Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

Small acid-soluble proteins (SASPs) play an important role in protection of DNA in dormant bacterial endospores against damage by heat, UV radiation or enzymic degradation. In the genome of the strict anaerobe Clostridium acetobutylicum, five genes encoding SASPs have been annotated and here a further sixth candidate is suggested. The ssp genes are expressed in parallel dependent upon Spo0A, a master regulator of sporulation. Analysis of the transcription start points revealed a sigma(G) or a sigma(F) consensus promoter upstream of each ssp gene, confirming a forespore-specific gene expression. SASPs were termed SspA (Cac2365), SspB (Cad 522), SspD (Cad 620), SspF (Cac2372), SspH (Cad 663) and Tlp (Cad 487). Here it is shown that with the exception of Tip, every purified recombinant SASP is able to bind DNA in vitro thereby protecting it against enzymic degradation by DNase I. Moreover, SspB and SspD were specifically cleaved by the two germination-specific proteases GPR (Cad 275) and YyaC (Cac2857), which were overexpressed in Escherichia coli and activated by an autocleavage reaction. Thus, for the first time to our knowledge, GPR-like activity and SASP specificity could be demonstrated for a YyaC-like protein. Collectively, the results assign SspA, SspB, SspD, SspF and SspH of C. acetobutylicum as members of alpha/beta-type SASPs, whereas Tip seems to be a non-DNA-binding spore protein of unknown function. In acetic acid-extracted proteins of dormant spores of C. acetobutylicum, SspA was identified almost exclusively, indicating its dominant biological role as a major alpha/beta-type SASP in vivo.