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1.
Environ Sci Pollut Res Int ; 24(1): 52-65, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27234828

ABSTRACT

Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible Pars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.


Subject(s)
Arsenites/chemistry , Biosensing Techniques , Deinococcus/metabolism , Escherichia coli/metabolism , Luciferases, Bacterial/metabolism , Arsenites/metabolism , Deinococcus/genetics , Environmental Monitoring/methods , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Genes, Reporter , Luciferases, Bacterial/genetics , Metals, Heavy/toxicity , Promoter Regions, Genetic , Water/chemistry , Water Pollutants, Chemical
2.
Adv Healthc Mater ; 4(7): 1076-83, 2015 May.
Article in English | MEDLINE | ID: mdl-25676134

ABSTRACT

The fast development of sensitive molecular diagnostic tools is currently paving the way for a personalized medicine. A new class of ultrasensitive magnetic resonance imaging (MRI) T2-contrast agents based on magnetosomes, magnetite nanocrystals biomineralized by magnetotactic bacteria, is proposed here. The contrast agents can be injected into the blood circulation and detected in the picomolar range. Purified magnetosomes are water-dispersible and stable within physiological conditions and exhibit at 17.2 T a transverse relaxivity r2 four times higher than commercial ferumoxide. The subsequent gain in sensitivity by T2(*) -weighted imaging at 17.2 T of the mouse brain vasculature is evidenced in vivo after tail vein injection of magnetosomes representing a low dose of iron (20 µmoliron kg(-1)), whereas no such phenomenon with the same dose of ferumoxide is observed. Preclinical studies of human pathologies in animal models will benefit from the combination of high magnetic field MRI with sensitive, low dose, easy-to-produce biocompatible contrast agents derived from bacterial magnetosomes.


Subject(s)
Brain/pathology , Ferrosoferric Oxide/chemistry , Magnetosomes/chemistry , Nanostructures/chemistry , Animals , Contrast Media/chemistry , Dextrans/chemistry , Magnetic Resonance Imaging/methods , Magnetics/methods , Magnetite Nanoparticles/chemistry , Magnetosomes/metabolism , Magnetospirillum/metabolism , Mice , Molecular Imaging/methods , Nanoparticles/chemistry
3.
J Am Chem Soc ; 128(15): 5209-18, 2006 Apr 19.
Article in English | MEDLINE | ID: mdl-16608357

ABSTRACT

In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron-Sulfur clusters, and the surface-exposed [4Fe4S] cluster has an unusual His(Cys)3 ligation. When this Histidine (H184 in Desulfovibrio fructosovorans) is changed into a cysteine or a glycine, a distal cubane is still assembled but the oxidative activity of the mutants is only 1.5 and 3% of that of the WT, respectively. We compared the activities of the WT and engineered enzymes for H2 oxidation, H+ reduction and H/D exchange, under various conditions: (i) either with the enzyme directly adsorbed onto an electrode or using soluble redox partners, and (ii) in the presence of exogenous ligands whose binding to the exposed Fe of H184G was expected to modulate the properties of the distal cluster. Protein film voltammetry proved particularly useful to unravel the effects of the mutations on inter and intramolecular electron transfer (ET). We demonstrate that changing the coordination of the distal cluster has no effect on cluster assembly, protein stability, active-site chemistry and proton transfer; however, it slows down the first-order rates of ET to and from the cluster. All-sulfur coordination is actually detrimental to ET, and intramolecular (uphill) ET is rate determining in the glycine variant. This demonstrates that although [4Fe4S] clusters are robust chemical constructs, the direct protein ligands play an essential role in imparting their ability to transfer electrons.


Subject(s)
Hydrogenase/chemistry , Hydrogenase/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Desulfovibrio/enzymology , Desulfovibrio/genetics , Electrochemistry , Electron Spin Resonance Spectroscopy , Hydrogen-Ion Concentration , Hydrogenase/antagonists & inhibitors , Hydrogenase/genetics , Imidazoles/chemistry , Imidazoles/metabolism , Iron-Sulfur Proteins/genetics , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Solutions
4.
J Biol Inorg Chem ; 9(5): 636-42, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15175937

ABSTRACT

The kinetics of the activation and anaerobic inactivation processes of Desulfovibrio gigas hydrogenase have been measured in D(2)O by FTIR spectroelectrochemistry. A primary kinetic solvent isotope effect was observed for the inactivation process but not for the activation step. The kinetics of these processes have been also measured after replacement of a glutamic residue placed near the active site of an analogous [NiFe] hydrogenase from Desulfovibrio fructosovorans. Its replacement by a glutamine affected greatly the kinetics of the inactivation process but only slightly the activation process. The interpretation of the experimental results is that the rate-limiting step for anaerobic inactivation is the formation from water of a micro-OH(-) bridge at the hydrogenase active site, and that Glu25 has a role in this step.


Subject(s)
Desulfovibrio gigas/enzymology , Hydrogenase/chemistry , Mutagenesis, Site-Directed , Anaerobiosis , Binding Sites , Glutamic Acid/chemistry , Hydrogen/chemistry , Hydrogenase/genetics , Hydrogenase/metabolism , Isotopes , Kinetics , Metalloproteins/chemistry , Metalloproteins/metabolism , Solvents , Spectroscopy, Fourier Transform Infrared/methods
5.
J Biol Chem ; 279(11): 10508-13, 2004 Mar 12.
Article in English | MEDLINE | ID: mdl-14688251

ABSTRACT

Kinetic, EPR, and Fourier transform infrared spectroscopic analysis of Desulfovibrio fructosovorans [NiFe] hydrogenase mutants targeted to Glu-25 indicated that this amino acid participates in proton transfer between the active site and the protein surface during the catalytic cycle. Replacement of that glutamic residue by a glutamine did not modify the spectroscopic properties of the enzyme but cancelled the catalytic activity except the para-H(2)/ortho-H(2) conversion. This mutation impaired the fast proton transfer from the active site that allows high turnover numbers for the oxidation of hydrogen. Replacement of the glutamic residue by the shorter aspartic acid slowed down this proton transfer, causing a significant decrease of H(2) oxidation and hydrogen isotope exchange activities, but did not change the para-H(2)/ortho-H(2) conversion activity. The spectroscopic properties of this mutant were totally different, especially in the reduced state in which a non-photosensitive nickel EPR spectrum was obtained.


Subject(s)
Glutamic Acid/chemistry , Hydrogenase/chemistry , Hydrogenase/genetics , Protons , Aspartic Acid/chemistry , Binding Sites , Catalysis , Catalytic Domain , Crystallography, X-Ray , Desulfovibrio/enzymology , Electron Spin Resonance Spectroscopy , Hydrogen/chemistry , Hydrogen/metabolism , Hydrogen-Ion Concentration , Kinetics , Mutagenesis, Site-Directed , Mutation , Oxidation-Reduction , Oxygen/metabolism , Plasmids/metabolism , Spectroscopy, Fourier Transform Infrared , Time Factors , X-Ray Diffraction
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