Characterization of novel glycosyl hydrolases discovered by cell wall glycan directed monoclonal antibody screening and metagenome analysis of maize aerial root mucilage

Abstract

An indigenous maize landrace from the Sierra Mixe region of Oaxaca, Mexico exhibits extensive formation of aerial roots which exude large volumes of a polysaccharide-rich gel matrix or “mucilage” that harbors diazotrophic microbiota. We hypothesize that the mucilage associated microbial community carries out multiple functions, including disassembly of the mucilage polysaccharide. In situ, hydrolytic assay of the mucilage revealed endogenous arabinofuranosidase, galactosidase, fucosidase, mannosidase and xylanase activities. Screening the mucilage against plant cell wall glycan-specific monoclonal antibodies recognized the presence of carbohydrate epitopes of hemicellulosic polysaccharides like xyloglucan (both non-fucosylated and fucosylated), xylan (both substituted and unsubstituted xylan domains) and pectic-arabinogalactans, all of which are potential carbon sources for mucilage microbial residents. Mucilage metagenome annotation using MG-RAST identified the members forming the microbial community, and gene fragments with predicted functions associated with carbohydrate disassembly. Data from the in situ hydrolytic activity and monoclonal antibody screening assays were used to guide the selection of five full length genes with predicted glycosyl hydrolase function from the GenBank database that were similar to gene fragments of high relative abundance in the mucilage metagenomes. These five genes were then synthesized for recombinant production in Escherichia coli. Here we report the characterization of an α-N-arabinofuranosidase (GH51) and an oligosaccharide reducing-end xylanase (GH8) from Flavobacterium johnsoniae; an α-L-fucosidase (GH29) and a xylan β-1,4 xylosidase (GH39) from Spirosoma linguale, and a β-mannosidase (GH2) from Agrobacterium fabrum. Biochemical characterization of these enzymes revealed a β-Mannosidase that also exhibits a secondary activity towards the cleavage of galactosyl residues. We also describe two xylanases (GH8 and GH39) from underexplored glycosyl hydrolase families, one thermostable α-L-Fucosidase (GH29) and a thermostable α-N-Arabinofuranosidase (GH51).

Identification of Nitrogen Fixation Genes in Lactococcus Isolated from Maize Using Population Genomics and Machine Learning

“…Although previous investigations into the carbohydrate utilization capabilities of diazotrophic L. lactis revealed their possession of homologs to a diverse array of GH genes with various monosaccharide specificities [27], Pan-GWAS and RF modeling identified BNF-association for GH homologs with predicted alpha-1,2-mannosidase and reducing-end xylose-releasing exo-oligoxylanase activities that correspond to GH families 92 and 8, respectively (Table S4). While the determined importance of these specific activities suggests that mucilage-associated L. lactis may utilize xylose and mannose to generate energy for BNF, the detection of these genes by prediction methods used here also corroborates a previous study reporting a high relative abundance of genes conferring these specific activities in the mucilage microbiome [91]. Taken together, the identification of these two GH homologs as important factors associated with the BNF phenotype supports the hypothesis that diazotrophic L. lactis derive energy for BNF from mucilage polysaccharide derivatives.…”

Identification of quantitative trait loci associated with nitrogen use efficiency in winter wheat

“…Previous studies of cereal crops have searched for novel NUE traits and alleles in adapted breeding materials [15], landraces [16][17], and wheat wild relatives [18]. While these authors have successfully identified QTL, genes, and genotypes conferring high NUE, additional sources of genetic variation likely still exist within currently unexplored germplasm.…”

Fungal communities (Mycobiome) as potential ecological indicators within confluence stretch of Ganges and Yamuna Rivers, India

“…Error correction of the reads was achieved with Canu, to give good quality long reads (Koren et al 2017). The analysis of taxonomic and functional diversity was carried out using MG-RAST (Metagenomic Rapid Annotation using Subsystem Technology) with an E value cutoff 1 × e −5 and sequence identity of 60% (Pozzo et al 2018). Further, the taxonomic and functional analysis was based on the OTU table downloaded from the MG-RAST server.…”