Uncovering microbial inter-domain interactions in complex communities

Abstract

Interactions between unicellular eukaryotes and bacteria are difficult to characterize in the environment owing to their large number and inherently microscopic scale. Although particular co-occurrences can be recovered through targeted approaches, e.g. single-cell sequencing or fluorescence in situ hybridization, the vast majority of the interactions remain unseen. Here, we discuss Emulsion, Paired Isolation and Concatenation polymerase chain reaction (epicPCR) as a tool to uncover these interactions in very high throughput. Originally developed for taxonomy-to-function linkage in bacterial communities, epicPCR has the potential to recover the complete interaction network in a given environment at single-cell resolution. This approach relies on the encapsulation of protistan single cells in emulsion droplets that can subsequently be gelified into beads. In this way, encapsulated cells can be exposed to lysis reagents and further phylogenetic paired marker amplification. A bacterium that physically co-occurs with the eukaryote will be jointly trapped, and the amplification will generate a concatenated PCR product containing physically coupled taxonomic markers from both partners, creating a link. Further amplification and sequencing enable the construction of an association pattern with statistically verified physical co-occurrences. Here, we discuss the potential, challenges and limitations of epicPCR. We argue that the microscopic scale at which epicPCR operates, the high throughput it delivers and its exploratory nature make it an unparalleled approach to unravel associations between microbes directly from environmental samples. This article is part of a discussion meeting issue ‘Single cell ecology’.

Single cell ecology

“…The progression of the microbe-microbe interaction research theme, and indeed many of the themes of this issue, ultimately rests on the need to develop new methods. Florenza et al grasp this challenge head on and discuss in detail the tailoring and application of emulsion, paired isolation and concatenated PCR (epicPCR) [121] for large-scale sampling of co-association between protists and associated prokaryotes [122]. This represents an exciting prospect that can potentially allow the study of co-associations on a huge scale, a scale tractable for valid statistical analysis, and removes the requirement for expensive nucleotide amplification and sequencing processes that would be required for single cell 'omic approaches.…”

Catch me if you can: Capturing extracellular DNA transformation in mixed cultures via Hi-C sequencing

“…epicPCR (emulsion, paired isolation, and concatenation PCR) is an alternative method to Hi-C sequencing for recovering linked phylogenetic and functional genetic information from millions of cells in a single analysis [66]. epicPCR is limited by the requirement of prior knowledge of sequences of the target genes before performing the qPCR and by the biases introduced by amplifying the 16S rRNA gene [67, 68]. To increase sensitivity, combining Hi-C sequencing and epicPCR would be interesting for identifying transformation events in mixed cultures and identifying new potential natural competent bacterial species.…”

EpicPCR-Directed Cultivation of a Candidatus Saccharibacteria Symbiont Reveals a Type IV Pili-dependent Epibiotic Lifestyle

“…Recently, EpicPCR has been adopted to detect interspecies relationships with close spatial interaction, which can be co-isolated in droplet emulsions. EpicPCR detections of phage-bacteria 28 infection and bacteria-protozoan 29 endosymbionts have been demonstrated. According to these studies, we envision that epicPCR can also be used for detecting bacterial epibiotic relationships, especially CPR-host symbionts in a microbial community.…”