Abstract
Bacterial endophytes that colonize Populus trees have been shown to contribute to nutrient acquisition, prime immunity responses and directly or indirectly responsible for increase in 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 has been limited due to the predominance of contaminating plant material and DNA. 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 analysis including assembly, contamination removal and completeness estimation was 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 which have been further inferred to determine effective conditions (such as use of complex heteropolysaccharides as energy source, low pH, and antibiotics) which could facilitate future cultivation efforts for these bacteria. In summary, this study provides a proof-of-concept for the modified enrichment protocol for separation and isolation of endophytic bacteria sample, followed by single-cell genomics analysis to select, amplify and analyze genomes of uncultured bacteria.
