Enrichment of root endophytic bacteria from Populus deltoides and single-cell genomics analysis
Sagar M. Utturkar, W. Nathan Cude, Michael S. Robeson, Zamin K. Yang, Dawn M. Klingeman, Miriam L. Land, Steve L. Allman, Tse-Yuan S. Lu, Steven D. Brown, Christopher W. Schadt, Mircea Podar, Mitchel J. Doktycz, and Dale A. Pelletier
15 July 2016, Applied and Environmental Microbiology 82(18): 5698-5708, DOI: 10.1128/AEM.01285-16
Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses and directly, or indirectly increase both above- and below-ground biomass. Endophytes are embedded within plant material and physical separation and isolation is a difficult task. 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 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 ability 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 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. Importance Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts.
Utturkar SM, Cude WN, Robeson MS 2nd, Yang ZK, Klingeman DM, Land ML, Allman SL, Lu TS, Brown SD, Schadt CW, Podar M, Doktycz MJ and Pelletier DA. (2016). Enrichment of root endophytic bacteria from Populus deltoides and single-cell genomics analysis. Applied and Environmental Microbiology, DOI: 10.1128/AEM.01285-16