Regulation of class D beta-lactamase gene expression in Ralstonia pickettii

摘要

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 qenes 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 mutatinq the-35 promoter sequence.Deletion of the sequences located upstream of the promoter reqions 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-11)and bla_(OXA-60)genes,respectively.Analysis of the genetic environment of both bla_(OXA)genes revealed several ORFs that were inactivated by homoloqous 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 homoloqy with known regulatory proteins.7>a/is-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.