In Vivo Assembly and Arrangement of the DNA Translocase SpoIIIE During Chromosome Segregation and Membrane Fission in B. Subtilis

Abstract

SpoIIIE, a DNA translocase motor protein of the SpoIIIE/FtsK family, is responsible for chromosome segregation during bacterial cell division. In sporulating Bacillus subtilis, SpoIIIE aggregates as a focus at the mid-sporulating septum to export a circular chromosome from the mother into the daughter cell. The architecture of SpoIIIE at the focus and its mechanism of operation for the DNA translocation and the membrane fission are still controversial. Here, we directly visualize the SpoIIIE architecture at the macromolecular scale and developed a novel genetic tool to determine its operational mode during DNA translocation and membrane fission in vivo. Using a combination of these genetic tools together with quantitative single molecule imaging and fluorescence recovery after photobleaching, we demonstrate that SpoIIIE assembles as two complexes (∼40nm size), one in the mother and one in the daughter cell. In these complexes there are enough molecules to form either two or four hexamers. Moreover, we demonstrate that the mother cell-SpoIIIE is able to translocate DNA and accomplish sporulation, even in absence of daughter cell-SpoIIIE. On the other hand, SpoIIIE from both mother and daughter cells mediate the septal membrane fission. Altogether, our results indicate that two hexamers of SpoIIIE assembles into a DNA crossing channel across the sporulation septum for chromosome segregation and septal membrane fission.