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Metabolism of L (−)-Carnitine by Enterobacteriaceae Under Aerobic

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Metabolism of L (−)-Carnitine by Enterobacteriaceae Under Aerobic FEMS Microbiology Letters 174 (1999) 295^301 Metabolism of L(3)-carnitine by Enterobacteriaceae under aerobic conditions Downloaded from https://academic.oup.com/femsle/article/174/2/295/502865 by guest on 29 September 2021 Thomas ElMner a, Andrea PreuMer a, Ulrich Wagner b, Hans-Peter Kleber a;* a Institut fuër Biochemie, Universitaët Leipzig, Talstr. 33, D-04103 Leipzig, Germany b Institut fuër Zoologie, Fakultaëtfuër Biowissenschaften, Pharmazie und Psychologie, Universitaët Leipzig, Talstr. 33, D-04103 Leipzig, Germany Received 11 January 1999; received in revised form 16 March 1999; accepted 16 March 1999 Abstract Different Enterobacteriaceae, such as Escherichia coli, Proteus vulgaris and Proteus mirabilis, are able to convert L(3)- carnitine, via crotonobetaine, into Q-butyrobetaine in the presence of carbon and nitrogen sources under aerobic conditions. Intermediates of L(3)-carnitine metabolism (crotonobetaine, Q-butyrobetaine) could be detected by thin-layer chromato- graphy. In parallel, L(3)-carnitine dehydratase, carnitine racemasing system and crotonobetaine reductase activities were determined enzymatically. Monoclonal antibodies against purified CaiB and CaiA from E. coli O44K74 were used to screen cell-free extracts of different Enterobacteriaceae (E. coli ATCC 25922, P. vulgaris, P. mirabilis, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae) grown under aerobic conditions in the presence of L(3)-carnitine. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Enterobacteriaceae; Carnitine; Trimethylammonium compound; Carnitine dehydratase; Crotonobetaine reductase 1. Introduction Bacteria are able to metabolise L(3)-carnitine in di¡erent ways [9] using this quaternary ammonium L(3)-carnitine (R(3)-3-hydroxy-4-trimethylami- compound as sole source of carbon and nitrogen nobutyrate) is a ubiquitously occurring compound (e.g. Pseudomonas sp.; [10]) or only as sole source in nature. In eukaryotes, L(3)-carnitine is essential of carbon (e.g. Acinetobacter sp.; [11,12]) under for the transport of long-chain fatty acids through aerobic conditions. Enterobacteriaceae are able to the inner mitochondrial membrane [1,2]. In bacteria, convert L(3)-carnitine via crotonobetaine into Q-bu- the physiological function of L(3)-carnitine is un- tyrobetaine in the presence of carbon and nitrogen known. In addition to glycine betaine, one of the sources under anaerobic conditions, but they do not most widely distributed osmoprotectants, L(3)- assimilate the carbon skeleton and nitrogen [13,14]. carnitine was shown to serve as osmoprotectant Two enzymes, L(3)-carnitine dehydratase (EC in Escherichia coli [3] and other microorganisms 4.2.1.89) and crotonobetaine reductase, were found [4^8]. in E. coli to catalyse this reaction sequence [15,16]. A carnitine racemase activity able to convert D(+)-car- * Corresponding author. Tel.: +49 (341) 97 36992; nitine into L(3)-carnitine was subsequently also Fax: +49 (341) 97 36998; E-mail: [email protected] postulated [17]. Studies using whole cells of E. coli 0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(99)00151-2 FEMSLE 8746 29-4-99 296 T. ElMner et al. / FEMS Microbiology Letters 174 (1999) 295^301 have shown that these enzymes are inducible in the 2.2. Cultivation of microorganisms and cell disruption presence of L(3)-carnitine or crotonobetaine under anaerobic conditions [18]. Although a role as exter- The strains were inoculated from agar slopes into nal electron acceptor of anaerobic respiration like complex media and cultivated as preculture under nitrate or fumarate [19] is postulated for crotonobe- aerobic conditions at 30³C up to the middle of the taine [14], the precise function of this two-step path- exponential growth phase. The complex media con- way is still unknown. The stimulation of anaerobic tained 17 g pancreatic peptone, 3 g yeast extract and growth of Enterobacteriaceae by crotonobetaine sup- 5 g NaCl per litre of deionised water. Cultivation ports this hypothesis [20]. was carried out in 1000 ml Erlenmeyer £asks con- L(3)-carnitine dehydratase has been puri¢ed and taining 250 ml complex medium supplemented either characterised [15]. A still unknown cofactor essential with 0.5% carnitine, 0.5% crotonobetaine or 0.5% Q- for enzyme activity was separated during puri¢cation butyrobetaine on a rotary shaker (175 rpm) at 30³C. Downloaded from https://academic.oup.com/femsle/article/174/2/295/502865 by guest on 29 September 2021 procedure. The addition of this low molecular mass Growth of cells was followed by measuring the ap- e¡ector ( 6 1000 Da) caused reactivation of the parent absorbance of the culture at 600 nm. Cells apoenzyme. It is not possible to replace the e¡ector were harvested at the end of the exponential growth with known coenzymes or cofactors involved in de- phase by centrifugation at 5000Ug for 15 min and hydration (hydration) reactions. The caiB gene-en- washed twice with 67 mM phosphate bu¡er (pH 7.5). coding L(3)-carnitine dehydratase was isolated by Cells were disrupted by grinding with Alcoa and oligonucleotide screening from a genomic library of protein was extracted with 10 mM phosphate bu¡er E. coli [21]. CaiB belongs to an operon, which con- (pH 7.5). Cell-free extracts were obtained by cen- sists of six open reading frames (caiTABCDE) [22]. trifugation at 15 000Ug for 45 min. Current studies have shown that caiA encodes cro- Cultivation under anaerobic conditions was car- tonobetaine reductase, which converts, together with ried out at 37³C in 1000-ml £asks ¢lled to the neck CaiB and the unknown cofactor, crotonobetaine into with medium as described above and stoppered air- Q-butyrobetaine [23]. Crotonobetaine reductase has tight. been puri¢ed and characterised [23]. The cai operon from E. coli O44K74 was induced by the global reg- 2.3. Enzyme assays ulatory proteins CRP and FNR and repressed by the histone-like protein H-NS [22,24]. L(3)-carnitine dehydratase assay was carried out The aim of our studies was to investigate the oc- according to Jung et al. [15]. The conversion of currence of carnitine metabolising enzymes by Enter- D(+)-carnitine into L(3)-carnitine was determined obacteriaceae under aerobic conditions. as described by Jung and Kleber [17]. Crotonobe- taine reductase activity was determined according to Dickie and Weiner [25] with crotonobetaine as 2. Materials and methods substrate [16]. Protein concentrations were determined according 2.1. Microorganisms to Bradford [26] using bovine serum albumin as standard. The speci¢c activity was de¢ned as Wmol E. coli O44K74, E. coli ATCC 25922, Proteus mi- substrate conversion per min mg protein. rabilis, Proteus vulgaris, Citrobacter freundii, Kleb- siella pneumoniae, Klebsiella oxytoca, Hafnia alvei, 2.4. Preparation of cofactor Morganella morganii, Providencia rettgeri, Serratia marcescens, Enterobacter agglomerans and Entero- E. coli O44K74 was grown at 37³C in complex bacter cloacae were used in the experiments [13]. medium supplemented with 0.13% L-carnitine, 0.2% All strains were obtained from `Institut fuër Medizi- fumarate and 1% glycerol under anaerobic condi- nische Mikrobiologie und Infektionsepidemiologie', tions. Cultivation was carried out in 1000 ml Erlen- Universitaët Leipzig. meyer £asks ¢lled to neck with medium and stop- FEMSLE 8746 29-4-99 T. ElMner et al. / FEMS Microbiology Letters 174 (1999) 295^301 297 pered air-tight. Cells were harvested at the end of the 2.8. SDS-PAGE and Western blotting exponential growth phase by centrifugation (5000Ug; 15 min), suspended in aqua dest. and dis- Antigen samples were separated by 12% SDS- rupted by two passages through a French pressure PAGE [28] and transferred onto 0.2 Wm nitrocellu- cell (SLM Instruments, Urbana, USA) operating at lose membranes (Serva, Heidelberg, Germany) [29]. 20 000 psi. Unbroken cells and debris were removed Following transfer, membranes were stained for pro- by centrifugation at 15 000Ug for 45 min at 4³C. tein with Ponceau S (0.2 mg ml31 in 2% acetic acid). Proteins were separated from cofactor by ultra¢ltra- After destaining, blots were blocked overnight at 4³C tion using an Amicon YM 01 membrane at 4³C. in PBS/0.2% Tween-20. Hybridoma supernatants (di- Afterwards cofactor was concentrated by lyophilisa- luted 1:3 in PBS/0.1% Tween-20) were incubated tion (Christ, Germany). Cofactor was suspended in with the membrane for 1 h at room temperature. aqua dest. and stored at 320³C. The negative mab 9D5 was employed for speci¢city Downloaded from https://academic.oup.com/femsle/article/174/2/295/502865 by guest on 29 September 2021 control. Immunoreactive bands were detected by per- 2.5. Determination of trimethylammonium compounds oxidase-conjugated goat anti-mouse IgG (Dianova) diluted 1:500 in PBS/0.1% Tween-20 and visualised Carnitine and the other quaternary ammonium ¢nally via 3,3P-diaminobenzidine tetrahydrochloride compounds were examined by thin-layer chromatog- as chromogen. raphy. Adsorbents, solute systems and Rf values have already been described [11]. 2.9. Chemicals 2.6. Puri¢cation of CaiA and CaiB L(3)-carnitine, D(+)-carnitine and crotonobetaine were generous gifts from Sigma Tau, Rome, Italy. Q- CaiA has been puri¢ed to electrophoretic homo- Butyrobetaine was a gift from Lonza AG, Basel, geneity from E. coli BL21(DE3) containing the Switzerland. Carnitine acetyltransferase was pur- plasmid pT7-7CaiA [23]. CaiB has been puri¢ed to chased from Boehringer, Mannheim, Germany. All electrophoretic homogeneity from a cell-free extract other chemicals were of analytical grade. of E. coli O44K74 according to Jung et al. [15]. 2.7. Preparation of monoclonal antibodies 3. Results and discussion Monoclonal antibodies against CaiA and CaiB 3.1. Metabolism of carnitine under aerobic conditions were obtained according to Preusser et al. [23]. Fe- male mice were immunised with puri¢ed CaiB and The ability of di¡erent Enterobacteriaceae to me- CaiA, which was covalently linked with biotin.
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