A novel AAA+ ATPase required for sporulation and stress response in Bacillus anthracis.

UNLABELLED: AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase-related Bacillus anthracis protein BAS0518 (BA PrkA) which has 88% amino acid homology to Bacillus subtilis PrkA. Conserved domain analysis showed that BA PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on AlphaFold 3 predicted structures, we classified BA PrkA as part of Clade III of the AAA+ superfamily. Our results suggest that BA PrkA has negligible protease and kinase activities under in vitro conditions. Nonetheless, BA PrkA was found to play a significant role in sporulation. It is temporally expressed during sporulation Stages II to VI. A null mutant lacking BA PrkA exhibits severe sporulation defects, with reduced spore viability and downregulation of genes related to spore integuments formation. These defects were reversed in a complementation strain expressing BA PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions and showed a 13-fold induction of BA prkA in wild-type B. anthracis when exposed to 1 M NaCl. Analysis of the BA PrkA interactome revealed enrichment of two proteins, ProA and EzrA, which are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BA PrkA is essential for sporulation and resistance to osmotic stress.

IMPORTANCE: Sporulation and stress adaptation are vital for the survival of Bacillus anthracis in harsh environments. We characterize a novel AAA+ ATPase, B. anthracis BAS0518 (BA PrkA), which shares 88% similarity with Bacillus subtilis PrkA. Analysis of BA PrkA null mutant shows that BA PrkA is essential for proper spore development and stress resilience. BA PrkA is expressed during sporulation Stages II-VI, and its deletion causes defective, less viable spores, reduced osmotic stress tolerance, and downregulation of key sporulation genes. Interaction analyses identified ProA and EzrA, proteins linked to stress response and sporulation, as potential partners. These findings establish BA PrkA as a regulatory link between stress adaptation and sporulation in B. anthracis.