Studies of the processing of the protease which initiates degradation of small, acid-soluble proteins during germination of spores of Bacillus species

Abstract

Three mutant forms of the protease (GPR) that initiates degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus species have been generated. In one variant (GPR delta), the putative pro sequence removed in conversion of the GPR zymogen (termed P46) to the active enzyme (termed P41) was deleted. GPR delta was expressed in both Escherichia coli and Bacillus subtilis as a polypeptide of 41 kDa (P41) which was active both in vivo and in vitro. The other two variants had changes in the sequence around the site where the pro sequence is removed, making this sequence even more like that recognized and cleaved by GPR in its SASP substrates. One of these variants (GPRS) was synthesized as P46S in both B. subtilis and E. coli, but P46S was processed to P41S earlier in B. subtilis sporulation than was wild-type P46. The second variant (GPREI) was made as P46EI but underwent extremely rapid processing to P41EI in both E. coli and B. subtilis. Expression of elevated (> 100-fold) levels of GPR delta or GPREI blocked sporulation at the time of synthesis of glucose dehydrogenase. Expression of elevated levels of GPRS or low levels (< 20% of the wild-type level) of GPR delta or GPREI did not retard sporulation, but the SASP level in the resultant spores was greatly reduced. Prolonged incubation of P41 delta, P41EI, or wild-type P41, either in vivo or with purified proteins in vitro, resulted in a second self-cleavage event generating a 39-kDa polypeptide termed P39. The sequence in the P(41)-->P(39) cleavage site was also quite similar to that recognized and cleaved by GPR in SASP. Together, these results strongly support a model in which activation of GPR during sporulation by conversion of P(46) to P(41) is a self-processing event triggered by a change in the spore core environment (i.e., dehydration) which precludes attack of the active P(41) on its SASP substrates. However, in the first minutes of spore germination, rapid spore core hydration allows rapid attack of active GPR on SASP.

Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis.

“…3). In particular, the amino acid sequence (93 TDLAPEG 99) around the insert was very similar to that of the germination-specific protease (GPR) cleavage site in the GPR zymogen (TDLAVEA) (18).…”

Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

“…These results suggest that either little, if any, P 46 is autoprocessed to P 41 during sporulation of strain DPS104 (spoVA) or the hydration level of the spoVA spore core is still low enough to preclude P 41 activity on ␣/␤-type SASP. It is also possible that the binding of ␣/␤-type SASP to DNA in the spoVA spores helps protect these proteins against GPR digestion (19,20,53) (but see Discussion).…”

“…B. subtilis GPR is synthesized as a zymogen with a molecular mass of ϳ46 kDa (termed P 46 ) beginning at approximately the third hour of sporulation (53) and is autoprocessed to the ϳ41-kDa active protease (P 41 ) about 2 h later. This latter autoprocessing is triggered synergistically by Ca-DPA accumulation in the forespore and by decreases in the developing forespore's core pH and water content, conditions that preclude attack of P 41 on its SASP substrates (19,20,53). C. perfringens also contains a gpr gene encoding a protein with high similarity to B. subtilis GPR, including the highly conserved P 46 3 P 41 autoprocessing site (53).…”

An α/β-Type, Small, Acid-Soluble Spore Protein Which Has Very High Affinity for DNA Prevents Outgrowth of Bacillus subtilis Spores

“…Previous work has shown that DNAbound ␣/␤-type SASP are very resistant to digestion by GPR compared to free ␣/␤-type SASP (21). To determine whether the increased affinity of SspC ⌬11-D13K for DNA results in reduced degradation by GPR in vitro compared to that of wildtype SspC, purified protein-DNA complexes were made of each ␣/␤-type SASP and supercoiled pUC19 plasmid DNA followed by the addition of partially purified recombinant Bacillus megaterium GPR (9). Under these conditions (10 mM Tris-HCl [pH 7.4]-150 mM NaCl-2 mM CaCl 2 at 37°C), approximately 90% of wild-type SspC is digested in 30 min, while less than 75% of SspC ⌬11-D13K is cleaved in the same time (Fig.…”