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Cysteine coordination of Pb(II) is involved in thePbrR-dependent... » Isaúde
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BMC microbiology [electronic resource]
2012-06-19 03:32:51

Cysteine coordination of Pb(II) is involved in thePbrR-dependent activation of the lead-resistancepromoter, PpbrA, from Cupriavidus metalliduransCH34

Descrição: Background:The pbr resistance operon from Cupriavidus metallidurans CH34 plasmid pMOL30 confersresistance to Pb(II) salts, and is regulated by the Pb(II) responsive regulator PbrR, which is aMerR 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 betweenthe 35 and 10 sequences, and activates transcription of resistance genes as a consequenceof binding the appropriate metal. Cysteine residues in these regulators are essential for metalion coordination and activation of expression from their cognate promoter. In this study weinvestigated the interaction of PbrR with the promoter for the structural pbr resistance genes,PpbrA, effects on transcriptional activation of altering the DNA sequence of PpbrA, andeffects on Pb(II)-induced activation of PpbrA when cysteine residues in PbrR were mutatedto serine.Results:Gel retardation and footprinting assays using purified PbrR show that it binds to, and protectsfrom DNase I digestion, the PpbrA promoter, which has a 19 bp spacer between its 35 and10 sites. Using beta-galactosidase assays in C. metallidurans, we show that when PpbrA ischanged to an 18 bp spacer, there is an increase in transcriptional activation both in thepresence and absence of Pb(II) salts up to a maximum induction equivalent to that seen in thefully-induced wild-type promoter. Changes to the 10 sequence of PpbrA from TTAAAT tothe consensus E. coli 10 sequence (TATAAT) increased transcriptional activation fromPpbrA, whilst changing the 10 sequence to that of the Tn501 mer promoter (TAAGGT) alsoincreased the transcriptional response, but only in the presence of Pb(II). Individual PbrRmutants C14S, C55S, C79S, C114S, C123S, C132S and C134S, and a double mutantC132S/C134S, were tested for Pb(II) response from PpbrA, using beta-galactosidase assays inC. metallidurans. The PbrR C14S, C79S, C134S, and C132S/C134S mutants were defectivein Pb(II)-induced activation of PpbrA.Conclusions:These data show that the metal-dependent activation of PbrR occurs by a similar mechanismto that of MerR, but that metal ion coordination is through cysteines which differ from thoseseen in other MerR family regulators, and that the DNA sequence of the 10 promoter affectsexpression levels of the lead resistance genes.

Identificador: doi:10.1186/1471-2180-12-109
Volume: 0
Página: 1 a

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