title: Analysis of catabolite control protein A-dependent repression in Staphylococcus xylosus by a genomic reporter gene system.
abstract: A single-copy reporter system for Staphylococcus xylosus has been developed, that uses a promoterless version of the endogenous beta-galactosidase gene lacH as a reporter gene and that allows integration of promoters cloned in front of lacH into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of S. xylosus promoters by the catabolite control protein CcpA. To test if lacH is a suitable reporter gene, beta-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the malRA maltose utilization operon promoter and autoregulation of the ccpA promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (cres), from promoter regions of several S. xylosus genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, P(vegII). For that purpose, cre sequences were placed at position +3 or +4 within the transcribed region of P(vegII). Measurements of beta-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of ccpA completely abolished glucose-dependent regulation. Therefore, the tested cres functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in S. xylosus was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in S. xylosus. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.
authors.affiliation: Mikrobielle Genetik, Universität Tübingen, D-72076 Tübingen, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Jankovic
authors.forename: O
authors.initials: O
authors.lastname: Egeter
authors.forename: R
authors.initials: R
authors.lastname: Brückner
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D016680:Genome, Bacterial; D005941:Glucokinase; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009876:Operon; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013210:Staphylococcus / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009004:Monosaccharide Transport Proteins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; C120827:glcU protein, Staphylococcus xylosus; C084528:malR protein, bacteria; D008320:Maltose; C096446:GlkA protein, Bacteria; D005941:Glucokinase; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases; D001616:beta-Galactosidase