An adapted method for Cas9-mediated editing reveals the species-specific role of β-glucoside utilization driving competition between Klebsiella species

Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. The system allowed efficient and precise editing via enhanced lambda Red expression and functionally-validated complementation with the use of universal bookmark targets in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition. * ImportanceCas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, K. michiganensis and K. grimontii. We then adapted a recently developed protocol for functional complementation based on universal bookmark targets applicable to all tested species. In summary, species specific adaptation of state-of-the-art genetic tools allow efficient gene deletion and complementation in types strains as well as natural isolates of KoSC members to study microbial interactions.