The SmtB/ArsR family of prokaryotic metalloregulators are winged-helix transcriptional repressors that collectively provide resistance to a wide range of both biologically required and toxic heavy metal ions. CmtRMtb is a recently described CdII/PbII regulator expressed in M. tuberculosis that is structurally distinct from the wellcharacterized SmtB/ArsR CdII/PbII sensor, S. aureus plasmid pI258-encoded CadC. From functional analyses and a multiple sequence alignment of CmtR homologs, CmtRMtb is proposed to bind PbII and CdII via coordination by Cys57, Cys61 and Cys102 [Cavet et al. (2003) J. Biol. Chem. 278, 44560-44566]. To better understand the mechanism how CmtRMtb utilizes specific metal ions to perform transcriptional repressor function, both CmtRMtb and its homolog in S. coelicolor A3(2) (CmtRSc) were studied. We establish here that both wild-type and C102S CmtRMtb are homodimers and bind CdII and PbII via formation of cysteine thiolate-rich coordination bonds. UV-Vis optical spectroscopy and 113Cd NMR spectroscopy (δ=480 ppm) suggest two or three thiolate donors, while 111mCd perturbed angular correlation (PAC) spectroscopy establish an unusual trigonal pyramidal coordination eometry. C102S CmtRMtb binds CdII and ZnII with only ≈ 10-20 fold lower affinity relative to wild-type CmtRMtb, but ≈ 100-1000-fold lower for PbII. Quantitative investigation of CmtR-cmt O/P binding equilibria using fluorescence anisotropy reveals that Cys57 and Cys61 anchor the coordination complex with Cys102 functioning as a key allosteric ligand, while playing only an accessory role in stabilizing the metal complex in the free protein. Similar metal titration experiments were carried out with a putative CmtR homolog from S. coelicolor A3(2) (CmtRSc) and a double cysteine substitution mutant C110G/C111S CmtRSc. The implications of these findings on the evolution of distinct metal sensing sites in a family of homologous proteins are discussed.

Salmonella enterica is an important cause of food poisoning and is responsible for approximately a billion human infections each year. Disease manifestation in humans varies from severe systemic enteric (typhoid) fever to self-limiting gastroenteritis depending upon the infecting S. enterica serovar. S. Typhimurium is responsible for acute gastroenteritis in humans but causes a typhoid-like disease in mice and thus serves as an important model for studying the pathogenesis of systemic salmonellosis. Following ingestion, S. Typhimurium employs a variety of virulence mechanisms to survive within its host and establishes infection in the intestinal tract by invading the epithelial cells. Recent studies have revealed the importance of sulfur compounds in the intestine, such as tetrathionate and thiosulfate for the disease progression. S. Typhimurium is capable of utilising these sulfur compounds as terminal electron acceptors for its anaerobic respiration and thus gains a growth advantage over host microbiota during infection. However, the regulation of sulfur availability within S. Typhimurium and the mechanisms involved in mitigating cellular sulfide toxicity are not well-defined. During this study, we have identified the sstRA operon in S. Typhimurium encoding a deduced SmtB/ArsR family of transcriptional regulatory protein (SstR) and a deduced rhodanese-family sulfurtransferase (SstA) and demonstrated a role in mitigating the effects of cellular sulfide toxicity. SstR has been confirmed to act as a transcriptional repressor from the sstRA operator-promoter and the SstR-dependent repression is alleviated by low pH and sulfide stress (sodium thiosulfate), consistent with a role for SstR in sensing sulfide stress to trigger gene expression. Electrophoretic mobility shift assays confirm binding of purified SstR to the sstRA operator-promoter region. Furthermore, a conserved pair of cysteine residues within SstR was identified to be crucial for alleviating SstR-mediated repression, with the substitution of either cysteine causing constitutive repression. This is consistent with SstR inducer-responsiveness involving a thiol-based redox switch. Importantly, S. Typhimurium mutants lacking the sstRA operon have reduced tolerance to sulfide stress, consistent with the sstRA operon having a role in cellular sulfide detoxification. Work is continuing to further characterise the roles of sstR and sstA in S. Typhimurium on their contributions to infections.

This study focuses on engineering whole cell-based biosensors for heavy metal detection. Through the exploitation of metalloregulatory proteins, fabrication of metal ion-responsive biosensors is achieved. Metalloregulatory proteins of the SmtB/ArsR family including arsenite-responsive ArsR, cadmium-responsive CadC, zinc-responsive CzrA, and nickel-responsive NmtR were evaluated as biosensor sensing modules. Characterization of these four metal sensing modules was accomplished through quantification of a reporter green fluorescence protein (gfp) gene. As such, biosensors pCTYC-r34ArsR-pL(ArsOvN)GFP and pCTYC-r34CadC-pL(CadOv1)GFP displayed excellent gfp expression and sensitivity to As(III) and Cd (II), respectively. These two biosensors were consequently selected and successfully implemented in soil bacterium Pseudomonas putida. Lastly, a proof of concept arsenite-responsive genetic toggle switch is proposed utilizing PurRcelR467 (PC47), a cellobiose-responsive gene, and an LAA degradation tag. Overall, this study expands the bank of metalloregulatory bioparts for heavy metal sensing in the aim of constructing an optimized water monitoring system.