Control of the Phosphorylation State of the HPr Protein of the Phosphotransferase System in <i>Bacillus subtilis</i>: Implication of the Protein Phosphatase PrpC

Abstract

In the Gram-positive bacterium Bacillus subtilis as well as in other firmicutes, the HPr protein of the phosphotransferase system (PTS) has two distinct phosphorylation sites, His-15 and Ser-46. These sites are phosphorylated by the Enzyme I of the PTS and by the ATP-dependent HPr kinase/phosphorylase, respectively. As a result, the phosphorylation state of HPr reflects the nutrient supply of the cell and is in turn involved in several responses at the levels of transport activity and expression of catabolic genes. Most important, HPr(Ser-P) serves as a cofactor for the pleiotropic transcription regulator CcpA. In addition to the proteins that phosphorylate HPr, those that are involved in the dephosphorylation are important in controlling the overall HPr phosphorylation state and the resulting regulatory and physiological outputs. In this study, we found that in addition to the phosphorylase activity of the HPr kinase/phosphorylase, the serine/threonine protein phosphatase PrpC uses HPr(Ser-P) as a target.

Genetic Dissection of Specificity Determinants in the Interaction of HPr with Enzymes II of the Bacterial Phosphoenolpyruvate:Sugar Phosphotransferase System inEscherichia coli

“…Of these lysates, 1 g total protein of each was loaded on SDS-15% polyacrylamide gels. After separation, the proteins were blotted to a polyvinyl difluoride (PVDF) membrane and HPr was detected using polyclonal rabbit antibodies directed against B. subtilis HPr as described previously (33). These experiments were carried out twice.…”

In vitro Phosphorylation of Key Metabolic Enzymes from <i>Bacillus subtilis:</i> PrkC Phosphorylates Enzymes from Different Branches of Basic Metabolism

“…Therefore, we assayed the phosphorylation of HPr in the presence of PrkC and [ ␥ - PrkC was capable of phosphorylating HPr. Both HPrK (with its phosphorylase activity) and PrpC, a protein phosphatase encoded in an operon with PrkC, are able to dephosphorylate HPr(Ser-P) [Mijakovic et al, 2002;Singh et al, 2007]. Therefore, we considered the possibility that Ser-46 of HPr was the phosphorylation site used by HPrK as well as by PrkC.…”

“…This phosphatase is able to dephosphorylate HPr(Ser-P) in B . subtilis and Mycoplasma pneumoniae [Halbedel et al, 2006;Singh et al, 2007].…”

Carbon Catabolite Repression in Bacillus subtilis : Quantitative Analysis of Repression Exerted by Different Carbon Sources

“…In the presence of glucose, HPr, and presumably also Crh, is phosphorylated by the bifunctional HPr kinase/phosphorylase (HPrK/P) on Ser46, whereas this site is less phosphorylated in cells growing in the absence of sugars (27,32). Dephosphorylation of HPr(Ser-P) is catalyzed by the phosphorylase activity of HPrK/P (31) and, to some extent, by the protein phosphatase PrpC (48). In vitro experiments suggested that the two antagonistic activities of HPrK/P are regulated by metabolites.…”