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Bacterial competition drives the evolution of antibacterial mechanisms, yet how new activities arise remains poorly understood. A major route to innovation is the reuse of pre-existing genetic systems, whereby conserved protein modules are repurposed in new biological contexts to generate new capabilities. Here, we show that the ParB-CTPase fold, a conserved nucleotide-binding module best known for its role in chromosome segregation, can be functionally repurposed as an antibacterial toxin. We identify ToxB, a ParB-like domain embedded within the polymorphic toxin region of contact-dependent inhibition systems and show that it functions as a potent antibacterial effector. Structural and biochemical analyses reveal that ToxB retains the core architecture of the ParB-CTPase fold but lacks DNA-binding capability and preferentially binds ATP. This shift in nucleotide specificity underpins a distinct mode of action, in which ATP binding and hydrolysis trigger rapid nucleoid compaction, chromosome segregation defects, oxidative stress, cell chaining, and ultimately cell lysis. ToxB also exhibits toxic activity in plant cells, suggesting that it targets conserved cellular processes. Together, these findings provide direct experimental evidence that the ParB-NTPase fold is biologically versatile and can be repurposed for biological roles fundamentally distinct from its ancestral function in DNA segregation. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 221776/Z/2/Z, 227755/Z/23/Z Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/X01097X/1 Diamond Light Source, MX32728

Firmicutes are gram-positive bacteria with important roles in human health, disease, and industry. However, more than a quarter of genes in the model firmicute Bacillus subtilis remain completely uncharacterized, including numerous core phylum-specific genes. Here, we design a compact pooled CRISPRi library targeting all protein-coding genes in B. subtilis and test its growth in ~150 stress conditions. Using data from this screen as a hypothesis generator, we perform targeted experiments that reveal that YneF, a conserved essential firmicute protein, plays a role in the SRP co-translational protein secretion pathway. We also demonstrate that ECF-transporters play a previously unknown but broadly conserved role in cell wall homeostasis, perform an unbiased analysis of amino acid crossfeeding, and make additional discoveries about bacterial competition and about the cell wall of B. subtilis. In addition to these major contributions to our understanding of B. subtilis (and gram-positive firmicutes in general), this work provides a rich dataset that will nucleate future studies of uncharacterized genes and presents a framework for accessible full-genome functional genomic screens in other bacteria. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R35 GM118061 The University of Queensland, https://ror.org/00rqy9422