In vitro reconstitution of bacterial cell division

Bacterial cells are not a well-mixed bag of molecules. Instead, they have a precisely controlled intracellular organization. Their building blocks assemble into complex structures at the right time and place in an apparent well-orchestrated manner. Due to their small size, however, it has been extremely difficult to study how this organization and how it is achieved. Accordingly, we still know very little about how the content of bacterial cells, their proteins and lipids, come together to fulfill their exact roles. For example, bacteria proliferate by cell division, where the mother cell splits into two daughter cells of equal size. This process is performed by a complex machinery consisting of more than a dozen different molecular components. As this machine is essential for bacterial survival, it is also the target of many antibiotic drugs that were designed to inhibit cell division and kill bacteria. Despite being so important for the life of bacteria and its role as a target for antibiotics, we still do not know how exactly the bacterial cell division machinery is assembled and how it operates. In this project, instead of looking at a complex machine inside of a tiny bacterial cell, we will rebuild it from scratch. We will prepare the building blocks of the bacterial cell division machinery and study how they self-organize using high-resolution microscopy. This approach gives us full control over the reaction conditions and direct access to the behavior of the individual components. Eventually, we will find out how the bacterial cell division machinery is built and how it operates.

Bacteria, much like human and animal cells, possess intricately organized interiors. However, their tiny size makes it exceptionally challenging to study how their internal components work together in living cells. This is especially important for understanding how bacteria divide-a process central to their survival and a prime target for antibiotics. In our project, we set out to tackle this challenge by reconstructing part of the bacterial cell division machinery outside of living cells. Using purified components, we recreated the activity of key proteins involved in this process: FtsZ, a tubulin-like protein; FtsA, an actin-related protein; and a portion of FtsN, a protein essential for activating the division machinery. Together, these proteins form dynamic filaments on the cell membrane that organize into a structure called the Z-ring. This ring is crucial for bacterial cell division, as it coordinates the activity of proteins that reshape the bacterial cell wall to divide the cell into two. To uncover the details of how these proteins self-organize, we developed new experimental tools and techniques. Here's what we discovered: 1. Behavior of FtsA on membranes: We investigated how FtsA binds to membranes, how long it stays attached, and how it interacts with itself to form larger complexes called oligomers. Importantly, we found that FtsN plays a key role in promoting this oligomerization, which is likely essential for organizing the division machinery. 2. FtsZ filaments in action: Using advanced microscopy techniques, we visualized the dynamic behavior of FtsZ filaments at a level of detail never achieved before. These experiments showed that FtsZ filaments don't form stable bundles; instead, they interact only transiently. Additionally, we discovered that the flexibility and density of these filaments influence how they interact and organize themselves. 3. A self-organizing Z-ring: Our experimental data supported a theoretical model predicting that FtsZ filaments undergo "treadmilling"-a process where one end grows while the other shrinks. This treadmilling helps align filaments and ensures that misaligned ones disappear, ultimately leading to the formation of the Z-ring at the center of the dividing cell. By combining these insights, we were able to gather precise, quantitative information about bacterial cell division, ranging from the behavior of individual proteins to the organization of the large-scale filament assemblies. This research not only deepens our understanding of how bacteria divide but also opens the door to new opportunities in medicine. Understanding the mechanics of cell division could guide the development of novel antibiotics that target this process. Furthermore, our work represents an important step toward fully reconstituting bacterial cell division in a test tube-an achievement that could revolutionize the study of bacterial physiology and drug development.