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Title: Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis

Abstract

Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. In this paper, we present a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Finally, comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.

Authors:
 [1];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1324106
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 82; Journal Issue: 18; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES

Citation Formats

Utturkar, Sagar M., Cude, W. Nathan, Robeson, Jr., Michael S., Yang, Zamin Koo, Klingeman, Dawn Marie, Land, Miriam L., Allman, Steve L., Lu, Tse-Yuan S., Brown, Steven D., Schadt, Christopher Warren, Podar, Mircea, Doktycz, Mitchel J., and Pelletier, Dale A. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis. United States: N. p., 2016. Web. doi:10.1128/AEM.01285-16.
Utturkar, Sagar M., Cude, W. Nathan, Robeson, Jr., Michael S., Yang, Zamin Koo, Klingeman, Dawn Marie, Land, Miriam L., Allman, Steve L., Lu, Tse-Yuan S., Brown, Steven D., Schadt, Christopher Warren, Podar, Mircea, Doktycz, Mitchel J., & Pelletier, Dale A. Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis. United States. doi:10.1128/AEM.01285-16.
Utturkar, Sagar M., Cude, W. Nathan, Robeson, Jr., Michael S., Yang, Zamin Koo, Klingeman, Dawn Marie, Land, Miriam L., Allman, Steve L., Lu, Tse-Yuan S., Brown, Steven D., Schadt, Christopher Warren, Podar, Mircea, Doktycz, Mitchel J., and Pelletier, Dale A. Fri . "Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis". United States. doi:10.1128/AEM.01285-16. https://www.osti.gov/servlets/purl/1324106.
@article{osti_1324106,
title = {Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis},
author = {Utturkar, Sagar M. and Cude, W. Nathan and Robeson, Jr., Michael S. and Yang, Zamin Koo and Klingeman, Dawn Marie and Land, Miriam L. and Allman, Steve L. and Lu, Tse-Yuan S. and Brown, Steven D. and Schadt, Christopher Warren and Podar, Mircea and Doktycz, Mitchel J. and Pelletier, Dale A.},
abstractNote = {Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. In this paper, we present a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Finally, comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.},
doi = {10.1128/AEM.01285-16},
journal = {Applied and Environmental Microbiology},
number = 18,
volume = 82,
place = {United States},
year = {2016},
month = {7}
}

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