scholarly journals Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Y. Verastegui ◽  
J. Cheng ◽  
K. Engel ◽  
D. Kolczynski ◽  
S. Mortimer ◽  
...  

ABSTRACTSoil 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 (12C) or stable-isotope-labeled (13C) 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), andCaulobacterales(PhenylobacteriumandAsticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. BothActinomycetalesandCaulobacterales(Phenylobacterium) were associated with metabolism of cellulose, andAlphaproteobacteriawere associated with the metabolism of arabinose; members of the orderRhizobialeswere 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 the13C-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.IMPORTANCEThe 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.

2014 ◽  
Vol 60 (7) ◽  
pp. 469-476 ◽  
Author(s):  
Lee J. Pinnell ◽  
Eric Dunford ◽  
Patrick Ronan ◽  
Martina Hausner ◽  
Josh D. Neufeld

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 [13C]glucose and [13C]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.


2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yu-Te Lin ◽  
Yu-Fei Lin ◽  
Isheng J. Tsai ◽  
Ed-Haun Chang ◽  
Shih-Hao Jien ◽  
...  

2021 ◽  
Vol 309 ◽  
pp. 107285
Author(s):  
Mengyu Gao ◽  
Jinfeng Yang ◽  
Chunmei Liu ◽  
Bowen Gu ◽  
Meng Han ◽  
...  

CATENA ◽  
2022 ◽  
Vol 209 ◽  
pp. 105828
Author(s):  
Chao Yang ◽  
Kangjia Li ◽  
Jipeng Sun ◽  
Weiyi Ye ◽  
Hao Lin ◽  
...  

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