title: A novel mode of regulation of the Staphylococcus aureus catabolite control protein A (CcpA) mediated by Stk1 protein phosphorylation.
abstract: The Staphylococcus aureus serine/threonine protein kinase Stk1 (also known as PknB) affects different key pathways such as cell wall metabolism, antibiotic susceptibility, and regulation of virulence. Here we report that the catabolite control protein A (CcpA), a highly conserved regulator of carbon catabolite repression and virulence in a number of gram-positive pathogens, was efficiently phosphorylated in vitro and in vivo by Stk1 in S. aureus, whereas the CcpA homologues of Bacillus subtilis and Bacillus anthracis were not affected by the Stk1 orthologue PrkC. Mass spectrometry and mutational analyses identified Thr-18 and Thr-33 as the phosphoacceptors; both are located in the DNA binding domain of this protein. Electrophoretic mobility shift assays demonstrated that the CcpA DNA binding activity was completely abrogated for the phosphorylated CcpA. The physiological relevance of CcpA phosphorylation was assessed by generating CcpA phosphoablative (T18A/T33A) or phosphomimetic (T18D/T33D) mutants. In contrast to the wild-type and phosphoablative ccpA alleles, introduction of the phosphomimetic ccpA allele in a ΔccpA mutant failed to restore the parental biofilm formation profile and the transcription of citZ and hla to levels seen with the wild type. The strong up regulation of ccpA transcripts and CcpA level in the ccpA mutant trans-complemented with the phosphomimetic CcpA variant suggest furthermore that CcpA acts as a negative regulator of its own expression. Together, these findings demonstrate that Stk1-driven phosphorylation of CcpA inhibits its DNA binding activity toward its regulon in S. aureus, representing a novel regulatory mechanism of CcpA activity in addition to the well known regulation via HprKP/Hpr in this clinically important pathogen.
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier II, CNRS UMR 5235, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Jade
authors.initials: J
authors.lastname: Leiba
authors.forename: Torsten
authors.initials: T
authors.lastname: Hartmann
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Cluzel
authors.forename: Martin
authors.initials: M
authors.lastname: Cohen-Gonsaud
authors.forename: Frédéric
authors.initials: F
authors.lastname: Delolme
authors.forename: Markus
authors.initials: M
authors.lastname: Bischoff
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000502:physiology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018441:Biofilms / Q000254:growth & development*; D004926:Escherichia coli / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D013211:Staphylococcus aureus / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*; D037521:Virulence Factors / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D037521:Virulence Factors; C069286:catabolite control proteins, bacteria; D017346:Protein Serine-Threonine Kinases; C546823:Stk1 protein, Staphylococcus aureus