AUTOPROCESSING OF THE PROTEASE THAT DEGRADES SMALL, ACID-SOLUBLE PROTEINS OF SPORES OF BACILLUS SPECIES IS TRIGGERED BY LOW PH, DEHYDRATION, AND DIPICOLINIC ACID

The sequence-specific protease (termed GPR) that degrades small, acid-soluble proteins (SASP) during germination of spores of Bacillus species is synthesized during sporulation as an inactive precursor termed P-46. Approximately 2 h later in sporulation, P-46 is converted proteolytically to a smaller form, termed P-41, which is active in vitro, but which does not act significantly on SASP in vivo until spore germination is initiated. While it appears likely that P-46-->P-41 conversion is an autoprocessing event, the mechanisms regulating P-46-->P-41 conversion in vivo are not clear. In this work we found that P-46-->P-41 conversion in vitro was stimulated tremendously in an allosteric manner by pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus Ca2+ but not by Ca2+ in combination with a variety of DPA analogs. The processing reaction stimulated by Ca2+-DPA was seen at pH 5.1 but not at pH 6.2 or 7, and under these conditions P-46-->P-41 conversion exhibited a linear time course and was a first-order reaction, indicative of an intramolecular autoprocessing reaction. At pH 5.1, P-46-->P-41 conversion was stimulated markedly by very high ionic strength. At pHs from 5.1 to 6.6, P-46-->P-41 conversion also occurred when P-46 was dehydrated to similar to 54% relative humidity. This processing was stimulated markedly when dehydration was carried out in the presence of DPA and NaCl but was greatly decreased when dehydration was to 10, 33, or 75% relative humidity. Since previous work has shown that P-46-->P-41 processing in vivo takes place (i) after a fall in forespore pH to 6.3 to 6.9 and approximately in parallel with (ii) DPA accumulation by the forespore and (iii) dehydration of the forespore, our new findings in vitro suggest that these three changes may synergistically trigger P-46-->P-41 autoprocessing in the developing forespore. Presumably the conditions in vivo during this autoprocessing preclude significant attack of the P-41 generated on its SASP substrates.