A set of genetic tools for use in Clostridioides difficile and related species

The Clostridia are a phylogenetically diverse group of anaerobic, spore-forming bacteria that include species of medical, veterinary and industrial importance. The last two decades have seen major advances in our understanding of Clostridial biology despite the difficulties of anaerobic microbiology and the challenges associated with limited genetic tools. Effort has largely focused on the human pathogen Clostridioides difficile , but many of the methods developed have also proven useful in other species. Here, we present a collection of new genetic tools, including an array of promoters of varying strength, that we have characterized in C. difficile , the food spoilage bacterium Clostridium sporogenes and industrially important Clostridium saccharoperbutylacetonicum . We also present a set of modular plasmids that allow expression of proteins with a variety of tags, including for protein purification and fluorescence microscopy and a method for genetic barcoding of C. difficile to facilitate competitive index experiments. We make these tools available in the hope that they will prove useful to the community in support of our growing understanding of these important bacteria.