Degradation of small, acid-soluble spore proteins during germination ofBacilus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtifis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2-to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression ofB. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -Vmutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.Approximately 10 to 20% of the protein of spores of Bacillus species is degraded to amino acids in the first minutes of spore germination (21). The proteins degraded in this process are a group of small, acid-soluble spore proteins (SASP), and SASP degradation is initiated by a sequencespecific protease termed GPR (10,18,21); the gene coding for GPR (termed gpr) has been cloned and sequenced from both Bacillus megaterium and Bacillus subtilis (27). Expression of gpr begins only in the developing forespore in the third hour of sporulation at or slightly before the time of synthesis of its SASP substrates (11,27). Studies of GPR from B. megaterium (11) have shown that it is a tetramer of identical subunits which are first synthesized as an apparent 46-kDa polypeptide (termed P46), which is converted to an apparent 41-kDa species (termed P41) -2 h later in sporulation. During the first minutes of spore germination, P41 is converted to an apparent 40-kDa species (termed P40); P40 is then slowly degraded to completion in an energy-requiring process as spore germination and outgrowth proceed. The processing of P46 to P41 appears to be an important step in the regulation of GPR activity, because the tetrameric enzyme composed of P46 is inactive both in vivo and in vitr...

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination ofgpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major od/,-type SASP. These data suggest that (i) a$/,-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the a$d-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of o/,-type SASP on the chromosome is detrimental to normal spore outgrowth.Approximately 10 to 20% of the protein of dormant spores of Bacillus species is degraded to free amino acids in the first minutes of spore germination (22). These amino acids can support much of the protein synthesis early in the development of the germinating and outgrowing spore (22). The proteins degraded in this process are a group of small, acid-soluble proteins (SASP) of two types, al and y (22). There are multiple ao/-type SASP in spores of Bacillus species, and the amino acid sequences of these proteins have been highly conserved both within and across species (22). The aJ,-type SASP are associated with spore DNA and play a key role in the resistance of spores to UV light (22, 23). Bacillus spores have only one y-type SASP, and the primary sequence of this protein has not been as highly conserved in evolution as has those of a/P-type SASP. -y-Type SASP are not associated with DNA in vivo and have no known function other than generating amino acids by their degradation during spore germination.The great majority, if not all, SASP degradation during spore germination is initiated by a single protease, which has been termed GPR (8,9,22). This enzyme is an endoprotease specific for a pentapeptide sequence found once or twice in all SASP. GPR is initially synthesized during sporulation as a 46-kDa precursor (termed P...

No abstract

The first ϳ10% of spores released from sporangia (early spores) during Bacillus subtilis sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ϳ24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca 2؉ but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.Spores of Bacillus and Clostridium species are dormant and extremely resistant to a variety of agents, including high temperatures, radiation, and many chemicals (19,31). This extreme spore resistance has major applied implications, because spores of Bacillus and Clostridium species are vectors for food spoilage and a number of diseases, some of which are food borne. As a consequence, there is much interest in the mechanisms of spore resistance as well as methods for spore inactivation.The most commonly used method for spore inactivation is wet heat treatment, which is extremely effective, even though spores are generally resistant to temperatures ϳ40°C higher than their growing-cell counterparts (4,19,31). Factors involved in spores' extreme resistance to wet heat include (i) the protection of spore DNA by its saturation with the ␣/␤-type small, acid-soluble spore proteins (SASPs); (ii) the thick proteinaceous spore coat layer; (iii) the thick layer of cortex peptidoglycan surrounding the spore core; (iv) the high level of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and its associated divalent cations, mostly Ca 2ϩ , in the spore core; and, most importantly, (v) the low water content of the core of spores suspended in water (4,5,19,31,32). High spore wet heat resistance is acquired late in sporulation, largely in parallel with the uptake of DPA by the developing forespore and the final decrease in spore core water content. However, the precise time of acquisition of full spore heat resistance during sporulation is not completely clear because (i) precise measurements of wet heat resistance are usually carried out only on purified spores; (ii) analysis of acquisition of wet heat resistance can be carried out only with spore populations, and the lack of precise synchrony in sporulation of cell populations can complicate ...

No abstract