Dataset Information

Expression profiling of Cellvibrio japonicus using plant mannans

ABSTRACT: Plant mannans are a component of lignocellulose that may possess diverse compositions changing both in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides, specifically mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has ten predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential components of the mannan degradation apparatus in this bacterium. Transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode Carbohydrate Active Enzymes (CAZymes) when mannan substrates were actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme genes in the context of mannan utilization. Growth analysis of the mutant strains indicated that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α- galactosidase, and mannosidase, respectively, were influential to the deconstruction of galactomannan. Furthermore, our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the efficient and complete degradation of this hemicellulose by bacteria.

ORGANISM(S): Cellvibrio japonicus

PROVIDER: GSE206864 | GEO | 2022/06/28

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Background: Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to an innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved. Results: Combined bioinformatic and transcriptomic analyses implicated three Glycoside Hydrolase Family 5 Subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here. Conclusions: Through a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural-functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatics predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.

Dataset Information

Expression profiling of Cellvibrio japonicus using plant mannans

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets