Fabian Müller

Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase.

Thiosulphate is one of the products of the initial step of the elemental sulphur oxidation pathway in the thermoacidophilic archaeon Acidianus ambivalens. A novel thiosulphate:quinone oxidoreductase (TQO) activity was found in the membrane extracts of aerobically grown cells of this organism. The enzyme was purified 21‐fold from the solubilized membrane fraction. The TQO oxidized thiosulphate with tetrathionate as product and ferricyanide or decyl ubiquinone (DQ) as electron acceptors. The maximum specific activity with ferricyanide was 73.4 U (mg protein)−1 at 92°C and pHu20036, with DQ it was 397u2003mU (mg protein)−1 at 80°C. The Km values were 2.6u2003mM for thiosulphate (kcatu2003=u2003167u2003s−1),u200a 3.4u2003mMu200a foru200a ferricyanideu200a andu200a 5.87u2003µM for DQ. The enzymic activity was inhibited by sulphite (Kiu2003=u20035u2003µM), metabisulphite, dithionite and TritonX‐100, but not by sulphate or tetrathionate. A mixture of caldariella quinone, sulfolobus quinone and menaquinone was non‐covalently bound to the protein. No other cofactors were detected. Oxygen consumption was measured in membrane fractions upon thiosulphate addition, thus linking thiosulphate oxidation to dioxygen reduction, in what constitutes a novel activity among Archaea. The holoenzyme was composed of two subunits of apparent molecular masses of 28 and 16u2003kDa. The larger subunit appeared to be glycosylated and was identical to DoxA, and the smaller was identical to DoxD. Both subunits had been described previously as a part of the terminal quinol:oxygen oxidoreductase complex (cytochrome aa3).

An Extracellular Tetrathionate Hydrolase from the Thermoacidophilic Archaeon Acidianus Ambivalens with an Activity Optimum at pH 1.

Background: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO2-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. Results: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9u2009h and final cell densities of up to 8u2009×u2009108/ml. TTH activity (≈0.28u2009U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54u2009kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a β-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. Conclusion: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.

Innovationsmanagement in regionalen Netzwerken: Ansätze für das strategische Kompetenz-Management kleiner und mittelständischer Unternehmen

Innovationen werden in Forschung und Praxis als zentrale Faktoren fur die Sicherung der Wettbewerbsfahigkeit kleiner und mittelstandischer Unternehmen (KMU) angesehen. Denn sie sind starker als (diversifizierte) Grosunternehmen auf Ruckflusse aus einem zu jederzeit ausgeglichenen Produktportfolio angewiesen und mussen — bei immer kurzer werdenden Produktlebenszyklen — kontinuierlich neue Produkte auf den Markt bringen (vgl. Meyer 2006: 210).