High resolution analysis of proteome dynamics during <i>bacillus subtilis</i> sporulation

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins, and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (termed blue, brown, green, and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue is negatively correlated with modules brown and green, containing ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green, and might also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation

Sleeping beauty wakes up: A quest for the developmental program steering Bacillus subtilis spore germination and outgrowth

The genera Bacillus and Clostridium form Endospores - the dormant, multilayered, highly resistant structures - in response to nutrient starvation. In presence of nutrients the spores germinate and grow out as normal vegetative cells. The Vegetative cells emerging from spores of pathogenic species additionally pose health risks and since germination itself is a highly heterogeneous process, it is challenging to attain microbial safety via the routine sterilization regimes of food and medical sectors. Therefore, the aspects of spore survival and revival are scientifically important in microbiology research. The ‘One-pot’ method described in this thesis allows for the proteome-wide analysis of the spore forming microorganisms. With identification of over 1000 proteins from the spores of Bacillus subtilis, Bacillus cereus and Clostridioides difficile the method presents a minimal sample loss. With a provision to identify immunogenic markers and putative target proteins the method is useful for the time-course studies to understand proteome turnover in the cells or spores. The studies described further, provide information about the predecessor proteins that may have important function in transition from dormant spore to the cell. An integrative analysis of the dynamics of the transcriptome and proteome of germinating spores contributes to the understanding of the planning and execution of germination. With no protein synthesis observed in a dormant spore, the sporulation conditions and the spore maturation period do not affect protein expression during spore germination. The molecular role of heat activation and dynamic interplay of mRNA and proteins during germination remain the topic of further research