The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study ofDrosophila-colonizingOrbaceae

Abstract

ABSTRACTToolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids inEscherichia coli, a strain ofSodalis praecaptivusthat colonizes insects, and aRosenbergiellaisolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the familyOrbaceaethat were isolated from several fly species. EngineeredOrbaceaestrains were able to colonizeDrosophila melanogasterand could be visualized in fly guts.Orbaceaeare common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how theDrosophilamicrobiome affects fly health. Thus, this work provides foundational genetic tools for studying new host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species.IMPORTANCETo fully understand how microbes have evolved to interact with their environments, one must be able to modify their genomes. However, it can be difficult and laborious to discover which genetic tools and approaches work for a new isolate. Bacteria from the recently describedOrbaceaefamily are common in the microbiomes of insects. We developed the Pathfinder plasmid toolkit for testing the compatibility of different genetic parts with newly cultured bacteria. We demonstrate its utility by engineeringOrbaceaestrains isolated from flies to express fluorescent proteins and characterizing how they colonize theDrosophila melanogastergut.Orbaceaeare widespread inDrosophilain the wild but have not been included in laboratory studies examining how the gut microbiome affects fly nutrition, health, and longevity. Our work establishes a path for genetic studies aimed at understanding and altering interactions between these and other newly isolated bacteria and their hosts.