Yongqiang Gao1, Rocio Del Carmen Barajas-Ornelas1, Jeremy D. Amon1, Fernando H. Ramírez-Guadiana1, Assaf Alon2, Kelly P. Brock3, Debora S. Marks3,4, Andrew C. Kruse2 and David Z. Rudner1 1Department of Microbiology, Harvard Medical School, Boston, Massachusetts 02115, USA; 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA; 3Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA; 4Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA Corresponding author: rudnerhms.harvard.edu Abstract

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth in response to nutrients. The small molecule dipicolinic acid (DPA) plays a central role in both the stress resistance of the dormant spore and its exit from dormancy during germination. The spoVA locus is required for DPA import during sporulation and has been implicated in its export during germination, but the molecular bases are unclear. Here, we define the minimal set of proteins encoded in the Bacillus subtilis spoVA operon required for DPA import and demonstrate that these proteins form a membrane complex. Structural modeling of these components combined with mutagenesis and in vivo analysis reveal that the C and Eb subunits form a membrane channel, while the D subunit functions as a cytoplasmic plug. We show that point mutations that impair the interactions between D and the C–Eb membrane complex reduce the efficiency of DPA import during sporulation and reciprocally accelerate DPA release during germination. Our data support a model in which DPA transport into spores involves cycles of unplugging and then replugging the C–Eb membrane channel, while nutrient detection during germination triggers DPA release by unplugging it.

Received February 14, 2022. Accepted May 16, 2022.