The long-term goal of this application is to understand the working mechanism of a supramolecular machinery, termed divisome, in carrying out bacterial cell division. The divisome is composed of more than thirty proteins that are orchestrated to assemble and function at the correct time and space to ensure successful cytokinesis. Understanding key rate-limiting steps in this process and how different proteins coordinate with each other is important to provide potential new antimicrobial targets for treating bacterial infections. In the past a major focus of the field is on the role of the essential component of the divisome, a ring-like structure (Z-ring) formed by the tubulin homolog protein FtsZ, in generating a mechanic force to constrict the membrane actively. The roles of the other two components of the divisome, septal cell wall peptidoglycan (PG) synthesis machinery and chromosome segregation machinery, are thought to follow the active contraction of Z-ring. Recently accumulating evidence suggests that the Z-ring may not be the main force generator but a key regulator/mediator, and that cell wall synthesis and chromosome segregation machineries have larger roles in driving septum closure than Z-ring contraction. The project described here will examine this hypothesis using a combination of single-molecule imaging, genetic, biochemical and structural methods.
The first Aim i s to determine the role of the Z-ring in the spatiotemporal organization of septal PG proteins. The dynamics and organizations in wild-type FtsZ cells will then be compared to those in mutant cells harboring Z-rings with altered structures, dynamics, GTPase activity, and protein-proteins interactions.
The second Aim i s to determine the role of septal PG synthesis in driving septum closure. Septal PG synthesis will be systematically perturbed using targeted mutations and drug treatments, and the corresponding time-dependent septum closure rate, septal PG incorporation rate, septa morphology and composition will be measured using a variety of single-molecule imaging and biochemical methods.
The third Aim i s to determine the structural basis responsible for the coordination between chromosome segregation and septum closing. Molecular interfaces between a suite of proteins that interact with each other and anchor the Z-ring to the chromosome will be determined using genetic, biochemical, computational and structural methods. The expected outcomes of the project are: (1) a high-resolution structural and dynamic model of the full E. coli divisome, (2) a redefined roles and relative contributions of the three major divisome components in cytokinesis, (3) molecular insight into the working mechanism of the divisome as a whole, and (4) a set of innovative imaging- based tools and assays enabling bacterial cell biologists.

Public Health Relevance

The goal of this study is to provide knowledge for the bacterial cell division process using E. coli as a model system. As this process is essential for survival and conserved across the bacterial kingdom, a better understanding will promote more effective development of new antibiotics targeting this process to combat infectious diseases caused by pathogenic bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086447-07
Application #
9199098
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Deatherage, James F
Project Start
2011-04-01
Project End
2020-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
7
Fiscal Year
2017
Total Cost
$342,630
Indirect Cost
$131,130
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205
Yang, Xinxing; Lyu, Zhixin; Miguel, Amanda et al. (2017) GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science 355:744-747
Coltharp, Carla; Xiao, Jie (2017) Beyond force generation: Why is a dynamic ring of FtsZ polymers essential for bacterial cytokinesis? Bioessays 39:1-11
Buss, Jackson A; Peters, Nick T; Xiao, Jie et al. (2017) ZapA and ZapB form an FtsZ-independent structure at midcell. Mol Microbiol 104:652-663
Woldemeskel, Selamawit Abi; McQuillen, Ryan; Hessel, Alex M et al. (2017) A conserved coiled-coil protein pair focuses the cytokinetic Z-ring in Caulobacter crescentus. Mol Microbiol 105:721-740
Xiao, Jie; DufrĂȘne, Yves F (2016) Optical and force nanoscopy in microbiology. Nat Microbiol 1:16186
Xiao, Jie; Goley, Erin D (2016) Redefining the roles of the FtsZ-ring in bacterial cytokinesis. Curr Opin Microbiol 34:90-96
Lyu, Zhixin; Coltharp, Carla; Yang, Xinxing et al. (2016) Influence of FtsZ GTPase activity and concentration on nanoscale Z-ring structure in vivo revealed by three-dimensional Superresolution imaging. Biopolymers 105:725-34
Coltharp, Carla; Buss, Jackson; Plumer, Trevor M et al. (2016) Defining the rate-limiting processes of bacterial cytokinesis. Proc Natl Acad Sci U S A 113:E1044-53
Buss, Jackson; Coltharp, Carla; Shtengel, Gleb et al. (2015) A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction dynamics. PLoS Genet 11:e1005128
Coltharp, Carla; Yang, Xinxing; Xiao, Jie (2014) Quantitative analysis of single-molecule superresolution images. Curr Opin Struct Biol 28:112-21

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