Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker’s yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.     

Signal amplification between Gbetagamma release and PI3Kgamma-mediated PI(3,4,5)P3 formation monitored by a fluorescent Gbetagamma biosensor protein and repetitive two component total internal reflection/fluorescence redistribution after photobleaching analysis

Phosphoinositide 3-kinase gamma (PI3Kgamma) is activated by Gbetagamma release after stimulation of Galpha i -coupled receptors, involving a recruitment of the enzyme to the plasma membrane via interaction of the regulatory subunit p101 or p87 with Gbetagamma. The receptor-mediated release of Gbetagamma was, however, insufficient to elicit a translocation of p101 observable by classical fluorescence microscopy approaches. Since the mobilities of plasma membrane-associated and cytosolic proteins differ strongly, small changes in the amount of plasma membrane association should be detectable by an altered diffusional behavior. Here, changes in mobility were monitored by fluorescence redistribution after photobleaching (FRAP) which was repetitively applied before and after stimulation of cells. To combine the advantages of total internal reflection (TIR) illumination, which preferentially excites fluorophors located at or near the plasma membrane, with that provided by the mobility information, we developed a combined TIR/FRAP setup which enabled us to point bleach parts of an image that was observed under TIR illumination. For FRAP data analysis, we introduce a convolution-based method and a global two component model. Using this TIR/FRAP approach, an increased plasma membrane association of the fluorescent Gbetagamma-binding domain of p101 after Gbetagamma release by G protein-coupled receptor stimulation could be detected and quantified. By comparing the translocation efficiency of this domain with that of YFP-GRP1(PH), a biosensor for the PI3Kgamma product PI(3,4,5)P3, we evaluate the signal amplification between Gbetagamma release and PI(3,4,5)P3 formation after activation of Galpha i -coupled receptors.     

Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution.

Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, and M5. We describe novel approaches to the detection of epitope-tagged proteins via fluorescence resonance energy transfer on beads. We have synthesized and characterized biotinylated and fluorescein-labeled FLAG peptides and examined the binding of FLAG peptides to commercial streptavidin beads using flow cytometric analysis. A requirement of assay development is the elucidation of parameters that characterize the binding interactions between component systems. We have thus compiled a set of Kd values determined from a series of equilibrium binding experiments with beads, peptides, and antibodies. We have defined conditions for binding biotinylated and fluoresceinated FLAG peptides to beads. Site occupancies of the peptides were determined to be on the order of several million sites per bead and Kd values in the 0.3-2.0 nM range. The affinity for antibody attachment to peptides was determined to be in the low nanomolar range (less than 10 nM) for measurements on beads and solution. We demonstrate the applicability of this methodology to assay development, by detecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase fusion protein. These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, proteomic, or clinical applications.     

Isolation from Agricultural Soil and Characterization of a Sphingomonas sp. Able To Mineralize the Phenylurea Herbicide Isoproturon

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.     

Facies pattern and genesis of the Jebel Rheris biostromes (Givetian,Eastern Anti-Atlas,Morocco)

Summary During Givetian times, the Jebel Rheris area was situated in a transitional zone at the northern margin of Gondwana, between the emerged Ougnate High in the north and the Mader Basin in the south. A facies pattern developed from stacked or amalgamated coral-stromatoporoid biostromes in the northern near-shore area to an alternating biostrome–crinoidal grainstone succession, which passed over a low angle slope setting towards the south to a pure crinoidal grainstone facies with abundant slumping structures. Finally in the south, a basinal turbiditic facies evolved. In the shallow sea, biostromes probably developed due to the lack of a ‘binder guild’ in the fossil community, which hampered the establishment of mound-like structures, stable enough to resist high-energy storm events. Repeated termination of the coral-stromatoporoid growth is attributed to transgressions. During suitable conditions, colonisation of the sea floor proceeded in three phases: a) cluster settlement; pioneer communities, mostly consisting of tabulate corals and domical to bulbous stromatoporoids, started growing in laterally delimited clusters; b) lateral dispersion; from these centres, settlement prograded laterally, until large areas of the sea-floor were covered; c) vertical accretion; the organisms more and more grew on each other, causing a homogeneous vertical expansion. A significant difference of this up to 200 m thick biostrome—crinoidal grainstone succession compared to continuously growing reefs is the fact that communities repeatedly had to start with the colonisation stage, thus could not reach a mature or climax stage.     

Landscape patterns of indicator plants for soil acidity in the Bavarian Alps

Aim Electronic distribution atlases and lists of ecological indicator values are becoming important tools in plant geography. In this contribution, we combine a geographical and an ecological data bank, and map out patterns of indicator value spectra (instead of single or average values) across a physiographically complex landscape. For our study, we select indicators of soil pH and carbonate content as key environmental factors that strongly affect overall plant diversity patterns in the temperate zone. Our goal is to relate the distribution and diversity of plant groups that are indicators of soil pH and carbonate content to environmental controls at the landscape‐scale, and thus contribute to a causal understanding of species pools. Location We studied the Bavarian Alps, which represent the German portion of the Northern Alps. Methods Based on the existing floristic survey, we calculated relative frequencies of nine classes of indicator plants for soil pH and carbonate content in grid cells. The resulting attribute matrix (cells by indicator class frequencies) was subjected to principal components analysis and to k‐means clustering. Results were compared and mapped out in the grid array of the whole region, resulting in continuous and discrete representations of species pool structure. We used a geographical information system to derive physiographical landscape properties from a geological map and a digital elevation model, and analysed their statistical relationship with the shapes of indicator spectra. Results and Main conclusions Averages of indicator values for soil pH and carbonate content follow the geological structure quite closely. Surprisingly, the diversity of indicator plant groups does not appear to be a function of geological or topographic heterogeneity. Rather, it seems to be related to areas of high elevation with uniform geology. The effect is a matter of additional acidophytes in high mountain areas and, in the high calcareous Alps, extreme calciphytes, while species with intermediate requirements are rarer than usual. For explanation, we suggest two facts: (1) a frequent lack of mature soils at high elevations and (2) particularities in soil genetic processes occurring under the harsh climatic conditions of high mountains.