APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2003, p. 186–190 0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.1.186–190.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 1
Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid Rene´ van Herwijnen,1 Dirk Springael,2 Pieter Slot,1 Harrie A. J. Govers,1 and John R. Parsons1* Department of Environmental and Toxicological Chemistry (IBED/MTC), University of Amsterdam, 1018WV Amsterdam, The Netherlands,1 and Vlaamse Instelling voor Technologisch Onderzoek (Vito), B-2400 Mol, Belgium2 Received 29 April 2002/Accepted 25 October 2002
Anthracene, together with other polycyclic aromatic hydrocarbons (PAHs), is a persistent and toxic soil contaminant (9, 11, 15). Pollution by PAHs is usually found on the sites of gas factories and wood preservation plants. Bioremediation is an economically and environmentally attractive solution for cleaning those sites (12). In order to achieve an efficient bioremediation process it is important that the bacteria involved perform a complete degradation pathway so that potentially toxic metabolites do not accumulate in the soil (13). If this is not the case, it is important to know which metabolites are expected to accumulate so other strains able to degrade those metabolites can be added to the soil if necessary. Consequently, it is important to acquire more information on the metabolic pathways of PAH-degrading bacteria. In this study, we examined the pathway for anthracene degradation by Mycobacterium sp. strain LB501T to determine whether metabolites are expected to accumulate. Strain LB501T utilizes anthracene as a sole carbon and energy source. It was isolated from contaminated soil based on adherence to hydrophobic membranes containing sorbed PAHs (2). LB501T grows primarily as a biofilm on solid anthracene. It is suggested that formation of this biofilm is a response of the bacteria to optimize the bioavailability of the substrate (24). At present the only known productive pathway for bacterial degradation of anthracene (7) proceeds through 3-hydroxy-2naphthoic acid, 2,3-dihydroxynaphthalene, and further through a pathway similar to the naphthalene degradation pathway (4). Species known to perform this pathway are from the genera Pseudomonas, Sphingobium, Nocardia, Rhodococcus, and Mycobacterium (4, 7, 8, 10, 21, 22). Degradation from anthracene to 3-hydroxy-2-naphthoic acid proceeds through dioxygenation
(1, 10, 22) and dehydration by which 1,2-dihydroxyanthracene is formed. This compound is cleaved by meta-ring cleavage and the cleavage product is further degraded to 2-hydroxy-3-naphthaldehyde and then to 2-hydroxy-3-naphthoic acid. The ring cleavage product is also converted into the side product 6,7benzocoumarin (7). A recent paper (17) proposed that 6,7benzocoumarin is an intermediate in a cometabolic pathway of anthracene before it is degraded by ring fission enzymes. Which metabolites would be formed from 6,7-benzocoumarin was not suggested. Apart from this pathway some new pathways have been proposed recently. Two dead-end products from anthracene detected for Mycobacterium sp. PYR-1 are 9,10-anthraquinone and 1-methoxy-2-hydroxyanthracene (17). ortho-cleavage of 1,2-dihydroxyanthracene into 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid has also been reported for Mycobacterium sp. PYR-1 and a Rhodococcus species (5, 17). No pathway for further degradation of these compounds has been given. In order to get more information on the degradation pathway of anthracene by LB501T we analyzed extracts of cultures of this strain for metabolites using gas chromatography-mass spectrometry (GC-MS) analysis. We also used mutants that were generated by exposure of the wild-type strain to UV light and which had lost their ability to grow on anthracene but were still able to degrade it. Those mutants were assumed to be blocked at certain steps in the degradation pathway of anthracene, and therefore we assumed that extracts of cultures of those mutants would show other or more metabolites of the pathway. We propose a degradation pathway for anthracene by Mycobacterium sp. strain LB501T on the basis of the metabolites identified.
* Corresponding author. Mailing address: Department of Environmental and Toxicological Chemistry (IBED/MTC), University of Amsterdam, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands. Phone: 31(0)205256580. Fax: 31(0)205256522. E-mail:
[email protected].
Bacterial strains and growth conditions. Mycobacterium sp. strain LB501T was isolated from a PAH-contaminated soil with the use of hydrophobic membranes containing PAHs. Bacteria isolated by this method are different from those
MATERIALS AND METHODS
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Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.
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TABLE 1. Semiquantitative abundances of the identified metabolites Compound
Peak
OPA PCA 6,7-Benzocoumarin cis-4-(2-Hydroxynaphth-3-yl)-2-oxobut-3-enoic acid 1,2-Dihydroxyanthracene 3-Hydroxy-2-naphthoic acid
A B C D E F
Abundancea in strain: LB501T
VM841
VM842
VM843
VM844
349.1 3.7 27.5 24.9 T T
25.9 T 21.2 11.2 52.9
11.8 T 10.5 8.1 20.0
35.3 T 72.4 13.3
21.7 T 9.5 10.6
0.32
T
a
The abundances are given as percentages of peak area relative to the average peak area of two contaminant peaks which were available in all chromatograms in the same ratio. T, peak was only observable as a trace.
et al. (R. van Herwijnen, P. Wattiau, D. Springael, L. Daal, L. Jonker, H. A. J. Govers, and J. R. Parsons, unpublished data) in 50 ml of acetone with 2.6 g of K2CO3 and 3 ml of iodomethane. This mixture was refluxed for 4 h. After refluxing, the acetone was evaporated to dryness and the samples were redissolved in hexane, filtered through a paper filter, and concentrated. A cleanup with a Florisil column was performed on all samples. The Florisil was activated at 600°C for 14 h and deactivated with 10% (wt/wt) water before use. After addition of the sample to the column, the column (0.8 g) was first eluted with 10 ml of pentane and then with 10 ml of dichloromethane. The fractions were collected separately and concentrated after adding 1 ml of 2,2,4-trimethylpentane. These samples were analyzed by GC-MS. GC-MS was performed with an HP 5890A GC equipped with an HP 5970 Mass Selective detector or a Thermoquest Trace GC equipped with a Finnigan Trace MS (ionization energy, 70 eV), each containing a J&W DB-5 (60 m by 0.32 mm) column. To monitor the concentration of OPA, 800 l of culture was sampled in duplicate for each sample point, 100 l of 4 M H2SO4 was added, and 100 l of a 0.73-mg ml⫺1 salicylic acid solution was added before analysis as an internal standard. Analysis was performed by high-performance liquid chromatography with a Waters 600E system controller equipped with a Waters 717 plus autosampler and a Waters 848 Tunable Absorbance detector (wavelength, 212 nm). The column used was a Lichrospher RP18 (125 by 4.0 mm, with a 5-m pore size). The mobile phase consisted of acetonitrile (A) and a 4-g liter⫺1 sodium acetate solution acidified with formic acid to pH 4 (B). The initial ratio was 5% A and 95% B maintained for 7 min, and then A was increased to 100% in 2 min and maintained for 11 min. Next, the ratio was brought back to the initial conditions in 5 min and stabilized for 15 min for the next analysis. Chemicals. All organic solvents were glass-distilled grade or high-performance liquid chromatography grade (Rathburn, Walkerburn, Scotland). Anthracene was supplied by Fluka (Zwijndrecht, The Netherlands), OPA was supplied by Chemservice (West Chester, Pa.), and PCA, 3-hydroxy-2-naphthoic acid, and iodomethane were supplied by Aldrich (Steinheim, Germany). Indole, citrate, pyruvate, and sodium acetate (p.a.) were supplied by Acros (Geel, Belgium). Florisil (60-100 mesh ASTM), K2CO3 (p.a.), glucose, fumarate, benzoic acid, succinate, and Na2SO4 (p.a.) were provided by Merck (Darmstadt, Germany).
RESULTS AND DISCUSSION Identification of metabolites. The GC-MS chromatograms of samples from cultures of the wild-type and mutant strains exposed to anthracene were compared with samples of cultures grown on glucose only and with chemical controls, which consisted of medium with anthracene that was incubated for the same period as the cultures. The metabolites found were not present in any of the controls.
TABLE 2. Mass spectra of the anthracene metabolite 6,7-benzocoumarin formed by Mycobacterium sp. strain LB501T compared to spectra from the literature Compound (source or reference)
6,7-Benzocoumarin 6,7-Benzocoumarin 5,6-Benzocoumarin 7,8-Benzocoumarin
Fragments (m/z [assignment, relative intensity]) ⫹
(this study, peak C) ...............................................196(M , 100), 168(-CO, 48), 139(-CO-CHO, 56), 98(7), 84(6), 70(18), 63(7) (5) ............................................................................196(M⫹, 100), 168(-CO, 41), 139(-CO-CHO, 55), 98(8), 84(7), 70(21), 63(6) (19) ..........................................................................196 (M⫹, 49), 168 (-CO, 100), 139 (-CO-CHO, 89), 70 (12), 63 (15) (19) ..........................................................................196(M⫹, 66), 168(-CO, 100), 139(-CO-CHO, 89), 89(12), 63(15)
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obtained with liquid enrichment cultures (2). LB501T is able to utilize anthracene as its sole carbon and energy source. Apart from the wild-type strain, four UV-generated mutant strains were used, named VM841 to VM844. For the generation of the UV mutant strains, a dilution series of a culture of LB501T, grown on Tris minimal medium containing anthracene as sole carbon source, was plated on Luria-Bertani rich medium agar. Immediately after plating, the plates were exposed to UV light (254 nm, with a distance to lamp of about 3 cm) for 4 s, leading to a survival rate of 10%. Colonies growing on the plates after incubation at 30°C for 1 week were purified twice on Luria-Bertani medium and checked by replica plating for the ability to utilize anthracene as sole carbon source by comparing growth on Tris minimal medium containing either glucose or anthracene. Mutants impaired for growth on anthracene (Ant⫺ mutants) were checked afterwards for the ability to transform anthracene by spraying colonies grown on glucose with anthracene in diethyl ether solution, creating a layer of anthracene crystals over the agar plate. We examined the formation of halos around the colonies after incubation at 30°C for 1 to 2 weeks. Ant⫺ mutants able to form halos were retained for anthracene metabolite analysis. To identify metabolites, cultures were grown in 100 ml of phosphate-buffered minimal medium containing 0.1% (vol/vol) trace element solution (23). Wildtype cultures grown for metabolite analysis contained solid anthracene at a concentration of 11 g liter⫺1. The cultures of the mutants contained 0.4% (wt/wt) glucose as growth substrate and 1 g of solid anthracene liter⫺1. The wild-type culture in which the concentration of o-phthalic acid (OPA) was monitored during growth contained 1 g of solid anthracene liter⫺1. All cultures were incubated in the dark at 25°C on a rotary shaker. Preparation of cell extracts and assay. Cell extracts were prepared according to the method of Phillips et al. (18). Two milliliters of bacterial culture was centrifuged and the pellet was resuspended in 1 ml of minimal medium. Cell were lysed by the addition of 20 l of toluene. Cell debris was removed by centrifugation at 16,000 ⫻ g for 30 s, and the supernatant was immediately used for the experiments. Deactivated cell extracts were made by boiling the extract for 15 to 30 min. Transformation of protocatechuic acid (PCA) or OPA by cell extracts was monitored with a Unicam UV500 UV-visible spectrometer at 290 and 275 nm, respectively. Assays were performed in optical glass cuvettes in 1 ml of cell extract with 10 l of a saturated solution of PCA at a pH of 7. Controls consisted of cell extract without substrate, deactivated cell extract with substrate, and deactivated cell extract without substrate. Analytical procedure. To identify the metabolites, the entire cultures were filtered over glass wool to remove the excess of PAH crystals. The filtered culture was extracted four times with 50 ml of ethyl acetate, twice at neutral pH and twice after acidification to pH 1 with 4 M H2SO4. The samples were concentrated after extraction and dried over a column with Na2SO4 which was rinsed with 20 ml of pentane. The eluent was concentrated and derivatized with iodomethane. Derivatization was performed in accordance with the method of van Herwijnen
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TABLE 3. Mass spectra of tentatively identified anthracene metabolites formed by Mycobacterium sp. strain LB501T Compound
Fragments (m/z [assignment, relative intensity]) ⫹
Derivatized cis-4-(2-hydroxynapth-3-yl)-2-oxobut-3-enoic acid (peak D) ..................270(M , 31), 239(-OCH3, 5), 223(43), 211(-OCH3-CO, 100), 196(-OCH3-CO-CH3, 14), 180(-OCH3-CO-OCH3 , 16), 168(14), 139(14), 76(12) Derivatized 1,2-dihydroxyanthracene (peak E) .............................................................238(M⫹, 3), 223(-CH3, 100), 206(2), 193(-CH3-OCH2, 30), 176(-OCH3-OCH3, 44), 165(29), 151(-OCH3-OCH3-C2H, 11), 139(3), 126(-OCH3-OCH3-C2H-C2H, 3), 111(3), 99(1), 88(13), 75(4)
is consistent with the known degradation pathway of anthracene as presented by Evans and others (4, 5, 7, 16, 17). This pathway proceeds through dihydroxylation at the 1,2 position, meta-cleavage of the 1,2-dihydroxyanthracene, and transfor-
FIG. 1. Proposed degradation pathway for anthracene by Mycobacterium sp. strain LB501T. Compounds D and E were identified tentatively, the compound in a closed box (C) was identified by comparison with a spectrum from the literature, and the compounds between brackets were not identified in our samples. A to F, compounds found in peaks A to F of GC-MS chromatograms.
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GC-MS chromatograms of derivatized samples from cultures of the wild type contained four peaks of possible metabolites. The semiquantitative abundances of the peaks are given in Table 1. Two of these could be identified with the use of authentic samples (A and B), one was identified using a spectrum from the literature (C), and one was identified tentatively (D) (Table 2). Peak A had the same retention time and mass spectrum as an authentic sample of derivatized OPA. This peak was very high in concentration relative to the other peaks. Peak B was a small peak which could be identified with the use of an authentic sample of derivatized PCA. Peak C was identified as derivatized 6,7-benzocoumarin because the mass spectrum (Table 2) showed the same fragmentation as the mass spectrum (Table 2) described by Dean-Ross et al. (5). Mass spectra of 5,6-benzocoumarin and 7,8-benzocoumarin (Table 2), which were also found in the literature (19), showed a fragmentation pattern different from the spectrum in our sample. Peak D was tentatively identified as cis-4-(2-hydroxynaphth-3-yl)-2-oxobut-3-enoic acid (CHOE) (Table 3), which is known as a product of meta-cleavage of 1,2-dihydroxyanthracene. The fragmentation of this compound is consistent with the possible fragments such as m/z 31 (-OCH3) of a derivatized hydroxy group and 28 (-CO) of a carboxy group. Peaks with similar mass spectra have been observed in samples from the cometabolic degradation of phenanthrene, fluoranthene, and anthracene by a Sphingomonas species (van Herwijnen et al., unpublished data). Those peaks were also ascribed to ring-cleavage products. Traces of two more compounds (E and F) were detected in samples of the wildtype strain after they had been detected in samples of mutants (Table 1). Chromatograms from cultures of the mutants VM841 and VM842 contained a peak (peak E) with an M⫹ of 238 which could be tentatively identified as derivatized dihydroxyanthracene (Table 3). Aromatic dioxygenation was tested by incubation of a culture growing on anthracene with indole. When indole has been dioxygenated, a blue stain of indigo should be observed in the culture (6, 25). After some days of incubation a blue stain was indeed observed, showing the ability of LB501T to perform dioxygenation. On the basis of known degradation pathways (4, 5, 7, 17) and the observation of CHOE and 6,7-benzocoumarin in our samples, we proposed that the compound responsible for peak E is derivatized 1,2dihydroxyanthracene. Chromatograms from cultures of the mutants VM843 and VM844 contained a very small peak (peak F) which was identified with the use of a derivatized authentic sample as derivatized 3-hydroxy-2-naphthoic acid. Interpretation of degradation pathway. The observation of CHOE, 6,7-benzocoumarin, and 3-hydroxy-2-naphthoic acid in samples from cultures of the wild type growing on anthracene
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mation of the cleavage product into 6,7-benzocoumarin. The original papers propose 6,7-benzocoumarin to be a dead-end product (4, 7), but a recent paper (17) proposed 6,7-benzocoumarin as an intermediate in the degradation pathway. We cannot determine the status of this compound from our results. It could be a dead-end product but also an intermediate in the route to 3-hydroxy-2-naphthoic acid. The pathway proceeds from CHOE through 3-hydroxy-2-naphthoic acid. The known pathway (4, 7) gives degradation from 3-hydroxy-2-naphthoic acid to 2,3-dihydroxynaphthalene and subsequently through a pathway similar to that for naphthalene metabolism to form salicylate and catechol. The observation of OPA and PCA in our samples suggests that there is also a novel pathway through those compounds (Fig. 1) which proceeds through a PCAdegrading pathway. A cleavage reaction similar to that of 1-hydroxy-2-naphthoic acid in the Aeromonas degradation pathway of phenanthrene (4) can be proposed to perform the cleavage of 3-hydroxy-2-naphthoic acid. Aldolase reactions of the cleavage product, such as those proposed for the degradation of fluorene by Arthobacter sp. strain F101 (3) and fluoranthene by Mycobacterium sp. strain PYR-1 (14), and subsequently a dehydrogenase reaction would give OPA. Known metabolic pathways of OPA through PCA enter the central metabolism as pyruvate by meta-cleavage of PCA or as succinate by orthocleavage of PCA (20). Both reactions are possible, and distinction between the two cannot be made from our data. Transformation and formation of intermediates. We tested growth of LB501T on several possible intermediates. OPA, PCA, 3-hydroxy-2-naphthoic acid, citrate, fumarate, benzoic acid, pyruvate, and succinate were tested to see if they could serve as growth substrates. No growth was observed on 3-hydroxy-2-naphthoic acid, citrate, fumarate, benzoic acid, and succinate. Pyruvate showed good growth and cultures with PCA showed very poor growth. Cultures with OPA showed no visible increase in optical density, but an increase in the amount of cells was observed microscopically. Since OPA was very high in concentration in the samples of the wild-type strain and very poor growth of LB501T was observed on this compound, we monitored the concentration
of the metabolite during growth on anthracene. We monitored two cultures of the wild-type strain (experiment) in comparison with two bacterial controls growing on glucose and two chemical controls (medium with anthracene without bacteria). With this information we wanted to confirm that OPA was really formed from anthracene and that it was not a dead-end product. The concentration of OPA in the experimental cultures is given in Fig. 2. Formation of OPA was not observed in any of the controls. Comparison of the experiment cultures with the controls shows that OPA is formed from anthracene by LB501T during growth, and the decreasing concentration towards the end of the experiment demonstrates that it is not a dead-end product. Experiments with cell extracts showed no degradation of OPA, probably due to a low amount or a low activity of OPA-degrading enzymes. The observations that there is very poor growth on OPA and that in a batch culture growing on anthracene the concentration of OPA decreases towards the end show that transformation of this compound occurs but that it is a very slow process. To examine the transformation of PCA by this bacterium, we performed experiments with cell extracts in which we compared bacteria grown on anthracene and glucose. Transformation of PCA could only be observed in cell extracts of cultures grown on anthracene. In assays with cell extracts from cultures grown on glucose or with any control, transformation of PCA could not be observed. This observation shows that enzymes responsible for degradation of PCA are induced during growth on anthracene and this may explain the poor growth on PCA. In conclusion, the observation of the degradation of anthracene through OPA and PCA suggests a partially new pathway for anthracene-utilizing bacteria. The complete degradation pathway makes Mycobacterium sp. LB501T a useful candidate for soil remediation experiments. ACKNOWLEDGMENTS We are grateful to Bas van de Sande and Merel van der Mark for their practical assistance in metabolite analysis and Annemie Ryn-
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FIG. 2. Concentration of OPA in cultures of Mycobacterium sp. LB501T growing on anthracene (OPA) in comparison with the biomass production (optical density [OD]). The bacterial controls and the chemical controls showed no formation of OPA. Duplicate cultures yielded similar results.
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gaert, Joeri Cuvelier, and Jan Wenzel for assistance in the generation and selection of mutant strains. This work was financed with grants from the EC under the projects BIOVAB (BIO4-CT97-2015) and BIOSTIMUL (QLK3-199-00326).
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ERRATUM Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid Rene´ van Herwijnen, Dirk Springael, Pieter Slot, Harrie A. J. Govers, and John R. Parsons Department of Environmental and Toxicological Chemistry (IBED/MTC), University of Amsterdam, 1018WV Amsterdam, The Netherlands, and Vlaamse Instelling voor Technologisch Onderzoek (Vito), B-2400 Mol, Belgium Volume 69, no. 1, p. 186–190, 2003. Page 188, Fig. 1: The seventh structure (below F, left of A, and above B) should be as follows:
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