From the outset, we dedicated substantial efforts to streamline protocols for cultivating and genetically engineering Bdellovibrio bacteriovorus (e.g. improved methods for precise gene knockouts, expression systems, inducible CRISPRi, and microplate-based predation assays). Early on, we also optimized live fluorescence microscopy tools to visualize B. bacteriovorus in real time at high spatial and temporal resolution.
A key finding was how chromosomal regions move and organize during the predator’s lifecycle. We confirmed that the chromosome is highly compacted in free-swimming attack-phase cells, which can exclude small soluble proteins from the nucleoid region. Once inside the prey, B. bacteriovorus grows as a filament containing multiple copies of its genome, and we discovered that replication initiates asynchronously, with multiple replication forks active at different times and starting from different ori copies. Meanwhile, chromosome segregation begins as soon as new copies are synthesized, decoupling it from the final multiple division step. These data led us to propose a concurrent replication–segregation model, compatible with the production of odd or even number of daughter cells.
In parallel, we investigated how cell polarity is established and maintained in a predatory bacterium. We identified and tracked the localization of pole-associated proteins (DivIVA and RomR) and uncovered their distinct, dynamic cell cycle–dependent distribution. RomR was established as early pole organizer, connecting prey-attacking nanomachines with signaling pathways and intracellular organization starting in the mother cell. We further advanced our understanding of predatory bacterial cell organization by developing a proximity-labeling approach to reveal in vivo protein networks throughout the predatory lifecycle.
Finally, we explored how prey features (such as size and envelope integrity) affect B. bacteriovorus proliferation. We showed that prey size and content differentially influence predator progeny number and growth rate, respectively, while typical envelope perturbations in E. coli do not prevent predation. Toward the end of the project, we introduced B. exovorus as a new model organism with a distinct predatory lifestyle, further broadening the scope of our findings. Overall, these achievements have already resulted in several peer-reviewed publications, specialized protocols, and multiple conference presentations, thereby disseminating our results across the bacterial cell biology community.
