Crystal structures of Staphylococcus aureus sortase A and its substrate complex

The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys(184) and Arg(197) residues and at a greater distance from the imidazolium side chain of His(120). All three residues, His(120), Cys(184), and Arg(197), are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria.     

Age at onset in Huntington’s disease is modified by the autophagy pathway: implication of the V471A polymorphism in Atg7

Huntington’s disease (HD) is caused by an expansion of a polyglutamine repeat of more than 35 units in the huntingtin protein. The expanded repeat length is inversely correlated with the age at onset (AAO); however, additional genetic factors apart from the expanded CAG repeat length can modify the course and the AAO in HD. Aberrations in macroautophagy have been observed in Huntington, Alzheimer, Parkinson, motor neuron and prion diseases. Therefore, we hypothesized that polymorphisms in autophagy-related (Atg) genes might contribute to the variation in the AAO. We initially tested eight single nucleotide polymorphisms in five Atg genes (Atg3, Atg5, Atg7, Atg16L1 and Beclin-1) for their frequency of ≥1%. Subsequently, we investigated the polymorphisms Atg7 V471A and Atg16L1 T281A for a disease-modifying effect in more than 900 European HD patients (including 2 populations consisting of 346 German patients and 327 patients of Italian descent). One polymorphism in the Atg7 gene that substitutes alanine for valine (V471A) showed a significant effect on the AAO (P = 0.0050) and was associated with an earlier disease onset of 4 years. Our results further support the important pathophysiological role of autophagy in HD.     

Members of the NF90/NFAR protein group are involved in the life cycle of a positive-strand RNA virus

A major issue of current virology concerns the characterization of cellular proteins that operate as functional components of the viral multiplication process. Here we describe a group of host factors designated as 'NFAR proteins' that are recruited by the replication machinery of bovine viral diarrhea virus, a close relative of the human pathogen hepatitis C virus. The NFAR proteins associate specifically with both the termini of the viral RNA genome involving regulatory elements in the 5' and 3' non-translated regions. Modification of the protein interaction sites in the 3' non-translated region yielded viral RNAs that were replication deficient. Viral replication was also inhibited by RNAi approaches that reduced the concentration of RNA helicase A, a member of the NFAR group, in the host cell's cytoplasm. Further experimental data suggest that NFAR proteins mediate a circular conformation of the viral genome that may be important for the coordination of translation and replication. Because NFAR proteins are presumed components of the antiviral response, we suspect that viral recruitment may also serve to weaken cellular defense mechanisms.     

The sxa2-dependent inactivation of the P-factor mating pheromone in the fission yeast Schizosaccharomyces pombe

Haploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P-factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2-dependent carboxypeptidase that inactivates P-factor by removal of the C-terminal leucine residue.     

Food purchases: Impacts from the consumers’ point of view investigated with a modular LCA

The goal of this research work was to assist consumers in considering environmental aspects of food consumption. A simplified, modular LCA approach has been used to evaluate the impacts from the consumers’ point of view. Comparative LCA’s have been calculated for five single aspects of decisions: type of agricultural practice, origin, packaging material, type of preservation, and consumption. The inventory for one module includes the environmental impacts related to one particular product characteristic. The modular LCA allows one to investigate the trade-offs among different decision parameters. It could be shown that most of the decision parameters might have an influence on the overall impact of a vegetable product. Greenhouse production and vegetables transported by air cause the highest surplus environmental impact. For meat products, the agricultural production determines the overall environmental impact. The total impact for vegetable or meat purchases may vary by a factor of eight or two-and-a-half. Different suggestions for consumers have been ranked according to the variation of average impacts, due to a marginal change of behaviour. Avoiding air-transported food products leads to the highest decrease of environmental impacts.     

Anaerobic Mineralization of Quaternary Carbon Atoms: Isolation of Denitrifying Bacteria on Dimethylmalonate

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the α-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans^T), and three strains isolated from freshwater ditches were affiliated with the β-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae^T and Acidovorax facilis^T, respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 × 10⁴ dimethylmalonate-utilizing cells ml⁻¹, representing up to 0.4% of the total culturable nitrate-reducing population.     

When genes meet nomenclature: tortoise phylogeny and the shifting generic concepts of Testudo and Geochelone

We used a five-gene data set (mtDNA: 12S rRNA, 16S rRNA, cyt-b; nDNA: Cmos, Rag2) comprising approximately two-thirds of all extant testudinid species and, for the first time, including all five Testudo species to investigate the question of whether all western Palaearctic testudinids are monophyletic. Further, we examined whether the recently suggested allocation of the African Geochelone pardalis in the otherwise exclusively South African genus Psammobates and of the Malagasy G. yniphora in the monotypic genus Angonoka is justified in the face of considerable morphological evidence against such placements. Our phylogenetic analyses do not support the paraphyly and generic break-up of Testudo, as suggested by previous papers using a smaller taxon sampling and mtDNA data only. We propose a continued usage of the generic name Testudo for all five western Palaearctic tortoise species. Within Testudo, two monophyletic subclades are present, one containing T. hermanni+T. horsfieldii, and the other comprising (T. kleinmanni+T. marginata)+T. graeca. Nomenclaturally, we demonstrate that Eurotestudo Lapparent de Broin et al., 2006, which was recently erected with the type species T. hermanni, is an objective junior synonym of Chersine Merrem, 1820 and Medaestia Wussow, 1916. Recognition of a monotypic genus Angonoka for G. yniphora is unwarranted according to both our re-analysis of sequence data and morphological data. Acknowledging the strong morphological similarity between G. yniphora and G. radiata, we suggest placing both species into the genus Astrochelys. Although sequence data for only one of the three Psammobates species was available for analysis, there is currently no cause to challenge the monophyly of this genus as established on the basis of morphological evidence. Thus, we hypothesize that G. pardalis is sister to a monophyletic Psammobates. In light of the clear morphological gap between G. pardalis and Psammobates species, the recognition of a distinct genus Stigmochelys for the former seems justified.     

Poly(3-Hydroxybutyrate) Granules at the Early Stages of Formation Are Localized Close to the Cytoplasmic Membrane in Caryophanon latum

Localization of newly synthesized poly(3hydroxybutyrate) (PHB) granules was determined by confocal laser scanning fluorescence microscopy of Nile red-stained cells and by transmission electron microscopy (TEM). PHB granules of Nile red-stained living cells of Caryophanon latum at the early stages of PHB accumulation were frequently found at or close to the cytoplasmic membrane. TEM analysis of the same culture revealed electron-translucent globular structures resembling PHB granules that were nonrandomly distributed in the cell lumen but were frequently found at or close to the cytoplasmic membrane. Immunogold labeling using PHB-specific antiserum confirmed that the electron-translucent structures represented PHB granules. Electron microscopy examination of PHB granules after cell lysis revealed that PHB granules were often associated with membrane vesicles. Nonrandom localization of PHB granules was also found in Beijerinckia indica. Cells of this species harbored one PHB granule at each cell pole. Our results show that newly synthesized PHB granules often are close to or even in physical contact with the cytoplasmic membrane. Possible explanations for this unexpected finding and a hypothetical model of PHB granule formation in C. latum are discussed.