Aromatic Ring-hydroxylating Dioxygenase Research Articles

DARHD: A sequence database for aromatic ring-hydroxylating dioxygenase analysis and primer evaluation

Biodegradation of aromatic compounds is ubiquitous in the environment and important for controlling organic pollutants. Aromatic ring-hydroxylating dioxygenases (ARHDs) are responsible for the first and rate-limiting step of aerobic biodegradation of aromatic compounds. The ARHD α subunit is a good biomarker for studying functional microorganisms in the environment, however their diversity and corresponding primer coverage are unclear, both of which require a comprehensive sequence database for the ARHD α subunit. Here amino acid sequences of the ARHD α subunit were collected, and a total of 103 sequences were selected as seed sequences that were distributed in 72 bacterial genera with 34 gene names. Based on both homolog search and keyword confirmation against the GenBank, a sequence database of ARHD (DARHD) has been established and 6367 highly credible sequences were retrieved. DARHD contained 407 bacterial genera capable of degrading 38 aromatic substrates, and intricate relationships among the gene name, aromatic substrate and microbial taxa were observed. Thereafter, a total of 136 pairs of primers were collected and assessed. Results showed coverages of most published primers were low. Our research provides new insights for understanding the diversity of ARHD α subunit, and gives guidance on the design and application of primers in the future.

Transcriptomic Analysis of Degradative Pathways for Azo Dye Acid Blue 113 in Sphingomonas melonis B-2 from the Dye Wastewater Treatment Process.

Background: Acid Blue 113 (AB113) is a typical azo dye, and the resulting wastewater is toxic and difficult to remove. Methods: The experimental culture was set up for the biodegradation of the azo dye AB113, and the cell growth and dye decolorization were monitored. Transcriptome sequencing was performed in the presence and absence of AB113 treatment. The key pathways and enzymes involved in AB113 degradation were found through pathway analysis and enrichment software (GO, EggNog and KEGG). Results: S. melonis B-2 achieved more than 80% decolorization within 24 h (50 and 100 mg/L dye). There was a positive relationship between cell growth and the azo dye degradation rate. The expression level of enzymes involved in benzoate and naphthalene degradation pathways (NADH quinone oxidoreductase, N-acetyltransferase and aromatic ring-hydroxylating dioxygenase) increased significantly after the treatment of AB113. Conclusions: Benzoate and naphthalene degradation pathways were the key pathways for AB113 degradation. NADH quinone oxidoreductase, N-acetyltransferase, aromatic ring-hydroxylating dioxygenase and CYP450 were the key enzymes for AB113 degradation. This study provides evidence for the process of AB113 biodegradation at the molecular and biochemical level that will be useful in monitoring the dye wastewater treatment process at the full-scale treatment.

Insights from comparative proteomic analysis into degradation of phenanthrene and salt tolerance by the halophilic Martelella strain AD-3

A halophilic PAHs-degrading strain, Martelella AD-3, was previously isolated from highly saline petroleum-contaminated soil. In this study, label-free proteomics were performed to identify differentially expressed proteins (DEPs) under Group P (phenanthrene +5% salinity) and Group G (glycerol +1% salinity), which would help to reveal the mechanism of phenanthrene degradation and salt tolerance. A total of 307 up-regulated DEPs were found in Group P, including 17 phenanthrene degradation proteins. Among these phenanthrene-degrading proteins, the ferredoxin of aromatic ring-hydroxylating dioxygenase (RHD) was up-regulated by 110-fold and gentisate 1,2-dioxygenases (GDOs) were only expressed in Group P. Besides, we also found nine high salt stress response proteins, including ectoine synthase and transport protein of compatible (osmoprotectant) solutes, were differentially up-regulated. These results indicate that strain AD-3 mainly relied on RHD and dihydrodiol dehydrogenase to degrade phenanthrene, and accumulated compatible solutes for resistance to salt stress. This study provides strong theoretical guidance for understanding the degradation of phenanthrene by strain AD-3 in high salt environments.

The genomic attributes of Cd-resistant, hydrocarbonoclastic Bacillus subtilis SR1 for rhizodegradation of benzo(a)pyrene under co-contaminated conditions.

Bacillus subtilis SR1 is a metal resistant, polyaromatic hydrocarbon-degrading bacterium isolated from petroleum contaminated sites. This study reports the characteristics of the genome of the isolate containing one circular chromosome (4,093,698 bp) annotated into 4155 genes and 4095 proteins. The genome analysis confirmed the presence of multiple catabolic genes: aromatic ring-hydroxylating dioxygenase (COG2146), aromatic ring hydroxylase (COG2368), catechol 2, 3 dioxygenase (COG2514), 4-hydroxybenzoate decarboxylase (COG0043), carboxymuconolactone decarboxylase (COG0599) responsible for the catabolism of aromatic hydrocarbons along with the genes for biosurfactant production and functional genes (czcD and cadA) for resistance to cadmium, zinc, and cobalt. Gas Chromatography-Mass spectroscopy analysis revealed up to 35% in-vitro degradation of benzo(a)pyrene after 21 days of growth along with the production of different intermediate metabolites. The pot trial analysis in the greenhouse condition validated the rhizodegradation of BaP, which was significantly higher in the presence of plant-microbe association (85%) than degradation in bulk soil (68%).

Microbial community composition and PAHs removal potential of indigenous bacteria in oil contaminated sediment of Taean coast, Korea

The tidal flats near Sinduri beach in Taean, Korea, have been severely contaminated by heavy crude oils due to the Korea's worst oil spill accident, say the Hebei Spirit Oil Spill, in 2007. Crude oil compounds, including polycyclic aromatic hydrocarbons (PAHs), pose significant environmental damages due to their wide distribution, persistence, high toxicity, mutagenicity, and carcinogenicity. Microbial community of Sinduri beach sediments samples was analyzed by metagenomic data with 16S rRNA gene amplicons. Three phyla (Proteobacteria, Firmicutes, and Bacteroidetes) accounted for approximately ≥93.0% of the total phyla based on metagenomic analysis. Proteobacteria was the dominant phylum in Sinduri beach sediments. Cultivable bacteria were isolated from PAH-enriched cultures, and bacterial diversity was investigated through performing culture characterization followed by molecular biology methods. Sixty-seven isolates were obtained, comprising representatives of Actinobacteria, Firmicutes, α- and γ-Proteobacteria, and Bacteroidetes. PAH catabolism genes, such as naphthalene dioxygenase (NDO) and aromatic ring hydroxylating dioxygenase (ARHDO), were used as genetic markers to assess biodegradation of PAHs in the cultivable bacteria. The ability to degrade PAHs was demonstrated by monitoring the removal of PAHs using a gas chromatography mass spectrometer. Overall, various PAH-degrading bacteria were widely present in Sinduri beach sediments and generally reflected the restored microbial community. Among them, Cobetia marina, Rhodococcus soli, and Pseudoalteromonas agarivorans were found to be significant in degradation of PAHs. This large collection of PAH-degrading strains represents a valuable resource for studies investigating mechanisms of PAH degradation and bioremediation in oil contaminated coastal environment, elsewhere.

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community.

Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.

Characterization of polycyclic aromatic hydrocarbons degradation and arsenate reduction by a versatile Pseudomonas isolate

A Pseudomonas isolate, designated PAHAs-1, was found capable of reducing arsenate and degrading polycyclic aromatic hydrocarbons (PAHs) independently and simultaneously. This isolate completely reduced 1.5 mM arsenate within 48 h and removed approximately 100% and 50% of 60 mg l−1 phenanthrene and 20 mg l−1 pyrene within 60 h, respectively. Using PAHs as the sole carbon source, however, this isolate showed a slow arsenate reduction rate (4.62 μM h−1). The presence of arsenic affected cell growth and concurrent PAHs removal, depending on PAH species and arsenic concentration. Adding sodium lactate to the medium greatly enhanced the arsenate reduction and pyrene metabolism. The presence of the alpha subunit of the aromatic ring-hydroxylating dioxygenase (ARHD) gene, arsenate reductase (arsC) and arsenite transporter (ACR3(2)) genes supported the dual function of the isolate. The finding of latter two genes indicated that PAHAs-1 possibly reduced arsenate via the known detoxification mechanism. Preliminary data from hydroponic experiment showed that PAHAs-1 degraded the majority of phenanthrene (>60%) and enhanced arsenic uptake by Pteris vittata L. (from 246.7 to 1187.4 mg kg−1 As in the fronds). The versatile isolate PAHAs-1 may have potentials in improving the bioremediation of PAHs and arsenic co-contamination using the plant-microbe integrated strategy.

Isolation and Functional Gene Analyses of Aromatic-Hydrocarbon-Degrading Bacteria from a Polychlorinated-Dioxin-Dechlorinating Process

Aerobic aromatic-hydrocarbon-degrading bacteria from a semi-anaerobic microbial microcosm that exhibited apparent complete dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were isolated through enrichment and plating culture procedures with dibenzofuran as the model substrate. By 16S rRNA gene sequence comparisons, these dibenzofuran-degrading isolates were identified as being members of the phyla Actinobacteria, Firmicutes, and Proteobacteria, among which those of the genera Paenibacillus and Rhizobium were most abundant. All of the isolates utilized naphthalene as the sole carbon and energy source and degraded dibenzofuran metabolically or co-metabolically; however, they hardly attacked monochlorinated dibenzofuran and dibenzo-p-dioxin. By PCR cloning and sequencing, genes predicted to encode aromatic-ring-hydroxylating dioxygenase (AhDO) were detected in all test isolates. Real-time quantitative PCR assays with specific primer sets detected approximately 105 copies of the AhDO large subunit genes g−1 wet wt in the microcosm from which the isolates were obtained. This order of the copy number corresponded to approximately 1% of the 16S rRNA gene copies from “Dehalococcoides” and its relatives present as potent dechlorinators. These results suggest that aerobic AhDO-containing bacteria co-exist and play a role in the oxidative degradation of less chlorinated and completely dechlorinated products in the PCDD/F-dechlorinating process, thereby achieving the apparent complete dechlorination of PCDD/Fs.

Horizontal Transfer of PAH Catabolism Genes in Mycobacterium: Evidence from Comparative Genomics and Isolated Pyrene-Degrading Bacteria

Biodegradation of high molecular weight polycyclic aromatic hydrocarbons (PAHs), such as pyrene and benzo[a]pyrene, has only been observed in a few genera, namely fast-growing Mycobacterium and Rhodococcus. In M. vanbaalenii PYR-1, multiple aromatic ring hydroxylating dioxygenase (ARHDOs) genes including pyrene dioxygenases nidAB and nidA3B3 are localized in one genomic region. Here we examine the homologous genomic regions in four other PAH-degrading Mycobacterium (strains JLS, KMS, and MCS, and M. gilvum PYR-GCK), presenting evidence for past horizontal gene transfer events. Seven distinct types of ARHDO genes are present in all five genomes, and display conserved syntenic architecture with respect to gene order, orientation, and association with other genes. Duplications and putative integrase and transposase genes suggest past gene shuffling. To corroborate these observations, pyrene-degrading strains were isolated from two PAH-contaminated sediments: Chattanooga Creek (Tennessee) and Lake Erie (western basin). Some were related to fast-growing Mycobacterium spp. and carried both nidA and nidA3 genes. Other isolates belonged to Microbacteriaceae and Intrasporangiaceae presenting the first evidence of pyrene degradation in these families. These isolates had nidA (and some, nidA3) genes that were homologous to Mycobacterial ARHDO genes, suggesting that horizontal gene transfer events have occurred.

Diversity of aromatic ring-hydroxylating dioxygenase gene in Citrobacter

Genetic and functional diversity of Citrobacter spp. for their abilities to degrade aromatic compounds was evaluated to develop mixed cultures or a consortium for bioremediation technology. Thirty Citrobacter strains isolated from various effluent treatment plants were found to degrade a range of aromatic compounds: phenol, benzoate, hydroxy benzoic acid and biotransform mono-chlorophenols and di-chlorophenol within 24 to 48h of incubation at 30°C. Sequence similarity and phylogeny of the ARHD gene transcripts (730 nucleotides) depicted their diversity within 9 Citrobacter strains: HPC255, HPC369, HPC560, HPC570, HPC784, HPC1196, HPC1216, HPC1276 and HPC1299. Here, the degree of associations varied up to 84% with (i) ARHD α-sub unit (SU), (ii) LSU of Phenylpropionate dioxygenase (PDO), (iii) Phenol hydroxylase α-SU, (iv) Benzoate 1,2-dioxygenase, α-SU, (v) Naphthalene dioxygenase LSU, etc. This study has provided basic information, which can be used to develop a consortium of bacteria with mutually beneficial characteristics.

Enzymatic Properties of Terephthalate 1,2-Dioxygenase ofComamonassp. Strain E6

The tphA1 II and tphA2 II A3 II genes of Comamonas sp. E6 perhaps code for the terephthalate (TPA) 1,2-dioxygenase (TPADO). To characterize E6 TPADO, these genes were expressed in a His-tagged form in Escherichia coli, and the recombinant proteins were purified. TPADO activity was reconstituted from TphA1 II and TphA2 II A3 II, indicating that TPADO consists of a reductase (TphA1 II) and a terminal oxygenase component (TphA2 II and TphA3 II). TphA1(II) contains FAD, and the presence of a plant-type [2Fe-2S] cluster was suggested. These results indicate that TPADO is a class IB aromatic ring-hydroxylating dioxygenase. NADH and NADPH were effective as electron donors for TphA1 II, but NADPH appeared to be the physiological electron donor, based on the kinetic parameters. TPADO showed activity only toward TPA, and Fe2+ was required for it. The Km values for TPA and the Vmax were determined to be 72+/-6 microM and 9.87+/-0.06 U/mg respectively.

Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia

BackgroundPolycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination.ResultsGenes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus.ConclusionThese results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.

Characterization of DitA3, the [Fe 3S 4] ferredoxin of an aromatic ring-hydroxylating dioxygenase from a diterpenoid-degrading microorganism

DitA3, a small soluble ferredoxin, is a component of a ring-hydroxylating dioxygenase involved in the microbial degradation of the diterpenoid, dehydroabietic acid. The anaerobic purification of a heterologously expressed his-tagged DitA3 yielded 20 mg of apparently homogeneous recombinant protein, rcDitA3, per liter of cell culture. Each mole of purified rcDitA3 contained 2.9 equivalents of iron and 4.2 equivalents of sulfur, indicating the presence of a single [Fe 3S 4] cluster. This conclusion was corroborated by UV-Visible absorption ( ε 412 = 13.4 mM − 1cm − 1 ) and EPR ( g x,y = 2.00 and g z = 2.02) spectroscopies. The reduction potential of rcDitA3, determined using a highly oriented parallel graphite (HOPG) electrode, was − 177.0 ± 0.5 mV vs. the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, pH 7.0, 22 °C). This potential is similar to those of small, soluble Rieske-type ferredoxin components of aromatic-ring dihydroxylating dioxygenases. In contrast to these Rieske-type ferredoxins, DitA3 appears to exist as a dimer in solution. The dimeric ferredoxin may be more stable or may increase the catalytic efficiency of the dioxygenase by delivering the two reducing equivalents required for turnover of the oxygenase.

Degradation of chlorobenzenes by a strain of Acidovorax avenae isolated from a polluted aquifer

A subsurface microbial community was isolated from a polluted site of Suquía River (Córdoba—Argentina), acclimated during 15 days in aerobic conditions using 1,2-dichlorobenzene (1,2-DCB) as the sole carbon source. From this acclimated community, we isolated and identified by 16S rDNA analysis a strain of Acidovorax avenae, which was able to perform the complete biodegradation of 1,2-DCB in two days affording stoichiometric amounts of chloride. This pure strain was also tested for biodegradation of chlorobenzene (CB); 1,3-DCB and 1,4-DCB, giving similar results to the experiments using 1,2-DCB. The aromatic-ring-hydroxylating dioxygenase (ARHDO) α-subunit gene core, encoding the catalytic site of the large subunit of chlorobenzene dioxygenase, was detected by PCR amplification and confirmed by DNA sequencing. These results suggest that the isolated strain of A. avenae could use a catabolic pathway, via ARHDO system, leading to the formation of chlorocatecols during the first steps of biodegradation, with further chloride release and subsequent paths that showed complete substrate consumption.

Molecular characterization of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp.

Polycyclic aromatic hydrocarbon (PAH)-degrading genes nidA and nidB that encode the α and β subunits of the aromatic ring-hydroxylating dioxygenase have been cloned and sequenced from Mycobacterium vanbaalenii PYR-1 [Khan et al., Appl. Environ Microbiol. 67 (2001) 3577–3585]. In this study, the presence of nidA and nidB in 12 other Mycobacterium or Rhodococcus strains was investigated. Initially, all strains were screened for their ability to degrade PAHs by a spray plate method, and for the presence of the dioxygenase Rieske center region by polymerase chain reaction (PCR). Only Mycobacterium sp. PAH 2.135 (RJGII-135), M. flavescens PYR-GCK (ATCC 700033), M. gilvum BB1 (DSM 9487) and M. frederiksbergense FAn9T (DSM 44346), all previously known PAH degraders, were positive in both tests. From the three positive strains, complete open reading frames of the nidA and nidB genes were amplified by PCR, using primers designed according to the known nidA and nidB sequences from PYR-1, cloned in the pBAD/Thio-TOPO vector and sequenced. The sequences showed >98% identity with the M. vanbaalenii PYR-1 nidA and nidB genes. Southern DNA–DNA hybridization using nidA and nidB probes from PYR-1 revealed that there is more than one copy of nidA and nidB genes in the strains PYR-1, BB1, PYR-GCK and FAn9T. However, only one copy of each gene was observed in PAH2.135.

Detection of known and novel genes encoding aromatic ring-hydroxylating dioxygenases in soils and in aromatic hydrocarbon-degrading bacteria

Primers were designed and successfully used to screen aromatic hydrocarbon-degrading bacteria for the presence of class II aromatic ring-hydroxylating dioxygenase (RHD) genes and to amplify novel RHD genes from DNA extracted from soil using the polymerase chain reaction. Two previously undiscovered groups of genes encoding putative class II RHDs, designated the S and T clusters, were found in RHD different soil samples. Only one of 70 RHD gene fragments amplified from these soil samples could be assigned to a cluster of previously reported RHD genes. These results suggest that distinct and potentially numerically dominant groups of as-yet unrecognized aromatic hydrocarbon-degrading bacteria exist in soils.

Dehalogenation, denitration, dehydroxylation, and angular attack on substituted biphenyls and related compounds by a biphenyl dioxygenase.

The attack by the bph-encoded biphenyl dioxygenase of Burkholderia sp. strain LB400 on a number of symmetrical ortho-substituted biphenyls or quasi ortho-substituted biphenyl analogues has been investigated. 2,2'-Difluoro-, 2,2'-dibromo-, 2,2'-dinitro-, and 2,2'-dihydroxybiphenyl were accepted as substrates. Dioxygenation of all of these compounds showed a strong preference for the semisubstituted pair of vicinal ortho and meta carbons, leading to the formation of 2'-substituted 2,3-dihydroxybiphenyls by subsequent elimination of HX (X = F, Br, NO(2), or OH). All of these products were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenases of Burkholderia sp. strain LB400 or of Rhodococcus globerulus P6. Dibenzofuran and dibenzodioxin, which may be regarded as analogues of doubly ortho-substituted biphenyls or diphenylethers, respectively, were attacked at the "quasi ortho" carbon (the angular position 4a) and its neighbor. This shows that an aromatic ring-hydroxylating dioxygenase of class IIB is able to attack angular carbons. The catechols formed, 2,3,2'-trihydroxybiphenyl and 2,3,2'-trihydroxydiphenylether, were further metabolized by 2,3-dihydroxybiphenyl 1,2-dioxygenase. While angular attack by the biphenyl dioxygenase was the main route of dibenzodioxin oxidation, lateral dioxygenation leading to dihydrodiols was the major reaction with dibenzofuran. These results indicate that this enzyme is capable of hydroxylating ortho or angular carbons carrying a variety of substituents which exert electron-withdrawing inductive effects. They also support the view that the conversions of phenols into catechols by ring-hydroxylating dioxygenases, such as the transformation of 2,2'-dihydroxybiphenyl into 2,3,2'-trihydroxybiphenyl, are the results of di- rather than of monooxygenations. Lateral dioxygenation of dibenzofuran and subsequent dehydrogenation and extradiol dioxygenation by a number of biphenyl-degrading strains yielded intensely colored dead-end products. Thus, dibenzofuran can be a useful chromogenic indicator for the activity of the first three enzymes of biphenyl catabolic pathways.

Occurrence of Two Resin Acid-Degrading Bacteria and a Gene Encoding Resin Acid Biodegradation in Pulp and Paper Mill Effluent Biotreatment Systems Assayed by PCR.

> Abstract We examined the distribution of two dehydroabietic acid-degrading bacteria, Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35, in biotreatment systems for pulp and paper mill effluents (PPMEs) using PCR assays. These two bacteria were first isolated from two PPME biotreatment systems and can degrade both dehydroabietic acid (DhA) and other abietane resin acids. We also examined the distribution of a catabolic gene, ditA1, encoding the alpha subunit of an aromatic ring-hydroxylating dioxygenase involved in DhA degradation by BKME-9. PCR primers specific for the 16S rDNA of BKME-9 and of DhA-35 and specific for ditA1 were used. Among 3 laboratory- and 17 full-scale PPME biotreatment systems, 10 contained phylotype BKME-9, 3 contained phylotype DhA-35, and 11 contained ditA1, indicating the wider distribution of phylotype BKME-9 than of phylotype DhA-35. Both phylotype BKME-9 and ditA1 were detected in the biotreatment system from which BKME-9 was originally isolated in 1994, suggesting the persistance of BKME-9 in that biotreatment system. The detection limit of the PCR assay was one cell per PCR reaction, which corresponds to one BKME-9 cell per 6 x 10(7) total sludge bacteria. A competitive PCR assay indicated that ditA1 ranged from 51 to 250 copies/mg of dry biomass. BKME-9 appears to contribute to the biodegration of resin acids in some PPME biotreatment systems. Using degenerate PCR primers and touchdown PCR, we obtained from our DhA-degrading strain collection six DNA sequences putatively homologous to that of ditA1. Cluster analysis of these DNA sequences suggests that ditA1 encodes a representative of a novel class of dioxygenase enzymes.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p114.html

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase

The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD+-dependent dehydrogenase (bedD) required to convert benzene into catechol. Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26. In vitro translation analysis showed bedD to code for a polypeptide of ca. 39 kDa. Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth. The bedD gene product was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase. The dehydrogenase encoded by bedD) was overexpressed in Escherichia coli and purified. It was found to utilize NAD+ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol. Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.