A tetrameric SpoVA2 membrane complex is required for DPA transport into <i>Bacillus anthracis</i> spores

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 <i>Bacillus subtilis</i>, the <i>spoVA</i> 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 <i>Bacillus anthracis</i> and other members of the <i>Bacillus cereus</i> group have two <i>spoVA</i> loci. The <i>spoVA1</i> operon resembles <i>B. subtilis spoVA</i>, while the <i>spoVA2</i> locus is more distantly related and only shares the three core genes <i>C2</i>, <i>D2</i>, and <i>Eb2</i>. Here, we show that <i>spoVA2</i> 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 <i>spoVA2</i> 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 <i>B. anthracis</i> sporulation and essential in <i>B. subtilis</i> engineered to express <i>spoVA2</i> in place of its native <i>spoVA</i> 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 <i>Bacillus anthracis</i>. Our understanding of the molecular mechanisms underlying these processes has principally been informed by studies in the non-pathogenic model <i>Bacillus subtilis</i>. Here, we identify and characterize a membrane complex in <i>B. anthracis</i> that is critical for spore resistance and spore germination that is absent from <i>B. subtilis</i>. 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 <i>B. anthracis</i> sporulation and germination will facilitate the development of strategies for more effective disease prevention and treatment.