fmicb-07-01851 November 17, 2016 Time: 15:41 # 1 ORIGINAL RESEARCH published: 21 November 2016 doi: 10.3389/fmicb.2016.01851 The Chromobacterium violaceum ArsR Arsenite Repressor Exerts Tighter Control on Its Cognate Promoter Than the Escherichia coli System Letícia M. Arruda, Lummy M. O. Monteiro and Rafael Silva-Rocha* Systems and Synthetic Biology Lab, Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil 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 Edited by: Michael Benedik, to micromolar concentrations of arsenite. To understand the molecular basis of Texas A&M University, USA this phenomenon, we analyzed the interaction between ArsR regulators and their Reviewed by: promoter target sites as well as induction of the system at saturating concentrations Jan Roelof Van Der Meer, University of Lausanne, Switzerland of the regulators. In vivo titration experiments indicate that ArsR from C. violaceum Jun-Jie Zhang, has stronger binding affinity for its target promoter than the regulator from E. coli Indiana University School of Medicine, does. Additionally, arsenite induction experiments at saturating regulator concentration USA demonstrates that although the Pars/arsR system from E. coli displays a gradual *Correspondence: Rafael Silva-Rocha response to increasing concentration of the inducer, the system from C. violaceum has [email protected] 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 Specialty section: This article was submitted to element from an environmental bacterium with potentially enhanced performance that Microbiotechnology, Ecotoxicology could be further explored for the construction of an arsenic biosensor. and Bioremediation, a section of the journal Keywords: regulatory network, arsenic response system, cis-regulatory elements, ars operon, ArsR/SmtB family Frontiers in Microbiology Received: 29 August 2016 Accepted: 03 November 2016 INTRODUCTION Published: 21 November 2016 Citation: Bacteria that thrive in environments contaminated with toxic compounds are usually endowed Arruda LM, Monteiro LMO and with diverse molecular mechanisms related to tolerance to these chemicals (Top and Springael, Silva-Rocha R (2016) 2003; Permina et al., 2006). From a physiological point of view, mechanisms for resistance to The Chromobacterium violaceum stressors are usually energy dependent and should be tightly regulated in order to avoid wasting ArsR Arsenite Repressor Exerts valuable resources when stressors are absent (Nojiri et al., 2004; Diaz et al., 2013). Many bacteria Tighter Control on Its Cognate Promoter Than the Escherichia coli have evolved molecular mechanisms to handle exposure to toxic metals and metalloids. The genes System. Front. Microbiol. 7:1851. encoding such systems are usually transcriptionally controlled by a plethora of regulatory systems doi: 10.3389/fmicb.2016.01851 that coordinate gene expression in response to the presence of the cognate chemical. Frontiers in Microbiology | www.frontiersin.org 1 November 2016 | Volume 7 | Article 1851 fmicb-07-01851 November 17, 2016 Time: 15:41 # 2 Arruda et al. Tighter Control of ars System Several regulatory systems dedicated to the sensing of toxic Although changing the circuit design can improve the metals and metalloids have been characterized in bacteria efficiency of the biosensor, the utilization of molecular (Busenlehner et al., 2003; Kaur et al., 2006; Permina et al., components with intrinsically enhanced transcriptional 2006; Reyes-Caballero et al., 2011). Genomic studies have performance when induced with the target compound demonstrated that most of these systems are broadly distributed could lead to a system with superior behavior. With this among different bacterial phyla (Paez-Espino et al., 2009). reasoning in mind, we aimed to identify a natural system For instance, transcription factors belonging to the SmtB/ArsR that displayed enhanced transcriptional response to arsenite family have been expensively characterized for their role in (AsIII). We focused on the ars system from Chromobacterium sensing and controlling gene expression in response to divalent violaceum, a gram-negative bacterium with a low arsenic metals (e.g., zinc, nickel, and cadmium) or toxic metalloids tolerance level but endowed with a fully functional Pars and (e.g., arsenic and antimonite) (Busenlehner et al., 2003; Eicken ArsR regulatory system (Azevedo et al., 2008; Silva-Rocha et al., 2003; Qin et al., 2007). Members of this family are et al., 2013a). We demonstrate that the ars system from usually small (∼100 aa) regulatory proteins that repress gene C. violaceum has superior arsenic induction performance expression by blocking access of RNA polymerase to target when compared to the chromosomal prototype system in promoters in the absence of the cognate inducer (VanZile Escherichia coli and that these differences can be traced to et al., 2000; Kar et al., 2001; Morita et al., 2001; Cavet et al., the binding affinity of ArsR regulators to their DNA targets 2002). These proteins usually act as dimers that bind to target and to the occurrence of a stronger transcriptional response DNA sequences in the apo form. Once in complex with their under inducing conditions in the C. violaceum system. Taken specific target metal/metalloid, the regulators strongly decrease together, the results shown here demonstrate the potential of their affinity for DNA allowing dissociation from the promoter environmental bacteria as a reservoir of molecular components and subsequently, gene expression activation (Kar et al., 1997; with enhanced performance for biosensor design, as well as a Morita et al., 2002, 2003; Eicken et al., 2003; Chauhan et al., characterization of a novel arsenic ArsR transcription factor in 2009). bacteria. Understanding the molecular mechanisms behind the transcriptional response to toxic metals and metalloids in bacteria has led to a growing interest in repurposing these MATERIALS AND METHODS systems to construct biosensors for detection of these chemicals in the environment (Stocker et al., 2003; Siddiki et al., 2011; Bacterial Strains, Plasmids, and Growth Cortés-Salazar et al., 2013; Chen et al., 2014). Such efforts have Conditions been made for the detection of arsenic, a highly abundant and Bacterial strains, plasmids, and primers used in this study are extremely toxic metalloid released to the environment as a listed in Table 1. E. coli DH5a cells were used for cloning result of anthropogenic activity (Matschullat, 2000; Mandal procedures. E. coli W3110 was used as the wild type strain, and Suzuki, 2002). In comparison to analytical chemistry whereas E. coli AW3110 (1ars operon) was used as the mutant methods, biosensors (which encompass a biological sensing host for testing the circuits. E. coli strains were grown at 37◦C in component and easily detectable output) would provide reliable, LB media (Sambrook et al., 1989) or M9 minimal media (6.4 g/L specific, and an inexpensive means for the in situ detection Na2HPO4·7H2O, 1.5 g/L KH2PO4, 0.25 g/L NaCl, and 0.5 g/L of target compounds in environmental samples (Baeumner, NH4Cl) supplemented with 2 mM MgSO4, 0.1 mM casamino 2003). acid, and 1% glycerol as the sole carbon source. When required, Constructed biosensors for environmental purposes have kanamycin (Km, 50 mg/mL) or chloramphenicol (34 mg/mL) generally coupled well-characterized components (usually from was added to the media to ensure plasmid retention. When cells bacteria) that are responsive to the target compound with a were grown in minimal media, antibiotics were used at half reporter gene that gives rise to a colorimetric, luminescent, or concentrations. For induction experiments, benzoic acid (Sigma– fluorescent output. In this sense, developed biosensors proved to Aldrich, St. Louis, MO, USA) and sodium arsenite (Sigma– be useful tools for arsenite and arsenate detection in groundwater Aldrich) were used at different concentrations. (Siegfried et al., 2012) and river water (Siegfried et al., 2015) as a low cost, suitable and transportable alternative to detect the Plasmid and Strain Construction metalloid to prevent or diminish arsenic exposure. Furthermore, For analysis of the ars system under negative feedback, the advent of biological circuit design approaches in the field of DNA fragments containing the Pars/arsR elements from synthetic biology has allowed the re-wiring of basic molecular C. violaceum and E. coli were PCR amplified from genomic DNA components (regulators, DNA binding sites, and operators), using primers 5arscvEco/3arscvBam and 5arsecEco/3arsecBam, which can be reinserted into the host cell to give rise to sensors respectively (Table 1). DNA fragments were amplified using with enhanced performance (Stocker et al., 2003; Trang et al., Phusion High-Fidelity DNA polymerase (New England Biolabs, 2005; Fernandez et al., 2016; Merulla and van der Meer, 2016). Ipswich, MA, USA) according to the manufacturer’s protocol. In fact, the addition of ArsR operator downstream of its target The resulting