BioProject

The diversity and ecology of cellulose decomposers in forest soils has remained largely uncharted due to the challenges to studying highly complex soil communities. These communities are integral to carbon and nutrient cycling and, from a biotechnological perspective, represent a reservoir of novel enzymes for biomass conversion. We used stable isotope probing (SIP) to investigate the cellulolytic community, tracing the incorporation of 13C-labeled cellulose into microbial biomass in soil microcosms. We utilized phylogenetic gene markers (16S rRNA and ITS) and metagenomic libraries to analyze the 13C-enriched DNA, coupled with analysis of genomes of cellulose-degrading bacteria that we isolated and from single-cell whole-genome amplification. We observed a number of organisms exhibiting significant cellulolytic activity, both bacteria (Streptomycetaceae, Oxalobacteracea, Janthinobacterium, Spartobacteria) and fungi (Sebacinales, Orbiliaceae, Chaetomium). We found broad differences between the cellulolytic community of organic and mineral soil layers, with taxa from the mineral soil mainly associated with novel, poorly characterized phyla (Verrucomicrobia, TM7 and FBP) or unclassifiable. SIP significantly improved metagenomic assembly by reducing community complexity, as <1% of reads from controls were assembled compared to 15-27% of reads from 13C-enriched DNA. The abundance of carbohydrate-active enzymes (CAZymes) in metagenomes derived from 13C-enriched DNA was four-fold greater than in controls. Many of the CAZymes more abundant in 13C-enriched metagenomes (9 out of 27) were classified as endo-cellulolytic, while some of the most highly enriched (between 10 and 33-fold) are previously unreported CAZymes, representing strong candidate cellulases or accessory enzymes for cellulase activity. Peroxidase genes, typically involved in lignin decomposition, were also enriched (14-fold more abundant), supporting the hypothesis that cellulolytic organisms commonly possess lignin-modifying enzymes. Our research yielded approximately one hundred taxonomically diverse, full-length CAZyme genes that are functionally-linked to cellulose degradation, which we have begun characterizing in vitro. We demonstrated the efficacy of SIP plus bioinformatic approaches for targeted discovery of cellulases using ecological principles. And, as a result, we also produced one of the most comprehensive accounts, to date, of the major cellulolytic fungi and bacteria from soil, revealing the role of a number of uncultured organisms.