Expanding the genetic toolbox of the obligate predatory bacterium with inducible gene expression and CRISPR interference.

is an obligate predatory bacterium that invades the periplasm of diderm prey bacteria, where it elongates and produces multiple daughter cells through nonbinary division. Investigating the molecular determinants of this lifecycle is challenging because deleting genes required for predation also impairs survival. Furthermore, the scarcity of robust conditional gene expression systems has restricted functional studies in this bacterium. Here, we address these limitations by expanding the genetic toolbox for . First, we analysed the relative strength of a series of promoters, providing new resources to fine-tune gene expression. We then established an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible expression system that can be activated during both the attack and growth phases of the predator. Finally, we designed a CRISPR interference (CRISPRi) module for IPTG-inducible gene knockdown, enabling rapid and targeted depletion. As a proof of principle, CRISPRi-mediated silencing of the cell curvature gene reproduced the straight phenotype of the deletion mutant. Likewise, depletion of the tubulin homologue FtsZ-which we showed is essential for survival-blocked cell division within the first replicative cycle, yielding filamentous progeny still able of exiting the prey cell. This highlights the intriguing potential of uncoupling key cell cycle and predatory processes. Overall, these tools significantly broaden the scope of genetic manipulation in and open new avenues for in-depth investigation of its noncanonical biology.