Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination

Abstract

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...

Role of the Nfo and ExoA Apurinic/Apyrimidinic Endonucleases in Repair of DNA Damage during Outgrowth of Bacillus subtilis Spores

“…The results described in this paper also implicate ␣/␤-type SASP in protecting the germinating and outgrowing spores from DNA damage, since the mutation frequency was higher in outgrown ␣ Ϫ ␤ Ϫ spores than in outgrown wild-type spores and was significantly increased in ␣ Ϫ ␤ Ϫ spores by mutations in nfo and exoA. However, the resistance conferred by ␣/␤-type SASP to dormant spores against several DNA-damaging factors is ultimately lost during spore outgrowth (17,23,24), and thus the protective effect postulated here would operate only until ␣/␤-type SASP are degraded by the specific germination protease germination protease in outgrowth (21,28). Indeed, spore resistance to UV radiation persists well after the initiation of spore germination (21).…”

“…However, the resistance conferred by ␣/␤-type SASP to dormant spores against several DNA-damaging factors is ultimately lost during spore outgrowth (17,23,24), and thus the protective effect postulated here would operate only until ␣/␤-type SASP are degraded by the specific germination protease germination protease in outgrowth (21,28). Indeed, spore resistance to UV radiation persists well after the initiation of spore germination (21). A recent study revealed that loss of ExoA and Nfo did not sensitize growing cells or wild-type or ␣ Ϫ ␤ Ϫ spores to H 2 O 2 and t-butylhydroperoxide (20).…”

Dissecting interactions between nucleosides and germination receptors in Bacillus cereus 569 spores

“…Following the activation of these receptors, spores release dipicolinic acid, calcium and amino acids (Moir, 2006;Setlow, 2003). Subsequently, the core of the spores becomes hydrated, and the spore cortex and sporespecific proteins are hydrolysed (Chirakkal et al, 2002;Sanchez-Salas et al, 1992;Setlow & Waites, 1976). Approximately 30 min after addition of germinants, the newly formed vegetative cells start to divide (Levinson & Hyatt, 1966;Setlow, 2003).…”

DNA in dormant spores of Bacillus species is in an A‐like conformation

“…If, as I have suggested in this review, A-like DNA can exist in vivo, why has it not been seen in other systems? The answer is probably that it rarely exists in significant amounts, because the presence of A-like DNA (or at least a/p-type SASP bound to the A-like DNA) results in drastic reductions in RNA synthesis and therefore cell growth, and in the case of E. coli results in increased spontaneous mutation and often cell death (Sanchez-Salas et al, 1992;Setlow et ai., 1991;Setlow, J.K., Setlow, B., and Setlow, P., unpublished results). Conversion of a cell's genome to an A-like conformation in a dormant bacterial spore, in which gene expression as well as metabolic activity is absent, may not present such a risk.…”