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Research Paper
Degradation of sulfonamide antibiotics by Microbacterium sp. strain BR1 – elucidating the downstream pathway Benjamin Ricken1, Oliver Fellmann1, Hans-Peter E. Kohler2, Andreas Scha¨ffer3,4, Philippe Franc¸ois-Xavier Corvini1,4 and Boris Alexander Kolvenbach1 1 Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gruendenstrasse 40, 4132 Muttenz, Switzerland 2 Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Microbiology, Eawag, U¨berlandstrasse 133, 8600 Du¨bendorf, Switzerland 3 Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany 4 State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Hankou Road 22, 210093 Nanjing, China
Microbacterium sp. strain BR1 is among the first bacterial isolates which were proven to degrade sulfonamide antibiotics. The degradation is initiated by an ipso-substitution, initiating the decay of the molecule into sulfur dioxide, the substrate specific heterocyclic moiety as a stable metabolite and benzoquinone imine. The latter appears to be instantaneously reduced to p-aminophenol, as that in turn was detected as the first stable intermediate. This study investigated the downstream pathway of sulfonamide antibiotics by testing the strain’s ability to degrade suspected intermediates of this pathway. While p-aminophenol was degraded, degradation products could not be identified. Benzoquinone was shown to be degraded to hydroquinone and hydroquinone in turn was shown to be degraded to 1,2,4-trihydroxybenzene. The latter is assumed to be the potential substrate for aromatic ring cleavage. However, no products from the degradation of 1,2,4-trihydroxybenzene could be identified. There are no signs of accumulation of intermediates causing oxidative stress, which makes Microbacterium sp. strain BR1 an interesting candidate for industrial waste water treatment.
Introduction Sulfonamide antibiotics used in veterinary and in human medicine are released into the environment because they are only slowly metabolized. Once released, they pose an environmental risk, as their presence might entail the propagation of antibiotic resistant bacteria [1]. In this regard, wastewater treatment plants (WWTPs) are of special interest, as they are known to be hotspots for the propagation of antibiotic resistance genes [2]. In consequence, efforts have been made to remove sulfonamide antibiotics in WWTPs. Elimination approaches based on adsorption of sulfonamide antibiotics, such as sulfamethoxazole, to activated carbon had limited efficiency due to unfavorable physico-chemical Corresponding author: Corvini, P.-X. ([email protected]) http://dx.doi.org/10.1016/j.nbt.2015.03.005 1871-6784/ß 2015 Published by Elsevier B.V.
properties of these compounds [3]. Also, other treatment technologies, such as ozonation or photo-Fenton reactions, are not desirable alternatives, because in these reactions side-products with unknown toxicities might be formed [4]. Furthermore, these treatment technologies need special equipment and require additional energy. Therefore, several research groups have focused on investigating sulfonamide biodegradation in activated sludge systems. In the course of this work, bacteria capable to partially mineralize sulfonamide antibiotics were isolated and two research groups recently reported that the isolates Microbacterium sp. strain BR1 and Achromobacter denitrificans PR1 were able to aerobically degrade a number of structurally related sulfonamide antibiotics [5,6]. Other sulfonamide degrading isolates [7,8] might have similar abilities. www.elsevier.com/locate/nbt
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Research Paper FIGURE 1
Overview on possible transformations of p-aminophenol as found in previous studies. p-Aminophenol (AP) can be either converted to trihydroxybenzene (THB) and directly undergo ring cleavage. It has been also proposed, that THB is first converted to 2-hydroxy-1,4-benzoquinone (HBQ) and hydroquinone (HQ), before the ring was cleaved. Furthermore the formation of p-benzoquinone imine (BQI) can be transformed to benzoquinone (BQ). The ring cleavage occurs either via HQ or HQ and THB.
ipso-Substitution initiates the fragmentation of the sulfonamide molecule in Microbacterium sp. strain BR1 giving rise to p-benzoquinone imine, sulfur dioxide, and the heterocyclic moiety 3amino-5-methylisoxazole (3A5MI) [5]. Other authors investigating the aerobic degradation pathway of sulfonamide antibiotics were also able to identify 3A5MI as a stable metabolite in the medium [5–10] and Majewsky [11] was able to show that 3A5MI did not exhibit any antibiotic activity. The fate of the aniline moiety, which is present in most of the currently available sulfonamide antibiotics on the market, has not been thoroughly investigated yet. Although it can be eventually mineralized [5,8,12,13], intermediates have not been identified so far and it remains unclear how exactly it is metabolized. As p-aminophenol is prone to auto-oxidize, toxic quinones that are likely to impair the viability of the sulfonamide-degrading microorganism may occur as intermediates [13,14]. In Microbacterium sp. strain BR1, p-aminophenol appeared to be one of the first intermediates after ipso-hydroxylation initiated the fragmentation of the sulfonamide antibiotic. The instability of p-aminophenol due to its strong tendency to autoxidize has hampered research efforts for elucidation of degradation pathways. Nonetheless, several pathways were proposed in previous studies [15–17]. A summary of possible pathways based on a literature survey is shown in Fig. 1 [16–20]. However, as these pathways are partially contradictory, many open questions with regard to the degradation of p-aminophenol still remain. Moreover, due to the chemical reactivity of the relevant intermediates 2
it is difficult for some steps to establish to which extent they are catalyzed by enzymes or occurring spontaneously. The present study aimed at the elucidation of the downstream degradation pathway of sulfonamides. Therefore, catabolism of p-aminophenol in Microbacterium sp. strain BR1 was studied by incubating potential degradation intermediates, such as p-benzoquinone [16], hydroquinone, and 1,2,4-trihydroxybenzene [15], with resting cells and crude cell extracts of Microbacterium sp. strain BR1.
Materials and methods Unless stated otherwise, all reagents were of analytical grade and obtained from Sigma–Aldrich (Buchs, Switzerland).
Preparation of resting cells of Microbacterium sp. strain BR1 Microbacterium sp. strain BR1 cells were grown on 25% (vol/vol) Standard I medium (Merck, Grogg Chemie, Stettlen-Deisswil, Switzerland) supplemented with 1 mM SMX. The cultures were incubated on a rotary shaker (Multitron; InforsHT, Bottmingen, Switzerland) at 130 rpm at 288C for 48 hours. Subsequently, cells were washed by centrifugation at 8000 g at 48C for 10 min (Avanti Centrifuge J-25-I, Beckmann Coulter, CA, USA). The supernatant was discarded, and cell pellets were suspended in 20 mM Bis–Tris buffer (pH 7.0 at 48C). This washing step was repeated twice, before re-suspending the cells at an OD600 of 30. The cells stored at 208C did not show decrease in degradational activity for a period of one month.
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SMX concentration was determined using a modified Griess nitrite detection test. Briefly, 10 mL of samples was mixed in 96 well plates with 100 mL of reagent A (0.5% NaNO2 in 0.5 M acetic acid). After 2 min, 120 mL of reagent B (one volume 0.3% 1-naphthol in 30% acetic acid [w/v] which was diluted with 25 volumes of 1 M NaOH) was added. Absorption of the sample at 520 nm was compared to the linear fit of an SMX calibration series with concentrations from 0 to 125 mM.
SMX degradation activity testing To confirm that cells were actively degrading SMX and the necessary enzymes were expressed, cell biomass was brought to an OD600 of 0.5. Of this cell suspension, 0.5 mL was then incubated with 100 mM SMX for 1 hour at room temperature. The remaining SMX concentration was determined photometrically before and after the incubation. Usually, cells degraded between 40 and 60% of initially applied SMX within 1 hour.
Preparation of crude cell extract 5 mL of cell suspensions with an OD600 of 30 was thawed in a Thermomixer comfort heating block (Vaudaux-Eppendorf, Basel, Switzerland) at 378C and 300 rpm for 15 min, before adding 1.5 mg/mL lysozyme (70,000 U/mg; Fluka, Buchs, Switzerland) and another 60 min of incubation. The incubated suspension was centrifuged for 15 min at 8000 g and 48C, and the pellet was re-suspended in 10 mL of 20 mM Bis–Tris buffer (pH 7 at 48C), before adding glass beads (106 mm) to 10% (w/v) and sonication on ice with a Labsonic M device (Sartorius, Goettingen, Germany) for 60 min with 80% amplitude and 0.6 s/s duty cycle. A magnetic stir bar was used at 600 rpm to achieve homogenous sonication. After sonication, cell debris was removed from the extract by centrifugation at 60,000 g at 48C for 20 min. Protein content of the crude cell extracts was determined using Pierce BCA protein assay kit (Thermo Scientific, Olten, Switzerland) and bovine serum albumin was used as a reference.
Incubations with different substrates and sample extraction Substrates, that is, p-aminophenol, hydroquinone, benzoquinone, 1,2,4-trihydroxybenzene, were prepared as stock solutions at a concentration of 4 g/L in methanol (HPLC-grade, J.T. Baker, Munich, Germany). Stock solutions were extemporarily diluted 1:10 with methanol. Incubations with whole cells and crude cell extracts were both performed in a total volume of 4.4 mL, a substrate concentration of 100 mM. The cell suspension was diluted to achieve an OD600 of 5, while crude cell extracts were used undiluted. At different intervals, 0.4 mL of sample was drawn, and rapidly mixed with 1 mL ethyl acetate (J.T. Baker, Munich, Germany) and ca. 50 mg of NaCl. In parallel, 0.4 mL of sample was drawn and mixed with ethyl acetate, NaCl and 50 mL of 6 M HCl (Riedel de Hae¨n, Seelze, Germany). In fact, neutral extraction was necessary for some substrates and metabolites, while others could only be recovered by acidic extraction. The organic fractions were removed and dried over NaSO4. The extraction of the aqueous phase was repeated twice with 1 mL of solvent. The solvent fractions were combined and evaporated to dryness under a gentle nitrogen stream prior to re-dissolution in 80 mL acetonitrile (HPLC grade, J.T. Baker, Munich, Germany). Extracts were derivatized by
adding 20 mL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA, Fluka, Buchs, Switzerland) and incubating the mixture for 45 min at 758C. Samples were subjected to GC–MS analysis after cooling and vortexing.
GC–MS analysis Samples were analyzed on a 7890A series Agilent gas chromatograph (Basel, Switzerland) equipped with a Zebron ZB-5MS column, (30 m by 0.25 mm, 0.25 m m film thickness, Phenomenex) coupled to an Agilent 5975C series mass spectrometer. The carrier gas was helium (1 mL/min). The injection volume was 1 mL (split 1:30). The temperature program was 708C for 3 min, 88C per minute to 2508C; the injector temperature was 1008C; the interface temperature 2808C. The mass selective detector (EI) was operated in the scan mode (mass range m/z 50–600) with an electron energy of 70 eV.
Results and discussion To rule out autooxidation of substrates to be mistaken for apparent degradation, buffer controls were set up for all substrates. In contrast to these controls in which the substrates remained stable, the concentrations of tested substrates (p-aminophenol, benzoquinone, and 1,2,4-trihydroxybenzene) were shown to decrease when incubated with Microbacterium sp. strain BR1 (Fig. 2). No intermediates could be identified in p-aminophenol degradation assays, neither by resting cells nor by cell extracts (Fig. 2a,d). Nonetheless, resting cells and crude cell extracts degraded benzoquinone and concomitantly hydroquinone was formed (Fig. 2b,e). Likewise, the degradation of hydroquinone led to the formation of 1,2,4-trihydroxybenzene (Fig. 2c,f). Interestingly, while incubations of benzoquinone with whole cells and cell extracts yielded quantifiable amounts of hydroquinone, only traces of 1,2,4-trihydroxybenzene could be found in these samples (data not shown). This may be attributed to lower intermediate concentrations of hydroquinone (see Fig. 2b,e) compared to the experiments where hydroquinone was added as a substrate at a concentration of 100 mM. The instability of 1,2,4-trihydroxybenzene may even exacerbate the diminished recovery from samples. Moreover, degradation of 1,2,4-trihydroxybenzene did not occur faster in biological samples than in the buffer controls (data not shown). Additionally, neither maleylacetic acid nor 3-oxoadipic acid, that is, the expected products of ring cleavage of 1,2,4-trihydroxybenzene and its metabolic successor, were identified in the extracts of the reaction mixtures. Based on the transformation reactions reported here on the one hand and on findings from previous studies on the other hand, several possibilities exist, two of which deserve closer attention. In the first proposal, benzoquinone imine, resulting from sulfonamide degradation, may be reduced by reductase activity, but also non-enzymatically by NADH, to yield p-aminophenol [21], which, in turn, might be transformed by a lyase to hydroquinone [15] followed by a further enzymatic oxidation to yield 1,2,4-trihydroxybenzene. Hydroquinone hydroxylase activity has been shown here for Microbacterium sp. strain BR1 and has also previously been reported for Candida parapsilosis CBS604 [22]. However, there is no definite proof yet for a lyase activity as postulated elsewhere [15]. Although p-aminophenol was slowly degraded by Microbacterium sp. strain BR, transformation products to support
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Photometrical determination of SMX
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Research Paper FIGURE 2
Metabolization of assumed intermediates of the downstream degradation pathway of sulfamethoxazole by resting cells and cell extracts of Microbacterium sp. strain BR1. (a) degradation of p-aminophenol by resting cells; (b) degradation of benzoquinone and formation of hydroquinone by resting cells; (c) degradation of hydroquinone and formation of 1,2,4-trihydroxybenzene by resting cells; (d) degradation of p-aminophenol by cell extracts; (e) degradation of benzoquinone and formation of hydroquinone by cell extracts; (f) degradation of hydroquinone and formation of 1,2,4-trihydroxybenzene by cell extracts. Open symbols correspond to negative controls carried out in buffer, while closed symbols correspond to resting cell experiments. Circles indicate the degradation of the added substrate, while triangles show the formation of products. In data points marked with an asterisk (*), the given compounds were not detectable.
the validity of this transformation step could not be identified so far. Compared to the slow kinetic in resting cells, the degradation of p-aminophenol with crude Microbacterium sp. strain BR1 cell extracts was fast (Fig. 2a,d) and also the process of degradation of hydroquinone was slightly faster with crude cell extracts compared to whole cells (Fig. 2c,f). The apparent lower degradation rates of p-aminophenol and hydroquinone might be explained with low uptake rates of both compounds. The log P value of paminophenol for example is 0.013 0.216 and of hydroquinone 0.620 0.203 (predicted properties, calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02, copyright 1994–2015 ACD/Labs). The contrary behavior of benzoquinone was already observed by Gimmler [23] who described the significant faster uptake of benzoquinone compared to hydroquinone into Porphyridium cells. This was explained by the increase of permeability, induced by the binding of benzoquinone to membrane proteins. Therefore, in the case of benzoquinone the uptake barrier may be reduced and thus its degradation by whole cells can occur faster. The second pathway proposed here (Fig. 3) is rationally justified by our experimental data and is also in agreement with data previously reported in the literature [5]. It differs from the first one in the aspect that p-benzoquinone imine is directly hydrolyzed to yield ammonia and benzoquinone. It was shown that benzoquinone could be reduced to hydroquinone by Microbacterium sp. strain BR1 and there is precedence for such a reaction in the literature [17,24]. The described p-benzoquinone reductase by 4
Zhang is FMN and NADPH dependent [24]. In our experiments, none of the two cofactors was added to the crude cell extracts, but 88 mM of benzoquinone was degraded and 17 mM of hydroquinone was formed within 1 hour. Taking into account that no benzoquinone degradation was observed in the abiotic control, and assuming a stoichiometric relation of one reduction equivalent to reduce one benzoquinone, 88 mM of reducing agents would be needed to carry out this reaction. But not more than 0.9 mmol NADH/g biomass dry weight can be expected for aerobe bacteria [25], which would correlate to less than 20 mM in our setup, even if the total amount of NADH has been extracted remained during cell disruption. Furthermore the NADH dependent SMX monooxygenase was not active if no NADH has been added to the crude cell extract. Thus a NADH dependent reductase is unlikely the responsible protein for the benzoquinone transformation. Other, NAD(P)H independent enzymes which are capable to reduce quinones to quinols are malate:quinone or succinate:quinone dehydrogenases [26]. Finally, a hydroxylation step may yield 1,2,4-trihydroxybenzene as the substrate for ring cleavage. Whether the presence of paminophenol in incubations of cell extracts with sulfonamides is an artifact due to non-enzymatic reduction by the added NADH remains to be elucidated [27]. Ring cleavage of 1,2,4-trihydroxybenzene by intradiol dioxygenases was shown to occur in several microorganisms [28–30]. Therefore, it seems reasonable to propose that sulfonamide transformation in Microbacterium sp. strain BR1 proceeded via
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FIGURE 3
Proposed degradation pathway of SMX by Microbacterium sp. strain BR1. The oxidation of SMX (a) at the ipso position leads to an instable intermediate (b) after electron rearrangement which subsequently fragments to 3A5MI (h), p-benzoquinone imine (c), and SO2 as previously postulated [5]. Upon hydrolysis to benzoquinone (d), the latter may be reduced to hydroquinone (e) which is then further transformed to 1,2,4-trihydroxybenzene (f). Alternatively, p-benzoquinone imine may be reduced to p-aminophenol (g), which could then be further transformed to hydroquinone by hydrolase activity.
1,2,4-trihydroxybenzene as the ring cleavage substrate, and that p-aminophenol most likely was not a physiological intermediate in sulfonamide degradation. Our results indicate that p-benzoquinone imine and benzoquinone, compounds with redoxcycling activity, are intermediates in the degradation pathway for sulfonamides and that Microbacterium sp. strain BR1 appears
to harbor biocatalytic tools to transform these potentially toxic intermediates.
Acknowledgement This work was supported by the Swiss National Science Foundation grant number 310030_146927.
References [1] Heuer H, Smalla K. Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months. Environ Microbiol 2007;9:657–66. http://dx.doi.org/10.1111/j.1462-2920.2006.01185.x. [2] Jury KL, Khan SJ, Vancov T, Stuetz RM, Ashbolt NJ. Are sewage treatment plants promoting antibiotic resistance? Crit Rev Environ Sci Technol 2011;41:243–70. http://dx.doi.org/10.1080/10643380902772589. ¨ hler M, Sterkele B, Joss A, Siegrist H, Traber J, et al. Einsatz von [3] Zwickenpflug B, Bo Pulveraktivkohle zur Elimination von Mikroverunreinigungen aus kommuna¨ bendorf 2010. lem Abwasser;Du [4] Huber MM, Canonica S, Park G-Y, von Gunten U. Oxidation of pharmaceuticals during ozonation and advanced oxidation processes. Environ Sci Technol 2003;37:1016–24. [5] Ricken B, Corvini PFX, Cichocka D, Parisi M, Lenz M, Wyss D, et al. ipsoHydroxylation and subsequent fragmentation – a novel microbial strategy to eliminate sulfonamide antibiotics. Appl Environ Microbiol 2013;79:5550–8. http://dx.doi.org/10.1128/aem.00911-13. [6] Reis PJM, Reis AC, Ricken B, Kolvenbach BA, Manaia CM, Corvini PFX, et al. Biodegradation of sulfamethoxazole and other sulfonamides by Achromobacter denitrificans PR1. J Hazard Mater 2014. http://dx.doi.org/10.1016/j.jhazmat. 2014.08.039. [7] Tappe W, Herbst M, Hofmann D, Koeppchen S, Kummer S, Thiele BB, et al. Degradation of sulfadiazine by Microbacterium lacus strain SDZm4 isolated from lysimeters previously manured with slurry from sulfadiazine medicated pigs. Appl Environ Microbiol 2013;79:2572–7. http://dx.doi.org/10.1128/aem.03636-12. [8] Topp E, Chapman R, Devers-Lamrani M, Hartmann A, Marti R, Martin-Laurent F, et al. Accelerated biodegradation of veterinary antibiotics in agricultural soil following long-term exposure, and isolation of a sulfamethazine-degrading sp.. J Environ Qual 2013;42:173. http://dx.doi.org/10.2134/jeq2012.0162. ¨ ller E, Schu ¨ssler W, Horn H, Lemmer H. Aerobic biodegradation of the [9] Mu sulfonamide antibiotic sulfamethoxazole by activated sludge applied as cosubstrate and sole carbon and nitrogen source. Chemosphere 2013;92:1–10. http://dx.doi.org/10.1016/j.chemosphere.2013.02.070.
[10] Hartig C. Analytik, Vorkommen und Verhalten aromatischer Sulfonamide in der aquatischen Umwelt; 2000. [11] Majewsky M, Wagner D, Delay M, Bra¨se S, Yargeau V, Horn H. Antibacterial activity of sulfamethoxazole transformation products (TPs): general relevance for sulfonamide TPs modified at the para position. Chem Res Toxicol 2014. http://dx.doi.org/10.1021/tx500267x. [12] Bouju H, Ricken B, Beffa T, Corvini PF-X, Kolvenbach BA. Isolation of bacterial strains capable of sulfamethoxazole mineralization from an acclimated membrane bioreactor. Appl Environ Microbiol 2012;78:277–9. http://dx.doi.org/ 10.1128/AEM.05888-11. [13] Monks TJ, Hanzlik RP, Cohen GM, Ross D, Graham DG. Quinone chemistry and toxicity. Toxicol Appl Pharmacol 1992;112:2–16. [14] Bolton JL, Trush MA, Penning TM, Dryhurst G, Monks TJ. Role of quinones in toxicology. Chem Res Toxicol 2000;13:135–60. http://dx.doi.org/10.1021/ tx9902082. [15] Takenaka S, Okugawa S, Kadowaki M, Murakami S, Aoki K. The metabolic pathway of 4-aminophenol in Burkholderia sp. strain AK-5 siffers from that of aniline and aniline with C-4 substituents. Appl Environ Microbiol 2003;69:5410–3. http://dx.doi.org/10.1128/AEM.69.9.5410. [16] Min S, Risheng Y, Yahua Y, Shengsong D, Wenxia G. Degradation of 4-aminophenol by hydrogen peroxide oxidation using enzyme from Serratia marcescens as catalyst. Front Environ Sci Eng China 2007;1:95–8. http://dx.doi.org/ 10.1007/s11783-007-0018-0. [17] Zhang S, Sun W, Xu L, Zheng X, Chu X, Tian J, et al. Identification of the paranitrophenol catabolic pathway, and characterization of three enzymes involved in the hydroquinone pathway, in Peudomonas sp. 1-7. BMC Microbiol 2012;12:27. http://dx.doi.org/10.1186/1471-2180-12-27. [18] Takenaka S, Koshiya J, Okugawa S, Takata A, Murakami S, Aoki K. Fe-superoxide dismutase and 2-hydroxy-1,4-benzoquinone reductase preclude the auto-oxidation step in 4-aminophenol metabolism by Burkholderia sp. strain AK-5. Biodegradation 2011;22:1–11. http://dx.doi.org/10.1007/s10532-010-9369-5.
www.elsevier.com/locate/nbt 5 Please cite this article in press as: Ricken, B. et al., Degradation of sulfonamide antibiotics by Microbacterium sp. strain BR1 – elucidating the downstream pathway, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.03.005
NBT-770; No of Pages 6 RESEARCH PAPER
Research Paper
[19] Chauhan A, Samanta SK, Jain RK. Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway. J Appl Microbiol 2000;88:764–72. http://dx.doi.org/10.1046/j.1365-2672.2000.01018.x. [20] Zhao JS, Singh A, Huang XD, Ward OP. Biotransformation of hydroxylaminobenzene and aminophenol by Pseudomonas putida 2NP8 cells grown in the presence of 3-nitrophenol. Appl Environ Microbiol 2000;66:2336–42. [21] Kwon SJ, Yang H, Jo K, Kwak J. An electrochemical immunosensor using paminophenol redox cycling by NADH on a self-assembled monolayer and ferrocene-modified Au electrodes. Analyst 2008;133:1599–604. http:// dx.doi.org/10.1039/b806302h. [22] Eppink MH, Cammaart E, Van Wassenaar D, Middelhoven WJ, van Berkel WJ. Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604. Eur J Biochem 2000;267:6832–40. [23] Gimmler H. Different uptake and binding of benzoquinone and hydroquinone in unicellular algae and type a chloroplasts. Z Pflanzenphysiol 1976;78:76–81. http://dx.doi.org/10.1016/S0044-328X(76)80142-0. [24] Zhang J-J, Liu H, Xiao Y, Zhang X-E, Zhou N-Y. Identification and characterization of catabolic para-nitrophenol 4-monooxygenase and para-benzoquinone reductase from Pseudomonas sp. strain WBC-3. J Bacteriol 2009;191:2703–10. http://dx.doi.org/10.1128/jb.01566-08.
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[25] Wimpenny JW. A. Firth, Levels of nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide in facultative bacteria and the effect of oxygen. J Bacteriol 1972;111:24–32. [26] Kabashima Y, Sone N, Kusumoto T, Sakamoto J. Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3. J Bioenerg Biomembr 2013;45:131–6. http://dx.doi.org/10.1007/s10863-012-9485-5. [27] Walter A, Wu J, Flechsig G, Haake D, Wang J. Redox cycling amplified electrochemical detection of DNA hybridization: application to pathogen E. coli bacterial RNA. Anal Chim Acta 2011;689:29–33. http://dx.doi.org/10.1016/ j.aca.2011.01.014.Redox. [28] Kolvenbach BA, Dobrowinski H, Fousek J, Vlcek C, Scha¨ffer A, Gabriel FLP, et al. An unexpected gene cluster for downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3. Appl Microbiol Biotechnol 2012;93:1315–24. http://dx.doi.org/10.1007/s00253-011-3451-8. [29] Kwon D-Y, Suma Y, Tiwari M, Choi J, Kang L-W, Lee J-K, et al. Decomposition of aromatic hydrocarbon intermediates by recombinant hydroxyquinol 1,2-dioxygenase from Arthrobacter chlorophenolicus A6 and its structure characterization. Int Biodeterior Biodegrad 2014. http://dx.doi.org/10.1016/j.ibiod.2014.03.002. ´ ska D. Intradiol dioxygenases – the key [30] Guzik U, Hupert-Kocurek K, Wojcieszyn enzymes in xenobiotics degradation. In: Chamy R, Rosenkranz F, editors. Biodegrad. Hazard. Spec. Prod.. InTech; 2013. p. 129–53.
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Research Paper
Degradation of sulfonamide antibiotics by Microbacterium sp. strain BR1 – elucidating the downstream pathway Benjamin Ricken1, Oliver Fellmann1, Hans-Peter E. Kohler2, Andreas Scha¨ffer3,4, Philippe Franc¸ois-Xavier Corvini1,4 and Boris Alexander Kolvenbach1 1 Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gruendenstrasse 40, 4132 Muttenz, Switzerland 2 Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Microbiology, Eawag, U¨berlandstrasse 133, 8600 Du¨bendorf, Switzerland 3 Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany 4 State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Hankou Road 22, 210093 Nanjing, China
Microbacterium sp. strain BR1 is among the first bacterial isolates which were proven to degrade sulfonamide antibiotics. The degradation is initiated by an ipso-substitution, initiating the decay of the molecule into sulfur dioxide, the substrate specific heterocyclic moiety as a stable metabolite and benzoquinone imine. The latter appears to be instantaneously reduced to p-aminophenol, as that in turn was detected as the first stable intermediate. This study investigated the downstream pathway of sulfonamide antibiotics by testing the strain’s ability to degrade suspected intermediates of this pathway. While p-aminophenol was degraded, degradation products could not be identified. Benzoquinone was shown to be degraded to hydroquinone and hydroquinone in turn was shown to be degraded to 1,2,4-trihydroxybenzene. The latter is assumed to be the potential substrate for aromatic ring cleavage. However, no products from the degradation of 1,2,4-trihydroxybenzene could be identified. There are no signs of accumulation of intermediates causing oxidative stress, which makes Microbacterium sp. strain BR1 an interesting candidate for industrial waste water treatment.
Introduction Sulfonamide antibiotics used in veterinary and in human medicine are released into the environment because they are only slowly metabolized. Once released, they pose an environmental risk, as their presence might entail the propagation of antibiotic resistant bacteria [1]. In this regard, wastewater treatment plants (WWTPs) are of special interest, as they are known to be hotspots for the propagation of antibiotic resistance genes [2]. In consequence, efforts have been made to remove sulfonamide antibiotics in WWTPs. Elimination approaches based on adsorption of sulfonamide antibiotics, such as sulfamethoxazole, to activated carbon had limited efficiency due to unfavorable physico-chemical Corresponding author: Corvini, P.-X. ([email protected]) http://dx.doi.org/10.1016/j.nbt.2015.03.005 1871-6784/ß 2015 Published by Elsevier B.V.
properties of these compounds [3]. Also, other treatment technologies, such as ozonation or photo-Fenton reactions, are not desirable alternatives, because in these reactions side-products with unknown toxicities might be formed [4]. Furthermore, these treatment technologies need special equipment and require additional energy. Therefore, several research groups have focused on investigating sulfonamide biodegradation in activated sludge systems. In the course of this work, bacteria capable to partially mineralize sulfonamide antibiotics were isolated and two research groups recently reported that the isolates Microbacterium sp. strain BR1 and Achromobacter denitrificans PR1 were able to aerobically degrade a number of structurally related sulfonamide antibiotics [5,6]. Other sulfonamide degrading isolates [7,8] might have similar abilities. www.elsevier.com/locate/nbt
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Research Paper FIGURE 1
Overview on possible transformations of p-aminophenol as found in previous studies. p-Aminophenol (AP) can be either converted to trihydroxybenzene (THB) and directly undergo ring cleavage. It has been also proposed, that THB is first converted to 2-hydroxy-1,4-benzoquinone (HBQ) and hydroquinone (HQ), before the ring was cleaved. Furthermore the formation of p-benzoquinone imine (BQI) can be transformed to benzoquinone (BQ). The ring cleavage occurs either via HQ or HQ and THB.
ipso-Substitution initiates the fragmentation of the sulfonamide molecule in Microbacterium sp. strain BR1 giving rise to p-benzoquinone imine, sulfur dioxide, and the heterocyclic moiety 3amino-5-methylisoxazole (3A5MI) [5]. Other authors investigating the aerobic degradation pathway of sulfonamide antibiotics were also able to identify 3A5MI as a stable metabolite in the medium [5–10] and Majewsky [11] was able to show that 3A5MI did not exhibit any antibiotic activity. The fate of the aniline moiety, which is present in most of the currently available sulfonamide antibiotics on the market, has not been thoroughly investigated yet. Although it can be eventually mineralized [5,8,12,13], intermediates have not been identified so far and it remains unclear how exactly it is metabolized. As p-aminophenol is prone to auto-oxidize, toxic quinones that are likely to impair the viability of the sulfonamide-degrading microorganism may occur as intermediates [13,14]. In Microbacterium sp. strain BR1, p-aminophenol appeared to be one of the first intermediates after ipso-hydroxylation initiated the fragmentation of the sulfonamide antibiotic. The instability of p-aminophenol due to its strong tendency to autoxidize has hampered research efforts for elucidation of degradation pathways. Nonetheless, several pathways were proposed in previous studies [15–17]. A summary of possible pathways based on a literature survey is shown in Fig. 1 [16–20]. However, as these pathways are partially contradictory, many open questions with regard to the degradation of p-aminophenol still remain. Moreover, due to the chemical reactivity of the relevant intermediates 2
it is difficult for some steps to establish to which extent they are catalyzed by enzymes or occurring spontaneously. The present study aimed at the elucidation of the downstream degradation pathway of sulfonamides. Therefore, catabolism of p-aminophenol in Microbacterium sp. strain BR1 was studied by incubating potential degradation intermediates, such as p-benzoquinone [16], hydroquinone, and 1,2,4-trihydroxybenzene [15], with resting cells and crude cell extracts of Microbacterium sp. strain BR1.
Materials and methods Unless stated otherwise, all reagents were of analytical grade and obtained from Sigma–Aldrich (Buchs, Switzerland).
Preparation of resting cells of Microbacterium sp. strain BR1 Microbacterium sp. strain BR1 cells were grown on 25% (vol/vol) Standard I medium (Merck, Grogg Chemie, Stettlen-Deisswil, Switzerland) supplemented with 1 mM SMX. The cultures were incubated on a rotary shaker (Multitron; InforsHT, Bottmingen, Switzerland) at 130 rpm at 288C for 48 hours. Subsequently, cells were washed by centrifugation at 8000 g at 48C for 10 min (Avanti Centrifuge J-25-I, Beckmann Coulter, CA, USA). The supernatant was discarded, and cell pellets were suspended in 20 mM Bis–Tris buffer (pH 7.0 at 48C). This washing step was repeated twice, before re-suspending the cells at an OD600 of 30. The cells stored at 208C did not show decrease in degradational activity for a period of one month.
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SMX concentration was determined using a modified Griess nitrite detection test. Briefly, 10 mL of samples was mixed in 96 well plates with 100 mL of reagent A (0.5% NaNO2 in 0.5 M acetic acid). After 2 min, 120 mL of reagent B (one volume 0.3% 1-naphthol in 30% acetic acid [w/v] which was diluted with 25 volumes of 1 M NaOH) was added. Absorption of the sample at 520 nm was compared to the linear fit of an SMX calibration series with concentrations from 0 to 125 mM.
SMX degradation activity testing To confirm that cells were actively degrading SMX and the necessary enzymes were expressed, cell biomass was brought to an OD600 of 0.5. Of this cell suspension, 0.5 mL was then incubated with 100 mM SMX for 1 hour at room temperature. The remaining SMX concentration was determined photometrically before and after the incubation. Usually, cells degraded between 40 and 60% of initially applied SMX within 1 hour.
Preparation of crude cell extract 5 mL of cell suspensions with an OD600 of 30 was thawed in a Thermomixer comfort heating block (Vaudaux-Eppendorf, Basel, Switzerland) at 378C and 300 rpm for 15 min, before adding 1.5 mg/mL lysozyme (70,000 U/mg; Fluka, Buchs, Switzerland) and another 60 min of incubation. The incubated suspension was centrifuged for 15 min at 8000 g and 48C, and the pellet was re-suspended in 10 mL of 20 mM Bis–Tris buffer (pH 7 at 48C), before adding glass beads (106 mm) to 10% (w/v) and sonication on ice with a Labsonic M device (Sartorius, Goettingen, Germany) for 60 min with 80% amplitude and 0.6 s/s duty cycle. A magnetic stir bar was used at 600 rpm to achieve homogenous sonication. After sonication, cell debris was removed from the extract by centrifugation at 60,000 g at 48C for 20 min. Protein content of the crude cell extracts was determined using Pierce BCA protein assay kit (Thermo Scientific, Olten, Switzerland) and bovine serum albumin was used as a reference.
Incubations with different substrates and sample extraction Substrates, that is, p-aminophenol, hydroquinone, benzoquinone, 1,2,4-trihydroxybenzene, were prepared as stock solutions at a concentration of 4 g/L in methanol (HPLC-grade, J.T. Baker, Munich, Germany). Stock solutions were extemporarily diluted 1:10 with methanol. Incubations with whole cells and crude cell extracts were both performed in a total volume of 4.4 mL, a substrate concentration of 100 mM. The cell suspension was diluted to achieve an OD600 of 5, while crude cell extracts were used undiluted. At different intervals, 0.4 mL of sample was drawn, and rapidly mixed with 1 mL ethyl acetate (J.T. Baker, Munich, Germany) and ca. 50 mg of NaCl. In parallel, 0.4 mL of sample was drawn and mixed with ethyl acetate, NaCl and 50 mL of 6 M HCl (Riedel de Hae¨n, Seelze, Germany). In fact, neutral extraction was necessary for some substrates and metabolites, while others could only be recovered by acidic extraction. The organic fractions were removed and dried over NaSO4. The extraction of the aqueous phase was repeated twice with 1 mL of solvent. The solvent fractions were combined and evaporated to dryness under a gentle nitrogen stream prior to re-dissolution in 80 mL acetonitrile (HPLC grade, J.T. Baker, Munich, Germany). Extracts were derivatized by
adding 20 mL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA, Fluka, Buchs, Switzerland) and incubating the mixture for 45 min at 758C. Samples were subjected to GC–MS analysis after cooling and vortexing.
GC–MS analysis Samples were analyzed on a 7890A series Agilent gas chromatograph (Basel, Switzerland) equipped with a Zebron ZB-5MS column, (30 m by 0.25 mm, 0.25 m m film thickness, Phenomenex) coupled to an Agilent 5975C series mass spectrometer. The carrier gas was helium (1 mL/min). The injection volume was 1 mL (split 1:30). The temperature program was 708C for 3 min, 88C per minute to 2508C; the injector temperature was 1008C; the interface temperature 2808C. The mass selective detector (EI) was operated in the scan mode (mass range m/z 50–600) with an electron energy of 70 eV.
Results and discussion To rule out autooxidation of substrates to be mistaken for apparent degradation, buffer controls were set up for all substrates. In contrast to these controls in which the substrates remained stable, the concentrations of tested substrates (p-aminophenol, benzoquinone, and 1,2,4-trihydroxybenzene) were shown to decrease when incubated with Microbacterium sp. strain BR1 (Fig. 2). No intermediates could be identified in p-aminophenol degradation assays, neither by resting cells nor by cell extracts (Fig. 2a,d). Nonetheless, resting cells and crude cell extracts degraded benzoquinone and concomitantly hydroquinone was formed (Fig. 2b,e). Likewise, the degradation of hydroquinone led to the formation of 1,2,4-trihydroxybenzene (Fig. 2c,f). Interestingly, while incubations of benzoquinone with whole cells and cell extracts yielded quantifiable amounts of hydroquinone, only traces of 1,2,4-trihydroxybenzene could be found in these samples (data not shown). This may be attributed to lower intermediate concentrations of hydroquinone (see Fig. 2b,e) compared to the experiments where hydroquinone was added as a substrate at a concentration of 100 mM. The instability of 1,2,4-trihydroxybenzene may even exacerbate the diminished recovery from samples. Moreover, degradation of 1,2,4-trihydroxybenzene did not occur faster in biological samples than in the buffer controls (data not shown). Additionally, neither maleylacetic acid nor 3-oxoadipic acid, that is, the expected products of ring cleavage of 1,2,4-trihydroxybenzene and its metabolic successor, were identified in the extracts of the reaction mixtures. Based on the transformation reactions reported here on the one hand and on findings from previous studies on the other hand, several possibilities exist, two of which deserve closer attention. In the first proposal, benzoquinone imine, resulting from sulfonamide degradation, may be reduced by reductase activity, but also non-enzymatically by NADH, to yield p-aminophenol [21], which, in turn, might be transformed by a lyase to hydroquinone [15] followed by a further enzymatic oxidation to yield 1,2,4-trihydroxybenzene. Hydroquinone hydroxylase activity has been shown here for Microbacterium sp. strain BR1 and has also previously been reported for Candida parapsilosis CBS604 [22]. However, there is no definite proof yet for a lyase activity as postulated elsewhere [15]. Although p-aminophenol was slowly degraded by Microbacterium sp. strain BR, transformation products to support
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Research Paper
Photometrical determination of SMX
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Research Paper FIGURE 2
Metabolization of assumed intermediates of the downstream degradation pathway of sulfamethoxazole by resting cells and cell extracts of Microbacterium sp. strain BR1. (a) degradation of p-aminophenol by resting cells; (b) degradation of benzoquinone and formation of hydroquinone by resting cells; (c) degradation of hydroquinone and formation of 1,2,4-trihydroxybenzene by resting cells; (d) degradation of p-aminophenol by cell extracts; (e) degradation of benzoquinone and formation of hydroquinone by cell extracts; (f) degradation of hydroquinone and formation of 1,2,4-trihydroxybenzene by cell extracts. Open symbols correspond to negative controls carried out in buffer, while closed symbols correspond to resting cell experiments. Circles indicate the degradation of the added substrate, while triangles show the formation of products. In data points marked with an asterisk (*), the given compounds were not detectable.
the validity of this transformation step could not be identified so far. Compared to the slow kinetic in resting cells, the degradation of p-aminophenol with crude Microbacterium sp. strain BR1 cell extracts was fast (Fig. 2a,d) and also the process of degradation of hydroquinone was slightly faster with crude cell extracts compared to whole cells (Fig. 2c,f). The apparent lower degradation rates of p-aminophenol and hydroquinone might be explained with low uptake rates of both compounds. The log P value of paminophenol for example is 0.013 0.216 and of hydroquinone 0.620 0.203 (predicted properties, calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02, copyright 1994–2015 ACD/Labs). The contrary behavior of benzoquinone was already observed by Gimmler [23] who described the significant faster uptake of benzoquinone compared to hydroquinone into Porphyridium cells. This was explained by the increase of permeability, induced by the binding of benzoquinone to membrane proteins. Therefore, in the case of benzoquinone the uptake barrier may be reduced and thus its degradation by whole cells can occur faster. The second pathway proposed here (Fig. 3) is rationally justified by our experimental data and is also in agreement with data previously reported in the literature [5]. It differs from the first one in the aspect that p-benzoquinone imine is directly hydrolyzed to yield ammonia and benzoquinone. It was shown that benzoquinone could be reduced to hydroquinone by Microbacterium sp. strain BR1 and there is precedence for such a reaction in the literature [17,24]. The described p-benzoquinone reductase by 4
Zhang is FMN and NADPH dependent [24]. In our experiments, none of the two cofactors was added to the crude cell extracts, but 88 mM of benzoquinone was degraded and 17 mM of hydroquinone was formed within 1 hour. Taking into account that no benzoquinone degradation was observed in the abiotic control, and assuming a stoichiometric relation of one reduction equivalent to reduce one benzoquinone, 88 mM of reducing agents would be needed to carry out this reaction. But not more than 0.9 mmol NADH/g biomass dry weight can be expected for aerobe bacteria [25], which would correlate to less than 20 mM in our setup, even if the total amount of NADH has been extracted remained during cell disruption. Furthermore the NADH dependent SMX monooxygenase was not active if no NADH has been added to the crude cell extract. Thus a NADH dependent reductase is unlikely the responsible protein for the benzoquinone transformation. Other, NAD(P)H independent enzymes which are capable to reduce quinones to quinols are malate:quinone or succinate:quinone dehydrogenases [26]. Finally, a hydroxylation step may yield 1,2,4-trihydroxybenzene as the substrate for ring cleavage. Whether the presence of paminophenol in incubations of cell extracts with sulfonamides is an artifact due to non-enzymatic reduction by the added NADH remains to be elucidated [27]. Ring cleavage of 1,2,4-trihydroxybenzene by intradiol dioxygenases was shown to occur in several microorganisms [28–30]. Therefore, it seems reasonable to propose that sulfonamide transformation in Microbacterium sp. strain BR1 proceeded via
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FIGURE 3
Proposed degradation pathway of SMX by Microbacterium sp. strain BR1. The oxidation of SMX (a) at the ipso position leads to an instable intermediate (b) after electron rearrangement which subsequently fragments to 3A5MI (h), p-benzoquinone imine (c), and SO2 as previously postulated [5]. Upon hydrolysis to benzoquinone (d), the latter may be reduced to hydroquinone (e) which is then further transformed to 1,2,4-trihydroxybenzene (f). Alternatively, p-benzoquinone imine may be reduced to p-aminophenol (g), which could then be further transformed to hydroquinone by hydrolase activity.
1,2,4-trihydroxybenzene as the ring cleavage substrate, and that p-aminophenol most likely was not a physiological intermediate in sulfonamide degradation. Our results indicate that p-benzoquinone imine and benzoquinone, compounds with redoxcycling activity, are intermediates in the degradation pathway for sulfonamides and that Microbacterium sp. strain BR1 appears
to harbor biocatalytic tools to transform these potentially toxic intermediates.
Acknowledgement This work was supported by the Swiss National Science Foundation grant number 310030_146927.
References [1] Heuer H, Smalla K. Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months. Environ Microbiol 2007;9:657–66. http://dx.doi.org/10.1111/j.1462-2920.2006.01185.x. [2] Jury KL, Khan SJ, Vancov T, Stuetz RM, Ashbolt NJ. Are sewage treatment plants promoting antibiotic resistance? Crit Rev Environ Sci Technol 2011;41:243–70. http://dx.doi.org/10.1080/10643380902772589. ¨ hler M, Sterkele B, Joss A, Siegrist H, Traber J, et al. Einsatz von [3] Zwickenpflug B, Bo Pulveraktivkohle zur Elimination von Mikroverunreinigungen aus kommuna¨ bendorf 2010. lem Abwasser;Du [4] Huber MM, Canonica S, Park G-Y, von Gunten U. Oxidation of pharmaceuticals during ozonation and advanced oxidation processes. Environ Sci Technol 2003;37:1016–24. [5] Ricken B, Corvini PFX, Cichocka D, Parisi M, Lenz M, Wyss D, et al. ipsoHydroxylation and subsequent fragmentation – a novel microbial strategy to eliminate sulfonamide antibiotics. Appl Environ Microbiol 2013;79:5550–8. http://dx.doi.org/10.1128/aem.00911-13. [6] Reis PJM, Reis AC, Ricken B, Kolvenbach BA, Manaia CM, Corvini PFX, et al. Biodegradation of sulfamethoxazole and other sulfonamides by Achromobacter denitrificans PR1. J Hazard Mater 2014. http://dx.doi.org/10.1016/j.jhazmat. 2014.08.039. [7] Tappe W, Herbst M, Hofmann D, Koeppchen S, Kummer S, Thiele BB, et al. Degradation of sulfadiazine by Microbacterium lacus strain SDZm4 isolated from lysimeters previously manured with slurry from sulfadiazine medicated pigs. Appl Environ Microbiol 2013;79:2572–7. http://dx.doi.org/10.1128/aem.03636-12. [8] Topp E, Chapman R, Devers-Lamrani M, Hartmann A, Marti R, Martin-Laurent F, et al. Accelerated biodegradation of veterinary antibiotics in agricultural soil following long-term exposure, and isolation of a sulfamethazine-degrading sp.. J Environ Qual 2013;42:173. http://dx.doi.org/10.2134/jeq2012.0162. ¨ ller E, Schu ¨ssler W, Horn H, Lemmer H. Aerobic biodegradation of the [9] Mu sulfonamide antibiotic sulfamethoxazole by activated sludge applied as cosubstrate and sole carbon and nitrogen source. Chemosphere 2013;92:1–10. http://dx.doi.org/10.1016/j.chemosphere.2013.02.070.
[10] Hartig C. Analytik, Vorkommen und Verhalten aromatischer Sulfonamide in der aquatischen Umwelt; 2000. [11] Majewsky M, Wagner D, Delay M, Bra¨se S, Yargeau V, Horn H. Antibacterial activity of sulfamethoxazole transformation products (TPs): general relevance for sulfonamide TPs modified at the para position. Chem Res Toxicol 2014. http://dx.doi.org/10.1021/tx500267x. [12] Bouju H, Ricken B, Beffa T, Corvini PF-X, Kolvenbach BA. Isolation of bacterial strains capable of sulfamethoxazole mineralization from an acclimated membrane bioreactor. Appl Environ Microbiol 2012;78:277–9. http://dx.doi.org/ 10.1128/AEM.05888-11. [13] Monks TJ, Hanzlik RP, Cohen GM, Ross D, Graham DG. Quinone chemistry and toxicity. Toxicol Appl Pharmacol 1992;112:2–16. [14] Bolton JL, Trush MA, Penning TM, Dryhurst G, Monks TJ. Role of quinones in toxicology. Chem Res Toxicol 2000;13:135–60. http://dx.doi.org/10.1021/ tx9902082. [15] Takenaka S, Okugawa S, Kadowaki M, Murakami S, Aoki K. The metabolic pathway of 4-aminophenol in Burkholderia sp. strain AK-5 siffers from that of aniline and aniline with C-4 substituents. Appl Environ Microbiol 2003;69:5410–3. http://dx.doi.org/10.1128/AEM.69.9.5410. [16] Min S, Risheng Y, Yahua Y, Shengsong D, Wenxia G. Degradation of 4-aminophenol by hydrogen peroxide oxidation using enzyme from Serratia marcescens as catalyst. Front Environ Sci Eng China 2007;1:95–8. http://dx.doi.org/ 10.1007/s11783-007-0018-0. [17] Zhang S, Sun W, Xu L, Zheng X, Chu X, Tian J, et al. Identification of the paranitrophenol catabolic pathway, and characterization of three enzymes involved in the hydroquinone pathway, in Peudomonas sp. 1-7. BMC Microbiol 2012;12:27. http://dx.doi.org/10.1186/1471-2180-12-27. [18] Takenaka S, Koshiya J, Okugawa S, Takata A, Murakami S, Aoki K. Fe-superoxide dismutase and 2-hydroxy-1,4-benzoquinone reductase preclude the auto-oxidation step in 4-aminophenol metabolism by Burkholderia sp. strain AK-5. Biodegradation 2011;22:1–11. http://dx.doi.org/10.1007/s10532-010-9369-5.
www.elsevier.com/locate/nbt 5 Please cite this article in press as: Ricken, B. et al., Degradation of sulfonamide antibiotics by Microbacterium sp. strain BR1 – elucidating the downstream pathway, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.03.005
NBT-770; No of Pages 6 RESEARCH PAPER
Research Paper
[19] Chauhan A, Samanta SK, Jain RK. Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway. J Appl Microbiol 2000;88:764–72. http://dx.doi.org/10.1046/j.1365-2672.2000.01018.x. [20] Zhao JS, Singh A, Huang XD, Ward OP. Biotransformation of hydroxylaminobenzene and aminophenol by Pseudomonas putida 2NP8 cells grown in the presence of 3-nitrophenol. Appl Environ Microbiol 2000;66:2336–42. [21] Kwon SJ, Yang H, Jo K, Kwak J. An electrochemical immunosensor using paminophenol redox cycling by NADH on a self-assembled monolayer and ferrocene-modified Au electrodes. Analyst 2008;133:1599–604. http:// dx.doi.org/10.1039/b806302h. [22] Eppink MH, Cammaart E, Van Wassenaar D, Middelhoven WJ, van Berkel WJ. Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604. Eur J Biochem 2000;267:6832–40. [23] Gimmler H. Different uptake and binding of benzoquinone and hydroquinone in unicellular algae and type a chloroplasts. Z Pflanzenphysiol 1976;78:76–81. http://dx.doi.org/10.1016/S0044-328X(76)80142-0. [24] Zhang J-J, Liu H, Xiao Y, Zhang X-E, Zhou N-Y. Identification and characterization of catabolic para-nitrophenol 4-monooxygenase and para-benzoquinone reductase from Pseudomonas sp. strain WBC-3. J Bacteriol 2009;191:2703–10. http://dx.doi.org/10.1128/jb.01566-08.
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New Biotechnology Volume 00, Number 00 March 2015
[25] Wimpenny JW. A. Firth, Levels of nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide in facultative bacteria and the effect of oxygen. J Bacteriol 1972;111:24–32. [26] Kabashima Y, Sone N, Kusumoto T, Sakamoto J. Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3. J Bioenerg Biomembr 2013;45:131–6. http://dx.doi.org/10.1007/s10863-012-9485-5. [27] Walter A, Wu J, Flechsig G, Haake D, Wang J. Redox cycling amplified electrochemical detection of DNA hybridization: application to pathogen E. coli bacterial RNA. Anal Chim Acta 2011;689:29–33. http://dx.doi.org/10.1016/ j.aca.2011.01.014.Redox. [28] Kolvenbach BA, Dobrowinski H, Fousek J, Vlcek C, Scha¨ffer A, Gabriel FLP, et al. An unexpected gene cluster for downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3. Appl Microbiol Biotechnol 2012;93:1315–24. http://dx.doi.org/10.1007/s00253-011-3451-8. [29] Kwon D-Y, Suma Y, Tiwari M, Choi J, Kang L-W, Lee J-K, et al. Decomposition of aromatic hydrocarbon intermediates by recombinant hydroxyquinol 1,2-dioxygenase from Arthrobacter chlorophenolicus A6 and its structure characterization. Int Biodeterior Biodegrad 2014. http://dx.doi.org/10.1016/j.ibiod.2014.03.002. ´ ska D. Intradiol dioxygenases – the key [30] Guzik U, Hupert-Kocurek K, Wojcieszyn enzymes in xenobiotics degradation. In: Chamy R, Rosenkranz F, editors. Biodegrad. Hazard. Spec. Prod.. InTech; 2013. p. 129–53.
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