3-mercaptopropionatedioxygenase,acysteine ... thioether 3,3-thiodipropionic acid (tdp)2 and its...

3-Mercaptopropionate Dioxygenase, a CysteineDioxygenase Homologue, Catalyzes the Initial Step of3-Mercaptopropionate Catabolism in the 3,3-ThiodipropionicAcid-degrading Bacterium Variovorax paradoxus*□S

Received for publication, September 2, 2008, and in revised form, November 3, 2008 Published, JBC Papers in Press, November 10, 2008, DOI 10.1074/jbc.M806762200

Nadine Bruland, Jan Hendrik Wubbeler, and Alexander Steinbuchel1

From the Institut fur Molekulare Mikrobiologie und Biotechnologie, Westfalische Wilhelms-Universitat Munster, Corrensstrasse 3,Munster D-48149, Germany

The thioether 3,3-thiodipropionic acid can be used as precur-sor substrate for biotechnological synthesis of 3-mercaptopro-pionic acid-containing polythioesters. Therefore, the hithertounknown catabolism of this compound was elucidated to engi-neer novel and improved polythioester biosynthesis pathways inthe future. Bacteria capable of using 3,3-thiodipropionic acid asthe sole source of carbon and energy for growth were enrichedfrom the environment. From eleven isolates, TBEA3, TBEA6,and SFWTweremorphologically and physiologically character-ized. Their 16 S rDNAs and other features affiliated these iso-lates to the �-subgroup of the proteobacteria. Tn5::mobmutagenesis of isolate Variovorax paradoxus TBEA6 yieldedten mutants fully or partially impaired in growth on 3,3-thio-dipropionic acid. Genotypic characterization of two 3,3-thio-dipropionic acid-negative mutants demonstrated the involve-ment of a bacterial cysteine dioxygenase (EC 1.13.11.22)homologue in the further catabolism of the 3,3-thiodipropionicacid cleavage product 3-mercaptopropionic acid. Detection of3-sulfinopropionate in the supernatant of one of these mutantsduring cultivation on 3,3-thiodipropionic acid as well as in vivoand in vitro enzyme assays using purified protein demonstratedoxygenation of 3-mercaptopropionic acid to 3-sulfinopropi-onate by this enzyme; cysteine and cysteamine were not used assubstrate. Beside cysteine dioxygenase and cysteamine dioxyge-nase, this 3-mercaptopropionic acid dioxygenase is the thirdexample for a thiol dioxygenase and the first report about themicrobial catabolism of 3-mercaptopropionic acid. Insertion ofTn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionateduring cultivation on 3,3-thiodipropionic acid, indicating thatthis compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA.

The thioether 3,3-thiodipropionic acid (TDP)2 and its esterare effective non-toxic antioxidants (1), and they are thereforewidely used as antioxidant and stabilizer in food, for food pack-aging, and for various technical applications. Experiment withrats showed thatTDPwas rapidly adsorbed after oral intake andexcreted in the urine (2). In technical applications esters of TDPare important stabilizers of polyolefins (1), and polymer-boundTDP is used to replacemethyl sulfide for the reductive quench-ing of ozonolysis reactions (3). Recently, the biotechnologicalproduction ofmedium- and long-chain dialkyl 3,3-thiodipropi-onate antioxidants by a lipase-catalyzed esterification of 3,3-thiodipropionic acid in the absence of solvents was reported. Incontrast to the chemical production of TDP ester, the biotech-nological process does not require any materials with deleteri-ous effects on health and environment (4). Another biotechno-logical process using TDP as primary product is the microbialproduction of polythioesters (PTEs) (5). In addition to 3-mer-captopropionic acid Ralstonia eutropha is able to use theorgano sulfur compounds TDP and 3,3-dithiodipropionic acid(DTDP) as precursor substrates for production of copolymersof 3-hydroxybutyrate and 3MP (6). In contrast to 3MP theapplication of TDP and DTDP has numerous advantages,because they have a lower toxicity and they are odorless, inex-pensive, and available on a large scale. Until today the use ofTDP and DTDP as precursor substrates are limited to R. eutro-pha, and biotechnological production of PTE using the recom-binant Escherichia coli strain JM109 pBPP1 (7) is only possiblewhen 3MP is added to the media. Because 3MP is incorporatedinto the polymer if TDP or DTDP is supplied as precursor sub-strate, it is assumed that these compounds are enzymaticallycleaved into 3MP and 3-hydroxypropionate, or two molecules3MP, respectively (6). The corresponding TDP- and/or DTDP-cleaving enzymes as well as the microbial catabolism of theintermediate 3MP are still unknown. The identification of suchenzymes could help to engineer the recombinant E. coli JM109

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) EF641108, EU825700,EU441166, EU441167, and EU449952.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2 and Tables S1–S3.

1 To whom correspondence should be addressed: Tel.: 49-251-833-9821; Fax:49-251-833-8388; E-mail: [email protected].

2 The abbreviations used are: TDP, thioether 3,3-thiodipropionic acid; PTE, poly-thioester; DTDP, 3,3-dithiodipropionic acid; Cdo, cysteine dioxygenase; MSM,mineral salts medium; IPTG, isopropyl 1-thio-�-D-galactopyranoside; HPLC,high-performance liquid chromatography; GC/MS, gas chromatography/mass spectrometry; RT, reverse transcription; MES, 4-morpholineethanesulfo-nic acid; ORF, open reading frame; Ssi, sulfate starvation-induced; Bis-Tris,2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; X-Gal,5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside; 3SP, 3-sulfinopropi-onic acid; 3MP, 3-mercaptopropionic acid; Rt, retention time.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 1, pp. 660 –672, January 2, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

660 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 1 • JANUARY 2, 2009

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pBPP1 toward TDP- andDTDP-based PTE production. There-fore, the corresponding genes could be used to improve thealready established BPEC pathway by heterologous expression(7). For 3MP it is known that it occurs naturally as an interme-diate during microbial degradation of the osmoprotectant di-methylsulfoniopropionate and during the biotransformation ofthe sulfur-containing amino acids methionine and homocys-teine in anoxic coastal sediments (8–11). However, to ourknowledge no reports on the pathways or enzymes for furthermetabolism of 3MP in bacteria have been published. In con-trast, the catabolism of cysteine, the structural analogue of3MP, is well known in bacteria (12, 13). In addition to the wellinvestigated cysteine degradation pathways, a novel pathwaywas recently reported in eubacteria byDominy et al. (14), whichinvolvesacysteinedioxygenase (Cdo,EC1.13.11.20).ThisFe2�-dependent enzyme catalyzes the irreversible oxidation of thesulfhydryl group of cysteine to cysteine sulfinic acid. Cdos arewell known in eukaryotes and play an important role by reduc-ing the cysteine pool and increasing the levels of importantmetabolites such as taurine and sulfates (15). The physiologicalfunction of this enzyme is not completely understood in bacte-ria. In addition to the important role in regulating the steady-state cysteine levels, also an important role of this enzyme dur-ing changes of bacterial life cycles was suggested (14), becausemany of the bacteria possessing a Cdo undergo a complex lifecycle involving morphological changes. For example, in cells ofBacillus subtilis expression of the cdo gene is up-regulated dur-ing transition from the vegetative state to sporulation (14).To our knowledge there are so far also no reports about bac-

teria that utilize the organic thioether TDP as sole source ofcarbon and energy. Only its use as a sulfur source was describedfor Mycobacterium goodii X7B (16). However, this bacteriumcould not growwithTDP in the absence of an additional carbonsource, and degradation products of TDP were also notreported. To engineer the PTE biosynthesis in the futuretoward to use TDP as precursor substrates, we investigated thecatabolism of TDP. The isolation and characterization of bac-teria capable of using TDP as the sole source of carbon andenergy are described in this study. In addition, Tn5::mobmutagenesis was carried out with one of these new isolates,Variovorax paradoxus strain TBEA6, to identify genes possiblyinvolved in TDP catabolism.

EXPERIMENTAL PROCEDURES

Isolation of Strains Capable of Using TDP as Sole Source ofCarbon and Energy—Samples from different soils, activatedsludge, and freshwater tank sediment were incubated in min-eral salts medium (MSM) containing 3 g/liter TDP as the solesource of carbon and energy at 30 °C for 3–5 days (17). Aliquotsof these cultures were then plated on the same medium solidi-fied with 1.5% (w/v) agar, single colonies were isolated, and thebest growing colonies were chosen for further studies.Bacterial Strains and Cultivation Conditions—All bacterial

strains used in this study are listed in supplemental Table S1. R.eutropha H16, strains of Variovorax paradoxus and isolateTBEA3 were cultivated at 30 °C in nutrient broth orMSM sup-plemented with 0.1 g/liter yeast extract under aerobic condi-tions on a rotary shaker at an agitation of 130 rpm. Strains of

E. coli were cultivated in Luria-Bertani (LB) medium or M9medium supplemented with yeast extract (0.1 g/liter) at 37 °Cunder the same conditions (18). Carbon sources were suppliedas filter-sterilized stock solutions as indicated in the text. Formaintenance of plasmids, antibiotics were prepared accordingto Sambrook et al. (18) and added to the media at the followingconcentrations (�g/ml): ampicillin (75), kanamycin (50), chlor-amphenicol (34), and tetracycline (12.5). In E. coli heterologousexpression of genes under the control of a lac-promotor wasinduced by addition of 1 mM IPTG to LB medium.Chemicals—Bulk TDP was provided by Bruno Bock Che-

mische Fabrik GmbH&Co. KG. Organic thiochemicals of highpurity grade were purchased from Acros Organics (Geel, Bel-gium) or Sigma-Aldrich (Steinheim, Germany) (Fig. 1).3-Sulfinopropionate was synthesized according to Jolles-Bergeret (19); the procedure was modified by one repetitionof the alkaline cleavage of the intermediate bis-(2-carboxyeth-yl)sulfone. Starting from 111 g of sodium formaldehyde sul-foxylate (purity,�98%) plus 108ml of acrylic acid (99.5%), 119 gof the intermediate bis-(2-carboxyethyl)sulfone were chemi-cally synthesized. After alkaline scission, precipitation, andwashing procedures, 99 g of the disodium salt of 3SP, with apurity of �90%, were finally obtained. Synthesis and purity ofthe substance was confirmed by HPLC and GC/MS.

FIGURE 1. Structural formula of organic thiochemicals used in thisstudy.

3-Mercaptopropionate Dioxygenase in V. paradoxus

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Strain Identification—API 20NE identification test (bio-Merieux, Marcy-l‘Etoile, France) and Bactident oxidase teststripes (Merck KgA, Darmstadt, Germany) were used accord-ing to themanufacturer’s instructions. Presence of catalase wastested using 3% (v/v) H2O2.The 16 S rRNA gene was amplified from total genomic DNA

by PCR using Primers 27f and 1525r (20). The PCR product waspurified using the NucleoTrap kit (Machery and Nagel, Duren,Germany) and applied as template for sequencing in which thefollowing primers were utilized: 27f, 357f, 803f, 907r, 1114f,1385r, and 1525r (20). The assembled sequence was comparedwith the GenBankTM data base and the Ribosomal DatabaseProject using the Blast and SIMILARITY-RANK (RibosomalDatabase Project) algorithms (21, 22). A phylogenetic tree wasconstructed using ClustalX (23).Transposon Mutagenesis and Characterization of Tn5::mob-

induced Mutants—Insertional mutagenesis of V. paradoxusstrain TBEA6 with transposon Tn5::mob was performed asdescribed previously by using the suicide plasmid pSUP5011,which was delivered from E. coli S17-1 to the recipient by con-jugation during spot agar mating (24, 25). Transconjugantswere selected on MSM plates containing 0.5% (w/v) gluconateplus kanamycin. Transconjugants impaired in growth on TDPwere then identified by plating on MSM agar plates containing0.3% (w/v) TDP plus kanamycin or 0.5% (w/v) gluconateplus kanamycin. For genotypic characterization of theTn5::mob-induced mutants, genomic DNA was isolated (26)and digested with SalI or BamHI. The genomic DNA fragmentswere then ligated to pBluescriptSK� DNA, which was linear-ized with the same restriction endonuclease; the ligation prod-ucts were then transformed into CaCl2-competent E. coliTop10 cells. Transformants were selected on LB medium con-taining ampicillin plus kanamycin, and hybrid plasmids weresubsequently isolated and sequenced using the primers M13forward, M13 reverse, and IS50L (supplemental Table S3).Analysis of Cell-free Supernatants and Sulfur Organic Com-

pounds by HPLC—Concentrations of TDP, 3MP, cysteinesulfinic acid, cysteamine, hypotaurine, and 3SP were analyzedby HPLC.HPLC analysis of TDP, 3MP, cysteamine, and 3SP was car-

ried out in a LaChrom Elite� HPLC apparatus (VWR-HitachiInternational GmbH, Darmstadt, Germany) consisting of aMetacarb 67H advanced C column (Varian, Palo Alto, CA, Bio-Rad Aminex equivalent) and a 22350 VWR-Hitachi columnoven. The primary separation mechanism includes ligandexchange, ion exclusion, and adsorption. A VWR-Hitachirefractive index detector (Type 2490) with an active flow celltemperature control and automated reference flushing elimi-nating temperature effects on the refractive index baseline wasused for detection. Aliquots of 20 �l of cell-free supernatants,solutions of organic sulfur compounds or enzyme assay wereinjected and elutedwith 0.005 N sulfuric acid (H2SO4) in doubledistilled water at a flow rate of 0.8 ml/min. Online integrationand analysis was done with EZ Chrome Elite Software (VWRInternational GmbH, Darmstadt, Germany). Cysteamine wasdetected under the same conditions using double distilledwater as mobile phase. Detection of hypotaurine and cysteinesulfinic acid was carried out in a Kontron Instrument (Neu-

fahrn, Germany). After derivatization with OPA reagent (27)using a Smartline Autosampler 3900 (Knauer Advanced Scien-tific Instruments, Berlin, Germany), 20 �l of the reaction wasinjected onto aNovapackC18 reversed-phase column (Knauer)and monitored fluorometrically at 330/450 nm (excitation/emission) by using a model 1046A fluorescence detector(Hewlett Packard). Substances were identified by comparisonof their retention times to those of standard organic acids. Thedetection limit for hypotaurine is �20 �M and 10 �M for cys-teine sulfinic acid.QuantitativeAnalysis of PolyhydroxyalkanoicAcid andTheir

Compositions by GC—Lyophilized cell material was subjectedto methanolysis in the presence of methanol and sulfuric acid(MeOH: 85%, v/v; H2SO4: 15%, v/v) for 4 h at 100 °C, and theresultingmethylesters of the polyhydroxyalkanoic acid constit-uents were characterized by gas chromatography using an Agi-lent 6850 GC (Agilent Technologies, Waldbronn, Germany)equipped with a BP21 capillary column (50 m by 0.22 mm; filmthickness, 250 nm; SGE, Darmstadt, Germany) and a flame ion-ization detector (Agilent Technologies). A 2-�l portion of theorganic phase was analyzed after split injection (split ratio, 1:5);a constant hydrogen flow of 0.6 ml/min was used as carrier gas.The temperatures of the injector and detector were 250 °C and220 °C, respectively. The following temperature program wasapplied: 120 °C for 5 min, increase of 3 °C/min to 180 °C, andincrease of 10 °C/min to 220 °C and 220 °C for 31 min. Sub-stances were identified by comparison of their retention timesto those of standard fatty acid methyl ester.Analysis of 3SP Production by GC/MS—Lyophilized cells,

cell-free supernatants, or aliquots of synthesized 3SP were sub-jected to methanolysis as described above, and the resultingmethylesters of the organic acidswere characterized by coupledGC/MS using an HP6890 gas chromatograph equipped with amodel 5973 EIMSDmass-selective detector (Hewlett Packard).A 2-�l portion of the organic phase was analyzed after splitlessinjection employing a BP21 capillary column (50mby 0.22mm;film thickness, 250 nm; SGE). Helium (0.6 ml/min) was used ascarrier gas. The temperatures of the injector and detector were250 °C and 240 °C, respectively. The same temperature pro-gram as described for GC analysis was applied. Data were eval-uated using the NIST Mass Spectral Search program.3Isolation of RNA and RT-PCR—Total RNAwas isolated from

V. paradoxus strain TBEA6 by using the Qiagen RNeasy-Kitaccording to themanufacturer’s instructions. RT-PCRwas per-formed using the Qiagen “One step RT-PCR” Kit according tothe manufacturer’s instructions. To recognize PCR productsbased on DNA contaminations in isolated RNA, a control withaddition of RNA after the reverse transcription step was done.DNA Isolation and Manipulation—Chromosomal DNA of

strains of V. paradoxus and R. eutropha H16 was isolatedaccording to Marmur (26). Plasmid DNA was isolated fromE. coli and V. paradoxus strains using the GeneJETTM plasmidminiprep kit from Fermentas (St. Leon-Rot, Germany) accord-ing to the manufacturer’s manual. DNA was digested withrestriction endonucleases under conditions described by the

3 S. Stein, A. Levitsky, O. Fateev, and G. Mallard (1998). The NIST Mass SpectralSearch Program, Windows-Software Version 1.6d.




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manufacturer or according to Sambrook et al. (18). PCR werecarried out in an Omnigene HBTR3CM DNA thermal cycler(Hybaid, Heidelberg, Germany) using Platinum� Taq DNAPolymerase (Invitrogen). PCR products were isolated from anagarose gel and purified using the NucleoTrap kit (Macheryand Nagel, Duren, Germany) according to the manufacturer’sinstructions. T4-DNA-Ligase was purchased from Invitrogen.Primers were synthesized by MWG-Biotech AG (Ebersberg,Germany).Transfer ofDNA—Competent cells ofE. coli strainswere pre-

pared and transformed by the CaCl2 procedure (18).DNA Sequencing and Sequence Data Analysis—DNA

sequencing was done in a Li-Cor 4000L automatic sequencingapparatus (Li-COR Inv., Biotechnology Division, NE, USA)using the Thermo long read cycle sequencing Kit (EpicenterTechnologies, WI, USA) and IRD 800-labeled oligonucleotides(MWG-Biotech, Ebersberg, Germany). BlastX was used fordetermination of nucleotide identity (21).DNA-DNA Hybridization—Southern hybridization was car-

ried out by the method described by Oelmuller et al. (29) at atemperature of 68 °C.Genome Walking—For sequencing of flanking genomic

regions of known sequences a PCR-based directional genomewalking method (30) was performed.Cloning of cdoVp, cdoARe, and cdoBRe—The cdoVp, cdoARe,

and cdoBRe genes were amplified from total genomicDNAofV.paradoxus strain TBEA6 or R. eutropha strain H16 by PCRusing Taq DNA polymerase (Invitrogen) and the following oli-gonucleotides: cdo(NdeI), cdo(XhoI), cdoA(XbaI), cdoA(XhoI),cdoB(ApaI), and cdoB(HindIII) (supplemental Table S3). PCRproducts were isolated from agarose gels using the NucleoTrapkit (Machery and Nagel) and ligated with pGEMTeasy� DNA(Promega, Madison, WI). Ligation products were transformedinto CaCl2-competent cells, and transformants were selectedon LB agar plates containing IPTG, X-Gal, plus ampicillin. Forheterologous expression in theT7 promoter/polymerase-basedexpression vector pET23a (Novagen, Madison, WI), cdoVp wasobtained by digestion of hybrid plasmid pGEMTeasy�::cdoVpwith restriction endonucleases NdeI and XhoI and purifiedfrom an agarose gel using the NucleoTrap kit (Machery andNagel). After ligation into expression vector pET23a,whichwaslinearized with the same restriction endonucleases, the ligationproduct was used for transformation of CaCl2-competent cellsof E. coli Top10. After selection of transformants using LBmedia containing ampicillin, the hybrid plasmids were isolated,analyzed by sequencing, and transformed to CaCl2-competentcells of E. coli (DE3) strains BL21 pLysS and Rosetta pLysS(Novagen, Madison, WI).For complementation studies and heterologous expression

in the broad host vector pBBR1MCS-3 (31), cdoVp, cdoARe, andcdoBRe were obtained from pGEMTeasy� vector, which weredigested with the respective restriction endonuclease andpurified from an agarose gel using the NucleoTrap kit(Machery and Nagel). The purified genes were subsequentlyligated into pBBR1MCS-3, which was linearized with thesame restriction endonucleases, and the ligation productswere transformed to CaCl2-competent cells of E. coli S17-1and E. coli Top10. Transformants were selected on LB

medium containing tetracycline, IPTG, plus X-Gal. Thehybrid plasmids pBBR1MCS-3::cdoVp, pBBR1MCS-3::cdoARe, and pBBR1MCS-3::cdoBRe were then conjugatedinto the transposon-induced mutants 2/5 and 13/33 fromE. coli S17-1.Preparation of Crude Extracts—Cells from 50- to 500-ml cul-

tures were harvested by centrifugation (20 min, 4 °C, and2,800 � g), washed twice, and resuspended in 50 mM NaPO4buffer (pH 7.6). Cells were disrupted by sonification in a Sono-puls GM200 apparatus (Bandelin, Berlin, Germany) with anamplitude of 16 �m (1 min/ml) while cooling in an NaCl/icebath. Soluble protein fractions of crude extracts were obtainedin the supernatants after 1-h centrifugation at 100,000 � g and4 °C and were used for enzyme purifications.Immobilized Metal Chelate Affinity Chromatography—To

obtain purified hexahistidine-tagged fusion CdoVp, His SpinTrap affinity columns (GE Healthcare, Uppsala, Sweden) wereused according to the instructions of the manufacturer withminor modifications. Tris-HCl (0.1 M, pH 7.6) was used asbuffer component instead of sodium phosphate, and for thewashing step a buffer containing 40 mM imidazole was applied.Thewashing stepwas repeated three times, and the elution stepwas repeated two times.Enzyme Assay—Standard in vitro activity of cysteine dioxy-

genase was assayed by incubating 3 �g of purified CdoVp for 30min at 30 °C in the presence of the following components: 10mM cysteine, 10 mM cysteamine, or 5 mM 3MP, 400 �M(NH4)2Fe(SO4)2 � 6H2O, 12.5 �M bathocuproine disulfonateand MES buffer (62 mM, pH 6.3). The reaction was stopped by10-min incubation at 95 °C. Negative controls were done withdenatured protein. The reaction products 3SP, hypotaurine,and cysteine sulfinic acid were analyzed by HPLC.For in vivo testing of recombinant cysteine dioxygenase

activity in recombinant E. coli strains, cells were cultivated inM9mediumcontaining 1% (v/v) glycerol at 30 °C. Expression ofthe recombinant proteinwas induced by addition of 1mM IPTGafter 6 h of cultivation. Subsequently, the substrate 3MP wasadded to a final concentration of 0.1% (v/v), and the cells weregrown for additional 24 h before they were harvested andwashed twicewith 0.9% (w/v)NaCl. Finally, the cell pellets werelyophilized. Analysis of the reaction product 3SP was done byGC/MS.Data Deposition—Nucleotide sequences have been depos-

ited in the GenBankTM data base under the following Gen-BankTM accession numbers: EF641108, 16 S rDNA gene of iso-late TBEA6; EU825700, 16 S rDNA gene of isolate TBEA3;EU441166, 16 S rDNA gene of isolate SFWT; EU441167, con-tiguous sequence comprising bugC, fox, bugA, cdo, and ahpD;and EU449952, contiguous sequence comprising act, acd, andpartial sequence of bugB.

RESULTS

Isolation of TDP-utilizing Bacteria and Taxonomic Affilia-tion of Isolate TBEA6—From different samples taken from soil,compost, sewage sludge, or the sediment of a fresh water tank,bacterial strains capable of using TDP as sole source of carbonand energy for growth were enriched and isolated (supplemen-tal Table S1). The best growing isolates were designated as




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TBEA6, TBEA3, and SFWT; they were further characterized bymethods of polyphasic taxonomy and by analysis of the 16 SrDNA sequences to unravel their phylogenetic position. Allthree isolates were Gram-negative, oxidase, and catalase-posi-tive, and best growth was observed at 30 °C, although isolatesTBEA6 and SFWT grew also very slowly at 4 °C. Coloniesof TBEA6 and SFWT were yellow-pigmented, and the motile

cells are short rods with a cell lengthof 1.5 �m. Using the API 20NEidentification test system, reductionof nitrate to nitrite and positiveassimilation of L-arabinose, D-man-nitol, gluconate, and malate werenoticed for these two strains. Posi-tive urease reaction was only identi-fied for isolate SFWT. The API20NE test system identified isolatesTBEA6 and SWFT as strains of Ral-stonia pickettii and TBEA3 as astrain of Comamonas acidovorans.The 16 S rRNA sequences of strainsTBEA6 and SFWT showed high lev-els of sequence similarities (99%) tovarious V. paradoxus strains. V.paradoxus is not yet contained inthe available API 20NE test system.The 16 S rDNA nucleotide se-quence of strain TBEA3 exhibitedthe highest sequence similarity(98.9%) with those of an unculturedbacterium. Fig. 2 shows a phyloge-netic tree providing an idea on thephylogenetic classifications of thethree isolates.Physiological Characterization of

Tn5::mob-induced Mutants of V.paradoxus Strain TBEA6 ShowingImpaired Growth on TDP—Al-though all three isolates were able touse TDP as sole source of carbonand energy for growth, no growthwas observed with 3MP, or DTDP,or the putative cleaving product3-hydroxypropionate. All three iso-lates utilized also the organic sulfurcompounds taurine and 3SP,whereas homocysteine was used forgrowth only by isolate TBEA6. Cys-teamine (0.1%; w/v) was also tested,but none of the investigated strainsused this compound as carbon andenergy source. Good growth wasobserved for strain SFWT when 0.1(w/v) cysteamine was added toMSM agar plates containing gluco-nate (1%; w/v), whereas strainTBEA6 showed weak growth. Allstrains accumulated poly(3-hy-

droxybutyrate), but no accumulation of PTE such as poly(3MP)or poly(3MP-co-3-hydroxybutyrate) occurred as revealed byGC analysis of cells cultivated under conditions permissive forsynthesis and accumulation of PTEs.Both V. paradoxus strains exhibited good access to standard

methods of molecular biology. They could for example betransformed by the broad host vector pBBR1MCS-3 (31).

FIGURE 2. Phylogenetic tree based on 16 S rDNA sequences, showing the positions of the TDP utilizingisolates TBEA6, SFWT, and TBEA3 within the �-proteobacteria. T. mimigardefordensis was used as an out-group. Bootstrap values are shown at the branch points. Accession numbers are given in parentheses. Bar, 0.01substitutions per nucleotide positions.




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Because isolate TBEA6 exhibited in contrast to isolate SFWTsusceptibility to the antibiotics kanamycin and tetracycline,isolate TBEA6 was chosen for further investigations and sub-jected to transposon mutagenesis employing plasmidpSUP5011 harboring Tn5::mob to identify genes coding forenzymes essential for catabolism of TDP (24). Ten transposon-induced mutants showing the phenotypes TDP-negative (1/8,1/20, 2/5, 4/5, 1/31, 2/34, and 13/33) or TDP-leaky (1/1, 1/9,and 2/41) were isolated. Growth of thesemutants onMSMagarplates containing 0.2% (w/v) gluconate, acetate, propionate,taurine, or succinate as sole source of carbon and energy wasnot affected, thus indicating that in these mutants specificallygenes involved in the catabolism of TDP were most probablyinactivated by insertion of Tn5::mob.To analyze if thesemutants are still able to degradeTDP, or if

they are impaired in the utilization of intermediates of TDPcatabolism and to identify putative intermediates, mutants andwild type were grown in Erlenmeyer flask without baffles con-taining 1% (w/v) TDP plus 0.2% (w/v) sodium succinate as car-bon sources in 300 ml of MSM. Samples were taken every 48 h,and aliquots of cell-free supernatants were analyzed by HPLC(Fig. 3). The initial concentration of TDP was decreasing in allcultures except in those of mutants 1/8 and 1/20. In the super-natant of mutant 1/1 accumulation of 3SP was observed byHPLC and GC/MS analysis, and the conversion of TDP to3-sulfinopropionate was analyzed quantitatively in an addi-tional experiment. Therefore, the wild type and this mutantwere cultivated in an Erlenmeyer flask with baffles containing0.2% (w/v) sodium succinate plus 1% (w/v) TDP in 50 ml ofMSM, and the cell-free supernatants were analyzed by HPLCfor contents of TDP and 3SP every 24 h (Fig. 4). The results ofthis experiment (Fig. 4A) clearly demonstrated that the increas-ing concentrations of 3SP during the time course of the exper-iment with the mutant correlated with decreasing concentra-tions of TDP.Whereas thewild type utilizedTDP at a rate of 1.2mM/h anddid not form3SP,mutant 1/1 utilizedTDPat a rate of0.32mM/h and formed 3SP at a rate of 0.33mM/h. For detection

of other putative intermediates GC/MS analyses of the culturewere performed. For this, 1 ml of the culture was taken every24 h, and the cell-containingmaterial was analyzed uponmeth-anolysis. Beside 3SP, small amounts of 3-hydroxypropionateand 3MP could be detected in the culture of mutant 1/1 (Fig.5A). Whereas 3MP and DTDP could also be detected in cul-tures of the wild type, identification of 3-hydroxypropionateand 3SP failed (Fig. 5B).In contrast to the wild type and to all other mutants, mutant

1/1 was unable to use 3SP as sole source of carbon and energyfor growth when provided inMSM. Themutant was grown in abaffled Erlenmeyer flask containing 0.5% (w/v) 3-sulfinopropi-onate, 0.5 (w/v) TDP, or 0.5% (w/v) sodium succinate as solesource of carbon and energy in 50 ml of MSM. Growth wasquantified by measuring the optical density at 600 nm, and theconcentrations of sodium succinate, TDP, and 3SP were deter-mined by HPLC. The results are displayed in Fig. 4B andrevealed that growth on 3SP andTDP is strongly affected in this

FIGURE 3. Utilization of TDP by the wild-type and Tn5::mob-inducedmutants affected in growth on TDP of V. paradoxus TBEA6. Cells wereincubated in MSM containing 1% (w/v) TDP plus 0.2% (w/v) sodium succinate.At the indicated time samples were withdrawn and centrifuged, and thesupernatants were analyzed by HPLC for TDP contents. Œ, mutant 1/8; �,mutant 1/20; *, mutant 2/34; �, mutant 4/5; F, mutant 2/5; ‚, mutant 2/41; f,mutant 1/1; E, mutant 1/9; �, mutant 1/31; �, wild type.

FIGURE 4. A, utilization of TDP and formation of 3SP by the Tn5::mob-inducedmutant 1/1 and the wild type. Cells were incubated in MSM containing 1%(w/v) TDP plus 0.2% (w/v) sodium succinate. At the indicated time sampleswere withdrawn and centrifuged, and the supernatants were analyzed byHPLC for TDP and 3SP contents. �, TDP utilization by mutant 1/1; f, 3SPformation by mutant 1/1; Œ, TDP utilization by the wild type. B, utilization ofsodium succinate, TDP, and 3SP as the sole source of carbon and energy bymutant 1/1. Cells were incubated in MSM containing 1% (w/v) sodium succi-nate, TDP, or 3SP. At the indicated time samples were withdrawn, and growthwas determined by measuring the optical density at 600 nm. In addition, thesamples were centrifuged, and the supernatants were analyzed by HPLC forTDP and 3SP contents. �, growth of mutant 1/1 on sodium succinate; Œ,growth of mutant 1/1 on TDP; f, growth of mutant 1/1 on 3SP; �, content of3SP; �, content of TDP; F, formation of 3SP in supernatant of cells growingwith TDP as carbon source.




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mutant. Whereas sodium succinate was completely utilizedduring the first 24 h of cultivation (data not shown), the con-centration of 3SP was not decreasing during the time course ofthe experiment, and the optical density remained at 0.3. TheTDP concentration was decreasing very weakly, and in thesame supernatant a weak accumulation of 3SP was detected.The optical density of this culture remained constant at �0.3.Molecular Characterization of Tn5::mob-induced Mutants

and Identification of Two Chromosomal DNA Regions Harbor-ing Genes Required for TDP Catabolism—The insertions ofTn5::mob into the genomes of these mutants were confirmed

by Southern hybridization using ApaI-digested DNA isolatedfrom themutants and a digoxygenin-labeledKm resistance cas-sette derived from Tn5::mob DNA as probe. To map the inser-tions of Tn5::mob in these mutants, SalI- or BamHI-restrictedgenomic DNA fragments conferring kanamycin resistancewere cloned in E. coli strain Top10. Sequencing of these DNAfragments using oligonucleotides hybridizing to the terminalregion of IS50L and themultiple cloning site of the used cloningvector pBluescriptSK� revealed only four different open read-ing frames (ORFs) in which Tn5::mob had been inserted (Table1). In mutants 1/8 and 2/34 Tn5::mob was mapped in a geneputatively coding for an FAD-linked oxidoreductase exhibiting55% identical amino acids to FAD-linked oxidase domain pro-tein of Acidovorax avenae subsp. citrulli AAC00–1 (fox). Inmutants 1/20 and 4/5 the transposon was mapped in an ORFshowing high homologies (46% identical amino acids) to a puta-tive exported protein of Burkholderia xenovorans LB400. Dueto the sequence similarities to representatives of the gene familyofBordetella uptake genes (bug), coding for extracytoplasmaticsolute receptor proteins, this gene (bugA) is also a putativemember of this gene family, which is strongly overrepresentedin several �-proteobacteria (32). In mutants 13/33 and 2/5 thetransposonwasmapped in a putative gene coding for a cysteinedioxygenase (cdo). The respective sequence exhibited 68%identical amino acids to a type I Cdo of Verminephrobactereiseniae EF01–2. In mutant 1/1, the transposon insertion waslocalized in an ORF showing high sequence homology (56%identical amino acids) to a predicted family III acyl-CoA-trans-ferase ofMagnetospirillium gryphiswaldenseMSR-1.In addition to the genes in which Tn5::mob was mapped,

several putative ORFs were detected when the regionsupstream and downstream of these insertions were sequencedby the PCR-based directional genomewalkingmethod (30) andanalyzed. Detailed analysis of these sequences revealed that thefoxVp, bugAVp, and cdoVp genes are contiguous, whereas therewere no hints that the gene coding for the putative acyl-CoA-transferase is also adjacent to the other gene cluster. In total, thesequences of a 5.1-kbp and of a 3.2-kbp region were unraveled,respectively. Downstream of the cdoVp gene in the first genecluster an ORF showing 63% identical amino acids to the alky-lhydroperoxidase core (ahpD) of Burkholderia xenovoransLB400 was detected. Downstream of actVp a gene putativelycoding for an acyl-CoA-dehydrogenase (acd) was identified.Furthermore, two other homologous genes of the Bordetellauptake gene family were identified: one (bugC) was identifiedupstream of the putative oxidoreductase gene in the first clus-ter; the other was detected downstream of the putative acylCoA-dehydrogenase (bugB). The gene organization is summa-rized in Fig. 6.The amino acid sequences of the putative Bug homologues

were analyzed using the TatP, SignalIP, and the TargetIP algo-rithm (www.cbs.dtu.dk/services) (33, 34) and revealed a highprobability for signal peptides. Whereas BugB and BugC pro-teins are probably secreted by the twin arginine translocationpathway (TAT), BugA shows no TATmotif or TAT signal pep-tide. Upstream of cdoVp a putative promoter sequence wasidentified employing the 1999 Neural Network Promotor Pre-

FIGURE 5. GC/MS analyses of samples withdrawn from the growth exper-iment of V. paradoxus TBEA6 wild-type and mutant 1/1 on MSM contain-ing sodium succinate (0.2%, w/v) and TDP (1%, w/v). For detection of puta-tive intermediates, 1 ml of the cell culture was lyophilized and used for GC/MSanalysis upon methanolysis. A, sample of mutant 1/1 after 72 h of incubation.The following compounds could be identified by mass spectra analysis: 1,propanoic acid 3-methoxymethylester (Rt, 6.7 min); 2, sulfuric acid (Rt, 7.5min); 3, malonic acid (Rt, 8.5 min); 4, methyl 3,3-dimethoxypropionate (Rt, 8.6min); 5, 3-mercaptopropionic acid dimethylester (Rt, 10.6 min); 6, 3-sulfin-opropionic acid methylester (Rt, 18.7 min); and 7, thiodipropionic acid di-methylester (Rt, 24.7 min). B, sample of TBEA6 wild type after 48 h of incuba-tion, the following compounds could be identified by mass spectra analysis: 1,3-hydroxybutyric acid methylester (Rt, 7.0 min); 2, sulfuric acid (Rt, 7.5 min); 3,trimethoxybutane (Rt, 8.0 min); 4, methyl 3,3-dimethoxypropionate (Rt, 8.6min); 5, pentanoic acid (Rt, 9.7 min); 6, 3-mercaptopropionic acid dimeth-ylester (Rt, 10.6 min); 7, thiodipropionic acid dimethylester (Rt, 24.9 min); 8,dithiodipropionic acid dimethylester (Rt, 32.33 min); and 9, hexadecanoicacid (Rt, 33.7).




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diction version 2.2 of the promoter prediction (www.fruitfly.org/seq_tools/promotor.html) software (35).Transcriptional Analysis of Genes Putatively Involved in TDP

Catabolism—Transcriptional analysis of genes identified bytransposon mutagenesis and putatively involved in TDPmetabolism were performed by RT-PCR to determine if cdoVpand actVp are constitutively transcribed, or if the expression isinduced in the presence of TDP in the wild type. Furthermore,RT-PCR was carried out with RNA isolated from the mutants4/5 and 2/34 to analyze whether the phenotype of thesemutants is caused by transposon insertion in the respectivegene, or if a polar effect caused by insertion of Tn5::mob in thegenes adjacent to cdoVp is responsible for the observed pheno-type. Because the strainswere grown inMSMmedia containing

10 mM MgSO4, the results of theRT-PCR can also be used to deter-mine if the expression of the genes isindependent of limited concentra-tions of sulfate, as previouslydescribed for sulfate starvation-in-duced (Ssi) proteins (36). Using thesame RNA, bugAVp was amplifiedfrom RNA of mutant 2/34 and foxVpwas amplified from RNA of mutant4/5. Successful amplification ofboth genes indicated independenttranscription of these genes andconfirms the genotype. For the samereason RT-PCR analysis of the actVpand acdVp genes was performedusing RNA isolated from mutant1/1. The results revealed that cdoVpand actVp are transcribed in the wildtype irrespectively of having usedsuccinate or TDP as the sole sourceof carbon and energy. This indi-cated that transcription of bothgenes is constitutive and is notinduced in the presence of TDP.Transcription of cdoVp was alsodetected in RNA isolated frommutants 4/5 or 2/34. In contrast, noRT-PCR product of acdVp wasdetected under all used conditions.To exclude non-efficient RT-PCR,

cdoVp was analyzed as a positive control in the same reactionmixtures and could be amplified in all cases. Results are sum-marized in Fig. 6.Analysis of Primary Structure of the Cdo Translational

Product—Multiple sequence alignments of the putative V.paradoxus Cdo and of Cdo homologues of different species,showing high sequence similarities, revealed several highly con-served residues already described for eukaryotic Cdo proteins.This enzyme belongs to the cupin protein superfamily, andtherefore the consensus sequences of two cupin motifs wereidentified in the amino acid sequences (37). Cupin motif 1(GX5HXHX3,4EX6G) is highly conserved in the V. paradoxusTBEA6 Cdo homologue except of glutamate, which is replaced

FIGURE 6. Localization of nine Tn5::mob insertions in two regions of the genomes of independentmutants of V. paradoxus strain TBEA6 showing fully or partially impaired growth on TDP and transcrip-tion analyses. Genes are shown as arrows. The positions of Tn5::mob insertions in the respective mutants areindicated. The results of transcriptional analysis of the genes are summarized in the lines below the respectivegenome region. RNA was isolated from the wild type of V. paradoxus strain TBEA6 cells growing in MSMcontaining 1% (w/v) TDP or 1% (w/v) sodium succinate or from cells of transposon-induced mutants growingin MSM containing 1% (w/v) TDP plus 0.2% (w/v) sodium succinate. �, transcript detectable; �, transcript notdetectable; n.t., not tested. Upper part: sequence analysis of Tn5::mob insertions of eight transposon-inducedmutants revealed three different adjacent genes putatively involved in TDP metabolism. cdo, putative gene ofcysteine dioxygenase; bug, putative gene for an extracytoplasmic solute receptor protein (Bordetella uptakegene); fox, putative gene for an FMN-dependent oxidoreductase. Lower part: neighborhood of the gene actputatively coding for an acyl-CoA-transferase/carnithin dehydratase in which Tn5::mob was mapped in mutant1/1. Downstream of the act gene, a gene coding for an acyl-CoA-dehydrogenase (acd) and a gene putativelycoding for an extracytoplasmic solute receptor protein (bug, Bordetella uptake gene) were located.

TABLE 1Identification and characterization of genes into which Tn5::mob was inserted in transposon-induced TDP-negative or TDP-leaky mutants ofV. paradoxus strain TBEA6

Mutant Phenotype Localization of transposon Highest homology (identical amino acids)1/1 TDP leaky and

3SP-negativeactVp, Acyl-CoA-transferase family III 56% (Magnetospirillium gryphiswaldenseMSR-1)

2/5 TDP-negative cdoVp, cysteine dioxygenase type I 68% (Verminephrobacter eiseniae EF01-2)4/5 TDP-negative bugVp, putative exported protein 46% (Burkholderia xenovorans LB 400)1/8 TDP-negative foxVp, putative FAD-linked oxidoreductase 55% (Acidovorax avenae subsp. citrulli AAC00-1)1/9 TDP-leaky bugVp, putative exported protein 46% (Burkholderia xenovorans LB 400)13/33 TDP-negative cdoVp, cysteine dioxygenase type I 68% (Verminephrobacter eiseniae EF01-2)1/20 TDP-negative bugVp, putative exported protein 46% (Burkholderia xenovorans LB 400)1/31 TDP-negative Intergenic region between the coding sequence for

the FAD-linked oxidoreductase and the putativeexported protein

2/34 TDP-negative foxVp, putative FAD-linked oxidoreductase 55% (Acidovorax avenae subsp. citrulli AAC00-1)2/41 TDP-leaky Localization of transposon was not determined




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by alanine or glycine. A common characteristic of eukaryoticCdo proteins is the replacement of the highly conserved gluta-mate within motif 1 by cysteine, and the formation of athioether bond between the thiol sulfur of cysteine and the C�of a nearby tyrosine residue is proposed (38–40). The secondcupin motif (GDX4PXGX2HX3N) is less conserved in Cdo pro-teins from eukaryotes (39). However, in the sequence of the V.paradoxus TBEA6 CdoVp, all conserved residues of the cupinmotif 2 are present.In addition to the two cupin motifs some other highly con-

served residues occur in Cdo proteins. In eukaryotes the resi-dues Tyr-58 and Arg-60 are located at the active site, and theyare supposed to be directly involved in substrate coordination(40). A substitution of the equivalent of the highly conservedArg-60 in bacterial Cdo proteins by glutamine, as previouslyreported (14), occurs obviously also in theV. paradoxusTBEA6CdoVp and in other bacterial Cdo proteins used for thismultiplesequence alignment (Fig. 7A). The highly conservedTyr-58waspresent in all sequences used for this multiple alignment. Addi-tionally highly conserved residues of eukaryotic Cdo proteins,also found in V. paradoxus and the other Cdo sequences, areSer-153, His-155, and Tyr-157, which probably form a catalytictriad (14, 39, 40).

BlastX searches using the cdoVp nucleotide sequence of V.paradoxus strain TBEA6 as query, revealed two putative genesfor Cdo in some bacteria (21). Whereas the translational prod-uct of one shows high sequence similarity (�50% identicalamino acids) to Cdo of V. paradoxus (Fig. 7A), the other showslower sequence similarity (�30% identical amino acids). Theamino acid sequences of lower sequence similarities to Cdo ofV. paradoxuswere also aligned, and the results revealed that thehighly conserved residues of Cdo proteins are also found inthese sequences (Fig. 7B). As described for Cdo proteins fromeukaryotes, the highly conserved glutamate (Glu) of cupinmotif 1 is replaced by cysteine (Cys-93) instead of glycine asoften occurring in paralogues. The cupin motif 2 is less con-served. In some cases the highly conserved serine (Ser-153) res-idue within the catalytic triad is replaced by histidine, and theequivalent of rat Tyr-58 is substituted by glutamine. Arg-60 ishighly conserved in contrast to the paralogue where Arg-60 issubstituted by glutamine.CDO Enzyme Activity Assays in Recombinant Strains—In

vivo assays were carried out using 3MP as substrate and ana-lyzed by GC/MS. In addition to E. coli BL21(DE3)pLysSpET23a::cdoVp and E. coli Top 10 pBBR1MCS-3::cdoVp twoother E. coli strains containing the putative cdoA and cdoB

FIGURE 7. Multiple sequence alignment of Cdo homologues from various bacteria. The boxes indicate highly conserved regions known for Cdo (11). Withinthe cupin motif the highly conserved residues are highlighted. A, multiple sequence alignment of Cdo of V. paradoxus TBEA6 with homologues showing highsequence similarities (�50%). The highly conserved glutamic acid is replaced by alanine or glycine. B, multiple sequence alignment of cdo paralogues of B.pertussis Tohama I, R. eutropha H16, and V. eiseniae EF01–2. The glutamic acid residue of motif 1 is replaced by cysteine.




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genes of R. eutropha H16 were also applied. E. coli strainBL21(DE3)pLysS harboring pET23a was used as negative con-trol. Cells were cultivated in M9 medium supplemented with0.1 g/liter yeast extract and containing 1% (v/v) glycerol as car-bon source at 30 °C. After 6 h of cultivation expression of therecombinant protein was induced by addition of 1 mM IPTG,and the substrate 3MP was added to a final concentration of0.1% (v/v). Cells were harvested after 24 h of cultivation andwashed twice with saline, before the cell pellets and thesupernatants were lyophilized and analyzed by GC/MS. Theputative reaction product 3SP was detected in cell pelletsand in the supernatants of E. coli BL21(DE3)pLysS harboringpET23a::cdoVp (Fig. 8) and also of E. coli Top 10 harboringpBBR1MCS-3::cdoARe. No 3SP was detected in the cell pel-let and supernatants of the negative control E. coli

BL21(DE3)pLysS harboring pET23a(supplemental Fig. S1) and in thoseof the recombinant E. coli strainsTop 10 harboring pBBR1MCS-3::cdoVporTop10pBBR1MCS-3::cdoBRe.Enzyme activity was determined

in vitro using cysteine, cysteamine,and 3MP as substrates. The puta-tive cdo gene of V. paradoxusstrain TBEA6 was heterologouslyexpressed using the T7-promotor/polymerase-based expression vec-tor pET23a and E. coli BL21(DE3)-pLysS as host strain. The CdoVpwas purified to electrophoretichomogeneity as hexahistidine-tagged protein by immobilizedmetal chelate affinity chromatogra-phy and applied to the in vitroenzyme assay (supplemental Fig.S2). Enzyme activity was only deter-mined when 3MP was supplied assubstrate. When cysteine or cys-teamine where used as substrateneither of the expected reactionproducts cysteine sulfinic acid orhypotaurine could be detected,although the assays were done atdifferent substrate concentrations(0.5–20 mM) and even with up to 10�g of purified enzyme. The oxygen-ation of 3MP was monitored withdifferent concentrations of 3MP(50–500 �M) and revealed a sigmoi-dal dependence on substrate con-centration, indicating that this reac-tion did not fit Michaelis-Mentenkinetics. The enzyme activity satu-rated at 300 �M 3MP, and concen-trations higher than 500 �Mresulted in a significant decrease ofthe activity. The addition of cys-teamine to the in vitro assays using

3MP as substrate also resulted in a clearly lower activity, andconcentrations above 10 mM completely inhibited the enzyme.In contrast, the addition of cysteine gave an activating effect.The enzyme activity was also determined in a Bis-Tris/Tris-buffer system at different pH values (pH 5–9) and revealedhighest activities at pH 7 (data not shown).Phenotypic Complementation of Mutants—Several at-

tempts were undertaken to complement the TDP-negativephenotype of the cdoVp mutants 2/5 and 13/33. The hybridplasmids pBBR1MCS-3::cdoARe, pBBR1MCS-3::cdoBRe, andpBBR1MCS-3::cdoVp were transferred to mutants 2/5 and13/33 by conjugation, and transconjugants were selected onMSM containing 0.3% (w/v) TDP and tetracycline. After 3days, growth on MSM agar plates containing TDP as solesource for carbon and energy was observed for mutant 13/33

FIGURE 8. GC/MS analysis of a sample withdrawn from the in vivo assay employing a recombinant strainof E. coli and using 3MP as substrate. Cells of E. coli BL21(DE3)pLysS pET23a::cdoVp were grown in M9-me-dium containing 1% (w/v) glycerol as carbon source. After 6 h of incubation the T7-promotor was induced byaddition of 1 mM IPTG, and subsequently 0.1% (v/v) 3MP was added. The cells were harvested after 48 h,washed twice with sterile saline, and lyophilized. The dry cell pellet was used for GC/MS analysis. A, GC-chromatogram, the following compounds could be identified by mass spectra analysis: 1, sulfuric acid (Rt, 7.6min); 2, pentanoic acid (Rt, 9.8 min); 3, 3-sulfinopropionic acid methylester (Rt, 19.4); 4, dodecanoic acid meth-ylester (Rt, 26.1 min); 5, methyltetradecanoic acid (Rt, 32.6 min); 6, hexadecanoic acid methylester (Rt, 40.2); and7, octadecanoic acid methylester (Rt, 52,9). B, mass spectrum of 3SP.




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harboring hybrid plasmid pBBR1MCS-3::cdoARe, whereasno growth was observed for mutant 13/33 harboring plasmidpBBR1MCS-3 or the hybrid plasmids pBBR1MCS-3::cdoBReor pBBR1MCS-3::cdoVp.

DISCUSSION

In total, eleven different bacterial strains capable of usingTDP as sole source of carbon and energy were isolated (supple-mental Table S1). Besides Schlegelella thermodepolymerans,which shows weak growth on TDP, this is the first report aboutbacteria capable of utilizing this thioether (41). Phylogeneticanalysis revealed that the best growing isolates enriched in thisstudy also belong to the family of Comamonadaceae (Fig. 2).High sequence similarities of the 16 S rRNA genes of �99% tothe 16 S rRNA gene of V. paradoxus indicated that the isolatesTBEA6 and SFWT are closely related to this species. Strains ofV. paradoxus are commonly known for the use of a large varietyof organic compounds (42). In addition to the utilization ofvarious sugars and amino acids, degradation of complex sub-strates like 2,4-dichlorophenoxyacetic acid or 3-nitotyrosinewas reported (43, 44). Desulfurization of toluen sulfonate andthe use of a broad variety of arylsulfonates, alkylsulfonates, andsulfate esters as sulfur sources byV. paradoxusDSM30034wasalso described recently (45). Furthermore, sulfolane is used assole source of carbon, sulfur, and energy by V. paradoxusWP1(46). Isolate TBEA3 exhibited only low sequence similarity tocultured and characterized bacteria. This isolate probably rep-resents a new taxon within theComamonadaceae,4 and studieson its taxonomic affiliation will be published separately.Transposon mutagenesis using Tn5::mob was done to eluci-

date themetabolismofTDP inV. paradoxus strainTBEA6.Tenindependent Tn5::mob-induced mutants affected in growth onTDPwere isolated and further characterized. Although growthon TDPwas highly or completely diminished, HPLC analysis ofthe supernatants revealed that TDP is still degraded by most ofthese mutants. Only mutants 1/8 and 1/20 were fully impairedin the degradation of TDP. In addition, with the exception ofmutant 1/1, all other mutants show growth on 3SP.In two mutants (2/5 and 13/33) the transposon was mapped

in a gene putatively coding for a cysteine dioxygenase. ThecdoVp gene is transcribed during growth on TDP and on succi-nate indicating that it is not induced by TDP but presumablyconstitutively expressed. In addition, the presence of 1 mMMgSO4 in the medium showed that transcription is probablynot repressed by inorganic sulfate as previously described forSsi proteins (36). Cysteine dioxygenase catalyzes the oxidationof the free thiol group of cysteine, which is thereby oxidized tocysteine sulfinic acid. Due to the structural similarity of 3MP tocysteine and because 3SP is accumulated by mutant 1/1 duringgrowth on succinate and TDP, we suppose that 3MP is used assubstrate by the Cdo of V. paradoxus strain TBEA6. Thisassumption is strongly supported by the detection of 3SP in invivo and in vitro assays using a recombinant E. coli strainexpressing cdoVp and using 3MP as substrate. Advanced in vitroassays applying purifiedCdo clearly demonstrated that oxygen-ation of 3MP depended on this enzyme. Highest enzyme activ-

ities were achieved applying 50–500 �M 3MP; concentrationshigher than 500 �M led to a significant decrease of Cdo activity.Enzyme activity showed a sigmoidal dependence on the sub-strate concentration and did obviously not display Michaelis-Menten kinetics, because it was also reported for Cdo fromother bacteria (14). 3MP is a highly reactive substance and inhi-bition of the enzymeby concentrations above 0.5mM is explain-able by the reducing properties of this thiol. Cysteamine con-centration above 5mM seems to have a similar effect as revealedby addition of different concentrations of cysteamine to thestandard assay using 3MP as substrate. Because hypotaurinecould not be detected when the enzyme was incubated withcysteamine, the latter was obviously not used as substrate.The enzyme-catalyzed oxidation of 3MP is very unusual and

has not been described for any characterized Cdo, yet. In con-trary, all so far characterized Cdo are reported as highly specificfor cysteine, and structurally related thiols were not used assubstrate for oxygenation (14, 47). Furthermore, 3MP wasshown to be a strong inhibitor of rat Cdo and amoderate inhib-itor of bacterial Cdo (14, 47). Although analysis of the primarystructure of the V. paradoxus Cdo revealed highly conservedresidues known for various cysteine dioxygenases, there aresome notable differences to described eukaryotic and prokary-otic Cdo. The highly conserved glutamate residue (Glu) isreplaced by the non-polar residue alanine in cupin motif 1,whereas cupinmotif 2 is almost completely conserved. Replace-ment of the highly conserved residue Arg-60 by glutamine wasalso reported for some bacterial Cdo (14); however, until nowtheir function as cysteine dioxygenase is based on theoreticalconsiderations only (14).Althoughwe observed an activating effect of cysteine when it

was applied to the enzyme assay, it was not possible to detectthe reaction product cysteine sulfinic acid.We have also shownthat cysteamine is no substrate for this enzyme (see above).Therefore, and because the primary structure of the CdoVphomologue of V. paradoxus TBAE6 exhibits significant differ-ences, this enzyme is not a cysteine dioxygenase but a thioldioxygenase with an alternative substrate range. This assump-tion is also supported by a lacking pleiotropic phenotype of thetwo transposon-induced cdoVp insertion mutants isolated inthis study.In eukaryotes a second thiol dioxygenase is known since

1966, however, the gene was identified only recently (48, 49).The enzyme catalyzes the oxidation of cysteamine, and likeCdoit is amember of the cupin superfamily (49). Some studies char-acterized this enzyme as highly specific for cysteamine, othersreported only low substrate specificity (49, 50). Besides cys-teamine, oxidation of homocysteamine, mercaptoethanol, and3-mercaptopropionic acid by this enzyme has been shown.Although there are no reports about further thiol dioxygen-

ase reactions in bacteria, it is likely that bacteria also possessmore than one thiol dioxygenase. Because we could also notfind any hint of oxygenation of cysteamine by CdoVp, it is pro-posed that the dioxygenase described in this study is a new typeof thiol dioxygenases. Due to the substrate it is referred to as3-mercaptopropionate dioxygenase.3MP, derived from biological and abiotic reactions, occurs

widespread in coastal sediments. Kiene and Taylor (8, 51) even4 N. Bruland and A. Steinbuchel, unpublished results.




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suggested that 3MP is a centralmetabolite in both catabolic andassimilatory sulfurmetabolism in general. Therefore, the abilityto utilize 3MP could be a beneficial trait for soil bacteria. Fur-thermore, degradation of this compound releases the sulfurmoiety back into the sulfur cycle. In R. eutropha and in severalother bacteria in silico analysis identified an additional geneputatively coding for a cysteine dioxygenase. These paraloguegenes show low sequence similarity to each other, and the align-ment of the primary structure revealed that one of the peculiardifferences between these paralogues is the replacement of theconserved glutamate residue in motif 1. Although one paral-ogue cdo gene product showed replacement of glutamate byglycine or alanine, the other showed substitution by cysteine asdescribed for eukaryoticCdos.Different substitutionswere alsofound for the highly conserved residues Tyr-58, Arg-60, andSer-153 in the Cdo of rat.Because characterization of these paralogue genes is also

based on theoretical considerations, it would be interesting tocharacterize and compare the substrate ranges of theseenzymes in the future. For R. eutrophaH16 the function of oneparalogue gene was analyzed in this study. The cdoARe geneproduct showed high sequence similarity to the CdoVp of V.paradoxus TBEA6. The glutamate residue was replaced by gly-cine in CdoARe. With this enzyme oxidation of 3MP was dem-onstrated in the in vivo assays, and heterologous expression ofcdoARe in the Tn5::mob-inducedmutant 13/33 ofV. paradoxusshowing transposon insertion in cdoVp gene restored growth onTDP.As shown in this study, mutant 1/1 accumulates 3SP, and

growth of themutant in this compound is completely impaired.Therefore, it is very likely that the actVp translational product isinvolved in the further degradation of 3SP. Due to the highsequence similarities of the putative gene product to family IIIacyl-CoA-transferases, we suppose that 3SP is activated by link-age to CoA. The resulting 3SP-CoA thioester is then probablyfurthermetabolized in one ormore steps to SO3 andpropionate(Fig. 9). A homologous gene product is putatively involved inMarinomonas sp. in degradation of dimethylsulfoniopropi-onate. The dddD gene product of this bacterium also showshigh sequence homologies to family III acyl-CoA-transferase,and itwas predicted to catalyze the addition ofCoAas the initialstep in dimethylsulfoniopropionate degradation (52). Tran-scription of actVp is also not induced byTDP or limited concen-trations of inorganic sulfate in V. paradoxus strain TBEA6 asdemonstrated by RT-PCR. Transcription of the gene down-stream of the actVp putatively coding for acyl-CoA-dehydro-genase was not detected in mutants 1/1 and 2/5 or in the wildtype under all used conditions, indicating that this gene is prob-ably not involved inTDPdegradation and that a polar effect canbe excluded.A putative bug homologue was identified upstream of

cdoVp, and in two mutants the transposon was mapped inthis gene. Although numerous orthologous bug genes wereidentified in genomes of several �-proteobacteria (32, 53),the function of most of them is still unknown. The bughomologous bctC gene of Bordetella pertussis was identifiedas part of a tripartite tricarboxylate transporter operon.Besides its function as an extracytoplasmic solute receptor,

the protein is also part of the signaling cascade leading toup-regulation of the operon in the presence of their sub-strates (28). Although some bug homologues were identifiedas part of a tripartite tricarboxylate transporter system, mostof them are not linked to any transport system (53). In the R.eutrophaH16 genome 154 bug homologous genes were iden-tified, and an additionally regulatory role for the bug homo-logues was suggested, because the majority of these geneswere located immediately adjacent or one or two coding

FIGURE 9. Proposed pathway of the catabolism of TDP in V. paradoxusstrain TBEA6. Initially, TDP is cleaved by a yet unidentified enzyme (1) into3-hydroxypropionate and 3-mercaptopropionate. The latter is then oxy-genated by a cysteine dioxygenase (2) thereby yielding 3-sulfinopropi-onate. After addition of coenzyme A by a family III acyl-CoA transferase (3),the sulfur moiety is putatively removed by a desulfinase (4) resulting inpropionyl-CoA, which is then further metabolized. Whereas enzyme reac-tions (2) and (3) are based on experimental data and on general predic-tions of the respective enzymes, the proposed reactions (1) and (4) arehypothetically and are based on theoretical considerations and indirectexperimental evidence.




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sequences distant from genes for transcriptional regulators(53).The role of the bug homologues in TDP degradation is still

unknown, although impaired growth of mutants 1/20 and 2/34on TDP provides evidence that this gene is involved in the uti-lization of this thioether. Because no adjacent genes for trans-port proteins were identified, a sole transport function isunlikely. TDP and limited concentrations of inorganic sulfatehave also no inducing effect on the transcription of bugA asrevealed by RT-PCR. Identification of two additional bughomologues corresponds with the finding that the representa-tives of this gene family are overrepresented in several genomesof �-proteobacteria (32). In addition, putative signal sequencesfor leader peptides were identified in all three bug sequences sothat the gene products are probably localized in the periplasma.InsertionofTn5::mob in a geneputatively coding for anFAD-

dependent oxidoreductase impaired growth ofV. paradoxus onTDP. Because polar effects can be excluded according to RT-PCR analysis, further studies have to elucidate the physiologicalrole of this enzyme in TDP metabolism.

Acknowledgment—We thankBrunoBockChemische FabrikGmbH&Co. Kommanditgesellschaft for the provision of bulk TDP.

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Nadine Bruland, Jan Hendrik Wübbeler and Alexander SteinbüchelVariovorax paradoxusAcid-degrading Bacterium

the Initial Step of 3-Mercaptopropionate Catabolism in the 3,3-Thiodipropionic 3-Mercaptopropionate Dioxygenase, a Cysteine Dioxygenase Homologue, Catalyzes

doi: 10.1074/jbc.M806762200 originally published online November 10, 20082009, 284:660-672.J. Biol. Chem.

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3-mercaptopropionatedioxygenase,acysteine ... thioether 3,3-thiodipropionic acid (tdp)2 and its...

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