Abstract
Environmental bacteria are endowed with several regulatory systems that have potential applications in biotechnology. In this report, we characterize the arsenic biosensing features of the ars response system from Chromobacterium violaceum in the heterologous host Escherichia coli. We show that the native Pars/arsR system of C. violaceum outperforms the chromosomal ars copy of E. coli when exposed to micromolar concentrations of arsenite. To understand the molecular basis of this phenomenon, we analyzed the interaction between ArsR regulators and their promoter target sites as well as induction of the system at saturating concentrations of the regulators. In vivo titration experiments indicate that ArsR from C. violaceum has stronger binding affinity for its target promoter than the regulator from E. coli does. Additionally, arsenite induction experiments at saturating regulator concentration demonstrates that although the Pars/arsR system from E. coli displays a gradual response to increasing concentration of the inducer, the system from C. violaceum has a steeper response with a stronger promoter induction after a given arsenite threshold. Taken together, these data demonstrate the characterization of a novel arsenic response element from an environmental bacterium with potentially enhanced performance that could be further explored for the construction of an arsenic biosensor.
Highlights
Bacteria that thrive in environments contaminated with toxic compounds are usually endowed with diverse molecular mechanisms related to tolerance to these chemicals (Top and Springael, 2003; Permina et al, 2006)
We demonstrate that the ars system from C. violaceum has superior arsenic induction performance when compared to the chromosomal prototype system in Escherichia coli and that these differences can be traced to the binding affinity of ArsR regulators to their DNA targets and to the occurrence of a stronger transcriptional response under inducing conditions in the C. violaceum system
In order to compare the performance of the ars system from C. violaceum with that of E. coli, we cloned the Pars/arsR elements from both organisms into a GFP reporter vector and introduced the resulting construct into E. coli
Summary
Bacteria that thrive in environments contaminated with toxic compounds are usually endowed with diverse molecular mechanisms related to tolerance to these chemicals (Top and Springael, 2003; Permina et al, 2006). Transcription factors belonging to the SmtB/ArsR family have been expensively characterized for their role in sensing and controlling gene expression in response to divalent metals (e.g., zinc, nickel, and cadmium) or toxic metalloids (e.g., arsenic and antimonite) (Busenlehner et al, 2003; Eicken et al, 2003; Qin et al, 2007) Members of this family are usually small (∼100 aa) regulatory proteins that repress gene expression by blocking access of RNA polymerase to target promoters in the absence of the cognate inducer (VanZile et al, 2000; Kar et al, 2001; Morita et al, 2001; Cavet et al, 2002). Once in complex with their specific target metal/metalloid, the regulators strongly decrease their affinity for DNA allowing dissociation from the promoter and subsequently, gene expression activation (Kar et al, 1997; Morita et al, 2002, 2003; Eicken et al, 2003; Chauhan et al, 2009)
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