title: Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.
abstract: Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.
authors.affiliation: a Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
authors.forename: Lee J
authors.initials: LJ
authors.lastname: Pinnell
authors.forename: Eric
authors.initials: E
authors.lastname: Dunford
authors.forename: Patrick
authors.initials: P
authors.lastname: Ronan
authors.forename: Martina
authors.initials: M
authors.lastname: Hausner
authors.forename: Josh D
authors.initials: JD
authors.lastname: Neufeld
mesh_terms: D020561:Alphaproteobacteria / Q000201:enzymology / Q000235:genetics; D001110:Arctic Regions; D001419:Bacteria / Q000201:enzymology / Q000235:genetics*; D041963:Bacteroidetes / Q000201:enzymology / Q000235:genetics; D020562:Betaproteobacteria / Q000201:enzymology / Q000235:genetics; D002247:Carbon Isotopes; D002482:Cellulose / Q000378:metabolism*; D041862:Chloroflexi / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015723:Gene Library; D006026:Glycoside Hydrolases / Q000235:genetics*; D056186:Metagenomics; D020957:Nunavut; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012987:Soil / Q000737:chemistry*; D012988:Soil Microbiology
chemical_list: D002247:Carbon Isotopes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012987:Soil; D002482:Cellulose; D006026:Glycoside Hydrolases
keywords: Arctic; arctique; cellulose; metagenomics; métagénomique; soil; sol; stable-isotope probing; toundra; traçage par isotope stable; tundra