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. 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.

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.

FIG 1

Comparison of bacterial 16S rRNA read abundance percentage, at the phylum level, between enriched and unenriched endosphere samples. Enrichment significance was determined via the use of the QIIME script group_significance.py and reported using FDR-adjusted P values. *** and *, P < 0.01 and P < 0.1, respectively.

FIG 2

Phylogenetic analysis of generated SAGs. A bootstrapped maximum likelihood tree created by concatenation alignment of 18 commonly used marker genes is shown. Bootstrap values are indicated by colored dots on each node: black dots (80 to 100), gray dots (50 to 80), and white dots (<50).

FIG 3

Summary of presence/absence of candidate genes for biotin biosynthesis pathway in Armatimonadetes sp. SAG E2G8 and complete genomes of Fimbriimonas ginsengisoli Gsoil 348 and Chthonomonas calidirosea T49, DSM 23976.

FIG 4

Diagram of the urease gene clusters present in the Planctomycetes sp. SAG E9H3. Various components of the gene cluster are indicated in separate boxes.