Two new autotrophic carbon fixation cycles have been recently described in Crenarchaeota. The 3-hydroxypropionate/4-hydroxybutyrate cycle using acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme has been identified for (micro)aerobic members of the Sulfolobales. The dicarboxylate/4-hydroxybutyrate cycle using oxygen-sensitive pyruvate synthase and phosphoenolpyruvate carboxylase as carboxylating enzymes has been found in members of the anaerobic Desulfurococcales and Thermoproteales. However, Sulfolobales include anaerobic and Desulfurococcales aerobic autotrophic representatives, raising the question of which of the two cycles they use. We studied the mechanisms of autotrophic CO 2 fixation in the strictly anaerobic Stygiolobus azoricus (Sulfolobales) and in the facultatively aerobic Pyrolobus fumarii (Desulfurococcales). The activities of all enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle were found in the anaerobic S. azoricus. In contrast, the aerobic or denitrifying P. fumarii possesses all enzyme activities of the dicarboxylate/4-hydroxybutyrate cycle. We conclude that autotrophic Crenarchaeota use one of the two cycles, and that their distribution correlates with the 16S rRNA-based phylogeny of this group, rather than with the aerobic or anaerobic lifestyle.
The monoterpenes are the most important contribution to the olfactory profile of wine due to their low odour threshold. These and other aroma-active substances do not generally exist in a free form but are conjugated to mono-or disaccharides, thereby forming water-soluble and odourless complexes. Enzymes that cleave the sugar moieties from the precursors can, therefore, have a major impact on the sensory profile of wine, as they release the volatile aroma compounds. For this reason, we searched for wine yeasts producing glycosidases which are active under oenological conditions. A collection of 100 wine yeasts were screened for glycosidase activities in whole cells and in culture supernatants. Kinetic parameters were determined spectrophotometrically with synthetic model substrates, and hydrolysis of natural glycosides was detected by thin-layer chromatography. A yeast isolate, AS1, was identified as a new Wickerhamomyces anomalus strain which hydrolysed a number of synthetic and natural glycosides under oenological conditions. Citronellol-and nerol-glucosides, among the most frequently occurring aroma precursors in wine, were also cleaved. In contrast to a commercial β-glucosidase, whole cells of W. anomalus AS1 catalysed deglycosylation of arbutin and salicin directly in a white and a red wine. Besides the formation of intra-and extracellular glucoside hydrolases, strain AS1 exhibited arabinosidase and xylosidase activities which are also essential for the release of flavour compounds. Even with limited functionality at oenological conditions, the glycoside hydrolase activities of W. anomalus AS1 may improve aroma development, provided that the reaction occurs over a longer period, as it is the case during wine-making.
Different strains of the genus Lactobacillus can be regularly isolated from must and wine samples. By various physiological activities, they can improve or reduce the wine quality. Lactobacillus hilgardii that is known to survive under harsh wine conditions is classified as a spoilage bacterium, e.g. due to the production of histamine. Many lactobacilli form an S-layer as the outermost cell wall component which has been found to facilitate the colonization of special ecological niches. A detailed understanding of the properties related to their S-layer proteins is necessary to improve the knowledge of the interactions between different bacterial cells and with the surrounding environments. The S-layer protein from the wine-related L. hilgardii strain B706 has been isolated and its gene sequence determined. The deduced amino acid sequence corresponds to a 41 kDa protein with an isoelectric point of 9.6 without additional posttranslational modifications after splitting off the leader peptide. The complete protein is organized in a 32 amino acids signal sequence for membrane translocation, a positively charged N-terminal domain that binds to the cell wall and a negatively charged C-terminal domain. When the S-layer was removed, the corresponding L. hilgardii B706 cells became more sensitive to bacteriolytic enzymes and some wine-related stress conditions. From a practical point of view, the S-layer may be considered as a target for the inhibition of food-spoiling lactobacilli.
The genus Saccharomyces comprises very closely related species. This high degree of relationship makes a simple identification and differentiation of strains difficult since these species are hardly discriminable by their morphological and physiological features. A sequence analysis of ribosomal DNA and the corresponding internal transcribed spacers can only rarely be successfully applied. In this study, we proved the applicability of a novel DNA fingerprinting method, the SAPD-PCR (specifically amplified polymorphic DNA) and of MALDI-TOF-MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) fingerprinting with the MALDI Biotyper for the differentiation of species belonging to the genus Saccharomyces. It was possible with SAPD-PCR to create specific banding patterns for all Saccharomyces species. Different strains of the same species produced nearly the same banding patterns. Specific and reproducible reference spectra could be generated for each of the strains with the MALDI Biotyper. Therefore, SAPD-PCR and MALDI-TOF-MS can be fast and reliable tools to identify these related Saccharomyces species which are applied in many biotechnological processes.
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