Towards a complete structure of the intact bacterial cytokinesis machine
EMSL Project ID
47422
Abstract
All bacteria need to grow and divide in order to proliferate in the environment. This involves duplication of their genetic material, partitioning the copies to daughter cells, and finally separation of the offspring from the mother cell. This last process, called cytokinesis, is not well understood at the molecular level, even in the most well studied species, Escherichia coli. At least 10 proteins are required specifically for cell division in this species, as inactivation of any one of them results in undivided cells containing all their duplicated chromosomes. Cellular localization and protein-protein interaction studies have shown that these proteins, which include homologs of actin and tubulin, are all recruited to a membrane-bound protein complex at the site of cell division. This complex, called the divisome, acts in a concerted and highly regulated way to divide the cell, and can thus be considered to be a protein machine. Despite knowing the individual players and some of their enzymatic activities, we understand little about the molecular structure of the divisome, how many sub-complexes are present, and how this structure changes over time from initial assembly through the stages of constriction and ultimately cell separation. As many divisome components are highly conserved among diverse bacterial species, understanding the process in E. coli and other tractable model systems such as Bacillus subtilis will provide key insights into how all bacteria divide and proliferate. Furthermore, as some bacterial divisome proteins are conserved in chloroplasts, knowledge of bacterial cell division has implications for the improvement of photosynthesis efficiency. This proposal is designed to characterize the bacterial divisome structurally, by coordinating the unique proteomics and microscopy capabilities of the EMSL. Mass spectrometry (MS) of bacteria synchronized with respect to assembly and function of their divisomes will be used to quantify the amount of each protein in the divisome at different points in the cell division cycle and assess the packing arrangement of the various components. Native and crosslinking MS will permit generation of a subunit interaction map, which can then be tested with extracts from cells depleted for individual divisome proteins. Proteomics will also be used to identify protein modifications such as phosphorylation or methylation that may appear during the time course of divisome function. The MS and proteomics approaches, which are not easily available to us at UT-Houston, will be complemented by high-resolution imaging of protein structure in situ using genetically encodable tags. This imaging approach also needs EMSL expertise and capabilities, as the access to transmission EM and flow cytometric technical expertise at UT-Houston is far from ideal. These studies, initially to be done with the most tractable model systems, should be ultimately useful for the study of any bacterial species.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2012-10-01
End Date
2013-09-30
Status
Closed
Released Data Link
Team
Principal Investigator