<Emphasis Type="Italic">para</Emphasis>-Nitrophenol 4-monooxygenase and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-nitrocatechol in <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. PnpA (PNP 4-monooxygenase) and PnpB (para-benzoquinone reductase) were shown to be involved in the initial steps of PNP catabolism via hydroquinone. We demonstrated here that PnpA also catalyzed monooxygenation of 4-nitrocatechol (4-NC) to hydroxyquinol, probably via hydroxyquinone. It was the first time that a single-component PNP monooxygenase has been shown to catalyze this conversion. PnpG encoded by a gene located in the PNP degradation cluster was purified as a His-tagged protein and identified as a hydroxyquinol dioxygenase catalyzing a ring-cleavage reaction of hydroxyquinol. Although all the genes necessary for 4-NC metabolism seemed to be present in the PNP degradation cluster in strain WBC-3, it was unable to grow on 4-NC as a sole source of carbon, nitrogen and energy. This was apparently due to the substrate’s inability to trigger the expression of genes involved in degradation. Nevertheless, strain WBC-3 could completely degrade both PNP and 4-NC when PNP was used as the inducer, demonstrating its potential in bioremediation of the environment polluted by both 4-NC and PNP.     

PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring-cleavage dioxygenase.

The pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6-dichlorohydroquinone (2,6-DCHQ). The pathway converting PCP to 2,6-DCHQ has been established previously; however, the pathway beyond 2,6-DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2, 6-DCHQ conversion. In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring-cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6-DCHQ to 2-chloromaleylacetate.     

Structural characterization of 2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase (PcpA) from Sphingobium chlorophenolicum,a new type of aromatic ring‐cleavage enzyme

PcpA (2,6‐dichloro‐p‐hydroquinone 1,2‐dioxygenase) from Sphingobium chlorophenolicum, a non‐haem Fe(II) dioxygenase capable of cleaving the aromatic ring of p‐hydroquinone and its substituted variants, is a member of the recently discovered p‐hydroquinone 1,2‐dioxygenases. Here we report the 2.6 Å structure of PcpA, which consists of four βαβββ motifs, a hallmark of the vicinal oxygen chelate superfamily. The secondary co‐ordination sphere of the Fe(II) centre forms an extensive hydrogen‐bonding network with three solvent exposed residues, linking the catalytic Fe(II) to solvent. A tight hydrophobic pocket provides p‐hydroquinones access to the Fe(II) centre. The p‐hydroxyl group is essential for the substrate‐binding, thus phenols and catechols, lacking a p‐hydroxyl group, do not bind to PcpA. Site‐directed mutagenesis and kinetic analysis confirm the critical catalytic role played by the highly conserved His10, Thr13, His226 and Arg259. Based on these results, we propose a general reaction mechanism for p‐hydroquinone 1,2‐dioxygenases.     

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls (PCBs)

The microbial degradation of polychlorinated biphenyls (PCBs) has been extensively conducted by many workers, and the following general results have been obtained. (1) PCBs are degraded oxidatively by aerobic bacteria and other microorganisms such as white rot fungi. PCBs are also reductively dehalogenated by anaerobic microbial consortia. (2) The biodegradability of PCBs is highly dependent on chlorine substitution, i.e., number and position of chlorine. The degradation and dehalogenation capabilities are also highly strain dependent. (3) Biphenyl-utilizing bacteria can cometabolize many PCB congeners to chlorobenzoates by biphenl-catabolic enzymes. (4) Enzymes involved in the PCB degradation were purified and characterized. Biphenyl dioxygenase, ring-cleavage dioxygenase, and hydrolase are crystallized, and two ring-cleavage dioxygenases are being solved by x-ray crystallography. (5) The bph gene clusters responsible for PCB degradation are cloned from a variety of bacterial strains. The structure and function are analyzed with respect to the evolutionary relationship. (6) The molecular engineering of biphenyl dioxygenases is successfully performed by DNA shuffling, domain exchange, and subunit exchange. The evolved enzymes exhibit wide and enhanced degradation capacities for PCBs and other aromatic compounds.     

Hydroquinone degradation via reductive dehydroxylation of gentisyl-CoA by a strictly anaerobic fermenting bacterium

Anaerobic degradation of hydroquinone was studied with the fermenting bacterium strain HQGö1. The rate of hydroquinone degradation by dense cell suspensions was dramatically accelerated by addition of NaHCO₃. During fermentation of hydroquinone in the presence of ¹⁴C-Na₂CO₃ benzoate was formed as a labelled product, indicating an initial ortho-carboxylation of hydroquinone to gentisate. Gentisate was activated to the corresponding CoA-ester in a CoA ligase reaction at a specific activity of 0.15 mol x min^–1 x mg protein^–1. Gentisyl-CoA was reduced to benzoyl-CoA with reduced methyl viologen as electron donor by simultaneous reductive elimination of both the ortho and meta hydroxyl group. The specific activity of this novel gentisyl-CoA reductase was 17 nmol x min^–1 x mg protein^–1. Further degradation to acetate was catalyzed by enzymes which occur also in other bacteria degrading aromatic compounds via benzoyl-CoA.     

An unexpected gene cluster for downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3

In silico analysis of nucleotide sequences flanking the recently found hydroquinone dioxygenase in Sphingomonas sp. strain TTNP3 revealed a gene cluster that encodes a hydroquinone catabolic pathway. In addition to the two open-reading frames encoding the recently characterized hydroquinone dioxygenase, the cluster consisted of six open-reading frames. We were able to express the three open-reading frames, hqdC, hqdD, and hqdE, and demonstrated that the three gene products, HqdC, HqdD, and HqdE had 4-hydroxymuconic semialdehyde dehydrogenase, maleylacetate reductase, and intradiol dioxygenase activity, respectively. Surprisingly, the gene cluster showed similarities to functionally related clusters found in members of the β- and γ-proteobacteria rather than to those found in other members of the genus Sphingomonas sensu latu.     

Ubiquitination and Endocytosis of Plasma Membrane Proteins: Role of Nedd4/Rsp5p Family of Ubiquitin-Protein Ligases

In addition to its well-known role in recognition by the proteasome, ubiquitin-conjugation is also involved in downregulation of membrane receptors, transporters and channels. In most cases, ubiquitination of these plasma membrane proteins leads to their internalization followed by targeting to the lysosome/vacuole for degradation. A crucial role in ubiquitination of many plasma membrane proteins appears to be played by ubiquitin-protein ligases of the Nedd4/Rsp5p family. All family members carry an N-terminal Ca²⁺-dependent lipid/protein binding (C2) domain, two to four WW domains and a C-terminal catalytic Hect-domain. Nedd4 is involved in downregulation of the epithelial Na⁺ channel, by binding of its WW domains to specific PY motifs of the channel. Rsp5p, the unique family member in S. cerevisiae, is involved in ubiquitin-dependent endocytosis of a great number of yeast plasma membrane proteins. These proteins lack apparent PY motifs, but carry acidic sequences, and/or phosphorylated-based sequences that might be important, directly or indirectly, for their recognition by Rsp5p. In contrast to polyubiquitination leading to proteasomal recognition, a number of Rsp5p targets carry few ubiquitins per protein, and moreover with a different ubiquitin linkage. Accumulating evidence suggests that, at least in yeast, ubiquitin itself may constitute an internalization signal, recognized by a hypothetical receptor. Recent data also suggest that Nedd4/Rsp5p might play a role in the endocytic process possibly involving its C2 domain, in addition to its role in ubiquitinating endocytosed proteins. Recieved: 19 January 2000/Revised: 6 April 2000     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

Purification and partial characterization of the extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase,in the fluorene degradation pathway from Rhodococcus sp. strain DFA3

Type II extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α₄β₄ heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe²⁺ increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2′-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The K_m and k_cat/K_m values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 μM and 1.5 × 10^?2 μM^?1sec^?1, and for 2,2′,3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10^?2 μM^?1sec^?1, respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.     

Recent advances in understanding resin acid biodegradation: microbial diversity and metabolism

Resin acids are tricyclic diterpenoids that are found in the oleoresin of coniferous trees. Resin-acid-degrading microorganisms are ubiquitous in the environment. The bacterial isolates that grow on resin acids as sole organic substrates are physiologically and phylogenetically diverse, and include psychrotolerant, mesophilic, and thermophilic bacteria. Recent studies of the biodegradation of resin acids by these organisms have demonstrated that in gram-negative bacteria, distinct biochemical pathways exist for the degradation of abietane- and pimerane-type resin acids. One of these organisms, Pseudomonas abietaniphila BKME-9, harbors a convergent pathway that channels the nonaromatic abietanes and dehydroabietic acid into 7-oxodehydroabietic acid. This dioxygenolytic pathway is encoded by the recently cloned and sequenced dit gene cluster. The dit cluster encodes the ferredoxin and the α- and β-subunits of a new class of ring-hydroxylating dioxygenases as well as an extradiol ring-cleavage dioxygenase. Although it was previously thought that resin acids are very recalcitrant under anoxic conditions, recent investigations have demonstrated that they are partially metabolized under anoxic conditions by undefined microorganisms. The anaerobic degradation of resin acids principally generates aromatized and decarboxylated products (such as retene) that are thought to persist in the environment. Received: 9 April 1999 / Accepted: 1 July 1999     

Identification of the<Emphasis Type="Italic">bphC</Emphasis> gene for<Emphasis Type="Italic">meta</Emphasis>-cleavage of aromatic pollutants from a metagenomic library derived from lake waters

Useful genes can be screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs were extracted by the SDS-based freezing-thawing method, and then further purified using an UltraClean^TM kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested withEcoR I,BamH I, andSac II inEscherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13∼15 kb in size. ThebphC genes responsible for degradation of aromatic hydrocarbons viameta-cleavage were identified from the metagenomic libraries by colony hybridization using thebphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene (bphC), capable of degradation of 2,3-DHBP, was cloned and its nucleotide sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results indicated that thebphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.     

A Nag-like dioxygenase initiates 3,4-dichloronitrobenzene degradation via 4,5-dichlorocatechol in Diaphorobacter sp. strain JS3050

The chemical synthesis intermediate 3,4-dichloronitrobenzene (3,4-DCNB) is an environmental pollutant. Diaphorobacter sp. strain JS3050 utilizes 3,4-DCNB as a sole source of carbon, nitrogen and energy. However, the molecular determinants of its catabolism are poorly understood. Here, the complete genome of strain JS3050 was sequenced and key genes were expressed heterologously to establish the details of its degradation pathway. A chromosome-encoded three-component nitroarene dioxygenase (DcnAaAbAcAd) converted 3,4-DCNB stoichiometrically to 4,5-dichlorocatechol, which was transformed to 3,4-dichloromuconate by a plasmid-borne ring-cleavage chlorocatechol 1,2-dioxygenase (DcnC). On the chromosome, there are also genes encoding enzymes (DcnDEF) responsible for the subsequent transformation of 3,4-dichloromuconate to β-ketoadipic acid. The fact that the genes responsible for the catabolic pathway are separately located on plasmid and chromosome indicates that recent assembly and ongoing evolution of the genes encoding the pathway is likely. The regiospecificity of 4,5-dichlorocatechol formation from 3,4-DCNB by DcnAaAbAcAd represents a sophisticated evolution of the nitroarene dioxygenase that avoids misrouting of toxic intermediates. The findings enhance the understanding of microbial catabolic diversity during adaptive evolution in response to xenobiotics released into the environment.     

Cis-2′, 3′-Dihydrodiol production on flavone B-Ring by Biphenyl Dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli

Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2′ and C3′ on the B-ring of flavone, resulting in the formation of flavone cis-2′, 3′-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Diversity of 16S rRNA and dioxygenase genes detected in coal‐tar‐contaminated site undergoing active bioremediation

Aims: In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation‐independent communities is available. Methods and Results: Coal‐tar‐contaminated soil was collected, which consisted of 122·5 mg g^?1 total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal‐tar‐contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (α‐subunit) common to all PAH dioxygenase enzymes and (iii) β‐subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha‐, Epsilon‐ and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of α‐subunit and β‐subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe‐2S] cluster binding site suggested that these gene fragments encode for α‐subunit of dioxygenase gene. Conclusions: Sequencing of the cloned libraries representing α‐subunit gene fragments (Rf1) and β‐subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal‐tar‐contaminated soil. Significance and Impact of the Study: The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.     

Characterization of a para-nitrophenol catabolic cluster in Pseudomonas sp. strain NyZ402 and construction of an engineered strain capable of simultaneously mineralizing both para- and ortho-nitrophenols

Pseudomonas sp. strain NyZ402 was isolated for its ability to grow on para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy, and was shown to degrade PNP via an oxidization pathway. This strain was also capable of growing on hydroquinone or catechol. A 15, 818 bp DNA fragment extending from a 800-bp DNA fragment of hydroxyquinol 1,2-dioxygenase gene (pnpG) was obtained by genome walking. Sequence analysis indicated that the PNP catabolic gene cluster (pnpABCDEFG) in this fragment shared significant similarities with a recently reported gene cluster responsible for PNP degradation from Pseudomonas sp. strain WBC-3. PnpA is PNP 4-monooxygenase converting PNP to hydroquinone via benzoquinone in the presence of NADPH, and genetic analysis indicated that pnpA plays a key role in PNP degradation. pnpA1 present in the upstream of the cluster (absent in the cluster from strain WBC-3) encodes a protein sharing as high as 55% identity with PnpA, but was not involved in PNP degradation by either in vitro or in vivo analyses. Furthermore, an engineered strain capable of growing on PNP and ortho-nitrophenol (ONP) was constructed by introducing onpAB (encoding ONP monooxygenase and ortho-benzoquinone reductase which catalyzed the transformation of ONP to catechol) from Alcaligenes sp. strain NyZ215 into strain NyZ402.     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

Multiple and flexible roles of facultative anaerobic bacteria in microaerophilic oleate degradation

Anaerobic degradation of long-chain fatty acids (LCFA) involves syntrophic bacteria and methanogens, but facultative anaerobic bacteria (FAB) might have a relevant role as well. Here we investigated oleate degradation by a syntrophic synthetic co-culture of Syntrophomonas zehnderi (Sz) and Methanobacterium formicicum (Mf) and FAB (two oleate-degrading Pseudomonas spp. I1 + I2). Sz + Mf were first cultivated in a continuous bioreactor under strict anaerobic conditions. Thereafter, I1 + I2 were inoculated and microaerophilic conditions were provided. Methane and acetate were the main degradation products by Sz + Mf in anaerobiosis and by Sz + Mf + I1 + I2 in microaerophilic conditions. However, acetate production from oleate was higher in microaerophilic conditions (5% O₂) with the four microorganisms together (0.41 ± 0.07 mmol day⁻¹) than in anaerobiosis with Sz + Mf (0.23 ± 0.05 mmol day⁻¹). Oleate degradation in batch assays was faster by Sz + Mf + I1 + I2 (under microaerophilic conditions) than by Sz + Mf alone (under strict anaerobic conditions). I1 + I2 were able to grow with oleate and with intermediates of oleate degradation (hydrogen, acetate and formate). This work highlights the importance of FAB, particularly Pseudomonas sp., in anaerobic reactors treating oleate-based wastewater, because they accelerate oleate conversion to methane, by protecting strict anaerobes from oxygen toxicity and also by acting as alternative hydrogen/formate and acetate scavengers for LCFA-degrading anaerobes.     

A novel light‐dependent selection marker system in plants

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin‐degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring‐cleavage extradiol dioxygenase in the Exiguobacterium sp. 255‐15; however, unlike other extradiol dioxygenases, Mn²⁺and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light‐dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high‐density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.     

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.