Lead (Pb) is a widely distributed, environmentally persistent, toxic metal. Most bacteria that are tolerant or resistant to lead either precipitate Pb in an insoluble form, or actively export it . Although some metal efflux ATPases, such as ZntA from Escherichia coli, and CadA from Staphylococcus aureus plasmid pI258, can export Pb(II) as well as Zn(II) and Cd(II) [2, 3], the only characterized bacterial Pb(II) specific resistance system is from Cupriavidus (formerly Wautersia and Ralstonia) metallidurans CH34 [4, 5] - a Gram-negative, multiply metal-resistant, β-proteobacterium originally isolated from a decantation basin at a Belgian zinc production plant (and originally identified as Alcaligenes eutrophus CH34; ). Over 150 genes in CH34 are involved in metal resistance, of which at least 70 are carried on the plasmids pMOL28 (171 kb) or pMOL30 (234 kb), and the remainder are carried on the 3.92 Mb chromosome or on a 2.58 Mb second chromosome . Plasmid pMOL30 carries the czc (Cd(II), Zn(II), Co(II)), mer (Hg(II)), sil (Ag(I)), cop (Cu(II)) and pbr Pb(II) resistance operons [4, 8].
The pbr lead resistance operon from pMOL30 was originally predicted to contain structural genes which encode PbrT, a putative Pb(II) uptake protein belonging to the ILT (Iron Lead Transporter) family , a P-type efflux ATPase (PbrA), a predicted inner–membrane protein (PbrB), a predicted prelipoprotein signal peptidase PbrC and a Pb(II) binding protein, PbrD. The regulator of the pbr operon was shown to be PbrR, which is a MerR family regulator [4, 10] Subsequent work has shown that the pbr operon also contains an interrupted orf; pbrU upstream of pbrT[11, 12] which is predicted to encode a putative inner membrane (Major Facilitator Family MFS1) permease gene, which is probably inactive, but still part of the pbr operon; and that PbrB/PbrC is a fusion protein [11, 12], and encodes an inner membrane bound undecaprenyl pyrophosphate (C55-PP) phosphatase . The pbr operon contains a predicted MerR-like promoter from which pbrRTU are transcribed on one DNA strand, and the pbrABCD genes are transcribed as a polycistronic message on the other [4, 12]. The most recent work on the mechanism of lead resistance encoded by the pMOL30 pbr operon has proposed a model where Pb2+ induces expression of the pMOL30-encoded PbrABCD by PbrR, as well as expression of zinc and cadmium efflux ATPase homologs ZntA and CadA which are carried on the chromosome or second chromosome. Each of these three ATPases is involved in exporting Pb2+ into the periplasm where inorganic phosphates produced by PbrB are involved in precipitating Pb2+ as insoluble lead phosphate. This model finds no role for PbrT, C, and D, yet some reports suggest PbrC may be required for the maturation or activity of phosphatase in the periplasm. PbrR from pMOL30 (Rmet_5946) is related to several other PbrR-like regulators that have been identified in the C. metallidurans CH34 chromosome, including pbrR2 (Rmet_2303 also known as pbr691[13, 14] which is believed to regulate a cadA and a pbrC homolog on the chromosome, and pbrR3 (Rmet_3456 also known as pbr710) believed to regulate a zntA homolog on the second chromosome, both of which are believed to be involved in Pb2+ export . There is evidence for only very low levels of cross-regulation of the pMOL30 PpbrA promoter by PbrR2 or PbrR3 .
Other metal-sensing MerR family members include those responding to cadmium (CadR; [16, 17]), copper (CueR; [18–20], ActP; , SctR; ), zinc (ZntR, [23, 24]; ZccR (Zn, Co, Cd), ) and gold (GolS, ). Metal-sensing MerR family regulators share many common features: they bind to and activate gene expression from promoters with unusually long spacer sequences of 19-20 bp between the −35 and −10 sequences, and contain cysteine and other amino acids that are essential in coordinating metals and activating gene expression [10, 16, 20, 27–29].
The objectives of this study were to 1) Characterize the interaction between PbrR and the pbrA promoter, and study the effects on transcription of shortening the 19 bp spacer between the −35 and −10 sequences, and altering the −10 sequence of PpbrA; and 2) to investigate the importance of cysteine residues in PbrR activation of PpbrA in response to Pb(II) ions. To this end each of the cysteine residues in PbrR (C14, C55, C79, C114, C123, C132 and C134) were individually changed to serine residues and a double mutant (C132S, C134S) was created. The effects of these mutations on in vivo transcriptional activation in response to Pb(II) were determined in C. metallidurans using β-galactosidase assays.