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Keywords:

  • protein phosphorylation;
  • phosphoenolpyruvate-dependent phosphotransferase system (PTS);
  • nitrogen-PTS;
  • EIIANtr;
  • ptsN;
  • yhbJ

Abstract

The nitrogen-related phosphotransferase system (Ntr-PTS) is a paralogous system working in parallel to the well-known carbohydrate:PTS. In a chain of phosphotransfer reactions, EINtr and NPr (PtsO) deliver phosphoryl groups to the EIIANtr (PtsN) protein. EIIANtr is implicated in important regulatory processes such as the σE-dependent cell envelope stress response and regulation of K+ uptake. Phosphorylation is believed to trigger the output of EIIANtr in these regulations. EIIANtr is encoded within the gene cluster ptsN–yhbJ–ptsO, which is highly conserved in Proteobacteria. In this study, we investigated the phosphorylation of the Escherichia coli EIIANtr protein in vivo by 32P-labeling. We show that EIIANtr is readily phosphorylated in wild-type cells. This phosphorylation occurs at a single site, the histidine 73 in EIIANtr. YhbJ and NPr are dispensable for this phosphorylation. A detailed analysis revealed that both the energy coupling phosphotransferases of the Ntr-PTS as well as the ‘sugar’-PTS contribute to the phosphorylation of EIIANtr, suggesting cross talk between both systems.