Identification of proteins in ribosomal fractions isolated from sporulating Bacillus subtilis

The aim of the project was to investigate polysome profiles and analyse ribosomes of sporulating Bacillus subtilis strains, WT and a triple knock-out (3KO) strain lacking three paralogs of ribosomal proteins, by mass spectrometry, depending on the sample preparation conditions. B. subtilis, a gram positive spore forming bacterium, was induced to sporulate and harvested three hours post sporulation induction, approximately soon after asymmetric septation, respectively. Three conditions were analysed: 1) proteins were cross-linked by addition of DSP to the culture just before harvesting, and the cells were lysed by membrane solubilisation with digitonin, 2) cell lysis was performed by membrane solubilisation with digitonin with no prior cross-linking, 3) cell lysis was performed by grinding with pestle and mortar with addition of aluminium oxide. To provide additional comparisons, analogous experiments were performed for the cultures collected in logarithmic growth phase and two hours post sporulation induction, before asymmetric septation (lysis by membrane solubilisation with digitonin and grinding respectively). For the 3KO strain, cultures were harvested 3hours post sporulation induction and lysates were prepared by grinding.