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-beta-glucanases, beta-glucosidases, beta-cellobiohydrolases, beta-galactosidase, and beta-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 beta-galactosidases/alpha-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 active-site 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).

FIG. 1.

GH-related domains in functionally characterized GHs. Some proteins exhibit a multidomain architecture. Serine-rich regions are highlighted.

FIG. 2.

Dendrogram of compositional sequence similarities calculated by comparison of frequencies of tetranucleotides in DNA fragments from fosmids of L. terrestris and A. caliginosa libraries versus fully sequenced bacterial chromosomes (chr) and plasmids.

FIG. 3.

Phylogenetic analysis of bacterial 16S rRNA gene fragments ca. 600 to 620 bp in length from enrichments originating from A. caliginosa and L. terrestris. The tree was constructed by the neighbor-joining method. Clones sequenced in this work are in bold. Sequences with identities of >99.5% were taken as belonging to a single OTU and are shown as one ID number. Values in parentheses are numbers of clones. Numbers at nodes are bootstrap values greater than 50% and were calculated by the Kimura two-parameter, maximum-likelihood, and Bayesian treeing methods, respectively; branching topologies that are not supported by either method are marked by a dash at the corresponding node.

FIG. 4.

Phylogenetic affiliations of bacterial 16S and eukaryotic 28S rRNA clones in the libraries derived from cellulose enrichment cultures established with casts of L. terrestris and A. caliginosa as inocula.