The small molecule dipicolinic acid (DPA) plays a critical role in bacterial spore resistance during dormancy and in the exit from quiescence during germination. In Bacillus subtilis, the spoVA locus is required for DPA import into the developing spore and has been implicated in its release during germination. The SpoVAC (C) and SpoVAEb (Eb) proteins form a membrane complex through which DPA transits, and the SpoVAD (D) protein binds the cytoplasmic face of the complex, resembling a plug. The human pathogen Bacillus anthracis and other members of the Bacillus cereus group have two spoVA loci. The spoVA1 operon resembles B. subtilis spoVA, while the spoVA2 locus is more distantly related and only shares the three core genes C2, D2, and Eb2. Here, we show that spoVA2 is critical for DPA import and spore heat resistance, while either locus is sufficient for DPA export during germination. We report that a fourth protein (called NJ2) encoded in the spoVA2 locus is part of the C2/D2/Eb2 complex and is predicted to bind the extracytoplasmic face of the membrane complex, resembling a cap. We show that NJ2 is important for DPA import during B. anthracis sporulation and essential in B. subtilis engineered to express spoVA2 in place of its native spoVA locus. Finally, we identified an Eb2 mutant that bypasses the requirement for NJ2 in DPA import and show that the mutation impairs DPA export, providing additional support for a direct role of SpoVA complexes in the release of DPA during germination.IMPORTANCESpore resistance and exit from dormancy during germination are central to the transmission and pathogenesis of endospore-forming pathogens like Bacillus anthracis. Our understanding of the molecular mechanisms underlying these processes has principally been informed by studies in the non-pathogenic model Bacillus subtilis. Here, we identify and characterize a membrane complex in B. anthracis that is critical for spore resistance and spore germination that is absent from B. subtilis. We show that this complex is required for the accumulation of dipicolinic acid in the spore core during sporulation and functions in its release from the core during germination. A deeper understanding of the molecular mechanisms of B. anthracis sporulation and germination will facilitate the development of strategies for more effective disease prevention and treatment.
Yu et al. (Thu,) studied this question.