Evidence for direct interaction between enzyme I-Ntr and aspartokinase to regulate bacterial oligopeptide transport

Bradyrhizobium japonicum transports olgopeptides and the heme precursor del ta -aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutant s disrupted in the lysC and ptsP genes were identified based on the inabili ty to use prolyl-glycyl-glycine as a proline source and were defective in [ C-14]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B, japonicum genome. However, RNase protection and in trans complementa tion analysis showed that lysC and ptsP are transcribed separately, and tha t both genes are involved in oligopeptide transport. Aspartokinase, encoded by lysC, catalyzes the phosphorylation of aspartate for synthesis of three amino acids, but the lysC strain is not an amino acid auxotroph, The ptsP gene encodes Enzyme I-Ntr (EINtr), a paralogue of Enzyme I of the phosphoen olpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experi ments indicated that purified recombinant aspartokinase and EINtr interact directly with each other. Expression of ptsP in trans from a multicopy plas mid complemented the lysC mutant, suggesting that aspartokinase normally af fects Enzyme I-Ntr in a manner that can be compensated for by increasing th e copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EINtr, but it was phosphorylated by ATP in the presence of cell extracts. T his phosphorylation was inhibited in the presence of aspartokinase. The fin dings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regu lating the phosphorylation state of EINtr.