Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria

Pinnell, Lee J.; Dunford, Eric A.; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D.

doi:10.1139/cjm-2014-0193

Cited by 30 publications

(20 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Bacterial cellulose has similar mechanical properties to plant cell walls, particularly in traits correlated to enzymatic degradation, which include comparable polymer length (3,000–9,000 units) and crystallinity (80–90% crystalline) (Chanliaud et al, 2002). Bacterial cellulose was previously used in SIP applications (El Zahar Haichar et al, 2007; Pinnell et al, 2014). …”

Section: Introductionmentioning

confidence: 99%

Long-Term Enrichment of Stress-Tolerant Cellulolytic Soil Populations following Timber Harvesting Evidenced by Multi-Omic Stable Isotope Probing

et al. 2017

View full text Add to dashboard Cite

Soil management is vital for maintaining the productivity of commercial forests, yet the long-term impact of timber harvesting on soil microbial communities remains largely a matter of conjecture. Decomposition of plant biomass, comprised mainly of lignocellulose, has a broad impact on nutrient cycling, microbial activity and physicochemical characteristics of soil. At “Long-term Soil Productivity Study” sites in California dominated by Ponderosa pine, we tested whether clear-cut timber harvesting, accompanied by varying degrees of organic matter (OM) removal, affected the activity and structure of the cellulose-degrading microbial populations 16 years after harvesting. Using a variety of experimental approaches, including stable isotope probing with 13C-labeled cellulose in soil microcosms, we demonstrated that harvesting led to a decrease in net respiration and cellulolytic activity. The decrease in cellulolytic activity was associated with an increased relative abundance of thermophilic, cellulolytic fungi (Chaetomiaceae), coupled with a decreased relative abundance of cellulolytic bacteria, particularly members of Opitutaceae, Caulobacter, and Streptomycetaceae. In general, harvesting led to an increase in stress-tolerant taxa (i.e., also non-cellulolytic taxa), though our results indicated that OM retention mitigated population shifts via buffering against abiotic changes. Stable-isotope probing improved shotgun metagenome assembly by 20-fold and enabled the recovery of 10 metagenome-assembled genomes of cellulolytic bacteria and fungi. Our study demonstrates the putative cellulolytic activity of a number of uncultured taxa and highlights the mineral soil layer as a reservoir of uncharacterized diversity of cellulose-degraders. It also and contributes to a growing body of research showing persistent changes in microbial community structure in the decades following forest harvesting.

show abstract

Section: Introductionmentioning

confidence: 99%

Long-Term Enrichment of Stress-Tolerant Cellulolytic Soil Populations following Timber Harvesting Evidenced by Multi-Omic Stable Isotope Probing

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Each reaction mixture consisted of ~7 ng of DNA template in order to minimize potential amplification bias (26, 30, 76), yielding 3 to 4 µg of amplified DNA. Positive-control DNA from the kit and negative controls without DNA were run in parallel.…”

Section: Methodsmentioning

confidence: 99%

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

Verastegui

Cheng

Engel

et al. 2014

mBio

118

View full text Add to dashboard Cite

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 (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), 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 13C-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.

show abstract

“…The predominant bacterium in the heavy DNA fraction of BHS soil showed 98% identity to Caulobacter henricii. Caulobacteraceae are particularly predominant in cellulose enrichment and SIP experiments (23,34,36,37), and although the cellulolytic ability of Caulobacter henricii appears to be untested, closely related species of Caulobacter can be grown on crystalline cellulose or gluco-oligosaccharides (38,39). The Caulobacter crescentus genome possesses genes encoding cellulases, xylanases, and xylosidases (40).…”

Section: -37) Previous Studies Also Commonly Identifiedmentioning

confidence: 99%

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

Wang

Sharp

Jones

et al. 2015

Appl Environ Microbiol

103

162

View full text Add to dashboard Cite

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of L-guluronic acid, D-glucose, and D-glycero-D-mannoheptose.13 C-labeled EPS and 13 C-labeled cellulose were purified from bacterial cultures grown on [ 13 C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from 13 C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo-or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species. Some major phyla of the domain Bacteria, like the Proteobacteria and Actinobacteria, are well-known from cultivation studies. Others, like Acidobacteria, Verrucomicrobia, and Planctomycetes, have few cultured species, although they make up large proportions of the bacterial 16S rRNA genes detected in environments such as soil (1). One theory proposed to explain the difficulty in culturing bacteria from these phyla is that most are K selected as opposed to r selected. Typical r-selected bacteria grow rapidly on simple monomeric substrates in nutrient-rich environments, conditions presented by most microbiological media (2, 3). On the other hand, K-selected bacteria have a more efficient cell metabolism and strong competitive ability and grow slowly on recalcitrant, complex substances (3-5). Several studies have demonstrated that members of difficult-to-culture phyla like Acidobacteria, Verrucomicrobia, and Planctomycetes can be cultivated using polysaccharides, such as xylan, xanthan, and pectin, as energy sources (e.g., see references 6 to 8). The heteropolysaccharide gellan, which is often used as a gelling agent as an alternative to agar, can also act as an energy source for some soil bacteria (9), in part explaining the observation that it improves cultu...

show abstract

Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria

Cited by 30 publications

References 48 publications

Long-Term Enrichment of Stress-Tolerant Cellulolytic Soil Populations following Timber Harvesting Evidenced by Multi-Omic Stable Isotope Probing

Long-Term Enrichment of Stress-Tolerant Cellulolytic Soil Populations following Timber Harvesting Evidenced by Multi-Omic Stable Isotope Probing

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

Contact Info

Product

Resources

About