New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis

Abstract

β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.

Identification and Characterization of a Halotolerant, Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota

“…The transformants were pooled and the metagenomic library was plated and screened for lipolytic activity on 3% tributyrin 2ϫ yeast-tryptone (YT) agar plates containing 100 g/ml ampicillin. Beta-glucosidases were sought on 2ϫ YT agar containing 0.5% esculin (Sigma-Aldrich) and 0.1% ammonium iron (III) citrate (Sigma-Aldrich) (12). Cellulolytic activity was detected by plating the library on 2ϫ YT agar plates containing 1 g/liter insoluble synthetic AZCL-HE-cellulose (azurine-cross-linked hydroxypropyl cellulose; Megazyme) and 100 g/ml ampicillin.…”

Host-specific assemblages typify gut microbial communities of related insect species

“…Supporting evidence in this regard would be the discovery of nitrogen fixation in termites (Benemann 1973), its association with hindgut bacteria (Yamada et al 2007; Potrikus & Breznak 1977; Kudo et al 1998; Ohkuma et al 1999), and identification of genes underlying nitrogen fixation and essential amino acid biosynthesis in hindgut bacteria in various termite species (Wertz et al 2012; Isanapong et al 2012). Additionally, ‘lower’ termites and Cryptocercus are aided in their trophic specialization on wood by lignocellulosic hindgut microbes that include both bacteria (Hongoh 2010; Mattéotti et al 2011; Abt et al 2012) and protists (Tartar et al 2009; Scharf et al 2011; Carpenter et al 2011; Tamschick & Radek 2013). These protists are themselves hosts to intra- and extra-cellular bacterial symbionts (Hongoh et al 2008a; Hongoh et al 2008b; Desai & Brune 2012; Strassert et al 2012).…”

In silico mining for alkaline enzymes from metagenomic dna data of gut microbes of the lower termite Coptotermes gestroi in Vietnam

“…It is already well-known that cellulases and hemicellulases are abundant in the symbiotic organisms in the gut of termites to degrade cellulose and hemicellulose [14,17]. Diversity and of lignocellulose-degrading alkaline enzymes and their function in the termite gut microbial community have been reported [17].…”