UC San Diego

Many cellular processes in bacteria transpire at a scale of ~1-2 µm that are difficult to study by optical microscopy alone due to limitations imposed by diffraction properties of light. Hence, visualization of cellular structures at a high spatial resolution in the native cellular milieu in bacteria is a poorly explored field. My thesis aims to study the process of sporulation in a Gram-positive bacterium, Bacillus subtilis. During conditions of nutrient deprivation, B. subtills undergoes a developmental pathway that culminates in the production of two cells with different sizes and fates, the smaller forespore and the larger mother cell. I have studied different stages of sporulation in B. subtilis using novel modalities in the field of cryo-electron microscopy, namely cryo-focused ion beam milling coupled with cryo-electron tomography (or cryo-FIB-ET). Cryo-FIB-ET allows visualization of macromolecules inside the cell at a resolution of a few nanometers. Using this technique, I have elucidated important principles governing the following processes during sporulation:

(1) Polar cell division: We demonstrate that FtsAZ filaments, the major orchestrator of cell division in bacteria are localized uniformly around the leading edge of the invaginating septum during vegetative growth but present only on the mother cell side during sporulation, a process mediated by a sporulation-specific protein, SpoIIE. This asymmetry in divisome localization during sporulation dictates not only the septal thickness but also the diverse fate of the two daughter cells.

(2) Engulfment: We study the role of cell wall remodeling and chromosome translocation during engulfment. We show that the mother cell membrane migrates in finger-like projections around the forespore due to uneven cell wall degradation. We also show that the turgor pressure generated by the forespore chromosome inflates the forespore and helps it maintain its well-rounded shape. These studies have thrown light on spatiotemporal regulation of important complexes dictate architectural transformations during engulfment.

Overall, our studies have added more cases to support the observation that several complex traits to regulate critical cellular processes like division, cell migration, DNA dynamics, cell shape etc. that were previously thought to be a characteristic of only eukaryotes are also present in prokaryotes and that cryo-FIB-ET will likely be the tool of the future to probe these and other processes at a molecular detail.