Evidence for direct interaction between enzyme I(Ntr) and aspartokinase to regulate bacterial oligopeptide transport.
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).
King
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Department of Biochemistry and Center for Microbial Pathogenesis, State University of New York at Buffalo, Buffalo, New York 14214, USA.
O'Brian
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MR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001222:Aspartate Kinase / Q000235:genetics / Q000378:metabolism*; D001395:Azotobacter / Q000201:enzymology / Q000235:genetics; D001692:Biological Transport; D020369:Bradyrhizobium / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009842:Oligopeptides / Q000378:metabolism*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C049251:prolyl-glycyl-glycine; D000255:Adenosine Triphosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C116188:lysC aspartokinase; D001222:Aspartate Kinase; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D000409:Alanine