Anoxic Androgen Degradation by the Denitrifying BacteriumSterolibacterium denitrificans via the 2,3-seco Pathway

Po-Hsiang Wang,a Chang-Ping Yu,b Tzong-Huei Lee,c Ching-Wen Lin,a Wael Ismail,d Shiaw-Pyng Wey,e An-Ti Kuo,a Yin-Ru Chianga

Biodiversity Research Center, Academia Sinica, Taipei, Taiwana; Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, Chinab; Graduate Institute ofPharmacognosy, Taipei Medical University, Taipei, Taiwanc; Biotechnology Program, College of Graduate Studies, Arabian Gulf University, Manama, Kingdom of Bahraind;Department of Medical Imaging and Radiological Sciences, College of Medicine, Chang-Gung University, Kweishan, Taiwane

The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerningthe anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In thiswork, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C19 andro-gens. By using a 13C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstratedthat S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the iden-tification of a C17 intermediate, we propose that the A-ring cleavage may be followed by the removal of a C2 side chain at C-5 of17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activitiesof the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenicactivity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminateandrogens. The A-ring cleavage intermediate (<500 �M) did not exhibit androgenic activity, whereas the sterane-containingintermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteo-bacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, andboth of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acidcan be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C19

androgens.

The presence of steroid hormones in the environment has be-come a major issue in environmental science and policy be-

cause these compounds (e.g., estrogens at concentrations above10 ng/liter) alter diverse physiological functions, including repro-duction and development, in animals, especially the aquatic spe-cies (1–5). Due to their hydrophobicity and strong adsorption tosediments, they are poorly soluble in water and have only limitedbioavailability in natural environments (6). Conventionally, es-trogens have attracted considerable attention due to their struc-tural stability, persistence in the environment, and extremelystrong endocrine-disrupting activity (1, 5, 7–9). Recent studiesalso documented masculinization of freshwater wildlife exposedto androgens in polluted rivers (10, 11).

Steroid hormones are discharged into the environment via var-ious routes. They are excreted through the urinary tracts of verte-brates after modifications (e.g., glucuronide and sulfate conjuga-tions) (12, 13). They are also detected in animal feces (14). Animalmanure thus contains large amounts of steroid hormones, and theland application of livestock manure as fertilizer is considered oneof the major sources of steroid hormones released to the environ-ment (15). Municipal sewage biosolids are also an importantsource of steroid hormones in the environment (16). In addition,it has been reported that steroid hormones contaminate aquaticenvironments through leaching from manure-treated agriculturalfields (17).

Steroid hormones have been detected in effluents from waste-water treatment plants and rivers worldwide at concentrations inthe ng/liter range (18–24). For example, Chang et al. (19) docu-mented five classes of steroid hormones occurring at dischargesites and in rivers in Beijing, China. According to their investiga-tions, androgens were the most abundant steroids and were de-

tected in total concentrations of up to 480 ng/liter in rivers and1,887 ng/liter at discharge sites. These data suggest the inefficientremoval of androgens in wastewater treatment plants.

Biodegradation has been proposed as a crucial mechanism forremoving steroid hormones from the environment and engi-neered systems (12, 25, 26). In the last few decades, various andro-gen-degrading aerobic bacteria were isolated and characterized(27–29). Coulter and Talalay first established the oxygen-depen-dent pathway (the 9,10-seco pathway) for the degradation of tes-tosterone by aerobes (30). This is the most widely studied pathwayfor aerobic androgen biodegradation (28). In contrast to the casefor the well-investigated aerobic degradation pathways, there isstill a lack of knowledge about androgen-degrading anaerobes andthe biochemical mechanisms involved in anoxic androgen bio-degradation (31, 32). Recent studies indicated that anoxic sedi-ments and soil may be reservoirs for steroid compounds (6, 15).Some evidence suggests that steroids in soil could be mineralizedby microbial activity even when oxygen is not available (33–35). Inaddition, a recent study on the elimination of steroid hormones ina municipal sewage treatment plant found that many of the de-

Received 24 November 2013 Accepted 18 March 2014

Published ahead of print 21 March 2014

Editor: H. Nojiri

Address correspondence to Yin-Ru Chiang, yinru915@gate.sinica.edu.tw.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03880-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03880-13

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tected androgens (for example, 80% of detected androsta-1,4-diene-3,17-dione [ADD]) were eliminated by microbial activitiesin the anaerobic tanks (22).

In addition to the environmental concerns, microbial steroidbiodegradation has also potential applications in pharmacy andmedicine (36, 37). Numerous studies have reported the microbialproduction of androst-4-en-3,17-dione (AD) and ADD from in-expensive cholesterol and phytosterols (27, 38). Additional inter-est is due to the potential applications of steroid-transformingenzymes with high regio- and stereo-specificity in the industrialsynthesis of steroids (37).

We recently proposed an anaerobic testosterone degradationpathway (the 2,3-seco pathway) adopted by a gammaproteobacte-rium, Steroidobacter denitrificans (Sdo. denitrificans) DSMZ 18526(39). The oxygenase-independent mechanism of the steroidal A-ring cleavage is highly comparable to that operating in anoxiccyclohexanol catabolism by Alicycliphilus denitrificans DSMZ14773 (39). However, there is a dearth of information concerningthe carbon removal mechanisms involved in anoxic testosteronecatabolism. Furthermore, the androgenic activity of the interme-diates needs to be determined. The distribution of the 2,3-secopathway among testosterone-degrading anaerobes also remainsunclear. Prior to this study, Sdo. denitrificans DSMZ 18526 was theonly known testosterone-degrading anaerobe (40–42).

BLAST analysis has indicated that Sterolibacterium strains canbe detected in the natural environment and engineered systems(see Fig. S1 in the supplemental material). Among them, Steroli-bacterium denitrificans (S. denitrificans) DSMZ 13999, a betapro-teobacterium, was reported to degrade cholesterol both underoxic and anoxic conditions (43–46). In this study, the ability of S.denitrificans to degrade C18 and C19 steroid hormones under an-oxic conditions was investigated. We applied a 13C-metabolomicapproach to show that S. denitrificans uses the 2,3-seco pathway toanaerobically degrade androgens. The androgenic activities of thebacterial culture and the identified intermediates were then as-sessed.

MATERIALS AND METHODSChemicals and bacterial strains. [4-14C]testosterone was obtained fromPerkin-Elmer. [2,3,4-13C]testosterone was purchased from Isosciences.The chemicals used were of analytical grade and were purchased fromMallinckrodt Baker, Merck, or Sigma-Aldrich. 3,17-Dihydroxy-9,10-seco-androsta-1,3,5(10)-triene-9-one, 3-hydroxy-9,10-seco-androsta-1,3,5(10)-triene-9,17-dione, 1-testosterone, androst-1-en-3,17-di-one, 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (2,3-SAOA), and1,17-dioxo-2,3-seco-androstan-3-oic acid were produced as described else-where (39, 47). Sterolibacterium denitrificans DSMZ 13999 was purchasedfrom the Deutsche Sammlung für Mikroorganismen und Zellkulturen(DSMZ), Braunschweig, Germany. Comamonas testosteroni ATCC 11996was obtained from the Bioresource Collection and Research Center(BCRC), Hsinchu, Taiwan.

Androgen and estrogen utilization kinetics. The preparation of thedenitrifying media and the anoxic in vivo assays were performed in ananaerobic chamber containing 95% nitrogen and 5% hydrogen gas. S.denitrificans (resting-cell assays; total proteins, 294 �g/ml) was incubatedwith individual androgens (ADD, androsterone [ADR], epiandros-terone [EADR], 19-nor-4-androsten-3,17-diol [NADL], or testoster-one) or estrogens (17�-estradiol, 17�-estradiol, estriol, estrone, or 17�-ethinylestradiol) at initial concentrations ranging from 5 to 50 mg/literunder denitrifying conditions. Hydroxypropyl-�-cyclodextrin (cyclodex-trin) (0.5% [wt/vol]) was added to the culture medium prepared accord-ing to a published procedure (46). Substrate utilization kinetics tests were

performed in a series of 2.2-ml Eppendorf tubes containing 1 ml of restingcell suspension. The steroid stock solutions were prepared in dimethylsulfoxide (DMSO), and the final DMSO concentration was adjusted to0.5% (vol/vol). The resulting cell suspension was anaerobically incubatedat 28°C with shaking (180 rpm) for 2 h. We measured the amount ofresidual steroids using high-pressure liquid chromatography (HPLC) anddetermined the protein content of the S. denitrificans cell suspension usingthe bicinchoninic acid (BCA) assay. The determination of Monod kineticparameters was carried out as described before (48).

Anaerobic growth of S. denitrificans on [4-14C]testosterone. In thefollowing experiments involving steroid quantification, 17�-ethinylestra-diol (final concentration, 50 �M), which cannot be utilized by S. denitri-ficans, was added to bacterial cultures to serve as an internal control. In adenitrifying fed-batch culture (100 ml), S. denitrificans cells were incu-bated with 1 mM testosterone containing [4-14C]testosterone (1 � 108

dpm). Nitrate (10 mM) was added to the culture when the nitrate addedinitially (10 mM NaNO3) was consumed. The fed-batch culture (initialtotal proteins, 32 �g/ml) was carried out in a 125-ml glass bottle sealedwith a rubber stopper. The headspace (25 ml) of the culture was connectedto a 125-ml glass bottle containing 100 ml of 5 M NaOH, which trapped14CO2 produced by the S. denitrificans cells. A Tygon tube (2 mm in innerdiameter and 25 cm long) was used for the connection. Every 12 h (0 to 60h), samples (0.5 ml) were withdrawn from the 5 M NaOH solution. Tenminutes before each sampling, nitrogen gas (ca. 50 ml) was used as acarrier gas to expel the residual 14CO2 from the bacterial culture at a flowrate of 5 ml/min. At the same time intervals, samples (2.5 ml) were with-drawn from the bacterial culture. The culture samples (0.5-ml samples, 3replicates) were extracted with the same volume of ethyl acetate threetimes to isolate the residual [4-14C]testosterone from the water fraction.The ethyl acetate was evaporated, and the residue was redissolved in 0.5 mlof ethanol. The water fraction, ethanol, and 5 M NaOH samples (0.1 ml)were added to 1.9 ml of Ultima Gold high-flash-point LSC scintillationcocktail reagent (Perkin-Elmer), and the amount of 14C was determinedby liquid scintillation counting (Tri-Carb 2900 TR liquid scintillationanalyzer [Perkin-Elmer]). The remaining ethanol samples (0.4-ml sam-ples, 3 replicates) were concentrated to 50 �l, and the testosterone in theethanol samples was quantified by HPLC. The protein content and nitrateconcentration in the samples were determined as described below. After60 h of incubation, the bacterial cells were harvested twice by centrifuga-tion (10,000 � g for 20 min), and the remaining cells were recovered bypassing the supernatant through a 0.22-�m nitrocellulose membrane fil-ter (Millipore). The S. denitrificans cells were lyophilized for 1 week, andthe freeze-dried biomass was weighed.

Anoxic transformation of [2,3,4-13C]testosterone by the batch cul-tures. In the following biotransformation assays, S. denitrificans was firstanaerobically grown in fed-batch cultures (nitrate was continuously sup-plied). The amounts of residual substrates (testosterone, cholesterol, orpalmitate [49]) in the denitrifying S. denitrificans cultures were monitoredusing HPLC. The utilization of last two substrates can avoid the HPLCdetection of C19 catabolic intermediates in the precultures. We then usedthe log-phase S. denitrificans cultures to transform steroid substrates in abatch mode (no additional nitrate was given during the incubation).

After the consumption of 2 mM testosterone, 10 ml of the anoxic S.denitrificans culture (200 ml) was transferred into two 12-ml glass bottles.The two cultures were subsequently fed with 2 mM testosterone (unla-beled testosterone and [2,3,4-13C]testosterone were mixed in a 1:1 molarratio) under denitrifying conditions. From an S. denitrificans culture con-taining 5 mM nitrate, the sample (1 ml) was withdrawn after 4 h of incu-bation at 28°C. From another batch culture containing 15 mM nitrate,the sample (1 ml) was withdrawn after 16 h of denitrifying incubation.The ethyl acetate-extractable intermediates were identified using ul-traperformance liquid chromatography– high-resolution mass spec-trometry (UPLC-HRMS).

Production of 6�-hydroxyandrost-4-en-3,17-dione. S. denitrificans(2 liters) was anaerobically grown with 2 mM testosterone. After the con-

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sumption of 1 mM testosterone, 1 mM 3-mercaptopropionate (an inhib-itor of acyl coenzyme A [acyl-CoA] dehydrogenase [50, 51]) was added tothe culture, and the incubation was continued for an additional 10 h.Separation of ethyl acetate extracts was carried out using thin-layer chro-matography (TLC) and HPLC. The structural elucidation of the HPLC-purified intermediate was performed with nuclear magnetic resonance(NMR) spectroscopy and UPLC-HRMS.

Quantification of steroids and androgenic activity in a denitrifyingS. denitrificans culture. After consumption of 2 mM cholesterol, a S.denitrificans culture (500 ml; total proteins, 264 �g/ml) was used to trans-form 0.5 mM testosterone. The samples (5 ml) were withdrawn every 1 h(0 to 10 h). The steroid intermediates extracted from the cultural samples(4 ml) were quantified using HPLC. The steroids extracted from the re-maining samples (1 ml) were dissolved in 10 �l of DMSO, and theirandrogenic activity was tested using the lacZ-based assay.

Growth of S. denitrificans under various oxygen concentrations. AnS. denitrificans culture (250 ml) containing potassium phosphate buffer(20 mM, pH 7.0), 10 mM NH4Cl (the nitrogen source), and 10 mMNaNO3 (the potential electron acceptor) was first anaerobically grown on1 mM palmitic acid (45). After palmitic acid was completely consumed,0.5 mM testosterone and 10 mM nitrate were added to the bacterial cul-ture. Portions (50 ml) of the resulting culture were transferred to three500-ml glass bottles and were incubated under various concentrations ofoxygen (headspace, 450 ml; 0, 5, and 20% [vol/vol]). Two cultures wereprepared in an anaerobic chamber. In the case of the microaerobic cul-ture, 24 ml of oxygen gas was injected into the anoxic headspace afterpassage through a 0.22-�m membrane filter (Millipore). Samples (1 ml)were retrieved every 12 h (0 to 72 h) to measure the consumption oftestosterone and nitrate and to detect the production of the ring cleavageintermediates.

Aerobic growth of S. denitrificans and C. testosteroni in the absenceof nitrate. S. denitrificans was aerobically grown in a phosphate-bufferedculture without nitrate (50 ml). After the consumption of 2 mM testos-terone (optical density at 600 nm [OD600] � 0.8 [optical path, 1 cm]),additional testosterone (2 mM) was added to the S. denitrificans culture.Testosterone (2 mM) was added to a C. testosteroni culture (OD600 � 1.0)grown in tryptic soy broth (BD Difco). Samples (1 ml) were withdrawnevery 2 h (0 to 10 h), and the steroid metabolites were identified usingUPLC-HRMS.

TLC. The steroids were separated on silica gel aluminum thin-layerchromatography (TLC) plates as described elsewhere (39).

HPLC quantification of androgens and estrogens in bacterial cul-tures. The steroids extracted from the culture samples were separated andquantified using a reversed-phase Hitachi HPLC system. The separationwas achieved on an analytical RP-C18 column [Luna 18(2), 5 �m, 150 by4.6 mm; Phenomenex) incubated at 35°C. The mobile phase included amixture of two solvents, A (0.1% aqueous trifluoroacetic acid) and B(acetonitrile containing 0.1% trifluoroacetic acid). The separation wasperformed at a flow rate of 1.0 ml/min with a gradient from 30% to 60%B within 60 min. The steroids were detected in the range of 200 to 300 nmusing a photodiode array detector. The quantification of steroids wasdone from their respective peak areas using a standard curve of individualstandards. Data shown are the means � standard errors (SE) from threeexperimental measurements.

UPLC-HRMS. The ethyl acetate-extractable samples and the HPLC-purified compound were analyzed using UPLC-HRMS as described be-fore (39).

NMR spectroscopy. The 1H- and 13C-NMR spectra were recorded at27°C using a Bruker AV600_GRC 600-MHz NMR instrument. Chemicalshifts (�) were recorded and shown as ppm values with deuterated meth-anol (99.8%; 1H, � � 3.31 ppm; 13C, � � 49.0 ppm) as the solvent andinternal reference.

Measurement of protein content and nitrate concentration. The cellsuspension (0.1 ml) was centrifuged at 10,000 � g for 10 min. Aftercentrifugation, the pellet was resuspended in 1 ml of reaction reagent

(Pierce BCA protein assay kit; Thermo Scientific). The protein contentwas determined using a BCA protein assay according to manufactur-er’s instructions with bovine serum albumin as the standard. The su-pernatant (0.1 ml) was diluted with 0.9 ml double-distilled water, andthe nitrate was determined using the cadmium reduction method ac-cording to manufacturer’s instructions (HI93728-01 nitrate reagentkit; Hanna Instruments).

Transformation of yeast cells with the plasmid pRR-AR-5Z. Theyeast Saccharomyces cerevisiae host stain (BY4727, trp) was a gift fromWen-Hsiung Li, Biodiversity Research Center, Academia Sinica, Taiwan.The plasmid pRR-AR-5Z (23058) was purchased from Addgene. Thetransformation was performed according to an electroporation protocol(52). The resulting transformants were grown on selective agar platescontaining 6.7 g/liter yeast nitrogen base without amino acids (BD Difco),20 g/liter glucose, and 1.92 g/liter SC-Trp (Sunrise Science). The presenceof the plasmid pRR-AR-5Z in the yeast cells was confirmed using PCR.

lacZ-based yeast androgen bioassay. The yeast androgen bioassaywas conducted as described by Fox et al. (53) with slight modifications.Briefly, the individual androgens or cell extracts were dissolved in DMSO,and the final concentration of DMSO in the assays (200 �l) was 1% (vol/vol). The resulting DMSO solutions (2 �l) were added to yeast cultures(198 �l; initial OD600, 0.5) in a 96-well microtiter plate. The �-galactosi-dase activity was determined after 18 h of incubation at 30°C. The yeastsuspension (25 �l) was added to Z buffer (225 �l) containing o-nitrophe-nol-�-D-galactopyranoside (2 mM), and the reaction mixtures were incu-bated at 37°C for 30 min. The reactions were stopped by adding 100 �l of1 M sodium carbonate, and the amount of nitrophenol product was mea-sured at 420 nm on a plate spectrophotometer. Data shown are themeans � SE for three replicates.

RESULTSUtilization of androgens and estrogens by S. denitrificans. An-drogens and estrogens often detected in the environment (18–20,22–24) were tested for the substrate utilization pattern of S. deni-trificans. In this study, cyclodextrin (0.5% [wt/vol]), a carrier mol-ecule, was added to all cell suspensions and bacterial cultures toimprove the solubility of steroids. Addition of cyclodextrin canavoid underestimation of the substrate utilization rate due to poorsolubility of steroids in media. The maximum specific substrateutilization rates (qm) and the estimated half-velocity constants(Ks) for these androgens are shown in Fig. 1. Testosterone was themost suitable steroid substrate for S. denitrificans. In the presenceof cyclodextrin, the qm and Ks values for testosterone were 4.67 mgsubstrate/mg protein/day and 79.41 mg/liter, respectively. In theabsence of cyclodextrin, the qm value for testosterone was 0.34 mgsubstrate/mg protein/day (data not shown). In contrast, EADRseems to be less favorable. We observed no substrate utilization forC18 steroids, including NADL and estrogens. The data indicatedthat under denitrifying conditions, S. denitrificans could utilizeC19 androgens, but not C18 steroids, as an energy source. How-ever, it was unclear if S. denitrificans mineralized androgens in theabsence of oxygen or if it just transformed testosterone to othercompounds.

S. denitrificans mineralizes testosterone during denitrifyinggrowth. Separation of S. denitrificans cells from the residual tes-tosterone by centrifugation was not successful. Therefore, the cul-ture samples were extracted with ethyl acetate to recover the re-sidual testosterone from the aqueous fraction. As shown in Fig. 2,the decrease in the residual [4-14C]testosterone in the mediumwas accompanied by the accumulation of radioactive 14C in thebacterial biomass and an increase in the amount of trapped 14CO2

(Fig. 2A). After 60 h of anaerobic growth of S. denitrificans, the

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total 14C recovered was 75% (14% 14C remained mainly as testos-terone in the medium, 20% 14C was trapped by the 5 M NaOHsolution, and 41% 14C was assimilated in biomass) (Fig. 2A).Given the stoichiometric results shown below (0.32 mM testoster-one [32%] was assimilated in biomass, and 0.57 mM testosterone[57%] was mineralized), most of the lost 14C may due to the in-complete trapping of 14CO2 using NaOH solution.

The anaerobic growth of S. denitrificans (measured as the in-crease in the protein concentration) was accompanied by a de-crease of residual testosterone and the consumption of nitrate(Fig. 2B). At the end of exponential growth phase (60 h), approx-imately 158 mg of dry cell mass per liter was produced, accountingfor the consumption of 0.89 mM testosterone and 10.3 mM ni-trate. Synthesis of bacterial biomass (158 mg/liter) can account forthe consumption of 0.32 mM testosterone and 1.2 mM nitratebased on the assimilation equation (using C4H7O3 as the empiri-cal formula for S. denitrificans biomass [45]): 20C19H28O2 77NO3

77H 14H2O ¡ 95C4H7O3 38.5N2.

Considering the testosterone and nitrate consumed for bio-mass synthesis, 0.57 mM testosterone and 9.1 mM nitrate shouldhave been consumed in the dissimilation process. These data arein good agreement with the theoretical stoichiometry for com-plete oxidation of testosterone: C19H28O2 20NO3

20H ¡19CO2 10N2 24H2O.

The complete oxidation of 1 mol testosterone yields 100 molelectrons. On the other hand, the reduction of 1 mol nitrate tomolecular nitrogen (N2) requires 5 mol electrons. An electronrecovery of 124% was calculated.

Identification of catabolic intermediates. To investigate the13C-labeled intermediates involved in anoxic testosterone degra-dation, we incubated two log-phase S. denitrificans cultures (10ml) with [2,3,4-13C]testosterone. The two batch cultures con-tained different nitrate concentrations. The steroid substrate wascomposed of [2,3,4-13C]testosterone and unlabeled testosterone.Therefore, pairs of molecular adduct ions (with an m/z differenceof 3) were observed in the mass spectra of testosterone-derived

FIG 1 Monod degradation kinetics of androgens by resting cell suspensions of S. denitrificans. The increase of bacterial biomass within 2 h was negligible (�3%).The solid lines represent fitted Monod kinetic curves. The data are from one representative experiment of four individual experiments.

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intermediates (see Fig. S2 in the supplemental material). After 4 hof anaerobic incubation, we detected a total of eight 13C-labeledcompounds in the S. denitrificans culture containing 5 mM nitrate(see Fig. S2A in the supplemental material). Note that the polar/ionic compounds such as 2,3-SAOA provided weak ion signalswith the atmospheric-pressure chemical ionization (APCI) method.These steroid compounds were identified by reference to theUPLC-HRMS behavior of steroid standards. The mass spectra ofthe 13C-labeled intermediates are given in Fig. S2B in the supple-mental material. Of these, 1-testosterone (Fig. S2B3), 2,3-SAOA(Fig. S2B4), and androst-1-en-3,17-dione (Fig. S2B7) are charac-teristic intermediates involved in the 2,3-seco pathway (39). Inter-estingly, a saturated steroid, androstan-17�-ol-3-one (Fig. S2B8),was observed in the anoxic S. denitrificans culture. This compoundwas produced from testosterone by a reduction reaction, whichrequires electrons. The appearance of this reductive compoundmight contribute to the NADH/NAD cycle in S. denitrificanscells.

After 16 h of denitrifying incubation, a 13C-labeled interme-diate with 17 carbons was identified in another batch culture(10 ml) containing 15 mM nitrate (Fig. 3). According to its

electron spray ionization (ESI) mass spectrum, this compoundis labeled with only one 13C (Fig. 3B), suggesting the removal oftwo 13C atoms from testosterone. We assumed that the C-3 andC-4 of this C17 intermediate were removed because (i) testos-terone, the steroid substrate, was labeled with three 13C at C-2,C-3, and C-4 and (ii) in the case of 2,3-SAOA, the single bondbetween C-2 and C-3 was broken. So far, we cannot produce asufficient amount of this C17 intermediate for NMR analysis.However, some clues suggest that this compound might have achemical structure as shown in Fig. 3C. First, the ring cleavageproduct, 2,3-SAOA, has a carboxylic group at its C-3 whichcould be activated by a coenzyme A (CoA) ligation reaction.After the activation, a CH3-CO-SCoA molecule could be re-moved via the retro-aldol reaction, which results in a carbonylgroup at the C-5 position. Second, this C17 compound was notobserved when 1 mM 3-mercaptopropionate (an inhibitor ofacyl-CoA dehydrogenase) was added to the in vivo biotransfor-mation assay, suggesting that this intermediate may result from

FIG 2 Denitrifying growth of S. denitrificans DSMZ 13999 on testosterone.(A) Assimilation and mineralization of [4-14C]testosterone in the bacterialculture. Symbols: p, ethyl acetate-extractable 14C; Œ, assimilated 14C; �,trapped 14CO2. (B) Nitrate consumption and testosterone utilization in thesame bacterial culture. Symbols: �, bacterial growth; �, residual total testos-terone; Œ, nitrate consumption. The data shown are the means � SE of threeexperimental measurements.

FIG 3 UPLC-HRMS analysis of a 13C-labeled intermediate produced by thedenitrifying S. denitrificans. (A) UPLC chromatogram of the ethyl acetate ex-tract. (B) ESI mass spectra of the C17 intermediate. The predicted elementalcomposition was calculated using MassLynx mass spectrometry software (Wa-ters). (C) Expected chemical structure. Critical carbon atoms in this com-pound are numbered according to the steroidal carbon numbering system. �,13C atom.

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reactions similar to �-oxidation. Third, during the anaerobicincubation of S. denitrificans with testosterone, the 17-hydroxylintermediates are usually more dominant than their 17-ketostructures (see Fig. 5 and Fig. S2 in the supplemental material).

Anaerobic incubation of S. denitrificans with testosteroneand 3-mercaptopropionate. In a denitrifying culture (2 liters), S.denitrificans was grown with unlabeled testosterone and 3-mer-captopropionate (1 mM) for 10 h. Accumulation of 2,3-SAOAand an unprecedented intermediate occurred. The accumulationof the A-ring cleavage intermediate (2,3-SAOA) implies the par-ticipation of �-oxidation or similar reactions in the following cat-abolic steps. The other intermediate was purified by liquid-liquidextraction, TLC, and HPLC. The molecular formula of the HPLC-purified compound was analyzed using UPLC-HRMS. A proto-nated molecular ion ([M H]; m/z 303.1968) was observed inits APCI mass spectrum (Fig. 4A). The molecular formula of thiscompound was determined to be C19H26O3. Based on the NMRanalysis (see the legend to Fig. S3 in the supplemental material fora detailed explanation), this steroid product was characterized as6�-hydroxyandrost-4-en-3,17-dione (Fig. 4B).

Sequential appearance of the catabolic intermediates in thedenitrifying S. denitrificans culture. We incubated a S. denitrifi-cans batch culture (500 ml) with testosterone to track the sequen-tial appearance of the catabolic intermediates and to determinethe androgenic activity in the bacterial culture (Fig. 5). 1-Dehy-drotestosterone was the first accumulated intermediate, and itspeak appeared after 2 h of incubation. The strong positive slopefor 2,3-SAOA indicates that it is the last detectable product. 1-Tes-tosterone behaved like intermediates between 1-dehydrotestos-teorne and 2,3-SAOA. The ethyl acetate extracts of the S. denitri-ficans culture were tested for their androgenic activity (Fig. 5).After 3 h of incubation, androgenic activity started to steadilydecrease over time. Note that the accumulation of 2,3-SAOA didnot result in an increase of androgenic activity.

Androgenic activities of the intermediates involved in the2,3-seco pathway. We then determined the androgenic activi-ties of the individual intermediates using a lacZ-based yeast

androgen assay. A total of seven intermediates were tested. Theresults showed that testosterone, 1-dehydrotestosterone (DT),and AD exhibited comparable androgenic activity (Fig. 6).However, 1-testosterone, androstan-17�-ol-3-one, and ADDexhibited relatively weak activity. 2,3-SAOA, like estradiol (thenegative control), had no detectable androgenic activity even ata concentration of 500 �M. These results are consistent withthose for the ethyl acetate extracts of the S. denitrificans culture(Fig. 5), indicating that under anoxic conditions, S. denitrificanscan eliminate the androgenic activity of testosterone by openingits sterane structure.

Testosterone degradation by S. denitrificans grown undervarious oxygen concentrations. Under oxic conditions, testoster-one was exhausted after 36 h of incubation. The slowest testoster-one utilization was observed when oxygen was not available, withtestosterone exhausted after 72 h of incubation (Fig. 7A). No ap-

FIG 4 Structural elucidation of 6�-hydroxyandrost-4-en-3,17-dione pro-duced by S. denitrificans. (A) APCI mass spectrum. (B) Key heteronuclearmultiple-bond correlation (HMBC) spectrum.

FIG 5 Time course of intermediate production and androgenic activity of adenitrifying S. denitrificans culture. The A420 of the negative control, DMSO(1%, vol/vol), was set to zero.

FIG 6 Response of the yeast androgen bioassay to the individual interme-diates. The results are from one representative of four individual experi-ments.

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parent nitrate consumption was observed in the aerobic and mi-croaerobic cultures, whereas in the anaerobic culture, 5.8 mMnitrate consumption accompanied the utilization of 0.47 mM tes-tosterone (Fig. 7A). It is worth mentioning that the characteristicring cleavage intermediate of the 9,10-seco pathway, 3,17-dihy-droxy-9,10-seco-androsta-1,3,5(10)-triene-9-one, was not de-tected in the aerobic and microaerobic S. denitrificans cultures. Incontrast, the production of 2,3-SAOA was observed in all bacterialcultures (Fig. 7B).

To investigate if the presence of nitrate might repress theexpression of the catabolic genes involved in the classic 9,10-seco pathway, we aerobically grew S. denitrificans with testoster-

one and without nitrate. Comamonas testosteroni, which is knownto degrade testosterone via the 9,10-seco pathway (28), was alsotested for comparison (see Fig. S4A in the supplemental material).S. denitrificans still produced 1-testosterone and 2,3-SAOA in theabsence of nitrate (see Fig. S4B in the supplemental material),indicating that nitrate is not repressing the expression of the S.denitrificans catabolic genes involved in the 9,10-seco pathway andconfirming that the 2,3-seco pathway is the sole catabolic strategyfor testosterone degradation in S. denitrificans.

DISCUSSION

Our results on [4-14C]testosterone mineralization, stoichiometry,and 13C-metabolomics indicate the complete degradation of tes-tosterone by S. denitrificans. We detected more assimilated 14Csignals (41%) than the assimilated testosterone (32%) calculatedbased on the theoretical assimilation equation. This may be be-cause (i) polar catabolic intermediates derived from testosteroneremain in the aqueous fraction after ethyl acetate extractionand/or (ii) individual carbons of the testosterone molecule havedifferential metabolic fates. It was demonstrated that Mycobacte-rium tuberculosis preferentially utilized different portions of thecholesterol molecule for energy generation and biosynthetic pur-poses (54). There is a large gap (25%) in the total 14C recovery. Inaddition to the incomplete capture of 14CO2, volatile metabolitesproduced in testosterone metabolism may not be trapped by theNaOH solution.

Based on the UPLC-HRMS data and the sequential appear-ance of the abundant intermediates in the denitrifying cultures,we found that S. denitrificans DSMZ 13999 also uses the 2,3-secopathway to degrade testosterone. We identified a C17 intermediateusing UPLC-HRMS. The loss of two 13C signals implies the re-moval of C-3 and C-4 from testosterone. This intermediate mightbe transformed from 2,3-SAOA by a series of CoA ligation, dehy-drogenation, hydration, and retro-aldol reactions (Fig. 8A).Highly similar reactions have been demonstrated in the transfor-mation of CoA ester of pregn-4-en-3-one-20-carboxylic acid toandrost-4-en-3,17-dione (Fig. 8B) (55–57). In the cholesterol-de-grading Mycobacterium tuberculosis H37Rv, the �-ketoacyl-CoAthiolase enzyme encoded by fadA5 catalyzes the thiolysis of C27

and C24 steroid-CoA esters (58). One propionyl-CoA moiety andone acetyl-CoA moiety are thus released through two cycles of�-oxidation. However, the additional propionyl-CoA cannot bereleased from pregn-4-en-3-one-20-carboxyl-CoA by a conven-tional �-oxidation due to the presence of the cyclopentane D ring.The two following steps, catalyzed by an acyl-CoA dehydrogenaseand an enoyl-CoA hydratase, will produce a tertiary alcohol (59),which cannot be oxidized to a keto group (Fig. 8B). Thus, themicrobial transformation of 17-hydroxypregn-4-en-3-one-20-carboxyl-CoA to androst-4-en-3,17-dione proceeds by a retro-aldol reaction (55). These reactions have also been detected inmicroorganisms grown under anoxic conditions (56). One mayenvisage that S. denitrificans removes the C2 side chain at C-5 of2,3-SAOA via similar reactions to yield 17-hydroxy-2,5-seco-3,4-dinorandrost-1,5-dione (2,5-SDAD) and acetyl-CoA (Fig. 8A).

In the presence of 3-mercaptopropionate, anaerobically grownS. denitrificans produced and accumulated 2,3-SAOA and 6�-hy-droxyandrost-4-en-3,17-dione. However, the C17 intermediatewas not detected. It seems that this 6�-hydroxysteroid is a by-product accumulating when the native catabolic flow via reactionssimilar to �-oxidation is hindered. The data suggest that in the

FIG 7 Testosterone catabolism by S. denitrificans proceeds via the 2,3-secopathway regardless of oxygen conditions. (A) Testosterone and nitrate con-sumption in the S. denitrificans cultures. Symbols: �, testosterone remainingin the anaerobic culture; �, testosterone in the microaerobic culture; Œ, tes-tosterone in the aerobic culture; Œ, nitrate consumption under anaerobicconditions; �, nitrate consumption under microaerobic conditions; o, ni-trate consumption under aerobic conditions. (B) 2,3-SAOA production inthree S. denitrificans cultures. Representative data from one of three indepen-dent experiments are shown.

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normal catabolic steps, the A-ring cleavage might be followed bythe B-ring activation via a 6-hydroxylation. The anaerobic hy-droxylation occurring at the steroidal B ring was never reportedbefore, although it is known that anaerobic microbial transforma-tion of bile acids involves 7�-dehydroxylation (60). The mecha-nism of the anaerobic 6�-hydroxylation remains to be unraveled.It might be catalyzed by an enzyme similar to steroid C25 dehy-drogenase via a direct hydroxylation (61) or by a protein similar to2-cyclohexenone hydratase protein through the addition of waterto a CAC bond (62). It is worth mentioning that the latter enzymeis bifunctional and also responsible for the subsequent dehydro-genation reaction. The corresponding substrate containing theCAC bond and the 6-keto product were never detected in S. deni-trificans cultures. It is therefore likely that 6�-hydroxyandrost-4-en-3,17-dione is produced from androst-4-en-3,17-dione by ananaerobic hydroxylation. Furthermore, at least seven proteinswith high similarity to the molybdenum-containing subunit of

steroid C25 dehydrogenase were identified in the S. denitrificansgenome (61). The anaerobic 6�-hydroxylation could be catalyzedby one of these molybdoproteins.

In this work, the androgenic activity of the intermediates in-volved in the 2,3-seco pathway was investigated for the first time.Our results revealed that only 2,3-SAOA (�500 �M), the ringcleavage intermediate, exhibited no androgenic activity. In addi-tion, we showed that during the denitrifying growth of S. denitri-ficans, the biotransformation of testosterone to 2,3-SAOA was ac-companied by an apparent decrease of its androgenic activity. It isknown that numerous bacteria can transform testosterone toother androgens (32). Our results (Fig. 6) indicated that the bio-transformation of testosterone to other steroids (e.g., ADD) mayreduce its androgenic activity, but the cleavage of the steranestructure can drastically reduce the androgenic activity of testos-terone.

It has been proposed that proteobacteria play a crucial envi-

FIG 8 (A) Proposed pathway of anoxic testosterone degradation by the denitrifying bacteria. Note that the 6-hydroxysteroid is produced by S. denitrificansDSMZ 13999 only when 3-mercaptopropionate is present. �, intermediates detected in S. denitrificans cultures (this study). †, intermediates detected in Sdo.denitrificans DSMZ 18526 (39, 41). (B) The established retro-aldol reaction involved in the 9,10-seco pathway for the oxic cholesterol catabolism (56).

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ronmental role in the aerobic degradation of androgens (29), andall studied androgen-degrading aerobes utilized the 9,10-secopathway to degrade androgens (28). In this study, we observed theproduction of 2,3-SAOA by S. denitrificans regardless of oxygenconditions. The data indicate that this oxygen-independent tes-tosterone catabolic pathway operates under not only anoxic butalso oxic conditions.

One of the major challenges in environmental microbiology isto detect the function of microorganisms in situ and, if possible, toidentify the pathways employed for the degradation of environ-mentally relevant compounds. In general, signature intermediatesof degradation pathways have to be highly specific for key reac-tions of the respective catabolic pathways (63). Therefore, thecommon intermediates, including DT, AD, and ADD, are notsuitable to serve as signature intermediates. On the other hand,1-oxygenated steroids are highly characteristic in the 2,3-secopathway but they were accumulated only in in vitro enzyme assays(47), suggesting the inappropriateness of using these intermedi-ates as signature intermediates for in situ environmental studies.In the case of microbial androgen degradation, we suggest the useof ring cleavage products [2,3-SAOA for the 2,3-seco pathway and3,17-dihydroxy-9,10-seco-androsta-1,3,5(10)-triene-9-one forthe 9,10-seco pathway] as the signature intermediates because (i)the two ring cleavage intermediates are highly specific for the re-spective catabolic pathways, (ii) the two ring cleavage intermedi-ates possess distinguishable molecular mass, UPLC/HPLC, andUV absorption behaviors which facilitate HPLC-UV or UPLC-MSidentification, (iii) the two ring cleavage intermediates are oftenabundant in testosterone-grown bacterial cultures, and (iv) thering cleavage intermediates exhibit no androgenic activity. So far,information about the functional genes involved in the 2,3-secopathway is lacking. The in situ detection of these ring cleavagemetabolites could be a powerful tool enabling detection of micro-bial androgen degradation in the natural environment and engi-neered systems.

Conclusions. The 2,3-seco pathway is used in all studiedcases for anaerobic catabolism of C19 androgens. In addition,some evidence indicates that this pathway also functions undermicroaerobic and aerobic conditions. Our current study pavesthe way for the elucidation of the downstream steps in anoxicandrogen biodegradation. Furthermore, this work provides thegroundwork for culture-independent in situ studies of micro-bial degradation of androgens by signature metabolite profil-ing.

ACKNOWLEDGMENTS

This research was funded by the National Science Council of Taiwan(NSC 100-2311-B-001-032-MY3). We acknowledge the support of theCAS/SAFEA International Partnership Program for Creative ResearchTeams (KZCX2-YW-T08).

We thank the Small Molecule Metabolomics core facility sponsored bythe Institute of Plant and Microbial Biology (IPMB), Academia Sinica, forUPLC-MS analysis.

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