Manganese is an essential nutrient for all organisms and is a limiting nutrient for many pathogenic bacteria, which respond to low manganese concentrations by producing proteins associated with virulence. Metalloregulatory proteins such as the streptococcal cell adhesion regulator (ScaR) from Streptococcus gordonii and manganese transport regulator (MntR) from Bacillus subtilis are responsible for mediating the cellular response to varying manganese concentrations by binding DNA at cognate operator sequences. ScaR and MntR are related by sequence similarity and function to the diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae, which is an iron-dependent regulatory protein. The work proposed here is directed at understanding how ScaR and MntR bind manganese specifically over iron, despite their structural similarities to DtxR and the chemical similarities between manganese and iron. Also, it is of interest to determine how the manganese activates MntR and ScaR for DNA binding. A variety of biophysical techniques will be used. X-ray crystallography will be the principal technique, and the primary aims of this proposal are to determine the crystal structures of MntR and Scar alone, bound to metal ions and in ternary complexes with manganese and DNA. In addition, quantitative studies will be performed to measure the affinity and specificity of MntR and ScaR, and site-directed mutants of these proteins, to their cognate metal ions and operator sequences. In this way, a fuller description of the activation and specificity of ScaR and MntR will be obtained.