New Metabolites in Dibenzofuran Cometabolic Degradation by a

Oct 12, 2009 - A biphenyl (BP)-utilizing bacterium, designated B6-2, was isolated from soil and identified as Pseudomonas putida. BP- grown B6-2 cells...
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Environ. Sci. Technol. 2009, 43, 8635–8642

New Metabolites in Dibenzofuran Cometabolic Degradation by a Biphenyl-Cultivated Pseudomonas putida Strain B6-2 Q I N G G A N G L I , †,‡ X I A O Y U W A N G , †,‡ GUANGBO YIN,† ZHONGHUI GAI,‡ HONGZHI TANG,‡ CUIQING MA,† Z I X I N D E N G , ‡ A N D P I N G X U * ,‡ State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of China, and MOE Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

Received July 5, 2009. Revised manuscript received September 29, 2009. Accepted October 3, 2009.

A biphenyl (BP)-utilizing bacterium, designated B6-2, was isolated from soil and identified as Pseudomonas putida. BPgrown B6-2 cells were capable of transforming dibenzofuran (DBF) via a lateral dioxygenation and meta-cleavage pathway. The ring cleavage product 2-hydroxy-4-(3′-oxo-3′H-benzofuran2′-yliden)but-2-enoic acid (HOBB) was detected as a major metabolite. B6-2 growing cells could also cometabolically degrade DBF using BP as a primary substrate. A recombinant Escherichia coli strain DH10B (pUC118bphABC) expressing BP dioxygenase, BP-dihydrodiol dehydrogenase, and dihydroxybiphenyl dioxygenase was shown to be capable of transforming DBF to HOBB. Using purified HOBB that was produced by the recombinant as the substrate for B6-2, we newly identified a series of benzofuran derivatives as metabolites. The structures of these metabolites indicate that an unreported HOBB degradation pathway is employed by strain B6-2. In this pathway, HOBB is proposed to be transformed to 2-oxo-4-(3′oxobenzofuran-2′-yl)butanoic acid and 2-hydroxy-4-(3′oxobenzofuran-2′-yl)butanoic acid (D4) through two sequential double-bond hydrogenation steps. D4 is suggested to undergo reactions including decarboxylation and oxidation to produce 3-(3′oxobenzofuran-2′-yl)propanoic acid (D6). 3-Hydroxy-3-(3′oxobenzofuran-2′-yl)propanoic acid (D7) and 2-(3′-oxobenzofuran2′-yl)acetic acid (D8) would represent metabolites involved in the processes of beta- and alpha-oxidation of D6, respectively. D7 and D8 are suggested to be transformed to their respective products 3-hydroxy-2,3-dihydrobenzofuran-2-carboxylic acid (D10) and 2-(3′-hydroxy-2′,3′-dihydrobenzofuran-2′-yl)acetic acid. D10 is proposed to be transformed to salicylic acid (D14) via 2,3dihydro-2,3-dihydroxybenzofuran, 2-oxo-2-(2′-hydroxyphenyl)acetic acid and 2-hydroxy-2-(2′-hydroxyphenyl)acetic acid. Further experimental results revealed that B6-2 was capable of growing with D14 as the sole carbon source. Because

* Corresponding author phone: +86-21-34206647; fax: +86-2134206723.; e-mail: [email protected]. † Shandong University. ‡ Shanghai Jiao Tong University. 10.1021/es901991d CCC: $40.75

Published on Web 10/12/2009

 2009 American Chemical Society

benzofuran derivatives may have biological, pharmacological, and toxic properties, the elucidation of this new pathway should be significant from both biotechnological and environmental views.

Introduction Dibenzofuran (DBF) is a component of crude oil, creosote, and coal tar. It has been used as a model compound for studying the microbial degradation of polycyclic aromatic compounds (PACs) and polychlorinated DBFs (PCDFs). PCDFs are notorious byproducts formed during the synthesis of chloroaromatic compounds, the incineration of domestic and industrial wastes, and the manufacture of pulp and paper (1-5). The search for microorganisms able to grow with DBF has led to the isolation of several bacteria belonging to the genera Sphingomonas, Terrabacter, and Rhodococcus (5-10). By now, only those bacteria degrading DBF via the angular dioxygenation and meta-cleavage pathway have been reported to be capable of utilizing DBF as the sole carbon and energy source. Because PACs including DBF are widespread and usually coexist in the polluted environments, cometabolic degradation of some PACs by bacteria growing with some others should be common, and there have been many studies on the cometabolic degradation of DBF via lateral dioxygenation and meta-cleavage pathways by naphthalene-, fluorene-, and biphenyl (BP)-utilizing strains (11-19). Previous studies (14, 17) showed that the transformation of DBF up to ring cleavage (an upper pathway of DBF degradation) in BP-cultivated Burkholderia xenovorans strain LB400 was catalyzed by BP-degrading enzymes. BP dioxygenase (BphA) of LB400 attacked DBF mainly at the 1,2-C position to produce 1,2-dihydroxy-1,2-dihydrodibenzofuran, which was dehydrogenated by BP-dihydrodiol dehydrogenase (BphB) and then dioxygenolytically cleaved by dihydroxybiphenyl dioxygenase (BphC). The ring cleavage product 2-hydroxy-4(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid (HOBB), a benzofuran derivative, was detected most frequently as the final product in many strain cultures. A few strains have also been reported to completely degrade HOBB via salicylic acid (11-13). However, except for salicylic acid, the degradation metabolites beyond HOBB have not been reported by now. Many naturally occurring or synthetic 2-substituted benzofurans with relatively simple structures are well-known to exhibit broad biological and pharmacological activities, such as anti-inflammation and imaging of beta-amyloid plaques in the brains of patients with Alzheimer’s disease (20-22). On the other hand, because benzofuran is toxic and carcinogenic (23), its derivatives may have similar properties. It is known that bioaccumulation of toxic compounds and their metabolites through food-chain is a threat to human and animal health. Therefore, xenobiotics and their metabolites produced by the most important xenobiotic-degraders, microorganisms, should be of special concern. From these points of view, it is of great interest to characterize the HOBB degradation pathway, which may guide us to develop a microbial tool to produce valuable chemicals from DBF and know better about the metabolites of DBF that would accumulate in the environments. In this study, we describe the production of HOBB from DBF by a BP-utilizing Pseudomonas putida strain B6-2. Using the purified HOBB as the substrate for B6-2, we propose a series of benzofuran derivatives as metabolites. VOL. 43, NO. 22, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Experimental Section Chemicals. DBF, N-methyl-N-nitrosourea, 2,3-dihydroxybiphenyl and chromatographic grade of ethyl acetate were purchased from Sigma-Aldrich. Salicylic acid was obtained from Toyo Kasei Kogyo Co., Ltd. Isolation and Identification of BP-Utilizing Strain B62. The isolation process was performed in a mineral salts medium (MSM) which contained (per liter of distilled water) 3.7 g of KH2PO4, 5.2 g of K2HPO4 · 3H2O, 2.0 g of NH4Cl, 1.0 g of Na2SO4, 0.1 g of MgSO4, and 1 mL of trace metal solution (24). A BP-contaminated soil sample was added to MSM supplemented with BP crystal and incubated aerobically at 30 °C on a reciprocal shaker at 180 rpm. After 10 times of sequential transfers, one isolate, designated B6-2, was isolated from the culture and characterized by various taxonomic tests at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ). 16S rDNA was sequenced by Shanghai Sangon Biotechnology Co., Ltd., China. The similarity search was performed with the nucleotide blast program (25) at the National Center for Biotechnology Information (NCBI) Web site (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). Cloning of Upper Pathway Genes for BP Metabolism. Genomic DNA was isolated from strain B6-2 using a Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI). DNA fragmentation was carried out using a HydroShear DNA shearing device (GeneMachines, U.S.). The fragments with sizes between 6 and 8 kb were purified with a QIAquick Gel Extraction Kit (QIAGEN, Germany), blunted with T4 DNA polymerase (TaKaRa), and ligated into a pUC118 Hinc II/ BAP vector (TaKaRa) with T4 DNA ligase (Promega) according to the manufacturers’ instructions. The constructed plasmid was transformed into Escherichia coli DH10B (26) by electroporation according to a standard procedure (27). The transformants were cultivated on Luria-Bertani (LB) agar plates containing 372 mg/L ampicillin and 0.1 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG). After 12-h incubation, the transformants were screened according to the appearance of blue and yellow colors after spraying with indole (28) and 2,3-dihydroxybiphenyl, respectively. The ability of the isolates to yield a yellow product from BP was further tested. Sequencing of the inserted DNA fragment and the similarity search with the blastX program were performed as shown above. Degradation of DBF by Strain B6-2 and the Recombinant E. coli Cells. Strain B6-2 was cultured in either MSM with 15 mmol/L BP as the sole source of carbon and energy or LB medium. The recombinant E. coli cells were cultured in LB medium containing 372 mg/L ampicillin and 1 mmol/L IPTG at 30 °C on a reciprocal shaker at 180 rpm. Cell suspensions of B6-2 and the recombinant E. coli strain were prepared separately by centrifugating the cultures in late exponential phase at 4120 × g for 15 min, washing cell pallets twice with MSM, and resuspending cells in MSM to get different turbidities. The B6-2 cell growth was tested with 1 mmol/L DBF, 1 mmol/L HOBB, 5 mmol/L salicylic acid, 15 mmol/L BP, and 15 mmol/L BP plus 3 mmol/L DBF separately, using the BP-grown B6-2 cell suspension with a turbidity at 620 nm of 0.15. Studies on DBF (0.2 mmol/L) degradation by the BP- and LB medium-grown B6-2 cell suspensions with a turbidity at 620 nm of 5 and by the recombinant E. coli cell suspension with a turbidity at 620 nm of 10 were also carried out. The cell growth in culture without substrate and the degradation of substrates in autoclaved cell suspensions were detected as controls. In every test, triplicate samples and controls were examined. The bacterial growth was verified by measuring the increase in the turbidity of cultures. After adding two volumes of ethanol to the whole cultures, the residual substrate concentrations were determined using high-performance liquid 8636

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chromatography (HPLC). Major metabolites of DBF were analyzed using HPLC-mass spectrometry (HPLC-MS). A major metabolite D1 was purified from the recombinant E. coli cell suspension exposed to DBF following a method for preparing HOBB described by Becher et al. (11). The purified D1 was derivatized with an ethereal solution of diazomethane which was generated from N-methyl-N-nitrosourea according to a previously reported method (29) and then analyzed by high-resolution mass spectrometry (HR-MS). Degradation of D1 by Strain B6-2. D1 was added to BPgrown B6-2 cell suspension with a turbidity at 620 nm of 5 at a final concentration of 0.2 mmol/L. At each incubation time of 4, 8, 16, and 24 h, a quarter of the culture was sampled and centrifugated. Metabolites were extracted from the supernatants with ethyl acetate after acidification to pH 2.0 with 1.25 mol/L H2SO4. The extracts were dried over Na2SO4 and evaporated to dryness under nitrogen gas. One portion of the sample was dissolved in acetonitrile and analyzed by HPLC and HPLC-MS. A second portion was derivatized with an ethereal solution of diazomethane and then analyzed by HR-MS. A third portion was also dissolved in acetonitrile, and major metabolites were purified by preparative HPLC on a SHIMADZU LC-10A liquid chromatograph coupled to a SPD-10Avp UV detector equipped with a Zorbax Eclipse XDB-C18 PrepHT column (250 mm × 21.2 mm × 7 µm; Agilent). The mobile phase was acetonitrile-1 mmol/L H2SO4 (14:86) at a flow rate of 12 mL/min, and the eluent was monitored at 254 nm. All data were processed and analyzed by Class-VP 6.12. 1H nuclear magnetic resonance (NMR) and 13 C NMR spectra analyses of a purified product D6 were carried out. Studies on the cometabolic degradation of purified metabolites by strain B6-2 were also carried out, and their products were extracted from the cultures following the method shown above and analyzed by HPLC and HPLCMS. Control samples without substrate were prepared and analyzed using HPLC and HR-MS. The degradation of D1 by LB medium-grown B6-2 cell suspension was also tested. Analytical Methods. Analytical HPLC and HPLC-MS were carried out using instruments as described by Gai et al. (4). ThemobilephaseforHPLCanalysiswaseithermethanol-water (70:30) at a flow rate of 0.5 mL/min (for the DBF degradation samples) or acetonitrile-1 mmol/L H2SO4 (14:86) at a flow rate of 0.7 mL/min (for the HOBB degradation samples), in which 1 mmol/L H2SO4 was substituted by 0.5% (v/v) formic acid for HPLC-MS analysis. HR-MS was performed on a GC-MS system (Waters GCT mass spectrometer, coupled to an Agilent HP6890 gas chromatograph) equipped with a J&W DB-5MS column. The oven temperature program started at 100 °C for 2 min, and then ramped to 190 at 15 °C/min, and to 280 at 5 °C/min, which was kept for 3 min. 1 H NMR (300.13 MHz) and 13C NMR (75.46 MHz) spectra were obtained on a BRUKER AVANCE 300 spectrometer with TMS as an internal standard and CD3OD as the solvent. Nucleotide Sequence Accession Number. The nucleotide sequences of bphABC and 16S rDNA reported in this paper have been submitted to GenBank under accession numbers FJ715926 and EF672104, respectively.

Results Characterization of Strain B6-2. According to the carbon utilization pattern (SI Table S1) and the physical and biochemical characteristics (SI Table S2), strain B6-2 was assigned to Pseudomonas sp. The pattern of cellular fatty acids synthesized by strain B6-2 was typical for the RNAgroup I of the genus Pseudomonas. The riboprint pattern of strain B6-2 showed the highest similarity (0.74) to that of P. putida (DSM50198) in the DuPont Identification Library. The 16S rDNA sequence of strain B6-2 showed 100% similarity to those of P. putida strains F1 and CD2 (GenBank accession

FIGURE 1. Degradation of DBF and production of HOBB by BP-grown B6-2 cells, and degradation of DBF and HOBB by LB medium-grown B6-2 cells. All of the cell suspensions were prepared with a turbidity at 620 nm of 5.0. The autoclaved BP-grown cell suspension exposed to DBF served as a control. Concentrations of DBF (0) and HOBB (9) in BP-grown cell suspensions, DBF (O) and HOBB (∆) in LB medium-grown cell suspensions, and DBF (2) in the autoclaved BP-grown cell suspension were tested. Data are the mean and standard deviation of independent triplicates. numbers EF204236 and AM930519, respectively). Therefore, strain B6-2 was assigned to P. putida. It was deposited in China Center for type Culture Collection under accession number CCTCC M207029. Characterizaiton of the Upper Pathway Genes for BP Degradation. A library of about 72 000 recombinant clones containing B6-2 genomic DNA fragments with sizes between 6 and 8 kb was constructed. One colony assigned to E. coli DH10B (pUC118bphABC) was obtained by screening the DNA library. DNA sequencing and similarity search results indicate that E. coli DH10B (pUC118bphABC) contains a 5974-bp DNA fragment comprising bphA, bphB, and bphC. bphA consists of four genes and the encoded proteins are a large (BphA1) and a small (BphA2) subunits of a terminal dioxygenase, a ferredoxin (BphA3) and its reductase (BphA4). The similarities of BphA1, BphA2, BphB, BphC from B6-2 with the corresponding parts from Pseudomonas pseudoalcaligenes strain KF707 (strain LB400) (GenBank accession numbers: M83673 for those of KF707, and NC_007953 for those of LB400) are 92% (90%), 98% (98%), 99% (99%), and 100% (99%), respectively. BphA3 and BphA4 of B6-2 are identical with those of KF707 and LB400 (30, 31). Degradation of DBF by B6-2 and E. coli Strain DH10B (pUC118bphABC). DBF did not support B6-2 cell growth, and was not degraded by LB medium-cultivated B6-2 cells. However, it was degraded rapidly by BP-grown B6-2 cells (Figure 1 and SI Figure S1). During incubation, a major metabolite D1 was detected. The amount of D1 increased to its maximum after about 5 h incubation, and decreased slowly almost to under detection limit thereafter. D1 was identified as HOBB by comparing its molecular mass, absorption maximum, and the HR-MS analytical data of its methyl derivative (SI Figure S2) with those reported in the literatures (11, 15). Two other products, D2 and D3 (with the same molecular mass of 184), had very similar ultraviolet-visible (UV-vis) spectra (SI Figure S3) with those reported for 1-hydroxydibenzofuran and 4-hydroxydibenzofuran, respectively. D2 and D3 should be spontaneously dehydrated products of 1,2-dihydroxy-1,2-dihydrodibenzofuran and 3,4dihydroxy-3,4-dihydrodibenzofuran, respectively (1, 14, 32). There were also a series of metabolites showing very similar UV-vis spectra. The identification of these metabolites is described in the following sections. E. coli DH10B (pUC118bphABC) was also capable of transforming DBF. After 12 h incubation, about 63% of 0.2 mmol/L DBF was transformed. HOBB, 1-hydroxydibenzofuran and 4-hydroxydibenzofuran were also identified as metabolites.

The growth of B6-2 on BP and on BP plus DBF is shown in Figure 2. When BP served as the sole carbon source, within 20 h, the culture turbidity at 620 nm increased from 0.15 to its maximum 2.06, and the concentration of BP decreased from 15 to 0.6 mmol/L. With additional 3 mmol/L DBF in the culture, the rates of cell growth and BP consumption, and the maximum culture turbidity decreased, which might be due to the inhibition by DBF or its metabolites on B6-2 cell growth. Identification of HOBB Degradation Products. About 86 mg HOBB was prepared from the DH10B (pUC118bphABC) cell suspension exposed to 200 mg DBF. B6-2 could not grow with the purified HOBB as the sole source of carbon and energy. HOBB could be transformed by BP-grown B6-2 cells, but could not by LB medium-cultivated B6-2 cells (Figure 1). During HOBB degradation by B6-2, several metabolites showing almost the same UV-vis spectra were detected by HPLC (SI Figure S4). The UV-vis spectra of four major metabolites assigned to D4, D6, D7, and D8 are shown in Figure 3A. Based on the similar UV-vis spectra, they are proposed to be a series of compounds having a common conjugated structure. HR-MS analysis of the methylated HOBB-degradation sample revealed the presence of eleven compounds produced from HOBB, including D4M, D6M, D7M, and D8 M (Figure 3B, the letter “M” in all of the assigned names indicating the methylation of the original compounds). None of these products was detected from the cell suspension without HOBB (SI Figure S5). Eight of them were newly identified as HOBB degradation products, and their HR-MS data are shown in Figure 4. The proposed structure of each metabolite is shown with each spectrum. Almost all of the mass spectra shown in Figure 4 have common fragments corresponding to the formulas C8H5O2 (theoretical: 133.0290), C7H5O2 (theoretical: 121.0290), C7H5O (theoretical: 105.0340), and C6H4O (theoretical: 92.0262) (some with differences of one or two H atoms), indicating that a structure similar to 2-substituted benzofuran-3-one should be a moiety of each compound. In addition, as shown in Figure 4, the fragments corresponding to losses of CH3O and CO2CH3 (some with differences of one or two H atoms) were commonly detected, indicating that each of these molecules contained a carboxylic acid methyl ester functional group. The acidic property of the original metabolites was consistent with the experimental result that they could be separated by HPLC only using an acidic mobile phase. D4M had a molecular ion (M+) at m/z 250.0834, corresponding to the formula C13H14O5 (theoretical: 250.0841). D4 had a molecular mass of 236 by HPLC-MS analysis. Therefore, D4M should be a monomethylated derivative of D4. A hydroxylated alkyl acid is proposed to be the side chain of benzofuranone according to the presence of ions at m/z 172.0490 (M+ -CO2CH3-H2O-H, theoretical: 172.0524), 160.0567 (M+ -CO2CH3-H2O-CH, theoretical: 160.0524), 147.0437 (M+ -CO2CH3-H2O-CH-CH, theoretical: 147.0446), and 134.0384 (M+ -CO2CH3-H2O-CH-CH-CH, theoretical: 134.0368). The formula of D4 had four hydrogen atoms more than HOBB. In the HOBB degradation experiment, D4 appeared in the 4 and 8 h samples, but disappeared after 16 h incubation accompanying the exhaustion of HOBB (SI Figure S4). Accordingly, D4 is proposed to be an upper pathway metabolite of HOBB. Based on the above experimental results, D4 is suggested to be 2-hydroxy-4-(3′oxobenzofuran-2′-yl)butanoic acid. D6, D7, and D8 could be detected from BP-grown B6-2 cell suspension exposed to the purified D4, whereas D7 and D8 could be detected from the suspension exposed to the purified D6. The exact molecular mass of D6M (220.0751) corresponded to the formula C12H12O4 (theoretical: 220.0736). D6M should be a monomethylated derivative as D6 had a molecular mass of 206 according to the HPLC-MS analytical VOL. 43, NO. 22, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Growth of stain B6-2 with utilization of BP (A), and with BP plus DBF accompanying the formation of HOBB (B). The growth of cells without substrate and degradation of substrates in autoclaved cultures served as controls. Turbidities (optical density at 620 nm) of cultures with (2) and without (∆) substrates were monitored. Concentrations of BP (9), DBF (1) and HOBB (O) in growing cell cultures, and BP (0) and DBF (]) in autoclaved cell cultures were tested. Data are the mean and standard deviation of independent triplicates.

FIGURE 3. A. UV-vis spectra of the HOBB metabolites D4, D6, D7, and D8 detected by HPLC. The mobile phase was acetonitrile and 1 mmol/L H2SO4 at a ratio of 14:86. B. A portion of the total ion current chromatogram from the HR-MS analysis of the diazomethane derivatized HOBB-degradation sample. result. The fragment ions of D4M and D6M in the low-mass range (em/z 190) were similar, and the value of the difference in their molecular masses corresponded to a CHOH group. According to the above data, D6 is suggested to be 3-(3′oxobenzofuran-2′-yl)propanoic acid. About 3 mg of D6 was prepared and analyzed using 1H NMR and 13C NMR. The proton signals, one methine (δ 3.28, 1H, m), two methylene (δ 2.12, 2H, m; δ 2.45, 2H, m), and four aromatic protons (δ 7.05-7.11, 2H, m; δ 7.61, 1H, d; δ 7.68, 1H, t, J ) 8.4 Hz) were detected. The signal of carboxylic proton was not detected under our experimental conditions. Eleven carbon signals in the 13C NMR (δ, 27.61, 30.89, 104.88, 113.54, 119.67, 122.36, 124.83, 139.54, 170.97, 175.57, and 200.46) were detected. The NMR analytical results further confirmed D6 as 3-(3′oxobenzofuran-2′-yl)propanoic acid. D8M had an exact molecular mass of 206.0555 corresponding to the formula C11H10O4 (theoretical: 206.0579). It was a monomethylated derivative because D8 had a molecular mass of 192 detected by HPLC-MS. By calculation, D8M and D6M had a difference of a CH2 group. In addition, D8M had a similar MS profile in the low-mass range (em/z 147) with those of D4M and D6M. Therefore, D8 is suggested to be 2-(3′-oxobenzofuran2′-yl)acetic acid. D7 had a molecular mass of 222 according to the HPLC-MS analytical result. D7M had an exact molecular mass of 236.0719 corresponding to the elemental composition C12H12O5 (theoretical: 236.0685), indicating it 8638

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to be monomethylated D7. The value of the difference between the molecular masses of D6M and D7M corresponded to an O atom. The fragment ions of D7M at m/z 218.0558 (M+ -H2O, theoretical: 218.0579), 186.0294 (M+ -OCH3-H2O-H, theoretical: 186.0317), 163.0418 (M+ -CO2CH3-CH2, theoretical: 163.0395), and 159.0399 (M+ -CO2CH3-H2O, theoretical: 159.0446) were detected, indicating it to be a hydroxylated derivative of D6M at the side chain. Accordingly, D7 is suggested to be 3-hydroxy-3-(3′oxobenzofuran-2′-yl)propanoic acid. D5M was a relatively minor product of HOBB, and the peak correlative with it in the HPLC profile was not examined. The molecular ion of D5M at m/z 248.0703 corresponded to the formula C13H12O5 (theoretical: 248.0685). Though the relative intensities were different, most of the ions of D5M and D4M were almost the same. D5 was two hydrogen atoms less than D4 while two hydrogen atoms more than HOBB. Fragments indicating the appearance of a hydroxyl group were not found in the MS profile. Accordingly, D5M is proposed to be the methyl ester of 2-oxo-4-(3′-oxobenzofuran-2′-yl)butanoic acid. The molecular ion of D9M was m/z 208.0738, corresponding to the empirical formula C11H12O4 (theoretical: 208.0736). D9M and D8M should be structurally similar compounds with a difference of two hydrogen atoms in their formulas by comparing their HR-MS data, which suggests that D9M is the methyl ester of 2-(3′-hydroxy-2′,3′-dihydrobenzofuran-2′-yl)acetic acid. HR-MS analysis showed another compound D10M (M+ at m/z 208.0765) with the same empirical formula as D9M. D10M had fragments at m/z 145.0268 (theoretical: 145.0290) and 133.0307 (theoretical: 133.0290) corresponding to losses of CH3O + CH3O + H and CO2CH3 + CH3 + H, respectively, from the molecular ion. Therefore, there should be two CH3 groups bonded to an alcoholic hydroxyl group and a carboxylic group, respectively, in D10M. Accordingly, D10M is suggested to be dimethylated 3-hydroxy-2,3-dihydrobenzofuran-2-carboxylic acid. The exact mass of D11M was 182.0597, corresponding to the empirical formula C9H10O4 (theoretical: 182.0579). It showed a highly similar MS profile with that of methyl 2-hydroxy-2-(4′-hydroxyphenyl)acetic acid in the NIST MS database (commercially available from the National Institute of Standards and Technology). As a product of HOBB, its hydroxyl group bonded to the benzene ring should be present at the 2′ position. Therefore, D11M is suggested to be the methyl ester of 2-hydroxy-2-(2′-hydroxyphenyl)acetic acid. Besides the ions resulting from losses of CH3O + H2 (m/z 149.0242, theoretical: 149.0239) and CO2CH3 (m/z 123.0458, theoretical: 123.0446), HR-MS data for D11M included ions at m/z 164.0448 (M+ -H2O, theoretical: 164.0473), 134.0351

FIGURE 4. HR-MS data and proposed structures of newly characterized HOBB-degradation metabolites labeled in Figure 3B. The identification of chemicals was shown in the text. (M+ -CH3O-OH, theoretical: 134.0368) and 105.0372 (M+ -CO2CH3-H2O, theoretical: 105.0340), matching the suggested structure.

D12 had a base peak ion at m/z 134.0379 (C8H6O2, M+, theoretical: 134.0368), and fragment ions at m/z 105.0338 (C7H5O, M+ -CHO, theoretical: 105.0340) and 76.0300 (C6H4, VOL. 43, NO. 22, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Proposed DBF degradation pathway by strain B6-2. The partial pathway enclosed in the solid lines has not been reported previously. The compounds enclosed in the dashed lines are proposed based on indirect evidence as described in the text. Chemical designations: D5, 2-oxo-4-(3′-oxobenzofuran-2′-yl)butanoic acid; D4, 2-hydroxy-4-(3′-oxobenzofuran-2′-yl)butanoic acid; D6, 3-(3′-oxobenzofuran-2′-yl)propanoic acid; D7, 3-hydroxy-3-(3′-oxobenzofuran-2′-yl)propanoic acid; D8, 2-(3′-oxobenzofuran-2′-yl)acetic acid; D9, 2-(3′-hydroxy-2′,3′-dihydrobenzofuran-2′-yl)acetic acid; D10, 3-hydroxy-2,3-dihydrobenzofuran-2-carboxylic acid; D11, 2-hydroxy-2-(2′-hydroxyphenyl)acetic acid; D12R, 2,3-dihydro-2,3-dihydroxybenzofuran; D13R, 2-oxo-2-(2′-hydroxyphenyl)acetic acid; D14, salicylic acid. M+ -CHO-CHO, theoretical: 76.0313) (SI Figure S2). The fragmentation pattern of D12 matched well with that of benzofuran-3(2H)-one in the NIST MS database. HR-MS analysis of D13 (SI Figure S2) revealed a molecular ion at m/z 148.0176 (C8H4O3, theoretical: 148.0160), a base peak ion at m/z 120.0211 (C7H4O2, M+ -CO, theoretical: 120.0211) and another important ion at m/z 92.0257 (C6H4O, M+ -CO-CO, theoretical: 92.0262). D13 was identified as benzofuran-2,3-dione by comparing its MS profile with those in the NIST MS database. D14M had a molecular ion at m/z 152.0488 (C8H8O3, theoretical: 152.0473) and fragment ions at m/z 120.0209 (C7H4O2, M+ -CH3O-H, theoretical: 120.0211) and 92.0252 (C6H4O, M+ -CO2CH3-H, theoretical: 92.0262) (SI Figure S2). It was identified as methylated salicylic acid by comparing its MS profile with those of the authentic compound and the compound in the NIST MS database. Further experimental result revealed that salicylic acid could support the growth of strain B6-2.

Discussion In the present study, the isolation and characterization of P. putida strain B6-2 that utilized BP as the sole source of carbon and energy are described. BP-grown B6-2 cells transformed DBF and HOBB quickly. Compared with the BP-grown cell suspension of Ralstonia sp. SBUG 290 (turbidity at 600 nm of 5) that cometabolically degraded DBF at a rate of up to 0.0029 mmol L-1 h-1 (11), the BP-grown B6-2 cell suspension (turbidity at 620 nm of 5) degraded DBF at a higher rate of 0.0083 mmol L-1 h-1 (Figure 1). DBF could also be degraded cometabolically by B6-2 growing with BP as a primary substrate. The presence of DBF somewhat inhibited B6-2 cell growth (Figure 2), probably due to the toxic properties of DBF and their metabolites. Degradation of DBF and HOBB by LB medium-cultivated B6-2 cells and cell growth with 8640

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DBF and HOBB were not detected, which seemed to be a common phenomenon in the case of DBF degradation by BP-utilizing strains (11, 12). Our further experimental results indicate that the presence of 10 g/L of either glucose or citric acid in MSM can greatly repress the BP (15 mmol/L) degradation rate but not the B6-2 cell growth rate (data not shown). This may be due to the so-called catabolic repression frequently occurring during microbial degradation of xenobiotics that are not preferred growth substrates (33-35). BphA1s are responsible for recognition and binding of substrates. Therefore, they are crucial for the ranges of aromatic compounds catalyzed by BphAs (36, 37). The sequence similarities of BphA1s among B6-2, LB400 and KF707 are 95% (LB400 and KF707), 92% (B6-2 and KF707), and 90% (B6-2 and LB400). The majorities of the different amino acids are concentrated between 234 and 377 (SI Figure S6), where a few amino acid exchanges can largely change the ranges of substrates and their oxygenation sites by BphAs (31, 36). It may be these differences that lead to the dioxygenation of DBF at the lateral and angular positions by BphA of LB400 (14, 17), the dioxygenation at the lateral but not the angular position by BphA of B6-2, and inability of KF707 to attack DBF (31). Using HOBB as the substrate for B6-2, we detected a series of metabolites. According to the analytical results, a pathway of DBF degradation by strain B6-2 is proposed in Figure 5. D5 and D4 may be derived from HOBB through two sequential double-bond hydrogenation steps. Analogous reactions have been reported during carbazole and BP metabolism by Pseudomonas cepacia strain F297 (15) and DBF metabolism by Sphingomonas sp. strain HH69 (5). In the later case, a side chain reduction product 3-(4′-oxochroman-2′-yl)lactic acid was further transformed to 2-(4′oxochroman-2′-yl)acetic acid (5). In our experiment, D4 is

proposed to undergo similar reactions including decarboxylation and oxidation to produce D6. As transformation products of D6, D7, and D8 would represent metabolites involved in the beta- and alpha-oxidation pathways, respectively. D9 may be the hydrogenative reduction product of D8. D7 may be transformed to D10 via an undetected beta-oxidation product 3-oxobenzofuran-2-carboxylic acid to which a similar hydrogenative reduction may have happened. The chain-reducing and shortening reactions have also been described in the formation of several chromone derivatives from DBF via 3-(4′-oxochroman-2′-yl)pyruvic acid (5) and in the cases of 4-chlorobiphenyl (38) and carbazole metabolisms (39). Although the alpha-oxidation pathway was reported most frequently in the degradation of branched chain fatty acids, such as phytanic acid (40), sequential alphaoxidation of the unbranched side chains of 1-phenyldodecane and 1-phenylnonane by Nocardia salmonicolor has also been postulated (41). Because D9 was a minor metabolite and the subsequent transformation was not studied, we could not determine whether it was also transformed by the alphaoxidation route to D10. On the other hand, although it had not been proved, it was considered that hydrolyzation was an important reaction for HOBB transformation (13). In our study, we could not exclude the possibility that a small portion of HOBB was transformed to D10 via reactions including the release of pyruvate by a hydrolase of strain B6-2, just like the production of 3-hydroxy-2,3-dihydrobenzothiophene-2-carboxylic acid from the ring-cleavage product of dibenzothiophene by strain F297 (15). The structurally similar compounds of D11, D12, and D13 in the benzothiophene degradation culture incubated with P. putida strain RE204 have been identified (42). By comparing with the benzothiophene degradation route, we suggest that D10 is decarboxylated and oxygenated to 2,3-dihydro2,3-dihydroxybenzofuran (D12R, Figure 5). D12R was not detected, and was proposed to easily undergo abiotic dehydration to produce D12 during sample preparation. D12R is proposed to be transformed via dehydrogenation, ring open, and oxygenation to 2-oxo-2-(2′-hydroxyphenyl)acetic acid (D13R, Figure 5). D13R was not detected in our study either and was shown indirectly by the identification of its abiotic transformation product D13. D13R may be reduced to D11 and subsequently transformed to salicylic acid (D14) in a similar way with the sequential transformation of D5 to D4 and D6. Because B6-2 was capable of growing with salicylic acid as the sole carbon source, partial mineralization of DBF by B6-2 was conceivable. These newly characterized DBF metabolites may have potential biological activities and toxicities. Therefore, much concern should be paid to this new pathway from both biotechnological and environmental views.

Acknowledgments We gratefully acknowledge financial support from National Natural Science Foundation of China (Grant Nos. 20977061 and 30821005) and National High Technology Research and Development Program of China (Grant Nos. 2007AA061101 and 2007AA10Z401). We also acknowledge the financial support from Key Basic Research Program of Shanghai (Grant No. 09JC1407700). X.W. and G.Y. contributed equally to this work.

Supporting Information Available Tables showing the utilization of carbons by strain B6-2 (Table S1) and its physical and biochemical characteristics (Table S2), and Figures showing portions of the HPLC profiles of DBF degradation samples (Figure S1), the HR-MS data of dimethylated HOBB, D12, D13, and D14M (Figure S2), the UV-vis spectra of 1-hydroxydibenzofuran and 4-hydroxydibenzofuran (Figure S3), the HPLC profiles of HOBB

degradation samples (Figure S4), portions of the total ion current chromatograms from HR-MS analyses of the diazomethane derivatized HOBB-degradation sample and control sample (Figure S5), and comparison of the large subunits of terminal dioxygenases among KF707, LB400, and B6-2 (Figure S6) are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Cerniglia, C. E.; Morgan, J. C.; Gibson, D. T. Bacterial and fungal oxidation of dibenzofuran. Biochem. J. 1979, 180, 175–185. (2) Yunker, M. B.; Cretney, W. J.; Ikonomou, M. G. Assessment of chlorinated dibenzo-p-dioxin and dibenzofuran trends in sediment and crab hepatopancreas from pulp mill and harbor sites using multivariate- and index-based approaches. Environ. Sci. Technol. 2002, 36, 1869–1878. (3) Peek, D. C.; Butcher, M. K.; Shields, W. J.; Yost, L. J.; Maloy, J. A. Discrimination of aerial deposition sources of polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran downwind from a pulp mill near Ketchikan, Alaska. Environ. Sci. Technol. 2002, 36, 1671–1675. (4) Gai, Z. H.; Yu, B.; Li, L.; Wang, Y.; Ma, C. Q.; Feng, J. H.; Deng, Z. X.; Xu, P. Cometabolic degradation of dibenzofuran and dibenzothiophene by a newly isolated carbazole-degrading Sphingomonas sp. strain. Appl. Environ. Microbiol. 2007, 73, 2832–2838. (5) Fortnagel, P.; Harm, H.; Wittich, R. M.; Krohn, S.; Meyer, H.; Sinnwell, V.; Wilkes, H.; Francke, W. Metabolism of dibenzofuran by Pseudomonas sp. strain HH69 and the mixed culture HH27. Appl. Environ. Miocrobiol. 1990, 56, 1148–1156. (6) Harms, H.; Wilkes, H.; Wittich, R.; Fortnagel, P. Metabolism of hydroxydibenzofurans, methoxydibenzofurans, acetoxydibenzofurans, and nitrodibenzofurans by Sphingomonas sp. strain HH69. Appl. Environ. Microbiol. 1995, 61, 2499–2505. (7) Wilkes, H.; Wittich, R.; Timmis, K. N.; Fortnagel, P.; Francke, W. Degradation of chlorinated dibenzofurans and dibenzo-pdioxins by Sphingomonas sp. strain RW1. Appl. Environ. Microbiol. 1996, 62, 367–371. (8) Schmid, A.; Rothe, B.; Altenbuchner, J.; Ludwig, W.; Engesser, K. H. Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360. J. Bacteriol. 1997, 179, 53–62. (9) Nojiri, H.; Kamakura, M.; Urata, M.; Tanaka, T.; Chung, J. S.; Takemura, T.; Yoshida, T.; Habe, H.; Omori, T. Dioxin catabolic genes are dispersed on the Terrabacter sp. DBF63 genome. Biochem. Biophys. Res. Commun. 2002, 296, 233–240. (10) Aly, H. A.; Huu, N. B.; Wray, V.; Junca, H.; Pieper, D. H. Two angular dioxygenases contribute to the metabolic versatility of dibenzofuran-degrading Rhodococcus sp. strain HA01. Appl. Environ. Microbiol. 2008, 74, 3812–3822. (11) Becher, D.; Specht, M.; Hammer, E.; Francke, W.; Schauer, F. Cometabolic degradation of dibenzofuran by biphenyl-cultivated Ralstonia sp. strain SBUG 290. Appl. Environ. Microbiol. 2000, 66, 4528–4531. (12) Stope, M. B.; Becher, D.; Hammer, E.; Schauer, F. Cometabolic ring fission of dibenzofuran by gram-negative and gram-positive biphenyl-utilizing bacteria. Appl. Microbiol. Biotechnol. 2002, 59, 62–67. (13) Wesche, J.; Hammer, E.; Becher, D.; Burchhardt, G.; Schauer, F. The bphC gene-encoded 2,3-dihydroxybiphenyl-1,2-dioxygenase is involved in complete degradation of dibenzofuran by the biphenyl-degrading bacterium Ralstonia sp. SBUG 290. J. Appl. Microbiol. 2005, 98, 635–645. (14) Seeger, M.; Camara, B.; Hofer, B. Dehalogenation, denitration, dehydroxylation, and angular attack on substituted biphenyls and related compounds by a biphenyl dioxygenase. J. Bacteriol. 2001, 183, 3548–3555. (15) Grifoll, M.; Selifonov, S. A.; Gatlin, C. V.; Chapman, P. J. Actions of a versatile fluorene-degrading bacterial isolate on polycyclic aromatic compounds. Appl. Environ. Microbiol. 1995, 61, 3711– 3723. (16) Selifonov, S. A.; Slepen’kin, A. V.; Adanin, V. M.; Nefedova, M.; Starovoitov, I. I. Oxidation of dibenzofuran by Pseudomonas strains harboring plasmids of naphthalene degradation. Mikrobiologiia. 1991, 60, 67–71. (17) Mohammadi, M.; Sylvestre, M. Resolving the profile of metabolites generated during oxidation of dibenzofuran and chlorodibenzofurans by the biphenyl catabolic pathway enzymes. Chem. Biol. 2005, 12, 835–846. VOL. 43, NO. 22, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

8641

(18) Debruyn, J. M.; Sayler, G. S. Microbial community structure and biodegradation activity of particle-associated bacteria in a coal tar contaminated creek. Environ. Sci. Technol. 2009, 43, 3047–3053. (19) Johnsen, A. R.; de Lipthay, J. R.; Reichenberg, F.; Sorensen, S. J.; Andersen, O.; Christensen, P.; Binderup, M. L.; Jacobsen, C. S. Biodegradation, bioaccessibility, and genotoxicity of diffuse polycyclic aromatic hydrocarbon (PAH) pollution at a motorway site. Environ. Sci. Technol. 2006, 40, 3293–3298. (20) Ono, M.; Kawashima, H.; Nonaka, A.; Kawai, T.; Haratake, M.; Mori, H.; Kung, M. P.; Kung, H. F.; Saji, H.; Nakayama, M. Novel benzofuran derivatives for PET imaging of beta-amyloid plaques in Alzheimer’s disease brains. J. Med. Chem. 2006, 49, 2725– 2730. (21) Boschelli, D. H.; Kramer, J. B.; Khatana, S. S.; Sorenson, R. J.; Connor, D. T.; Ferin, M. A.; Wright, C. D.; Lesch, M. E.; Imre, K.; Okonkwo, G. C. Inhibition of E-selectin-, ICAM-1-, and VCAM-1-mediated cell adhesion by benzo[b]thiophene-, benzofuran-, indole-, and naphthalene-2-carboxamides: identification of PD 144795 as an antiinflammatory agent. J. Med. Chem. 1995, 38, 4597–4614. (22) Kadieva, M. G.; Oganesyan, E. T. Methods for the synthesis of benzofuran derivatives (review). Chem. Heterocycl. Compd. 1997, 33, 1245–1258. (23) NTP toxicology and carcinogenesis studies of benzofuran (CAS No. 271-89-6) in F344/N rats and B6C3F1 mice (gavage studies). Natl. Toxicol. Program. Tech. Rep. Ser. 1989, 370, 1189. (24) Li, L.; Li, Q.; Li, F.; Shi, Q.; Yu, B.; Liu, F.; Xu, P. Degradation of carbazole and its derivatives by a Pseudomonas sp. Appl. Microbiol. Biotechnol. 2006, 73, 941–948. (25) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403– 410. (26) Durfee, T.; Nelson, R.; Baldwin, S.; Plunkett, G, 3rd; Burland, V.; Mau, B.; Petrosino, J. F.; Qin, X.; Muzny, D. M.; Ayele, M. The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse. J. Bacteriol. 2008, 190, 2597–2606. (27) Molecular Cloning: A Laboratory Manual, 3rd, ed.; Sambrook, J., Russell, D. W., Eds.; Cold Spring Harbor Laboratory Press: New York, 2001. (28) Lee, N. R.; On, H. Y.; Jeong, M. S.; Kim, C. K.; Park, Y. K.; Ka, J. O.; Min, K. H. Characterization of biphenyl biodegradation, and regulation of biphenyl catabolism in Alcaligenes xylosoxydans. J. Microbiol. 1997, 35, 141–148. (29) Arensdorf, J. J.; Focht, D. D. A meta cleavage pathway for 4-chlorobenzoate, an intermediate in the metabolism of 4-chlorobiphenyl by Pseudomonas cepacia P166. Appl. Environ. Microbiol. 1995, 61, 443–447.

8642

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 22, 2009

(30) Taira, K.; Hirose, J.; Hayashida, S.; Furukawa, K. Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707. J. Biol. Chem. 1992, 267, 4844–4853. (31) Furukawa, K.; Suenaga, H.; Goto, M. Biphenyl dioxygenases: functional versatilities and directed evolution. J. Bacteriol. 2004, 186, 5189–5196. (32) Hammer, E.; Krowas, D.; Schafer, A.; Specht, M.; Francke, W.; Schauer, F. Isolation and characterization of a dibenzofurandegrading yeast: identification of oxidation and ring cleavage products. Appl. Environ. Microbiol. 1998, 64, 2215–2219. (33) Duetz, W. A.; Marques, S.; de Jong, C.; Ramos, J. L.; van Andel, J. G. Inducibility of the TOL catabolic pathway in Pseudomonas putida (pWW0) growing on succinate in continuous culture: evidence of carbon catabolite repression control. J. Bacteriol. 1994, 176, 2354–2361. (34) Dinamarca, M. A.; Aranda-Olmedo, I.; Puyet, A.; Rojo, F. Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures. J. Bacteriol. 2003, 185, 4772–4778. (35) Moreno, R.; Rojo, F. The target for the Pseudomonas putida Crc global regulator in the benzoate degradation pathway is the BenR transcriptional regulator. J. Bacteriol. 2008, 190, 1539– 1545. (36) Kumamaru, T.; Suenaga, H.; Mitsuoka, M.; Watanabe, T.; Furukawa, K. Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase. Nat. Biotechnol. 1998, 16, 663–666. (37) Mondello, F. J.; Turcich, M. P.; Lobos, J. H.; Erickson, B. D. Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation. Appl. Environ. Microbiol. 1997, 63, 3096– 3103. (38) Masse, R.; Messier, F.; Peloquin, L.; Ayotte, C.; Sylvestre, M. Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls. Appl. Environ. Microbiol. 1984, 47, 947–951. (39) Gieg, L. M.; Otter, A.; Fedorak, P. M. Carbazole degradation by Pseudomonas sp. LD2: metabolic characteristics and the identification of some metabolites. Environ. Sci. Technol. 1996, 30, 575–585. (40) Jansen, G. A.; Wanders, R. J. Alpha-oxidation. Biochim. Biophys. Acta 2006, 1763, 1403–1412. (41) Sariaslani, F. S.; Harper, D. B.; Higgins, I. J. Microbial degradation of hydrocarbons. Catabolism of 1-phenylalkanes by Nocardia salmonicolor. Biochem. J. 1974, 140, 31–45. (42) Eaton, R. W.; Nitterauer, J. D. Biotransformation of benzothiophene by isopropylbenzene-degrading bacteria. J. Bacteriol. 1994, 176, 3992–4002.

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