Modulation of Metabolism and Switching to Biofilm Prevail over Exopolysaccharide Production in the Response of Rhizobium alamii to Cadmium

Heavy metals such as cadmium (Cd2+) affect microbial metabolic processes. Consequently, bacteria adapt by adjusting their cellular machinery. We have investigated the dose-dependent growth effects of Cd2+ on Rhizobium alamii, an exopolysaccharide (EPS)-producing bacterium that forms a biofilm on plant roots. Adsorption isotherms show that the EPS of R. alamii binds cadmium in competition with calcium. A metabonomics approach based on ion cyclotron resonance Fourier transform mass spectrometry has showed that cadmium alters mainly the bacterial metabolism in pathways implying sugars, purine, phosphate, calcium signalling and cell respiration. We determined the influence of EPS on the bacterium response to cadmium, using a mutant of R. alamii impaired in EPS production (MSΔGT). Cadmium dose-dependent effects on the bacterial growth were not significantly different between the R. alamii wild type (wt) and MSΔGT strains. Although cadmium did not modify the quantity of EPS isolated from R. alamii, it triggered the formation of biofilm vs planktonic cells, both by R. alamii wt and by MSΔGT. Thus, it appears that cadmium toxicity could be managed by switching to a biofilm way of life, rather than producing EPS. We conclude that modulations of the bacterial metabolism and switching to biofilms prevails in the adaptation of R. alamii to cadmium. These results are original with regard to the conventional role attributed to EPS in a biofilm matrix, and the bacterial response to cadmium

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

Preparation and pharmacological characterization of [76Br]-meta-bromobenzylguanidine ([76Br]MBBG)

[76Br]-meta-Bromobenzylguanidine ([76Br]MBBG) was prepared from the iodinated analog (MIBG) and [76Br]NH4 using a Cu+-assisted halogen exchange reaction. [76Br]MBBG was produced in a 60–65% radiochemical yield with a specific activity of 20 MBq/nmol. In rats, biodistribution kinetic studies showed a high uptake of [76Br]MBBG in heart tissues with its maximum of 5% ID/g at 2 h p.i.; whereas 4 h p.i., the maximum of the heart-to-lung concentration ratio of 8 was observed. Metabolic studies in rats indicated that [76Br]MBBG was rapidly metabolized in plasma. However in heart tissue, 25 h p.i., 85% of the radioactivity still represented unchanged radiotracer. Pharmacological studies in rats showed that the myocardial uptake of [76Br]MBBG was similar to that of norepinephrine. After pretreatment of the rats, the uptake of [76Br]MBBG was reduced 4 h p.i. to the following values: after desipramine (DMI) to 37%, after dexamethasone (DXM) to 88% and after 6-hydroxydopamine (6-OHDA) to 16%. These preliminary results suggest that [76Br]MBBG can be useful for the assessment of heart catecholamine reuptake disorders with PET

Simple rules govern the diversity of bacterial nicotianamine-like metallophores

International audienceIn metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in $Staphylococcus\ aureus$, $Pseudomonas\ aeruginosa$ and $Yersinia\ pestis$ respectively. Depending on the species, these metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or $\alpha$-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored substrate specificity of CntM by combining bioinformatics and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity was mainly due to the amino acid at position 33 ($S.\ aureus$ numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from $P.\ aeruginosa$ preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity towards $\alpha$- ketoacids is largely governed by a single residue at position 150 of CntM ($S.\ aureus$ numbering): an aspartate at this position ensures selectivity towards pyruvate whereas an alanine leads to the consumption of both pyruvate and $\alpha$-ketoglutarate. Modifying this residue in $P.\ aeruginosa$ led to a complete reversal of selectivity. Thus, opine-type metallophore diversity is governed by the absence/presence of a $cntK$ gene encoding a histidine racemase, and the amino acid residue at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by $Paenibacillus\ mucilaginosus$, which was confirmed $in\ vitro$ and called bacillopaline

A bioluminescent arsenite biosensor designed for inline water analyzer

International audienceWhole-cell biosensors based on the reporter gene system can offer rapid detection of trace levels of organic or metallic compounds in water. They are well characterized in laboratory conditions, but their transfer into technological devices for the surveillance of water networks remains at a conceptual level. The development of a semi-autonomous inline water analyzer stumbles across the conservation of the bacterial biosensors over a period of time compatible with the autonomy requested by the end-user while maintaining a satisfactory sensitivity, specificity, and time response. We focused here on assessing the effect of lyophilization on two biosensors based on the reporter gene system and hosted in Escherichia coli. The reporter gene used here is the entire bacterial luciferase lux operon (luxCDABE) for an autonomous bioluminescence emission without the need to add any substrate. In the cell-survival biosensor that is used to determine the overall fitness of the bacteria when mixed with the water sample, lux expression is driven by a constitutive E. coli promoter P-rpoD. In the arsenite biosensor, the arsenite-inducible promoter P-ars involved in arsenite resistance in E. coli controls lux expression. Evaluation of the shelf life of these lyophilized biosensors kept at 4 degrees C over a year evidenced that about 40 % of the lyophilized cells can be revived in such storage conditions. The performances of the lyophilized biosensor after 7 months in storage are maintained, with a detection limit of 0.2 mu M arsenite for a response in about an hour with good reproducibility. These results pave the way to the use in tandem of both biosensors (one for general toxicity and one for arsenite contamination) as consumables of an autonomous analyzer in the field

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

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

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