Isosurface rendered representation of sporulating Bacillus subtilis cell with membranes, cell wall, ribosomes and coat structures highlighted. Image credit: Khanna et al. (CC BY 4.0)
Much of what happens in biology occurs at scales so small that the microscopy methods traditionally used by biologists cannot visualize them. One such process is bacterial sporulation: in stressful conditions, bacteria like Bacillus subtilis can divide to produce a smaller cell called a forespore, which the larger mother cell then engulfs. The forespore matures into a hardy spore, which is able to survive in harsh environments and only transform into an active bacterium when conditions improve.
Bacterial cells are surrounded by a stiff layer of a material called peptidoglycan. This wall sits outside of the bacterium’s thin flexible membrane and determines the bacterium’s shape. At the beginning of sporulation, the forespore is separated from the mother cell by a peptidoglycan wall. Engulfment of the spore by the mother cell requires a dramatic change in the shape of this partition. Microbiologists had thought that all the rigid peptidoglycan must be degraded to allow the partition to deform flexibly during engulfment; however, no one had yet observed the tiny structures involved.
Khanna et al. directly visualized sporulation in B. subtilis using a technique called cryo-electron tomography (or cryo-ET for short). In cryo-ET, samples are cooled to low temperatures and then imaged with a beam of electrons. Cryo-ET requires thin samples, thinner even than most bacteria. By combing cryo-ET with another methodology that allowed them to focus in on thin sections of their sample, Khanna et al. generated high resolution images, which provided a look at forespore engulfment in unprecedented detail.
These images revealed that the peptidoglycan wall separating the mother cell from forespore is not completely degraded: a thin layer of peptidoglycan persists. Comparing these images to cryo-ET images of cells treated with drugs that block the production of peptidoglycan suggested a new engulfment mechanism. This includes a cycle of peptidoglycan production and degradation that accompanies the advancing mother cell membrane as it surrounds the forespore during engulfment. Khanna et al. could also see that the mother cell’s membrane formed finger-like projections as it moved around the forespore, something that was not visible with previous techniques.
This detailed engulfment mechanism is an important advance in the understanding of bacterial spore formation. Additionally, Khanna et al. have generated a collection of images, methods and analyses that may prove useful to a wide community of biologists attempting to understand sporulation and other fundamental processes that occur on a small scale.
