Role of SpoIIIE during chromosome segregation during sporulation in <i>B. subtilis</i> and experimental setup.

<p>(a) Formation of the asymmetric sporulation septum (brown disc) divides the cell into mother cell and forespore compartments, and traps one fourth of the chromosome (black ribbon) inside the forespore (upper panel). Packaging of the remainder of the chromosome into the nascent spore (lower panel) is achieved by SpoIIIE (green disc), a septal-bound double-stranded DNA motor of the FtsK family. (b) SpoIIIE is composed of a membrane-spanning domain (orange), a 134-residue unstructured linker (brown), and a motor domain responsible for directional DNA translocation (green). A single membrane bilayer is represented by yellow circles and black sticks. (c–d) Models for the architecture of the DNA translocating complex. (c) The DNA channel model suggested that SpoIIIE hexamers on either side of fused septa assemble to form a DNA-conducting channel. (d) The sequence-directed DNA exporter model proposed that specific interactions between SpoIIIE-γ and SRS sequences (blue arrows) lead to the establishment of active SpoIIIE hexamers (green) present exclusively on the mother cell side of the septum. Inactive SpoIIIE subunits are shown in red and are located in this model on the forespore side. (e) Left panel shows a conventional epi-fluorescence image of sporulating <i>B. subtilis</i> cells, in which SpoIIIE (green) assembles in diffraction-limited foci. Membranes are shown in red. Right panel shows a PALM image of a sporulating cell undergoing sporulation, in which the green spot represents a probability density reconstruction of the localization of SpoIIIE. Scale bar, 1 µm. (f) PALM acquisition procedure. A culture of <i>B. subtilis</i> pre-incubated with the DNA intercalator sytox-green and fiducial marks are introduced into a microfluidics device (left panel). Cells are flattened by using flow force and chromosomes are imaged by epi-fluorescence microscopy. SpoIIIE is imaged by PALM (middle panel), and finally a membrane dye is introduced and an epi-fluorescence image is obtained (right panel). (g) Cells were automatically detected and their contour (dotted lines) calculated from DNA images (left image). Individual SpoIIIE proteins were detected by PALM microscopy (middle panel), and membrane images used to classify bacteria according to their stage in the cell cycle (red, dividing; blue, vegetative/pre-divisional; green, sporulating). Middle panel shows the raw image of a single frame displaying two individual single-molecules. Scale bar, 1 µm.