DNA binding by full length Spo0A in B. subtilis requires phosphorylation dependent dimerization [8, 9] . However, it was shown that the isolated DBD is capable of binding to legitimate targets of the full length protein [10] . Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene (PabrB) of B. subtilis. PabrB is commonly used as a high-affinity control in binding assays with the B. subtilis Spo0A or Spo0A-DBD protein [43, 44] . It is noteworthy that we failed to identify a homolog of abrB in C. difficile using BLAST, indicating that potential indirect regulation by Spo0A cannot occur through abrB in C. difficile as it does in B. subtilis. We found that C. difficile Spo0A-DBD bound with high affinity to PabrB (Fig. 2D and E) . We performed electrophoretic mobility shift assays (EMSAs) using radiolabeled PabrB and increasing amounts of purified C. difficile Spo0A-DBD that was purified using a C-terminal 66His-tag. The addition of protein leads to a dose-dependent retardation of the DNA fragment with an apparent K D of ,50 nM. In the same range of protein concentrations, no binding was observed for a negative control (a DNA fragment of B. subtilis citG [45] ) (Fig. 2E) , suggesting that binding was specific for the abrB promoter region.