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Metabolism of L(−)-carnitine by Enterobacteriaceae under aerobic conditions
FEMS Microbiology Letters 174 (1999) 295^301
Metabolism of L(3)-carnitine by Enterobacteriaceae under aerobic conditions Thomas ElMner a , Andrea PreuMer a , Ulrich Wagner b , Hans-Peter Kleber a; * b
a Institut fuër Biochemie, Universitaët Leipzig, Talstr. 33, D-04103 Leipzig, Germany Institut fuër Zoologie, Fakultaët fuër 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 chromatography. 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 L(3)-carnitine (R(3)-3-hydroxy-4-trimethylaminobutyrate) is a ubiquitously occurring compound in nature. In eukaryotes, L(3)-carnitine is essential for the transport of long-chain fatty acids through the inner mitochondrial membrane [1,2]. In bacteria, the physiological function of L(3)-carnitine is unknown. In addition to glycine betaine, one of the most widely distributed osmoprotectants, L(3)carnitine was shown to serve as osmoprotectant in Escherichia coli [3] and other microorganisms [4^8]. * Corresponding author. Tel.: +49 (341) 97 36992; Fax: +49 (341) 97 36998; E-mail: [email protected]
Bacteria are able to metabolise L(3)-carnitine in di¡erent ways [9] using this quaternary ammonium compound as sole source of carbon and nitrogen (e.g. Pseudomonas sp.; [10]) or only as sole source of carbon (e.g. Acinetobacter sp.; [11,12]) under aerobic conditions. Enterobacteriaceae are able to convert L(3)-carnitine via crotonobetaine into Q-butyrobetaine in the presence of carbon and nitrogen sources under anaerobic conditions, but they do not assimilate the carbon skeleton and nitrogen [13,14]. Two enzymes, L(3)-carnitine dehydratase (EC 4.2.1.89) and crotonobetaine reductase, were found in E. coli to catalyse this reaction sequence [15,16]. A carnitine racemase activity able to convert D(+)-carnitine into L(3)-carnitine was subsequently also 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: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 5 1 - 2
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have shown that these enzymes are inducible in the presence of L(3)-carnitine or crotonobetaine under anaerobic conditions [18]. Although a role as external electron acceptor of anaerobic respiration like nitrate or fumarate [19] is postulated for crotonobetaine [14], the precise function of this two-step pathway is still unknown. The stimulation of anaerobic growth of Enterobacteriaceae by crotonobetaine supports this hypothesis [20]. L(3)-carnitine dehydratase has been puri¢ed and characterised [15]. A still unknown cofactor essential for enzyme activity was separated during puri¢cation procedure. The addition of this low molecular mass e¡ector ( 6 1000 Da) caused reactivation of the apoenzyme. It is not possible to replace the e¡ector with known coenzymes or cofactors involved in dehydration (hydration) reactions. The caiB gene-encoding L(3)-carnitine dehydratase was isolated by oligonucleotide screening from a genomic library of E. coli [21]. CaiB belongs to an operon, which consists of six open reading frames (caiTABCDE) [22]. Current studies have shown that caiA encodes crotonobetaine reductase, which converts, together with CaiB and the unknown cofactor, crotonobetaine into Q-butyrobetaine [23]. Crotonobetaine reductase has been puri¢ed and characterised [23]. The cai operon from E. coli O44K74 was induced by the global regulatory proteins CRP and FNR and repressed by the histone-like protein H-NS [22,24]. The aim of our studies was to investigate the occurrence of carnitine metabolising enzymes by Enterobacteriaceae under aerobic conditions.
2. Materials and methods 2.1. Microorganisms E. coli O44K74, E. coli ATCC 25922, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Hafnia alvei, Morganella morganii, Providencia rettgeri, Serratia marcescens, Enterobacter agglomerans and Enterobacter cloacae were used in the experiments [13]. All strains were obtained from `Institut fuër Medizinische Mikrobiologie und Infektionsepidemiologie', Universitaët Leipzig.
2.2. Cultivation of microorganisms and cell disruption The strains were inoculated from agar slopes into complex media and cultivated as preculture under aerobic conditions at 30³C up to the middle of the exponential growth phase. The complex media contained 17 g pancreatic peptone, 3 g yeast extract and 5 g NaCl per litre of deionised water. Cultivation was carried out in 1000 ml Erlenmeyer £asks containing 250 ml complex medium supplemented either with 0.5% carnitine, 0.5% crotonobetaine or 0.5% Qbutyrobetaine on a rotary shaker (175 rpm) at 30³C. Growth of cells was followed by measuring the apparent absorbance of the culture at 600 nm. Cells were harvested at the end of the exponential growth phase by centrifugation at 5000Ug for 15 min and washed twice with 67 mM phosphate bu¡er (pH 7.5). Cells were disrupted by grinding with Alcoa and protein was extracted with 10 mM phosphate bu¡er (pH 7.5). Cell-free extracts were obtained by centrifugation at 15 000Ug for 45 min. Cultivation under anaerobic conditions was carried out at 37³C in 1000-ml £asks ¢lled to the neck with medium as described above and stoppered airtight. 2.3. Enzyme assays L(3)-carnitine dehydratase assay was carried out according to Jung et al. [15]. The conversion of D(+)-carnitine into L(3)-carnitine was determined as described by Jung and Kleber [17]. Crotonobetaine reductase activity was determined according to Dickie and Weiner [25] with crotonobetaine as substrate [16]. Protein concentrations were determined according to Bradford [26] using bovine serum albumin as standard. The speci¢c activity was de¢ned as Wmol substrate conversion per min mg protein. 2.4. Preparation of cofactor E. coli O44K74 was grown at 37³C in complex medium supplemented with 0.13% L-carnitine, 0.2% fumarate and 1% glycerol under anaerobic conditions. Cultivation was carried out in 1000 ml Erlenmeyer £asks ¢lled to neck with medium and stop-
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pered air-tight. Cells were harvested at the end of the exponential growth phase by centrifugation (5000Ug; 15 min), suspended in aqua dest. and disrupted by two passages through a French pressure cell (SLM Instruments, Urbana, USA) operating at 20 000 psi. Unbroken cells and debris were removed by centrifugation at 15 000Ug for 45 min at 4³C. Proteins were separated from cofactor by ultra¢ltration using an Amicon YM 01 membrane at 4³C. Afterwards cofactor was concentrated by lyophilisation (Christ, Germany). Cofactor was suspended in aqua dest. and stored at 320³C. 2.5. Determination of trimethylammonium compounds Carnitine and the other quaternary ammonium compounds were examined by thin-layer chromatography. Adsorbents, solute systems and Rf values have already been described [11]. 2.6. Puri¢cation of CaiA and CaiB CaiA has been puri¢ed to electrophoretic homogeneity from E. coli BL21(DE3) containing the plasmid pT7-7CaiA [23]. CaiB has been puri¢ed to electrophoretic homogeneity from a cell-free extract of E. coli O44K74 according to Jung et al. [15].
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2.8. SDS-PAGE and Western blotting Antigen samples were separated by 12% SDSPAGE [28] and transferred onto 0.2 Wm nitrocellulose membranes (Serva, Heidelberg, Germany) [29]. Following transfer, membranes were stained for protein with Ponceau S (0.2 mg ml31 in 2% acetic acid). After destaining, blots were blocked overnight at 4³C in PBS/0.2% Tween-20. Hybridoma supernatants (diluted 1:3 in PBS/0.1% Tween-20) were incubated with the membrane for 1 h at room temperature. The negative mab 9D5 was employed for speci¢city control. Immunoreactive bands were detected by peroxidase-conjugated goat anti-mouse IgG (Dianova) diluted 1:500 in PBS/0.1% Tween-20 and visualised ¢nally via 3,3P-diaminobenzidine tetrahydrochloride as chromogen. 2.9. Chemicals L(3)-carnitine, D(+)-carnitine and crotonobetaine were generous gifts from Sigma Tau, Rome, Italy. QButyrobetaine was a gift from Lonza AG, Basel, Switzerland. Carnitine acetyltransferase was purchased from Boehringer, Mannheim, Germany. All other chemicals were of analytical grade.
2.7. Preparation of monoclonal antibodies
3. Results and discussion
Monoclonal antibodies against CaiA and CaiB were obtained according to Preusser et al. [23]. Female mice were immunised with puri¢ed CaiB and CaiA, which was covalently linked with biotin. Generation of hybridomas and immunoglobulin isotyping were performed as described previously [27]. Hybridoma supernatants were assayed for speci¢c monoclonal antibodies (mabs) using enzyme-linked immunosorbent assays (ELISA) [23]. Each hybridoma was cloned twice by limiting dilution prior to characterisation of secreted mabs. Anti-CaiA-mab and anti-CaiB-mab were selected for immunological enzyme analyses after cloning and stabilisation of related hybridomas. The speci¢city of the isolated mabs to the antigens was also tested by immunoblotting experiments [23].
3.1. Metabolism of carnitine under aerobic conditions The ability of di¡erent Enterobacteriaceae to metabolise L(3)-carnitine under aerobic conditions is summarised in Table 1. Under aerobic conditions P. vulgaris and P. mirabilis are able to convert L(3)-carnitine via crotonobetaine into Q-butyrobetaine as under anaerobic conditions [13]. In contrast to E. coli ATCC 25922, E. coli O44K74 showed no metabolism of L(3)-carnitine. E. coli O44K74 possesses an inducible, active and carrier-mediated uptake system for trimethylammonium compounds, which is repressed just as the expression of L(3)carnitine dehydratase by oxygen [30]. But our studies have shown that carnitine metabolism is also possible in genera Escherichia and Proteus under aerobiosis. This implies that the carrier-mediated carnitine uptake must be active under aerobiosis. C. freundii
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Table 1 Metabolism of L(3)-carnitine by di¡erent Enterobacteriaceae under aerobic conditions Strain
Degradation of L(3)-carnitine
E. E. P. P. C. P.
3 + + + + +
coli O44K74 coli ATCC 25922 mirabilis vulgaris freundii rettgeri
Formation of metabolites
and P. rettgeri convert L(3)-carnitine into Q-butyrobetaine under anaerobic conditions in presence of carbon and nitrogen sources [13]. In contrast to anaerobic conditions, formation of crotonobetaine and Q-butyrobetaine could not be observed under aerobiosis. Both strains degrade L(3)-carnitine completely. Up to now the degradation pathway could not be identi¢ed, because neither L(3)-carnitine dehydrogenase nor L(3)-carnitine dehydratase were detectable. Other Enterobacteriaceae (E. agglomerans, E. cloacae, K. pneumoniae, K. oxytoca, H. alvei, M. morganii and S. marcescens) were also investigated, but they showed no metabolism of L(3)-carnitine similarly as described under anaerobic conditions [13]. 3.2. Occurrence of carnitine metabolising enzymes In E. coli O44K74 it was shown that L(3)-carnitine dehydratase and crotonobetaine reductase are inducible enzymes detectable in cells grown anae-
Crotonobetaine
Q-Butyrobetaine
3 + + + 3 3
3 + + + 3 3
robically in the presence of crotonobetaine or L(3)-carnitine [18]. For the ¢rst time we could detect these carnitine metabolising enzymes in cell-free extracts of di¡erent Enterobacteriaceae grown under aerobic conditions in presence of L(3)-carnitine (Table 2). L(3)-carnitine dehydratase was detectable in cellfree extracts of P. mirabilis, P. vulgaris and E. coli. In contrast to E. coli ATCC 25922, E. coli O44K74 shows only very small L(3)-carnitine dehydratase activity without cofactor supplementation. Isolated cofactor from anaerobic grown E. coli O44K74 increased not only the L(3)-carnitine dehydratase activity in cell-free extracts from E. coli O44K74 but also in E. coli ATCC 25922 and P. mirabilis. This indicates the existence of a very similar or same cofactor by other Enterobacteriaceae under aerobiosis. Only the crude extract of P. vulgaris showed cofactor saturation. Absence of metabolism of L(3)-carnitine in E. coli O44K74 (Table 1) is obviously caused by the lacking cofactor. In C. freundii the expression of
Table 2 Speci¢c activities of L(3)-carnitine dehydratase, crotonobetaine reductase and the D(+)-carnitine racemasing system in cell-free extracts from di¡erent Enterobacteriaceae grown aerobically in presence of L(3)-carnitine Strain
Speci¢c activity (mU mg31 ) L(3)-carnitine dehydratase
E. E. P. P. C. P.
coli O44K74 0.6 coli ATCC 25922 61.1 mirabilis 34.4 vulgaris 75.0 freundii ^ rettgeri ^
(25.8)a (138.3) (19.8) (6.1) (207.5) (71.0)
L(3)-carnitine dehydratase +cofactor
Crotonobetaine reductase+cofactor
D(+)-carnitine racemasing system+cofactor
8.2 74.3 51.9 74.0 ^ ^
^ 4.6 1.2 5.1 ^ ^
^ ^ 0.1 3.4 ^ ^
(231.0) (647.8) (2708.7) (901.1) (1215.7) (2171.6)
(27.6) (70.2) (20.7) (1.2) (20.5) (75.1)
(15.9) (13.4) (10.2) (10.5) (51.7) (6.5)
The same amount of cofactor was added to all performed incubation mixtures. a Speci¢c activities of corresponding enzymes in cell-free extracts from di¡erent Enterobacteriaceae grown under anaerobic conditions in presence of L(3)-carnitine.
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Fig. 1. Evidence of CaiB in cell-free extracts of di¡erent Enterobacteriaceae by monoclonal antibodies. Cell-free extracts were separated by SDS-PAGE, blotted onto nitrocellulose membranes and immunostained with antibodies against CaiB from E. coli O44K74 (cf. Section 2.7). Puri¢ed CaiB shows two additional bands at 25 and 20 kDa due to fragments of CaiB (cf. lane 8). BOEHRINGER protein standard as shown were : triosephosphate isomerase, 26 600; aldolase, 39 200; glutamate dehydrogenase, 55 600; fructose-6-phosphate kinase, 85 200; L-galactosidase, 116 400.
L(3)-carnitine dehydratase and crotonobetaine reductase seems to be repressed by oxygen. Cell-free extracts of E. agglomerans, E. cloacae, K. oxytoca, K. pneumoniae, H. alvei, M. morganii and S. marcescens were also investigated, but no L(3)-carnitine dehydratase activity could be detected even with cofactor supplementation. In contrast to the L(3)-carnitine dehydratase, addition of the cofactor was always essential for evidence of D(+)-carnitine racemasing system and the crotonobetaine reductase (Table 2). The reason for the discrepancies are probably very
low concentrations of the cofactor and/or the enzymes or the a¤nity of cofactor to L(3)-carnitine dehydratase is higher than to crotonobetaine reductase and carnitine racemasing system. D(+)-carnitine racemasing system and crotonobetaine reductase were tested additionally in cell-free extracts of E. cloacae and K. pneumoniae, but no signi¢cant activity could be found. Under anaerobic conditions E. coli, P. mirabilis, P. vulgaris and C. freundii convert L(3)-carnitine into Q-butyrobetaine almost quantitatively [13]. These re-
Fig. 2. Evidence of CaiA in cell-free extracts of di¡erent Enterobacteriaceae by monoclonal antibodies. Cell-free extracts were separated by SDS-PAGE, blotted onto nitrocellulose membranes and immunostained with antibody against CaiA from E. coli O44K74 (cf. Section 2.7). Protein standard cf. Fig. 1.
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sults were achieved by thin-layer chromatography and are compatible with detected enzyme activities (cf. Table 2). In comparison, the enzyme activities under anaerobiosis are up to 50-fold higher than under aerobic conditions. Obviously, the transcription of the cai operon is reduced under aerobiosis. This supports the hypothesis of crotonobetaine as external electron acceptor of anaerobic respiration [14]. Besides L(3)-carnitine (cf. Table 2) crotonobetaine also acts as inducer under aerobic conditions. In the presence of crotonobetaine the following L(3)-carnitine dehydratase activities (with addition of cofactor) were obtained: E. coli O44K74 (23.9 mU mg31 ), E. coli ATCC 25922 (16.5 mU mg31 ), P. mirabilis (60.1 mU mg31 ) and P. vulgaris (64.2 mU mg31 ). Q-Butyrobetaine does not show any e¡ect on expression of carnitine metabolising enzymes under aerobiosis similar to anaerobic conditions [18].
against CaiB was observed [23]. Screening of cellfree extracts by means of a monoclonal antibody against CaiA is shown in Fig. 2. In E. coli O44K74 caiA is expressed only under anaerobic conditions. However, the expression of caiA is realised under aerobic conditions by E. coli ATCC 25922, P. mirabilis and P. vulgaris. In summary, the expression of caiB under aerobic conditions could be veri¢ed by E. coli O44K74, E. coli ATCC 25922 and P. vulgaris using antibodies and L(3)-carnitine dehydratase activity by measuring in cell-free extracts. Although L(3)-carnitine dehydratase activity was detected enzymatically in P. mirabilis, no cross reactivity with anti-CaiB-mab was observed. CaiA is expressed under aerobiosis only in E. coli ATCC 25922, P. mirabilis and P. vulgaris. In further investigations, we intend to carry out regulation studies in order to understand the aerobic/anaerobic shift.
3.3. Detection of carnitine metabolising enzymes with monoclonal antibodies
Acknowledgments
It is supposed that structural analogies exist between CaiB and CaiA of di¡erent Enterobacteriaceae. That's why di¡erent cell-free extracts were screened with monoclonal antibodies against CaiB obtained from E. coli O44K74. CaiB from E. coli O44K74 has a relative molecular mass of 85 kDa and is a dimer consisting of two identical subunits (45 kDa) [15]. Monoclonal antibody raised against CaiB from E. coli O44K74 showed only reactivity with the puri¢ed subunit of CaiB while no reaction against CaiA was observed. Anti-CaiB-mab recognises proteins from E. coli ATCC 25922 and P. vulgaris (Fig. 1). Cell-free extract from C. freundii also showed a low cross reactivity with anti-CaiB-mab. Obviously the amount of L(3)-carnitine dehydratase was not su¤cient for an enzymatic determination. These results suggest that the subunits of CaiB in E. coli ATCC 25922, P. vulgaris and C. freundii have nearly the same molecular mass as in E. coli O44K74. CaiA from E. coli O44K74 has a native molar mass of 164.4 kDa and is composed of four identical subunits with relative molar masses of 41.5 kDa [23]. By Western blotting anti-CaiA-mab was found to recognise the subunit of CaiA while no reaction
This work was supported by the Deutsche Forschungsgemeinschaft grant No. K/A 911/1-2. The authors thank Stan Theophilou for help with the manuscript.
References [1] Bremer, J. (1963) Carnitine in intermediary metabolism - the biosynthesis of palmitoylcarnitine by cell subfractions. J. Biol. Chem. 238, 2774^2779. [2] Fritz, I.B. and Yue, K.T.N. (1963) Long chain acylcarnitine acyltransferase and the role of acylcarnitine derivatives in the catalytic increase of fatty acid oxidation induced by carnitine. J. Lipid Res. 4, 279^288. [3] Jung, H., Jung, K. and Kleber, H.-P. (1990) L-Carnitine metabolism and osmotic stress response in Escherichia coli. J. Basic Microbiol. 30, 409^413. [4] Jebbar, M., Champion, C., Blanco, C. and Bonnassie, S. (1998) Carnitine acts as a compatible solute in Brevebacterium linens. Res. Microbiol. 149, 211^219. [5] Kappes, R.M. and Bremer, E. (1998) Response of Bacillus subtilis to high osmolarity: uptake of carnitine, crotonobetaine and Q-butyrobetaine via ABC-transport system Opu C. Microbiology 144, 83^90. [6] Kets, E.P.W., Galinski, E.A. and De Bont, J.A.M. (1994) Carnitine : a novel compatible solute in Lactobacillus plantarum. Arch. Microbiol. 162, 243^248.
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T. ElMner et al. / FEMS Microbiology Letters 174 (1999) 295^301 [7] Lucchesi, G.I., Lisa, T.A., Casale, C.H. and Domenech, C.E. (1995) Carnitine resembles choline in the induction of cholinesterase, acid phosphatase, and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosa. Curr. Microbiol. 30, 55^60. [8] Smith, L.T. (1996) Role of osmolytes in adaptation of osmotically stressed and chill-stressed Listeria monocytogenes grown in liquid media and on processed meat surfaces. Appl. Environ. Microbiol. 62, 3088^3093. [9] Kleber, H.-P. (1997) Bacterial carnitine metabolism. FEMS Microbiol. Lett. 147, 1^9. [10] Aurich, H., Kleber, H.-P. and Schoëpp, W. (1967) An inducible carnitine dehydrogenase from Pseudomonas aeruginosa. Biochim. Biophys. Acta 139, 505^507. [11] Kleber, H.-P., Seim, H., Aurich, H. and Strack, E. (1977) Verwertung von Trimethylam-moniumverbindungen durch Acinetobacter calcoaceticus. Arch. Microbiol. 112, 201^206. [12] Seim, H., Loëster, H., Claus, R., Kleber, H.-P. and Strack, E. (1982) Splitting of the C-N-bond in carnitine by an enzyme (trimethylamine forming) from membranes of Acinetobacter calcoaceticus. FEMS Microbiol. Lett. 15, 165^167. [13] Seim, H., Ezold, R., Kleber, H.-P. and Strack, E. (1980) Sto¡wechsel des L-Carnitins bei Enterobakterien. Z. Allg. Mikrobiol. 20, 591^594. [14] Seim, H., Loëster, H., Claus, R., Kleber, H.-P. and Strack, E. (1982) Stimulation of the anaerobic growth of Salmonella typhimorium by reduction of L-carnitine, carnitine derivatives and structure-related trimethylammonium compounds. Arch. Microbiol. 132, 91^95. [15] Jung, H., Jung, K. and Kleber, H.-P. (1989) Puri¢cation and properties of carnitine dehydratase from Escherichia coli - a new enzyme of carnitine metabolization. Biochim. Biophys. Acta 1003, 270^276. [16] Roth, S., Jung, K., Jung, H., Hommel, R.K. and Kleber, H.-P. (1994) Crotonobetaine reductase from Escherichia coli - a new inducible enzyme of anaerobic metabolization of L-(3)-carnitine. Antonie van Leeuwenhoek 65, 63^69. [17] Jung, H. and Kleber, H.-P. (1991) Metabolism of D-(+)-carnitine by Escherichia coli. Appl. Microbiol. Biotechnol. 35, 393^395. [18] Jung, K., Jung, H. and Kleber, H.-P. (1987) Regulation of Lcarnitine metabolism in Escherichia coli. J. Basic Microbiol. 27, 131^137.
301
[19] Haddock, B.A. and Jones, C.W. (1977) Bacterial respiration. Bacteriol. Rev. 41, 47^99. [20] Seim, H., Loëster, H., Claus, R., Kleber, H.-P. and Strack, E. (1982) Stimulation of the anaerobic growth of Salmonella typhimurium by reduction of L-carnitine, carnitine derivatives and structure-related trimethylammonium compounds. Arch. Microbiol. 132, 91^95. [21] Eichler, K., Schunck, W.-H., Kleber, H.-P. and MandrandBerthelot, M.-A. (1994) Cloning, nucleotide sequence and expression of the Escherichia coli gene encoding carnitine dehydratase. J. Bacteriol. 176, 2970^2975. [22] Eichler, K., Bourgis, F., Buchet, A., Kleber, H.-P. and Mandrand-Berthelot, M.-A. (1994) Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coli. Mol. Microbiol. 13, 775^786. [23] Preusser, A., Wagner, U., Elssner, T. and Kleber, H.-P. (1999) Crotonobetaine reductase from Escherichia coli consists of two proteins. Biochim. Biophys. Acta, in press. [24] Eichler, K., Buchet, A., Lemke, R., Kleber, H.-P. and Mandrand-Berthelot, M.-A. (1996) Identi¢cation and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli. J. Bacteriol. 178, 1248^1257. [25] Dickie, P. and Weiner, J.H. (1979) Puri¢cation and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli. Can. J. Biochem. 57, 813^ 821. [26] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [27] Wagner, U., Lachmann, I., Haëdge, D. and Droëssler, K. (1997) Development and characterization of monoclonal antibodies speci¢c for two di¡erent exoproteases of aeromonas salmonicida. J. Fish Dis. 20, 223^235. [28] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [29] Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350^4354. [30] Jung, H., Jung, K. and Kleber, H.-P. (1990) L-carnitine uptake by Escherichia coli. J. Basic Microbiol. 30, 507^514.
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Metabolism of L(3)-carnitine by Enterobacteriaceae under aerobic conditions Thomas ElMner a , Andrea PreuMer a , Ulrich Wagner b , Hans-Peter Kleber a; * b
a Institut fuër Biochemie, Universitaët Leipzig, Talstr. 33, D-04103 Leipzig, Germany Institut fuër Zoologie, Fakultaët fuër 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 chromatography. 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 L(3)-carnitine (R(3)-3-hydroxy-4-trimethylaminobutyrate) is a ubiquitously occurring compound in nature. In eukaryotes, L(3)-carnitine is essential for the transport of long-chain fatty acids through the inner mitochondrial membrane [1,2]. In bacteria, the physiological function of L(3)-carnitine is unknown. In addition to glycine betaine, one of the most widely distributed osmoprotectants, L(3)carnitine was shown to serve as osmoprotectant in Escherichia coli [3] and other microorganisms [4^8]. * Corresponding author. Tel.: +49 (341) 97 36992; Fax: +49 (341) 97 36998; E-mail: [email protected]
Bacteria are able to metabolise L(3)-carnitine in di¡erent ways [9] using this quaternary ammonium compound as sole source of carbon and nitrogen (e.g. Pseudomonas sp.; [10]) or only as sole source of carbon (e.g. Acinetobacter sp.; [11,12]) under aerobic conditions. Enterobacteriaceae are able to convert L(3)-carnitine via crotonobetaine into Q-butyrobetaine in the presence of carbon and nitrogen sources under anaerobic conditions, but they do not assimilate the carbon skeleton and nitrogen [13,14]. Two enzymes, L(3)-carnitine dehydratase (EC 4.2.1.89) and crotonobetaine reductase, were found in E. coli to catalyse this reaction sequence [15,16]. A carnitine racemase activity able to convert D(+)-carnitine into L(3)-carnitine was subsequently also postulated [17]. Studies using whole cells of E. coli
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have shown that these enzymes are inducible in the presence of L(3)-carnitine or crotonobetaine under anaerobic conditions [18]. Although a role as external electron acceptor of anaerobic respiration like nitrate or fumarate [19] is postulated for crotonobetaine [14], the precise function of this two-step pathway is still unknown. The stimulation of anaerobic growth of Enterobacteriaceae by crotonobetaine supports this hypothesis [20]. L(3)-carnitine dehydratase has been puri¢ed and characterised [15]. A still unknown cofactor essential for enzyme activity was separated during puri¢cation procedure. The addition of this low molecular mass e¡ector ( 6 1000 Da) caused reactivation of the apoenzyme. It is not possible to replace the e¡ector with known coenzymes or cofactors involved in dehydration (hydration) reactions. The caiB gene-encoding L(3)-carnitine dehydratase was isolated by oligonucleotide screening from a genomic library of E. coli [21]. CaiB belongs to an operon, which consists of six open reading frames (caiTABCDE) [22]. Current studies have shown that caiA encodes crotonobetaine reductase, which converts, together with CaiB and the unknown cofactor, crotonobetaine into Q-butyrobetaine [23]. Crotonobetaine reductase has been puri¢ed and characterised [23]. The cai operon from E. coli O44K74 was induced by the global regulatory proteins CRP and FNR and repressed by the histone-like protein H-NS [22,24]. The aim of our studies was to investigate the occurrence of carnitine metabolising enzymes by Enterobacteriaceae under aerobic conditions.
2. Materials and methods 2.1. Microorganisms E. coli O44K74, E. coli ATCC 25922, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Hafnia alvei, Morganella morganii, Providencia rettgeri, Serratia marcescens, Enterobacter agglomerans and Enterobacter cloacae were used in the experiments [13]. All strains were obtained from `Institut fuër Medizinische Mikrobiologie und Infektionsepidemiologie', Universitaët Leipzig.
2.2. Cultivation of microorganisms and cell disruption The strains were inoculated from agar slopes into complex media and cultivated as preculture under aerobic conditions at 30³C up to the middle of the exponential growth phase. The complex media contained 17 g pancreatic peptone, 3 g yeast extract and 5 g NaCl per litre of deionised water. Cultivation was carried out in 1000 ml Erlenmeyer £asks containing 250 ml complex medium supplemented either with 0.5% carnitine, 0.5% crotonobetaine or 0.5% Qbutyrobetaine on a rotary shaker (175 rpm) at 30³C. Growth of cells was followed by measuring the apparent absorbance of the culture at 600 nm. Cells were harvested at the end of the exponential growth phase by centrifugation at 5000Ug for 15 min and washed twice with 67 mM phosphate bu¡er (pH 7.5). Cells were disrupted by grinding with Alcoa and protein was extracted with 10 mM phosphate bu¡er (pH 7.5). Cell-free extracts were obtained by centrifugation at 15 000Ug for 45 min. Cultivation under anaerobic conditions was carried out at 37³C in 1000-ml £asks ¢lled to the neck with medium as described above and stoppered airtight. 2.3. Enzyme assays L(3)-carnitine dehydratase assay was carried out according to Jung et al. [15]. The conversion of D(+)-carnitine into L(3)-carnitine was determined as described by Jung and Kleber [17]. Crotonobetaine reductase activity was determined according to Dickie and Weiner [25] with crotonobetaine as substrate [16]. Protein concentrations were determined according to Bradford [26] using bovine serum albumin as standard. The speci¢c activity was de¢ned as Wmol substrate conversion per min mg protein. 2.4. Preparation of cofactor E. coli O44K74 was grown at 37³C in complex medium supplemented with 0.13% L-carnitine, 0.2% fumarate and 1% glycerol under anaerobic conditions. Cultivation was carried out in 1000 ml Erlenmeyer £asks ¢lled to neck with medium and stop-
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pered air-tight. Cells were harvested at the end of the exponential growth phase by centrifugation (5000Ug; 15 min), suspended in aqua dest. and disrupted by two passages through a French pressure cell (SLM Instruments, Urbana, USA) operating at 20 000 psi. Unbroken cells and debris were removed by centrifugation at 15 000Ug for 45 min at 4³C. Proteins were separated from cofactor by ultra¢ltration using an Amicon YM 01 membrane at 4³C. Afterwards cofactor was concentrated by lyophilisation (Christ, Germany). Cofactor was suspended in aqua dest. and stored at 320³C. 2.5. Determination of trimethylammonium compounds Carnitine and the other quaternary ammonium compounds were examined by thin-layer chromatography. Adsorbents, solute systems and Rf values have already been described [11]. 2.6. Puri¢cation of CaiA and CaiB CaiA has been puri¢ed to electrophoretic homogeneity from E. coli BL21(DE3) containing the plasmid pT7-7CaiA [23]. CaiB has been puri¢ed to electrophoretic homogeneity from a cell-free extract of E. coli O44K74 according to Jung et al. [15].
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2.8. SDS-PAGE and Western blotting Antigen samples were separated by 12% SDSPAGE [28] and transferred onto 0.2 Wm nitrocellulose membranes (Serva, Heidelberg, Germany) [29]. Following transfer, membranes were stained for protein with Ponceau S (0.2 mg ml31 in 2% acetic acid). After destaining, blots were blocked overnight at 4³C in PBS/0.2% Tween-20. Hybridoma supernatants (diluted 1:3 in PBS/0.1% Tween-20) were incubated with the membrane for 1 h at room temperature. The negative mab 9D5 was employed for speci¢city control. Immunoreactive bands were detected by peroxidase-conjugated goat anti-mouse IgG (Dianova) diluted 1:500 in PBS/0.1% Tween-20 and visualised ¢nally via 3,3P-diaminobenzidine tetrahydrochloride as chromogen. 2.9. Chemicals L(3)-carnitine, D(+)-carnitine and crotonobetaine were generous gifts from Sigma Tau, Rome, Italy. QButyrobetaine was a gift from Lonza AG, Basel, Switzerland. Carnitine acetyltransferase was purchased from Boehringer, Mannheim, Germany. All other chemicals were of analytical grade.
2.7. Preparation of monoclonal antibodies
3. Results and discussion
Monoclonal antibodies against CaiA and CaiB were obtained according to Preusser et al. [23]. Female mice were immunised with puri¢ed CaiB and CaiA, which was covalently linked with biotin. Generation of hybridomas and immunoglobulin isotyping were performed as described previously [27]. Hybridoma supernatants were assayed for speci¢c monoclonal antibodies (mabs) using enzyme-linked immunosorbent assays (ELISA) [23]. Each hybridoma was cloned twice by limiting dilution prior to characterisation of secreted mabs. Anti-CaiA-mab and anti-CaiB-mab were selected for immunological enzyme analyses after cloning and stabilisation of related hybridomas. The speci¢city of the isolated mabs to the antigens was also tested by immunoblotting experiments [23].
3.1. Metabolism of carnitine under aerobic conditions The ability of di¡erent Enterobacteriaceae to metabolise L(3)-carnitine under aerobic conditions is summarised in Table 1. Under aerobic conditions P. vulgaris and P. mirabilis are able to convert L(3)-carnitine via crotonobetaine into Q-butyrobetaine as under anaerobic conditions [13]. In contrast to E. coli ATCC 25922, E. coli O44K74 showed no metabolism of L(3)-carnitine. E. coli O44K74 possesses an inducible, active and carrier-mediated uptake system for trimethylammonium compounds, which is repressed just as the expression of L(3)carnitine dehydratase by oxygen [30]. But our studies have shown that carnitine metabolism is also possible in genera Escherichia and Proteus under aerobiosis. This implies that the carrier-mediated carnitine uptake must be active under aerobiosis. C. freundii
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Table 1 Metabolism of L(3)-carnitine by di¡erent Enterobacteriaceae under aerobic conditions Strain
Degradation of L(3)-carnitine
E. E. P. P. C. P.
3 + + + + +
coli O44K74 coli ATCC 25922 mirabilis vulgaris freundii rettgeri
Formation of metabolites
and P. rettgeri convert L(3)-carnitine into Q-butyrobetaine under anaerobic conditions in presence of carbon and nitrogen sources [13]. In contrast to anaerobic conditions, formation of crotonobetaine and Q-butyrobetaine could not be observed under aerobiosis. Both strains degrade L(3)-carnitine completely. Up to now the degradation pathway could not be identi¢ed, because neither L(3)-carnitine dehydrogenase nor L(3)-carnitine dehydratase were detectable. Other Enterobacteriaceae (E. agglomerans, E. cloacae, K. pneumoniae, K. oxytoca, H. alvei, M. morganii and S. marcescens) were also investigated, but they showed no metabolism of L(3)-carnitine similarly as described under anaerobic conditions [13]. 3.2. Occurrence of carnitine metabolising enzymes In E. coli O44K74 it was shown that L(3)-carnitine dehydratase and crotonobetaine reductase are inducible enzymes detectable in cells grown anae-
Crotonobetaine
Q-Butyrobetaine
3 + + + 3 3
3 + + + 3 3
robically in the presence of crotonobetaine or L(3)-carnitine [18]. For the ¢rst time we could detect these carnitine metabolising enzymes in cell-free extracts of di¡erent Enterobacteriaceae grown under aerobic conditions in presence of L(3)-carnitine (Table 2). L(3)-carnitine dehydratase was detectable in cellfree extracts of P. mirabilis, P. vulgaris and E. coli. In contrast to E. coli ATCC 25922, E. coli O44K74 shows only very small L(3)-carnitine dehydratase activity without cofactor supplementation. Isolated cofactor from anaerobic grown E. coli O44K74 increased not only the L(3)-carnitine dehydratase activity in cell-free extracts from E. coli O44K74 but also in E. coli ATCC 25922 and P. mirabilis. This indicates the existence of a very similar or same cofactor by other Enterobacteriaceae under aerobiosis. Only the crude extract of P. vulgaris showed cofactor saturation. Absence of metabolism of L(3)-carnitine in E. coli O44K74 (Table 1) is obviously caused by the lacking cofactor. In C. freundii the expression of
Table 2 Speci¢c activities of L(3)-carnitine dehydratase, crotonobetaine reductase and the D(+)-carnitine racemasing system in cell-free extracts from di¡erent Enterobacteriaceae grown aerobically in presence of L(3)-carnitine Strain
Speci¢c activity (mU mg31 ) L(3)-carnitine dehydratase
E. E. P. P. C. P.
coli O44K74 0.6 coli ATCC 25922 61.1 mirabilis 34.4 vulgaris 75.0 freundii ^ rettgeri ^
(25.8)a (138.3) (19.8) (6.1) (207.5) (71.0)
L(3)-carnitine dehydratase +cofactor
Crotonobetaine reductase+cofactor
D(+)-carnitine racemasing system+cofactor
8.2 74.3 51.9 74.0 ^ ^
^ 4.6 1.2 5.1 ^ ^
^ ^ 0.1 3.4 ^ ^
(231.0) (647.8) (2708.7) (901.1) (1215.7) (2171.6)
(27.6) (70.2) (20.7) (1.2) (20.5) (75.1)
(15.9) (13.4) (10.2) (10.5) (51.7) (6.5)
The same amount of cofactor was added to all performed incubation mixtures. a Speci¢c activities of corresponding enzymes in cell-free extracts from di¡erent Enterobacteriaceae grown under anaerobic conditions in presence of L(3)-carnitine.
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Fig. 1. Evidence of CaiB in cell-free extracts of di¡erent Enterobacteriaceae by monoclonal antibodies. Cell-free extracts were separated by SDS-PAGE, blotted onto nitrocellulose membranes and immunostained with antibodies against CaiB from E. coli O44K74 (cf. Section 2.7). Puri¢ed CaiB shows two additional bands at 25 and 20 kDa due to fragments of CaiB (cf. lane 8). BOEHRINGER protein standard as shown were : triosephosphate isomerase, 26 600; aldolase, 39 200; glutamate dehydrogenase, 55 600; fructose-6-phosphate kinase, 85 200; L-galactosidase, 116 400.
L(3)-carnitine dehydratase and crotonobetaine reductase seems to be repressed by oxygen. Cell-free extracts of E. agglomerans, E. cloacae, K. oxytoca, K. pneumoniae, H. alvei, M. morganii and S. marcescens were also investigated, but no L(3)-carnitine dehydratase activity could be detected even with cofactor supplementation. In contrast to the L(3)-carnitine dehydratase, addition of the cofactor was always essential for evidence of D(+)-carnitine racemasing system and the crotonobetaine reductase (Table 2). The reason for the discrepancies are probably very
low concentrations of the cofactor and/or the enzymes or the a¤nity of cofactor to L(3)-carnitine dehydratase is higher than to crotonobetaine reductase and carnitine racemasing system. D(+)-carnitine racemasing system and crotonobetaine reductase were tested additionally in cell-free extracts of E. cloacae and K. pneumoniae, but no signi¢cant activity could be found. Under anaerobic conditions E. coli, P. mirabilis, P. vulgaris and C. freundii convert L(3)-carnitine into Q-butyrobetaine almost quantitatively [13]. These re-
Fig. 2. Evidence of CaiA in cell-free extracts of di¡erent Enterobacteriaceae by monoclonal antibodies. Cell-free extracts were separated by SDS-PAGE, blotted onto nitrocellulose membranes and immunostained with antibody against CaiA from E. coli O44K74 (cf. Section 2.7). Protein standard cf. Fig. 1.
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sults were achieved by thin-layer chromatography and are compatible with detected enzyme activities (cf. Table 2). In comparison, the enzyme activities under anaerobiosis are up to 50-fold higher than under aerobic conditions. Obviously, the transcription of the cai operon is reduced under aerobiosis. This supports the hypothesis of crotonobetaine as external electron acceptor of anaerobic respiration [14]. Besides L(3)-carnitine (cf. Table 2) crotonobetaine also acts as inducer under aerobic conditions. In the presence of crotonobetaine the following L(3)-carnitine dehydratase activities (with addition of cofactor) were obtained: E. coli O44K74 (23.9 mU mg31 ), E. coli ATCC 25922 (16.5 mU mg31 ), P. mirabilis (60.1 mU mg31 ) and P. vulgaris (64.2 mU mg31 ). Q-Butyrobetaine does not show any e¡ect on expression of carnitine metabolising enzymes under aerobiosis similar to anaerobic conditions [18].
against CaiB was observed [23]. Screening of cellfree extracts by means of a monoclonal antibody against CaiA is shown in Fig. 2. In E. coli O44K74 caiA is expressed only under anaerobic conditions. However, the expression of caiA is realised under aerobic conditions by E. coli ATCC 25922, P. mirabilis and P. vulgaris. In summary, the expression of caiB under aerobic conditions could be veri¢ed by E. coli O44K74, E. coli ATCC 25922 and P. vulgaris using antibodies and L(3)-carnitine dehydratase activity by measuring in cell-free extracts. Although L(3)-carnitine dehydratase activity was detected enzymatically in P. mirabilis, no cross reactivity with anti-CaiB-mab was observed. CaiA is expressed under aerobiosis only in E. coli ATCC 25922, P. mirabilis and P. vulgaris. In further investigations, we intend to carry out regulation studies in order to understand the aerobic/anaerobic shift.
3.3. Detection of carnitine metabolising enzymes with monoclonal antibodies
Acknowledgments
It is supposed that structural analogies exist between CaiB and CaiA of di¡erent Enterobacteriaceae. That's why di¡erent cell-free extracts were screened with monoclonal antibodies against CaiB obtained from E. coli O44K74. CaiB from E. coli O44K74 has a relative molecular mass of 85 kDa and is a dimer consisting of two identical subunits (45 kDa) [15]. Monoclonal antibody raised against CaiB from E. coli O44K74 showed only reactivity with the puri¢ed subunit of CaiB while no reaction against CaiA was observed. Anti-CaiB-mab recognises proteins from E. coli ATCC 25922 and P. vulgaris (Fig. 1). Cell-free extract from C. freundii also showed a low cross reactivity with anti-CaiB-mab. Obviously the amount of L(3)-carnitine dehydratase was not su¤cient for an enzymatic determination. These results suggest that the subunits of CaiB in E. coli ATCC 25922, P. vulgaris and C. freundii have nearly the same molecular mass as in E. coli O44K74. CaiA from E. coli O44K74 has a native molar mass of 164.4 kDa and is composed of four identical subunits with relative molar masses of 41.5 kDa [23]. By Western blotting anti-CaiA-mab was found to recognise the subunit of CaiA while no reaction
This work was supported by the Deutsche Forschungsgemeinschaft grant No. K/A 911/1-2. The authors thank Stan Theophilou for help with the manuscript.
References [1] Bremer, J. (1963) Carnitine in intermediary metabolism - the biosynthesis of palmitoylcarnitine by cell subfractions. J. Biol. Chem. 238, 2774^2779. [2] Fritz, I.B. and Yue, K.T.N. (1963) Long chain acylcarnitine acyltransferase and the role of acylcarnitine derivatives in the catalytic increase of fatty acid oxidation induced by carnitine. J. Lipid Res. 4, 279^288. [3] Jung, H., Jung, K. and Kleber, H.-P. (1990) L-Carnitine metabolism and osmotic stress response in Escherichia coli. J. Basic Microbiol. 30, 409^413. [4] Jebbar, M., Champion, C., Blanco, C. and Bonnassie, S. (1998) Carnitine acts as a compatible solute in Brevebacterium linens. Res. Microbiol. 149, 211^219. [5] Kappes, R.M. and Bremer, E. (1998) Response of Bacillus subtilis to high osmolarity: uptake of carnitine, crotonobetaine and Q-butyrobetaine via ABC-transport system Opu C. Microbiology 144, 83^90. [6] Kets, E.P.W., Galinski, E.A. and De Bont, J.A.M. (1994) Carnitine : a novel compatible solute in Lactobacillus plantarum. Arch. Microbiol. 162, 243^248.
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T. ElMner et al. / FEMS Microbiology Letters 174 (1999) 295^301 [7] Lucchesi, G.I., Lisa, T.A., Casale, C.H. and Domenech, C.E. (1995) Carnitine resembles choline in the induction of cholinesterase, acid phosphatase, and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosa. Curr. Microbiol. 30, 55^60. [8] Smith, L.T. (1996) Role of osmolytes in adaptation of osmotically stressed and chill-stressed Listeria monocytogenes grown in liquid media and on processed meat surfaces. Appl. Environ. Microbiol. 62, 3088^3093. [9] Kleber, H.-P. (1997) Bacterial carnitine metabolism. FEMS Microbiol. Lett. 147, 1^9. [10] Aurich, H., Kleber, H.-P. and Schoëpp, W. (1967) An inducible carnitine dehydrogenase from Pseudomonas aeruginosa. Biochim. Biophys. Acta 139, 505^507. [11] Kleber, H.-P., Seim, H., Aurich, H. and Strack, E. (1977) Verwertung von Trimethylam-moniumverbindungen durch Acinetobacter calcoaceticus. Arch. Microbiol. 112, 201^206. [12] Seim, H., Loëster, H., Claus, R., Kleber, H.-P. and Strack, E. (1982) Splitting of the C-N-bond in carnitine by an enzyme (trimethylamine forming) from membranes of Acinetobacter calcoaceticus. FEMS Microbiol. Lett. 15, 165^167. [13] Seim, H., Ezold, R., Kleber, H.-P. and Strack, E. (1980) Sto¡wechsel des L-Carnitins bei Enterobakterien. Z. Allg. Mikrobiol. 20, 591^594. [14] Seim, H., Loëster, H., Claus, R., Kleber, H.-P. and Strack, E. (1982) Stimulation of the anaerobic growth of Salmonella typhimorium by reduction of L-carnitine, carnitine derivatives and structure-related trimethylammonium compounds. Arch. Microbiol. 132, 91^95. [15] Jung, H., Jung, K. and Kleber, H.-P. (1989) Puri¢cation and properties of carnitine dehydratase from Escherichia coli - a new enzyme of carnitine metabolization. Biochim. Biophys. Acta 1003, 270^276. [16] Roth, S., Jung, K., Jung, H., Hommel, R.K. and Kleber, H.-P. (1994) Crotonobetaine reductase from Escherichia coli - a new inducible enzyme of anaerobic metabolization of L-(3)-carnitine. Antonie van Leeuwenhoek 65, 63^69. [17] Jung, H. and Kleber, H.-P. (1991) Metabolism of D-(+)-carnitine by Escherichia coli. Appl. Microbiol. Biotechnol. 35, 393^395. [18] Jung, K., Jung, H. and Kleber, H.-P. (1987) Regulation of Lcarnitine metabolism in Escherichia coli. J. Basic Microbiol. 27, 131^137.
301
[19] Haddock, B.A. and Jones, C.W. (1977) Bacterial respiration. Bacteriol. Rev. 41, 47^99. [20] Seim, H., Loëster, H., Claus, R., Kleber, H.-P. and Strack, E. (1982) Stimulation of the anaerobic growth of Salmonella typhimurium by reduction of L-carnitine, carnitine derivatives and structure-related trimethylammonium compounds. Arch. Microbiol. 132, 91^95. [21] Eichler, K., Schunck, W.-H., Kleber, H.-P. and MandrandBerthelot, M.-A. (1994) Cloning, nucleotide sequence and expression of the Escherichia coli gene encoding carnitine dehydratase. J. Bacteriol. 176, 2970^2975. [22] Eichler, K., Bourgis, F., Buchet, A., Kleber, H.-P. and Mandrand-Berthelot, M.-A. (1994) Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coli. Mol. Microbiol. 13, 775^786. [23] Preusser, A., Wagner, U., Elssner, T. and Kleber, H.-P. (1999) Crotonobetaine reductase from Escherichia coli consists of two proteins. Biochim. Biophys. Acta, in press. [24] Eichler, K., Buchet, A., Lemke, R., Kleber, H.-P. and Mandrand-Berthelot, M.-A. (1996) Identi¢cation and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli. J. Bacteriol. 178, 1248^1257. [25] Dickie, P. and Weiner, J.H. (1979) Puri¢cation and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli. Can. J. Biochem. 57, 813^ 821. [26] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [27] Wagner, U., Lachmann, I., Haëdge, D. and Droëssler, K. (1997) Development and characterization of monoclonal antibodies speci¢c for two di¡erent exoproteases of aeromonas salmonicida. J. Fish Dis. 20, 223^235. [28] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [29] Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350^4354. [30] Jung, H., Jung, K. and Kleber, H.-P. (1990) L-carnitine uptake by Escherichia coli. J. Basic Microbiol. 30, 507^514.
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