Biochemical Characterization of Bacillus subtilis Spo0E mutants

DIAZ, A.R; STEPHENSON, S.; DIAZ AR, CORE LJ, JIANG M, MORELLI M, CHIANG CH, SZURMANT H, PEREGO M.

Conferencia; 3rd Conference on Functional Genomics of Gram Positive Microorganisms and 13th International Conference on Bacilli; 2005

Spo0A~P isthe essential response regulator and transcriptional factor for sporulationinitiation in Bacillus subtilis. The phosphorylation level of Spo0A in the cellis determined by the opposing activities of sensor histidine kinases andaspartyl phosphate phosphatases acting on the phosphorelay, the multicomponentsignal transduction system for sporulation initiation. The Spo0A~P isspecifically dephosphorylated by the three members of the Spo0E family ofphosphatases, Spo0E, YisI and YnzD. These are small proteins, ranging from 56or 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. Thismotif is indeed highly conserved among Spo0E orthologues identified in otherspore-forming Gram-positive bacteria and this suggested a critical role forthis sequence in Spo0E activity. We carried out an alanine scanning mutagenesisof 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 ofthe substitutions were first tested in an in vivo sporulation assay. Weobserved that the substitutions of E41 and C44 did not affect Spo0E activitywhile the T35A and Q40A mutants were partially inactive. Inactive proteinresulted from the alanine substitutions of residues I36, S39, L42, D43, L45 andI46.