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Self-Organization of the Bacterial Cell

Periodic Reporting for period 3 - SELFORGANICELL (Self-Organization of the Bacterial Cell)

Reporting period: 2019-04-01 to 2020-09-30

Bacteria, like eukaryotic cells, have precisely regulated intracellular anatomies. Due to their small size, however, obtaining quantitative information about the underlying protein dynamics has been extremely difficult to obtain from experiments in living cells. Accordingly, we still have a very limited understanding about molecular mechanisms underlying essential functions.

For example, bacterial cell division is initiated and controlled by a cytoskeletal structure called the Z-ring, which is organized by treadmilling filaments of the tubulin homolog FtsZ. This Z-ring not only recruits other proteins to the division site, it is also thought to distribute cell wall synthesis around the diameter of the cell. Despite being essential for the life of bacteria and its important role as a pharmacological target, we still do not know how the bacterial cell division machinery works.

In this proposal, we aimed to answer three outstanding questions regarding the mechanism of bacterial cell division and the function of the Z-ring: 1. How do FtsZ associated proteins modulate the organization of FtsZ filaments? 2. How do treadmilling FtsZ filaments organize cell division proteins in space and time? 3. How do peptidoglycan synthases build the cell wall? We will answer these questions in an in vitro reconstitution approach, where we combine protein biochemistry and high resolution, quantitative microscopy. This biochemical reverse engineering approach helps us to obtain quantitative information on different spatial scales, from the large-scale ensemble level down to single molecules. Eventually, it our research findings will help us understand the dynamic organization of the internal architecture of bacterial cell.
Using an in vitro reconstitution approach my research group studied the mechanism of bacterial cell division. Since the start 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 (Caldas et al., 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.

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., BioRxiv, 2020).

In sum, we believe that our aim of rebuilding the cell division machinery in vitro will not only help to understand this complex process better, but also to identify new targets and approaches for antibiotic therapies.
The Z-ring is formed from transient assemblies of treadmilling filaments that align on the membrane surface into a dynamic, coherent cytoskeletal structure19,46. The nature of these interactions, however, are so far not known. Previous approaches to visualize the dynamic lateral interactions between treadmilling filaments were limited by imaging techniques available. Fluorescence microscopy techniques such as TIRF and SPIM allow for fast imaging of protein dynamics, but their spatial resolution is not sufficient to resolve the behavior of individual filaments within dense cytoskeletal networks. Electron microscopy and standard atomic force microscopy experiments were able to shed light onto the organization of filaments at much higher resolution, but fast dynamic information is lost from these snapshots.

In first pilot experiments, we succeeded to use super-resolution STED (stimulated emission depletion) microscopy and use high-speed atomic force microscopy (HS-AFM) to visualize how treadmilling filaments laterally interact at high spatiotemporal resolution. This information will help us understand how dynamic, transient interactions contribute to the organization of membrane-bound FtsZ filaments from an isotropic, random distribution into a well-ordered, large structure. Following a quantitative characterization, we will develop a minimal model of active-matter to understand the emergence of order. In a collaboration with the group of Edouard Hannezo (IST Austria), we will develop Brownian dynamics simulations to recapitulate the observed behavior. We will establish a theoretical phase diagram, which can be quantitatively tested. Together, these experiments will not only offer us unprecedented insight into the dynamics of FtsZ protofilament interactions. Eventually, they will also help us understand the biophysical principles for how local behavior of nanometer-sized filaments results in the self-organization of a cytoskeletal structure on a much larger scale.