Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species

Abstract

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed oa/13-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., 850C for 30 min) which give little killing of wild-type spores of B. subtilis kill >99%o of spores which lack most a/13-type SASP (termed a-1-spores). Similar large differences in survival of wild-type and a-13-spores were found at 90, 80, 65, 22, and 10'C. After heat treatment (850C for 30 min) or prolonged storage (220C for 6 months) that gave >99%o killing of at-13-spores, 10 to 20%o of the survivors contained auxotrophic or asporogenous mutations.However, at-A-spores heated for 30 min at 850C released no more dipicolinic acid than similarly heated wild-type spores (<20%o of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (930C for 30 min) that gave .99%6 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 (850C, 30 min) and unheated wild-type spores and unheated cC 1 spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated a-0-spores had more than 10 single-strand breaks per 20 kb.These data suggest that binding of at/,3-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 13-spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for oc/13-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. Britain.tures to those at lower temperatures suggests that spores should be able to survive for years at common environmental temperatures (5, 24). Indeed, there are reports of spore survival over thousands of years (although obviously no details on spore population survival) (34). If spores are to survive for these extended times, their DNA must somehow be protected against the accumulation of potentially lethal damage during these periods, as the absence of metabolism and ATP in dormant spores precludes DNA repair (29,30,32). As noted above, spore DNA is protected against damage by UV radiation through the binding of a/1-type SASP. Other possible types of DNA damage are oxidative damage to bases and base loss through depurination; either of these types of DNA damage can lead to a mutagenic or lethal change in the DNA (7,10).DNA depurination appears to be of special significance, as this process is water catalyzed at neutral pH, with the rate of depurination rising dramaticall...

Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol

“…The a -b -spores were significantly more sensitive to this ethanol treatment than were their wild-type counterparts, with less than 0AE1% survival after 2 h (Fig. 4); however, there was only~35% killing of the a -b -spores after incubation for 2 h in water at 65°C (data not shown), similar to that found previously (Fairhead et al 1993). Spore coat removal has no significant effect on spore heat resistance (Gerhardt and Marquis 1989) and this was also the case for resistance of both wild-type (PS533) and a -b -(PS578) spores to 70% ethanol at 65°C (data not shown).…”

“…1b). In addition, the spores surviving treatment with HCl exhibited no increase in the level of mutations (Table 1), but the significant level of mutants in the untreated a -b -spores, as found previously, should be noted (Fairhead et al 1993;Fairhead et al 1994). Spore coats are an important component of spore resistance to some chemicals (Russell 1990;Bloomfield and Arthur 1994;McDonnell and Russell 1999;Setlow 2000;Tennen et al 2000), so the HCl resistance of decoated spores was examined.…”

“…However, incubation of wild-type B. subtilis spores for 2 h in 70% ethanol at 65°C resulted in~2 logs of killing (Fig. 4), while incubation for 2 h in water at this temperature gave no spore killing (data not shown), as found previously (Fairhead et al 1993). In contrast, growing wildtype cells (PS533) were > 99AE99% killed by a 1-min treatment with 70% ethanol at 65°C (data not shown).…”

“…In experiments with spores of strains FB72 or FB108, ethanol-treated samples were cooled rapidly on ice, centrifuged, washed once with water and treated with the non-nutrient germinant Ca 2+ -DPA to stimulate spore germination as described below. Aliquots were then plated on 2· YT agar (Tennen et al 2000) before incubating for 36-48 h at 37°C to determine spore viability (Fairhead et al 1993;Fairhead et al 1994;Tennen et al 2000); incubation for longer times gave no increase in spore viability. All spore killing experiments were performed at least twice on two independent spore preparations with essentially identical relative rates of killing of spores of different strains (< 20% difference in relative rates); spores of different strains whose killing was to be compared were always both prepared and tested together.…”

“…All spore killing experiments were performed at least twice on two independent spore preparations with essentially identical relative rates of killing of spores of different strains (< 20% difference in relative rates); spores of different strains whose killing was to be compared were always both prepared and tested together. Colonies formed from wild-type or a -b -spores which survived various treatments were picked onto minimal medium, sporulation or 2· YT agar plates as described previously (Fairhead et al 1993) and auxotrophic and asporogenous mutants enumerated.…”