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Regulation of class D -lactamase gene expression in Ralstonia pickettii

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

Correspondence
Thierry Naas
thierry.naas{at}bct.ap-hop-paris.fr

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 -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 -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 -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.

The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the bla_OXA-22 genetic environment reported in this paper is AF064820.