title: Transcriptional activator YesS is stimulated by histidine-phosphorylated HPr of the Bacillus subtilis phosphotransferase system.
abstract: In low GC content gram-positive bacteria, the HPr protein is the master regulator of carbon metabolism. HPr is a key component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system that interacts with and/or phosphorylates proteins relevant to carbon catabolite repression. HPr can be phosphorylated by two distinct kinases as follows: the bifunctional enzyme HPr kinase/Ser(P)-HPr phosphorylase (HprK/P) phosphorylating the serine 46 residue (Ser(P)-HPr) and acting as a phosphorylase on Ser(P)-HPr; and the PEP-requiring enzyme I (EI) generating histidine 15-phosphorylated HPr (His(P)-HPr). The various HPr forms interact with numerous enzymes and modulate their activity. By carrying out a genome-wide yeast two-hybrid screen of a Bacillus subtilis library, we identified a novel HPr-interacting protein, the transcriptional activator YesS, which regulates the expression of pectin/rhamnogalacturonan utilization genes. Remarkably, yeast tri-hybrid assays involving the ATP-dependent HprK/P and the PEP-dependent EI suggested that YesS interacts with HPr and His(P)-HPr but not with Ser(P)-HPr. These findings were confirmed by in vitro interaction assays using the purified HPr-binding domain of the YesS protein. Furthermore, pectin utilization and in vivo YesS-mediated transcriptional activation depended upon the presence of His(P)-HPr, indicating that HPr-mediated YesS regulation serves as a novel type of carbon catabolite repression. In the yeast two-hybrid assays, B. subtilis HprK/P and EI were active and specifically recognized their substrates. Both kinases formed long lived complexes only with the corresponding nonphosphorylatable mutant HPr. These findings suggest that two-hybrid assays can be used for the identification of unknown kinases of phosphorylated bacterial proteins detected in phosphoproteome analyses.
authors: Laboratoire de Microbiologie et Génétique Moléculaire, INRA-AgroParisTech-CNRS, 78850 Thiverval-Grignon.
authors: Sandrine
authors: S
authors: Poncet
authors: Laboratoire de Génétique Microbienne, Domaine de Vilvert, INRA, 78352 Jouy en Josas Cedex, France.
authors: Maryline
authors: M
authors: Soret
authors: Laboratoire de Génétique Microbienne, Domaine de Vilvert, INRA, 78352 Jouy en Josas Cedex, France.
authors: Peggy
authors: P
authors: Mervelet
authors: Laboratoire de Microbiologie et Génétique Moléculaire, INRA-AgroParisTech-CNRS, 78850 Thiverval-Grignon. Electronic address: Josef.Deutscher@grignon.inra.fr.
authors: Josef
authors: J
authors: Deutscher
authors: Laboratoire de Génétique Microbienne, Domaine de Vilvert, INRA, 78352 Jouy en Josas Cedex, France. Electronic address: Philippe.Noirot@jouy.inra.fr.
authors: Philippe
authors: P
authors: Noirot
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D009876:Operon; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D010770:Phosphotransferases; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases