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• W2050423538 abstract "The thioether 3,3-thiodipropionic acid can be used as precursor substrate for biotechnological synthesis of 3-mercaptopropionic acid-containing polythioesters. Therefore, the hitherto unknown catabolism of this compound was elucidated to engineer novel and improved polythioester biosynthesis pathways in the future. Bacteria capable of using 3,3-thiodipropionic acid as the sole source of carbon and energy for growth were enriched from the environment. From eleven isolates, TBEA3, TBEA6, and SFWT were morphologically and physiologically characterized. Their 16 S rDNAs and other features affiliated these isolates to the β-subgroup of the proteobacteria. Tn5::mob mutagenesis of isolate Variovorax paradoxus TBEA6 yielded ten mutants fully or partially impaired in growth on 3,3-thiodipropionic acid. Genotypic characterization of two 3,3-thiodipropionic acid-negative mutants demonstrated the involvement of a bacterial cysteine dioxygenase (EC 1.13.11.22) homologue in the further catabolism of the 3,3-thiodipropionic acid cleavage product 3-mercaptopropionic acid. Detection of 3-sulfinopropionate in the supernatant of one of these mutants during cultivation on 3,3-thiodipropionic acid as well as in vivo and in vitro enzyme assays using purified protein demonstrated oxygenation of 3-mercaptopropionic acid to 3-sulfinopropionate by this enzyme; cysteine and cysteamine were not used as substrate. Beside cysteine dioxygenase and cysteamine dioxygenase, this 3-mercaptopropionic acid dioxygenase is the third example for a thiol dioxygenase and the first report about the microbial catabolism of 3-mercaptopropionic acid. Insertion of Tn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionate during cultivation on 3,3-thiodipropionic acid, indicating that this compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA. The thioether 3,3-thiodipropionic acid can be used as precursor substrate for biotechnological synthesis of 3-mercaptopropionic acid-containing polythioesters. Therefore, the hitherto unknown catabolism of this compound was elucidated to engineer novel and improved polythioester biosynthesis pathways in the future. Bacteria capable of using 3,3-thiodipropionic acid as the sole source of carbon and energy for growth were enriched from the environment. From eleven isolates, TBEA3, TBEA6, and SFWT were morphologically and physiologically characterized. Their 16 S rDNAs and other features affiliated these isolates to the β-subgroup of the proteobacteria. Tn5::mob mutagenesis of isolate Variovorax paradoxus TBEA6 yielded ten mutants fully or partially impaired in growth on 3,3-thiodipropionic acid. Genotypic characterization of two 3,3-thiodipropionic acid-negative mutants demonstrated the involvement of a bacterial cysteine dioxygenase (EC 1.13.11.22) homologue in the further catabolism of the 3,3-thiodipropionic acid cleavage product 3-mercaptopropionic acid. Detection of 3-sulfinopropionate in the supernatant of one of these mutants during cultivation on 3,3-thiodipropionic acid as well as in vivo and in vitro enzyme assays using purified protein demonstrated oxygenation of 3-mercaptopropionic acid to 3-sulfinopropionate by this enzyme; cysteine and cysteamine were not used as substrate. Beside cysteine dioxygenase and cysteamine dioxygenase, this 3-mercaptopropionic acid dioxygenase is the third example for a thiol dioxygenase and the first report about the microbial catabolism of 3-mercaptopropionic acid. Insertion of Tn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionate during cultivation on 3,3-thiodipropionic acid, indicating that this compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA. The thioether 3,3-thiodipropionic acid (TDP) 2The abbreviations used are: TDP, thioether 3,3-thiodipropionic acid; PTE, polythioester; DTDP, 3,3-dithiodipropionic acid; Cdo, cysteine dioxygenase; MSM, mineral salts medium; IPTG, isopropyl 1-thio-β-d-galactopyranoside; HPLC, high-performance liquid chromatography; GC/MS, gas chromatography/mass spectrometry; RT, reverse transcription; MES, 4-morpholineethanesulfonic 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-β-d-galactopyranoside; 3SP, 3-sulfinopropionic acid; 3MP, 3-mercaptopropionic acid; Rt, retention time. and its ester are effective non-toxic antioxidants (1Scott G. Chem. Commun. 1968; 24: 1572-1574Google Scholar), and they are therefore widely used as antioxidant and stabilizer in food, for food packaging, and for various technical applications. Experiment with rats showed that TDP was rapidly adsorbed after oral intake and excreted in the urine (2Reynolds R.C. Astill B.D. Fassett W. Toxicol. Appl. Pharmacol. 1974; 28: 133-141Crossref PubMed Scopus (5) Google Scholar). In technical applications esters of TDP are important stabilizers of polyolefins (1Scott G. Chem. Commun. 1968; 24: 1572-1574Google Scholar), and polymer-bound TDP is used to replace methyl sulfide for the reductive quenching of ozonolysis reactions (3Appel R.B. Tomlinson I.A. Hill I. Synth. Commun. 1995; 25: 3589-3595Crossref Scopus (18) Google Scholar). Recently, the biotechnological production of medium- and long-chain dialkyl 3,3-thiodipropionate antioxidants by a lipase-catalyzed esterification of 3,3-thiodipropionic acid in the absence of solvents was reported. In contrast to the chemical production of TDP ester, the biotechnological process does not require any materials with deleterious effects on health and environment (4Weber N. Klein E. Vosmann K. J. Agric. Food Chem. 2006; 54: 2957-2963Crossref PubMed Scopus (8) Google Scholar). Another biotechnological process using TDP as primary product is the microbial production of polythioesters (PTEs) (5Lu¨tke-Eversloh T. Bergander K. Luftmann H. Steinbu¨chel A. Microbiology. 2001; 147: 11-19Crossref PubMed Scopus (163) Google Scholar). In addition to 3-mercaptopropionic acid Ralstonia eutropha is able to use the organo sulfur compounds TDP and 3,3-dithiodipropionic acid (DTDP) as precursor substrates for production of copolymers of 3-hydroxybutyrate and 3MP (6Lu¨tke-Eversloh T. Steinbu¨chel A. FEMS Microbiol. Lett. 2003; 221: 191-196Crossref PubMed Scopus (62) Google Scholar). In contrast to 3MP the application of TDP and DTDP has numerous advantages, because they have a lower toxicity and they are odorless, inexpensive, and available on a large scale. Until today the use of TDP and DTDP as precursor substrates are limited to R. eutropha, and biotechnological production of PTE using the recombinant Escherichia coli strain JM109 pBPP1 (7Liu S.-J. Steinbu¨chel A. Appl. Environ. Microbiol. 2000; 66: 739-743Crossref PubMed Scopus (62) Google Scholar) is only possible when 3MP is added to the media. Because 3MP is incorporated into the polymer if TDP or DTDP is supplied as precursor substrate, it is assumed that these compounds are enzymatically cleaved into 3MP and 3-hydroxypropionate, or two molecules 3MP, respectively (6Lu¨tke-Eversloh T. Steinbu¨chel A. FEMS Microbiol. Lett. 2003; 221: 191-196Crossref PubMed Scopus (62) Google Scholar). The corresponding TDP- and/or DTDP-cleaving enzymes as well as the microbial catabolism of the intermediate 3MP are still unknown. The identification of such enzymes could help to engineer the recombinant E. coli JM109 pBPP1 toward TDP- and DTDP-based PTE production. Therefore, the corresponding genes could be used to improve the already established BPEC pathway by heterologous expression (7Liu S.-J. Steinbu¨chel A. Appl. Environ. Microbiol. 2000; 66: 739-743Crossref PubMed Scopus (62) Google Scholar). For 3MP it is known that it occurs naturally as an intermediate during microbial degradation of the osmoprotectant dimethylsulfoniopropionate and during the biotransformation of the sulfur-containing amino acids methionine and homocysteine in anoxic coastal sediments (8Kiene R.P. Taylor B.F. Nature. 1988; 332: 148-150Crossref Scopus (58) Google Scholar, 9Kiene R.P. Taylor B.F. Appl. Environ. Microbiol. 1988; 54: 2208-2212Crossref PubMed Google Scholar, 10Yoch D.C. Appl. Environ. Microbiol. 2002; 68: 5804-5815Crossref PubMed Scopus (333) Google Scholar, 11Kiene R.P. Malloy K.D. Taylor B.F. Appl. Environ. Microbiol. 1990; 56: 156-161Crossref PubMed Google Scholar). However, to our knowledge no reports on the pathways or enzymes for further metabolism of 3MP in bacteria have been published. In contrast, the catabolism of cysteine, the structural analogue of 3MP, is well known in bacteria (12McFall E. Newman E.B. Escherichia coli and Salmonella: Cellular and Molecular Biology.in: ASM Press, Washington, DC1996: 358-374Google Scholar, 13Mihara H. Esaki N. Appl. Microbiol. Biotechnol. 2002; 60: 12-23Crossref PubMed Scopus (225) Google Scholar). In addition to the well investigated cysteine degradation pathways, a novel pathway was recently reported in eubacteria by Dominy et al. (14Dominy Jr., J.E. Simmons C.R. Karplus P.A. Gehring A.M. Stipanuk M.H. J. Bacteriol. 2006; 188: 5561-5569Crossref PubMed Scopus (80) Google Scholar), which involves a cysteine dioxygenase (Cdo, EC 1.13.11.20). This Fe2+-dependent enzyme catalyzes the irreversible oxidation of the sulfhydryl group of cysteine to cysteine sulfinic acid. Cdos are well known in eukaryotes and play an important role by reducing the cysteine pool and increasing the levels of important metabolites such as taurine and sulfates (15Chai S.C. Jerkins A.A. Banik J.J. Shalev I. Pinkham J.L. Uden P.C. Maroney M.J. J. Biol. Chem. 2004; 280: 9865-9869Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The physiological function of this enzyme is not completely understood in bacteria. In addition to the important role in regulating the steady-state cysteine levels, also an important role of this enzyme during changes of bacterial life cycles was suggested (14Dominy Jr., J.E. Simmons C.R. Karplus P.A. Gehring A.M. Stipanuk M.H. J. Bacteriol. 2006; 188: 5561-5569Crossref PubMed Scopus (80) Google Scholar), because many of the bacteria possessing a Cdo undergo a complex life cycle involving morphological changes. For example, in cells of Bacillus subtilis expression of the cdo gene is up-regulated during transition from the vegetative state to sporulation (14Dominy Jr., J.E. Simmons C.R. Karplus P.A. Gehring A.M. Stipanuk M.H. J. Bacteriol. 2006; 188: 5561-5569Crossref PubMed Scopus (80) Google Scholar). To our knowledge there are so far also no reports about bacteria that utilize the organic thioether TDP as sole source of carbon and energy. Only its use as a sulfur source was described for Mycobacterium goodii X7B (16Li F. Xu P. Feng J. Meng L. Zheng Y. Luo L. Ma C. Appl. Environ. Mirobiol. 2005; 71: 276-281Crossref PubMed Scopus (79) Google Scholar). However, this bacterium could not grow with TDP in the absence of an additional carbon source, and degradation products of TDP were also not reported. To engineer the PTE biosynthesis in the future toward to use TDP as precursor substrates, we investigated the catabolism of TDP. The isolation and characterization of bacteria capable of using TDP as the sole source of carbon and energy are described in this study. In addition, Tn5::mob mutagenesis was carried out with one of these new isolates, Variovorax paradoxus strain TBEA6, to identify genes possibly involved in TDP catabolism. Isolation of Strains Capable of Using TDP as Sole Source of Carbon and Energy—Samples from different soils, activated sludge, and freshwater tank sediment were incubated in mineral salts medium (MSM) containing 3 g/liter TDP as the sole source of carbon and energy at 30 °C for 3-5 days (17Schlegel H. Kaltwasser G.H. Gottschalk G. Arch. Mikrobiol. 1961; 38: 209-222Crossref PubMed Scopus (622) Google Scholar). Aliquots of these cultures were then plated on the same medium solidified with 1.5% (w/v) agar, single colonies were isolated, and the best 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 isolate TBEA3 were cultivated at 30 °C in nutrient broth or MSM supplemented with 0.1 g/liter yeast extract under aerobic conditions on a rotary shaker at an agitation of 130 rpm. Strains of E. coli were cultivated in Luria-Bertani (LB) medium or M9 medium supplemented with yeast extract (0.1 g/liter) at 37 °C under the same conditions (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.in: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998Google Scholar). Carbon sources were supplied as filter-sterilized stock solutions as indicated in the text. For maintenance of plasmids, antibiotics were prepared according to Sambrook et al. (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.in: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998Google Scholar) and added to the media at the following concentrations (μg/ml): ampicillin (75), kanamycin (50), chloramphenicol (34), and tetracycline (12.5). In E. coli heterologous expression of genes under the control of a lac-promotor was induced by addition of 1 mm IPTG to LB medium. Chemicals—Bulk TDP was provided by Bruno Bock Chemische Fabrik GmbH & Co. KG. Organic thiochemicals of high purity grade were purchased from Acros Organics (Geel, Belgium) or Sigma-Aldrich (Steinheim, Germany) (Fig. 1). 3-Sulfinopropionate was synthesized according to Jollás-Bergáret (19Jolles-Bergeret B. Eur. J. Biochem. 1974; 42: 349-353Crossref PubMed Scopus (17) Google Scholar); the procedure was modified by one repetition of the alkaline cleavage of the intermediate bis-(2-carboxyethyl)sulfone. Starting from 111 g of sodium formaldehyde sulfoxylate (purity, >98%) plus 108 ml of acrylic acid (99.5%), 119 g of the intermediate bis-(2-carboxyethyl)sulfone were chemically synthesized. After alkaline scission, precipitation, and washing procedures, 99 g of the disodium salt of 3SP, with a purity of ∼90%, were finally obtained. Synthesis and purity of the substance was confirmed by HPLC and GC/MS. Strain Identification—API 20NE identification test (bio-Márieux, Marcy-l’Etoile, France) and Bactident oxidase test stripes (Merck KgA, Darmstadt, Germany) were used according to the manufacturer’s instructions. Presence of catalase was tested using 3% (v/v) H2O2. The 16 S rRNA gene was amplified from total genomic DNA by PCR using Primers 27f and 1525r (20Devereux R. Willis S.G. Molecular Microbial Ecology Manual.in: Kluwer, The Netherlands1995: 509-522Google Scholar). The PCR product was purified using the NucleoTrap kit (Machery and Nagel, Du¨ren, Germany) and applied as template for sequencing in which the following primers were utilized: 27f, 357f, 803f, 907r, 1114f, 1385r, and 1525r (20Devereux R. Willis S.G. Molecular Microbial Ecology Manual.in: Kluwer, The Netherlands1995: 509-522Google Scholar). The assembled sequence was compared with the GenBank™ data base and the Ribosomal Database Project using the Blast and SIMILARITY-RANK (Ribosomal Database Project) algorithms (21Altschul S.F. Madden T.L. Scha¨ffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60220) Google Scholar, 22Maidak B.L. Olsen G.J. Larsen N. Overbeek R. McCaughey M.J. Woese C.R. Nucleic Acids Res. 1997; 25: 109-111Crossref PubMed Scopus (772) Google Scholar). A phylogenetic tree was constructed using ClustalX (23Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35619) Google Scholar). Transposon Mutagenesis and Characterization of Tn5::mob-induced Mutants—Insertional mutagenesis of V. paradoxus strain TBEA6 with transposon Tn5::mob was performed as described previously by using the suicide plasmid pSUP5011, which was delivered from E. coli S17-1 to the recipient by conjugation during spot agar mating (24Simon R. Priefer U. Pu¨hler A. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5658) Google Scholar, 25Friedrich B. Hogrefe C. Schlegel H.G. J. Bacteriol. 1981; 147: 198-205Crossref PubMed Google Scholar). Transconjugants were selected on MSM plates containing 0.5% (w/v) gluconate plus kanamycin. Transconjugants impaired in growth on TDP were then identified by plating on MSM agar plates containing 0.3% (w/v) TDP plus kanamycin or 0.5% (w/v) gluconate plus kanamycin. For genotypic characterization of the Tn5::mob-induced mutants, genomic DNA was isolated (26Marmur J. J. Mol. Biol. 1961; 3: 208-218Crossref Scopus (9014) Google Scholar) and digested with SalI or BamHI. The genomic DNA fragments were then ligated to pBluescriptSK- DNA, which was linearized with the same restriction endonuclease; the ligation products were then transformed into CaCl2-competent E. coli Top10 cells. Transformants were selected on LB medium containing ampicillin plus kanamycin, and hybrid plasmids were subsequently isolated and sequenced using the primers M13 forward, M13 reverse, and IS50L (supplemental Table S3). Analysis of Cell-free Supernatants and Sulfur Organic Compounds by HPLC—Concentrations of TDP, 3MP, cysteine sulfinic acid, cysteamine, hypotaurine, and 3SP were analyzed by HPLC. HPLC analysis of TDP, 3MP, cysteamine, and 3SP was carried out in a LaChrom Elite® HPLC apparatus (VWR-Hitachi International GmbH, Darmstadt, Germany) consisting of a Metacarb 67H advanced C column (Varian, Palo Alto, CA, Bio-Rad Aminex equivalent) and a 22350 VWR-Hitachi column oven. The primary separation mechanism includes ligand exchange, ion exclusion, and adsorption. A VWR-Hitachi refractive index detector (Type 2490) with an active flow cell temperature control and automated reference flushing eliminating temperature effects on the refractive index baseline was used for detection. Aliquots of 20 μl of cell-free supernatants, solutions of organic sulfur compounds or enzyme assay were injected and eluted with 0.005 n sulfuric acid (H2SO4) in double distilled water at a flow rate of 0.8 ml/min. Online integration and analysis was done with EZ Chrome Elite Software (VWR International GmbH, Darmstadt, Germany). Cysteamine was detected under the same conditions using double distilled water as mobile phase. Detection of hypotaurine and cysteine sulfinic acid was carried out in a Kontron Instrument (Neufahrn, Germany). After derivatization with OPA reagent (27Aboulmagd E. Oppermann-Sanio F.B. Steinbu¨chel A. Arch. Microbiol. 2000; 174: 297-306Crossref PubMed Scopus (94) Google Scholar) using a Smartline Autosampler 3900 (Knauer Advanced Scientific Instruments, Berlin, Germany), 20 μl of the reaction was injected onto a Novapack C18 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 comparison of their retention times to those of standard organic acids. The detection limit for hypotaurine is ∼20 μm and 10 μm for cysteine sulfinic acid. Quantitative Analysis of Polyhydroxyalkanoic Acid and Their Compositions by GC—Lyophilized cell material was subjected to methanolysis in the presence of methanol and sulfuric acid (MeOH: 85%, v/v; H2SO4: 15%, v/v) for 4 h at 100 °C, and the resulting methylesters of the polyhydroxyalkanoic acid constituents were characterized by gas chromatography using an Agilent 6850 GC (Agilent Technologies, Waldbronn, Germany) equipped with a BP21 capillary column (50 m by 0.22 mm; film thickness, 250 nm; SGE, Darmstadt, Germany) and a flame ionization detector (Agilent Technologies). A 2-μl portion of the organic 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 and 220 °C, respectively. The following temperature program was applied: 120 °C for 5 min, increase of 3 °C/min to 180 °C, and increase of 10 °C/min to 220 °C and 220 °C for 31 min. Substances were identified by comparison of their retention times to 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 subjected to methanolysis as described above, and the resulting methylesters of the organic acids were characterized by coupled GC/MS using an HP6890 gas chromatograph equipped with a model 5973 EI MSD mass-selective detector (Hewlett Packard). A2-μl portion of the organic phase was analyzed after splitless injection employing a BP21 capillary column (50 m by 0.22 mm; film thickness, 250 nm; SGE). Helium (0.6 ml/min) was used as carrier gas. The temperatures of the injector and detector were 250 °C and 240 °C, respectively. The same temperature program as described for GC analysis was applied. Data were evaluated using the NIST Mass Spectral Search program. 3S. Stein, A. Levitsky, O. Fateev, and G. Mallard (1998). The NIST Mass Spectral Search Program, Windows-Software Version 1.6d. Isolation of RNA and RT-PCR—Total RNA was isolated from V. paradoxus strain TBEA6 by using the Qiagen RNeasy-Kit according to the manufacturer’s instructions. RT-PCR was performed using the Qiagen “One step RT-PCR” Kit according to the manufacturer’s instructions. To recognize PCR products based on DNA contaminations in isolated RNA, a control with addition 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 isolated according to Marmur (26Marmur J. J. Mol. Biol. 1961; 3: 208-218Crossref Scopus (9014) Google Scholar). Plasmid DNA was isolated from E. coli and V. paradoxus strains using the GeneJET™ plasmid miniprep kit from Fermentas (St. Leon-Rot, Germany) according to the manufacturer’s manual. DNA was digested with restriction endonucleases under conditions described by the manufacturer or according to Sambrook et al. (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.in: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998Google Scholar). PCR were carried out in an Omnigene HBTR3CM DNA thermal cycler (Hybaid, Heidelberg, Germany) using Platinum® Taq DNA Polymerase (Invitrogen). PCR products were isolated from an agarose gel and purified using the NucleoTrap kit (Machery and Nagel, Du¨ren, Germany) according to the manufacturer’s instructions. T4-DNA-Ligase was purchased from Invitrogen. Primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). Transfer of DNA—Competent cells of E. coli strains were prepared and transformed by the CaCl2 procedure (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.in: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998Google Scholar). DNA Sequencing and Sequence Data Analysis—DNA sequencing was done in a Li-Cor 4000L automatic sequencing apparatus (Li-COR Inv., Biotechnology Division, NE, USA) using the Thermo long read cycle sequencing Kit (Epicenter Technologies, WI, USA) and IRD 800-labeled oligonucleotides (MWG-Biotech, Ebersberg, Germany). BlastX was used for determination of nucleotide identity (21Altschul S.F. Madden T.L. Scha¨ffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60220) Google Scholar). DNA-DNA Hybridization—Southern hybridization was carried out by the method described by Oelmu¨ller et al. (29Oelmu¨ller U. Kru¨ger N. Steinbu¨chel A. Friedrich C.G. J. Microbiol. Methods. 1990; 11: 73-84Crossref Scopus (121) Google Scholar) at a temperature of 68 °C. Genome Walking—For sequencing of flanking genomic regions of known sequences a PCR-based directional genome walking method (30Mishra R.N. Singla-Pareek S.J. Nair S. Sopory S.K. Reddy M.K. BioTechniques. 2002; 33: 830-834Crossref PubMed Scopus (45) Google Scholar) was performed. Cloning of cdoVp, cdoARe, and cdoBRe—The cdoVp, cdoARe, and cdoBRe genes were amplified from total genomic DNA of V. paradoxus strain TBEA6 or R. eutropha strain H16 by PCR using Taq DNA polymerase (Invitrogen) and the following oligonucleotides: cdo(NdeI), cdo(XhoI), cdoA(XbaI), cdoA(XhoI), cdoB(ApaI), and cdoB(HindIII) (supplemental Table S3). PCR products were isolated from agarose gels using the NucleoTrap kit (Machery and Nagel) and ligated with pGEMTeasy® DNA (Promega, Madison, WI). Ligation products were transformed into CaCl2-competent cells, and transformants were selected on LB agar plates containing IPTG, X-Gal, plus ampicillin. For heterologous expression in the T7 promoter/polymerase-based expression vector pET23a (Novagen, Madison, WI), cdoVp was obtained by digestion of hybrid plasmid pGEMTeasy®::cdoVp with restriction endonucleases NdeI and XhoI and purified from an agarose gel using the NucleoTrap kit (Machery and Nagel). After ligation into expression vector pET23a, which was linearized with the same restriction endonucleases, the ligation product was used for transformation of CaCl2-competent cells of E. coli Top10. After selection of transformants using LB media containing ampicillin, the hybrid plasmids were isolated, analyzed by sequencing, and transformed to CaCl2-competent cells 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 (31Kovach M.E. Gene (Amst.). 1995; 166: 175-176Crossref PubMed Scopus (2749) Google Scholar), cdoVp, cdoARe, and cdoBRe were obtained from pGEMTeasy® vector, which were digested with the respective restriction endonuclease and purified from an agarose gel using the NucleoTrap kit (Machery and Nagel). The purified genes were subsequently ligated into pBBR1MCS-3, which was linearized with the same restriction endonucleases, and the ligation products were transformed to CaCl2-competent cells of E. coli S17-1 and E. coli Top10. Transformants were selected on LB medium containing tetracycline, IPTG, plus X-Gal. The hybrid plasmids pBBR1MCS-3::cdoVp, pBBR1MCS-3::cdoARe, and pBBR1MCS-3::cdoBRe were then conjugated into the transposon-induced mutants 2/5 and 13/33 from E. coli S17-1. Preparation of Crude Extracts—Cells from 50- to 500-ml cultures were harvested by centrifugation (20 min, 4 °C, and 2,800 × g), washed twice, and resuspended in 50 mm NaPO4 buffer (pH 7.6). Cells were disrupted by sonification in a Sonopuls GM200 apparatus (Bandelin, Berlin, Germany) with an amplitude of 16 μm (1 min/ml) while cooling in an NaCl/ice bath. Soluble protein fractions of crude extracts were obtained in the supernatants after 1-h centrifugation at 100,000 × g and 4 °C and were used for enzyme purifications. Immobilized Metal Chelate Affinity Chromatography—To obtain purified hexahistidine-tagged fusion CdoVp, His Spin Trap affinity columns (GE Healthcare, Uppsala, Sweden) were used according to the instructions of the manufacturer with minor modifications. Tris-HCl (0.1 m, pH 7.6) was used as buffer component instead of sodium phosphate, and for the washing step a buffer containing 40 mm imidazole was applied. The washing step was repeated three times, and the elution step was repeated two times. Enzyme Assay—Standard in vitro activity of cysteine dioxygenase was assayed by incubating 3 μg of purified CdoVp for 30 min at 30 °C in the presence of the following components: 10 mm cysteine, 10 mm cysteamine, or 5 mm 3MP, 400 μm (NH4)2Fe(SO4)2 × 6H2O, 12.5 μm bathocuproine disulfonate and MES buffer (62 mm, pH 6.3). The reaction was stopped by 10-min incubation at 95 °C. Negative controls were done with denatured 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 in M9 medium containing 1% (v/v) glycerol at 30 °C. Expression of the recombinant protein was induced by addition of 1 mm IPTG after 6 h of cultivation. Subsequently, the substrate 3MP was added to a final concentration of 0.1% (v/v), and the cells were grown for additional 24 h before they were harvested and washed twice with 0.9% (w/v) NaCl. Finally, the cell pellets were lyophilized. Analysis of the reaction product 3SP was done by GC/MS. Data Deposition—Nucleotide sequences have been deposited in the GenBank™ data base under the following GenBank™ accession numbers: EF641108, 16 S rDNA gene of isolate TBEA6; EU825700, 16 S rDNA gene of isolate TBEA3; EU441166, 16 S rDNA gene of isolate SFWT; EU441167, contiguous sequence comprising bugC, fox, bugA, cdo, and ahpD; and EU449952, contiguous sequence comprising act, acd, and partial sequence of bugB. Isolation of TDP-utilizing Bacteria and Taxonomic Affiliation 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 carbon and energy for growth were enriched and isolated (supplemental Table S1). The best growing isolates were designated as TBEA6, TBEA3, and SFWT; they were further characterized by methods of polyphasic taxonomy and by analysis of the 16 S rDNA sequences to unravel their phylogenetic position. All three isolates were Gram-negative, oxidase, and catalase-positive, and best growth was observed at 30 °C, although isolates TBEA6 and SFWT grew also very slowly at 4 °C. Colonies of TBEA6 and SFWT were yellow-pigmented, and the motile cells are short rods with a cell length of 1.5 μm. Using the API 20NE identification test system, reduction of nitrat" @default.
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• W2050423538 date "2009-01-01" @default.
• W2050423538 modified "2023-10-03" @default.
• W2050423538 title "3-Mercaptopropionate Dioxygenase, a Cysteine Dioxygenase Homologue, Catalyzes the Initial Step of 3-Mercaptopropionate Catabolism in the 3,3-Thiodipropionic Acid-degrading Bacterium Variovorax paradoxus" @default.
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