Single-Step Production of a Recyclable Nanobiocatalyst for Organophosphate Pesticides Biodegradation Using Functionalized Bacterial Magnetosomes

Enzymes are versatile catalysts in laboratories and on an industrial scale; improving their immobilization would be beneficial to broadening their applicability and ensuring their (re)use. Lipid-coated nano-magnets produced by magnetotactic bacteria are suitable for a universally applicable single-step method of enzyme immobilization. By genetically functionalizing the membrane surrounding these magnetite particles with a phosphohydrolase, we engineered an easy-to-purify, robust and recyclable biocatalyst to degrade ethyl-paraoxon, a commonly used pesticide. For this, we genetically fused the opd gene from Flavobacterium sp. ATCC 27551 encoding a paraoxonase to mamC, an abundant protein of the magnetosome membrane in Magnetospirillum magneticum AMB-1. The MamC protein acts as an anchor for the paraoxonase to the magnetosome surface, thus producing magnetic nanoparticles displaying phosphohydrolase activity. Magnetosomes functionalized with Opd were easily recovered from genetically modified AMB-1 cells: after cellular disruption with a French press, the magnetic nanoparticles are purified using a commercially available magnetic separation system. The catalytic properties of the immobilized Opd were measured on ethyl-paraoxon hydrolysis: they are comparable with the purified enzyme, with Km (and kcat) values of 58 µM (and 178 s−1) and 43 µM (and 314 s−1) for the immobilized and purified enzyme respectively. The Opd, a metalloenzyme requiring a zinc cofactor, is thus properly matured in AMB-1. The recycling of the functionalized magnetosomes was investigated and their catalytic activity proved to be stable over repeated use for pesticide degradation. In this study, we demonstrate the easy production of functionalized magnetic nanoparticles with suitably genetically modified magnetotactic bacteria that are efficient as a reusable nanobiocatalyst for pesticides bioremediation in contaminated effluents

Coproporphyrin Excretion and Low Thiol Levels Caused by Point Mutation in the Rhodobacter sphaeroides S-Adenosylmethionine Synthetase Gene ▿ †

A spontaneous mutant of Rhodobacter sphaeroides f. sp. denitrificans IL-106 was found to excrete a large amount of a red compound identified as coproporphyrin III, an intermediate in bacteriochlorophyll and heme synthesis. The mutant, named PORF, is able to grow under phototrophic conditions but has low levels of intracellular cysteine and glutathione and overexpresses the cysteine synthase CysK. The expression of molybdoenzymes such as dimethyl sulfoxide (DMSO) and nitrate reductases is also affected under certain growth conditions. Excretion of coproporphyrin and overexpression of CysK are not directly related but were both found to be consequences of a diminished synthesis of the key metabolite S-adenosylmethionine (SAM). The wild-type phenotype is restored when the gene metK encoding SAM synthetase is supplied in trans. The metK gene in the mutant strain has a mutation leading to a single amino acid change (H145Y) in the encoded protein. This point mutation is responsible for a 70% decrease in intracellular SAM content which probably affects the activities of numerous SAM-dependent enzymes such as coproporphyrinogen oxidase (HemN); uroporphyrinogen III methyltransferase (CobA), which is involved in siroheme synthesis; and molybdenum cofactor biosynthesis protein A (MoaA). We propose a model showing that the attenuation of the activities of SAM-dependent enzymes in the mutant could be responsible for the coproporphyrin excretion, the low cysteine and glutathione contents, and the decrease in DMSO and nitrate reductase activities

An estimate of the volume of a single particle from TEM images.

<p>Scale bar is 0.2 µm. (<b>a</b>) Unprocessed TEM image of the functionalized magnetosomes. (<b>b</b>) Automatic particle counting and sizing analyzed by ImageJ. (<b>c</b>) Particles size distribution computed with 7 bins (boundaries are given in the table). The surface <i>S</i> is expressed in pixel².</p

Recycling of the biocatalyst.

<p>A 500 µl aliquot of magnetosomes suspension is immobilized onto the magnetic column. A 1 ml sample contaminated with 50 µM ethyl-paraoxon is passed through the column by flow gravity and recovered for <i>p</i>-nitrophenol spectrophotometric assay. The column is stored at room temperature with the magnetosomes and the experiment is repeated two more times at 2-hour intervals. Magnetosomes still magnetically retained on the column are stored at 4°C overnight and the entire set of experiments is repeated the next day. The hydrolysis activities are plotted for each repeat (100% activity expresses the complete degradation of ethyl-paraoxon by the purified enzyme) for day 1 (red) and day 2 (blue).</p

MamC-Opd assay in the magnetosome sample.

<p>(a) Western blot assay of purified and immobilized Opd. The purified protein deposits are 117, 58, 29, 15 and 7 ng, from left to right. The magnetosome membrane was dissolved in 4% SDS and two deposits of solubilized membrane proteins were made (15 and 10 µl). Expected molecular weights for Opd and MamC-Opd are 34.6 and 49.6 kDa respectively. (b) Calibration curve for Opd quantification obtained with GeneTools image analysis software from Syngene. The computed Opd quantities in the solubilized magnetosome membrane proteins are 17.3 and 9.6 ng for the 15 and 10 µl of solubilized magnetosome membrane protein deposits (from left to right).</p

Catalytic properties of MamC-Opd in the magnetosomes sample.

<p>Michaelis-Menten curves for immobilized (black) and purified (red) Opd. For each enzymatic assays, we used 26 ng of immobilized Opd (20 µl of magnetosomes suspension) and 200 ng of soluble Opd. The mean deviation between the fitted Michaelis-Menten curves (lines) and the experimental data points (circles) is 4.2% for both curves.</p

Functionalization of bacterial magnetosomes.

<p>(<b>a, b</b>) TEM image of magnetosomes in <i>Magnetospirillum magneticum</i> AMB-1 cells. The magnetite crystals are aligned within the cytoplasm of the cells on a cytoskeleton made of actin-like proteins. (<b>c</b>) Functionalization of the magnetosome membrane. The targeted enzyme (blue) is anchored on the lipid-coated magnetite crystals by fusion with a membrane protein (yellow).</p

Changing the Ligation of the Distal [4Fe4S] Cluster in NiFe Hydrogenase Impairs Inter- and Intramolecular Electron Transfers

International audienceIn 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

Genetically tailored magnetosomes used as MRI probe for molecular imaging of brain tumor

International audienceWe investigate here the potential of single step production of genetically engineeredmagnetosomes, bacterial biogenic iron-oxide nanoparticles embedded in a lipid vesicle, as a new tailorable magnetic resonance molecular imaging probe. We demonstrate in vitro the specific binding and the significant internalization into U87 cells of magnetosomes decorated with RGD peptide. After injection at the tail vein of glioblastoma-bearing mice, we evidence in the first 2 h the rapid accumulation of both unlabeled and functionalized magnetosomes inside the tumor by Enhanced Permeability and Retention effects. 24 h after the injection, a specific enhancement of the tumor contrast is observed on MR images only for RGD-labeled magnetosomes. Post mortem acquisition of histological data confirms MRI results with more magnetosomes found into the tumor treated with functionalized magnetosomes. This work establishes the first proof-of-concept of a successful bio-integrated production of molecular imaging probe for MRI

RGD-functionalized magnetosomes are efficient tumor radioenhancers for X-rays and protons

International audienceAlthough chemically synthesized ferro/ferrimagnetic nanoparticles have attracted great attention in cancer theranostics, they lack radio-enhancement efficacy due to low targeting and internalization ability. Herein, we investigated the potential of RGD-tagged magnetosomes, bacterial biogenic magnetic nanoparticles naturally coated with a biological membrane and genetically engineered to express an RGD peptide, as tumor radioenhancers for conventional radiotherapy and proton therapy. Although native and RGD-magnetosomes similarly enhanced radiation-induced damage to plasmid DNA, RGD-magnetoprobes were able to boost the efficacy of radiotherapy to a much larger extent than native magnetosomes both on cancer cells and in tumors. Combined to magnetosomes@RGD, proton therapy exceeded the efficacy of X-rays at equivalent doses. Also, increased secondary emissions were measured after irradiation of magnetosomes with protons versus photons. Our results indicate the therapeutic advantage of using functionalized magnetoparticles to sensitize tumors to both X-rays and protons and strengthen the case for developing biogenic magnetoparticles for multimodal nanomedicine in cancer therapy