Transcriptomic Analyses of Xylan Degradation by Prevotella bryantii and Insights into Energy Acquisition by Xylanolytic Bacteroidetes

Abstract

Enzymatic depolymerization of lignocellulose by microbes in the bovine rumen and the human colon is critical to gut health and function within the host. Prevotella bryantii B 1 4 is a rumen bacterium that efficiently degrades soluble xylan. To identify the genes harnessed by this bacterium to degrade xylan, the transcriptomes of P. bryantii cultured on either wheat arabinoxylan or a mixture of its monosaccharide components were compared by DNA microarray and RNA sequencing approaches. The most highly induced genes formed a cluster that contained putative outer membrane proteins analogous to the starch utilization system identified in the prominent human gut symbiont Bacteroides thetaiotaomicron. The arrangement of genes in the cluster was highly conserved in other xylanolytic Bacteroidetes, suggesting that the mechanism employed by xylan utilizers in this phylum is conserved. A number of genes encoding proteins with unassigned function were also induced on wheat arabinoxylan. Among these proteins, a hypothetical protein with low similarity to glycoside hydrolases was shown to possess endoxylanase activity and subsequently assigned to glycoside hydrolase family 5. The enzyme was designated PbXyn5A. Two of the most similar proteins to PbXyn5A were hypothetical proteins from human colonic Bacteroides spp., and when expressed each protein exhibited endoxylanase activity. By using site-directed mutagenesis, we identified two amino acid residues that likely serve as the catalytic acid/base and nucleophile as in other GH5 proteins. This study therefore provides insights into capture of energy by xylanolytic Bacteroidetes and the application of their enzymes as a resource in the biofuel industry.

Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

“…This task is made easier by the chromosomal arrangement of these genes, which are generally clustered on the chromosome (in both an operon-dependent and an operon-independent fashion), which would ease cloning and heterologous expression. Studies of other hemicellulolytic bacteria have also shown the same general trend in gene clustering (6,7,72,73).…”

“…Similar transcriptomic experiments have been shown to be particularly useful in predicting the function of various mono-and polysaccharide transporters (6,7,72,73). These proteins also have potential applications in biotechnology.…”

Polysaccharide Utilization Loci: Fueling Microbial Communities

“…1) through (meta)genomic, transcriptomic, and proteomic approaches (93)(94)(95)(96). PULs have been identified in species outside the Bacteroides genus in diverse environments, including the ruminal Prevotella bryantii (97), the freshwater dweller Flavobacterium johnsoniae (98), and the gut microbiota of the termite Pseudacanthotermes militaris (99). Distinctly, PULs from the gliding soil bacterium Cytophaga hutchinsonii lack individual susC-susD pairs, but instead, two such pairs are encoded elsewhere in the genome (100).…”

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

“…GH9 family enzymes have an (␣/␣) 6 -barrel fold, and this family of enzymes is known to contain members that exhibit different enzymatic activities, including endoglucanase (45), cellobiohydrolase (15), 1,4-␤-D-glucan glucohydrolase (43), ␤-glucosidase (40), and exo-␤-glucosaminidase (23). The GH5 family of enzymes have an (␤/␣) 8 -barrel fold, and this family also encodes diverse enzymatic activities, including endoglucanase (16), mannanase (20), mannosidase (20), xylanase (14), chitosanase (50), glucosidase (11), licheninase (25), fucosidase (61), galactanase (44), endoglycoceramidase (9), and mannan transglycosylase (6). The binding cleft in GH9 catalytic modules can hold six sugar molecules, among which four subsites (Ϫ4 to Ϫ1) are at the nonreducing end and the two remaining subsites are at the reducing end (ϩ1 to ϩ2) (45).…”