Effects of Carboxy-Terminal Modifications and pH on Binding of a Bacillus subtilis Small, Acid-Soluble Spore Protein to DNA

Variants of the wild-type Bacillus subtilis alpha/beta-type small, acid-soluble spore protein (SASP) SspC(wt) were designed to evaluate the contribution of C-terminal residues to these proteins' affinity for DNA. SspC variants lacking one to three C-terminal residues were similar to SspC(wt) in DNA binding, but removal of six C-terminal residues greatly decreased DNA binding. In contrast, a C-terminal extension of three residues increased SspC's affinity for DNA 5- to 10-fold. C-terminal and N-terminal changes that independently caused large increases in SspC-DNA binding affinity were combined and produced an additive effect on DNA binding; the affinity of the resulting variant, SspC(DeltaN11-D13K-C3), for DNA was increased >/==" BORDER="0">20-fold over that of SspC(wt). For most of the SspC variants tested, lowering the pH from 7 to 6 improved DNA binding two- to sixfold, although the opposite effect was observed with variants having additional C-terminal basic residues. In vitro, the binding of SspC(DeltaN11-D13K-C3) to DNA suppressed the formation of cyclobutane-type thymine dimers and promoted the formation of the spore photoproduct upon UV irradiation to the same degree as the binding of SspC(wt). However, B. subtilis spores lacking major alpha/beta-type SASP and overexpressing SspC(DeltaN11-D13K-C3) had a 10-fold-lower viability and far less UV and heat resistance than spores overexpressing SspC(wt). This apparent lack of DNA protection by SspC(DeltaN11-D13K-C3) in vivo is likely due to the twofold-lower level of this protein in spores compared to the level of SspC(wt), perhaps because of effects of SspC(DeltaN11-D13K-C3) on gene expression in the forespore during sporulation. The latter results indicate that only moderately strong binding of alpha/beta-type SASP to DNA is important to balance the potentially conflicting requirements for these proteins in DNA transcription and DNA protection during spore formation, spore dormancy, and spore germination and outgrowth.