Capacity of hoofed artiodactyl distal gut Bacteroides spp. to saccharify brown seaweed polysaccharides

Abstract

The ruminant gastrointestinal tract (GIT) contains a highly diverse microbial ecosystem in terms of both species and functional capacity. This community, whose evolutionary history is closely tied to that of their host, facilitates the digestion of recalcitrant plant materials through encoded carbohydrate active enzymes (CAZymes). The ability for ruminants to transform human-indigestible plant material into meat and milk have made them an invaluable asset to humans, with the demand for these products continually increasing in response to human population growth, affluence, and dietary preferences. The composition of the GIT microbiota is consequential for the host in a number of ways, including feed efficiency, health, and enteric methane emission. Thus, manipulating the structure of this community has been an area of interest for the livestock industry. Alternative feedstocks hold great potential in this regard. Macroalgae, or seaweeds, have been an attractive potential feed additive due to some species having anti-methanogenic properties. However, little is still known about the digestion of seaweed cell walls, which are distinct from those of terrestrial plants, by the GIT microbiota and seaweeds are commonly assumed to be non-digestible/non-nutritive. The discovery of CAZymes associated with seaweed polysaccharide saccharification in the human GIT microbiota suggests that these genes penetrated the mammalian gut microbiome via horizontal gene transfer (HGT) from seaweed-associated marine bacteria, and that this acquisition may have been an ancient event. Recent work in the Abbott Lab demonstrated that genes for CAZymes targeting carrageenan, a red seaweed polysaccharide, exist within the GIT microbiomes of geographically and taxonomically diverse ruminant and nonruminant animals. Similarly, cattle and Norwegian White lamb GIT microbiomes have been shown to saccharify alginate, a brown seaweed polysaccharide. These studies have cumulated in the proposal of the “latent traits hypothesis”, suggesting that seaweed-targeting CAZymes are ubiquitous in the animal GIT microbiota and persist in the absence of substrate, likely carried in the genomes of generalist bacterial members present in low abundance. This work raises questions about the evolution of dietary adaptations, timescales associated with such an acquisition, and the source of relevant GIT microbiota and CAZymes. Currently, it is unknown whether other ruminant species have the same plasticity in adapting to alginate, and if so, how this is accomplished. The thesis presented here investigates whether other ruminants and closely related pseudoruminants from order Artiodactyla harbor microorganisms and CAZymes capable of degrading brown seaweed polysaccharides. Fecal samples were collected from ruminants and pseudoruminants (n=1) at the Wilder Institute/Calgary Zoo and Saskatoon Forestry Farm Park & Zoo, and selectively enriched for microorganisms capable of growth on alginate. Genomic analysis of fecal community metagenomic data and bacterial isolate DNA revealed an alginate utilization pathway that remains conserved amongst all bacterial isolates from 4 different ruminant host species; Masai giraffe, rocky mountain goat, bighorn sheep, and moose. CAZymes identified in this pathway, including BxPLX, a sequence from Bacteroides xylanisolvens (Bx) and potential member of a new polysaccharide lyase (PL) family, are predicted to be involved in the saccharification of alginate. The crystal structure of BxPLX was solved in complex with potential substrates. In addition, the activities of the other PLs from this Alginate Utilization Pathway (AUL) were characterized by thin layer chromatography (TLC), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and NMR spectroscopy. The outcomes of this project will expand our knowledge of alginate-specific PLs, as well as provide new evolutionary and functional insights into the ability of ruminants and pseudoruminants to digest seaweed polysaccharides.