strain F274. degradation of fluorene by

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temperature-controlled cuvette containing 1 ml of K-Na phos- phate buffer (50 mM; pH ...... 27:1538-1542. 40. Wackett, L. P., L. D. Kwart, and D. T. Gibson. 1988.

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274. M Grifoll, S A Selifonov and P J Chapman Appl. Environ. Microbiol. 1994, 60(7):2438.

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JUIY 1994, p. 2438-2449 0099-2240/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Vol. 60, No. 7

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,

Evidence for a Novel Pathway in the Degradation of Fluorene by Pseudomonas sp. Strain F274t M. GRIFOLL,I* S. A. SELIFONOV,22t AND P. J. CHAPMAN3 Department of Microbiology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain1; Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, Florida 325142; and Environmental Research Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Florida 325613 Received 9 December 1993/Accepted 13 April 1994

Polycyclic aromatic hydrocarbons (PAHs) represent a chemical class of widespread priority pollutants that includes a variety of toxic and genotoxic compounds (13, 20, 22). PAHs are introduced into the environment mainly from coal gasification or liquefaction processes and from contamination associated with the transport, transformation, and use of fossil fuels and derivatives. Microbial degradation represents the primary route of transformation and removal of PAHs in the environment, and exploitation of this natural process in the remediation of contaminated sites has been presented as a promising alternative to physicochemical treatments (27). Fundamental to a comprehension of the environmental fate of chemicals and the development and effective application of bioremediative technologies is an understanding of processes used by bacteria to degrade these chemicals singly and in mixtures. While there exists a large body of literature documenting the reactions employed by bacteria which utilize and transform aromatic hydrocarbons such as naphthalene, biphenyl, and phenanthrene (6, 17), few studies of the degradation of naphthenoaromatic hydrocarbons (compounds containing both aromatic and methylenic moieties) have been done (3, 18, 33, 36). Consequently, the biochemical mechanisms by which they can be transformed and eventually mineralized are not well understood.

Recent work has pointed out that naphthenoaromatic compounds are substrates for arene-dioxygenases, which either dioxygenate the aromatic moiety, giving the corresponding cis-dihydrodiols, or catalyze monooxygenation of the benzylic methylenic groups to alcohols that are further dehydrogenated by nonspecific dehydrogenases to produce ketones (4, 33, 34, 36, 40). The role that this monooxygenating activity plays in the biodegradation of naphthenoaromatics in nature is still obscure.

Fluorene is one of the simplest naphthenoaromatic compounds, possessing two aromatic rings with a benzylic methylenic group in a central five-membered ring. It is a major component of fossil fuels and derivatives (about 7.6% of creosote PAHs [18a]) and, together with fluorenone and a number of its alkyl derivatives, has been detected in vehicle exhaust emissions, air particulates, and riverine and marine sediments (13, 20, 23). In addition, the chemical structure of fluorene bears obvious structural relationships to other pollutants of concern (carbazoles, dibenzothiophenes, dibenzofurans, and dibenzodioxins and their halogenated derivatives). Until now, few studies of the biodegradation of fluorene have been reported. An earlier report (18) suggested that the biodegradation of fluorene by an Arthrobacter sp. may proceed by two different pathways, one via monooxygenation at C-9 producing 9-fluorenone as a dead-end metabolite and the other involving dioxygenation and meta cleavage of one of the aromatic rings, with the possible involvement of a biological Baeyer-Villiger-type reaction responsible for dihydrocoumarin formation. A recent publication (30) reports biotransformation of fluorene by the fungus Cunninghamella elegans via monooxygenation at C-9 to give first fluorenol and then 9-fluorenone, with further monooxygenation to 2-hydroxyfluorenone. Other studies of fluorene transformation have com-

* Corresponding author. Mailing address: Department of Microbiology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain.

Phone: (343) 402-1482. Fax: (343) 411-0592. Electronic mail address:

[email protected] t Contribution no. 873 from the Environmental Research Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Fla. t Present address: Department of Biochemistry, University of Minnesota, St. Paul, Minn.

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A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,ladihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, l,la-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.

DEGRADATION OF FLUORENE BY PSEUDOMONAS STRAIN F274

VOL. 60, 1994

MATERIALS AND METHODS

Chemicals. PAHs and derivatives used in this study were purchased from Aldrich Chemical Co., Inc., Milwaukee, Wis. Diazomethane was generated by alkaline decomposition of Diazald (N-methyl-N-nitroso-p-toluenesulfonamide) (1). 3,4and 4,5-dihydroxyphthalic acids were kind gifts from R. W. Eaton (Environmental Research Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Fla.) (11). Media and reagents were purchased from Difco Laboratories, Detroit, Mich. Solvents were obtained from J. T. Baker, Inc. All chemicals and solvents were of the highest purity available. Media and supply of PAHs. The mineral salts medium used throughout this study was described previously (21). The pH was adjusted to 7.2 and the medium was sterilized prior to the addition of organic substrates. Solid medium was prepared with 1.5% agar. Water-soluble substrates were added at a final concentration of 5 mM. PAHs and analogs were added to liquid medium or phosphate buffer (cell suspensions) as crystals in excess of their aqueous solubility (1 g/liter), and the mixtures were sonicated for 20 min prior to inoculation. An exception was made in the experiments demonstrating growth at the expense of fluorene, in which this compound was added to sterile liquid medium in acetone solution (2%) to give a final concentration of 4 mM. Flasks were shaken at 200 rpm at 30°C for 24 h before inoculation to allow acetone removal. Characterization of fluorene-degrading strain F274. Strain F274 was isolated in this laboratory by means of enrichment cultures established in fluorene mineral salts medium inoculated with soil highly contaminated with creosote (American Creosote Works, Pensacola, Fla.). The isolate grew in solid mineral medium with fluorene as the sole source of carbon and energy. Bacteriological, growth, and biochemical tests were performed by standard methods (16). Growth substrates were assayed at a 5 mM concentration in mineral medium except for testosterone, which was supplied in excess (1 g/liter). The fatty acid methyl ester analysis of the lipids of this strain was performed by MIDI Laboratories (Newark, Del.) with cells grown for 24 h on Trypticase soy broth solidified with agar. Utilization of fluorene as a sole source of carbon and energy.

Growth at the expense of fluorene was established by demonstrating an increase in bacterial protein with a concomitant decrease in the concentration of fluorene. Fluorene utilization and accumulating metabolites were demonstrated by examination of extracts of the culture medium by high-pressure liquid chromatography (HPLC). A culture grown for 24 h in Luria-Bertani (LB) medium supplemented with 2 g of glucose per liter was used as the inoculum (1%). Replicate batch cultures were grown in 125-ml Erlenmeyer flasks containing 25 ml of mineral medium and fluorene (4 mM). Incubation was performed at 23°C with rotatory shaking (200 rpm). Uninoculated flasks and inoculated flasks without fluorene served as controls. Fluorene concentration, accumulation of metabolites, and protein concentration were determined at 24-h intervals over 9 days. Controls were analyzed at 4-day intervals. The protein concentration was determined for duplicate 1-ml samples by the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, Ill.) following treatment with 0.1 N NaOH at 70°C for 60 min. To assess fluorene disappearance and metabolite formation, the entire flask contents were extracted with ethyl acetate and neutral and acidic extracts were analyzed by HPLC as described below. Utilization of other aromatic compounds as sources of carbon and energy. Growth on aromatic compounds was screened with solid mineral medium. Phthalate, salicylate, dihydrocoumarin, p-hydroxybenzoate, and 2,2'-biphenol were supplied at 5 mM. 9-Fluorenone, naphthalene, biphenyl, anthracene, phenanthrene, acenaphthene, dihydroanthracene, dibenzofuran, dibenzothiophene, carbazole, fluoranthene, and pyrene were supplied by spraying the surface of inoculated mineral medium plates with a solution of the substrate in ethanol-free diethyl ether (5%) (24). Benzene, toluene, naphthalene, and biphenyl were supplied by incubating the plates exposed to vapors of the substrate under a bell jar. Growth was evidenced by a significant increase in bacterial mass on test plates compared with that on control plates incubated in the absence of substrates and, in the case of sprayed plates, also by clearing of the insoluble substrate adjacent to the inoculated cell mass. Plates were incubated at 23°C for 8 weeks. Results were confirmed by inoculating F274 into 125-ml flasks containing 25 ml of mineral medium with individual substrates supplied as described above. Cultures were analyzed for evidence of increases in protein content at 0, 15, and 30 days by the bicinchoninic acid method. Growth and harvesting of fluorene-induced cells. For smallscale experiments, cells were grown in 2,000-ml Erlenmeyer flasks containing, in 400 ml of mineral salts medium, L-arginine (5 mM) and fluorene crystals (1 g/liter). Incubation was carried out on a rotatory shaker at 23°C and 200 rpm. For preparative isolation and identification of metabolites, cells were grown in 13-liter bottles (Pyrex; Fisher catalog no. 02-887B) containing 8 liters of the same medium. Aeration and mixing were done by using magnetic stirring bars (2.5 by 10 cm) (Bel-Art) at approximately 300 rpm driven by magnetic stirrers (Thermolyne type 25500 Maxi-Stirrer) at room temperature (22°C). When late exponential phase was reached (approximately 48 h; A650 = 0.7), remaining fluorene crystals were removed by filtration through sterile glass wool and the cultures were incubated for an additional 30 min in order to reduce any remaining fluorene and metabolites. Cells were harvested by centrifugation, washed twice with 50 mM Na-K buffer (pH 7.2), and resuspended in a reduced volume of the same buffer. Washed-cell incubations in the presence of fluorene and analogs. Fluorene or other substrates were added (1 g/liter) either to 250-ml Erlenmeyer flasks containing 50 ml of buffer

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plemented work on degradation of biaryl ether structures. Engesser et al. (12) reported transformation of fluorene by a dibenzofuran-utilizing Brevibacterium strain (DPO 1361) via monooxygenation at position C-9 followed by angular dioxygenation at the 1,1a position. Recently, Monna et al. (26) suggested similar reactions by Staphylococcus auriculans DBF63, which was isolated for its ability to grow with dibenzofuran, suggesting both 1- and 4-hydroxyfluorenones as deadend products. We propose here a metabolic pathway of degradation of fluorene by a fluorene-degrading strain of Pseudomonas, on the basis of identification of ring oxidation and ring cleavage metabolites and the demonstration of key enzyme activities. Initial oxidation of the methylenic group (C-9) gives 9-fluorenone, which subsequently undergoes angular dioxygenation at the 1,1a position as shown by accumulation of 1,la-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4benzocoumarin suggests steps in the further catabolism of this compound which involve novel cleavage of the five-membered ring to form a substituted biphenyl, its conversion to phthalate, and further metabolism via 4,5-dihydroxyphthalate, protocatechuate, and 3-carboxy-cis,cis-muconate. Identification of products formed from several fluorene analogs gives additional support for the initial oxidation steps of the pathway.

2439

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column (25 m by 0.32 mm [inside diameter]) with 0.25-,um film thickness and helium as the carrier gas (linear velocity, 23 cm/s). The column temperature was held isothermally at 50°C for 1 min and then programmed to 290°C at a rate of 10°C/min. The mass spectrometer was operated at 70 eV of electron ionization energy. Injector, transfer line, and analyzer temperatures were set at 270, 300, and 300°C, respectively. NMR spectra (both 'H and `3C) were recorded on a GE QE 300 Plus NMR spectrometer at 300.56 and 75.6 MHz, respectively. Compounds were dissolved in CDCl3. Tetramethylsilane and the central signal of solvent at 77.0 ppm were used as references for 'H and '3C spectra, respectively. Preparation of cell extracts and demonstration of enzyme activities. Fluorene-induced cells were washed and resuspended in 50 mM phosphate buffer (pH 7.0) to give approximately 0.5 g (wet weight) of cells per ml. Cell extracts were prepared by passing the suspension twice through a French pressure cell at 14,000 to 20,000 lb/in2. Extracts were subjected to centrifugation at 47,800 x g for 40 min at 4°C, and the resultant clear supernatant was separated from pelleted cells and debris. Typically such extracts contained 7 mg of protein per ml. Cell extracts were examined for the presence of the following enzymes: 4,5-dihydroxyphthalate decarboxylase, 3,4-dihydroxyphthalate decarboxylase, protocatechuate 4,5-dioxygenase, protocatechuate 3,4-dioxygenase, and protocatechuate 2,3-dioxygenase. Assays were performed by monitoring disappearance of substrates and appearance of products by UVvisible spectrometry, on the basis of spectral properties reported by others for these enzyme systems (8, 11, 29, 31). Spectral changes at 30°C were recorded with a Perkin-Elmer Lambda 6 spectrophotometer. Reactions were monitored in a temperature-controlled cuvette containing 1 ml of K-Na phosphate buffer (50 mM; pH 7.0), 20 ,ul of cell extract, and 8 ,lI of 25 mM substrate (0.2 ,uM). Substrate was omitted from reference reaction mixtures. Protocatechuate dioxygenase activity was also examined by using extracts of cells grown with p-hydroxybenzoate. RESULTS

Taxonomic characterization of F274. Growth of strain F274 on solidified mineral medium, with fluorene supplied as crystals on the lid of the plate, was evident after 10 days at 23°C as characteristic pink mucoid colonies (1 mm in diameter), while on LB solid medium (2% glucose) beige mucoid colonies of about the same size were formed within 72 h. The isolate was a rod-shaped gram-negative bacterium, aerobic, motile, and nonfermentative. Fluorescent pigments were not produced in Bacto Pseudomonas agar F medium (Difco Laboratories), and cells grown on p-hydroxybenzoate showed ortho cleavage of protocatechuate. These and other physiological properties are listed in Table 1. The GC profile obtained from fatty acid methyl ester analysis of the total lipids of the strain showed the following composition: 14:0 (1.42%?, 15:0 (1.21%), 14:0 20H (9.24%), 16:19 (11.40%), 16:1' 1 (2.82%), 16:0 (12.12%), 17:1A9 (0.67%), 17:1All (4.46%), 18:1l' (55.66%), and 19:0oCP 11.12 (1.01%). No matches were found with any of the species assembled in the data base (MIDI Laboratories). Pseudomonas saccharophila and Sphingomonas capsulata had the most similar profiles. P. saccharophila is the only species of Pseudomonas possessing 2-hydroxymyristic acid (14:0 20H) as a major component. Pseudomonas picketii and Pseudomonas rubrisubalbicans also possess low concentrations of this fatty acid, but they differ considerably in others. On the other hand, the presence of

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(small-scale experiments) or to 2,000-ml Erlenmeyer flasks containing 400 ml of buffer, and the mixture was sonicated as described above. Fluorene-induced cells were added to give a suspension with an A650 of approximately 2.5. Flasks were placed in a rotatory shaker at 23°C and 200 rpm and incubated for 18 h. This time of incubation was chosen after monitoring the kinetics of accumulation of metabolites in small-scale experiments with incubations of up to 36 h. Controls without cells were included. At 0, 1, 3, 6, 18, and 36 h, 1-ml samples were removed, cells and particulate material were removed by centrifugation, and supernatants were analyzed by HPLC. The results obtained with these experiments indicated that the maximum accumulation of metabolites occurred at 18 h. Isolation and identification of metabolites. Washed-cell suspensions were centrifuged to remove cells and remaining undissolved fluorene (or analogs), and sodium chloride (5%, wt/vol) was added to the supernatant to improve extraction of metabolites. Supernatants were extracted three times with 1 volume of ethyl acetate, acidified to pH 2.5 with 6 N HCl, and then extracted again in the same manner. Extracts were dried over anhydrous Na2SO4 and concentrated by rotatory evaporation. Portions were treated with an excess of ethereal diazomethane, and the solvent was removed. For initial detection and characterization of metabolites, aliquots of residual neutral, methylated neutral, and methylated acidic extracts were redissolved in methylene chloride and analyzed by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). Replicate aliquots were dried, dissolved in methanol, and analyzed by HPLC. Neutral metabolites from fluorene were isolated by column liquid chromatography. Portions of a concentrated neutral extract (140 mg) were applied to an unactivated silica (J. T. Baker, Inc., no. 3405-01; 60/200-mesh) column (1 by 17 cm) preequilibrated with methylene chloride. The column was eluted successively with 100 ml of CH2Cl2, 100 ml of CH2Cl2ethyl acetate (9:1), and 100 ml of ethyl acetate. Elution of metabolites was monitored by thin-layer chromatography (TLC) (silica gel 60 F-254; 0.25-mm layer thickness; Merck), using either CH2Cl2 or ethyl acetate as the developing solvent. Appropriate fractions containing products were combined, concentrated, and dried, and after being weighed they were analyzed by HPLC and GC-MS. Acidic extracts contained only one major metabolite, which was characterized by the techniques described below. When authentic samples were available, metabolites were identified by comparing their UVvisible absorption spectra, mass spectra, and chromatographic properties (GC, HPLC, and TLC) with those observed for authentic compounds. When authentic materials were not available, identification was confirmed by nuclear magnetic resonance (NMR) analysis. Metabolites formed from structural analogs of fluorene were identified directly by HPLC and GC-MS analysis of whole neutral extracts and verified by coinjection of authentic samples. When authentic compounds were not available, structures were suggested by MS fragmentation patterns. Analytical methods. Reversed-phase HPLC was performed with a Hewlett-Packard model 1090 chromatograph equipped with a diode array UV-visible detector set at 207 nm. Separation was achieved on a C18 octyldecyl silane Hypersil column (10 cm by 2.1 mm; 5-,um particle size) with a linear gradient of acetonitrile (10 to 79% [vol/vol] in 23 min) in 50 mM KH2PO4 (pH 3.5). The injection volume was 10 ,Il. GC-MS analyses were performed with a Hewlett-Packard 5890 series II gas chromatograph with a 5971 mass selective detector. Compounds were separated on an HP-5 capillary

APPL. ENvIRON. MICROBIOL.

TION OF FLUORENE BY PSEUDOMONAS STRAIN F274

TABLE 1. Morphological and biochemical characteristics of Pseudomonas sp. strain F274

cis-octadecenoic and 2-hydroxymyristic acids as major components is characteristic of the genus Sphingomonas (39, 43). However, the biochemical characteristics of strain F274 (Table 1) do not allow assignment of this organism to the genus Sphingomonas. According to these results, strain F274 has been tentatively identified as a species of Pseudomonas. Utilization of fluorene. Utilization of fluorene as a sole source of carbon and energy by Pseudomonas sp. strain F274 was confirmed by its complete removal from fluorene-mineral salts medium, with a corresponding increase in bacterial protein (Fig. 1). After a 24-h lag phase, the concentration of fluorene recovered from cultures decreased slightly during the second day and dramatically over the next 3 days (from 4 to 0.15 mM). Concomitantly, bacterial biomass increased from 0.47 to 330 pug of protein per ml. These values indicate a conversion of approximately 670 ,ug of fluorene to 660 ,ug (dry weight) of cells (assuming protein to be about 50% of cell dry weight) (28). Given that cell dry weight is approximately 50% carbon (28), there appears to be cellular assimilation of fluorene with an efficiency of about 50%. The generation time during exponential growth was estimated to be about 9 h. During the stationary phase, fluorene decreased slowly to levels no longer detectable by HPLC. During growth, an acidic metabolite (VII) was observed in the culture medium. This metabolite was later identified as phthalic acid (see below) when its concentration was estimated at 12.2 nmol/ml at the end of the exponential phase (approximately 0.1% of the initial concentration of fluorene). No significant changes were observed in either fluorene concentration or biomass in controls. Growth on other aromatic compounds. In addition to utilizing fluorene, strain F274 was shown to grow with 9-fluorenone. Other aromatic compounds supporting growth were p-hydroxybenzoate and phenylacetate. Growth (or clearing, in the case of insoluble substrates) was not observed with benzene, toluene, naphthalene, biphenyl, acenaphthene, dihydroanthracene, anthracene, phenanthrene, fluoranthene, pyrene, diben-

Result

Characteristic

Bacteriological tests Gram ...........................Motility ............................+ Morphology ........................... Rods

Production of fluorescent pigments ............................Growth at 41°C ............................Growth tests

D-Glucose ................................L-Arabinose ...............................D-Mannose ..................................D-Mannitol ................................N-Acetylglucosamine ...........................Maltose ...........................D-Fructose ................................D-Gluconate ................................-

Caprate ............................Adipate ...........................L-Malate ...............................Citrate ...........................Phenyl acetate ............................+ p-Hydroxybenzoate ............................+ L-Arginine ................................+ Acetate ............................+ Testosterone ............................+ Biochemical tests Oxidase ...........................'+ .+ Nitrate reduction Denitrification .-

Arginine dihydrolase .-

Urease .Esculin hydrolysis .Gelatinase ..-

ortho cleavage of protocatechuate

.+

zofuran, dibenzothiophene, xanthene, carbazole, dihydrocoumarin, phthalate, 2,2'-biphenol, or salicylate. Kinetics and identification of fluorene metabolites. Analysis (HPLC) of supernatants removed at different times from incubations of washed cells with fluorene (I) showed the

a Cells grown in p-hydroxybenzoate.

- 50 so

400

5 A

0 CD

-0-X_

30

-

-

2

200

L.-

-

(a00

CD

=

40

-

300 -X

o

2441

_.)

m

0

_.

-20 :3

2

c_

lL

.100

Ir-

1

L

0

1

2

3

4

a

6

7

a

9

10

Days FIG. 1. Utilization of fluorene by Pseudomonas sp. strain F274 and accumulation of phthalic acid during growth in liquid mineral medium with fluorene as the sole source of carbon and energy at 23°C and 200 rpm. Growth is shown as an increase in cell protein (O). Fluorene (A) and phthalic acid (*) concentrations were determined by HPLC analyses of organic extracts from the cultures.

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,B-Galctoside

VOL. 60, 1994

2442

APPL. ENVIRON. MICROBIOL.

GRIFOLL ET AL.

presence of six major metabolites (II to VII) (Fig. 2). After 1 h of incubation, metabolites II (RI, 11.5 min) and III (R, 14.7 min) were major peaks, but they were not detected in later samples taken at 3, 6, 18, and 36 h. These transient metabolites presented exactly the same retention times and UV-visible spectra as those of authentic 9-fluorenol and 9-fluorenone,

respectively. The concentration of metabolite IV (R1, 5.8 min) increased to a maximum at 18 h and then decreased slowly

injector, giving, along with the major compound (approximately 80%; R,, 20.2 min; M+ at mlz = 214), two minor compounds (approximately 8% with an R, of 17.3 and 10% with an R, of 18.3 min). The first of these minor compounds showed a retention time and mass spectrum identical to those of authentic 9-fluorenone (M+ at m/z = 180). The second one

[196(M+,100%), 168(M+-CO,40%), 139(M+-CO-COH,31.5%), 113 (M+-CO-COH-HC==CH,3.5%)] corresponded to a phenolic derivative of fluorenone and was later identified as 1-hydroxyfluorenone, the sole product of dehydration of the major compound (35). MS (direct probe) of metabolite IV exhibited a molecular ion at an m/z of 214. This metabolite has been shown to be (+)-l,la-dihydroxy-1-hydro-9-fluorenone by an extensive structural characterization (35). The mass spectrum of metabolite VI exhibited a molecular ion with an m/z of 212. Major fragments (m/z 184, 156, and 128) were indicative of sequential elimination of three carbonyl groups. After treatment with diazomethane, a product that exhibited a molecular ion at an m/z of 226 was formed, indicative of the presence of a single methylatable functional group. No elimination of a hydroxyl, water, or carboxyl group =

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until it was undetectable at 36 h. The concentrations of metabolites V (R1, 6.4 min), VI (Re, 11.0 min), and VII (R, 0.9 min), which were first detected within 1 to 3 h of incubation, increased, reaching maxima at 18 h, and thereafter remained constant until the end of incubation. No product formation or significant loss of fluorene was observed in uninoculated controls. In order to obtain sufficient metabolites for purification and identification, a large-scale incubation was set up, using 2 liters of washed-cell suspension with 2 g of fluorene. Incubation was stopped at 18 h. The neutral extract of the supernatant weighed 320 mg, while the weight of the acidic extract was 127 mg. A portion of the neutral extract (280 mg) was purified by column chromatography. Separation was followed by TLC, and appropriate pooled fractions were further analyzed by HPLC and GC-MS. The first eluent, 50 ml of CH2Cl2, was discarded. The next fraction (50 ml of CH2Cl2) (17 mg) contained a mixture of fluorene (I) (73%), metabolite III (3%), and metabolite VI (24%). The third fraction (100 ml of CH2Cl2) (7 mg) showed a single spot by TLC (Rf, 0.6 in ethyl acetate) corresponding to metabolite VI. The next fraction (100 ml of CH2Cl2-ethyl acetate, 9:1) showed a low content of all metabolites present in the previous fractions and was not examined further. The last fraction (50 ml of ethyl acetate) (184 mg) contained a single spot on TLC (Rf, 0.4 in ethyl acetate), with a characteristic strong green fluorescence under 360-nm UV illumination, that corresponded to metabolite IV. Metabolite V was not detected. Mass spectral properties of the recovered metabolites are shown in Table 2. Metabolite III, present in fraction I, exhibited UV-visible and mass spectra identical to those of 9-fluorenone, with corresponding GC and HPLC retention times. Its identification was further confirmed by coelution during both GC and HPLC with an authentic sample. GC-MS analysis of chromatographically pure (by HPLC and TLC) metabolite IV showed thermal decomposition in the GC

was observed in the MS fragmentation of either metabolite VI or its methyl derivative. According to these properties, a hydroxy-benzocoumarin structure was suggested. Since the nonmethylated metabolite VI appeared to be unstable, only its methyl derivative was subjected to NMR analysis. 'H NMR analysis of methylated metabolite VI showed seven distinct aromatic proton signals and a resonance corresponding to a methoxy group at 3.997 ppm (3H, s) (Fig. 3). The aromatic proton signals can be assigned to two separated noninterrupted groups (four and three protons) as follows (8 ppm): H-1, 8.443 (JH-1,H-2 = 7.8 Hz; JH-1,H-3 = 1.4 Hz); H-2, 7.601 (JH-2,H-3 = 7.4 Hz; JH-2,H-4 = 1.0 Hz); H-3, 7.835 (JH-3,H-4 = 8.0 Hz); H-4, 8.128 (JH-4,H-5 = 0.4 Hz); H-5, 7.658 (JH-5,H-6 = 8.1 Hz; JH-5,H-7 = 1.2 Hz); H-6, 7.284 (JH-6,H-7 = 8.2 Hz); H-7, 7.059. No singlet aromatic proton signals typical of isolated protons were observed. 13C NMR analysis showed 14 nonequivalent carbon atom resonances: seven intensive sp2 aromatic carbon signals at 112.27, 114.24, 122.29, 124.36, 129.06, 130.73, and 134.92 ppm; one Sp3 carbon (methoxy) atom signal at 56.33 ppm; one weak lactone carbon atom signal at 172.0 ppm; and five sp2 quaternary carbon atom signals of low intensity at 121.35, 124.4, 135.04, 141.35, and 147.1 ppm. According to these properties, the methyl derivative of VI was identified as 8-methoxy-3,4-benzocoumarin (synonyms: 9-oxa-10-oxo-1OH-8-methoxyphenanthrene and 3'-methoxy-2'-hydroxybiphenyl-2-carboxylic acid 8-lactone). Therefore, metabolite VI is identified as 8-hydroxy3,4-benzocoumarin (synonyms: 9-oxa-10-oxo-1OH-phenanthr8-ol and 2',3'-dihydroxybiphenyl-2-carboxylic acid B-lactone). HPLC of the acidic extract showed only one major peak, which presented a retention time and UV-visible spectrum identical to those of metabolite VII, observed previously in supernatants and shown to be phthalic acid. Results obtained by GC-MS analysis of its methyl derivative are shown in Table 2 and are indistinguishable from those found for an authentic sample of the dimethyl ester of phthalic acid. Incubation of fluorene-induced washed cells with 9-fluorenone showed the same metabolites formed in a very similar pattern, which, together with the transient accumulation of 9-fluorenone during incubations with fluorene, suggests that this ketone is not a dead-end product (as suggested in the metabolism of fluorene by an Arthrobacter sp. [18]) but a true intermediate in the biodegradation of fluorene by Pseudomonas sp. strain F274. In addition, a small amount (approximately 6%) of a compound with an R, (15.3 min) and mass spectrum [m/z: 196(M+,100%), 181(M+-CH3,9%), 165(M+-OCH3,90%), 151(7%), 137(M+-CO-OCH3,7%), 125(9%), 123(7%), 107 (6%), 94(6%), 79(12.9%)] identical to those found for the methyl ester of 3,4-dimethoxybenzoic acid (obtained by diazomethane methylation of authentic protocatechuic acid) was also detected. Enzyme activities. Crude extracts from fluorene-induced cells showed the presence of protocatechuate 4,5-dioxygenase activity as demonstrated by UV-spectral changes showing the disappearance of protocatechuate (IX) (Xmax at 250 and 290 nm at pH 7) with accumulation of a product having a Xmax at 293 and 410 nm at pH 7 (Fig. 4A). The A410 increased after addition of NaOH to the reaction mixture. These spectral characteristics are those of 2-hydroxy-4-carboxy-cis,cis-muconic semialdehyde (X) (11). Crude extracts from cells grown withp-hydroxybenzoate as a sole carbon and energy source showed no protocatechuate 4,5-dioxygenase activity but instead catalyzed formation of a product with the UV-visible spectral properties of EB-carboxy-

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DEGRADATION OF FLUORENE BY PSEUDOMONAS STRAIN F274

2443

.

200

180160 200

250

_____200o

30

250

300

350

nm

30

120

E

60

40

25020

m200

0

300

250

300

350

nm

i

20

5 510 T1ime % (mrnin III I I I I

L.

I I 5 a %.O

a

15

)op

1000

B

800

900700

__20_2_0300_50_

6 0 0

GI

v

500

E

400: 300

1

200

1

200

250

300

350

nm

100'

5

10 Time (min. )

15

20 I

FIG. 2. HPLC elution profiles of supernatants from washed-cell suspensions of strain F274 with fluorene after 1 (A) and 18 (B) h of incubation. The UV-visible spectra of metabolites are displayed as insets. Compound I is fluorene. Other compounds have been identified as follows: II, 9-fluorenol; III, 9-fluorenone; IV, (+)1,la-hydroxy-1-hydro-9-fluorenone; VI, 8-hydroxy-3,4-benzocoumarin; and VII, phthalic acid. Isolation and identification of compound V were not possible because of its chemical instability.

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830

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APPL. ENVIRON. MICROBIOL.

TABLE 2. GC retention times and electron impact mass spectral properties [m/z (suggested fragments and percent relative intensity)] of metabolites formed from fluorene by Pseudomonas sp. strain F274 m/z (%)

Metabolite

R, (min)

III

17.20

180(M+,100), 152(M+-CO,30.0), 126(5.3), 90(2.0), 76(10.0), 63(5.0)

IV

Nonea

214(M+,2.0), 212(M+-H-H,3.8), 196(M+-H2O,100), 180(M+-OH-OH,70.0), 168(M+-H2O-CO,37.8), 152(M+-OH-OH-CO,40.5), 139(38.3), 128(38.0), 115(18.1), 102(17.0), 87(7.8), 75(12.2)

VI

21.03

212(M+,100), 184(M+-CO,13.5), 156(M+-CO-CO,17.2), 128(M+-CO-CO-CO,27.6), 106(3.4), 102(7.2), 92(3.0), 77(3.7), 75(3.5), 63(5.2), 51(4.9)

VI (Me)b

22.39

226(M+,100), 21 1(M+-CH3,2.7), 197(3.2), 195(1.4), 183(M+-CH3-CO,35.6), 168(2.6), 155(M+CH3-CO-CO,11.2), 139(5.0), 127(18.0), 113(2.6), 101(4.3), 77(4.4), 63(4.5)

VII (Me)

13.51

194(M+,8.5), 163(M+-CH30,100), 135(M+-COOCH3,4.4), 133(4.9), 120(1.6), 104(M+-COOCH3OCH3,3.12), 92(5.17), 77(10.6), 76(M+-COOCH3-COOCH3)

b

Me, analyzed as methyl derivative.

cis,cis-muconate, indicative of the presence of protocatechuate 3,4-dioxygenase (29). Crude extracts of cells grown in the presence of fluorene also showed 4,5-dihydroxyphthalate decarboxylase activity, as evidenced by UV-visible spectral changes (Fig. 4B) consistent with conversion first of 4,5-dihydroxyphthalate (VIII) (Xmax at 288 nm, shoulder at 250 nm) to protocatechuate (IX) and then conversion of this compound to 2-hydroxy-4-carboxy-cis,cismuconic semialdehyde (X) as in Fig. 4A; 3,4-dihydroxyphthalate was not acted upon under these reaction conditions. Detection of dihydroxyphthalate decarboxylase, a key enzyme of phthalate metabolism in Pseudomonas spp. (32), together with protocatechuate 4,5-dioxygenase indicates that Pseudomonas sp. strain F274 has the capability to further metabolize 3

5

4

phthalate. Therefore, accumulation of small amounts of this product during growth is not due to final product formation but rather to a problem of its permeability. Identification of metabolites from fluorene analogs. Washed-cell suspensions prepared from fluorene-induced cultures were incubated with dihydroanthracene, anthrone, xanthene, and dibenzofuran. In all cases, metabolite production was shown by HPLC analysis of the supernatants and GC-MS analysis of neutral extracts. Table 3 shows the MS data for the major metabolites, with suggested fragment identities. Major metabolites detected from dihydroanthracene were identified as anthrone and anthraquinone. Methylated acidic extracts (results not shown) contained three major unidentified compounds, indicating that degradation of dihydroanthracene

6

2

-Iv

7 8~a

ma

3.997 ppm

(3H, s, not shiown)

H-1H-I HH-4

-, -I

9. 0

.I

8. 5

H-3

I

8. 0

I

H-7

H-5,H, 1-2

I

H-6

I

7. 5

I

I

7

0

FIG. 3. 'H NMR spectrum and chemical structure of methylated metabolite VI.

I

I

6. 5

I

I

ppm

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"Direct probe.

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DEGRADATION OF FLUORENE BY PSEUDOMONAS STRAIN F274

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1.. 0 0 c

co n

co0

wavelength (nanometers)

FIG. 4. Metabolism of protocatechuate (A) and 4,5-dihydroxyphthalate (B) by extracts of fluorene-induced cells of Pseudomonas sp. strain F274. The first UV spectrum was recorded before addition of the cell extract, and subsequent spectra were recorded at 3-min intervals. goes beyond formation of the ketone and quinone. Extracts from anthrone incubations showed the presence of exactly the same products with the exception of anthrone itself. From xanthene, a major neutral metabolite was formed and identified as xanthone by comparison with an authentic sample. Finally, transformation of dibenzofuran by F274 yielded only one major neutral product detectable by HPLC. GC-MS results for the neutral extract after methylation showed the presence of four major peaks attributable to different extents of methylation of a trihydroxybiphenyl. In Table 3, fragmentation of the monomethoxy isomer is shown. The fragmentation

pattern beyond an mlz of 201, which is assigned as (M+-CH3), is consistent with the MS spectrum reported by Fortnagel et al. (14) for 2,2',3-trihydroxybiphenyl. Small amounts of the methyl ester of salicylic acid [R,, 9.8 min; m/z, 155 for M+ (53%)] were detected in diazomethane-treated acidic extracts.

DISCUSSION Pseudomonas sp. strain F274 grows with fluorene as a sole source of carbon and energy; growth is seen to enter the stationary phase as fluorene is depleted. The observed growth

TABLE 3. GC retention times, electron impact mass spectral properties [m/z (suggested fragmentation and percent relative intensity)], and identification of neutral metabolites formed from substrate analogs of fluorene by washed cells of fluorene-grown Pseudomonas sp. strain F274 Substrate

R, (min)

Abundance (%)

Fragments (mlz, %)

Dihydroanthracene

20.47

16.7

Anthrone

20.77

62.5

194(M+,100), 165(M+-CO-H,59.1), 139(8.8), 126(1.7), 115(3.4), 96(3.4), 89(2.3), 82(21.6), 69(6.8) 208(M+,100), 180(M+-CO,58.6), 152(M+CO-CO,41.4), 126(5.7), 113(1.1), 102(3.5), 90(4.7), 76(27.6)

Anthrone

20.77

80.0

208(M+,100), 180(M+-CO,58.6), 152(M+-COCO,41.4), 126(5.7), 113(1.1), 102(3.5), 90(4.7), 76(27.6)

Anthraquinone

Xanthene

19.81

100.0

196(M+,100), 168(M+-CO,35.2), 139(26.5), 114(2.5), 98(4.1), 92(6.3), 84(10.8)

9-Xanthenone

Dibenzofuran

18.03

23.8

216(M+,100), 201(M+-CH3,14.2), 183(M+CH3-H20,35.2), 173(M+-CH3-CO,8.3), 155(M+-CH3-H2O-CO,20.7), 145(M+CH3-CO-CO,1.7), 127(M+CH3-H20-CO-CO,14.2), 115(13.9), 91(4.6), 77(11.1)

2,2',3-Trihydroxybiphenyl (monomethyl derivative)

Identification

Anthraquinone

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wavelength (nanometers)

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GRIFOLL ET AL.

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I

II

III

IV

OH

0

0

OH

VI

COO'-

CENTRAL METABOLISM

COO'

OH

C02

o coo HO

OHCCO x

OH

CO

HO

Ix

VIII

FIG. 5. Schematic pathway proposed for the degradation of fluorene by Pseudomonas intermediates and have not been isolated.

yield suggests that this process occurs with a highly efficient assimilation of all substrate carbon. On the basis of identification of products formed from fluorene, in growth media and by washed-cell suspensions, a new pathway of degradation of fluorene is proposed. The salient features of this pathway are: (i) monooxygenation at C-9 to give 9-fluorenol (and, thereafter, 9-fluorenone), a reaction that appears to be necessary before (ii) dioxygenation at an angular site in a stereospecific manner, and (iii) novel cleavage of the five-membered ring to generate a substituted biphenyl, further degraded by reactions akin to those of biphenyl catabolism. According to the data on identification of the metabolites and enzymatic activities presented above, the following steps in the degradation of fluorene by Pseudomonas sp. strain F274 are suggested (Fig. 5). The first reaction fluorene undergoes is monohydroxylation at position 9 to give 9-hydroxyfluorene (II), which is then dehydrogenated to the corresponding ketone. 9-Fluorenone (III), which was previously described as a dead-end metabolite in the biodegradation of fluorene by an Arthrobacter sp. (18), is here a substrate for dioxygenatioh by an angular attack, giving 1,la-dihydroxy-1-hydro-9-fluorenone (IV). Since this dioxygenation produces a tertiary alcohol, further dehydrogenation cannot lead to formation of a 1,2diphenol, which is usually necessary before aromatic ring cleavage (6, 17). The problem of ring opening seems to be resolved in this strain by means of a novel cleavage of the five-membered ring. Identification of 8-hydroxy-3,4-benzocoumarin (in its methylated form, VI) suggests that metabolism of the angular diol may include a biological Baeyer-Vi liger reaction. Such reactions have been suggested previous y to explain metabolites formed during the catabolism of fluorene (18) and fluoranthene (41). Another type of reaction can be proposed as an alternative for the formation of this compound. Dehydrogenation of the 1-sec-alcohol group of the angular diol

cOO COO' VII

sp.

strain F274. Structures in brackets

are

proposed

(IV) would yield a 1,3- or 3-diketone-type compound whose ready hydrolysis at the C-9-C-10 bond would give 2-carboxy2',3'-dihydroxybiphenyl. The identified 8-hydroxy-3,4-benzocoumarin is in fact the 8-lactone of this compound and could be formed as a result of a reversible dehydration reaction similar to that described for 8-hydroxy-naphthalene-1-acetic acid (9). Dioxygenase-catalyzed cleavage of 2-carboxy-2',3'dihydroxybiphenyl at the C-1,2 bond and subsequent hydrolysis of the ring fission product, reactions analogous to those of biphenyl degradation, appear to account for the formation of phthalate (VII). According to enzyme assays, phthalate then appears to be dioxygenated at the 4 and 5 positions before undergoing decarboxylation to give protocatechuate, which is then cleaved by protocatechuate 4,5-dioxygenase, giving 2-hydroxy-4-carboxy-cis,cis-muconic semialdehyde (31). The finding of 4,5-dihydroxyphthalate decarboxylase, a key enzyme of phthalic acid degradation in Pseudomonas spp. (32), coupled with the demonstration of protocatechuate 4,5-dioxygenase activity, suggests that strain F274 apparently has the requisite genetic information for complete assimilation of the entire fluorene molecule. Additional support for the initial reactions proposed is provided from the results of incubations of cells with a number of substrate analogs of fluorene possessing internal benzylic methylene functions. With all of these substrates, monooxygenation of the benzylic methylene groups is observed; anthrone and anthraquinone are produced from dihydroanthracene, anthraquinone is produced from anthrone, and xanthone is produced from xanthene (Fig. 6). Monooxygenation precedes any further functionalization of these compounds. Recent reports have shown monooxygenation of benzylic methylenic groups in naphthenoaromatic compounds by some arene-dioxygenases. Schocken and Gibson (33) reported that cooxidation of acenaphthene by a biphenyl-degrading Beijer-

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0

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DEGRADATION OF FLUORENE BY PSEUDOMONAS STRAIN F274 0

Dihydroanthracene

0

0

0

0

0

Andirone

OHOH

Dibenzofuran

OH

FIG. 6. Chemical structures of neutral metabolites produced by Pseudomonas sp. strain F274 from fluorene analogs.

inckia strain apparently proceeds through two successive monooxygenation and dehydrogenation steps, suggesting acenaphthene quinone as a dead-end product. Since a diolaccumulating mutant of this strain (B8) also formed these products, oxygenation of acenaphthene to acenaphthenol and its subsequent oxygenation to give acenaphthenequinone were assumed to be due to biphenyl dioxygenase, but unambiguous proof is lacking. In recent work (34), Pseudomonas aeruginosa carrying the genes of naphthalene dioxygenase cloned from the naphthalene plasmid NAH7 was also shown to monooxygenate acenaphthene to 1-acenaphthenol, which then underwent a second monooxygenation to give a mixture of acenaphthene diols. Formation of 1-acenaphthenone from 1-acenaphthenol and acenaphtho-1,2-quinone from acenaphthene-1,2-diol occurred as a result of nonspecific dehydrogenase reactions. Acenaphthylene was both mono- and dioxygenated, giving, respectively, acenaphthenone and cis-acenaphthene-1,2-diol. The same system converted fluorene to 9-hydroxyfluorene (II), 9-fluorenone (III), and a mixture of phenols evidently resulting from the spontaneous dehydration of vicinal cis-dihydrodiols of both fluorene and fluorenone. The toluene dioxygenase of Pseudomonas putida Fl oxidizes both indane and indene, yielding 1-indanol and the pair cis-1,2-indandiol and 1-indanol, respectively (40). Washed-cell suspensions of P. putida further oxidized 1-indanol to 1-indanone. Wackett et al. (40) also reported hydroxylation of indane to 1-indanol by the naphthalene dioxygenase of a Pseudomonas sp. Similar monooxygenations of naphthenoaromatic compounds have been reported by other researchers (2-4, 37). In all of these cases, the observed monooxygenation activity seems to be due to the anomalous action of arene-dioxygenases at benzylic positions, generating secondary alcohols, and (through the action of ancillary alcohol dehydrogenases) subsequently accumulating ketones and quinones as dead-end products. Formation of 9-fluorenone as a terminal product in cultures of a fluorene-utilizingArthrobacter sp., in which the productive

pathway was initiated by 3,4-dioxygenation of the arene ring system (18), appears to be another example of an unproductive anomalous monooxygenation of the arene-dioxygenase. The productive pathway in the Arthrobacter sp. seems to be based on release of one molecule of pyruvate by reactions similar to those of the naphthalene and dibenzothiophene pathways (10, 25). Whether additional assimilatable carbon is supplied from the remainder of the fluorene structure is not yet clear. In the case of xanthone degradation by an Arthrobacter sp. (38), ring dioxygenation initiates a similar reaction sequence that not only enables release of a pyruvate fragment but also leads to more extensive degradation of the 4-hydroxycoumarin residue. In the present work, it can be seen that the angular dioxygenation mechanism supplies an alternative strategy for fluorene degradation that accommodates benzylic monooxygenation by providing reactions for 9-fluorenone catabolism. This type of attack was proposed as a first step in the bacterial oxidation of dibenzofuran and other biaryl ether structures. Fortnagel et al. (14, 15) reported that biodegradation of dibenzofuran by Pseudomonas sp. strain HH69 proceeds via angular dioxygenation followed by opening of the resulting hemiketal to give 2,2',3-trihydroxybiphenyl, which is further degraded by a biphenyl-type pathway to produce salicylate as an intermediate. Similar pathways have been proposed for the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1, on the basis of identification of 2,2',3-trihydroxydiphenyl and 2,2',3-trihydroxydiphenyl ether, respectively (42). Very recently, Bunz and Cook (5) have purified, from the same strain, a dioxygenase responsible for angular dioxygenation. This enzyme is postulated to catalyze dioxygenation of dibenzofuran at the 4,4a position to give an angular diol with a hemiketal structure which can undergo spontaneous ring opening and aromatization, yielding 2,2',3trihydroxybiphenyl. The angular dioxygenating enzyme from Pseudomonas strain F274 also acts on 9-fluorenone to give a product with UV maxima identical to those of 1,la-dihydroxy1-hydro-9-fluorenone (IV; Fig. 5). On the basis of identification of the angular diol 1,2dihydroxy-2-hydro-4-carboxyphenone, a product of the action of Pseudomonas sp. strain NSS2 on 4-carboxybenzophenone, Fortnagel et al. (15) suggested that 9-fluorenone underwent a similar angular dioxygenase attack. From either substrate, monophenols of the parent compound were also formed. Their formation was explained as due to spontaneous dehydration of the angular diols considered to be dead-end products. Engesser et al. (12) reported that transformation of fluorene by dibenzofuran-grown cells of Brevibactenium sp. strain DPO 1361 resulted in the f,rmation of an angular diol of 9-fluorenone. Here too, the presence of monohydroxyfluoren-9-one was explained as a consequence of dehydration of the angular diol. Monna et al. (26) described the formation of 9-fluorenone, 1-hydroxy-9-fluorenone, and 4-hydroxy-9-fluorenone from fluorene by dibenzofuran-grown cells of S. auniculans DBF63. These researchers suggested that angular dioxygenation followed by ready diol dehydration accounted for the presence of 1-hydroxy-9-fluorenone, apparently a dead-end

product. The remarkable stability of the angular diol of fluorenone has been described elsewhere (35), prompting the suggestion that the monophenolic products observed by others are more likely to be derived from cis-dihydrodiols. No evidence of phenolic derivatives of fluorene or 9-fluorenone has been obtained in the present work. The initial reactions of fluorene degradation, outlined here for Pseudomonas sp. strain F274, show similarity to the systems mentioned above. For example, dibenzofuran is shown here to

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Xanthene

2447

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GRIFOLL ET AL.

but does not catalyze monooxygenation of fluorene to 9-fluorenone. If one assumes a close similarity between this dioxygenase and the angular dioxygenase of strain F274, then monooxygenation of fluorene by F274 would have to be catalyzed by a different enzyme. Whether reductive dioxygenation of an arene ring leads to assimilation of the entire substrate or merely to a portion of its structure, it appears that monooxygenation of any accessible benzylic methylenic function can be anticipated. The present work together with other works cited here indicates that ketones and quinones can be anticipated as products from naphthenoaromatic hydrocarbons formed through fortuitous microbial action and also by reactions involved in more extensive degradation pathways. The presence of a number of naphthenoaromatic ketones in environmental samples (13, 20) or their formation during microbial degradation of mixed aromatic hydrocarbons, such as in cresote (7), may be due, at least in part, to these types of reactions. It is tempting to speculate that Pseudomonas sp. strain F274 has evolved an efficient route of fluorene utilization based on elements of degradation pathways for biphenyl and phthalic acid. This may have been accomplished by ensuring that 9-fluorenone formation occurs in a more specific and directed manner than through the fortuitous action of the type of arene-dioxygenase generally recognized as initiating attack on fully aromatic hydrocarbons. Angular dioxygenation of 9-fluorenone further guarantees the efficiency of such a pathway, provided that catabolism of the product of its action is possible and that other nonmetabolizable diols are not formed. These speculations imply roles and properties for the enzymes of this novel pathway that are under investigation. orenone

ACKNOWLEDGMENTS This research was supported in part by a postdoctoral fellowship (to M.G.) (PF91 39856347) from the Spanish Ministry of Education and Science and by Cooperative Agreement (CR-817770) between Environmental Research Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Fla., and the University of West Florida. We gratefully acknowledge S. Resnick for technical assistance in the isolation of the strain, B. Blattman and M. Downey for conducting HPLC and GC-MS analyses, J. E. Gurst for determining NMR spectra, MIDI Laboratories for performing the fatty acid methyl ester analysis of strain F274, and R. W. Eaton for helpful and stimulating discussions.

ADDENDUM After submission of the manuscript for publication, a similar pathway was described for catabolism of fluorene by a dibenzofuran- and fluorene-degrading strain of Brevibacterium sp. (strain DPO 1361) via angular dioxygenation (38a).

REFERENCES 1. Black, T. H. 1983. The preparation and reactions of diazomethane. Aldrichim. Acta 16:39. 2. Boyd, D. R., R. A. S. McMordie, N. D. Sharma, H. Dalton, P. Williams, and R. 0. Jenkins. 1989. Stereospecific benzylic hy-

droxylation of bicyclic alkenes by Pseudomonas putida: isolation of (+)-R-1-hydroxy-1,2-dihydronaphthalene, an arene hydrate of naphthalene from metabolism of 1,2-dihydronaphthalene. J.

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DEGRADATION OF FLUORENE BY PSEUDOMONAS STRAIN F274

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