An ever-growing threat to the health of our nation and the world is the increase in multi-drug resistant bacterial infections. To facilitate the design and development of new treatments, we need a better understanding of fundamental cell biological processes in bacteria. This project is designed to determine the mechanism by which the bacterial cytokinetic ring is assembled and stabilized using Escherichia coli as a model organism. Our hope is that this will lead to the discovery of new ways to disrupt the function of this essential structure. FtsZ is a structural homologue of the eukaryotic tubulin protein. Both FtsZ and tubulin form dynamic polymers that are required for a variety of vital cellular functions. In bacterial cells, FtsZ forms a ring-like structure (the Z- ring) at midcell. This structure is required for the recruitment of all other downstream components of the division machinery. In E. coli, Z-ring formation is directed to the midcell zone by the extensively characterized Min system. However, aside from this long-range, spatial regulatory system, many aspects of Z-ring formation remain mysterious. For example, it is not clear how FtsZ polymers coalesce into the Z-ring or what keeps this ring-like assemblage of dynamic polymers together once it forms. To begin addressing these issues, we initiated a study of Z-ring dynamics in live cells using time-lapse fluorescence microscopy. We noticed that midcell Z-rings occasionally fall apart in both non-constricting and constricting cells. Shortly after becoming destabilized, Z-rings reform exactly at their original position and resume the division process. Based on these observations we propose that mechanisms must be in place to: i) stabilize the Z-ring at midcell once it is formed and ii) mark the location of the Z-ring such that in instances when the ring pattern delocalizes, it can reform precisely where it was initially assembled. We hypothesize that divisome components with cell wall binding activity play key roles in these processes and that they do so by anchoring positive regulators of FtsZ polymerization to the cell wall. This would, in principle, create a tight zone within the midcell region where FtsZ polymerization is favored and thus serve to stabilize the dynamic structure and guide its reassembly. The goal of this proposal is to test this hypothesis and uncover the underlying molecular mechanisms. Preliminary evidence suggests a role for the EnvC cell wall binding protein in this process. We will investigate this further by studying Z-ring dynamics in EnvC+ and EnvC- cells. Since EnvC is entirely periplasmic, for it to function as proposed, additional factors are required to connect it with the Z-ring in the cytoplasm. We will search for these factors using a combined genetic and biochemical approach. Finally, since EnvC is not essential, we hypothesize that redundant Z-ring stabilization systems exist. We will investigate this using an unbiased genetic screen and a candidate approach focusing on other known components of the division machinery with cell wall binding activity. Overall, we anticipate that this project will shed significant new light on the process of cytokinesis in bacteria and reveal novel ways to disrupt the function of the division apparatus.

Public Health Relevance

Cell division is an essential process in all organisms, yet the mechanisms that promote division are still poorly understood. The focus of this proposal is to understand how bacterial proteins that associate with the cell wall stabilize the bacterial division machinery. Our goal is both to advance basic scientific knowledge and to identify new ways of disrupting the bacterial division machinery for the purpose of antibiotic development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F05-A (20))
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Sakalian, Michael
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Harvard University
Schools of Medicine
United States
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Peters, Nick T; Morlot, Cécile; Yang, Desirée C et al. (2013) Structure-function analysis of the LytM domain of EnvC, an activator of cell wall remodelling at the Escherichia coli division site. Mol Microbiol 89:690-701
Yang, Desiree C; Peters, Nick T; Parzych, Katherine R et al. (2011) An ATP-binding cassette transporter-like complex governs cell-wall hydrolysis at the bacterial cytokinetic ring. Proc Natl Acad Sci U S A 108:E1052-60