Characterization of regulatory element Rp3 of regulation of beta-lactamases from Ralstonia pickettii.
The two chromosomally encoded beta-lactamases, OXA-22 and OXA-60, from Ralstonia pickettii are inducible by beta-lactam molecules. Disruption of RP3 abolished induction of both beta-lactamases and the resistance to pH, osmolarity and survival in the stationary phase, suggesting that RP3 might be a global regulator. Interactions between RP3, OXA-22 and OXA-60 were investigated at a transcript and protein level using 5'-rapid amplification of cDNA ends experiments, real-time reverse transcription (RT)-PCR and footprinting assays. The rp3 gene was actively transcribed and the promoter sequences corresponded to a nontypical sigma(70)-type promoter. RT-PCR analysis showed that rp3 expression as well as that of the bla(OXA) genes was positively regulated: the level of transcripts of rp3, bla(OXA-22) and bla(OXA-60) genes were, respectively, increased 20-, 100- and 2000-fold upon imipenem induction. DNAse I footprinting showed that RP3 specifically bound to tandem repeats centered at positions -55.5 and -73.5 upstream from the bla(OXA-22) and bla(OXA-60) transcriptional start sites. Interestingly, the binding site at bla(OXA-60) overlapped the -35 region of the rp3 promoter, although the region essential for induction lies at the beginning of the orf-rp3. This result indicates that RP3 is most probably only one component of a novel regulatory system involved in the expression of beta-lactamases in R. pickettii.
Girlich
Delphine
D
Service de Bactériologie-Virologie, INSERM U914, Emerging Resistance to Antibiotics, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris and Université Paris XI, Bicêtre, France. dgirlich@yahoo.fr
Kolb
Annie
A
Naas
Thierry
T
Nordmann
Patrice
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000150:complications; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D016905:Gram-Negative Bacterial Infections / Q000209:etiology / Q000378:metabolism; D006801:Humans; D015378:Imipenem / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D049920:Ralstonia pickettii / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D015533:Transcriptional Activation / Q000187:drug effects; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D014157:Transcription Factors; D015378:Imipenem; C413942:beta-lactamase OXA-22; D001618:beta-Lactamases; C494141:beta-lactamase OXA-60, Ralstonia pickettii