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.

Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis

“…This is not an obstacle in settings such as fecal microbiome studies, where abundant organisms are present, but in other scenarios (e.g., in scenarios with low-biomass environmental samples and uncultured bacteria or in single cell analysis), the number of cells and the amount of genetic material can be limited (13)(14)(15)(16)(17). It is in these situations that whole-genome amplification (WGA) has the potential to play an important role in pathogen detection.…”

Analysis of single-cell genome sequences of bacteria and archaea

“…However, this approach is only applicable when library pools contain different organisms, as highly similar organisms would produce a high fraction of false positives, which would be flagged as contaminants. To our knowledge, this is the only currently available tool specifically designed for this analysis, though other similar approaches have been performed: for example, searching the contigs of a genome against all other genomes in the pool using blastn and removing those contigs that match above user-defined identity and length thresholds [37].…”

Simultaneous Discovery of Positive and Negative Interactions Among Root Microbiome Bacteria Using Microwell Recovery Arrays

“…Strain YR343 is a Gram-negative, plant growth promoting bacteria (PGPB) isolated from the rhizosphere of an eastern cottonwood Populus trichocarpa tree (27,28). As P. trichocarpa is a promising biofuel feedstock (29), uncovering interactions that influence the function of beneficial organisms in its rhizosphere has received intensive interest in recent years (30)(31)(32). Pantoea sp.…”