Proteomic response of Bacillus subtilis 3610 to nanoparticles exposition
The natural biotope of Bacillus subtilis is the upper layer of soil where it grows as a biofilm. To mimic this physiological development and study the impact of nanoparticles during the formation of a biofilm in a contaminated soil, we have studied the proteomic response of the ancestral strain Bacillus subtilis 3610, which is able to form biofilm contrary to the 168 laboratory strain. The bacteria were grown on soft agar plates containing n-ZnO, n-TiO2 or ZnSO4 metal ion.
Sample Processing Protocol
Cells were obtained from a well Bacillus subtilis strain 3610 cultures grown with or without stress, as described above. Cells were incubated in a 200 µl final volume of 20 mM phosphate buffer (pH 7.4) containing 15 µl of 30 mg/ml lysozyme and 4 µl of 1/50 benzonase solution for 20 min at 37°C. This was then centrifuged at 13,000 rpm at 4°C for 10 min. Total protein quantification was performed using the Bradford assay. Proteins were solubilized in SDS loading buffer before brief SDS-PAGE separation. They were then in-gel digested using trypsin before 2h nanoLC-MS/MS (Ultimate 3000 and LTQ-Orbitrap Velos pro, Thermo Scientific). Analytical duplicates were acquired.
Data Processing Protocol
Identification and quantification were performed using MaxQuant software (version 184.108.40.206) and Uniprot database (SwissProt, Bacillus subtilis (strain 168) taxonomy, February 2017 version). Default parameters were used, except that unqiue peptide was set to 1. 2 miscleavages were allowed, Carbamidomethyl (C) was chosen as fixed modification and Oxidation (M)and Acetyl (Protein N-term) as variable ones. Label-free quantification was performed and match between runs allowed. Analytical duplicates were summed.
Eymard-Vernain E, Luche S, Rabilloud T, Lelong C. Impact of nanoparticles on the Bacillus subtilis (3610) competence. Sci Rep. 2018 8(1):2978 PubMed: 29445231