title: Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.
abstract: Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the (13)C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential.
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: Verastegui
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: K
authors.initials: K
authors.lastname: Engel
authors.affiliation: Iogen Corporation, Ottawa, Ontario, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Kolczynski
authors.affiliation: Iogen Corporation, Ottawa, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Mortimer
authors.affiliation: Iogen Corporation, Ottawa, Ontario, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Lavigne
authors.affiliation: Iogen Corporation, Ottawa, Ontario, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Montalibet
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Romantsov
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Hall
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: B J
authors.initials: BJ
authors.lastname: McConkey
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: D R
authors.initials: DR
authors.lastname: Rose
authors.affiliation: Iogen Corporation, Ottawa, Ontario, Canada.
authors.forename: J J
authors.initials: JJ
authors.lastname: Tomashek
authors.affiliation: Iogen Corporation, Ottawa, Ontario, Canada.
authors.forename: B R
authors.initials: BR
authors.lastname: Scott
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada tcharles@uwaterloo.ca jneufeld@uwaterloo.ca.
authors.forename: T C
authors.initials: TC
authors.lastname: Charles
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada tcharles@uwaterloo.ca jneufeld@uwaterloo.ca.
authors.forename: J D
authors.initials: JD
authors.lastname: Neufeld
mesh_terms: D000192:Actinomycetales / Q000145:classification / Q000235:genetics; D042063:Caulobacteraceae / Q000187:drug effects / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D012245:Rhodospirillales / Q000145:classification / Q000235:genetics; D012988:Soil Microbiology