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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.
AuthorsDelphine Girlich, Thierry Naas, Patrice Nordmann
JournalMicrobiology (Reading, England) (Microbiology (Reading)) Vol. 152 Issue Pt 9 Pg. 2661-2672 (Sep 2006) ISSN: 1350-0872 [Print] England
PMID16946261 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • beta-Lactamases
Topics
  • Amino Acid Sequence
  • Base Sequence
  • Conserved Sequence
  • Enzyme Induction
  • Gene Expression Regulation, Bacterial
  • Genes, Bacterial
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Open Reading Frames
  • Promoter Regions, Genetic
  • Protein Structure, Tertiary
  • Ralstonia pickettii (enzymology, genetics, isolation & purification)
  • Recombination, Genetic
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • Transcription, Genetic
  • beta-Lactamases (analysis, biosynthesis, classification, genetics, metabolism)

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