Biochemical Characterization of Bacillus subtilis Spo0E mutants Affecting Spo0A~P dephosphorylation activity

DIAZ, A.R; STEPHENSON, S.; PEREGO, M.

Conferencia; Bacillus and other Gram Positives; 2005

Spo0A~P is the essential response regulator and transcriptional factor for sporulation initiation in Bacillus subtilis. The phosphorylation level of Spo0A in the cell is determined by the opposing activities of sensor histidine kinases and aspartyl phosphate phosphatases acting on the phosphorelay, the multicomponent signal transduction system for sporulation initiation. The Spo0A~P is specifically dephosphorylated by the three members of the Spo0E family of phosphatases, Spo0E, YisI and YnzD. These are small proteins, ranging from 56 or 57 amino acids as for YisI and YnzD respectively, to 85 amino acids of Spo0E, with an overall low level of sequence identity (29-34% of identical residues, 12-15% of conserved substitution). Particularly striking, however, is the sequence conservation of a pentapeptide within the sequence of the three proteins. The conserved sequence is flanked by two conserved amino acid residues upstream and two hydrophobic residues downstream that seem to constitute the signature for Spo0E like phosphatases: TIxxSQELDxHyHy. This motif is indeed highly conserved among Spo0E orthologues identified in other spore-forming Gram-positive bacteria and this suggested a critical role for this sequence in Spo0E activity. We carried out an alanine scanning mutagenesis of the signature sequence of the Spo0E protein. Residues T35, I36, Q40, E41, L42, D43, C44, L45 and I46 of Spo0E were mutated to alanine and the effect of the substitutions were first tested in an in vivo sporulation assay. We observed that the substitutions of E41 and C44 did not affect Spo0E activity while the T35A and Q40A mutants were partially inactive. Inactive protein resulted from the alanine substitutions of residues I36, S39, L42, D43, L45 and I46.