Parallel tracking by sequencing reveals the impact of restriction modification systems on transfer of an integrative and conjugative element

Horizontal gene transfer (HGT) is a promising avenue for microbiome engineering that enables DNA delivery to microbes in their native environment. Integrative and conjugative elements (ICEs), a class of genomically-integrated mobile elements, are ideal vectors for this purpose. Developing targeted ICE transfer for controlled microbiome engineering requires a better understanding of ICE mobility in complex communities. However, current methods for tracking horizontal gene transfer are not high throughput. To improve upon current methods, we developed a sequencing-based strategy to track and quantify ICE transfer in complex mixtures that we refer to as ICE-seq. This method was able to assess ICE transfer in a synthetic community of 150 recipients simultaneously and in combination with phenotypic assays demonstrated that restriction-modification (RM) systems in the donor and recipient alter ICE transfer rate with subspecies resolution by up to 10,000-fold. We propose that manipulating RM systems can be a strategy to engineer targeted ICE transfer for directed microbiome engineering. ImportanceTypical approaches to engineer microbiomes involve adding beneficial strains to existing microbiomes, for example the addition of probiotics to the human gut microbiome or soil amendments to improve plant health or for bioremediation. However, these beneficial bacteria often do not survive for extended periods of time, which limits their overall effectiveness. To circumvent this issue, we can transfer beneficial genes by native mechanisms into existing bacteria that are already in the target environment where they can then perform the beneficial function encoded by these genes. In this work, we developed a technique to track gene transfer into multiple recipients simultaneously. Furthermore, bacteria have innate defense systems to protect against invading DNA that can reduce this gene transfer. Our technique allowed us to identify the impact of these defense systems and their potential function for targeting gene transfer to specific recipients.