A Novel Steroid-Coenzyme A Ligase from Novosphingobiumsp. Strain Chol11 Is Essential for an Alternative DegradationPathway for Bile Salts

Onur Yücel,a Johannes Holert,b Kevin Christopher Ludwig,a Sven Thierbach,a Bodo Philippa

aWestfälische Wilhelms-Universität Münster, Institut für Molekulare Mikrobiologie und Biotechnologie,Münster, Germany

bUniversity of British Columbia, Department of Microbiology and Immunology, Life Sciences Institute,Vancouver, British Columbia, Canada

ABSTRACT Bile salts such as cholate are steroid compounds with a C5 carboxylicside chain and occur ubiquitously in vertebrates. Upon their excretion into soils andwaters, bile salts can serve as growth substrates for diverse bacteria. Novosphingo-bium sp. strain Chol11 degrades 7-hydroxy bile salts via 3-keto-7-deoxy-Δ4,6 metabo-lites by the dehydration of the 7-hydroxyl group catalyzed by the 7�-hydroxysteroiddehydratase Hsh2. This reaction has not been observed in the well-studied 9-10-secodegradation pathway used by other steroid-degrading bacteria indicating that strainChol11 uses an alternative pathway. A reciprocal BLASTp analysis showed thatknown side chain degradation genes from other cholate-degrading bacteria (Pseu-domonas stutzeri Chol1, Comamonas testosteroni CNB-2, and Rhodococcus jostii RHA1)were not found in the genome of strain Chol11. The characterization of a trans-poson mutant of strain Chol11 showing altered growth with cholate identified anovel steroid-24-oyl– coenzyme A ligase named SclA. The unmarked deletion ofsclA resulted in a strong growth rate decrease with cholate, while growth withsteroids with C3 side chains or without side chains was not affected. Intermedi-ates with a 7-deoxy-3-keto-Δ4,6 structure, such as 3,12-dioxo-4,6-choldienoic acid(DOCDA), were shown to be likely physiological substrates of SclA. Furthermore,a novel coenzyme A (CoA)-dependent DOCDA degradation metabolite with anadditional double bond in the side chain was identified. These results supportthe hypothesis that Novosphingobium sp. strain Chol11 harbors an alternativepathway for cholate degradation, in which side chain degradation is initiated bythe CoA ligase SclA and proceeds via reaction steps catalyzed by so-far-unknownenzymes different from those of other steroid-degrading bacteria.

IMPORTANCE This study provides further evidence of the diversity of metabolicpathways for the degradation of steroid compounds in environmental bacteria. Theknowledge about these pathways contributes to the understanding of the CO2-releasing part of the global C cycle. Furthermore, it is useful for investigating thefate of pharmaceutical steroids in the environment, some of which may act as endo-crine disruptors.

KEYWORDS CoA ligase, Novosphingobium, bile salts, steroid degradation

Bile salts are a subclass of steroids, which occur in all vertebrates where they act asemulsifiers of lipophilic nutrients in the intestine and affect lipid and energy

metabolism (1, 2). In addition, many further signaling functions of bile salts are currentlybeing discovered with regard to, e.g., microbiome-host interactions (3). Considerableamounts of bile salts are released into the environment, including approximately 400to 800 mg bile salts released per human per day via urine and feces (4). Additionally,

Received 7 July 2017 Accepted 6 October2017

Accepted manuscript posted online 20October 2017

Citation Yücel O, Holert J, Ludwig KC,Thierbach S, Philipp B. 2018. A novel steroid-coenzyme A ligase from Novosphingobium sp.strain Chol11 is essential for an alternativedegradation pathway for bile salts. ApplEnviron Microbiol 84:e01492-17. https://doi.org/10.1128/AEM.01492-17.

Editor Maia Kivisaar, University of Tartu

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Bodo Philipp,bodo.philipp@uni-muenster.de.

BIODEGRADATION

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some vertebrates release bile salts as pheromones (5, 6). In soils and waters, bile saltsare a carbon- and energy-rich substrate for heterotrophic bacteria. Accordingly, diverseGram-negative and Gram-positive bacteria, which can grow with bile salts as carbonand energy sources under aerobic conditions, have been isolated and investigated inrecent years (7, 8). Using the trihydroxy bile salt cholate (Fig. 1, compound I) as a modelcompound, two pathways for initiating bile salt degradation have been identified. Thefirst degradation pathway is extensively being investigated with Pseudomonas stutzeristrain Chol1 and Rhodococcus jostii strain RHA1 as well as with different strains ofComamonas testosteroni (9–12) and can be separated into four distinct reaction se-

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FIG 1 Section of the proposed cholate (I) degradation pathways in P. stutzeri Chol1 (blue) and Novosphingo-bium sp. strain Chol11 (yellow). In both strains, cholate is sequentially oxidized to 3-ketocholate (II) andΔ4-3-ketocholate (III). In P. stutzeri Chol1, this intermediate is further degraded via Δ1,4-3-ketocholate (IV), CoAester of 7�,12�-dihydroxy-3-oxochola-1,4,(22E)-triene-24-oate ([DHOCTO] V), CoA ester of THOCDO (VI),7�,12�-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde, ([DHOPDCA] VII), 7�,12�-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate ([DHOPDC] VIII), 7�,12�-dihydroxy-androsta-1,4-diene-3,17-dione ([12�-DHADD] IX),and 3,7,12-trihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione ([THSATD] X). In Novosphingobium sp.strain Chol11, Δ4-3-ketocholate (III) is degraded via 12�-hydroxy-3-oxo-4,6-choldienoic acid ([HOCDA] XI),3,12-dioxo-4,6-choldienoic acid ([DOCDA] XII), and 12�-hydroxy-androsta-1,4,6-triene-3,17-dione ([HATD] XIII).3,12�-Dihydroxy-9,10-secoandrosta-1,3,5(10),6-tetraene-9,17-dione ([DHSATD] XIV) is the next expected in-termediate, which cannot be metabolized by P. stutzeri Chol1. In Novosphingobium sp. strain Chol11,12�-DHADD can be converted into HATD by Hsh2.

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quences: partial oxidation of the A ring, stepwise degradation of the carboxylic sidechain, cleavage of the B ring between C-9 and C-10, and further degradation of theresulting 9,10-seco steroid. This degradation pathway proceeds via intermediates witha 3-keto-Δ1,4-diene structure of the steroid skeleton, which is formed by the initiatingoxidative reactions at the A ring.

In Proteobacteria, the side chain is usually completely degraded before the cleavageof the B ring (9, 13). In Actinobacteria, these reactions can occur concomitantly (14). Sidechain degradation is initiated by coenzyme A (CoA) activation catalyzed by a steroid-24-oyl-CoA ligase in the model organisms P. stutzeri Chol1, Pseudomonas putida strainDOC21, and C. testosteroni strain KF-1 (15). In strain DOC21, this enzyme (StdA1DOC21)also catalyzes the activation of C5 side chain degradation products with an oxidized Aring. Further degradation of the side chain proceeds by stepwise removal of anacetyl-CoA and a propionyl-CoA residue by modified �-oxidation reactions (9, 16). Forthis, the side chain is dehydrogenated at the Δ22 position and the resulting doublebond is hydrated. Biochemical and genetic evidences in Proteobacteria suggest thatacetyl-CoA is subsequently removed from the �-hydroxyacyl-CoA ester by an aldolyticcleavage reaction, leading to the formation of an aldehyde intermediate with ashortened C3 side chain (17). The aldehyde function is subsequently oxidized to thecorresponding acid, leading to the formation of 3-oxopregna-1,4-diene-20-carboxylatederivatives (OPDCs) with free carboxyl groups. In strain DOC21, a second steroidacyl-CoA ligase, StdA2DOC21, was shown to specifically activate the C3 side chain (15).The remaining side chain is removed via a mechanism similar to the first reaction cycle,ending with another aldolytic cleavage reaction splitting off propionyl-CoA (9, 15, 16).Homologs of StdA1 and StdA2 are also present in P. stutzeri Chol1 and C. testosteroniKF-1. In R. jostii RHA1, most side chain degradation reactions are thought to be similarto the progression in Proteobacteria, as it harbors two CoA ligases, CasG and CasI, whichcatalyze the CoA activation of cholate degradation intermediates with C5 and a C3 sidechains, respectively (18). However, the exact reaction sequence leading to the cleavageof acetyl-CoA from the side chain is currently unknown in R. jostii.

The complete removal of the side chain results in the formation of C19-androstadienediones (ADDs), which are further transformed into the aforementioned9,10-seco steroids (7, 12). This reaction is catalyzed by a monooxygenase, whichhydroxylates the C-9 atom, leading to the opening of the B ring and the simultaneousaromatization of the A ring. Further degradation of this 9,10-seco steroid proceeds viaopening of the aromatic A ring followed by hydrolytic cleavage of the resulting openrings (12). These reactions yield 2-hydroxyhexa-2,4-dienoic acid and derivatives ofH-methylhexahydroindanone-propanoates (HIPs), which consist of the remaining C andD rings plus a propionyl side chain derived from the former B ring (9). Further HIPdegradation reactions have only recently been discovered (19).

In the actinobacterium Dietzia sp. strain Chol2 and the Alphaproteobacteria Sphin-gomonas sp. strain Chol10 and Novosphingobium sp. strain Chol11, an alternativedegradation pathway has been detected (8). In these bacteria, cholate degradationproceeds via novel 3-keto-Δ4,6-diene metabolites with double bonds in the B rings,namely, 12�-hydroxy-3-oxo-4,6-choldienoic acid (HOCDA; XI in Fig. 1) and 3,12-dioxo-4,6-choldienoic acid (DOCDA; XII). To our knowledge, these metabolites are not formedduring bile salt degradation in any of the above-described model organisms, P. stutzeri.Chol1, P. putida DOC21, C. testosteroni, and R. jostii RHA1. The hydroxysteroid 7�-dehydratase Hsh2 has recently been identified as the key enzyme for formation of thesecompounds from cholate and other 7-hydroxy bile salts in Novosphingobium sp. strainChol11 (20). After the oxidation of the 3-hydroxy group and the introduction of adouble bond at the Δ4 position, the hydroxyl group at C-7 is removed by Hsh2,resulting in the formation of HOCDA (XI), which is further degraded to 12�-hydroxy-androsta-1,4,6-triene-3,17-dione (HATD; XIII). Dehydration of the 7-hydroxy group hasbeen shown to also proceed during the degradation of the 7�-hydroxy bile saltursodeoxycholate (20). Our studies suggest that this dehydration reaction is an oblig-atory step during the degradation of 7-hydroxy steroids in strain Chol11.

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In this study, we aimed at further exploring this alternative bile salt degradationpathway. For this, we applied bioinformatics analysis of the draft genome of strainChol11 as well as transposon and directed mutagenesis to identify further genes,proteins, and reaction steps involved in this pathway.

RESULTSIn silico genome analysis of Novosphingobium sp. strain Chol11. The genome of

Novosphingobium sp. strain Chol11 (EMBL database [EBI] accession no. OBMU01000001to OBMU01000010) comprises two chromosomes (chromosome 1 [2.54 Mb] and chro-mosome 2 [0.86 Mb]) and two plasmids (pSa [0.13 Mb] and pSb [0.13 Mb]) containing3,532 putative open reading frames. For identifying potential steroid degradationproteins, we analyzed the in silico proteome of strain Chol11 with hidden Markovmodels (HMMs) and BLASTp. This analysis identified 50 potential steroid degradationproteins encoded in the genome of strain Chol11, including 16 hits for putative steroidside chain degradation proteins, 14 hits for A/B ring degradation, 17 hits for C/D ringdegradation, and three hits for the degradation of 2-hydroxyhexa-2,4-dienoic acid (seeTable S1 in the supplemental material). Eight of those proteins are encoded onchromosome 1 (2.54-Mb chromosome) and 40 are encoded on chromosome 2 of strainChol11. While HMM hits for putative steroid degradation genes on chromosome 1 arescattered throughout the chromosome, three potential steroid degradation geneclusters can be identified on chromosome 2 (see Fig. S1). Cluster 1 contains fourpotential steroid degradation genes encoding homologs of the A/B ring degradationproteins KstD, KshA, and HsaD and a potential side chain degradation protein. Cluster2 contains 20 potential steroid degradation genes, including two sets of neighboringgenes encoding the potential A/B ring degradation proteins HsaA to -D and thepotential 2-hydroxyhexa-2,4-dienoic acid degradation proteins HsaE to -G. These hitsare accompanied by five genes presumably encoding side chain degradation proteinsand four genes presumably encoding C/D degradation proteins, as well as three genesencoding homologs of KshA. Cluster 3 contains nine potential steroid degradationgenes, including one gene encoding a homolog of KstD, as well as three genesencoding homologs of putative side chain degradation proteins and five genes encod-ing C/D degradation proteins.

The reciprocal BLASTp analysis was performed using characterized and hypotheticalsteroid degradation proteins of P. stutzeri strain Chol1, R. jostii RHA1, and C. testosteronistrain CNB-2 as query sequences and the proteome of strain Chol11 as the subject (Fig.2). In general, Chol11 has more homologs to steroid degradation proteins from theGram-negative strains Chol1 and CNB-2 than to proteins from the Gram-positive strainRHA1. While multiple homologs to proteins encoded in the cholesterol degradationgene cluster in strain RHA1 exist, only minor sequence similarities to proteins in thecholate degradation gene cluster in RHA1 can be found.

The proteome of strain Chol11 contains homologs of most key enzymes of steroidring degradation, such as KstD, KshA, HsaC, and HsaD, as well as homologs of most C/Dring degradation proteins (Fig. 2). Strikingly, the proteome of Chol11 does not containreciprocal BLAST hits of most known steroid side chain degradation proteins from strainChol1 or RHA1. In particular, there are no homologous proteins for Scd1AB, Shy1, Sal1,or Sad, which catalyze the release of acetyl-CoA from the C5 side chain of cholate inChol1, or homologs of Scd2AB or Sal2, which catalyze part of the subsequent releaseof propionyl-CoA from the steroid skeleton. These findings suggest that the steroid sidechain degradation in strain Chol11 may proceed via so-far-unknown reaction steps.

Characterization of the transposon mutant strain Chol11 Tn50KL. To identifypotential new side chain degradation genes in strain Col11, we subjected the wild typeto random mutagenesis by the insertion of the transposon mini-Tn5 Km1. Three of5,000 transposon mutants had altered growth phenotypes when growing with cholate.Transposon mutant strain Chol11 Tn50KL grew only poorly with cholate as the solecarbon and energy source (see Fig. S2) and was therefore further characterized.High-performance liquid chromatography-mass spectrometry (HPLC-MS) analyses re-

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vealed that HOCDA (XI) and DOCDA (XII) transiently accumulated in the culturesupernatants of strain Chol11 Tn50KL (Fig. 3, top). After 10 days of incubation, twounknown intermediates with absorption maxima at 290 nm and with molecular massesat 414 and 430 Da remained in culture supernatants (P1 and P2, respectively) (Fig. 3,bottom; see also Fig. S3A and B). Growth experiments with extracted supernatantsshowed that these compounds were not degraded by the wild-type strain Chol11 (notshown).

Plasmid sequencing of kanamycin-resistant Escherichia coli clones of a Chol11Tn50KL clone library revealed that the transposon was inserted into the gene nov2c230,which was renamed sclA (see below). Automatic annotation suggested that sclA codesfor an AMP-dependent synthetase and ligase with a molecular mass of approximately63 kDa.

Characterization of the mutant strain Chol11 �sclA. In the next step, weconstructed an unmarked sclA gene deletion mutant to rule out potential polar

FIG 2 Reciprocal BLASTp analysis of steroid degradation proteins of Novosphingobium sp. strain Chol11. The heat map shows BLASTsimilarities to characterized and hypothetical steroid degradation proteins from Pseudomonas stutzeri Chol1, Rhodococcus jostii RHA1, andComamonas testosteroni CNB-2. Characterized side chain degradation proteins are marked in boldface font.

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effects of the transposon in strain Chol11 Tn50KL. The growth rate of strain Chol11ΔsclA with cholate as the substrate was 5-fold lower than that of wild-type cells(�wild-type � 0.34 � 0.02 h�1 and �Chol11 ΔsclA � 0.07 � 0.01 h�1) (Fig. 4A). Similarto results for strain Chol11 Tn50KL, HPLC-MS analyses showed that Δ4-3-ketocholate(III), HOCDA (XI), and DOCDA (XII) accumulated transiently in culture supernatants,indicating that the initial A and B ring oxidation reactions of cholate degradation werenot affected in the deletion mutant. At the end of the exponential growth phase afterapproximately 10 days, cholate was no longer detectable. Also the dead-end interme-diates P1 and P2 accumulated in culture supernatants. So far, P1 and P2 have not beenidentified.

Strain Chol11 ΔsclA grew with deoxycholate with a significantly lower growth ratethan the wild-type strain Chol11 and did not reach a similar maximum cell density (seeFig. S4). This indicates that SclA is required for metabolizing both cholate and deoxy-cholate. In addition, we performed growth experiments with two cholate degradationintermediates from P. stutzeri Chol1, namely, 7�,12�-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC; VIII in Fig. 1) with a C3 side chain and 7�,12�-dihydroxy-androsta-1,4-diene-3,17-dione (12�-DHADD; IX) without any side chain (Fig. 4B). Thegrowth phenotype of the deletion mutant with DHOPDC (VIII) and 12�-DHADD (IX)resembled that of the wild type, indicating that the degradation of these substrates wasnot affected in Chol11 ΔsclA.

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FIG 4 (A) Growth of Novosphingobium sp. strain Chol11 (filled diamonds) and mutant strain Chol11 ΔsclA(open circles) with 1 mM cholate. (B) Growth of Novosphingobium sp. strain Chol11 (filled symbols) andmutant Chol11 ΔsclA (open symbols) with 1 mM DHOPDC (circles) or with 1 mM DHADD (diamonds).Error bars indicate standard deviations (n � 3).

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As sclA was annotated as a putative CoA ligase, we analyzed CoA ligase activities incell extracts of wild-type and mutant strains with cholate and its degradation interme-diates in the presence of CoA, ATP, and Mg2� using HPLC-MS. No CoA ester was formedby the wild type or mutant cell extracts with cholate or DHOPDC (VIII) as the substrates.In contrast, CoA esters of 3-ketocholate (II), Δ4-3-ketocholate (III), and DOCDA (XII) wereformed within 15 min of incubation in wild-type cell extracts (see Fig. S5). After 40 minof incubation, approximately 45% of DOCDA (XII) was transformed into DOCDA-CoA inthese assays. Further incubation led to a decrease of DOCDA-CoA accompanied by aconcomitant increase of free DOCDA (XII), indicating hydrolysis of the thioester bond(see Fig. S6). Without the addition of ATP, DOCDA-CoA was not formed (data notshown), suggesting that these reactions were catalyzed by an ATP-dependent CoAligase. In cell extracts of strain Chol11 ΔsclA, the formation of DOCDA-CoA was notobserved (Fig. S5). These results strongly suggest that sclA encodes a CoA ligaseresponsible for side chain activation of cholate degradation intermediates with C5 sidechains and oxidized A rings. In the extracts of cells grown with glucose, CoA activationof DOCDA was also measured (see Fig. S7), indicating that the presence of cholate is notrequired for the expression of this CoA ligase.

Several attempts to complement the sclA deletion mutant were made, including theexpression of an intact copy of sclA under the control of the lac promoter of Escherichiacoli or a putative promoter found in the upstream region of sclA. However, all attemptsfailed.

Characterization of SclA. To confirm the proposed CoA ligase activity of SclA, weproduced a His-tagged recombinant SclA protein in E. coli. The corresponding proteinband of the His-tagged recombinant protein on SDS-PAGE was consistent with thecalculated molecular mass of 63 kDa for SclA (see Fig. S8). The substrate spectrum ofthe purified recombinant SclA protein was characterized using cholate and cholatedegradation intermediates known from P. stutzeri Chol1 (21) and Novosphingobium sp.strain Chol11 (20). For this purpose, SclA was incubated with cholate (I), 3-ketocholate(II), Δ4-3-ketocholate (III), DOCDA (XII), 7�,12�,dihydroxy-3-oxochola-1,4,(22E)-triene-24-oate (DHOCTO; V), 7�,12�,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO; VI),and DHOPDC (VIII) as the substrates in the presence of CoA, ATP, and Mg2�. Thereaction mixtures were analyzed for substrate consumption and CoA ester formation byHPLC-MS after 45 min (see Fig. S9). In accordance with CoA ligase activity in cellextracts, the highest concentrations of CoA esters were measured with DOCDA (XII) asthe substrate (Fig. 5). Simultaneously, AMP formation and ATP depletion were ob-served, suggesting an AMP-forming CoA ligase (see Fig. S10). Compared to the amount

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FIG 5 Analysis of substrate spectra of SclA. Purified recombinant SclA was incubated with cholate (a [Iin Fig. 1]), deoxycholate (b), 3-ketocholate (c [II in Fig. 1]), Δ4-3-ketocholate (d [III in Fig. 1]), DOCDA (e [XIIin Fig. 1]), DHOCTO (f [V in Fig. 1]), and DHOPDC (g [VIII in Fig. 1]), and CoA ester formation after 45 minof incubation was analyzed by HPLC-MS. The amount of DOCDA-CoA formed after 45 min was set to100%, and CoA ester formation with other substrates was compared with the concentration of DOCDA-CoA. Integrated peak areas originated from extracted ion chromatograms in negative ion mode of MS(for cholyl-CoA, m/z 1,156.7; for deoxycholyl-CoA, m/z 1,140.7; for 3-ketocholyl-CoA, m/z 1,154.7; forΔ4-3-ketocholyl-CoA, m/z 1,152.7; for DOCDA-CoA, m/z 1,132.8; for DHOCTO-CoA, m/z 1,148.7; forDHOPDC, m/z 1,122.7) (see Fig. S11 in the supplemental material for mass and UV spectra) (n � 3).

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of DOCDA-CoA formed after 45 min, less than 2% of cholyl-CoA was formed withcholate as the substrate and approximately 32% and 83% of the respective CoA estersof 3-ketocholate (II) and Δ4-3-ketocholate (III). In contrast, no CoA ester formation wasobserved with DHOPDC (VIII). CoA ester formation with DHOCTO was lower than 5%.Δ1,4-3-Ketocholate (IV) was also activated with CoA; however, this substrate preparationalso contained a considerable amount of Δ4-3-ketocholate (III), which makes it difficultto make a clear statement on the activation level for this substrate. These resultsshowed that SclA predominantly activates C5 acyl side chains of steroids with a3-keto-Δ4-monoene or 3-keto-Δ4,6-diene structure of the steroid skeleton. On the basisof the results of genetic and biochemical analyses, we named this gene sclA for steroidCoA ligase 1. In vector control strains of E. coli, no CoA ligase activity was observed withDOCDA as the substrate in cell extracts excluding any CoA ligase activity derived fromthe host strain used for overexpression.

Analysis of side chain degradation in Novosphingobium sp. strain Chol11. In ourprevious studies, we identified HATD (XIII) as a cholate degradation intermediate ofNovosphingobium sp. strain Chol11, from which the carboxylic side chain had beencompletely removed (8). However, we did not detect any intermediates with a modifiedside chain in culture supernatants of this strain during growth with cholate or other bilesalts. As the discovery of SclA suggested that the degradation of DOCDA (XII) is initiatedby CoA activation, we performed enzyme assays with cell extracts of strain Chol11 toinvestigate further degradation of DOCDA-CoA. For this purpose, the above-mentionedCoA activation assays with DOCDA (XII; 384 Da) were performed in the presence of theartificial electron carrier phenazine methosulfate (PMS). Under these conditions, a novelintermediate eluting very closely to DOCDA (XII) was detected in reaction mixtures after300 min of incubation (Fig. 6A, top and middle). This compound (P3) had an absorptionmaximum at approximately 205 nm in addition to the maximum at 290 nm, and itsmolecular mass (382 Da) was 2 Da lighter (see Fig. S11A) than that of the initialsubstrate DOCDA (XII). Control assays revealed that the formation of this compounddepended on the presence of both CoA and PMS. In the presence of PMS and theabsence of CoA, another unknown compound (P4) was detected instead of the P3

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FIG 6 (A) Transformation of DOCDA (top) in cell extracts of Novosphingobium sp. strain Chol11. In thepresence of PMS, CoA, ATP, and Mg2�, DOCDA was converted into P3 (middle). In the presence of PMS,DOCDA was converted into P4 (bottom). P3 and P4 are predicted to be Δ22-DOCTRA [3,12-dioxo-4,6,(22E)-choltrienoic acid] and Δ1-DOCTRA (3,12-dioxo-1,4,6-choltrienoic acid), respectively. HPLC-MS data aredisplayed as basic peak chromatograms in negative mode of MS. (B) Chemical structures of DOCDA,Δ1-DOCTRA, Δ22-DOCTRA, and DOCTTRA [3,12-dioxo-1,4,6,(22E)-choltetraenoic acid].

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(Fig. 6A, bottom). This intermediate also had a molecular mass of 382 Da. However, itsabsorption spectra differed clearly from the first one. It had a UV spectrum with featurescharacteristic of the Δ1,4,6-triene intermediate HATD (XIII), exhibiting absorption max-ima at 226, 255, and 305 nm (Fig. S11B) (8). When PMS was replaced with NAD� in thepresence and absence of CoA, none of these compounds were formed. The molecularmasses of P3 and P4, as well as the dependency of their formation on PMS, indicate thatboth of these compounds have one CAC double bond more than the substrateDOCDA (XII). Supplying cell extracts with recombinant SclA increased the formation ofP3 (see Fig. S12A and B). Under these conditions, a novel intermediate was detected inreaction mixtures after 300 min of incubation (see Fig. S12B). Control assays revealedthat the formation of this compound depended on the presence of both CoA (Fig.S12C) and PMS. This compound (P5) had absorption maxima at approximately 215, 260,and 300 nm and its molecular mass (380 Da) was 4 Da lighter (see Fig. S13) than thatof the initial substrate DOCDA (XII). When extracts of cholate-grown cells were replacedwith extracts of glucose-grown cells, P4 was the main product in the reaction mixtureafter 300 of incubation (see Fig. S14). In contrast, P3 and P5 were not detected underthese conditions (Fig. S14). The absorption maximum at approximately 205 nm (see Fig.S15) suggests that P3 has an additional double bond in the C5 side chain. The fact thatthe formation of this double bond required CoA activation of the substrate alsosupports this suggestion. As in P. stutzeri Chol1, an acyl-CoA dehydrogenase (ACAD)might catalyze the �,�-dehydrogenation of the side chain. The exact position of thispotential double bond is unknown, but assuming a �-oxidation-like pathway, it wouldbe expected between C-22 and C-23. Accordingly, P3 was proposed to be 3,12-dioxo-4,6,(22E)-choltrienoic acid (Δ22-DOCTRA). In agreement with the suggested cholatedegradation sequence in strain Chol11, the compound P4 presumably has an additionaldouble bond at the Δ1 position and was thus identified as 3,12-dioxo-1,4,6-choltrienoicacid (Δ1-DOCTRA). The high similarity between the absorption spectra of this com-pound and those of the Δ1,4,6-compound HATD (XIII) supports this suggestion.

In the next step, enzyme assays were performed with purified P4 as the substrate.In the presence of CoA, ATP, and Mg2�, most of P4 was activated by SclA with CoAwithin 15 min (see Fig. S16A and C). When CoA activation assays were performed withcell extracts supplied with recombinant SclA and PMS, small amounts of P5 weredetected in the reaction mixture (Fig. S16B), indicating that P4 is the precursor of P5.Control assays revealed that the formation of this compound depended on thepresence of CoA (Fig. S16B). On the basis of mass and UV spectra, P5 was proposed tobe 3,12-dioxo-1,4,6,(22E)-choltetraenoic acid (DOCTTRA) (Fig. 6B).

DISCUSSION

Novosphingobium sp. strain Chol11 is proposed to have an alternative initial deg-radation route for 7-hydroxysteroids such as the bile salt cholate (compound I in Fig. 1).This pathway is characterized by the activity of the 7-hydroxysteroid dehydratase Hsh2as the key enzyme and 3-keto-7-deoxy-Δ4,6 steroids as key intermediates (20). In thisstudy, we identified SclA from strain Chol11 as a novel steroid CoA ligase, whichpreferentially activates the C5 acyl side chain of bile salt degradation intermediates witha 3-keto-7-deoxy-Δ4,6 structure of the steroid skeleton. In agreement with this function,an sclA deletion mutant had a strong phenotype during growth with cholate. Thesefindings support further the suggested cholate degradation pathway, which proceedsvia the 3-keto-Δ4,6-diene-7-deoxy intermediates HOCDA (XI) and DOCDA (XII) in strainChol11.

SclA comprises conserved residues for CoA, ATP, and AMP binding sites, indicatingan AMP-forming acyl-CoA ligase, which is in agreement with experimental results.Moreover, it contains a facl-like domain, which is characteristic for these enzymes andis also found in the steroid acyl-CoA ligases StdA1 from P. putida DOC21 and CasG fromR. jostii RHA1. SclA shares 38% sequence identity with StdA1 and 32% sequence identitywith CasG. The substrate specificity of CoA ligases involved in steroid side chaindegradation appears to be determined by the length of the side chain (15, 18). While

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the aforementioned StdA1 and CasG only activate side chains with five carbons, StdA2and CasI catalyze the CoA activation of side chains with three carbons in P. putidaDOC21 and R. jostii RHA1, respectively. In contrast to the influence of the side chain, itis so far unknown to what extent modifications on the steroid skeleton affect theactivity of CoA ligases. CasI exhibits different affinities to metabolites with variedconfigurations of the steroid skeleton (18). StdA1DOC21 is known to be able to activateboth cholate and 3-ketocholate (II) with CoA (15). In cell extracts of P. stutzeri Chol1,Δ1/4-3-ketocholate and Δ1,4-3-ketocholate are thioesterified with CoA, probably byStdA1Chol1, although the in vitro oxidation of cholate to Δ1,4-3-ketocholate does notrequire a thioesterification step (22). Our data strongly suggest that a 3-keto-7-deoxy-Δ4,6 steroid skeleton is the preferred substructure for SclA, indicating that DOCDA (XII)is the physiological substrate of this enzyme in strain Chol11. First, with purified SclA,the largest amounts of CoA thioesters were formed with DOCDA (XII), while only verysmall amounts of cholyl-CoA and 3-ketocholyl-CoA were formed. Second, CoA ligaseassays with cell extracts of strain Chol11 revealed no cholyl-CoA formation, whileDOCDA (XII) was activated; this is in agreement with the observation that CoA activa-tion is not required for the initial A and B ring-modifying reactions of cholate metab-olism, which form DOCDA (XII) from cholate (20). However, it must be noted that theamounts of CoA ester formed from Δ4-3-ketocholate and DOCDA differed only slightlyfrom each other. Therefore, it is possible that the dehydration reaction catalyzed byHsh2 can also occur after the activation of Δ4-3-ketocholate with CoA. In support of thispossibility, it has been shown that BaiE, which is a bile acid 7�-dehydratase fromClostridium scindens and shares 38% sequence identity with Hsh2, can use the CoA esterof Δ4-3-ketocholate as the substrate (23). According to the crystal structure analyses ofBaiE, the CoA moiety does not bind to the active site pocket of the enzyme.

The lack of SclA resulted in a dramatic growth rate decrease and caused theaccumulation of two novel but yet unidentified dead-end products. This phenotypesupports the hypothesis that a CoA activation step catalyzed by SclA is an essential partof the degradation sequence for bile salts in strain Chol11. However, similar to thedeletion of hsh2 (20), the deletion of sclA strongly impaired but did not abolish thegrowth of strain Chol11 with cholate. This phenotype is in contrast to that of P. stutzeriChol1, in which the deletion of genes involved in initial reactions of bile salt degrada-tion results in a complete lack of growth (9, 21, 22), and supports the notion that strainChol11 has a broad metabolic repertoire. Our bioinformatics analysis revealed that thegenome of strain Chol11 encodes several potential isoenzymes for some steroiddegradation reactions. For example, five putative homologs of KshA, which could act asisoenzymes, are encoded on chromosome 2 of strain Chol11. However, we did not findany gene that could potentially compensate for the loss of sclA. Nevertheless, wecannot rule out the possibility that genes for the so-far-unknown steroid CoA ligases orCoA transferases are among those encoding hypothetical proteins. Alternatively, theloss of SclA might be compensated for by unspecific activities of other CoA ligases orCoA transferases toward substrates of SclA. The reduced growth rate of the ΔsclAmutant supports the second possibility, as unspecific enzymes might plausibly com-pensate for the loss of sclA, allowing continued cholate metabolism though at asignificantly reduced rate.

Despite different complementation strategies, the phenotype of wild-type cellscould not be restored in mutant strain Chol11 ΔsclA. A sequencing analysis of regionsneighboring the deleted gene ruled out a frameshift. A potential reason for the lack ofa complementing effect of plasmid-encoded SclA could be that its expression in strainChol11 but not in E. coli has some structural requirements, which can only be providedwhen the gene is in its original genomic context. Nevertheless, the assigned functionof SclA was consistent with enzyme activities in cell extracts and with the purifiedrecombinant protein, as well as with the phenotype of the mutant.

Activity of some AMP-dependent acyl-CoA synthetases is proposed to be regulatedby the acetylation of a lysine residue, which inhibits the formation of an adenylateintermediate but not the thioesterification of preadenylated intermediates (24). Inter-

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estingly, the characterized steroid-24-oyl-CoA ligases, namely, StdA1 and CasG, as wellas the putative CoA ligase from P. stutzeri Chol1, encoded by C211_RS11125, contain anacetylation site lysine residue within a highly conserved PX4GK motif, which is requiredfor this posttranslational modification (25). The acetylation of these CoA ligases may bean important regulatory mechanism for bile salt metabolism in these strains, becauseit is the first step of the carboxylic side chain degradation. In contrast to these steroidCoA ligases, the PX4GK motif is not conserved in SclA (see Fig. S17), indicating potentialdifferences in the regulation of bile salt degradation.

It is currently unknown how the side chain of DOCDA is further degraded. In cellextracts, we detected two dehydrogenation reactions acting on DOCDA. While the firstone was CoA dependent, the second one was CoA independent. The next plausiblestep in the side chain degradation of DOCDA-CoA would be the formation of anenoyl-CoA compound catalyzed by an acyl-CoA dehydrogenase. This possibility issupported by the CoA-dependent dehydrogenation of DOCDA (XII) into a compound,whose spectroscopic properties are in agreement with Δ22-DOCTRA (P3) (Fig. 6B). Inother cholate-degrading Proteobacteria, e.g., P. stutzeri Chol1, P. putida DOC21, and C.testosteroni strain TA441, the side chains are completely removed before the B rings canbe opened (9, 13, 15). The fact that Chol11 forms the C19 steroid HATD (XIII) as a cholatedegradation intermediate, which has no side chain but an intact ring system, suggeststhat the side chain is also completely removed before the opening of the steroidskeleton in strain Chol11. As such, it may be possible that the deletion of sclA forces themutant strain to metabolize the steroid skeleton before metabolizing the C5 side chain.Apart from the aforementioned lower growth rate, the ΔsclA mutant also reached alower final optical density, indicating a lower molar growth yield. In agreement withthat, the accumulation of the dead-end metabolites P1 and P2 in the supernatant of thesclA mutant indicates that the degradation is incomplete. Thus, SclA appears to have anessential function for the optimal growth of strain Chol11 with bile salts.

The CoA-independent dehydrogenation was likely to be catalyzed by a Δ1-ketosteroid dehydrogenase, which converts DOCDA (XII) into Δ1-DOCTRA (P4) (Fig. 6B).It is unknown whether Δ1 and side chain dehydrogenation concomitantly occur instrain Chol11. However, the in vitro conversion of Δ1-DOCTRA into DOCTTRA (P5) (Fig.6B) in the presence of CoA indicates that the degradation of side chains can also occurafter the introduction of a double bond at the Δ1 position.

Despite the observation of Δ22-DOCTRA and DOCTTRA (Fig. 6B) as plausible degra-dation intermediates, there are neither physiological nor genomic hints of how the sidechain is further degraded. A common characteristic of sphingomonads is the complexarrangement of genes involved in degradation pathways, which are frequently scat-tered in several clusters (26, 27). In agreement with this complex localization, HMMsand reciprocal BLASTp analyses identified three putative steroid degradation geneclusters on chromosome 2 of strain Chol11 encoding several key enzymes presumablymediating steroid ring degradation. Moreover, several candidate genes involved insteroid metabolism were also found outside these cluster regions. However, homologsof known key enzymes involved in side chain degradation were not found. In P. stutzeriChol1, the removal of acetyl-CoA from the C5 side chain occurs via aldolytic cleavage(17). The resulting aldehyde intermediate is oxidized and subsequently activated withCoA. There are no aldolase (sal), aldehyde dehydrogenase (sad), or steroid-22-oyl-CoAligase (sdtA2) homologs present in the genome of strain Chol11. Nevertheless, theremoval of the side chain may be very similar to that in P. stutzeri strain Chol1, but theinvolved enzymes may not resemble those known from P. stutzeri Chol1 or otherwell-studied strains. In agreement with this, we could not find homologs for thecharacteristic heteromeric acyl-CoA dehydrogenases, which are responsible for desatu-ration of carboxylic side chains of steroids in other bacteria, such as P. stutzeri Chol1 (9)and Mycobacterium tuberculosis (28, 29), indicating that the formation of Δ22-DOCTRAmust have been catalyzed by a so-far-unknown enzyme system.

Another explanation for the lack of known genes for side chain degradation in strainChol11 might be that the actual reactions involved in this process are different from the

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known reactions for side chain removal in other bacteria. For example, a thiolyticcleavage of acetyl-CoA catalyzed by a �-keto-thiolase would directly form a CoA-activated C3 side chain intermediate and would not require an additional steroid-22-oyl-CoA ligase. Accordingly, we did not observe any CoA ligase activity toward DHOPDC(VIII) harboring a C3 carboxyl side chain in cell extracts of cholate- or DHOPDC-grownstrain Chol11.

In conclusion, this study further supports the hypothesis that strain Chol11 has analternative pathway for the degradation of 7-hydroxy bile salts and adds furtherevidence of the diversity of metabolic pathways for the degradation of steroid com-pounds in environmental bacteria. The knowledge about these pathways might beuseful for exploring the fate of pharmaceutical steroids in the environment, some ofwhich may act as endocrine disruptors (30).

MATERIALS AND METHODSBacterial strains, growth media, and growth experiments. Novosphingobium sp. strain Chol11 was

grown in the HEPES-buffered mineral medium B (MB) (31) with 1 mM cholate as described previously (8).Mutant strain Chol11 ΔsclA was grown in the same medium with 15 mM glucose or 1 mM cholate.Mutant strain Chol11 ΔsclA with plasmid pBBR1MCS-5, pBBR1MCS-5::sclA, or pBBR1MCS-5::sclA�P wasgrown in the same medium with 15 mM glucose in the presence of 60 �g · ml�1 gentamicin. StrainChol11 Tn50KL was initially grown in phosphate-buffered mineral medium (MMChol) (32) with 15 mMpropionate and 75 �g · ml�1 kanamycin; later, MMChol1 was replaced by MB for all further experimentswith strain Chol11 and its mutants. Escherichia coli was grown in lysogeny broth (LB) at 37°C and 200 rpm.LB was supplemented with gentamicin (30 �g · ml�1) for ST18 strains carrying pBBR1MCS-5 andpBBR1MCS-5::sclA, with chloramphenicol (30 �g · ml�1) for strain ST18(pDM4::230UpDown), and withkanamycin (50 �g · ml�1) for E. coli Tuner(pET28b::sclA) and E. coli S17-1 �pir pUT(mini-Tn5Km1) (33–35).Growth media for E. coli ST18 strains, which are auxotrophic for 5-aminolevulinic acid, were additionallysupplemented with 50 �g · ml�1 of this substance (36). Bacto agar (1.5% [wt/vol]; BD, Sparks, USA) andgrowth substrate were added to the aforementioned media for preparing respective solid media, onwhich each strain was maintained and transferred weekly. Growth substrates were 1 mM cholate (forstrain Chol1), 15 mM propionate (for strain Chol11 Tn50KL), and 15 mM glucose (for strain Chol11 ΔsclA).

All growth experiments were performed in 10-ml test tubes containing 3 to 5 ml of the respectivemedia at 30°C with orbital shaking at 200 rpm (Minitron; Infors HT, Einsbach, Germany). Growth wasfollowed by measuring the optical density at 600 nm (OD600) with a test tube photometer (CamspecM107; Spectronic Camspec, United Kingdom). For precultures of strain Chol11, test tubes containing 3 to5 ml medium with 1 mM cholate were seeded with the respective strains from agar plates. Preculturesof strain Chol11 Tn50KL were grown with 15 mM propionate in the presence of 75 �g · ml�1 kanamycin.Precultures of strain Chol11 ΔsclA were grown with 15 mM glucose in the presence of 1 mM cholate.Precultures of strain Chol11 ΔsclA with plasmid pBBR1MCS-5, pBBR1MCS-5::sclA, or pBBR1MCS-5::sclA�Pwere grown with 15 mM glucose in the presence of 60 �g · ml�1 gentamicin. All main cultures wereinoculated from precultures in late exponential phase to an OD600 of 0.01 to 0.02 and contained 1 mMbile salt (cholate or deoxycholate) or 1 mM cholate degradation intermediates DHOPDC (VIII) or12�-DHADD (IX) as carbon and energy sources.

Transposon mutagenesis and localization of transposon in genome. For transposon mutagen-esis, the suicide vector pUT(mini-Tn5Km1) (34) was mobilized into Novosphingobium sp. strain Chol11 bybiparental mating with donor strain E. coli S17-1 �pir. For this, main cultures of strains ST18 and Chol11were grown as described above. In late exponential phase, 1 � 109 cells of strain S17-1 �pir and 3 � 109

cells of strain Chol11 were harvested by centrifugation at 7,300 � g for 8 min, washed with 500 �l LB,and resuspended in 50 �l LB. Suspended cells were mixed by gentle pipetting and spread onto LB agarplates. After incubating at 30°C for 4 h, cells were resuspended in 2 ml of 0.9% NaCl, spread onto LB agarplates containing 75 �g · ml�1 kanamycin, and incubated at 30°C for 7 days.

For localizing the transposon in the chromosome of the transposon mutant strain Chol11 Tn50KL, agenomic library of this mutant strain was established. For this, genomic DNA of strain Chol11 Tn50KL waspurified with the Puregene tissue core kit B (Qiagen) and partially digested with SalI (FasDigest; ThermoFisher) at 37°C for 2 h. The resulting fragments were ligated into the SalI restriction site of the E. colishuttle vector pNV18Sm (37) using standard methods. The resulting plasmids were transformed into E.coli DH5�. For the selection of plasmids containing genomic DNA fragments with the transposon,transformed cells were spread onto LB agar plates containing 50 �g · ml�1 kanamycin and incubated at37°C overnight. Kanamycin-resistant colonies were analyzed by PCR using primer pair C/D (Table 1),which is specific for the mini-Tn5 transposon. Plasmids from a positive colony were purified and used asthe template DNA for sequencing the respective genomic fragments of strain Chol11. For this, primerpairs A/B (specific for pNV18Sm) and C/D (specific for the mini-Tn5 transposon) were used. Sequencingwas performed by Eurofins Genomics (Ebersberg, Germany). For sequencing the remaining parts of thegenomic fragment of Chol11, the primer pair E/F was used. The obtained sequences were compared withthose from genomic DNA of strain Chol11 to localize the transposon.

Construction of the unmarked deletion mutant Chol11 �sclA. To construct an unmarked deletionof sclA, two PCR products spanning parts of the up- and downstream regions of the gene were obtainedfrom genomic DNA of strain Chol11 using the primer pairs G/H and I/J (Table 1). The resulting fragments

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were used as the templates for a second splicing-by-overlap extension PCR (SOE-PCR) (38) with theprimer pair H/I. The product of this second PCR was digested with XhoI and XbaI and ligated into thecorresponding sites of the suicide vector pDM4 (34). The resulting plasmid was mobilized into strainChol11 as described previously (20). Transconjugants were streaked twice onto LB agar plates containing50 �g · ml�1 chloramphenicol before they were transferred onto LB agar plates containing 7% sucrosefor the selection of vector excision by a second crossover. Colonies were screened for gene deletion byPCR using primer pair H/I. Positive colonies were streaked twice onto LB agar plates containing 7%sucrose. Finally, the genomic DNA of mutant strain Chol1 ΔsclA was isolated and used as the templatefor PCR amplification with primer pair H/I, and the resulting DNA fragment was sequenced to confirm thedeletion.

For complementation of mutant strain Chol11 ΔsclA, fragments containing the open reading frame(ORF) of sclA with or without a putative promoter region were amplified using primer pairs K/L and M/N,respectively, and genomic DNA from strain Chol11 as the template. The resulting fragments (sclA�P andsclA) were digested with XbaI and EcoRI or HindIII and NcoI, respectively, and ligated into the corre-sponding sites of pBBR1MCS-5. The resulting plasmids pBBR1MCS-5::sclA and pBBR1MCS-5::sclA�P wereintroduced into strain Chol11 ΔsclA by biparental mating as described above. Transconjugants wereselected on solid minimal medium B with 15 mM glucose and 60 �g · ml�1 gentamicin.

Overexpression of SclA. A fragment containing the ORF of sclA was amplified using primer pair O/Pand genomic DNA of Chol11 as the template, and was digested with NcoI and HindIII and ligated intothe corresponding sites of the pET28b expression vector. The resulting plasmid pET28b::sclA wastransformed into E. coli Tuner(DE3), and plasmid-harboring strains were selected on LB agar plates with50 �g · ml�1 kanamycin. To produce polyhistidine (His)-tagged SclA, three 500-ml cultures with LB andkanamycin (20 �g · ml�1) were inoculated with precultures of strain Tuner(pET28b::sclA) grown in LB withkanamycin (20 �g · ml�1) at 37°C and 200 rpm and were subsequently incubated at 25°C and 160 rpm.To induce protein expression, 4 mM isopropyl-�-D-thiogalactopyranoside (IPTG; Carl Roth, Karlsruhe,Germany) was added after 2 h of incubation. At an OD600 of approximately 3 (after 16 of incubation), thecultures were harvested by centrifugation at 7,300 � g for 10 min at 4°C and washed with 20 mM Trisbuffer (pH 8) containing 150 mM NaCl.

After resuspending the pellets in the same buffer, cells were disrupted by sonication (Hielscher,Teltow, Germany) on ice twice for 3 min (amplitude, 60%; pulse cycle, 0.6) followed by centrifugation at17,000 � g for 30 min at 4°C. Supernatants were filtered through a 0.2-�m syringe filter. Cell-free lysateswere loaded onto 10-ml Ni2�-nitrilotriacetate agarose columns equilibrated with 20 mM Tris buffer (pH8.0) containing 150 mM NaCl and 10 mM imidazole.

His-tagged proteins were eluted with 20 to 30% elution buffer containing 20 mM Tris buffer (pH 8.0),150 mM NaCl, and 400 mM imidazole. Protein-containing fractions were pooled, washed, and concen-trated in 50 mM 3-morpholinopropane-1-sulfonate (MOPS) buffer (pH 7.8) using a centrifugal concen-trator (Vivaspin 20 protein concentrator; polyethersulfone [PES] membrane; molecular weight cutoff[MWCO], 10,000; Sartorius Stedim Biotech, Göttingen, Germany). The purified proteins were supple-mented with 10% glycerol and stored at �80°C.

Preparation of cell extracts and protein determination. For preparing cell extracts, 100- to 500-mlcultures (in Erlenmeyer flasks with baffles) of strains Chol11 and the mutant Chol11 ΔsclA were grownwith 1 mM cholate and with 15 mM glucose in the presence of 1 mM cholate, respectively, at 30°C and130 rpm. Cultures were harvested in mid-exponential growth by centrifugation at 8,800 � g for 10 minat 4°C. The resulting pellets were washed by resuspending in 15 ml 10 mM MOPS buffer (pH 7.8) andwere finally resuspended in 1.5 ml 50 mM MOPS (pH 7.8). Cells were disrupted by ultrasonication on icefor 10 min (amplitude, 60%; pulse cycle, 0.5) with intermittent incubations on ice every 4 min.Subsequently, cell debris was removed by centrifugation at 15,000 � g for 30 min at 4°C.

Protein concentrations from cell extracts and purified proteins were determined using the bicin-choninic acid (BCA) assay (Pierce, Thermo Scientific, Rockford, IL, USA) using bovine serum albumin as the

TABLE 1 Oligonucleotides used in this study

Designation Sequence Description

A 5=-AGGGTTTTCCCAGTCACGACGTT M13-fB 5=-GAGCGGATAACAATTTCACACAGG M13-revC 5=-CATTACGCTGACTTGACGGGAC Tn5 mini-fD 5=-ATCTTGTGCAATGTAACATCAGAG Tn5 mini-rE 5=-TATGGCATGACCGAGACATCG gen-outward-fF 5=-AGAGGGTGCGTTACATCGAT gen-outward-rG 5=-CCATGCGGAACTCTCCTGTGACTCTATTCCGCCGCCCGC nov2c230-up-fH 5=-TTTTTTCTAGAGCCCCGGAATGATGGCATTG nov2c230-up-rI 5=-TTTTTTCTCGAGACCTTGATGGCGTTTTCACG nov2c230-down-fJ 5=-ACAGGAGAGTTCCGCATGG nov2c230-down-rK 5=-TTTTTTAAGCTTGATCAGCGGACGAAAGGGAT sclA�promoter-fL 5=-TTTTTTCCATGGCAATCACGTTGTTGGCCCAG sclA�promoter-rM 5=-TTTTTTTCTAGATGTTCAGTTGTTGCCGGCCA sclA-fN 5=-TTTTTTGAATTCCGGCGGAATAGAGTCGTGTT sclA-rO 5=-TTTTTTCCATGGTGTTCAAGCAGAACGGCGAT nov2c230-overex-fP 5=-TTTTTTAAGCTTGTTGTTGCCGGCCAGTTCAG nov2c230-overex-r

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standard. The concentrations of purified proteins were determined by absorption measurements at 280nm using a Nanophotometer (Implen, Munich, Germany).

Enzyme assays. The reaction mixtures for the enzyme assays were buffered with 50 mM MOPS (pH7.8) and incubated on a shaker at 30°C and 450 rpm. Samples were withdrawn at defined time intervalsand analyzed immediately by HPLC-MS. For CoA activation assays with cell extracts, the reaction mixtureswere supplemented with 0.5 to 1 mM cholate (I in Fig. 1), 3-ketocholate (II), Δ4 3-ketocholate (III), DOCDA(XII), or DHOPDC (VIII) as the substrate. For the assays with purified recombinant SclA, the reactionmixtures were supplemented with deoxycholate or DHOCTO (V) as the substrate in addition to theabove-mentioned substrates. The reaction mixtures were also supplied with 2 mM CoA, 2 mM ATP, and3 mM Mg2� and started by the addition of cell extracts or recombinant SclA to final concentrations of0.7 to 1.0 and 0.3 mg protein · ml�1, respectively. The amount of DOCDA-CoA formed after 45 min wasset to 100%, and CoA ester formation with other substrates was compared to this value. For theinvestigation of side chain degradation, cell extracts of Novosphingobium sp. strain Chol11 wereincubated with 1 mM DOCDA (XII) in the presence of 2 mM CoA, 2 mM ATP, 3 mM Mg2�, 2 mM NAD�,and 0.25 mM phenazine methosulfate (PMS).

For the evaluation of the HPLC-MS results, the base peak chromatograms or extracted ion chro-matograms at defined mass ranges were analyzed.

Preparation of steroid compounds. The steroid compounds DHOCTO (V), DHOPDC (VIII), and12�-DHADD (IX) were produced with P. stutzeri Chol1 or its mutants as described previously (17). Toproduce Δ4-3-ketocholate (III), cell extracts of Chol11 Δhsh2 (�0.7 mg protein · ml�1 final concen-tration in the reaction mixture) were incubated with 2.5 mM 3-ketocholate (II) (Steraloids, RI, USA)in the presence of 0.25 mM PMS at 30°C for 5 h. Subsequently, the reaction mixture was incubatedat 99°C for 30 min to inactivate enzymes, was acidified to pH 3.5 with 25% HCl, and was extractedthree times with ethyl acetate. After drying over MgSO4, the solvent was evaporated under vacuumat 80°C and the extract was dried in a drying oven at 80°C for ca. 14 h. Finally, Δ4-3-ketocholate (III)was dissolved in 50 mM MOPS-buffer (pH 7.8) and stored at �20°C until use. To produce DOCDA,cell extracts of Novosphingobium sp. strain Chol11 (1 mg protein · ml�1 final concentration in reactionmixtures) were incubated with 2 mM cholate in the presence of 5 mM NAD� and 0.25 mM PMS overnight.The purification of DOCDA (XII) was achieved by following the same procedure described above forΔ4-3-ketocholate (III). To produce P4 (Δ1-DOCTRA), cell extracts of Novosphingobium sp. strain Chol11(1 mg protein · ml�1 final concentration in reaction mixtures) were incubated with 0.4 mM DOCDA in thepresence of 0.1 mM PMS and 2 mM NAD� overnight. After the organic extraction as described above,extracts were fractionated using a semipreparative HPLC as described below. After pooling the collectedfractions containing P4, purification was achieved by organic extraction as described above. Theconcentrations of purified metabolites were measured with a double-beam photometer (UV-2600;Shimadzu, Kyoto, Japan). For estimating the concentrations of purified Δ4-3-ketocholate (III) and Δ1,4-3-ketocholate (IV), a molar extinction coefficient of 14.7 cm�1 · mM�1 (�245 nm) was used (17). For DOCDA(XII) and P4, the molar extinction coefficients of 21.1 cm�1 · mM�1 (�290 nm) and 13.22 cm�1 · mM�1 (�300 nm),respectively, were used (39). The purity of metabolites was assessed by HPLC-MS analysis.

HPLC-MS analysis. All steroid compounds and culture supernatants were analyzed with an HPLC-MSsystem consisting of a Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific) with a UV-visible light diodearray detector and an ion trap mass spectrometer (Amazon speed; Bruker, Bremen, Germany) with anelectrospray ion source (ESI).

For analyzing bile salts and their degradation intermediates and for the fractionation of extractscontaining P4, a reversed-phase C18 column (150 mm by 3 mm, Eurosphere II, 100-5 C18; Knauer) at 25°Cwas used. Ammonium acetate buffer (10 mM, pH 6.7, eluent A) and acetonitrile (eluent B) were used aseluents with a flow rate of 0.3 ml · min�1. For the detection of cholate and its degradation intermediates,a gradient method was used starting with 10% eluent B for 2 min, increasing to 48% eluent B within 25min, and returning to 10% eluent B within 1 min, followed by an equilibration of 5 min at a flow rate of0.4 ml · min�1. For analyzing ATP and AMP in reaction mixtures, a polar reversed-phase C18 column (250mm by 4.6 mm by 4 �m; Phenomenex Synergi) was used. For the detection of ATP and AMP, a gradientmethod with a flow rate of 0.45 ml · min�1 was used starting with 0% eluent B for 5 min, increasing to70% eluent B within 10 min, keeping the concentration of eluent B at 70% for 5 min, and returning to10% eluent B for 1 min, followed by an equilibration of 6 min.

Ionization of samples was performed at alternating the ionization mode of ESI with the followingsettings: capillary voltage, 4,000 V; plate offset, 500 V; nebulizer pressure, 22.5 lb/in2; dry gas flow, 12liters · min�1; dry gas temperature, 200°C. MS was operated in ultrascan mode in a scan range of 50 to1,000 Da. For the evaluation of measurements, base peak chromatograms (BPC) or extracted ionchromatograms (EIC) with defined masses or mass ranges were used.

Bioinformatics analyses. Protein sequences were downloaded from the National Center for Bio-technology Information (NCBI) databases. Multiple sequence alignments were performed using theClustal Omega software (version 2.3). To identify potential steroid degradation proteins in strain Chol11,we used a set of 67 recently published hidden Markov models (HMMs) (https://github.com/MohnLab/mohn_lab_steroid_degradation_hmm_analysis_2015 [40]) representing 25 steroid degradation proteins.The proteome of strain Chol11 was searched using the program hmmsearch from the HMMER software(v3.1b1 [http://hmmer.org]) applying a maximum E value of 10�25 and a minimum coverage of 30%. TheR package genoPlotR (v 0.8.4 [41]) was used for analyzing the localization of predicted steroid degra-dation genes in the genome of strain Chol11.

To compare potential steroid degradation proteins in strain Chol11 with characterized and hypo-thetical steroid degradation proteins encoded in the steroid degradation gene clusters of P. stutzeri Chol1

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(42), C. testosteroni CNB-2 (43), and R. jostii RHA1 (44), we performed a reciprocal BLASTp analysis usingthe program BackBLAST (v1.0 [44]). BLASTp analysis was performed using a maximum E value of 10�30

and a minimum identity of 25%, leaving all other BLAST settings at the default.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01492-17.

SUPPLEMENTAL FILE 1, PDF file, 1.2 MB.

ACKNOWLEDGMENTSWe thank Rebekka Lülf and Karin Niermann for excellent experimental support.This work was funded by two grants of the Deutsche Forschungsgemeinschaft (DFG

projects PH71/3-2 and INST 211/646-1 FUGG) to B.P.

REFERENCES1. Hofmann AF, Hagey LR, Krasowski MD. 2010. Bile salts of vertebrates:

structural variation and possible evolutionary significance. J Lipid Res51:226 –246. https://doi.org/10.1194/jlr.R000042.

2. Vitek L, Haluzik M. 2016. The role of bile acids in metabolic regulation. JEndocrinol 228:R85–R96. https://doi.org/10.1530/JOE-15-0469.

3. Ridlon JM, Harris SC, Bhowmik S, Kang D-J, Hylemon PB. 2016. Conse-quences of bile salt biotransformations by intestinal bacteria. Gut Mi-crobes 7:22–39. https://doi.org/10.1080/19490976.2015.1127483.

4. Ridlon JM, Kang DJ, Hylemon PB. 2006. Bile salt biotransformations byhuman intestinal bacteria. J Lipid Res 47:241–259. https://doi.org/10.1194/jlr.R500013-JLR200.

5. Fine JM, Sorensen PW. 2005. Biologically relevant concentrations ofpetromyzonol sulfate, a component of the sea lamprey migratory pher-omone, measured in stream water. J Chem Ecol 31:2205–2210. https://doi.org/10.1007/s10886-005-6745-4.

6. Li K, Brant CO, Siefkes MJ, Kruckman HG, Li WM. 2013. Characterizationof a novel bile alcohol sulfate released by sexually mature male sealamprey (Petromyzon marinus). PLoS One 8:e68157. https://doi.org/10.1371/journal.pone.0068157.

7. Philipp B. 2011. Bacterial degradation of bile salts. Appl Microbiol Bio-technol 89:903–915. https://doi.org/10.1007/s00253-010-2998-0.

8. Holert J, Yücel O, Suvekbala V, Kulic Z, Möller H, Philipp B. 2014. Evidenceof distinct pathways for bacterial degradation of the steroid compoundcholate suggests the potential for metabolic interactions by interspeciescross-feeding. Environ Microbiol 16:1424 –1440. https://doi.org/10.1111/1462-2920.12407.

9. Holert J, Yücel O, Jagmann N, Prestel A, Möller HM, Philipp B. 2016.Identification of bypass reactions leading to the formation of one centralsteroid degradation intermediate in metabolism of different bile salts inPseudomonas sp. strain Chol1. Environ Microbiol 18:3373–3389. https://doi.org/10.1111/1462-2920.13192.

10. Horinouchi M, Hayashi T, Kudo T. 2012. Steroid degradation in Coma-monas testosteroni. J Steroid Biochem Mol Biol 129:4 –14. https://doi.org/10.1016/j.jsbmb.2010.10.008.

11. Mohn WW, Wilbrink MH, Casabon I, Stewart GR, Liu J, van der Geize R,Eltis LD. 2012. Gene cluster encoding cholate catabolism in Rhodococcusspp. J Bacteriol 194:6712– 6719. https://doi.org/10.1128/JB.01169-12.

12. Galán B, García-Fernández J, Felpeto-Santero C, Fernández-Cabezón L,García JL. 2017. Bacterial metabolism of steroids. In Rojo F (ed), Aerobicutilization of hydrocarbons, oils and lipids, handbook of hydrocarbon andlipid microbiology. Springer International Publishing, Cham, Switzerland.

13. Horinouchi M, Hayashi T, Koshino H, Malon M, Yamamoto T, Kudo T.2008. Identification of genes involved in inversion of stereochemistry ofa C-12 hydroxyl group in the catabolism of cholic acid by Comamonastestosteroni TA441. J Bacteriol 190:5545–5554. https://doi.org/10.1128/JB.01080-07.

14. Swain K, Casabon I, Eltis LD, Mohn WW. 2012. Two transporters essentialfor reassimilation of novel cholate metabolites by Rhodococcus jostiiRHA1. J Bacteriol 194:6720 – 6727. https://doi.org/10.1128/JB.01167-12.

15. Barrientos A, Merino E, Casabon I, Rodriguez J, Crowe AM, Holert J,Philipp B, Eltis LD, Olivera ER, Luengo JM. 2015. Functional analyses ofthree acyl-CoA synthetases involved in bile acid degradation in Pseu-domonas putida DOC21. Environ Microbiol 17:47– 63. https://doi.org/10.1111/1462-2920.12395.

16. Horinouchi M, Hayashi T, Koshino H, Malon M, Hirota H, Kudo T. 2014.Identification of 9alpha-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid and beta-oxidation products of the C-17 side chain incholic acid degradation by Comamonas testosteroni TA441. J SteroidBiochem Mol Biol 143:306 –322. https://doi.org/10.1016/j.jsbmb.2014.04.014.

17. Holert J, Kulic Z, Yücel O, Suvekbala V, Suter MJF, Möller HM, Philipp B. 2013.Degradation of the acyl side chain of the steroid compound cholate inPseudomonas sp strain Chol1 proceeds via an aldehyde intermediate. JBacteriol 195:585–595. https://doi.org/10.1128/JB.01961-12.

18. Casabon I, Swain K, Crowe AM, Eltis LD, Mohn WW. 2014. Actinobacterialacyl coenzyme A synthetases involved in steroid side-chain catabolism.J Bacteriol 196:579 –587. https://doi.org/10.1128/JB.01012-13.

19. Crowe AM, Casabon I, Brown KL, Liu J, Lian J, Rogalski JC, Hurst TE,Snieckus V, Foster LJ, Eltis LD, Rubin EJ. 2017. Catabolism of the last twosteroid rings in Mycobacterium tuberculosis and other bacteria. mBio8:e00321-17. https://doi.org/10.1128/mBio.00321-17.

20. Yücel O, Drees S, Jagmann N, Patschkowski T, Philipp B. 2016. Anunexplored pathway for degradation of cholate requires a 7alpha-hydroxysteroid dehydratase and contributes to a broad metabolic rep-ertoire for the utilization of bile salts in Novosphingobium sp. strainChol11. Environ Microbiol 18:5187–5203. https://doi.org/10.1111/1462-2920.13534.

21. Holert J, Jagmann N, Philipp B. 2013. The essential function of genes fora hydratase and an aldehyde dehydrogenase for growth of Pseudomo-nas sp. strain Chol1 with the steroid compound cholate indicates analdolytic reaction step for deacetylation of the side chain. J Bacteriol195:3371–3380. https://doi.org/10.1128/JB.00410-13.

22. Birkenmaier A, Holert J, Erdbrink H, Möller HM, Friemel A, Schoenen-berger R, Suter MJF, Klebensberger J, Philipp B. 2007. Biochemical andgenetic investigation of initial reactions in aerobic degradation of thebile acid cholate in Pseudomonas sp. strain Chol1. J Bacteriol 189:7165–7173. https://doi.org/10.1128/JB.00665-07.

23. Bhowmik S, Chiu HP, Jones DH, Chiu HJ, Miller MD, Xu Q, Farr CL, RidlonJM, Wells JE, Elsliger MA, Wilson IA, Hylemon PB, Lesley SA. 2016.Structure and functional characterization of a bile acid 7� dehydrataseBaiE in secondary bile acid synthesis. Proteins 84:316 –331. https://doi.org/10.1002/prot.24971.

24. Starai VJ, Celic I, Cole RN, Boeke JD, Escalante-Semerena JC. 2002.Sir2-dependent activation of acetyl-CoA synthetase by deacetylation ofactive lysine. Science 298:2390 –2392. https://doi.org/10.1126/science.1077650.

25. Casabon I, Crowe AM, Liu J, Eltis LD. 2013. FadD3 is an acyl-CoAsynthetase that initiates catabolism of cholesterol rings C and D inactinobacteria. Mol Microbiol 87:269 –283. https://doi.org/10.1111/mmi.12095.

26. Stolz A. 2009. Molecular characteristics of xenobiotic-degrading sphin-gomonads. Appl Microbiol Biotechnol 81:793– 811. https://doi.org/10.1007/s00253-008-1752-3.

27. Khara P, Roy M, Chakraborty J, Ghosal D, Dutta TK. 2014. Functionalcharacterization of diverse ring-hydroxylating oxygenases and inductionof complex aromatic catabolic gene clusters in Sphingobium sp. PNB.FEBS Open Bio 4:290 –300. https://doi.org/10.1016/j.fob.2014.03.001.

28. Wipperman MF, Yang M, Thomas ST, Sampson NS. 2013. Shrinking the

Steroid Degradation in Novosphingobium sp. Chol11 Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01492-17 aem.asm.org 15

on October 3, 2020 by guest

http://aem.asm

.org/D

ownloaded from

FadE proteome of Mycobacterium tuberculosis: insights into cholesterolmetabolism through identification of an �2�2 heterotetrameric acylcoenzyme A dehydrogenase family. J Bacteriol 195:4331– 4341. https://doi.org/10.1128/JB.00502-13.

29. Yang M, Lu R, Guja KE, Wipperman MF, St Clair JR, Bonds AC, Garcia-DiazM, Sampson NS. 2015. Unraveling cholesterol catabolism in Mycobacte-rium tuberculosis: ChsE4-ChsE5 �2�2 acyl-coA dehydrogenase initiates�-oxidation of 3-oxo-cholest-4-en-26-oyl CoA. ACS Infect Dis 1:110 –125.https://doi.org/10.1021/id500033m.

30. Hampl R, Kubatova J, Starka L. 2016. Steroids and endocrine disruptors–history,recent state of art and open questions. J Steroid Biochem Mol Biol 155:217–223.https://doi.org/10.1016/j.jsbmb.2014.04.013.

31. Jagmann N, Brachvogel HP, Philipp B. 2010. Parasitic growth of Pseu-domonas aeruginosa in co-culture with the chitinolytic bacterium Aero-monas hydrophila. Environ Microbiol 12:1787–1802. https://doi.org/10.1111/j.1462-2920.2010.02271.x.

32. Philipp B, Erdbrink H, Suter MJF, Schink B. 2006. Degradation of andsensitivity to cholate in Pseudomonas sp. strain Chol1. Arch Microbiol185:192–201. https://doi.org/10.1007/s00203-006-0085-9.

33. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM,Peterson KM. 1995. Four new derivatives of the broad-host-rangecloning vector PBBR1MCS, carrying different antibiotic-resistance cas-settes. Gene 166:175–176. https://doi.org/10.1016/0378-1119(95)00584-1.

34. Milton DL, OToole R, Horstedt P, WolfWatz H. 1996. Flagellin A isessential for the virulence of Vibrio anguillarum. J Bacteriol 178:1310 –1319. https://doi.org/10.1128/jb.178.5.1310-1319.1996.

35. de Lorenzo V, Herrero M, Jakubzik U, Timmis KN. 1990. Mini-Tn5transposon derivatives for insertion mutagenesis, promoter probing,and chromosomal insertion of cloned DNA in Gram-negative Eubac-teria. J Bacteriol 172:6568 – 6572. https://doi.org/10.1128/jb.172.11.6568-6572.1990.

36. Thoma S, Schobert M. 2009. An improved Escherichia coli donor strain for

diparental mating. FEMS Microbiol Lett 294:127–132. https://doi.org/10.1111/j.1574-6968.2009.01556.x.

37. Szvetnik A, Bihari Z, Szabo Z, Kelemen O, Kiss I. 2010. Genetic manipu-lation tools for Dietzia spp. J Appl Microbiol 109:1845–1852.

38. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. 1989. Site-directedmutagenesis by overlap extension using the polymerase chain-reaction.Gene 77:51–59. https://doi.org/10.1016/0378-1119(89)90358-2.

39. Mukherjee E, Banerjee S, Mahato SB. 1993. Transformation of cholic acidby Arthrobacter simplex. Steroids 58:484 – 490. https://doi.org/10.1016/0039-128X(93)90007-A.

40. Bergstrand LH, Cardenas E, Holert J, Van Hamme JD, Mohn WW. 2016.Delineation of steroid-degrading microorganisms through compara-tive genomic analysis. mBio 7:e00166-16. https://doi.org/10.1128/mBio.00166-16.

41. Guy L, Kultima JR, Andersson SGE. 2010. genoPlotR: comparative geneand genome visualization in R. Bioinformatics 26:2334 –2335. https://doi.org/10.1093/bioinformatics/btq413.

42. Holert J, Alam I, Larsen M, Antunes A, Bajic VB, Stingl U, Philipp B. 2013.Genome sequence of Pseudomonas sp. strain Chol1, a model organismfor the degradation of bile salts and other steroid compounds. GenomeAnnounc 1:e00014-12. https://doi.org/10.1128/genomeA.00014-12.

43. McLeod MP, Warren RL, Hsiao WWL, Araki N, Myhre M, Fernandes C,Miyazawa D, Wong W, Lillquist AL, Wang D, Dosanjh M, Hara H, Petrescu A,Morin RD, Yang G, Stott JM, Schein JE, Shin H, Smailus D, Siddiqui AS, MarraMA, Jones SJM, Holt R, Brinkman FSL, Miyauchi K, Fukuda M, Davies JE,Mohn WW, Eltis LD. 2006. The complete genome of Rhodococcus sp. RHA1provides insights into a catabolic powerhouse. Proc Natl Acad Sci U S A103:15582–15587. https://doi.org/10.1073/pnas.0607048103.

44. Ma YF, Zhang Y, Zhang JY, Chen DW, Zhu YQ, Zheng HJ, Wang SY, JiangCY, Zhao GP, Liu SJ. 2009. The complete genome of Comamonas testos-teroni reveals its genetic adaptations to changing environments. ApplEnviron Microbiol 75:6812– 6819. https://doi.org/10.1128/AEM.00933-09.

Yücel et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01492-17 aem.asm.org 16

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http://aem.asm

.org/D

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