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 (Apis mellifera) and bumble bees (Bombus sp.). Expressing fluorescent
proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella
apis, and Serratia strains enables us to
visualize how these bacteria colonize the bee gut. We also demonstrate
CRISPRi repression in B. apis and use Cas9-facilitated
knockout of an S. alvi 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.