Target transcription binding sites differentiate two groups of MerR-monovalent metal ion sensors

Abstract

SummaryThe evolution of bacterial regulatory circuits often involves duplication of genes encoding transcription factors that may suffer both modifications in their detected signals, as well as, rewiring of their target operators. This, and subsequent horizontal gene transfer events contribute to generate a diverse array of regulatory pathways. In Salmonella, two homologous transcription factors CueR and GolS are responsible for Cu and Au sensing and resistance respectively. They share similarities not only in their sequence but also in their target binding sites, although they cluster separately among MerRmonovalent metal sensors. Here, we demonstrate that CueR and GolS can selectively distinguish their target binding sites by recognizing bases at positions 3Ј and 3 of their cognate operators. Swap of these bases results in switching regulator dependency. The differences in promoter architecture plus the environmentally controlled regulator's cytoplasmic availability warrant intra-regulon regulator-operator selectivity, and the proper response to metal injury. Furthermore, the presence of the distinctive operators' bases is widely extended among the two groups of MerRmonovalent metal sensors, providing evidence of the co-evolution of these factors and their target operators. This approach allows the prediction of regulator's dependency and the identification of transcription modules among groups of homologous transcription factors.

A Single Serine Residue Determines Selectivity to Monovalent Metal Ions in Metalloregulators of the MerR Family

“…␤-Galactosidase assays of golB::lacZ and copA::lacZ transcriptional fusions were used to analyze the metal-mediated activation of GolS and CueR, respectively, essentially as described previously (13). The cells were grown overnight in LB medium supplemented with the addition of sublethal concentrations of different metal salts.…”

“…Two homologous monovalent metal ion sensors that coexist in Salmonella enterica were characterized: the nonselective CueR sensor, which responds to Cu(I), Ag(I), and Au(I) with similar sensitivities, and the Au sensor GolS, which discriminates Au(I) from Cu(I) or Ag(I) (10)(11)(12)(13). By domain-swapping and site-directed mutagenesis, we established that residues at positions 113 and 118 within the MBL are the main contributors to Au(I) selectivity (12).…”

Selective detection of gold using genetically engineered bacterial reporters

“…Western blot analysis of GolS was performed essentially as described (Perez Audero et al, 2010), but using rabbit polyclonal anti-GolS antibodies and the ECL Plus Western Blotting Detection System (Amersham). Protein concentration was determined by Bradford assay, using bovine serum albumin as standard.…”

“…All reagents, chemicals, and oligonucleotides were from Sigma-Aldrich Co. (pPgolB) pMC1871 derived plasmid with the golB promoter inserted upstream the lacZ gene (Perez Audero et al, 2010) (St. Louis, MO), except LB culture medium that was from Difco TM -BD Co. (Sparks, MD). All heavy metal salts were at least of analytical grade (purity greater than 95%).…”

Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System

“…We also tested the PhoP/PhoQ-unrelated transcriptional reporters tppB::lacZ and golT::lacZ as controls; tppB is a two-component OmpR/EnvZcontrolled gene, whereas golT is the transcriptional regulator of an operon involved in Salmonella resistance to transition metals (35)(36)(37). As shown in Fig.…”