PREVENTION OF DNA DAMAGE IN SPORES AND INVITRO BY SMALL, ACID-SOLUBLE PROTEINS FROM BACILLUS SPECIES

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85-degrees-C for 30 min) which give little killing of wild-type spores of B. subtilis kill >99% of spores which lack most alpha/beta-type SASP (termed alpha- beta- spores). Similar large differences in survival of wild-type and alpha- beta- spores were found at 90, 80, 65, 22, and 10-degrees-C. After heat treatment (85-degrees-C for 30 min) or prolonged storage (22-degrees-C for 6 months) that gave >99% killing of alpha- beta- spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha- beta- spores heated for 30 min at 85-degrees-C released no more dipicolinic acid than similarly heated wild-type spores (<20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93-degrees-C for 30 min) that gave greater-than-or-equal-to 99% killing of wild-type spores, <1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85-degrees-C, 30 min) and unheated wild-type spores and unheated alpha- beta- spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.