Using an in vitro reconstitution approach my research group studied the mechanism of bacterial cell division. In the course of this project, we were able to provide answers on three important questions:
1. How do FtsZ associated proteins modulate the organization of FtsZ filaments? The Z-ring has long been known to act as a dynamic scaffold for the assembly of the bacterial cell division machinery. What governs the architecture, dynamics and stability of this cytoskeletal structure was unclear, but FtsZ-associated proteins (Zaps) had been suggested to play an important role. We used an in vitro reconstitution approach to study the influence of the well-conserved protein ZapA on the self-organization of FtsZ and FtsA on lipid membranes. Combining high-resolution fluorescence microscopy with novel quantitative image analysis (Caldas et al., 2020), we found that ZapA aligns FtsZ filaments and stabilizes filament bundles in a highly cooperative manner. Despite its strong influence on their large-scale organization, we found that ZapA binds only transiently to FtsZ filaments and has no effect on their treadmilling velocity. Our results led to a new model for how FtsZ-associated proteins can increase the precision and stability of bacterial cell division. Furthermore, we propose that the ultrasensitive response of FtsZ filaments to ZapA is important for the directional maturation of the cell division machinery
Relevant publications:
• Caldas, P., Radler, P., Sommer, C. & Loose, M. in Methods in Cell Biology (Academic Press, 2020).
• Caldas, P. et al. Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA. Nat Commun 10, 5744 (2019).
2. How do treadmilling FtsZ filaments organize cell division proteins in space and time? The Z-ring not only defines the site of cell division, treadmilling of FtsZ filaments in this structure was also found to actively distribute cell wall synthesis around the cell thereby promoting the integrity of the new formed cell. To understand how FtsZ filament dynamics are coupled to cell wall synthesis, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division (Baranova et al., 2020). We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ-FtsA filaments. Remarkably, despite their directed motion on the ensemble level, individual peptides showed random diffusion and transient confinement. Our findings reveal that divisome proteins dynamically follow treadmilling FtsZ filaments by a diffusion-and-capture mechanism, which can help to establish a moving signaling zone at the division site in vivo. This work represents an important experimental demonstration for how transient interactions can lead to the self-organization of complex biological structures.
Relevant publications:
• Baranova, N. et al. Diffusion and capture permits dynamic coupling between treadmilling FtsZ filaments and cell division proteins. Nat Microbiol 16, 1–11 (2020).
• Baranova, N. & Loose, M. Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers. Methods Cell Biol 137, 355–370 (2017).
3. How do peptidoglycan synthases build the cell wall? Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. We developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we demonstrate this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay allows unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials (Hernández-Rocamora, Baranova et al., eLife, 2020).
Relevant publications:
• Hernandez-Rocamora, V. M.*, Baranova, N.* et al. Real-time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin-binding proteins. eLife 10, (2021).
In sum, by rebuilding parts of the cell division machinery in vitro we were able to understand this complex process better, and identified new targets and approaches for antibiotic therapies.
