Binding of small acid-soluble spore proteins from Bacillus subtilis changes the conformation of DNA from B to A.

Abstract

Small acid-soluble spore proteins (SASPs) appear 3-4 hr after the onset of sporulation in Gram-positive bacteria and constitute up to 20% of the protein of mature spores. Previous studies using Bacillus subtlis deletion mutants lacking SASP-a and -1S have shown that such mutations abolish the elevated resistance ofspores to UV radiation. Analyses using circular dichroism and Fourier-transform infrared spectroscopy now demonstrate that bining a/f-type SASPs to DNA in vitro causes a structural change in DNA, from the B to the A conformation. This may provide the basis whereby a/f8-type SASPs confer increased spore UV resistance in vivo-by changing spore DNA conformation, they alter DNA photochemistry such that UV irradiation produces spore photoproduct instead of the more lethal cyclobutane-type thymine dimers.genes (termed ssp genes) exhibit a remarkably high degree of conservation of their primary sequences throughout evolution (10), and any one ofthe a/a-type SASPs appears capable ofconferring UV resistance on bacterial spores (11). By using several purified a/,a-type SASPs, we have examined the effects of SASP on DNA conformation, and we report here that SASP binding to DNA in vitro induces a B -> A conformation change. It seems likely that this phenomenon underlies the ability of SASP to protect spore DNA against thymine-dimer formation. Given their evolutionary antiquity (10), the SASPs also may have as-yet-undiscovered protein relatives that use the same strategy to protect cells against UV. The high intrinsic UV resistance of obligate anaerobic bacteria (12), for example, may reflect just such a system. Among the dramatic changes accompanying sporulation in Gram-positive bacteria, a major increase in resistance to killing by UV radiation ranks high as a factor ensuring that the organism will survive dormancy (1). Detailed study of this phenomenon has shown that UV irradiation of spores produces no detectable cis-syn cyclobutane-type thymine dimers in DNA-in sharp contrast with the predominance of this lesion in other UV-irradiated in vivo systems. Instead, the DNA of UV-irradiated spores accumulates primarily 5-thyminyl-5,6-dihydrothymine ("spore photoproduct") (2,3). Germinating spores contain a specific enzyme that efficiently repairs spore photoproduct (4), thereby ensuring a high probability of survival after exposure to UV.It is clear that DNA conformation can significantly alter photoreactivity (5) and the type of UV-induced photoproducts (6). Thus, one simple explanation for the change in DNA photochemistry that occurs during sporulation invokes an alteration in DNA conformation. Indeed, to explain the altered photochemistry of spore DNA, it was suggested a number of years ago that DNA in bacterial spores is in the A conformation whose stereochemical constraints inhibit thymine dimer formation (7). Howe-, er, the relationship between A-DNA and UV-induced formation of spore photoproduct has never been proven, and it has been suggested (6, 8) that hydration is a more important determinant of D...

Structure of a protein–DNA complex essential for DNA protection in spores of Bacillus species

“…The DNA structure in the complex is consistent with circular dichroism and Fourier transform infrared studies (22); cryoelectron microscopy also indicates a change in the pitch of ␣/␤-type SASPbound DNA to 3.2 nm (23). The structure of DNA in the complex is also consistent with the effects of ␣/␤-type SASPs on DNA supercoiling (24), because the average number of basepairs per turn in the DNA in our structure is 11.5, similar to that in A-DNA, whereas this value is 10 in B-DNA 25 (SI Table 2).…”

Thermaerobacter marianensis gen. nov., sp. nov., an aerobic extremely thermophilic marine bacterium from the 11000 m deep Mariana Trench

“…The molecular determination of spoOA and ssp genes, which are involved in sporulation of many endospore-forming bacteria and have been used as molecular probes for the screening of spore-forming bacteria (Brown et al, 1994;Setlow, 1988;Mohr et al, 1991 ;Brill & Wiegel, 1997), was performed by PCR. The primers for amplification were designed and the PCR reactions were carried out as described by Brill & Wiegel (1997).…”

Synthesis and characterization of a 29‐amino acid residue DNA‐binding peptide derived from α/β‐type small, acid‐soluble spore proteins (SASP) of bacteria

“…3, one can see that the extent of helix formation in this solvent slightly exceeds that induced by DNA. The SASP-peptide alters the CD spectrum of DNA, but this effect differs significantly fror~t that observed with intact ~p-type SASP [13]. As shown in Fig.…”

“…As shown in Fig. 5, the amplitude of the long-wavelength CD band of the DNA increases by about 40%, but there is no indication of a shift in A,~ towards shorter wavelengths as occurs in the case of a B ~ A transition [13].…”

“…Since UV irradiation of purified DNA under conditions of low humidky also forms a significant amount of spore photoproduct [10], the altered DNA photochemistry in spores has been attributed to an alteration in DNA conformation: when the DNA undergoes a change from B-form (at high relative humidity) to A-form (at lower humidity) its photoproduct distribution switches from one consisting mostly of thymine dimers to one dominated by spore photoproduct. This leads to the obvious infemnc~ that ~,type SASP may provide a UV-protective effect to spores by causing a B ---> A conformation change upon binding to DNA [11,12], In vitro studies by Mohr et al [13] have borne out this supposition: both circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy demonstrate the proposed conformation change upon ~/fl-type SASP binding. -10 -5 1 5 10 15 20 25 30 (gl-6) 35 40 45 50 55 60 I l I I I I I I [ I I 1 I I We have now embarked on structural studies aimed at elucidating the mechanism of action of this novel family of DNA binding proteins.…”