Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

Hernández-Rocamora, Víctor M.; Baranova, Natalia S.; Peters, Katharina; Breukink, Eefjan; Loose, Martin; Vollmer, Waldemar

doi:10.1101/2020.08.02.233189

Cited by 1 publication

(1 citation statement)

References 65 publications

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“…S3b-S3c). We then decided to use this approach to measure the degree of FtsA self-interaction on supported lipid membranes by quantifying the change in donor fluorescence after photobleaching the acceptor fluorophore [32][33][34] (Fig. 3c-e and Fig.…”

Section: Ftsa R286w Allows For Enhanced Recruitment Of Ftsncyto To Ftsz Filamentsmentioning

confidence: 99%

In vitro reconstitution of divisome activation

Radler

Baranova

Caldas

et al. 2021

Preprint

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Bacterial cell division is coordinated by the Z-ring, a cytoskeletal structure of treadmilling filaments of FtsZ and their membrane anchors, FtsA and ZipA. For divisome maturation and initiation of constriction, the widely conserved actin-homolog FtsA plays a central role, as it links downstream cell division proteins in the membrane to the Z-ring in the cytoplasm. According to the current model, FtsA initiates cell constriction by switching from an inactive polymeric conformation to an active monomeric form, which then stabilizes the Z-ring and recruits downstream proteins such as FtsN. However, direct biochemical evidence for this mechanism is missing so far. Here, we used biochemical reconstitution experiments in combination with quantitative fluorescence microscopy to study the mechanism of divisome activation in vitro. By comparing the properties of wildtype FtsA and FtsA R286W, a gain-of-function mutant thought to mimic its active state, we found that active FtsA outperforms the wildtype protein in replicating FtsZ treadmilling dynamics, filament stabilization and FtsN recruitment. We could attribute these differences to a faster membrane exchange of FtsA R286W as well as its higher packing density below FtsZ filaments. Using FRET microscopy, we also show that binding of FtsN does not compete with, but promotes FtsA self-interaction. Together, our findings shed new light on the assembly and activation of the bacterial cell division machinery and the mechanism of how FtsA initiates cell constriction.

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Section: Ftsa R286w Allows For Enhanced Recruitment Of Ftsncyto To Ftsz Filamentsmentioning

confidence: 99%