TY - JOUR
T1 - Enrichment of root endophytic bacteria from populus deltoides and single-cell-genomics analysis
AU - Utturkar, Sagar M.
AU - Cude, W. Nathan
AU - Robeson, Michael S.
AU - Yang, Zamin K.
AU - Klingeman, Dawn M.
AU - Land, Miriam L.
AU - Allman, Steve L.
AU - Lu, Tse Yuan S.
AU - Brown, Steven D.
AU - Schadt, Christopher W.
AU - Podar, Mircea
AU - Doktycz, Mitchel J.
AU - Pelletier, Dale A.
N1 - Publisher Copyright:
© 2016, American Society for Microbiology. All Rights Reserved.
PY - 2016
Y1 - 2016
N2 - 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. Here, we describe 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. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.
AB - 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. Here, we describe 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. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria.
UR - http://www.scopus.com/inward/record.url?scp=84987861937&partnerID=8YFLogxK
U2 - 10.1128/AEM.01285-16
DO - 10.1128/AEM.01285-16
M3 - Article
C2 - 27422831
AN - SCOPUS:84987861937
SN - 0099-2240
VL - 82
SP - 5698
EP - 5708
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 18
ER -