Disruption of Protein BA_3317 Affects Asymmetric Division as an Exporter of Sporulation Signal Molecules in Bacillus anthracis

Background Bacillus anthracis forms dormant spores that constitute the primary infectious agent of anthrax. BA_3317, a membrane protein harboring a quorum-sensing (QS)-related AgrB domain, is essential for sporulation in B. anthracis . Methods We constructed an in-frame deletion mutant of BA_3317 in B. anthracis vaccine strain A16R. Sporulation efficiency was quantified, and mutant morphology was observed via confocal microscopy. To investigate the role of BA_3317 in spore germination, we performed secretion exchange experiments between A16R and ΔBA_3317 at T _0.5 , analyzed the transcriptional activity of spoIIE , and determined lecithinase activity after activating the plcR-papR QS system. Additionally, we identified the upstream regulators of BA_3317 using in vitro promoter pull-down assays. Results Deletion of BA_3317 severely reduced sporulation efficiency and ΔBA_3317 mutant was partially arrested at the asymmetric cell division stage. Cells secretion exchange experiments and spatial reporter assays revealed that BA_3317 exports a signal molecule required for sporulation, and its loss downregulated key sporulation gene spoIIE . The mutant also lacked lecithinase activity via the ectopically activated the plcR-papR QS system, which was restored by adding the PapR heptapeptide, indicating BA_3317 mediates peptide signal transport. BA_3317 expression is repressed by SpoVG prior to asymmetric division and positively regulated by GerE during late sporulation. Conclusion Our findings identify BA_3317 as a critical regulator of B. anthracis sporulation that functions as an exporter of sporulation signaling molecules. This study advances understanding of species-specific sporulation mechanisms in B. anthracis and provides a potential target for anthrax prevention and control.