title: A polysaccharide utilization locus from an uncultured bacteroidetes phylotype suggests ecological adaptation and substrate versatility.
abstract: Recent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an uncultured Bacteroidetes phylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: A K
authors.initials: AK
authors.lastname: Mackenzie
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: A E
authors.initials: AE
authors.lastname: Naas
authors.affiliation: Department of Plant Biology and Biotechnology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: S K
authors.initials: SK
authors.lastname: Kracun
authors.affiliation: Department of Plant Biology and Biotechnology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Schückel
authors.affiliation: Department of Plant Biology and Biotechnology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: J U
authors.initials: JU
authors.lastname: Fangel
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: J W
authors.initials: JW
authors.lastname: Agger
authors.affiliation: Department of Plant Biology and Biotechnology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: W G T
authors.initials: WG
authors.lastname: Willats
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: V G H
authors.initials: VG
authors.lastname: Eijsink
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway phil.pope@nmbu.no.
authors.forename: P B
authors.initials: PB
authors.lastname: Pope
mesh_terms: D000220:Adaptation, Biological; D000818:Animals; D041963:Bacteroidetes / Q000235:genetics* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D055664:Electrochemical Techniques; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D056186:Metagenomics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D012053:Reindeer; D012417:Rumen / Q000382:microbiology; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013538:Svalbard
chemical_list: D011134:Polysaccharides; D006026:Glycoside Hydrolases