Advancing Vibrio genetics: A platform for efficient genomic manipulation

Abstract Most non-model Vibrio species lack the genetic tools needed for targeted mutagenesis, which limits the ability to functionally characterize newly identified pathways. To address this challenge, we present here efficient, robust methods for genetically manipulating Vibrio species that rely on RecA-mediated homologous recombination and two well-characterized counterselection methods, galactokinase ( galK ) 2-Deoxy-D-galactose (DOG-2) toxicity and the rpsL R / rpsL S streptomycin susceptibility system, both of which are active across a broad range of Vibrio species. We further characterized two genus-specific conserved promoters capable of driving high-level ectopic expression across all tested species. These promoters were incorporated into two broadly applicable, conjugatively transferable suicide backbones designed to facilitate double homologous recombination. Using these systems, we successfully disrupted polar flagellar motility in multiple Vibrio species and introduced extensive modifications to both flagellar and secretory pathways in V. diazotrophicus . Notably, although the galK system exhibited broader applicability, the rpsL system proved to be more efficient in cases where a streptomycin resistant strain could be generated. We also developed two mobilizable replicative backbones that express pH-stable fluorescent proteins for use within the genus. Collectively, these tools expand the genetic toolkit available for both gene disruption and heterologous gene expression in non-model members of the Vibrionaceae . Importance Members of the Vibrionaceae are not only among the most abundant and ecologically influential microorganisms in marine ecosystems, but they also represent major drivers of disease across a wide range of hosts, including humans. However, identifying the genetic determinants of Vibrionaceae pathogenesis has remained challenging due to their halophilic growth requirements, restriction enzyme profiles, and resistance to expressing foreign proteins. Canonical counterselection pathways are largely ineffective in these species, which underscores the need for novel and efficient genetic tools to advance functional studies. This study adapts two strategies previously successful in other non-model organisms for use within the Vibrio genus and its close relatives. These methods therefore represent an essential step toward overcoming long-standing genetic barriers in Vibrio species and provide a framework to expand our understanding of biology in this important bacterial genus.