Programmed strain tagging and gene disruption throughout a diverse bacterial genus

Transposons are a convenient vehicle to insert DNA into a bacterial genome, but widely-used transposons such as Tn7 are not able to hit custom targets. Recently, CRISPR RNA-guided integrases have been shown to direct the insertion of a mini transposon to a chosen site using a short guide sequence. We adapted this system for the widespread plant-associated genus Sphingomonas, revealing flexibilities and limitations of the tool. We uniquely tagged five genetically diverse strains, both in neutral sites and in sites with phenotypic consequences, and we demonstrate the utility of the tags for quantitative strain tracking in complex bacterial populations. Although we initially focused on the attTn7 site recognized by Tn7 as an insertion site with minimal fitness effects, we discovered via a genus-wide search that this site disrupts potentially important genes in some strains. Therefore we validated an improved site for benign integration in Sphingomonas and used a construct with guides targeting this site to transform a heterogeneous Sphingomonas population, bypassing prior strain isolation. Using a novel rapid and economical transposon mapping method, we were able to identify correctly-tagged primary transformant colonies with novel genetic content, thus demonstrating a short cut towards the establishment of diverse tagged synthetic communities for the experimental study of bacterial natural variation.