Structure and Mechanism of Action of the Protease That Degrades Small, Acid-Soluble Spore Proteins during Germination of Spores of Bacillus Species

Between 10 and 20% of total spore protein is degraded to amino acids in the first minutes of germination of spores of Bacillus species (30). The proteins degraded are a group of small, acid-soluble proteins (SASP) unique to the spore stage of the life cycle. The α/β-type SASP, which are coded for by a multigene family, are bound to the dormant spore’s DNA and provide a significant component of spore resistance to heat, hydrogen peroxide, and UV radiation (28, 30, 31). The γ-type SASP, which are coded for by a single gene, are also in the spore core (the site of spore DNA) but are not bound to any macromolecule. SASP degradation at the beginning of spore germination both frees up the DNA for transcription and provides amino acids that are used for protein synthesis during subsequent development (22, 30). SASP degradation during spore germination is initiated by one or two endoproteolytic cleavages catalyzed by a sequence specific germination protease termed GPR which is synthesized during sporulation at about the same time as its SASP substrates (30). GPR is synthesized as a protein of 46 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this species, termed P46, is inactive (13). Two hours later in sporulation, P46 undergoes intramolecular autoprocessing which removes an N-terminal propeptide of 15 (Bacillus megaterium) or 16 (B. subtilis) amino acids; the new, fully active form of GPR is called P41 (13, 24). Both P46 and P41 are tetramers, and only the tetrameric form of P41 is active (12). The autoprocessing of P46 to P41 is triggered both in vivo and in vitro by low pH, dipicolinic acid, and dehydration (8). Although active in vitro, P41 carries out no SASP degradation during sporulation because the conditions inside the developing and dormant spore (in particular the dehydration and mineralization) are not favorable for enzymatic action. However, in the first minutes of spore germination, the spore core rehydrates, allowing rapid attack of GPR on SASP. Synthesis of GPR in an inactive form and its autoprocessing at the correct time in sporulation are essential to generate a fully resistant spore; a strain with a GPR variant that autoprocesses P46 to P41 ∼1 h earlier during sporulation produces spores that are more sensitive than wild-type spores to a variety of treatments, because the α/β-type SASP content of the mutant spores is reduced (6). In addition to the autoprocessing reaction converting P46 to P41, the P41 of B. megaterium also undergoes an additional autoprocessing to P39 with loss of an additional seven N-terminal residues (8). The enzymatic activities of P39 and P41 are identical, and P39 is not generated from P41 of B. subtilis. Consequently, the generation of P39 with B. megaterium GPR may not have any functional significance. While the role played by GPR in SASP degradation as well as its autoprocessing are reasonably well understood, little is known of the structural differences between P46 and P41 and the nature of GPR’s active site(s). Indeed, the precise class of protease to which GPR belongs has not been established. To obtain more information on these latter topics, we have analyzed the trypsin digestion products generated from P46 and P41 and have used a variety of techniques in an attempt to determine the class of protease to which GPR belongs. The latter data suggest that GPR is not a member of a previously described class of proteases.