Protein phosphatase PphA from Synechocystis sp.PCC 6803:the physiological framework of P_(II)-P dephosphorylation

摘要

The phosphorylated signal transduction protein P_(II) (P_(II)-P) in the cyanobacterium Synechocystis sp.strain PCC 6803 is dephosphorylated by PphA,a protein phosphatase of the 2C family (PP2C).In this study,the physiological conditions of P_(II)-P dephosphorylation were investigated with respect to the in vivo specificity of P_(II)-P towards PphA and the cellular abundance of PphA in cells growing under different nitrogen regimes.Furthermore,the consequences of impaired P_(II)-P dephosphorylation with respect to short-term inhibition of glutamine synthetase (GS) were studied.With a contribution of approximately 15 of total Mn~(2+)-dependent p-nitrophenyl phosphate hydrolysis activity,PphA has only a minor impact on the total PP2C activity in Synechocystis extracts.Nevertheless,residual P_(II)-P dephosphorylation in PphA-deficient cells could only be observed after prolonged incubation in the presence of ammonium.The abundance of PphA correlates with the phosphorylation state of P_(II) under nitrogen-replete conditions and is specifically enhanced by nitrite.Regulation of pphA expression operates at the post-transcriptional level.In the presence of nitrate/nitrite,PphA is present in molar excess over P_(II)-P,enabling the cells to rapidly dephosphorylate P_(II)-P in response to changing environmental conditions.A PphA-deficient mutant is not impaired in short-term inhibition of GS activity following ammonium treatment.Down-regulation of GS occurs by induction of gif genes (encoding GS inactivating factors 7 and 1 7),which is controlled by NtcA-mediated gene repression.Thus,impaired P_(II)-P dephosphorylation does not affect ammonium-prompted inactivation of NtcA.