title: How seryl-phosphorylated HPr inhibits PrfA, a transcription activator of Listeria monocytogenes virulence genes.
abstract: Listeria monocytogenes PrfA, a transcription activator for several virulence genes, including the hemolysin-encoding hly, is inhibited by rapidly metabolizable carbon sources (glucose, fructose, etc.). This inhibition is not mediated via the major carbon catabolite repression mechanism of gram-positive bacteria, since inactivation of the catabolite control protein A (CcpA) did not prevent the repression of virulence genes by the above sugars. In order to test whether the catabolite co-repressor P-Ser-HPr might be involved in PrfA regulation, we used a Bacillus subtilis strain (BUG1199) containing L. monocytogenes prfA under control of pspac and the lacZ reporter gene fused to the PrfA-activated hly promoter. Formation of P-Ser-HPr requires the bifunctional HPr kinase/phosphorylase (HprK/P), which, depending on the concentration of certain metabolites, either phosphorylates HPr at Ser-46 or dephosphorylates P-Ser-HPr. The hprKV267F allele codes for an HprK/P leading to the accumulation of P-Ser-HPr, since it has normal kinase, but almost no phosphorylase activity. Interestingly, introducing hprKV267F into BUG1199 strongly inhibited transcription activation by PrfA. Preventing the accumulation of P-Ser-HPr in the hprKV267F mutant by replacing Ser-46 in HPr with an alanine restored PrfA activity, while ccpA inactivation had no effect. Interestingly, disruption of ccpA in the hprK wild-type strain BUG1199 also led to inhibition of PrfA. The lowered lacZ expression in the ccpA strain is probably also due to elevated amounts of P-Ser-HPr, since it disappeared when Ser-46 in HPr was replaced with an alanine. To carry out its catalytic function in sugar transport, HPr of the phosphotransferase system (PTS) is also phosphorylated by phosphoenolpyruvate and enzyme I at His-15. However, P-Ser-HPr is only very slowly phosphorylated by enzyme I, which probably accounts for PrfA inhibition. In agreement with this concept, disruption of the enzyme I- or HPr-encoding genes also strongly inhibited PrfA activity. PrfA activity therefore seems to depend on a fully functional PTS phosphorylation cascade.
authors.affiliation: Microbiologie et Génétique Moléculaire, CNRS/INRA/INA-PG, Thiverval-Grignon, France.
authors.forename: Rana
authors.initials: R
authors.lastname: Herro
authors.forename: Sandrine
authors.initials: S
authors.lastname: Poncet
authors.forename: Pascale
authors.initials: P
authors.lastname: Cossart
authors.forename: Carmen
authors.initials: C
authors.lastname: Buchrieser
authors.forename: Edith
authors.initials: E
authors.lastname: Gouin
authors.forename: Philippe
authors.initials: P
authors.lastname: Glaser
authors.forename: Josef
authors.initials: J
authors.lastname: Deutscher
mesh_terms: D019943:Amino Acid Substitution; D019677:Artificial Gene Fusion; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D001427:Bacterial Toxins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D009154:Mutation; D010454:Peptide Termination Factors / Q000037:antagonists & inhibitors* / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000502:physiology*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D010454:Peptide Termination Factors; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D001616:beta-Galactosidase; C038705:hlyA protein, Listeria monocytogenes