1887

Abstract

, 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 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 , 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 and genes, respectively. Analysis of the genetic environment of both genes revealed several ORFs that were inactivated by homologous recombination. Disruption of ORF-RP3, located 190 bp upstream of 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. -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 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.

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2006-09-01
2019-11-12
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