Growth-dependent Phosphorylation of the PtsN (EIINtr) Protein of Pseudomonas putida

Abstract

The nitrogen-related branch of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) of Pseudomonas putida includes the ptsN gene encoding the EII Ntr (PtsN) enzyme. Although the implication of this protein in a variety of cellular functions has been observed in diverse bacteria, the physiological signals that bring about phosphorylation/dephosphorylation of the PtsN protein are not understood. This work documents the phosphorylation status of the EII Ntr enzyme of P. putida at various growth stages in distinct media. Culture conditions were chosen to include fructose (the uptake of which is controlled by the PTS) or glucose (a non-PTS sugar in P. putida) in minimal medium with casamino acids, ammonia, or nitrate as alternative nitrogen sources. To quantify the relative ratio of PtsN/PtsN ϳ P in live cells, we resorted to the in situ electrophoresis of whole bacteria expressing an E-epitope-tagged EII Ntr followed by the fractionation of the thereby released native proteome in a non-denaturing gel. Although the PtsN species phosphorylated in amino acid His 68 was detected under virtually all growth scenarios, the relative levels of the non-phosphorylated form varied dramatically depending on the growth phase and the nutrients available in the medium. The share of phosphorylated PtsN increased along growth in a fashion apparently independent of any trafficking of sugars. The large variations of non-phosphorylated PtsN in different growth conditions, in contrast to the systematic excess of the phosphorylated PtsN form, suggested that the P-free PtsN is the predominant signaling species of the protein.

Evidence of In Vivo Cross Talk between the Nitrogen-Related and Fructose-Related Branches of the Carbohydrate Phosphotransferase System of Pseudomonas putida

“…However, the source of the high-energy phosphate is unclear in this case, as the system was still repressed by the sugar in a ptsP (EI NtrϪ ) strain (9). Moreover, PϳEIIA Ntr accumulates during growth, even in the presence of glucose in the medium (27), while the maximum Pu activity occurs at stationary phase. As a result, the mechanisms that link such PTS proteins to Pu activity remain ambiguous.…”

“…The functions of such a branch of a PTS, which is alien to carbohydrate transport, and the signals that bring about the phosphorylation of the corresponding proteins in vivo are uncertain. Yet they seem to be related to sensing the metabolic balance between N and C (3,8,27,32,39), and therefore this branch is designated PTS Ntr . The only known mechanistic hint of the function of PTS Ntr is that PtsN checks the biosynthesis of branched-chain amino acids (20) by interacting directly with the K ϩ transporter TrkA (19) in Escherichia coli.…”

“…With this background, we set out to clarify the route of transfer of highenergy phosphate through the EI Ntr , NPr, and EIIA Ntr proteins of P. putida. To this end, we used a recently developed procedure for visualizing the relative proportions of phosphorylated and nonphosphorylated EIIA Ntr in intact cells (26,27). As shown below, by using various P. putida genetic backgrounds and growth conditions, we found that the phosphorylation state of EIIA Ntr was a suitable descriptor of the functioning of the whole ϳP transfer chain.…”

Phosphorylation-Dependent Mobility Shift of Proteins on SDS-PAGE is Due to Decreased Binding of SDS

“…9-11 Several regulatory roles of the nitrogen PTS were revealed in some bacteria. [12][13][14][15][16][17] The state of phosphorylation of proteins in this system is also crucial to the regulation phenomena.…”

Recent Advances in the Expression, Evolution, and Dynamics of Prokaryotic Genomes

“…K. Pflüger (Madrid, Spain) from V. de Lorenzo's group described a novel electrophoretic method to separate phosphorylated proteins directly from intact cells. The procedure was instrumental in monitoring the flow of high-energy phosphate through the abridged phosphoenolpyruvate-carbohydrate phosphotransferase system of Pseudomonas putida (76). This work suggests that the phosphoenolpyruvate-carbohydrate phosphotransferase system in this bacterium is not connected to sugar transport but rather serves as a sensor of the metabolic state of the cell.…”