Abstract
Bradyrhizobium japonicum transports oligopeptides and the heme precursor delta-aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutants disrupted in the lysC and ptsP genes were identified based on the inability to use prolyl-glycyl-glycine as a proline source and were defective in [(14)C]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in trans complementation analysis showed that lysC and ptsP are transcribed separately, and that 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) (EI(Ntr)), a paralogue of Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experiments indicated that purified recombinant aspartokinase and EI(Ntr) interact directly with each other. Expression of ptsP in trans from a multicopy plasmid complemented the lysC mutant, suggesting that aspartokinase normally affects Enzyme I(Ntr) in a manner that can be compensated for by increasing the copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EI(Ntr), but it was phosphorylated by ATP in the presence of cell extracts. This phosphorylation was inhibited in the presence of aspartokinase. The findings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regulating the phosphorylation state of EI(Ntr).
Highlights
Bacteria utilize small peptides as nutrients, chemoattractants, and quorum sensing signals, and their metabolism is a target for antibiotics [1,2,3]
Our interest in oligopeptide transport in the bacterium Bradyrhizobium japonicum is founded on studies of heme biosynthesis, where it has been demonstrated that the heme precursor ␦-aminolevulinic acid (ALA) is taken up by a system that transports oligopeptides [8]
We show that Enzyme INtr (EINtr) is involved in oligopeptide transport, and it, and perhaps the PTSNtr system as a whole, has a role in transport of a non-sugar solute
Summary
Bacteria utilize small peptides as nutrients, chemoattractants, and quorum sensing signals, and their metabolism is a target for antibiotics [1,2,3]. The Opp system binds peptides two to five peptides in length, with the highest affinity for tripeptides [6, 7] For both permease systems, the amino acid side chain appears not to be important for specificity, and these systems transport peptides independent of sequence. It is taken up by the Dpp system in E. coli [9] and S. typhimurium [10], but ALA is taken up in a dpp mutant if the opp system is activated [8]. Phenotypes of mutants in the parallel PTS system ( called the PTSNtr system), gene organization and the structure of EINtr, suggest a role for this system coordinating nitrogen and carbon metabolism. A novel function for the amino acid biosynthesis enzyme aspartokinase was implicated, and evidence for interaction between EINtr and aspartokinase is presented
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