Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids

Engineering the bacteria present in animal microbiomes promises to lead to breakthroughs in medicine and agriculture, but progress is hampered by a dearth of tools for genetically modifying the diverse species that comprise these communities. Here we present a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in this toolkit can be used to rapidly test various antibiotic resistance markers, promoters, fluorescent reporters, and other coding sequences in newly isolated bacteria. We demonstrate the utility of this toolkit in multiple species of Proteobacteria that are native to the gut microbiomes of honey bees (<i>Apis mellifera</i>) and bumble bees (B<i>ombus</i> sp.). Expressing fluorescent proteins in <i>Snodgrassella alvi, Gilliamella apicola, Bartonella apis,</i> and <i>Serratia</i> strains enables us to visualize how these bacteria colonize the bee gut. We also demonstrate CRISPRi repression in <i>B. apis</i> and use Cas9-facilitated knockout of an <i>S. alvi</i> adhesion gene to show that it is important for colonization of the gut. Beyond characterizing how the gut microbiome influences the health of these prominent pollinators, this bee microbiome toolkit (BTK) will be useful for engineering bacteria found in other natural microbial communities