Novel Intermediates of Acenaphthylene Degradation by Rhizobium sp ...

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Apr 17, 2006 - Laurie and Lloyd-Jones (13). Transposon Tn5 can ..... 180:2522–2530. 6. Grifoll, M., S. A. Selifenov, C. V. Galtin, and P. J. Chapman. 1995.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2006, p. 6034–6039 0099-2240/06/$08.00⫹0 doi:10.1128/AEM.00897-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 72, No. 9

Novel Intermediates of Acenaphthylene Degradation by Rhizobium sp. Strain CU-A1: Evidence for Naphthalene-1,8-Dicarboxylic Acid Metabolism Siriwat Poonthrigpun,1 Kobchai Pattaragulwanit,1,2* Sarunya Paengthai,1 Thanyanuch Kriangkripipat,1 Kanchana Juntongjin,1,2 Suthep Thaniyavarn,1,2 Amorn Petsom,3 and Pairoh Pinphanichakarn1,2 Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand1; National Research Center for Environmental and Hazardous Waste Management (NRC-EHWM), Faculty of Science, Chulalongkorn University, Bangkok, Thailand2; and Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand3 Received 17 April 2006/Accepted 18 July 2006

The acenaphthylene-degrading bacterium Rhizobium sp. strain CU-A1 was isolated from petroleum-contaminated soil in Thailand. This strain was able to degrade 600 mg/liter acenaphthylene completely within three days. To elucidate the pathway for degradation of acenaphthylene, strain CU-A1 was mutagenized by transposon Tn5 in order to obtain mutant strains deficient in acenaphthylene degradation. Metabolites produced from Tn5-induced mutant strains B1, B5, and A53 were purified by thin-layer chromatography and silica gel column chromatography and characterized by mass spectrometry. The results suggested that this strain cleaved the fused five-membered ring of acenaphthylene to form naphthalene-1,8-dicarboxylic acid via acenaphthenequinone. One carboxyl group of naphthalene-1,8-dicarboxylic acid was removed to form 1-naphthoic acid which was transformed into salicylic acid before metabolization to gentisic acid. This work is the first report of complete acenaphthylene degradation by a bacterial strain. samples of an industrial waste deposit, has been reported to utilize acenaphthylene as a sole source of carbon and energy. The strain A4 transformed acenaphthylene to 1,8-naphthalenedicarboxylic acid and utilized both compounds as sole carbon and energy sources. No metabolite from the oxidation of 1,8-naphthalenedicarboxylic acid was reported (11, 19). Pseudomonas aeruginosa PA01(pRE695), a recombinant strain carrying the naphthalene dioxygenase gene from plasmid NAH7, transformed acenaphthylene to cis-acenaphthene-1,2diol. Then, the nonspecific dehydrogenase activities in the host strain further oxidized cis-acenaphthene-1,2-diol to 1,2-acenaphthenequinone prior to spontaneous ring fission to form naphthalene-1,8-dicarboxylic acid (23). Like Sphingomonas sp. strain A4, no further oxidation of naphthalene-1,8-dicarboxylic acid was found. In this study, a novel Rhizobium strain (CU-A1) capable of utilizing acenaphthylene as a sole carbon and energy source was isolated and characterized. This strain was mutagenized by transposon Tn5, and accumulated intermediates of acenaphthylene degradation formed from those mutants were purified and identified. In addition to the previously proposed intermediates in degradation pathways of acenaphthylene mentioned earlier (23), we describe novel metabolites of acenaphthylene degradation formed from naphthalene-1,8-dicarboxylic acid. Based on these results, a complete pathway for the degradation of acenaphthylene is proposed for the first time.

Polycyclic aromatic hydrocarbons (PAHs) constitute a class of hazardous organic chemicals consisting of two or more fused benzene rings in various structural configurations. PAHs are ubiquitous pollutants in the environment (12). PAHs mostly occur as a result of fossil fuel combustion and as by-products of industrial processes (3). Due to their carcinogenicity, mutagenicity, and toxicity, PAHs are listed as priority toxic pollutants by the United States Environmental Protection Agency (10). Acenaphthylene, one of the listed PAHs, is found as a constituent of coal tar and tobacco smoke (15) and of organic contaminants in groundwater (31). This compound is well-known for causing adverse effects in humans as well as aquatic organisms (14). The metabolism of PAHs by pure cultures of microorganisms has been reported for more than 90 years. Various genera of bacteria, fungi, and algae have the ability to degrade PAHs (3, 27). In contrast, the oxidation of acenaphthylene by bacteria has rarely been reported. The first bacterium capable of acenaphthylene oxidation was a naphthalene-grown bacterium isolated from estuarine water polluted with oil which could cooxidize acenaphthylene to an unidentified quinone metabolite (4). Schocken and Gibson (22) reported on a Beijerinckia sp. that could cometabolize acenaphthylene with succinate by dioxygenating acenaphthylene to form cis-1,2-acenaphthenedihydrodiol and 1,2-dihydroxyacenaphthylene and finally to form acenaphthenequinone which cannot be further oxidized. Only Sphingomonas sp. strain A4, formerly known as Pseudomonas sp. strain A4, a bacterium isolated from soil

MATERIALS AND METHODS Media and growth conditions. Mineral medium (MM) was used for routine cultivation of the strain CU-A1 and the mutants thereof (9). Acenaphthylene and other PAHs were dissolved in dimethyl sulfoxide, and protocatechuic acid was dissolved in ethanol. All PAH solutions were filter sterilized through a polytetrafluoroethylene (PTFE) membrane cartridge (0.2-␮m pore size; Advantec

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. Phone: 66-2218-5070. Fax: 66-2252-7576. E-mail: Kobchai1a @yahoo.com. 6034

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Toyo, Tokyo, Japan). They were added to MM, pH 7.5, after autoclaving at a final concentration of 0.6 g/liter acenaphthylene, 1 g/liter protocatechuic acid, and 0.1 g/liter other PAHs. The strain CU-A1 and mutants thereof were routinely grown at 30°C with shaking at 200 rpm. Escherichia coli S17-1 was grown at 37°C in Luria-Bertani (LB) or 2⫻ yeast extract-tryptone medium (21). Agar plates were prepared by addition of 1.5% (wt/vol) Bacto agar. Kanamycin was added at the final concentration of 50 ␮g/ml. Isolation and identification of acenaphthylene-utilizing bacteria from soil. The strain CU-A1 was isolated from petroleum-contaminated soil from Thailand on the basis of its ability to grow on acenaphthylene as the sole carbon and energy source. Approximately 1 g of soil sample was added into 10 ml of MM supplemented with 6 mg of acenaphthylene and subsequently cultivated at 30°C for one week. The cultures were then transferred to fresh MM containing acenaphthylene and incubated under the same conditions. This step was repeated three times. After visible growth could be detected, the culture was spread on a MM agar plate supplemented with acenaphthylene vapor to isolate acenaphthylene-utilizing bacteria. Colonies grown on the MM plate were picked and inoculated in MM containing acenaphthylene. Their purity was confirmed by plating on LB agar. The acenaphthylene-degrading strain was identified according to Bergey’s Manual of Systematic Bacteriology (17). The 16S rRNA gene sequence of the isolate was obtained by direct sequencing of PCR-amplified 16S rRNA gene. Genomic DNA isolation was performed by standard protocols (21). Amplification of the 16S rRNA gene by PCR was carried out using 27f and 1492r primers (30). The PCR product was purified using a DNA extraction kit (QIAGEN, Germany) according to the manufacturer’s protocol and sequenced by the Dragon Genomic Center (Takara Bio, Japan). The obtained 16S rRNA gene sequence was compared with those available in GenBank using BlastN (1). Substrate utilization. Growth on acenaphthylene was determined by measuring the increase in bacterial number and the decrease in concentration of acenaphthylene. The strain CU-A1 and its mutants were cultivated in MM liquid medium containing 1 g/liter protocatechuic acid at 30°C with shaking at 200 rpm for 24 h followed by washing with 0.85% NaCl three times. The pellet was resuspended in 0.85% NaCl and starved at 30°C for 6 h. The cell suspension was then adjusted to an absorbance of 1.0 (A600). The substrate utilization was tested by inoculating 100 ␮l of the resting cell suspension in 5 ml MM containing an individual PAH (600 mg/liter of acenaphthylene, 200 mg/liter of naphthylene, and 100 mg/liter of other PAHs). Uninoculated samples and inoculated samples in the absence of substrate served as controls. After 0, 2, 4, and 7 days of incubation, cultures were analyzed in triplicate for an increase in bacterial cell number and for remaining substrate. Bacterial cell number was determined by viable plate count on LB agar. The remaining amount of substrate in each sample was extracted by ethyl acetate and analyzed by high-performance liquid chromatography (HPLC) (26). Transposon mutagenesis. Transposon Tn5 on the suicide vector pSUP2021 was conjugatively transferred from Escherichia coli S17-1 to Rhizobium sp. strain CU-A1 by a filter-mating technique (25). Exponential-phase cells of E. coli S17-1 as donor and Rhizobium sp. strain CU-A1 as recipient in LB medium were mixed at the donor-to-recipient ratio of 1:1 in a Microfuge tube and centrifuged. The pellet was resuspended in 50 ␮l of the remaining supernatant and transferred to a nitrocellulose membrane (0.45-␮m pore size; Sartorius, Germany) which was then placed on LB agar. After mating at 30°C for 18 to 24 h, the cell mixture was resuspended in 1 ml 0.85% NaCl, and a 100-␮l portion of an appropriately diluted solution was spread onto MM agar supplemented with protocatechuic acid and kanamycin. Following incubation at 30°C for 4 days, transconjugants were spotted onto master plates of the same medium composition. Selection of acenaphthylene-degrading defective mutants. To select acenaphthylene-degrading defective mutants, transconjugants were transferred from the master plate onto MM agar supplemented with kanamycin, and the carbon source acenaphthylene was applied as crystal on the lid. The selection plates were sealed with Parafilm and incubated at 30°C for up to one week. Mutants incapable of growing or having slow growth on the selection plates were picked from the master plates. Mutants could also be screened by the production of colored metabolites on selection plates. All selected mutants were subsequently confirmed by their inability to grow within three days in liquid MM containing kanamycin and acenaphthylene as the sole carbon and energy source. Extraction and purification of acenaphthylene degradation intermediates. For the isolation of intermediates, acenaphthylene degradation defective mutants were enriched in MM supplemented with protocatechuic acid and kanamycin as mentioned in the “Substrate utilization” section. The obtained cell pellet was transferred to MM containing 0.6 g/liter acenaphthylene. The culture was incubated at 30°C and 200 rpm for five days before extraction of intermediates. After incubation, the culture was acidified to pH 2 to 3 with 1 N HCl and then

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extracted twice with equal volumes of ethyl acetate. The ethyl acetate extracts were pooled and dried over anhydrous Na2SO4 and evaporated to dryness in a vacuum at 25°C. The residue was redissolved in methanol for further purification. Accumulated intermediates were purified by preparative thin-layer chromatography (TLC) (silica gel 60 F254; Merck, Germany) with hexane-ethyl acetateacetic acid (10:10:1) as the developing solvent. The major spot was scraped off the plates, eluted with ethyl acetate, and submitted to silica gel column chromatography. The column was eluted stepwise with 0 to 100% ethyl acetate in hexane with 10% increments for each step. Each fraction was dried over anhydrous Na2SO4, evaporated to dryness in vacuo at 25°C, and redissolved in methanol. The resulting intermediates were analyzed for their purity by TLC and reversedphase HPLC with an octyldecyl silane column (Shimadzu Scientific, Japan) (26) with some minor modifications for HPLC conditions. The mobile phase was a gradient of 40 to 80% methanol in water eluting for 30 min then at 80% methanol in water for another 15 min. Identification of intermediates. The purified intermediates were identified by mass spectrometry using a Trio 2000 mass spectrometer (Fisons Instruments, England). The electron impact mass spectra (EIMS) were measured at 70 eV. Intermediates were identified by comparing their EIMS with those of authentic compounds. Chemicals. All chemicals used in this study were obtained from Kanto Chemical, Tokyo, Japan, or Sigma-Aldrich, Steinheim, Germany. Bacteriological media were purchased from Difco Laboratories, Detroit, Michigan. Organic solvents were obtained from Merck, Darmstadt, Germany. All chemicals and solvents were of the highest purity commercially available. Nucleotide sequence accession number. The 16S rRNA gene sequence of the acenaphthylene-degrading isolate was deposited in GenBank under accession number AY947466.

RESULTS AND DISCUSSION Isolation and characterization of Rhizobium sp. strain CUA1. Acenaphthylene-degrading strain CU-A1 was obtained from petroleum-contaminated soil samples by enrichment culture techniques. After incubation for three days in MM liquid media supplemented with acenaphthylene, strain CU-A1 had changed the color of the media from pale yellow to dark yellow and the turbidity indicated the ability of this strain to utilize acenaphthylene as a sole source of carbon and energy. Strain CU-A1 is a rod-shaped, gram-negative, aerobic, motile, and non-spore-forming bacterium. On LB medium, this strain grew as a small white round colony with no Congo red adsorption into the colony on yeast extract mannitol Congo red agar. The analysis of the PCR-amplified 16S rRNA gene sequence revealed 96% similarities of this strain to Rhizobium galegae (accession no. X67226) (32) and Rhizobium mongolense (accession no. U89822) (28). Utilization of acenaphthylene as sole carbon and energy source. Rhizobium sp. strain CU-A1 was checked for its ability to grow on acenaphthylene as the sole source of carbon and energy. Utilization of acenaphthylene was demonstrated by a decrease of acenaphthylene with a concomitant increase in bacterial cell numbers (Fig. 1). After incubation for three days, the acenaphthylene concentration initially at 0.6 g/liter was reduced to an amount undetectable by HPLC, whereas the cell number of strain CU-A1 had increased from 6.16 to 8.79 log CFU/ml. In the control experiments without bacterial cells, the loss of 66% of the initial concentration of acenaphthylene occurred after seven days in the same system. This may be due to the physical properties of acenaphthylene, one of the smaller PAH molecules, which can be easily volatilized (2). Without acenaphthylene, the total cell count of strain CU-A1 remained unchanged throughout the experiment, which reflected no growth of strain CU-A1 in MM. In addition to

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FIG. 1. Growth profile of Rhizobium sp. strain CU-A1 cultured in MM supplemented with (Œ) and without (‚) acenaphthylene and acenaphthylene concentration with (■) and without (䊐) bacterial inoculation.

acenaphthylene, the strain CU-A1 also used naphthalene as the sole carbon and energy source but not acenaphthene, phenanthrene, anthracene, pyrene, fluorene, and fluoranthene (data not shown). Tn5 transposon mutagenesis and isolation of acenaphthylene degradation defective mutants. To study the metabolic intermediates of acenaphthylene degradation by the strain CU-A1, genes involved in the acenaphthylene degradation pathway should be inactivated and the resulting mutants should accumulate metabolites. We therefore induced mutagenesis of this strain using transposon Tn5 as described by Laurie and Lloyd-Jones (13). Transposon Tn5 can transpose into chromosomal DNA of Rhizobium sp. strain CU-A1 with the efficiency of approximately 4.7 ⫻ 10⫺5 per recipient cell, which was comparable to the efficiency obtained in Tn5 mutagenesis of Rhizobium meliloti (25). No spontaneous kanamycin-resistant mutants derived from Rhizobium sp. strain CU-A1 were found under the same conditions. Southern hybridization analysis revealed that Tn5 was randomly inserted at only one site in the chromosomal DNA of each transconjugant (data not shown). Three mutants incapable of growing on acenaphthylene as a sole carbon source in liquid medium

within three days, designated as A53, B1, and B5, were selected from about 15,000 transconjugants. Isolation and identification of acenaphthylene metabolites. The mutant strains A53, B1, and B5 were exposed to acenaphthylene for five days as described in Materials and Methods. These mutants accumulated five major metabolites that were different from those of wild-type strain CUA1, as shown by different spots on TLC analysis. These metabolites were purified by preparative TLC and silica gel column chromatography followed by HPLC analyses to confirm their purity. Compounds I and II were obtained from mutants B1 and B5, whereas compounds III, IV, and V were from mutant A53. After purification, all metabolites were subjected to EIMS analysis. Retention times from HPLC analyses and EIMS characteristics of these compounds are shown in Table 1. Mass spectral characteristics of metabolites I, II, III, and IV were similar to those of authentic compounds available in the Wiley mass spectra database and therefore identified as acenaphthenequinone, naphthalene-1,8-dicarboxylic acid, gentisic acid, and 1-naphthoic acid, respectively. Compound V showed a mass spectrum identical to 2-hydroxybenzoic acid (salicylic acid) or 3-hydroxybenzoic acid, which differ only in the substitution position of the hydroxyl group. This metabolite was identified as 2-hydroxybenzoic acid, as the wild-type strain CU-A1 was able to grow on 2-hydroxybenzoic acid but not on 3-hydroxybenzoic acid (data not shown). Growth of Rhizobium sp. strain CU-A1 and its Tn5 mutants on acenaphthylene and the identified metabolites. The abilities of mutant strains A53, B1, and B5 and wild-type strain CU-A1 to utilize acenaphthylene and intermediate compounds I, II, III, IV, and V as sole sources of carbon and energy are shown in Table 2. The strain CU-A1 grew well on all tested substrates. The time course of the disappearance of acenaphthylene and its metabolites during 96 h of incubation (Fig. 2) showed the correlation between depletion of acenaphthylene and the occurrence of compounds I to V. All identified metabolites occurred during acenaphthylene degradation and were rapidly utilized corresponding to the increase in cell growth to about 3 orders of magnitude within 96 h. On the other hand, no spontaneous degradative products of acenaphthylene could be detected at any time point in the uninoculated acenaphthylene control (data not shown).

TABLE 1. HPLC retention times and electron impact mass spectral properties of the acenaphthylene metabolites accumulated by Tn5 mutants of Rhizobium sp. strain CU-A1 Mutant

Compound

Rt (min)a

m/z of fragment ions (% relative intensity)

182 (M⫹, 17), 154 (M⫹-CO, 66), 126 (100), 99 (10), 98 (12), 87 (22), 75 (11), 74 (26), 63 (15) 198 (M⫹, 66), 154 (M⫹-CO2, 100), 127 (10), 126 (79), 99 (8), 98 (6), 87 (7), 77 (10), 76 (11), 74 (15), 63 (21) 154 (M⫹, 60), 136 (H⫹-H2O, 100), 108 (17), 80 (22) 172 (M⫹, 100), 155 (M⫹-OH, 53), 127 (M⫹-COOH, 72), 115 (18), 101 (8), 98 (5), 89 (4), 87 (6), 86 (5), 77 (12), 75 (11), 74 (12), 63 (14) 138 (M⫹, 15), 121 (10), 120 (M⫹-H2O, 12), 92 (M⫹-HCOOH, 81), 81 (11), 66 (16), 65 (48), 64 (71), 63 (100), 62 (36), 61 (13), 53 (53)

B1

I

12.44

B5

II

18.14

A53 A53

III IV

4.08 16.89

A53

V

19.89

a

Rt, retention time.

Identification

Acenaphthenequinone Naphthalene-1,8-dicarboxylic acid 2,5-Dihydroxybenzoic acid (gentisic acid) 1-Naphthoic acid 2-Hydroxybenzoic acid (salicylic acid)

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TABLE 2. Growth of Rhizobium sp. strain CU-A1 and its mutants on various substrates Growth of straina: Substrate

Acenaphthylene Acenaphthenequinone (I) Naphthalene-1,8-dicarboxylic acid (II) Gentisic acid (III) 1-Naphthoic acid (IV) Salicylic acid (V)

CU-A1

A53

B1

B5

⫹⫹ ⫹⫹ ⫹⫹

⫹ ⫹ ⫹

— ⫹ ⫹⫹

— — —

⫹⫹ ⫹⫹ ⫹⫹

— ⫹ —

⫹⫹ ⫹⫹ ⫹⫹

⫹⫹ ⫹⫹ ⫹⫹

a ⫹, ⌬log CFU ⱕ 1 order of magnitude; ⫹⫹, ⌬log CFU ⱖ 1 order of magnitude; —, no growth. ⌬log CFU is the difference between log CFU at day 6 and the initial log CFU.

Besides gentisic acid (III), A53 could accumulate 1-naphthoic acid (IV) and salicylic acid (V): IV and V could accumulate if gentisic acid accumulation inhibits degradation of these compounds further up the pathway. Mutant A53 grew moderately on acenaphthylene as well as on the other metabolites with the exception of gentisic acid and salicylic acid. By comparison with the naphthalene degradation pathways of Rhodococcus sp. strain B4 (7) and Pseudomonas sp. strain U2 (5), gentisic acid was classified as a metabolite in the lower pathway. These data suggested that A53 could use the noncarboxylated ring of 1-naphthoic acid as a source of carbon and energy but it is unable to metabolize the remaining ring. Mutants B1 and B5 could not grow on acenaphthylene, whereas mutant B5 could not utilize naphthalene-1,8-dicarboxylic acid (II), but B1 showed moderate growth on its accumulated metabolite, acenaphthenequinone (I). This might be due to some spontaneous conversion of acenaphthenequinone to naphthalene-1,8-dicarboxylic acid as reported previously (23). However, the spontaneous reaction may occur slowly, giving a low content of naphthalene-1,8-dicarboxylic acid which is not enough to support the maximum growth. All commercially available acenaphthylene preparations used as carbon sources in this work have been found to be contaminated with acenaphthene. Therefore, a control exper-

FIG. 3. HPLC elution profile of the extract of the medium from Rhizobium sp. strain CU-A1 cultivated in MM for 36 h in the presence of 600 mg/liter of acenaphthylene. Retention times are shown in parentheses. I, acenaphthenequinone; II, naphthalene-1,8-dicarboxylic acid; III, gentisic acid; IV, 1-naphthoic acid; V, salicylic acid; ACN, acenaphthylene; ACT, acenaphthene.

iment using acenaphthene as a sole carbon source for Rhizobium sp. strain CU-A1 was performed in parallel. HPLC patterns of the acid extract of acenaphthene-grown Rhizobium sp. strain CU-A1 revealed no degradation product after 36 h of cultivation (data not shown), whereas those of acenaphthylene showed all identified metabolites within the same cultivation period (Fig. 3). This result ensured that all metabolites were solely from acenaphthylene metabolism. Proposed pathway of acenaphthylene degradation in Rhizobium sp. strain CU-A1. Up to now, the complete degradation pathway for acenaphthylene in microorganisms has not been established and only two acenaphthylene-utilizing bacterial isolates have been reported (19, 24). Most published data on bacterial degradation of acenaphthylene reported naphthalene-1,8-dicarboxylic acid as an accumulated main product (11, 22). Our present work shows the proposed acenaphthylene degradation pathway in Rhizobium sp. strain CU-A1 (Fig. 4). Acenaphthenequinone (I) and naphthalene-1,8-dicarboxylic

FIG. 2. Time course of the disappearance of acenaphthylene and its metabolites during the growth of Rhizobium sp. strain CU-A1 for 96 h. F, growth of Rhizobium sp. strain CU-A1; 䊐, acenaphthylene; ■, acenaphthenequinone; ‚, naphthalene-1,8-dicarboxylic acid; Œ, 1-naphthoic acid; 〫, salicylic acid; E, gentisic acid.

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FIG. 4. Proposed pathway of acenaphthylene degradation by Rhizobium sp. strain CU-A1. Structures in brackets represent the hypothetical metabolites from this study. 1, acenaphthylene; 2, cis-acenaphthene-1,2-diol; 3, 1-hydroxy-2-ketoacenaphthene; 4, 1,2-dihydroxyacenaphthylene; 5, maleyl pyruvate; 6, fumaryl pyruvate; I, acenaphthenequinone; II, naphthalene-1,8-dicarboxylic acid; III, gentisic acid; IV, 1-naphthoic acid; V, salicylic acid.

acid (II) were identified as metabolites of acenaphthylene degradation. Therefore, the early oxidation steps of acenaphthylene in strain CU-A1 may occur in the same manner as reported in Beijerinckia sp. (22) and Pseudomonas aeruginosa recombinant strain PA01 harboring pRE695, a plasmid containing a naphthalene dioxygenase gene (nahA) (23). In contrast to the previous study which reported naphthalene-1,8dicarboxylic acid as a dead-end product of acenaphthene and acenaphthylene oxidation by Pseudomonas cepacia F297 (6) and P. aeruginosa PA01 and PA01(pRE695) (23), naphthalene-1,8-dicarboxylic acid may be decarboxylated at one of the carboxyl groups to 1-naphthoic acid (IV) by Rhizobium sp. strain CU-A1. This reaction may occur in the same manner as the oxidation of phenantherene-4,5-dicarboxylic acid, an intermediate of pyrene degradation by Mycobacterium sp. strain PYR-1 (8), Mycobacterium sp. strain KR2 (20), and Mycobacterium sp. strain AP1 (29), in which one carboxyl group was removed from the substrate resulting in the formation of phenanthrene-4-carboxylic acid. In the same manner as phenanthrene-4-carboxylic acid oxidation (20), 1-naphthoic acid might be dioxygenated at position 1,2 resulting in 1,2dihydroxynaphthalene, which would be further oxidized to trans-o-hydroxybenzilidene pyruvic acid, and finally to salicylic acid (V) which was also identified as an intermediate of acenaphthylene degradation by strain CU-A1. As another possibility, 1-naphthoic acid oxidation could occur in the same pathway as reported in Pseudomonas maltophilia (renamed as Stenotrophomonas maltophilia) CSV89. Here, 1-naphthoic acid was twice hydroxylated at the aromatic ring adjacent to the one bearing the carboxyl group, resulting in the formation of 1,2-dihydroxy-8-carboxynaphthalene. The resulting diol was further oxidized via 3-formyl salicylate and 2-hydroxyisophthalate to salicylate (18). Salicylic acid could be further oxidized to another newly identified accumulated intermediate (III) of acenaphthylene metabolism, gentisic acid. Gentisic acid was found to be the product of various kinds of PAH metabolism, such as the degradation of naphthalene via salicylic acid (5), and in fluorene degradation (6). Gentisic acid could be further oxidized

by strain CU-A1 to maleyl pyruvate and fumaryl pyruvate as reported in Ralstonia sp. strain U2 (5, 16). ACKNOWLEDGMENTS This work was supported by the Thailand Research Fund (TRG4580088 and RTA4580010) and a research grant from Chulalongkorn University to the Bioremediation Research Unit, Department of Microbiology, Faculty of Science. T.K. thanks the Ministry of University Affairs (MUA)-CU for a thesis grant. We thank Onruthai Pinyakong for many useful comments and discussion. REFERENCES 1. Altschul, S. F., T. L. Madden, A. A. Scha ¨ffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. 2. Ashok, B. T., and S. Saxena. 1995. Biodegradation of polycyclic aromatic hydrocarbons—a review. J. Sci. Ind. Res. 54:443–451. 3. Cerniglia, C. E. 1992. Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3:351–368. 4. Dean-Raymond, D., and R. Bartha. 1975. Biodegradation of some polynuclear aromatic petroleum components by marine bacteria. Dev. Ind. Microbiol. 16:97–110. 5. Fuenmayor, S. L., M. Wild, A. L. Boyes, and P. A. Williams. 1998. A gene cluster encoding steps in conversion of naphthalene to gentisate in Pseudomonas sp. strain U2. J. Bacteriol. 180:2522–2530. 6. Grifoll, M., S. A. Selifenov, C. V. Galtin, and P. J. Chapman. 1995. Actions of a versatile fluorene-degrading bacterial isolate on polycyclic aromatic compounds. Appl. Environ. Microbiol. 61:3711–3723. 7. Grund, E., B. Denecke, and R. Eichenlaub. 1992. Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4. Appl. Environ. Microbiol. 58:1874–1877. 8. Heitkamp, M. A., J. P. Freeman, D. W. Miller, and C. E. Cerniglia. 1988. Pyrene degradation by a Mycobacterium sp.: identification of a ring oxidation and ring fission products. Appl. Environ. Microbiol. 54:2556–2565. 9. Kasuga, K., H. Nojiri, H. Yamane, T. Kodama, and T. Omori. 1997. Cloning and characterization of the genes involved in the degradation of dibenzofuran by Terrabacter sp. strain DBF63. J. Ferment. Bioeng. 84:387–399. 10. Keith, L. H., and W. A. Telliard. 1979. Priority pollutants. I. A perspective view. Environ. Sci. Technol. 13:416–423. 11. Komatsu, T., T. Omori, and T. Kodama. 1993. Microbial degradation of polycyclic aromatic hydrocarbons acenaphthene and acenaphthylene by a pure bacterial culture. Biosci. Biotech. Biochem. 57:864–865. 12. Langworthy, D. E., R. D. Stapleton, G. S. Sayler, and R. H. Findlay. 2002. Lipid analysis of the response of a sedimentary microbial community to polycyclic aromatic hydrocarbons. Microb. Ecol. 43:189–198. 13. Laurie, A. D., and G. Lloyd-Jones. 1999. The phn genes of Burkholderia sp. strain RP007 constitute a divergent gene cluster for polycyclic aromatic hydrocarbon catabolism. J. Bacteriol. 181:531–540. 14. Lederer, W. H. 1985. Acenaphthylene. In W. H. Lederer (ed.), Regulatory

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