Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics
Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnolog...
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University of Waterloo
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ftunivwaterloo:oai:uwspace.uwaterloo.ca:10012/8282 2023-05-15T18:40:17+02:00 Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics Verastegui Pena, Yris Milusqui 2014-02-14 http://hdl.handle.net/10012/8282 en eng University of Waterloo http://hdl.handle.net/10012/8282 Cellulose tundra soil temperate rainforest soil agricultural soil stable-isotope probing glycosyl hydrolases metagenomics multiple displacement amplification denaturing gradient gel electrophoresis 16S rRNA gene high through-put sequencing cosmid library Biology Master Thesis 2014 ftunivwaterloo 2022-06-18T23:00:00Z Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial “indicator species” for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were ... Master Thesis Tundra University of Waterloo, Canada: Institutional Repository |
institution |
Open Polar |
collection |
University of Waterloo, Canada: Institutional Repository |
op_collection_id |
ftunivwaterloo |
language |
English |
topic |
Cellulose tundra soil temperate rainforest soil agricultural soil stable-isotope probing glycosyl hydrolases metagenomics multiple displacement amplification denaturing gradient gel electrophoresis 16S rRNA gene high through-put sequencing cosmid library Biology |
spellingShingle |
Cellulose tundra soil temperate rainforest soil agricultural soil stable-isotope probing glycosyl hydrolases metagenomics multiple displacement amplification denaturing gradient gel electrophoresis 16S rRNA gene high through-put sequencing cosmid library Biology Verastegui Pena, Yris Milusqui Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics |
topic_facet |
Cellulose tundra soil temperate rainforest soil agricultural soil stable-isotope probing glycosyl hydrolases metagenomics multiple displacement amplification denaturing gradient gel electrophoresis 16S rRNA gene high through-put sequencing cosmid library Biology |
description |
Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial “indicator species” for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were ... |
format |
Master Thesis |
author |
Verastegui Pena, Yris Milusqui |
author_facet |
Verastegui Pena, Yris Milusqui |
author_sort |
Verastegui Pena, Yris Milusqui |
title |
Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics |
title_short |
Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics |
title_full |
Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics |
title_fullStr |
Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics |
title_full_unstemmed |
Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics |
title_sort |
targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics |
publisher |
University of Waterloo |
publishDate |
2014 |
url |
http://hdl.handle.net/10012/8282 |
genre |
Tundra |
genre_facet |
Tundra |
op_relation |
http://hdl.handle.net/10012/8282 |
_version_ |
1766229585648680960 |