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Background  The  pbr  resistance operon from  Cupriavidus metallidurans  CH34 plasmid pMOL30 confers resistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is a MerR family activator. In other metal sensing MerR family regulators, such as MerR, CueR, and ZntR the cognate regulator binds to a promoter with an unusually long spacer between the −35 and −10 sequences, and activates transcription of resistance genes as a consequence of binding the appropriate metal. Cysteine residues in these regulators are essential for metal ion coordination and activation of expression from their cognate promoter. In this study we investigated the interaction of PbrR with the promoter for the structural  pbr  resistance genes, P pbrA , effects on transcriptional activation of altering the DNA sequence of P pbrA , and effects on Pb(II)-induced activation of P pbrA  when cysteine residues in PbrR were mutated to serine.    Results  Gel retardation and footprinting assays using purified PbrR show that it binds to, and protects from DNase I digestion, the P pbrA  promoter, which has a 19 bp spacer between its −35 and −10 sites. Using β-galactosidase assays in  C. metallidurans , we show that when P pbrA  is changed to an 18 bp spacer, there is an increase in transcriptional activation both in the presence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in the fully-induced wild-type promoter. Changes to the −10 sequence of P pbrA  from TTAAAT to the consensus  E. coli − 10 sequence (TATAAT) increased transcriptional activation from P pbrA , whilst changing the −10 sequence to that of the Tn 501 mer  promoter (TAAGGT) also increased the transcriptional response, but only in the presence of Pb(II). Individual PbrR mutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutant C132S/C134S, were tested for Pb(II) response from P pbrA , using β-galactosidase assays in  C. metallidurans . The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defective in Pb(II)-induced activation of P pbrA .    Conclusions  These data show that the metal-dependent activation of PbrR occurs by a similar mechanism to that of MerR, but that metal ion coordination is through cysteines which differ from those seen in other MerR family regulators, and that the DNA sequence of the −10 promoter affects expression levels of the lead resistance genes.

bmc microbiology

Cysteine coordination of Pb(II) is involved in the PbrR-dependent activation of the lead-resistance promoter, PpbrA, from Cupriavidus metallidurans CH34