Phylum level comparison of rumen microbiome associated with solid and liquid rumen phase from white tailed deer (n = 3) and elk (n = 15).

<p>Values shown represent the percent of sequences assigned to a particular phylum averaged over all samples of the same type.</p

Principle component analysis of 16S profiles from rumen contents collected from elk (Solid-blue, Liquid-Black), white tailed deer (Solid-Red, Liquid-Green).

<p>Principle component analysis of 16S profiles from rumen contents collected from elk (Solid-blue, Liquid-Black), white tailed deer (Solid-Red, Liquid-Green).</p

Diversity of Rumen Bacteria in Canadian Cervids

<div><p>Interest in the bacteria responsible for the breakdown of lignocellulosic feedstuffs within the rumen has increased due to their potential utility in industrial applications. To date, most studies have focused on bacteria from domesticated ruminants. We have expanded the knowledge of the microbial ecology of ruminants by examining the bacterial populations found in the rumen of non-domesticated ruminants found in Canada. Next-generation sequencing of 16S rDNA was employed to characterize the liquid and solid-associated bacterial communities in the rumen of elk (<i>Cervus canadensis</i>), and white tailed deer (<i>Odocoileus virginianus</i>). Despite variability in the microbial populations between animals, principle component and weighted UniFrac analysis indicated that bacterial communities in the rumen of elk and white tail deer are distinct. Populations clustered according to individual host animal and not the association with liquid or solid phase of the rumen contents. In all instances, <i>Bacteroidetes</i> and <i>Firmicutes</i> were the dominant bacterial phyla, although the relative abundance of these differed among ruminant species and between phases of rumen digesta, respectively. In the elk samples <i>Bacteroidetes</i> were more predominant in the liquid phase whereas <i>Firmicutes</i> was the most prevalent phyla in the solid digesta (P = 1×10⁻⁵). There were also statistically significant differences in the abundance of OTUs classified as <i>Fibrobacteres</i> (P = 5×10⁻³) and <i>Spirochaetes</i> (P = 3×10⁻⁴) in the solid digesta of the elk samples. We identified a number of OTUs that were classified as phylotypes not previously observed in the rumen environment. Our results suggest that although the bacterial diversity in wild North American ruminants shows overall similarities to domesticated ruminants, we observed a number of OTUs not previously described. Previous studies primarily focusing on domesticated ruminants do not fully represent the microbial diversity of the rumen and studies focusing on non-domesticated ruminants should be expanded.</p></div

Weighted-UniFrac comparison of microbial communities found in rumen samples based on Jaccard analysis of 97% similarity.

<p>The P-value is a measure of the significance with which the microbial communities compared differ. P-values of <0.001 are highly significant, 0.001–0.01 are significant, 0.01–0.05 are marginally significant, 0.05–0.1 are suggestive and >0.1 is not significant. P-value ranges indicate the upper and lower limits of the calculation when multiple samples were compared.</p

Microbial composition of wild ruminants assessed using Jaccard analysis of OTUs at 97% identity.

<p>Samples are labeled animal elk or white tailed deer - animal number - S (rumen solids) or L (rumen liquids).</p

Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

<div><p>Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The <i>Arabidopsis thaliana</i> genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the <i>At</i>BBE-like protein family (termed <i>At</i>BBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in <i>At</i>BBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for <i>At</i>BBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in <i>At</i>BBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.</p></div

<i>Ephedra sinica</i> unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.

<p>Each unigene is assigned an identifier, which corresponds to a database ID in the ESI-Velvet library. Percent amino acid identity between unigenes and queries is provided. Abbreviations: AAO4, aromatic aldehyde oxidase 4; Ab, <i>Atropa belladonna</i>; Am, <i>Antirrhinum majus</i>; AHAS, acetohydroxyacid synthase; ArAT, aromatic amino acid transaminase; At, <i>Arabidopsis thaliana</i>; BALDH, benzaldehyde dehydrogenase; BDH, benzaldehyde dehydrogenase; BL; benzoate CoA ligase; BZO, benzoyloxyglucosinolate; Ca, <i>Caffea arabica</i>; Ce, <i>Catha edulis</i>; CHD, cinnamoyl-CoA hydratase-dehydrogenase; 4CL, 4-coumaroyl-CoA ligase; Cm, <i>Cucumis melo</i>; CS, caffeine synthase; COR, codeinone reductase; Ds, <i>Datura stramonium</i>; Ec, <i>Eschscholzia californica</i>; Es, <i>Ephedra sinica</i>; KAT, 3-ketoacyl-CoA thiolasae; NMT, <i>N</i>-methyltransferase; PEANMT, phosphoethanolamine <i>N</i>-methyltransferase; PMT, putrescine <i>N</i>-methyltransferase; Ps, <i>Papaver somniferum</i>; PAL, L-phenylalanine ammonia lyase; PDC, pyruvate decarboxylase; Ph, <i>Petunia x hybrida</i>; PPA-AT, prephenate aminotransferase; PRMT, protein arginine <i>N</i>-methyltransferase; Pt, <i>Pinus taeda</i>; RED, reductase; SanR, sanguinarine reductase; Sl, <i>Solanum lycopersicon</i>; SUVH, histone lysine <i>N</i>-methyltransferase, H3L9-specific; TA, transaminase; ThDPC, thiamin diphosphate-dependent carboligase; TNMT, (<i>S</i>)-tetrahydroprotoberberine <i>N</i>-methyltransferase; TR, tropinone reducase.</p><p><i>Ephedra sinica</i> unigenes representing enzymes putatively involved in ephedrine alkaloid biosynthesis.</p

Transcriptome Profiling of Khat (<i>Catha edulis</i>) and <i>Ephedra sinica</i> Reveals Gene Candidates Potentially Involved in Amphetamine-Type Alkaloid Biosynthesis

<div><p>Amphetamine analogues are produced by plants in the genus <i>Ephedra</i> and by khat (<i>Catha edulis</i>), and include the widely used decongestants and appetite suppressants (1<i>S</i>,2<i>S</i>)-pseudoephedrine and (1<i>R</i>,2<i>S</i>)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and <i>Ephedra</i> species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for <i>Ephedra sinica</i> and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications.</p></div

Overlay of <i>At</i>BBE-like 28 and Phl p 4 I153V N158H.

<p>A: <i>re</i>-side: <i>At</i>BBE-like 28 is shown in green and yellow, Phl p4 (PDB: 4PWC) is shown in magenta. The chloride ion embedded in <i>At</i>BBE-like 28 is shown in green, bromide from Phl p 4 is shown in red. The oxygen pocket is highly conserved and in both structures occupied by oxygen surrogating halide ions. In Phl p4 the halide ion is complexed by the nitrogen of the peptide bond between Cys150 and Val149 and the nitrogen of the peptide bond between Val149 and Gly148. In <i>At</i>BBE-like 28 the halide ion is complexed by the corresponding residues, additionally His174 and Gln182 are involved in hydrogen bonds towards the chloride ion. B: <i>si</i>-side: The positions of the active site forming residues are conserved. Their nature has been changed, leading to two different active sites embedded in a very similar protein scaffold. Though the position of the isoalloxazine ring is highly conserved in the BBE-like family the C4 C6 axis of the plane N10 C4 C6 has been shifted 32° resulting in a displacement of N5 by 1.6 Å.</p