Добірка наукової літератури з теми "Bacillus mycoides SeITE01"

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite (SeO32−) into the less bioavailable elemental selenium (Se0) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of SeO32− exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with SeO32−. The results show substantial biochemical changes in SeITE01 cells when exposed to SeO32−. The initial uptake of SeO32− by SeITE01 cells (3h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against SeO32− are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of SeO32− to Se0. In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from SeO32− toxicity while it is converting into SeNPs.

This study is focused on biosynthesis and characterization of Selenium nanoparticles (SeNPs) by Bacillus mycoides SelTE01 (biogenic SeNPs), evaluating their ability as antimicrobial agents. In so doing, a comparison between biogenically and chemically synthesized SeNPs was carried out, in order to stress differences and similarities. During my project, I worked in the Environmental Microbiology Laboratory at University of Verona and in Biofilm Research Group at University of Calgary (Canada). At University of Verona, I synthesized biogenic SeNPs by Bacillus mycoides SelTE01 grown with Na2SeO3 and chemical SeNPs using L-cysteine, ascorbic acid or a mix of SDS and Na2S2O3. I also characterized both SeNPs using Dynamic Light Scattering (DLS), Z potential measurement, Scanning Electron Microscopy (SEM) analysis and Energy Disperse Spectrometer (EDS) analysis. In Biofilm Research Group in Calgary, I evaluated SeNPs antimicrobial activity against pathogenic biofilms, normally resistant to conventional methods of disinfection. I investigated SeNPs ability to inhibit biofilm formation, exposing pathogenic biofilms to different concentrations of SeNPs and using Minimum Biofilm Eradication Concentration (MBEC) test and Calgary Biofilm Device (CBD). MBEC test is a high throughput screening assay used to determine the efficacy of antimicrobials against biofilms. It’s based on use of CBD: particular 96-well plate in which one batch culture apparatus allows multiple species biofilms to be tested against a lot of variables. In particular, I used CBDs coated of hydroxyapatite (HA), component of bones and teeth. In so doing, I was able to verify that biogenic SeNPs have stronger antimicrobial activity than those chemically synthetized.

Selenium nanoparticles (SeNPs) are 10 to 400nm spheres composed of zero-valent selenium. SeNPs can be synthesized either chemically or biologically by microorganisms, plant extracts or enzymes. Biogenic SeNPs display a capping layer of organic molecules, which confer unique characteristics to such SeNPs, e.g. a major stability over time and a more efficient antimicrobial activity. Composition and role of the capping layer are mostly unknown and currently under investigation. In this study, environmental strains Bacillus mycoides SeITE01, Stenotrophomonas maltophilia SeITE02, Achromobacter sp. R2A, Ensifer sp. R2D and Lysinibacillus sp. R1E are considered, which are able to biosynthesize SeNPs. In the first section, SeNPs from the five bacteria are analyzed: microplate colorimetric assays are performed in order to quantify total carbohydrates, protein and lipids contents of such SeNPs capping layers. Moreover, SeNPs are treated with different protocols to remove part of the organic layer. Effect of such treatments on capping composition and SeNPs stability are studied and compared for all the five strains. In the second section, SeNPs produced by B. mycoides SeITE01 are analyzed from a proteomic point of view: biogenic SeNPs capping layer proteins are identified. Chemical SeNPs exposed to a SeITE01 cell free extract are analyzed as well. Identified proteins are compared in order to establish which proteins bind specifically biogenic SeNPs and are more probably involved in SeNPs formation. Finally, a model for SeNPs transport through SeITE01 cell wall is formulated, based on proteomic evidence. Native proteins activity assay and microscopy analysis are performed, in order to confirm the new model. In conclusion, studying the organic capping layer of biogenic SeNPs from different strains is of paramount importance to understand the effect of such molecules on SeNPs characteristics and formulate hypotheses on biosynthesis mechanism. SeITE01, SeITE02, R2A, R2D and R1E biosynthesized SeNPs show a different ratio of carbohydrates, proteins and lipids components of the capping layer and differently respond to treatments. Particularly, Gram-negative strains (SeITE02, R2A, R2D) show similar composition and respond to treatments in a similar fashion, while Gram-positive strains (SeITE01, R1E) show more variability. For SeITE01, proteomic and microscopy analyses led to a new model formulation for SeNPs transport outside the cell. Together with previous studies, this new hypothesis can contribute to a more complete vision of SeNPs synthesis in this aerobic strain.

Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02 are environmental bacterial isolates that rely on detoxification processes to transform selenite (SeO32-), a highly toxic and bioavailable chemical species of selenium, into insoluble and virtually nontoxic elemental selenium (Se0) with the formation of biogenic selenium nanoparticles (Bio-SeNPs). In the last decade, Bio-SeNPs have attracted attention for their interesting applications in the nanotechnology, industrial and medical fields not only due to their special physico-chemical features, but also for their attractive antimicrobial activities and anticancer properties. These worthwhile biotechnological traits are related to the presence on Bio-SeNPs of an external organic coating, whose composition and role are mostly unknown and currently under investigation. In the first part of this thesis, FTIR (Fourier Transform Infrared) spectroscopy was applied to study the SeO32- bio-reduction process analysing the bio-molecular composition of both SeITE01 and SeITE02 cells. The analysis was conducted during the diverse cellular growth phases and in different conditions, namely untreated (growth without the presence in the medium of sodium selenite Na2SeO3) and Se-treated (exposure to the stress factor SeO32-). Moreover, along with FTIR spectroscopic analyses, the biogenic intracellular and extracellular SeNPs bio-produced by the bacterial strains and collected with two extraction methods (vacuum filtration and pelleting processing) were examined also using DLS (Dynamic Light Scattering) measurements and TEM (Transmission Electron Microscopy) imaging. In the second part of this research, metabolomics was used to investigate the biological reduction and effect of Na2SeO3 on SeITE01 and SeITE02 cells by a LC-MS (Liquid Chromatography Mass-Spectrometry) approach. Both intracellular and extracellular metabolites and their concentration fluxes during a defined time course and in response to the exposure of bacterial cells to SeO32- were studied. From the results obtained with all the investigation techniques it was possible to observe and underline two distinct behaviors and trends assumed by the bacterial strains. SeITE01 cells showed substantial changes when exposed to SeO32-, activating a series of macromolecular responses and biochemical pathways to defend the cells against the toxic action of both the oxyanion and the Se nanostructures. Moreover, different FTIR spectral trends were acquired with regard to the organic coatings present on the surfaces of the biogenic SeNPs synthesized by this bacterium, and the most marked differences were recorded according to their different localization (intracellular or extracellular). The Bio-SeNPs production in this Gram-positive microorganism was also described by the data collected with the DLS and the zeta-potential measurements. The analyzed Bio-SeNPs showed an average dimension between 637 and 393 nm for the intracellular Bio-SeNPs, and 147 nm for the extracellular ones. Low value of negative potentials suggest a lower stability of these nanostructures in solution. On the contrary, the Gram-negative bacterium SeITE02 did not present drastic changes in the macromolecular composition after SeO32- exposure, and the variations recorded were mainly due to the maintenance of vital cellular functions. DLS data revealed an average size of 315 nm for the intracellular Bio-SeNPs and 160 nm for the extracellular ones, whilst negative potential values at or below -30 mV were recorded, indicating a remarkable stability of these biogenic Se nanostructures and a lower tendency to form aggregates and thus to precipitate. Thus, the results obtained in the course of this investigation revealed two distinct attitudes and responses on the part of the two microorganisms SeITE01 and SeITE02 to the SeO32- exposure and Bio-SeNPs production.