Mutational analysis of conserved residues in the putative DNA-binding domain of the response regulator Spo0A of Bacillus subtilis

The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is req uired for the expression of genes involved in the initiation of sporulation . Spo0A binds directly to and both activates and represses transcription fr om the promoters of several genes required during the onset of endospore fo rmation. The C-terminal 113 residues are known to contain the DNA-binding a ctivity of Spo0A. Previous studies identified a region of the C-terminal ha lf of Spo0A that is highly conserved among species of endospore-forming Bac illus and Clostridium and which encodes a putative helix-turn-helix DNA-bin ding domain, To test the functional significance of this region and determi ne if this motif is involved in DNA binding, we changed three conserved res idues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis , We then isolated and analyzed the five substitution-containing Spo0A prot eins for DNA binding and sporulation-specific gene activation. The S210A Sp o0A mutant exhibited no change from wild-type binding, although it was defe ctive in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activat ion, These data suggest that these conserved residues are important for tra nscriptional activation and that the E213 residue is involved in DNA bindin g.