title: Regulation of class D beta-lactamase gene expression in Ralstonia pickettii.
abstract: Ralstonia pickettii, an environmental bacterium that may also be responsible for human infections, produces two unrelated, inducible and chromosomally encoded oxacillinases, OXA-22 and OXA-60. In order to study the molecular basis of the induction process of these oxacillinase genes, the induction kinetics, the promoter/operator regions necessary for expression and induction, and the role of several ORFs located upstream and downstream of the bla(OXA) genes were investigated. The beta-lactamase production reached a maximal level after 1 h induction, returned to its basal level within the following 3 h and was then again inducible. Using 5'RACE experiments, the promoter sequences of both oxacillinases were determined. These sequences showed weak promoter activities, which could, however, be increased approximately 200-fold by mutating the -35 promoter sequence. Deletion of the sequences located upstream of the promoter regions did not modify the basal beta-lactamase expression in R. pickettii, but resulted in the lack of induction. A minimum of 240 and 270 bp upstream of the transcription initiation sites was required for inducible expression of the bla(OXA-22) and bla(OXA-60) genes, respectively. Analysis of the genetic environment of both bla(OXA) genes revealed several ORFs that were inactivated by homologous recombination. Disruption of ORF-RP3, located 190 bp upstream of bla(OXA-60) and divergently transcribed, abolished induction of both beta-lactamases. ORF-RP3, which encoded a polypeptide of 532 aa with an estimated molecular mass of 58.7 kDa, displayed no obvious sequence homology with known regulatory proteins. Trans-complementation of ORF-RP3 restored the basal and inducible expression of both oxacillinase genes, indicating that the induction of both enzymes was related to the presence of ORF-RP3. In addition to the loss of induction, inactivation of the ORF-RP3 in R. pickettii resulted in a complex pleiotropic phenotype, with increased lag phase and reduced survival after heat exposure, suggesting that ORF-RP3 might be a global regulator involved in unrelated regulatory pathways.
authors.affiliation: Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 Le Kremlin-Bicêtre, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Girlich
authors.affiliation: Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 Le Kremlin-Bicêtre, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Naas
authors.affiliation: Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 Le Kremlin-Bicêtre, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D004790:Enzyme Induction; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D049920:Ralstonia pickettii / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D011995:Recombination, Genetic; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000032:analysis / Q000096:biosynthesis / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001618:beta-Lactamases