title: Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.
abstract: EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the β-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha.
authors: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
authors: Katja
authors: K
authors: Karstens
authors: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
authors: Christopher P
authors: CP
authors: Zschiedrich
authors: Department of Molecular Physiology, Institute of Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
authors: Botho
authors: B
authors: Bowien
authors: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
authors: Jörg
authors: J
authors: Stülke
authors: Max F. Perutz Laboratories, Department of Microbiology, Immunobiology and Genetics, Center of Molecular Biology, University of Vienna, 1030 Vienna, Austria.|Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, 37077 Göttingen, Germany.
authors: Boris
authors: B
authors: Görke
mesh_terms: D026481:Blotting, Far-Western; D020118:Cupriavidus necator / Q000201:enzymology*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011755:Pyrophosphatases / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D010770:Phosphotransferases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D011755:Pyrophosphatases