Alanine Scanning Mutagenesis of the Spo0E phosphatase Domain Interacting with the Spo0A Response Regulator of Bacillus subtilis

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

Boca Raton, Florida, USA

Congreso; BLAST Meeting; 2005

Spo0A~P is the essential responseregulator and transcriptional factor for sporulation initiation in Bacillussubtilis. Thephosphorylation level of Spo0A in the cell is determined by the opposingactivities of sensor histidine kinases and aspartyl phosphate phosphatasesacting on the phosphorelay, the multicomponent signaltransduction system for sporulation initiation. The Spo0A~P is specificallydephosphorylated by the three members of the Spo0E family of phosphatases,Spo0E, YisI and YnzD. These are small proteins, ranging from56 or 57 amino acids as for YisI and YnzD respectively, to 85 amino acids ofSpo0E, with an overall low level of sequence identity (29-34% of identicalresidues, 12-15% of conserved substitution). Particularly striking, however, isthe sequence conservation of a pentapeptide within the sequence of the threeproteins. The conserved sequence is flanked by two conserved amino acidresidues upstream and two hydrophobic residues downstream that seem toconstitute the signature for Spo0E like phosphatases: TIxxSQELDxHyHy. This motif is indeed highlyconserved among Spo0E orthologues identified in other spore-formingGram-positive bacteria and this suggested a critical role for this sequence inSpo0E activity. We carried out an alanine scanning mutagenesis of the signaturesequence of the Spo0E protein. Residues T35, I36, Q40, E41, L42, D43, C44, L45and I46 of Spo0E were mutated to alanine and the effect of the substitutionswere first tested in an in vivo sporulation assay. We observed that thesubstitutions of E41 and C44 did not affect Spo0E activity while the T35A andQ40A mutants were partially inactive. Inactive protein resulted from thealanine substitutions of residues I36, S39, L42, D43, L45 and I46.