Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei.

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.

The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations.

The environmental yeast diversity of spontaneous cocoa bean fermentations in east Cuba was investigated. Seven fermentations, 25 equipment- and handling-related samples, and 115 environmental samples, such as flowers, leaf and cocoa pod surfaces, as well as drosophilid insects, were analysed. The basic fermentation parameters temperature and pH were recorded during five fermentations for at least six days. A total of 435 yeast isolates were identified by a combination of PCR-fingerprinting of genomic DNA with the M13 primer and sequence analysis of DNA from representative isolates, using the internal transcribed spacer region, the D1/D2 region of the large subunit rRNA gene, and an actin gene-encoding fragment, as required. Among 65 yeast species detected, Pichia manshurica and Hanseniaspora opuntiae were the most frequently isolated species, obtained from five and four fermentations, followed in frequency by Pichia kudriavzevii from two fermentations. Saccharomyces cerevisiae was isolated only occasionally. Cocoa fermentation yeast species were also present on processing equipment. The repeated isolation of a preliminarily as Yamadazyma sp. classified species, a group of strains similar to Saccharomycopsis crataegensis from fermentations and equipment, and the isolation of fifteen other potentially novel yeast species in low numbers provides material for further studies. Environmental samples showed higher yeast diversity compared to the fermentations, included the most frequent fermentation species, whereas the most frequently isolated environmental species were Candida carpophila, Candida conglobata, and Candida quercitrusa. Potential selective advantages of the most frequently isolated species were only partly explained by the physiological traits tested. For instance, tolerance to higher ethanol concentrations was more frequent in strains of Pichia spp. and S. cerevisiae compared to Hanseniaspora spp.; the ability to also assimilate ethanol might have conferred a selective advantage to Pichia spp. In contrast, high glucose tolerance was common among strains of Hanseniaspora spp., Torulaspora delbrueckii, and Candida tropicalis, among which only Hanseniaspora spp. were frequently isolated.

Acetobacter sicerae sp. nov., isolated from cider and kefir, and identification of species of the genus Acetobacter by dnaK, groEL and rpoB sequence analysis.

Five acetic acid bacteria isolates, awK9_3, awK9_4 ( = LMG 27543), awK9_5 ( = LMG 28092), awK9_6 and awK9_9, obtained during a study of micro-organisms present in traditionally produced kefir, were grouped on the basis of their MALDI-TOF MS profile with LMG 1530 and LMG 1531(T), two strains currently classified as members of the genus Acetobacter. Phylogenetic analysis based on nearly complete 16S rRNA gene sequences as well as on concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB indicated that these isolates were representatives of a single novel species together with LMG 1530 and LMG 1531(T) in the genus Acetobacter, with Acetobacter aceti, Acetobacter nitrogenifigens, Acetobacter oeni and Acetobacter estunensis as nearest phylogenetic neighbours. Pairwise similarity of 16S rRNA gene sequences between LMG 1531(T) and the type strains of the above-mentioned species were 99.7%, 99.1%, 98.4% and 98.2%, respectively. DNA-DNA hybridizations confirmed that status, while amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) data indicated that LMG 1531(T), LMG 1530, LMG 27543 and LMG 28092 represent at least two different strains of the novel species. The major fatty acid of LMG 1531(T) and LMG 27543 was C18 : 1ω7c. The major ubiquinone present was Q-9 and the DNA G+C contents of LMG 1531(T) and LMG 27543 were 58.3 and 56.7 mol%, respectively. The strains were able to grow on D-fructose and D-sorbitol as a single carbon source. They were also able to grow on yeast extract with 30% D-glucose and on standard medium with pH 3.6 or containing 1% NaCl. They had a weak ability to produce acid from d-arabinose. These features enabled their differentiation from their nearest phylogenetic neighbours. The name Acetobacter sicerae sp. nov. is proposed with LMG 1531(T) ( = NCIMB 8941(T)) as the type strain.

Gluconobacter cerevisiae sp. nov., isolated from the brewery environment.

Three strains, LMG 27748(T), LMG 27749 and LMG 27882 with identical MALDI-TOF mass spectra were isolated from samples taken from the brewery environment. Analysis of the 16S rRNA gene sequence of strain LMG 27748(T) revealed that the taxon it represents was closely related to type strains of the species Gluconobacter albidus (100 % sequence similarity), Gluconobacter kondonii (99.9 %), Gluconobacter sphaericus (99.9 %) and Gluconobacter kanchanaburiensis (99.5 %). DNA-DNA hybridization experiments on the type strains of these species revealed moderate DNA relatedness values (39-65 %). The three strains used d-fructose, d-sorbitol, meso-erythritol, glycerol, l-sorbose, ethanol (weakly), sucrose and raffinose as a sole carbon source for growth (weak growth on the latter two carbon sources was obtained for strains LMG 27748(T) and LMG 27882). The strains were unable to grow on glucose-yeast extract medium at 37 °C. They produced acid from meso-erythritol and sucrose, but not from raffinose. d-Gluconic acid, 2-keto-d-gluconic acid and 5-keto-d-gluconic acid were produced from d-glucose, but not 2,5-diketo-d-gluconic acid. These genotypic and phenotypic characteristics distinguish strains LMG 27748(T), LMG 27749 and LMG 27882 from species of the genus Gluconobacter with validly published names and, therefore, we propose classifying them formally as representatives of a novel species, Gluconobacter cerevisiae sp. nov., with LMG 27748(T) ( = DSM 27644(T)) as the type strain.

Acetobacter lambici sp. nov., isolated from fermenting lambic beer.

An acetic acid bacterium, strain LMG 27439(T), was isolated from fermenting lambic beer. The cells were Gram-stain-negative, motile rods, catalase-positive and oxidase-negative. Analysis of the 16S rRNA gene sequence revealed the strain was closely related to Acetobacter okinawensis (99.7 % 16S rRNA gene sequence similarity with the type strain of this species), A. ghanensis (99.6 %), A. syzygii (99.6 %), A. fabarum (99.4 %) and A. lovaniensis (99.2 %). DNA-DNA hybridization with the type strains of these species revealed moderate DNA-DNA hybridization values (31-45 %). Strain LMG 27439(T) was unable to grow on glycerol or methanol as the sole carbon source, on yeast extract with 10 % ethanol or on glucose-yeast extract medium at 37 °C. It did not produce acid from l-arabinose, d-galactose or d-mannose, nor did it produce 2-keto-d-gluconic acid, 5-keto-d-gluconic acid or 2,5-diketo-d-gluconic acid from d-glucose. It did not grow on ammonium as the sole nitrogen source and ethanol as the sole carbon source. These genotypic and phenotypic data distinguished strain LMG 27439(T) from established species of the genus Acetobacter, and therefore we propose this strain represents a novel species of the genus Acetobacter. The name Acetobacter lambici sp. nov. is proposed, with LMG 27439(T) ( = DSM 27328(T)) as the type strain.

Development of an ion-exchange chromatography method for monitoring the degradation of prebiotic arabinoxylan-oligosaccharides in a complex fermentation medium.

Arabinoxylan-oligosaccharides (AXOS) are a new class of prebiotics with promising health-promoting characteristics. However, the mechanism by which bacteria break down these compounds in the colon is still uncharacterized, due to their structural complexity. A new analytical method that offers structural information was developed to characterize AXOS degradation during fermentation. The method was based on the simultaneous determination of arabinose, xylose, xylo-oligosaccharides (XOS), and AXOS by applying high-performance anion-exchange chromatography with pulsed amperometric detection. To study the structural features of AXOS in solution without the use of spectroscopic techniques or standards, enzymatic-based reference degradation chromatograms were generated based on enzymes with known specificity. The new method for fingerprinting showed to be a powerful and fast tool to study AXOS degradation with high repeatability with respect to peak area, peak width at half height, and retention time (respective relative standard deviations of ≤3.1%, 2.8%, and 0.8%). This method was successfully applied to study the degradation kinetics of AXOS in a complex fermentation medium by Bifidobacterium longum LMG 11047. The results showed that this strain could use both the arabinose side chains and xylose backbones up to xylotetraose. The characterization of the degradation abilities of AXOS by colon bacteria will allow a better understanding of the beneficial effects of these prebiotics. Furthermore, if the appropriate enzymes are available to design the reference degradation chromatograms, this new method for the qualitative fingerprinting of AXOS breakdown can also be applied for the breakdown of other complex oligosaccharides and polysaccharides.