title: The interplay of the EIIA(Ntr) component of the nitrogen-related phosphotransferase system (PTS(Ntr)) of Pseudomonas putida with pyruvate dehydrogenase.
abstract: BACKGROUND
  Pseudomonas putida KT2440 is endowed with a variant of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS(Ntr)), which is not related to sugar transport but believed to rule the metabolic balance of carbon vs. nitrogen. The metabolic targets of such a system are largely unknown.
  
  
  METHODS
  Dielectric breakdown of P. putida cells grown in rich medium revealed the presence of forms of the EIIA(Ntr) (PtsN) component of PTS(Ntr), which were strongly associated to other cytoplasmic proteins. To investigate such intracellular partners of EIIA(Ntr), a soluble protein extract of bacteria bearing an E epitope tagged version of PtsN was immunoprecipitated with a monoclonal anti-E antibody and the pulled-down proteins identified by mass spectrometry.
  
  
  RESULTS
  The E1 subunit of the pyruvate dehydrogenase (PDH) complex, the product of the aceE gene, was identified as a major interaction partner of EIIA(Ntr). To examine the effect of EIIA(Ntr) on PDH, the enzyme activity was measured in extracts of isogenic ptsN(+)/ptsN(-)P. putida strains and the role of phosphorylation was determined. Expression of PtsN and AceE proteins fused to different fluorescent moieties and confocal laser microscopy indicated a significant co-localization of the two proteins in the bacterial cytoplasm.
  
  
  CONCLUSIONS
  EIIA(Ntr) down-regulates PDH activity. Both genetic and biochemical evidence revealed that the non-phosphorylated form of PtsN is the protein species that inhibits PDH.
  
  
  CONCLUSIONS
  EIIA(Ntr) takes part in the node of C metabolism that checks the flux of carbon from carbohydrates into the Krebs cycle by means of direct protein-protein interactions with AceE. This type of control might connect metabolism to many other cellular functions. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.
authors.affiliation: Centro Nacional de Biotecnologia, CSIC, Madrid, Spain.
authors.forename: Katharina
authors.initials: K
authors.lastname: Pflüger-Grau
authors.forename: Max
authors.initials: M
authors.lastname: Chavarría
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D047468:Immunoprecipitation; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+); D010731:Phosphoenolpyruvate Sugar Phosphotransferase System