Galactomannan Catabolism Conferred by a Polysaccharide Utilization Locus of Bacteroides ovatus

Abstract

A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) β-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by β-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two β-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (−1 and −2 subsites), including a conserved loop closing the active site beyond subsite −2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized β-mannanases, we propose a scheme of sequential action by the glycoside hydrolases encoded by the β-mannan PUL and involved in the β-mannan utilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the β-mannanase BoMan26A.

Alpha‐ and beta‐mannan utilization by marine Bacteroidetes

“…Based on the proteogenomic analyses in this study we are able to propose distinct pathways for α‐ and β‐mannan utilization in our marine model bacteria that are similar but not identical to those proposed by Cuskin and colleagues () and Bågenholm and colleagues () respectively. In the putative β‐mannan degradation pathways of Salegentibacter sp.…”

“…Hel_I_6, Leeuwenhoekiella sp. MAR_2009_132 and related PULs from Bacteroides ovatus (Bågenholm et al ., ). B .…”

“…We show that, even though marine polysaccharides often differ from their land plant counterparts, marine bacteria possess mannan‐inducible PULs that are highly conserved and resemble mannan‐specific PULs in human gut bacteria. Based on the data gathered on the mannan‐inducible proteins in this study and published homologues in human intestine Bacteroides (Cuskin et al ., ; Bågenholm et al ., ) we propose models for α‐ and β‐mannan degradation pathways in these two marine Bacteroidetes strains.…”

Molecular Mechanism by which Prominent Human Gut Bacteroidetes Utilize Mixed-Linkage Beta-Glucans, Major Health-Promoting Cereal Polysaccharides

“…Recently, comprehensive functional analysis has revealed the detailed molecular mechanisms by which individual PULs enable human gut Bacteroidetes to utilize predominant plant polysaccharides, including the matrix glycans, xyloglucan (Hemsworth et al, 2016; Larsbrink et al, 2014; Tauzin et al, 2016), xylan (Rogowski et al, 2015), β-mannan (Bågenholm et al, 2017), and rhamnogalacturonan II (Ndeh et al, 2017). Mixed-linkage β(1,3)/β(1,4)-glucans (MLGs, Fig.…”

Polysaccharide Utilization Loci: Fueling Microbial Communities

“…A pair of xylan-targeting PULs from B. ovatus, PUL-XylS and PUL-XylL, was found to encode enzymes tailored for individual plant ␤-xylans that varied in their composition and branching (79). Recently, the detailed genetic, biochemical, and enzyme structural characterization of a galactomannanspecific PUL from B. ovatus revealed the interplay of two mannan-specific SGBPs, two GH26 endo-␤-mannanases, and a GH36 exo-␣-galactosidase in the deconstruction of this plant cell wall polysaccharide (110,111). Among environmental bacteria, a complex chitin utilization locus from Flavobacterium johnsoniae has been extensively functionally characterized (112).…”