Abstract
The source of constriction required for division of a bacterial cell remains enigmatic. FtsZ is widely believed to be a key player, because in vitro experiments indicate that it can deform liposomes when membrane tethered. However in vivo evidence for such a role has remained elusive as it has been challenging to distinguish the contribution of FtsZ from that of peptidoglycan-ingrowth. To differentiate between these two possibilities we studied the early stages of division in Escherichia coli, when FtsZ is present at the division site but peptidoglycan synthesizing enzymes such as FtsI and FtsN are not. Our approach was to use correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) to monitor the localization of fluorescently labeled FtsZ, FtsI or FtsN correlated with the septal ultra-structural geometry in the same cell. We noted that the presence of FtsZ at the division septum is not sufficient to deform membranes. This observation suggests that, although FtsZ can provide a constrictive force, the force is not substantial at the onset of division. Conversely, the presence of FtsN always correlated with membrane invagination, indicating that allosteric activation of peptidoglycan ingrowth is the trigger for constriction of the cell envelope during cell division in E. coli.
Highlights
The source of constriction required for division of a bacterial cell remains enigmatic
To differentiate between these two possibilities we studied the early stages of division in Escherichia coli, when FtsZ is present at the division site but peptidoglycan synthesizing enzymes such as FtsI and FtsN are not
We cryogenically preserved Escherichia coli cells expressing a chromosomal copy of FtsZ-GFP by vitrification and imaged them by cryo-CLEM (Supplementary Fig. 2)
Summary
The source of constriction required for division of a bacterial cell remains enigmatic. In order to determine whether a divisome protein is contributing to constriction of the envelope in vivo, it is essential to be able to monitor its localization at the divisome, and at the same time the ultra-structure of the membranes (to detect small invaginations) To achieve this we have, in this study, used correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) as it is currently the only method that enables tracking of fluorescently labeled proteins in vivo and high-resolution cryo-electron microscopy (cryo-EM) imaging of membrane ultra-structure in the same cell[21,22] (Supplementary Fig. 1 for an overview of the workflow). Using this approach we have monitored the contributions of FtsZ, FtsI and FtsN during the initial stage(s) of envelope constriction
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