For C. difficile, we conclude that the Spo0A protein likely recognizes a motif that is similar to the B. subtilis Spo0A box on the basis of four lines of evidence; 1. All DNA binding/contacting residues are conserved (Fig. 2B) , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A (Fig. 2D) , 3. Mutagenesis of key residues in the B. subtilis Spo0A box reduces affinity of C. difficile Spo0A for DNA (Fig. 2E ) and 4. A B. subtilis Spo0A box has predictive value for DNA binding by C. difficile Spo0A (Fig. 3A) . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein. It is likely that differences do exist between the preferred binding sites for both proteins that will be evident when a comprehensive analysis is performed of in vivo DNA binding of C. difficile Spo0A; based on the limited data set of this study, a MEME analysis [58] already suggests possible differences in the extended Spo0A motif (W.K. Smits, unpublished observations). These differences may relate to the much higher AT content of C. difficile compared to B. subtilis (71 vs. 56.5%, respectively), or phosphorylation dependent dimerization, for instance.