Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro

Abstract

α/β-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the α/β-type wild-type SASP, SspC(wt). One mutation (SspC(Tyr)) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other α/β-type SASP. The other mutations changed residues conserved in all α/β-type SASP. In one (SspC(Ala)), residue 52 (Gly) was changed to Ala; in the second (SspC(Gln)), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspC(wt) and SspC(Tyr) interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major α/β-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspC(Ala) had no detectable effect on DNA properties in vivo or in vitro, while SspC(Gln) had effects intermediate between those of SspC(Ala) and SspC(Wt). Strikingly, neither SspC(Ala) nor SspC(Gln) bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of α/β-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.