Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro.
alpha/beta-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 alpha/beta-type wild-type SASP, SspCwt. One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP. The other mutations changed residues conserved in all alpha/beta-type SASP. In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), 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 SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt. Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.
Tovar-Rojo
F
F
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Setlow
P
P
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000502:physiology / Q000528:radiation effects; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012808:Sigma Factor; D013171:Spores, Bacterial / Q000235:genetics; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus