Hans-Peter Kleber

Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coii

The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H‐NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59311 Da (CaiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium.

Production of water-soluble surface-active exolipids by Torulopsis apicola

SummarySeveral Torulopsis yeasts were screened for production of extracellular surface-active compounds. One strain, Torulopsis apicola IMET 43747, was studied in greater detail. Both on nalkanes and on carbohydrates it produced a mixture of water-soluble biosurfactants with remarkable interfacial activities and surface-tension values around 30 mN m-1 and interfacial tension below 1 mN m-1. Most of the biosurfactants are produced in the late exponential and in the early stationary growth phase. Production was increased by using hydrophobic compounds as the carbon source. The yields on n-alkanes were influenced by the concentrations of both the carbon source and the yeast extract. The effects of one purified biosurfactant on microbial growth on nalkanes and its antibacterial and antiphagal activities reveal new physiological aspects of biosurfactant generation by T. apicola.

Purification and properties of carnitine dehydratase from Escherichia coli a new enzyme of carnitine metabolization

Carnitine dehydratase from Escherichia coli 044 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine. It has been purified 500-fold to electrophoretic homogeneity by chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, second phenyl-Sepharose and finally gel filtration on a Sephadex G-100 column. During the purification procedure a low-molecular-weight effector essential for enzyme activity was separated from the enzyme. The addition of this still unknown effector caused reactivation of the apoenzyme. The relative molecular mass of the apoenzyme has been estimated to be 85,000. It seems to be composed of two identical subunits with a relative molecular mass of 45,000. The purified and reactivated enzyme has been further characterized with respect to pH and temperature optimum (7.8 and 37-42 degrees C), equilibrium constant (Keq = 1.5 +/- 0.2) and substrate specifity. The enzyme is inhibited by thiol reagents. The Km value for crotonobetaine is 1.2.10(-2) M. gamma-Butyrobetaine, D(+)-carnitine and choline are competitive inhibitors of crotonobetaine hydration.

Verwertung von Trimethylammoniumverbindungen durch Acinetobacter calcoaceticus

The utilization of carnitine and carnitine derivatives (O-acylcarnitines, carnitine carboxylderivatives) and structure-related trimethylammonium-compounds (betaines and nitrogen-bases) by Acinetobacter calcoaceticus was studied by means of the control of growth and the quantitative detection of metabolites. The strain grew only on L-carnitine, L-O-acylcarnitines, and gamma-butyrobetaine as the sole carbon sources. The utilization of these compounds and the growth correlated with the cleavage of the C-N bond and thereby with the formation of trimethylamin. D-Carnitine was metabolized, if an additional carbon source, like L-carnitine, was present in the incubation mixture, or if the bacteria were preincubated with L- or DL-carnitine, but no growth was observed on D-carnitine as the sole carbon source. The bacteria oxidized choline to glycinebetaine in the presence of additional carbon sources, glycinebetaine itself was not assimilated. With regard to the catabolism of quaternary nitrogen compounds Acinetobacter calcoaceticus shows a different pathway in comparison with other bacterial species metabolizing carnitine.The utilization of carnitine and carnitine derivatives (O-acylcarnitines, carnitine carboxylderivatives) and structure-related trimethylammonium-compounds (betaines and nitrogen-bases) by Acinetobacter calcoaceticus was studied by means of the control of growth and the quantitative detection of metabolites. The strain grew only on l-carnitine, l-O-acylcarnitines, and γ-butyrobetaine as the sole carbon sources. The utilization of these compounds and the growth correlated with the cleavage of the C-N bond and thereby with the formation of trimethylamine. d-Carnitine was metabolized, if an additional carbon source, like l-carnitine, was present in the incubation mixture, or if the bacteria were preincubated with l-or dl-carnitine, but no growth was observed on d-carnitine as the sole carbon source. The bacteria oxidized choline to glycinebetaine in the presence of additional carbon sources, glycinebetaine itself was not assimilated. With regard to the catabolism of quaternary nitrogen compounds Acinetobacter calcoaceticus shows a different pathway in comparison with other bacterial species metabolizing carnitine.ZusammenfassungDie Verwertung von Carnitin und Carnitinderivaten (O-Acylcarnitine, Carnitincarboxyl-derivate) und strukturverwandten Trimethylammoniumverbindungen (Betaine und Stickstoffbasen) durch Acinetobacter calcoaceticus wurde anhand des Wachstums und des quantitativen Nachweises der Metabolite untersucht. Der Stamm wuchs auf l-Carnitin, l-O-Acylcarnitinen und γ-Butyrobetain als jeweils einziger C-Quelle. Der Verbrauch dieser Verbindungen und das Wachstum korrelierten mit der Spaltung der C-N-Bindung und mit dem gebildeten Trimethylamin. d-Carnitin wurde metabolisiert, wenn als zusätzliche C-Quelle l-Carnitin im Nährmedium vorhanden war, oder wenn die Bakterien mit l-oder dl-Carnitin vorinkubiert worden waren. Mit d-Carnitin als einziger C-Quelle wuchsen die Bakterien jedoch nicht. Die Bakterien oxidierten Cholin zu Glycinbetain in Gegenwart einer zusätzlichen C-Quelle, Glycinbetain selbst wurde nicht assimiliert. In Hinsicht auf den Abbau quaternärer Stickstoffverbindungen besitzt Acinetobacter calcoaceticus im Vergleich zu anderen Carnitin-verwertenden Bakterienarten einen für ihn charakteristischen Stoffwechselweg.

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine, carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT2 by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of γ-butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to γ-butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.

Biotransformation of D(+)–carnitine into L(−)–carnitine by resting cells of Escherichia coli O44 K74

l(−)‐carnitine was produced from d(+)‐carnitine by resting cells of Escherichia coli O44 K74. Oxygen did not inhibit either the carnitine transport system or the enzymes involved in the biotransformation process. Aerobic conditions led to higher product yield than anaerobic conditions. The biotransformation yield depended both on biomass and initial substrate concentrations used; the selected values for these variables were 4·30 g l−1 cells and 100 mmol l−1d(+)‐carnitine. Under these conditions the l(−)‐carnitine production rate was 0·55 g l−1 h−1, the process yield was 44%, and the productivity was 0·22 g l−1 h−1 after a 30 h incubation period. Crotonobetaine production, besides l(−)‐carnitine, showed that the action of more than one enzyme occurred during the biotransformation process. On the other hand, the addition of fumarate at high substrate concentrations (250 and 500 mmol l−1) led to a higher metabolic activity, which meant an increment of l(−)‐carnitine production.

Crotonobetaine reductase from Escherichia coli consists of two proteins.

Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)). CI has been purified to electrophoretic homogeneity from a cell-free extract of E. coli O44 K74. The purified protein shows l(-)-carnitine dehydratase activity and its N-terminal amino acid sequence is identical to the caiB gene product from E. coli O44 K74. The relative molecular mass of CI has been determined to be 86100. It is composed of two identical subunits with a molecular mass of 42600. The isoelectric point of CI was found to be 4.3. CII was purified from an overexpression strain in one step by ion exchange chromatography on Fractogel EMD TMAE 650(S). The N-terminal amino acid sequence of CII shows absolute identity with the N-terminal sequence of the caiA gene product, i.e. of the postulated crotonobetaine reductase. The relative molecular mass of the protein is 164400 and it is composed of four identical subunits of molecular mass 41500. The isoelectric point of CII is 5.6. CII contains non-covalently bound FAD in a molar ratio of 1:1. In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII. A still unknown low-molecular-mass effector described for the l(-)-carnitine dehydratase is also necessary for crotonobetaine reductase activity. Monoclonal antibodies were raised against the two components of crotonobetaine reductase.

[Interrelationships between carnitine metabolism and fatty acid assimilation in Pseudomonas putida (author's transl)].

The carnitine metabolism and some relations to the fatty acid metabolism were studied in Pseudomonas putida by means of control of growth, analysis of metabolites, and determination of enzyme activities. The strain grew on γ-butyrobetaine, D,L-and L-carnitine, glycinebetaine, choline, D,L-norcarnitine, D,L-γ-amino-β-hydroxybutyrate, and D,L-β-hydroxybutyrate. Although the strain used straight-chain fatty acids of 2–16 C-atoms, it was only able to grow on O-acyl-L-carnitines of 10 or more C-atoms in the acylgroup. Addition of carnitine stimulated the growth on long-chain fatty acids.The formation of trimethylamine increased, if L-carnitine or γ-butyrobetaine were the only carbon sources, and decreased, if these trimethylammonium compounds were carbon as well as nitrogen sources. L-Carnitine induced the carnitine dehydrogenase as well as the β-hydroxybutyrate dehydrogenase. γ-Butyrobetaine as carbon and nitrogen source induced the carnitine dehydrogenase, too. In the crude extract the specific activities of β-hydroxybutyrate dehydrogenase were 0.7 or 1.6 μmoles·min-1·mg-1 after growth on L-carnitine and D,L-β-hydroxybutyrate, respectively. The synthesis of both enzymes was repressed by glycinebetaine, glucose and long-chain fatty acids. Dependent on the nitrogen source L-carnitine was catabolized via two different pathways.ZusammenfassungDer Carnitinstoffwechsel und einige Beziehungen zum Fettsäurestoffwechsel wurden mittels der Wachstumskontrolle, der Bestimmung von Metaboliten und des Nachweises von Enzymaktivitäten in Pseudomonas putida untersucht. Der Stamm wuchs auf γ-Butyrobetain, D,L-und L-Carnitin, Glycinbetain, Cholin, D,L-Norcarnitin, D,L-γ-Amino-β-hydroxybutyrat und D,L-β-Hydroxybutyrat. Obwohl der Stamm unverzweigte Fettsäuren von 2–16 C-Atomen zu untzen vermag, konnte er nur auf O-Acyl-L-carnitinen von 10 oder mehr C-Atomen in der Acylgruppe wachsen. Zugabe von Carnitin stimulierte das Wachstum auf langkettigen Fettsäuren.Die Bildung von Trimethylamin stieg, wenn Carnitin oder λ-Butyrobetain nur C-Quellen waren, und sank, wenn diese Trimethylammoniumverbindungen sowohl C-als auch N-Quellen waren. L-Carnitin induzierte sowohl die Carnitindehydrogenase als auch die β-Hydroxybutyratdehydrogenase. λ-Butyrobetain als C-und N-Quelle induzierte ebenfalls die Carnitindehydrogenase. Im Rohextrakt betrug die spezifische Aktivität der β-Hydroxybutyratdehydrogenase entsprechend dem Wachstum auf L-Carnitin oder D,L-β-Hydroxybutyrat 0,7 oder 1,6 μMol · min-1 · mg-1. Glycinbetain, Glucose und langkettige Fettsäuren reprimierten die Synthese beider Enzyme. Abhängig von der N-Quelle wird L-Carnitin offensichtlich auf zwei unterschiedlichen Stoffwechselwegen abgebaut.The carnitine metabolism and some relations to the fatty acid metabolism were studied in Pseudomonas putida by means of control of growth, analysis of metabolites, and determination of enzyme activites. The strain grew on gamma-butyrobetaine, D,L- and L-carnitine, glycinebetaine, choline, D,L-norcarnitine, D,L-gamma-amino-beta-hydroxybutyrate, and D,L-beta-hydroxybuty-rate. Although the strain used straight-chain fatty acids of 2-16 C-atoms, it was only able to grow on O-acyl-L-carnitines of 10 or more C-atoms in the acyl-group. Addition of carnitine stimulated the growth on long-chain fatty acis. The formation of trimethylamine increased, if L-carnitine or gamma-butyrobetaine were the only carbon sources, and decreased, if these trimethylammonium compounds were carbon as well as nitrgen sources. L-Carnitine induced the carnitine dehydrogenase as well as the beta-hydroxybutyrate dehydrogenase, gamma-Butyrobetaine as carbon and nitrogen source induced the carnitine dehydrogenase, too. In the crude extract the specific activiteis of beta-hydroxybutyrate dehydrogenase were 0.7 or 1.6 mumoles.min-1.mg-1 after growth on L-carnitine and D,L-beta-hydroxybutyrate, respectively. The synthesis of both enzymes was repressed by glycinebetaine, glucose and long-chain fatty acis. Dependent on the nitrogen source L-carnitine was catabolized via two different pathways.

Crotonobetaine reductase fromEscherichia coli — a new inducible enzyme of anaerobic metabolization of L(-)-carnitine

Crotonobetaine reductase fromEscherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, γ-butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, γ-butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40–45 °C). The Km value for crotonobetaine was determined to be 1.1×10−2M. γ-Butyrobetaine,D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For γ-butyrobetaine (Ki=3×10−5M) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.

Metabolism of d(+)-carnitine by Escherichia coli

SummaryEscherichia coli 044 K74 grown under anaerobic conditions in the presence of l(−)-carnitine is able to convert d(+)-carnitine into the l(−)-enantiomer. This activity is repressed by electron acceptors such as oxygen and nitrate as well as by glucose. d(+)-Carnitine shows no effect on the induction or repression of the corresponding enzyme or enzyme system. Resting cells of E. coli 044 K74 were used for the formation of l(−)-carnitine from d(+)-carnitine. The maximum obtained yield was 50%. γ-Butyrobetaine was formed as a by-product.