Small, acid-soluble proteins bound to DNA protect Bacillus subtilis spores from being killed by freeze-drying

Abstract

Wild-type spores of BaciUlus subtilis were resistant to eight cycles of freeze-drying, whereas about 901% of spores lacking the two major DNA-binding proteins (small, acid-soluble proteins a and I8) were killed by three to four cycles of freeze-dryings, with significant mutagenesis and DNA damage accompanying the killing. This role for a/p-type small, acid-soluble proteins in spore resistance to freeze-drying may be important in spore survival in the environment. Dormant spores of Bacillus species are much more resistant than their growing counterparts to a variety of treatments, including heat, UV radiation, and chemicals such as hydrogen peroxide (6-8, 13). While many factors are involved in spore resistance to heat and hydrogen peroxide (6, 11, 13), one common factor is the saturation of spore DNA with a group of a/,-type small, acid-soluble proteins (a/,B-type SASP) (11, 13). These proteins are made in the forespore late in sporulation in amounts sufficient to completely cover the spore chromosome but are degraded in the first minutes of spore germination (12, 13). B. subtilis spores lacking the two major a/n-type SASP (termed o-4spores) are significantly more heat and hydrogen peroxide sensitive than are wild-type spores (5, 11). Studies

Mechanism of killing of spores of Bacillus anthracis in a high-temperature gas environment, and analysis of DNA damage generated by various decontamination treatments of spores of Bacillus anthracis ,Bacillus subtilis and Bacillus thuringiensis

“…Dry heat treatment also is reported to cause mutagenesis of spores of Bacillus atrophaeus, a very close relative of B. subtilis (Grinshpun et al 2010;Johansson et al 2011). However, desiccation alone gave no significant killing of wild-type B. subtilis spores and no mutagenesis (Table 1), as found previously (Fairhead et al 1994;Setlow 2006). Dry heat treatment of a À b À B. subtilis spores also produced a high level of Rif r mutants in the survivors, and desiccation alone gave an c. 20-fold increase in mutation frequency to Rif r over that seen with untreated spores (Table 4; Control for wet heat treatment), again as seen previously with a À b À spores when mutagenesis was determined from numbers of asporogenous and auxotrophic mutants and accumulation of DNA damage (Fairhead et al 1994;Setlow 2006).…”

“…However, desiccation alone gave no significant killing of wild-type B. subtilis spores and no mutagenesis (Table 1), as found previously (Fairhead et al 1994;Setlow 2006). Dry heat treatment of a À b À B. subtilis spores also produced a high level of Rif r mutants in the survivors, and desiccation alone gave an c. 20-fold increase in mutation frequency to Rif r over that seen with untreated spores (Table 4; Control for wet heat treatment), again as seen previously with a À b À spores when mutagenesis was determined from numbers of asporogenous and auxotrophic mutants and accumulation of DNA damage (Fairhead et al 1994;Setlow 2006). In contrast to the mutagenic effects of UV and dry heat on wild-type *Bacillus anthracis spores with or without HTGE were heat activated, germinated in 2xYT medium, and the germination and outgrowth of ≥140 individual spores were assessed as described in Methods.…”

The effect of hypochlorite on spores of Bacillus subtilis lacking small acid-soluble proteins

“…Previous studies have shown that heat (Fairhead et al 1993), hydrogen peroxide and freeze drying (Fairhead et al 1994) produce mutations in spores of the a-P-mutant but not in those of the wild type. When the effect of hypochlorite on the mutation rate of spores was examined mutants were also detected in spores of strain PS 361 but not in strain PS 346 (Table 1).…”

“…Many factors are involved in such resistance (Setlow 1994) but recent studies have shown that small acid-soluble proteins (SASP) of the a/P-type coat the DNA in wild-type spores of Bacillus suhtilis and that spores which lack the two major a/&type SASP (termed CI-,!spores) are significantly more sensitive to hydrogen peroxide , U.V. light (Mason and Setlow 1986;Fairhead and Setlow 1992), heat (Fairhead et al 1993) and freeze drying (Fairhead et al 1994). In particular, in vitro studies have shown that a/j-type SASP protect the DNA against enzymes and antimicrobial agents Setlow 1994).…”

The effect of acid shock on sporulating Bacillus subtilis cells

“…Compared with wild-type spores, mutant spores lacking the a ⁄ b-type SASP are u.v. and heat sensitive (Setlow 1994); they are also more sensitive to hydrogen peroxide , freeze drying (Fairhead et al 1994) and formaldehyde (Loshon et al 1999), and have decreased longevity (Fairhead et al 1993).…”