Prions are believed to spontaneously convert from a native, monomeric highly helical form (called PrP(c)) to a largely β-sheet-rich, multimeric and insoluble aggregate (called PrP(sc)). Because of its large size and insolubility, biophysical characterization of PrP(sc) has been difficult, and there are several contradictory or incomplete models of the PrP(sc) structure. A β-sheet-rich, soluble intermediate, called PrP(β), exhibits many of the same features as PrP(sc) and can be generated using a combination of low pH and/or mild denaturing conditions. Studies of the PrP(c) to PrP(β) conversion process and of PrP(β) folding intermediates may provide insights into the structure of PrP(sc). Using a truncated, recombinant version of Syrian hamster PrP(β) (shPrP(90-232)), we used NMR spectroscopy, in combination with other biophysical techniques (circular dichroism, dynamic light scattering, electron microscopy, fluorescence spectroscopy, mass spectrometry, and proteinase K digestion), to characterize the pH-driven PrP(c) to PrP(β) conversion process in detail. Our results show that below pH 2.8 the protein oligomerizes and conversion to the β-rich structure is initiated. At pH 1.7 and above, the oligomeric protein can recover its native monomeric state through dialysis to pH 5.2. However, when conversion is completed at pH 1.0, the large oligomer "locks down" irreversibly into a stable, β-rich form. At pH values above 3.0, the protein is amenable to NMR investigation. Chemical shift perturbations, NOE, amide line width, and T(2) measurements implicate the putative "amylome motif" region, "NNQNNF" as the region most involved in the initial helix-to-β conversion phase. We also found that acid-induced PrP(β) oligomers could be converted to fibrils without the use of chaotropic denaturants. The latter finding represents one of the first examples wherein physiologically accessible conditions (i.e., only low pH) were used to achieve PrP conversion and fibril formation.

The tonsil of the soft palate in pigs is a secondary lymphoid tissue that provides a first line of defense against foreign antigens entering by the mouth or nares. It has been known for a long time to be the site of colonization of important swine and zoonotic bacterial pathogens. Initially our understanding of microbes present at this site came from culture-based studies. Very recently, sequence-based approaches have been used to identify the core microbiome of the swine tonsil. Although animal to animal and herd to herd variation was detected in these studies, >90 of the organisms detected belonged to the phyla Proteobacteria and Firmicutes. Members of the family Pasteurellaceae appeared to be predominate in the tonsil; however, the relative proportions of Actinobacillus, Haemophilus, and Pasteurella varied. Members of the families Moraxellaceae, Fusobacteriaceae, Veillonellaceae, and Neisseriaceae were also seen as frequent residents of the tonsil.

Lichen symbioses are thought to be stabilized by the transfer of fixed carbon from a photosynthesizing symbiont to a fungus. In other fungal symbioses, carbohydrate subsidies correlate with reductions in plant cell wall-degrading enzymes, but whether this is true of lichen fungal symbionts (LFSs) is unknown. Here, we predict genes encoding carbohydrate-active enzymes (CAZymes) and sugar transporters in 46 genomes from the Lecanoromycetes, the largest extant clade of LFSs. All LFSs possess a robust CAZyme arsenal including enzymes acting on cellulose and hemicellulose, confirmed by experimental assays. However, the number of genes and predicted functions of CAZymes vary widely, with some fungal symbionts possessing arsenals on par with well-known saprotrophic fungi. These results suggest that stable fungal association with a phototroph does not in itself result in fungal CAZyme loss, and lends support to long-standing hypotheses that some lichens may augment fixed CO2 with carbon from external sources.

bHere, we report the first complete genome sequence of Actinobacillus suis, an important opportunistic pathogen of swine. By comparing the genome sequence of A. suis with those of other members of the family Pasteurellaceae, we hope to better understand the role of these organisms in health and disease in swine.A ctinobacillus suis is an important opportunistic pathogen of swine (10) that is able to cause disease in animals of all ages. In addition to a common polysaccharide (6), two O and three K serovars of A. suis have been described (11,12,14), and there are several lines of evidence to suggest that some strains have greater virulence potential than others (16,17). A. suis shares many virulence factors (e.g., ApxI and ApxII) with the closely related swine pathogen Actinobacillus pleuropneumoniae (7). Both of these organisms can cause an acute hemorrhagic pleuropneumonia, but A. suis has a broader host range than A. pleuropneumoniae and it can, in addition, cause septicemia, enteritis, meningitis, arthritis, skin lesions, and abortion (10). By comparing the A. suis genome sequence with available A. pleuropneumoniae sequences, we hope to identify genetic differences that begin to explain the unique tissue and host specificity of these pathogens and members of the family Pasteurellaceae that colonize the oropharyngeal cavity.Shotgun genome sequencing of the virulent A. suis serovar O2 strain H91-0380 was done by using 454 pyrosequencing at the McGill University and Génome Québec Innovation Centre and assembled using MIRA 3 (8). The contigs were organized by BLASTX analysis of their 3= and 5= ends (13) and by alignment with an OpGen AflII optical map (9). The gaps were closed by long-range PCR and primer walking. Using these approaches, a single contig totaling 2,484,940 bp was assembled and annotated using the NCBI automated prokaryotic genome annotation pipeline (http://www.ncbi.nlm.nih.gov/genomes/static /Pipeline.html); further analysis was done using RAST (1). A 305-bp region flanked by poly(A) repeats was not able to be sequenced despite numerous attempts.The A. suis H91-0380 genome has a GϩC content of 40.24%. It contains 2,249 coding sequences and has six complete rRNA operons. Chromosome alignment using progressiveMauve (5) revealed that the A. suis H91-0380 genome is very similar to that of A. pleuropneumoniae, especially serovar 3; there are many syntenic regions, but large segments have been rearranged.In addition to the apxI and apxII operons, putative virulence factors detected in the A. suis genome include 37 open reading frames (ORFs) associated with iron acquisition and metabolism, including hemoglobin receptor (3) and transferrin receptor (2) proteins, and 40 genes encoding 22 putative fimbrial and afimbrial adhesins, including homologues of a type IV fimbriae operon, a low-GϩC tad locus, genes encoding tangled pili, prepilins, and a fibronectin-binding protein, 11 outer membrane proteins (OMPs), and 5 autotransporters (ATs). Like other members of the family Pasteurellaceae, there is evidence t...

Lichen symbioses are generally thought to be stabilized by the transfer of fixed carbon compounds from a photosynthesizing unicellular symbiont to a fungus. In other fungal symbioses, carbohydrate subsidies correlate with genomic reductions in the number of genes for plant cell wall-degrading enzymes (PCWDEs), but whether this is the case with lichen fungal symbionts (LFSs) is unknown. We predicted genes encoding carbohydrate-active enzymes (CAZymes) and sugar transporters in 17 existing and 29 newly sequenced genomes from across the class Lecanoromycetes, the largest extant clade of LFSs. Despite possessing lower mean numbers of PCWDE genes compared to non-symbiont Ascomycota, all LFS genomes possessed a robust suite of predicted PCWDEs. The largest CAZyme gene numbers, on par with model species such as Penicillium, were retained in genomes from the subclass Ostropomycetidae, which are found in crust lichens with highly specific ecologies. The lowest numbers were in the subclass Lecanoromycetidae, which are symbionts of many generalist macrolichens. Our results suggest that association with phototroph symbionts does not in itself result in functional loss of PCWDEs and that PCWDE losses may have been driven by adaptive processes within the evolution of specific LFS lineages. The inferred capability of some LFSs to access a wide range of carbohydrates suggests that some lichen symbioses may augment fixed CO2 with carbon from external sources.