Like eukaryotic cells, bacteria have multiple cytoskeletal elements. One of these bacterial cytoskeleton elements, MreB, a homolog of eukaryotic actin, has been shown to be involved in maintaining cell shape and protein localization. The mechanism by which MreB controls cell wall synthesis to maintain cell shape or direct protein localization remains unclear. MreB resembles actin structurally, and both proteins form filaments within cells. However, MreB does not form a helical structure when purified as it does in vivo. Hundreds of interacting proteins are known for eukaryotic actin, but only one interacting partner (RodZ) has been confirmed for MreB, indicating there are probably unknown accessory proteins. In order to better understand MreB function, I propose to focus on identifying and characterizing its interacting partners. In particular, I have developed a bimolecular fluorescent complementation (BiFC) assay in Escherichia coli, which will enable the study of MreB-protein interactions in vivo. I will initially use the assay to perform a functional analysis of MreB filaments, though the expression of MreB point mutants with enhanced or reduced binding to itself, followed by in vitro polymerization assays and electron microscopy to confirm these results. The role of RodZ, a known binding partner, will also be looked at for its role in filament stabilization. Secondly, I will look at the role of MreB for proper cell growth. Iwill test the hypothesis that MreB acts as a scaffold for a cell wall synthesis complex. I will also being to explore the hypothesis that MreB interacts with cellar metabolic proteins to properly localize them. An alanine scanning mutagenesis will be performed on surface exposed residues of MreB to determine if there is a single interaction domain on MreB that other proteins bind. The results of this proposal will create new knowledge of the functionality of MreB, which can then be applied by the field to the creation of novel antimicrobial compounds.
The long-term goals of this research are to understand how the bacterial MreB cytoskeleton is organized and how it functions to help the cell maintain proper growth. The lessons we learn from these relatively simple model systems will be broadly applicable to other cell biology fields because all cells have cytoskeletal elements and need to coordinate cellular processes in order to grow properly. In addition, while highly conserved, the proteins of the bacterial cytoskeleton have elements that are unique to bacteria and therefore provide possibilities for new antimicrobials to be developed that target these specific interactions.