Cell division of Staphylococcus adopts a “popping” mechanism that mediates extremely rapid separation of the septum. Elucidating the structure of the septum is crucial for understanding this exceptional bacterial cell division mechanism. Here, the septum structure of Staphylococcus warneri is extensively characterized using high-speed time-lapse confocal microscopy, atomic force microscopy, and electron microscopy. The cells of S. warneri divide in a fast “popping” manner on a millisecond timescale. Our results show that the septum is composed of two separable layers, providing a structural basis for the ultrafast daughter cell separation. The septum is formed progressively toward the center with non-uniform thickness of the septal disk in radial directions. The peptidoglycan on the inner surface of double-layered septa is organized into concentric rings, which are generated along with septum formation. Moreover, this study signifies the importance of new septum formation in initiating new cell cycles. This work unravels the structural basis underlying the “popping” mechanism that drives S. warneri cell division and reveals a generic structure of the bacterial cell. IMPORTRANCE This work shows that the septum of Staphylococcus warneri is composed of two layers and the peptidoglycan on the inner surface of the double-layered septum is organized into concentric rings. Moreover, new cell cycles of S. warneri could be initiated before the previous cell cycle is complete. This work advances our knowledge about a basic structure of bacterial cell and provides the double layered structural information of septum for the bacterium that divide with the “popping” mechanism.

Braun’s lipoprotein (Lpp) plays a major role in stabilizing the integrity of the cell envelope in Escherichia coli , as it provides a covalent cross-link between the outer membrane and the peptidoglycan layer. An important challenge in elucidating the physiological role of Lpp lies in attaining a detailed understanding of its distribution on the peptidoglycan layer. Here, using atomic force microscopy, we visualized Lpp directly on peptidoglycan sacculi. Lpp is homogeneously distributed over the outer surface of the sacculus at a high density. However, it is absent at the constriction site during cell division, revealing its role in the cell division process with Pal, another cell envelope–associated protein. Collectively, we have established a framework to elucidate the distribution of Lpp and other peptidoglycan-bound proteins via a direct imaging modality.