Diversity of Glycosyl Hydrolases from Cellulose-Depleting Communities Enriched from Casts of Two Earthworm Species

Abstract

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-␤-glucanases, ␤-glucosidases, ␤-cellobiohydrolases, ␤-galactosidase, and ␤-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of ␤-galactosidases/␣-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and activesite architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).

The gut microbiota of the wood-feeding termite Reticulitermes lucifugus (Isoptera; Rhinotermitidae)

“…To our knowledge, only one member of the genus Comamonas has been isolated from a termite thus far (Chou et al 2007). The hydrolytic/cellulolytic activity of members of the family Comamonadaceae has been described for soil bacteria of the genera Variovorax and Delftia (Talia et al 2012); several glycosyl hydrolase-encoding genes originating from Comamonadaceae were recently detected in the metagenome from casts of two earthworm species (Beloqui et al 2010). We were also able to isolate facultative anaerobes of the family Enterobacteriaceae.…”

Metagenomic Analyses: Past and Future Trends

“…As sequence information is not required, this is the only strategy that bears the potential to identify entirely novel classes of genes encoding known or novel functions (18,27,38,42,96). Three different functiondriven approaches have been used to recover novel biomolecules: phenotypical detection of the desired activity (7,38,68), heterologous complementation of host strains or mutants (13,95,109,137), and induced gene expression (128,129,141).…”

“…In this way, 19 positive clones, of which one exhibited strong ␤-D-glucuronidase activity after cloning of the corresponding gene into an expression vector, were detected (38). Another example for an activity-based screen by direct detection of phenotypes was published by Beloqui et al (7). In order to identify novel glycosyl hydrolases, E. coli clones harboring metagenomic fosmid libraries derived from cellulose-depleting microbial communities of a fresh cast of earthworms were screened for their ability to hydrolyze p-nitrophenyl-␤-D-glucopyranoside and p-nitrophenyl-␣-L-arabinopyranoside.…”

Predominance of Trichoderma and Penicillium in cellulolytic aerobic filamentous fungi from subtropical and tropical forests in China, and their use in finding highly efficient β-glucosidase

“…In addition, β-glucosidase activities are generally measured using artificial substrates such as p-nitrophenyl-β-D-glycopyranoside (pNPG) or 4-methylumbelliferyl-β-D-glucuronide. When using these artificial substrates, most enzymes show higher catalytic activity toward artificial substrates than toward cellobiose [16][17][18][19][20]. However, UeBgl3A from Ustilago esculenta and a recombinant β-glucosidase from Caldicellulosiruptor saccharolyticus are exceptions, with higher catalytic activity toward cellobiose than toward artificial substrate [21,22].…”