Alanine mutants of the Spo0F response regulator modifying specificity for sensor kinases in sporulation initiation

Five single alanine substitution mutations in the Spo0F response regulator gave rise to mutant strains of Bacillus subtilis with seemingly normal spor ulation that nevertheless rapidly segregated variants blocked in sporulatio n. The basis for this deregulated phenotype was postulated to be increased phosphorylation of the Spo0A transcription factor, resulting from enhanced phosphate input or decreased dephosphorylation of the phosphorelay. Strains bearing two of these Spo0F mutant proteins, Y13A and I17A, retained a requ irement for KinA and KinB kinases in sporulation, whereas the remaining thr ee, L66A, I90A and H101A, gave strains that sporulated well in the absence of both KinA and KinB, Sporulation of strains bearing L66A and H101A mutati ons was decreased in a mutant lacking KinA, KinB and KinC, but the strain b earing the I90A mutation required the further deletion of KinD to lower its sporulation frequency, The affected residues, L-66, I-90 and H-101, are in volved in crucial hydrophobic contacts stabilizing the orientation of helix alpha 4 of Spo0F. The data are consistent with the notion that these three mutations alter the conformation of the beta 4-alpha 4 loop of Spo0F that is known to contain residues critical for KinA:Spo0F recognition. As this l oop has a propensity for multiple conformations, the spatial arrangement of this loop may play a critical role in kinase selection by Spo0F and might be altered by regulatory molecules interacting with Spo0F.