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.

Klorat som avfallsprodukt från pappersindustrin har lett till miljöproblem på grund av klorats toxiska verkan på växter och alger, och har även lett till bekymmer för människohälsan där klorat avfallet har kommit i kontakt med dricksvatten. För att åtgärda detta så har mycket forskning gjorts på bakterier med förmågan att reducera klorat till syrgas och kloridjoner, en anaerob process som vissa naturligt förekommande bakterier kan utföra. Med ökad kunskap om regleringen av denna kloratreducerande process, kan dessa bakterier utgöra en effektiv reningsprocess av pappersbrukens avloppsvatten.  Ideonella dechloratans är en sådan bakterie, den har ett genkluster som kodar för de kloratreducerande enzymerna. Nedströms för dessa finns en arsR-sekvens som tros att koda för en transkriptionsfaktor; och ytterligare information om denna transkriptionsfaktor kan möjligen bidra till förståelse av genuttrycket hos den kloratreducerande funktionen. Syftet med detta arbete är att transformera expressionsceller med förmågan att uttrycka arsR, så framtida försök kan göras för att identifiera potentiella bindningssäten för ArsR-proteinet. arsR-sekvensen amplifierades med primrar specifika för ändarna hos arsR, sekvensen ligerades i en pET-21a(+) vektor från Novagen och transformerades med BL21 (DE3) expressionsceller. Med en IPTG inducering kunde ett stort uttryck av olösligt ArsR observeras. Komplikationer med resultatet och framtida tillvägagångsätt diskuteras. / Chlorate as a waste product from the paper industry has caused environmental problems due its toxic effect on plants and algae and is also a concern for human health where chlorate has contaminated the tap water supplies. To address this issue, a great deal of research has been carried out on naturally occurring bacteria that can anaerobically reduce chlorate to oxygen and chloride ions. With additional knowledge of how this chlorate-reducing process is regulated, these bacteria may one day provide an effective purification process of wastewater from paper mills. Ideonella dechloratans is such a bacterium that has a gene cluster which encodes the chlorate-reducing enzymes. Downstream of this cluster is an arsR-sequence believed to encode a transcription factor, which could aid in the understanding of the gene expression for the chlorate-reducing operon. The goal of this research is to transform expression cells with the ability to express the arsR-sequence so that future trials can be made to identify any potential binding sites for the ArsR-protein. The arsR-sequence was amplified with primers specific to the ends of the arsR-sequence. The sequence was then ligated into a pET-21a(+) vector from Novagen and transformed with BL21 (DE3) expression cells. By IPTG inducing these transformants it was possible to observe a significant expression of insoluble ArsR. Complications with the outcome and future approaches are discussed.

Heavy metals from natural and man-made sources can be a great threat to human and animal life. As small inorganic ions they are challenging to detect, usually requiring expensive and complicated machinery. Several heavy metals can accumulate in the human body, leading to long term toxic effects on the nervous system. Many bacteria have developed strategies to survive in heavy metal rich environments. One of these strategies is a bacterial operon containing genes for detoxification mechanisms controlled by a promoter and a regulatory protein. In this work some of these promoter-protein pairs, Pars-ArsR, PcopA-CueR, PmerTPAD-MerR and PzntA-ZntR from Escherichia coli have been employed in the design and construction of a set of biosensors aimed at the detection of heavy metals in drinking water. Biosensors usually employ biological recognition elements, transducing the signal from these to produce an output that can be integrated into electronic circuitry. The sensors presented in this work focus on reducing complexity and on providing a controlled sensor reaction. The arsenic biosensor ‘AsGard’ is based on the Pars-ArsR pair and functions by making the dissociation of an ArsR-mCherry fusion protein from its binding site in the Pars promoter visible. In the cell, ArsR dissociates from Pars upon binding of trivalent arsenic ions. Immobilising the relevant part of the Pars sequence on a solid plastic support allows for the mobilisation of previously bound ArsR-mCherry proteins in the presence of arsenic to become the sensor output. The AsGard sensor detects arsenic within minutes in a concentration range overlapping with the arsenic thresholds for drinking water as set by the World Health Organisation. Additional prototype sensors are presented bringing a reporter gene under the control of the aforementioned promoters. These sensors have been tested in vivo and in vitro in a cell free transcription translation system and partially detect metal concentrations close to relevant ranges. The Pars based sensor is tuneable in vitro by modifying the ratio of the supplied regulatory protein ArsR and is able to detect arsenic well within the relevant range. Spinach2, a fluorescent RNA aptamer, may make future designs independent from translation, drastically reducing complexity of cell free biosensors based on cis-trans transcriptional regulation.

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.

Thiomonas sp. 3As, bactérie isolée d'eaux de drainage de mine (Carnoulès, France) contaminées par l'arsenic, a la capacité d'oxyder l'arsenic. Nous l'avons caractérisée aux niveaux physiologique et chimiotaxonomique, ce qui a montré qu'il s'agit d'une nouvelle espèce : "Thiomonas arsenitoxudans". L'arsénite axydase catalyse l'oxydation de l'arsénite (As(III)) en arséniate (As(V)), moins soluble et toxique. Nous avons montré chez cette bactérie que les gènes aoxAB codant pour les 2 sous-unités de cette enzyme sont co-transcrits ArsR/SmtB. Cet opéron est transcrit en présence d'As(III) et d'As(V). Nos résultats indiquent qu'ArsR-like (1) est stabilisé par l'As(III) ou l'As(V). Nos résultats suggèrent qu'ArsR-like a un mécanisme d'action différent de celui établi pour les autres membres de la famille ArsR/SmtB / Thiamonas sp. 3As, a bacterium isolated from acid mine drainage waters heavily loaded with arsenic (Carnoulès, France), has the capacity to oxidize arsenic. We characterized it both at the physiological and chemotaxonomical levels, which showed that it belongs to a new species : "thiomonas arsenitoxydans". The arsenite oxidase catalyses the oxidation of arsenite (As(III)) to arsenate (As(V)), less soluble and toxic. We showed that the genes aoxAB encoding the 2 sub-units of this enzyme are co-transcribed with the genes coding for 2 cytochromes c and a metalloregulator ArsR-like of the ArsR/SmtB family in "Tm. arsenitoxydans". This operon is transcribed in the presence of As(III)) or As(V). Our results indicaded that ArsR-like (1) is stabilized by As(III) or As(V), and (2) binds on the regulatory sequence of the aox operon in the presence of As(III) or As(V). From oour results, we suggest that the mechanism of ArsR-like is different from that of the other members of the ArsR/SmtB family.