Project description:Heavy metal accumulation in agricultural areas is a global environmental problem that affects microorganisms and plants, with serious implications for human health. This study aimed to investigate the molecular responses of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5 to cobalt stress. We evaluated bacterial growth and cell viability under cobalt stress and performed comparative proteomic and reverse genetics analyses. Cobalt significantly inhibited bacterial growth but did not cause cell death. Proteomic analysis in the presence of 2.5 mM CoCl2, which caused approximately 50% growth inhibition, revealed the induction of pathways related to iron uptake, carbohydrate metabolism, amino acid metabolism, quality control, and efflux. Knockout mutants for genes involved in these pathways (∆tbdR, ∆zwf, ∆pdhB, ∆argH and ∆czcC) confirmed the essential role of the CzcC efflux system in cobalt tolerance. Cobalt stress triggers molecular responses in G. diazotrophicus PAL5, with efflux systems playing a crucial role in stress tolerance.
Project description:The goal of this study was to investigate the effects of magnetic iron cobalt oxide nanoparticles (cobalt doped Fe3O4 nanoparticles with increasing amounts of cobalt) after pulmonary exposure while in parallel presenting a proteomics platform that is easily transferable to large-scale nanotoxicology screening as part of an integrated assessment and testing approach for regulation of nanoparticles. Bronchoalveolar lavage fluid (BALF) is a proximal biofluid that can be used to monitor airway inflammation and toxic responses in the lung. It is routinely sampled for differential lung diagnostics and has been discussed as a source for early detection of lung cancer. In order to assess effects of metal oxide nanoparticles upon inhalation, bronchoalveolar lavage fluid from mice dosed by single intratracheal instillation was collected and subjected to classical biocompatibility assays as well as proteome analysis. Magnetic oxide nanoparticles with iron and cobalt oxide (Fe3-xCoxO4) at different ratios (1:0, 3:1, 1:1, 1:3, 0:1) were tested at two concentrations (54 µg, 162 µg per animal) and two time points after instillation (day 1, day 3). As a positive control carbon black nanomaterial known to induce lung inflammation was included for both time points, but only at the high dosage (162 µg per animal). Proteomics experiments were divided into three parts – test of reproducibility, discovery and screening phase. The reproducibility of the newly introduced Evosep One LC system was evaluated by re-measuring of technical replicates (n=16). During the discovery phase, selected representative samples with 3 biological replicates per group (total n=9) including pure iron oxide nanoparticles, pure cobalt oxide nanoparticles and vehicle controls were subjected to in-depth proteome profiling by extensive pre-fractionation and including isobaric tandem mass tag (TMT) labeling following a classical LC-MS/MS setup. This step allowed us to identify affected pathways and generate hypotheses regarding mechanisms of the effects of nanoparticles. During screening, all samples of the study were measured label-free as single-shot injections separated on short gradients of 21 min using the robust, high-throughput Evosep One LC system. This step allowed a fast screening of the 5 different types of magnetic metal oxide nanoparticles on BALF, at two concentrations and two time points together with their representative controls (total n=166). All screening measurements were completed in only 2.7 days.
Project description:The goal was to use microarray to compare changes in the global transcription profiles between an engineered bacterial strain and one of its descendants subject to 600 generations of experimental evolution in batch culture. This comparison allowed us to identify a beneficial mutation that substantially increased expression of a novel cobalt transporter cassette in this descendant.
Project description:Two synthetic bacterial consortia (SC) composed by bacterial strains isolated from a natural phenanthrene-degrading consortium (CON), Sphingobium sp. AM, Klebsiella aerogenes B, Pseudomonas sp. Bc-h and T, Burkholderia sp. Bk and Inquilinus limosus Inq were grown in LMM supplemented with 200 mg/L of phenanthrene (PHN) during 72 hours in triplicate.