Testing the epicPCR workflow to reveal eukaryote-prokaryote interactions at single-cell resolution

• Physical interactions between microeukaryotes and their prokaryotic counterparts abound in nature but are laborious to characterize due to their large number and microscopic dimension. Emulsion, paired-isolation and concatenation PCR (epicPCR) has the potential to uncover these interactions at a large scale while maintaining the resolution of individual cells. In this approach, single eukaryotic cells are trapped inside polyacrylamide beads together with their physically associated bacterial partners. This is followed by molecular barcoding of taxonomic marker genes of both parts in a compartmentalized manner. In this study, we report the first iteration in the evaluation of this workflow when adapted to recover eukaryote-prokaryote associations via SSU rRNA gene linkage. Predatory associations between two cultured mixotrophic flagellates and their co-cultured bacterial cohort were our target. Three sets of experiments were carried out, in which the model flagellates were 1) encapsulated on their own, 2) with a bacterial community assembled artificially or 3) with a wastewater sample. In all cases, the most frequent associations between either of the mixotrophs and bacterial constituents were those that involved the most abundant bacterial taxa in the samples. This was most evident in experiment from the second set, where the strongest associations corresponded to those between the eukaryotes and the most abundant members of the mock bacterial community. This result points to the loss of single cell resolution at some point during the protocol. We hypothesize that the erosion of the compartmentalization principle might arise from two sources. First, the extreme polydispersity in droplet size of the polymerizing emulsion, combined with much smaller cell size and much higher population density of the bacteria relative to the eukaryotes, can cause random co-encapsulation of cells that are not physically attached. Second, the design of the barcoding reaction as implemented here might be prone to generate a large number of non-barcoded fragments susceptible to be spuriously tagged during nested PCR, ruining the signature of individual separation. Although technical limitations exist, avenues for further development remain open. The vast exploratory potential of the epicPCR technique justifies further research to overcome these technical constrains.

Subject headings

NATURVETENSKAP  -- Biologi -- Ekologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Ecology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

Keyword

eukaryote-prokaryote interactions

single-cell

epicPCR

Publication and Content Type

vet (subject category)

ovr (subject category)