University of Illinois Urbana-Champaign

Degradation of xylan by rumen Prevotella SPP

Dodd, Dylan

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Dodd_Dylan.pdf

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https://hdl.handle.net/2142/15567

Description

Title
Degradation of xylan by rumen Prevotella SPP
Author(s)
Dodd, Dylan
Issue Date
2010-05-14T20:50:40Z
Director of Research (if dissertation) or Advisor (if thesis)
Cann, Isaac K.
Doctoral Committee Chair(s)
Cann, Isaac K.O.
Committee Member(s)
  • Salyers, Abigail A.
  • Olsen, Gary J.
  • Orlean, Peter A.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2010-05-14T20:50:40Z
Keyword(s)
  • biofuels
  • rumen
  • microbiota
  • colon
  • xylan
  • xylanase
  • xylosidase
  • glucosidase
  • RNAseq
  • Prevotella
  • Bacteroides
  • Bacillus anthracis
  • glutamate racemase
  • alanine racemase
Abstract
Prevotella ruminicola 23 and Prevotella bryantii B14 are obligate anaerobic bacteria in the Bacteroidetes phylum that contribute to hemicellulose utilization within the bovine rumen. These bacteria efficiently degrade the hemicellulosic polymer, xylan, however the biochemical basis for deconstruction of this polysaccharide remains largely unknown. In the current study, genomic, transcriptomic, and biochemical analyses were employed to gain insight into the cellular machinery that these two bacteria elaborate to degrade the hemicellulosic polymer, xylan. These studies identified two previously uncharacterized glycoside hydrolase proteins from P. ruminicola 23: a bi-functional xylanase/ferulic acid esterase (xyn10D-fae1A) and a bi-functional beta-D-xylosidase/alpha-L-arabinofuranosidase (xyl3A). These two genes were expressed as recombinant proteins in Escherichia coli, and biochemical analyses of the purified proteins revealed that these two enzymes function synergistically to depolymerize xylan. Furthermore, directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. Subsequent bioinformatics studies with P. bryantii B14 identified four glycoside hydrolase (GH) family 3 genes in the genome for this organism. To test whether or not these genes encode proteins with redundant biochemical functions, the four genes were cloned and over-expressed as recombinant proteins in E. coli, and the biochemical properties of these proteins were assessed. The results from these studies indicated that each of the four purified proteins exhibited unique properties with one gene exhibiting beta-D-glucosidase activity, and the remaining three exhibiting beta-D-xylosidase activity. Further probing of the biochemical properties for the three beta-D-xylosidase enzymes identified salient differences in substrate specificities for these enzymes. Whole genome transcriptional profiling of P. bryantii cultures grown either on polymeric wheat arabinoxylan (WAX) or the constituent monosaccharides, xylose and arabinose (XA) identified a subset of genes that are highly induced on WAX compared to XA. The most highly induced genes formed an operon which contained putative outer membrane proteins analogous to the starch utilization system (Sus) previously identified in the prominent human gut symbiont, Bacteroides thetaiotaomicron. The arrangement of this gene cluster is highly conserved in other xylanolytic organisms within the Bacteroidetes phylum which suggests that the mechanism of xylan utilization involving these genes is conserved amongst these bacteria. A large number of genes encoding proteins of unknown function were also induced on WAX relative to XA which suggests that P. bryantii B14 may employ novel mechanisms for xylan degradation. A hypothetical gene predicted to encode a protein with low homology to glycoside hydrolase (GH) family 5 was upregulated during growth on WAX, and was cloned and heterologously expressed as a hexahistidine fusion protein in E. coli. Biochemical analysis of the purified protein revealed that it possesses endo-xylanase activity thus the enzyme was named PbXyn5A. Two of the most similar proteins in the GenBank database derive from the human colonic Bacteroides spp. (B. eggerthii ORF1299 and B. intestinalis ORF4213) and are also annotated as hypothetical proteins. The corresponding genes were expressed and the purified recombinant proteins also exhibited robust endo-xylanase activity. Mutational analysis revealed two carboxylic acid residues that were essential for activity in all three enzymes. These glutamic acid residues are conserved amongst the distantly related members of the GH family 5 which supports the assignment of these proteins as GH family 5 endo-xylanases. This study provides insight into polysaccharide degradation by P. bryantii B14 and reveals similarities in the mechanism for xylan degradation between human colonic and ruminant Bacteroidetes.
Graduation Semester
2010-5
Permalink
http://hdl.handle.net/2142/15567
Copyright and License Information
Copyright 2010 DYLAN DODD

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