.     

Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida

Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, alpha and beta; the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the alpha subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase alpha subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the alpha subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.     

Crystal structure of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1

Biphenyl dioxygenase is the enzyme that catalyzes the stereospecific dioxygenation of the aromatic ring. This enzyme has attracted the attention of researchers due to its ability to oxidize polychlorinated biphenyls, which is one of the serious environmental contaminants. We determined the crystal structure of the terminal oxygenase component of the biphenyl dioxygenase (BphA1A2) derived from Rhodococcus strain sp. RHA1 in substrate-free and complex forms. These crystal structures revealed that the substrate-binding pocket makes significant conformational changes upon substrate binding to accommodate the substrate into the pocket. Our analysis of the crystal structures suggested that the residues in the substrate-binding pocket can be classified into three groups, which, respectively, seem to be responsible for the catalytic reaction, the orientation/conformation of the substrate, and the conformational changes of the substrate-binding pocket. The cooperative actions of residues in the three groups seem to determine the substrate specificity of the enzyme.     

Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.     

The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 A by molecular replacement revealed the enzyme to share the same global alpha(3)beta(3) structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 A. An 11 A long water channel extends from the interface between the alpha and beta subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.     

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).     

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.     

Molecular biological characterization of biphenyl-degrading bacteria and identification of the biphenyl 2,3-Dioxygenase α-subunit genes

Bacterial isolates from soils contaminated with (chlorinated) aromatic compounds, which degraded biphenyl/chlorinated biphenyls (CB) and belonged to the genera Rhodococcus and Pseudomonas, were studied. Analysis of the 16S rRNA gene sequences was used to determine the phylogenetic position of the isolates. The Rhodococcus cells were found to contain plasmids of high molecular mass (220–680 kbp). PCR screening for the presence of the bphA1 gene, a marker indicating the possibility for induction of 2,3-dioxygenase (biphenyl/toluene dioxygenase subfamily), revealed the presence of the bphA1 genes with 99–100% similarity to the homologous genes of bacteria of the relevant species in all pseudomonad and most Rhodococcus isolates. A unique bphA1 gene, which had not been previously reported for the genus, was identified in Rhodococcus sp. G10. The absence of specific amplification of the bphA1 genes in some biphenyl-degrading bacteria (Rhodococcus sp. B7b, B106a, G12a, P2kr, P2(51), and P2m), as well as in an active biphenyl degrader Rhodococcus ruber P25, indicated the absence of the genes encoding the proteins of the biphenyl/toluene dioxygenase subfamily and participation of the enzymes other than this protein family in biphenyl/CB degradation.     

Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis

Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues, which were identified using a homology model of the alpha subunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline, for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2,4-dimethylaniline, a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2,4-dimethylaniline approximately 1.7-fold and 2.1-fold, respectively. Thus, it is shown that the alpha subunit of the terminal dioxygenase indeed plays a part in the substrate specificity as well as the activity of aniline dioxygenase. Interestingly, the equivalent residues of V205 and I248 have not been previously reported to influence the substrate specificity of other Rieske dioxygenases. These results should facilitate future engineering of the enzyme for bioremediation and industrial applications.     

Structure of the terminal oxygenase component of angular dioxygenase, carbazole 1,9a-dioxygenase

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. This reaction is an initial degradation reaction of the carbazole degradation pathway by various bacterial strains. Only a limited number of Rieske non-heme iron oxygenase systems (ROSs) can catalyze this novel reaction, termed angular dioxygenation. Angular dioxygenation is also involved in the degradation pathways of carbazole-related compounds, dioxin, and CARDO can catalyze the angular dioxygenation for dioxin. CARDO consists of a terminal oxygenase component (CARDO-O), and the electron transport components, ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R). CARDO-O has a homotrimeric structure, and governs the substrate specificity of CARDO. Here, we have determined the crystal structure of CARDO-O of Janthinobacterium sp. strain J3 at a resolution of 1.95A. The alpha3 trimeric overall structure of the CARDO-O molecule roughly corresponds to the alpha3 partial structures of other terminal oxygenase components of ROSs that have the alpha3beta3 configuration. The CARDO-O structure is a first example of the terminal oxygenase components of ROSs that have the alpha3 configuration, and revealed the presence of the specific loops that interact with a neighboring subunit, which is proposed to be indispensable for stable alpha3 interactions without structural beta subunits. The shape of the substrate-binding pocket of CARDO-O is markedly different from those of other oxygenase components involved in naphthalene and biphenyl degradation pathways. Docking simulations suggested that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation.     

Aerobic bioremediation of chlorobenzene source-zone soil in flow-through columns: performance assessment using quantitative PCR

Flow-through aquifer columns were operated for 12 weeks to evaluate the benefits of aerobic biostimulation for the bioremediation of source-zone soil contaminated with chlorobenzenes (CBs). Quantitative Polymerase Chain Reaction (qPCR) was used to measure the concentration of total bacteria (16S rRNA gene) and oxygenase genes involved in the biodegradation of aromatic compounds (i.e., toluene dioxygenase, ring hydroxylating monooxygenase, naphthalene dioxygenase, phenol hydroxylase, and biphenyl dioxygenase). Monochlorobenzene, which is much more soluble than dichlorobenzenes, was primarily removed by flushing, and biostimulation showed little benefit. In contrast, dichlorobenzene removal was primarily due to biodegradation, and the removal efficiency was much higher in oxygen-amended columns compared to a control column. To our knowledge, this is the first report that oxygen addition can enhance CB source-zone soil bioremediation. Analysis by qPCR showed that whereas the biphenyl and toluene dioxygenase biomarkers were most abundant, increases in the concentration of the phenol hydroxylase gene reflected best the higher dichlorobenzene removal due to aerobic biostimulation. This suggests that quantitative molecular microbial ecology techniques could be useful to assess CB source-zone bioremediation performance.     

Subtle Difference between Benzene and Toluene Dioxygenases of Pseudomonas putida

Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, α and β; the α subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the α subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase α subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the α subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.     

Changes in bacterial populations and in biphenyl dioxygenase gene diversity in a polychlorinated biphenyl-polluted soil after introduction of willow trees for rhizoremediation

The aim of this study was to analyze the structural and functional changes occurring in a polychlorinated-biphenyl (PCB)-contaminated soil ecosystem after the introduction of a suitable host plant for rhizoremediation (Salix viminalis). We have studied the populations and phylogenetic distribution of key bacterial groups (Alpha- and Betaproteobacteria, Acidobacteria, and Actinobacteria) and the genes encoding iron-sulfur protein alpha (ISPalpha) subunits of the toluene/biphenyl dioxygenases in soil and rhizosphere by screening gene libraries using temperature gradient gel electrophoresis. The results, based on the analysis of 415 clones grouped into 133 operational taxonomic units that were sequence analyzed (>128 kbp), show that the rhizospheric bacterial community which evolved from the native soil community during the development of the root system was distinct from the soil community for all groups studied except for the Actinobacteria. Proteobacteria were enriched in the rhizosphere and dominated both in rhizosphere and soil. There was a higher than expected abundance of Betaproteobacteria in the native and in the planted PCB-polluted soil. The ISPalpha sequences retrieved indicate a high degree of catabolic and phylogenetic diversity. Many sequences clustered with biphenyl dioxygenase sequences from gram-negative bacteria. A distinct cluster that was composed of sequences from this study, some previously described environmental sequences, and a putative ISPalpha from Sphingomonas wittichii RW1 seems to contain greater diversity than the presently recognized toluene/biphenyl dioxygenase subfamily. Moreover, the rhizosphere selected for two ISPalpha sequences that accounted for almost 60% of the gene library and were very similar to sequences harbored by Pseudomonas species.     

Evaluation of trichloroethylene degradation by E. coli transformed with dimethyl sulfide monooxygenase genes and/or cumene dioxygenase genes

Pseudomonas fluorescens IP01 grown on isopropylbenzene (cumene) and Acinetobacter sp. 20B grown on dimethyl sulfide (DMS) degraded up to 90% and 25% of 1.5 mg trichloroethylene (TCE)/l, respectively. Escherichia coli harboring the DMS monooxygenase genes from strain 20B, the cumene dioxygenase genes from strain IP01 and both oxygenase genes, degraded up to 50%, 75% and 88% of 75 mg TCE/l, respectively. The growth rates of the E. coli recombinants remained nearly unaffected by TCE at 15 150 mg/l. Thus, the E. coli recombinants were indicated to degrade high concentrations of TCE efficiently at least up to 150 mg l–1.     

Identification,cloning, and characterization of a multicomponent biphenyl dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.     

Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5 degrees C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 x 10(2) copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.     

Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 × 10² copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

New classification system for oxygenase components involved in ring-hydroxylating oxygenations

Batie et al. [Chemistry and Biochemistry of Flavoenzymes, 3, 543-556 (1991)] proposed a classification system for ring-hydroxylating oxygenases in which the oxygenases are grouped into three classes in terms of the number of constituent components and the nature of the redox centers. But in recent years, many ring-hydroxylating oxygenases have been newly identified and characterized, and found difficult to classify into these three classes. Typical examples are carbazole 1,9a-dioxygenase and 2-oxo-1,2-dihydroquinoline 8-monooxygenase, which have been classified into class III and class IB, respectively, from biochemical characteristics. However, a phylogenetic study showed that the terminal oxygenases of both are closely related to class IA. Because this discrepancy derived from counting all the components together, here we proposed a new scheme based on the homology of the amino acid sequences of the alpha subunits of the terminal oxygenase components. This new scheme strongly reflects the actual phylogenetic affiliation of the terminal oxygenase component. By comparing their sequences pairwise using the CLUSTAL W program, 54 oxygenase components were classified into 4 groups (groups I, II, III, and IV). While group I contains broad-range oxygenases sharing low homology, groups II, III, and IV contain some typical oxygenases: benzoate/toluate dioxygenases for group II, naphthalene/polycyclic aromatic hydrocarbon dioxygenases for group III, and benzene/toluene/biphenyl dioxygenases for group IV. Our new scheme is simple and powerful, since an oxygenase component can be nearly automatically grouped when the DNA sequence is available, and it fits very well with the phylogenetic affiliation.